BACKGROUND ART Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.","The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.","In conventional optical discs, only lands or only grooves were used to record recording marks.","However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.","a land/groove recording technique, has been brought into use as a format of optical discs.","An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).","However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.","In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.","Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).","To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.","However, only change of the duty ratio cannot bring about ample freedom.","Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.","2000-215511, referred to as Literature 1).","However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.","This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.","Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.","It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability."],[" BRIEF DESCRIPTION OF DRAWINGS FIG.","1 is a cross-sectional view that shows an optical disc according to the first embodiment of the invention FIG.","2 is a graph that shows dependency or a carrier upon the thickness of a record-assist layer in the optical disc according to the first embodiment of the invention; FIG.","3 is a graph that shows dependency of the signal amplitude upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.","4 is a graph that shows dependency of a cross talk signal upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.","5 is a cross-sectional view that shows an optical disc according to the second embodiment of the invention; FIG.","6 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; FIG.","7 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; and FIG.","8 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["TECHNICAL FIELD This invention relates to an optical recording medium and its manufacturing method especially suitable for application to an optical recording medium employing a land/groove recording technique.","BACKGROUND ART Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.","The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.","In conventional optical discs, only lands or only grooves were used to record recording marks.","However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.","a land/groove recording technique, has been brought into use as a format of optical discs.","An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).","However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.","In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.","Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).","To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.","However, only change of the duty ratio cannot bring about ample freedom.","Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.","2000-215511, referred to as Literature 1).","However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.","This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.","Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.","It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability.","DISCLOSURE OF INVENTION The Inventor made vigorous researches to overcome the above-discussed problems the conventional techniques involved.","The researches are outlined below.","The recording principle of conventional optical mediums having a multi-layered film of a recording layer for recording information signals and a record-assist layer for supporting the recording layer is presumed to rely upon that, when the temperature is raised higher than the crystallization temperature lower than the melting point of the recording layer by laser beams irradiated to the recording layer, minute crystals produced in the crystallized auxiliary layer, and crystal growth of the recording layer occurs from the minute crystals as the seed crystals.","This principle has been brought about because record marks are different in amplitude between optical discs having the auxiliary layer and optical discs without the auxiliary layer.","The Inventor put forward the researches on the record-assist layer under the above presumption.","That is, the Inventor recorded 2T pulses on samples common in duty ratio but variously different in thickness of the record-assist layer, and measured dependency of a carrier upon the recording power in lands and grooves, respectively.","As a result, the Inventor has found that the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film and that there is almost no change of the carrier in lands even when the thickness of the record-assist layer is changed.","On the other hand, as to the dependency of the carrier upon the recording power in grooves, the Inventor has found that, although the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film, when the record-assist layer is changed thickness, the carrier rises as the thickness decreases.","This tendency was observed in the range close to the optimum value.","Accordingly, the Inventor got inspiration of using the phenomenon confirmed by the above-explained experiment.","That is, in case the duty ratio and the recording power are fixed, dependency of the carrier upon the thickness of the record-assist layer arises particularly in grooves, ad the dependency upon the thickness does not almost appear in lands.","From this fact, the Inventor had the idea that the carriers can be brought into agreement between lands and grooves without the need of changing the duty ratio.","The Inventor made further researches on changes of the carrier in grooves.","That is, the Inventor calculated how the change of amplitude of the recording layer of GeTe alloy depends upon the thickness of the record-assist layer by the effective Fresnel coefficient method.","From the result of the calculation, the Inventor got the knowledge that the amplitude increases as the thickness of the record-assist layer decreases.","With reference to the value when the thickness of the record-assist layer was 30 nm, when the thickness was decreased from 20 nm to 10 nm, the increase of the amplitude was approximately 1 dB according to the calculation by the effective Fresnel coefficient method.","Through the researches by the Inventor, the above-explained phenomenon can be assumed to occur because grooves are closer to the optical pickup than lands and less affected by track grooves, and therefore become more liable to reflect the amplitude changes in response to changes of the reflectance due to the difference of the thickness.","In regard to the carrier changes on the part of the grooves, the amplitude changes of the reflectance by the change of the film structure are considered to be dominant.","The present invention has been made based on the above-explained researches.","To accomplish the object, the first aspect of the invention is an optical recording medium comprising: a disc substrate having formed a concavo-convex structure formed at least on one surface thereof; and a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, wherein d/4 BACKGROUND ART Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.\",\n \"The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.\",\n \"In conventional optical discs, only lands or only grooves were used to record recording marks.\",\n \"However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.\",\n \"a land/groove recording technique, has been brought into use as a format of optical discs.\",\n \"An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).\",\n \"However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.\",\n \"In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.\",\n \"Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).\",\n \"To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.\",\n \"However, only change of the duty ratio cannot bring about ample freedom.\",\n \"Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.\",\n \"2000-215511, referred to as Literature 1).\",\n \"However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.\",\n \"This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.\",\n \"Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.\",\n \"It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability.\"\n ],\n [\n \" BRIEF DESCRIPTION OF DRAWINGS FIG.\",\n \"1 is a cross-sectional view that shows an optical disc according to the first embodiment of the invention FIG.\",\n \"2 is a graph that shows dependency or a carrier upon the thickness of a record-assist layer in the optical disc according to the first embodiment of the invention; FIG.\",\n \"3 is a graph that shows dependency of the signal amplitude upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.\",\n \"4 is a graph that shows dependency of a cross talk signal upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.\",\n \"5 is a cross-sectional view that shows an optical disc according to the second embodiment of the invention; FIG.\",\n \"6 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; FIG.\",\n \"7 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; and FIG.\",\n \"8 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"TECHNICAL FIELD This invention relates to an optical recording medium and its manufacturing method especially suitable for application to an optical recording medium employing a land/groove recording technique.\",\n \"BACKGROUND ART Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.\",\n \"The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.\",\n \"In conventional optical discs, only lands or only grooves were used to record recording marks.\",\n \"However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.\",\n \"a land/groove recording technique, has been brought into use as a format of optical discs.\",\n \"An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).\",\n \"However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.\",\n \"In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.\",\n \"Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).\",\n \"To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.\",\n \"However, only change of the duty ratio cannot bring about ample freedom.\",\n \"Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.\",\n \"2000-215511, referred to as Literature 1).\",\n \"However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.\",\n \"This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.\",\n \"Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.\",\n \"It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability.\",\n \"DISCLOSURE OF INVENTION The Inventor made vigorous researches to overcome the above-discussed problems the conventional techniques involved.\",\n \"The researches are outlined below.\",\n \"The recording principle of conventional optical mediums having a multi-layered film of a recording layer for recording information signals and a record-assist layer for supporting the recording layer is presumed to rely upon that, when the temperature is raised higher than the crystallization temperature lower than the melting point of the recording layer by laser beams irradiated to the recording layer, minute crystals produced in the crystallized auxiliary layer, and crystal growth of the recording layer occurs from the minute crystals as the seed crystals.\",\n \"This principle has been brought about because record marks are different in amplitude between optical discs having the auxiliary layer and optical discs without the auxiliary layer.\",\n \"The Inventor put forward the researches on the record-assist layer under the above presumption.\",\n \"That is, the Inventor recorded 2T pulses on samples common in duty ratio but variously different in thickness of the record-assist layer, and measured dependency of a carrier upon the recording power in lands and grooves, respectively.\",\n \"As a result, the Inventor has found that the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film and that there is almost no change of the carrier in lands even when the thickness of the record-assist layer is changed.\",\n \"On the other hand, as to the dependency of the carrier upon the recording power in grooves, the Inventor has found that, although the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film, when the record-assist layer is changed thickness, the carrier rises as the thickness decreases.\",\n \"This tendency was observed in the range close to the optimum value.\",\n \"Accordingly, the Inventor got inspiration of using the phenomenon confirmed by the above-explained experiment.\",\n \"That is, in case the duty ratio and the recording power are fixed, dependency of the carrier upon the thickness of the record-assist layer arises particularly in grooves, ad the dependency upon the thickness does not almost appear in lands.\",\n \"From this fact, the Inventor had the idea that the carriers can be brought into agreement between lands and grooves without the need of changing the duty ratio.\",\n \"The Inventor made further researches on changes of the carrier in grooves.\",\n \"That is, the Inventor calculated how the change of amplitude of the recording layer of GeTe alloy depends upon the thickness of the record-assist layer by the effective Fresnel coefficient method.\",\n \"From the result of the calculation, the Inventor got the knowledge that the amplitude increases as the thickness of the record-assist layer decreases.\",\n \"With reference to the value when the thickness of the record-assist layer was 30 nm, when the thickness was decreased from 20 nm to 10 nm, the increase of the amplitude was approximately 1 dB according to the calculation by the effective Fresnel coefficient method.\",\n \"Through the researches by the Inventor, the above-explained phenomenon can be assumed to occur because grooves are closer to the optical pickup than lands and less affected by track grooves, and therefore become more liable to reflect the amplitude changes in response to changes of the reflectance due to the difference of the thickness.\",\n \"In regard to the carrier changes on the part of the grooves, the amplitude changes of the reflectance by the change of the film structure are considered to be dominant.\",\n \"The present invention has been made based on the above-explained researches.\",\n \"To accomplish the object, the first aspect of the invention is an optical recording medium comprising: a disc substrate having formed a concavo-convex structure formed at least on one surface thereof; and a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, wherein d/4 BACKGROUND OF INVENTION Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.","These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.","Most of these response and transform functions are linear in nature.","Many of these functions operate on a finite grid of data, frequently known as a data window.","While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.","Many application systems need non-linear functions.","Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.","These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.","Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.","The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.","What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.","The above mentioned finite grids of data are often sampled in real-time by sensor devices.","Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.","Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.","Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.","The consequence of this is that bits go unused.","By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.","Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.","What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.","Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.","There is a central problem associated with these representations, the accumulation of arithmetic errors.","This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.","Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.","The consequence of this is that as calculations progress, additional precision is required, but not available.","What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.","By way of example, many real-time DSP applications possess the following common constraints.","High speed input sample rates, often on the order of 100K to 100 Million samples per second.","High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.","Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.","Many of these applications are developed against a further constraint of short time-to-market requirement.","Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.","Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.","Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.","Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.","Commercial examples of MIMD products include the high-end products of the ADSP product line.","Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.","There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.","The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.","Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.","This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.","Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.","What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.","The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.","The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.","While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.","The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.","What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.","Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.","Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.","These computer have been largely constructed from arrays of FPGA's.","They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.","Such computer also tend to have problems with multiplication.","While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.","Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.","Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.","The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.","What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.","What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.","What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.","What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.","There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.","By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.","However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.","When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.","What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.","Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).","However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.","What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.","There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.","The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.","Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.","Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.","Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.","Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.","Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.","To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.","Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.","What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.","What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.","What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.","What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.","What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.","What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.","What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.","What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.","What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods."],[" SUMMARY OF INVENTION Certain embodiments of the invention solve all the above mentioned problems found in the prior art.","Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.","The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.","Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.","These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.","Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.","The logarithmic conversion of a zero yields a represented number with a minus-infinity.","The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.","Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.","Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.","Special-plus added to special-plus results in a special-plus.","When a special-minus number and a special-plus number are added, the result is a special-minus result.","This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.","In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.","A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.","Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.","This supports selecting input-output resources for optimal data bandwidth throughput.","They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.","The computation resource array may contain at least one instruction propagating resource.","The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.","The computation resource array may further contain at least one instruction processing resource.","Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.","The instruction processing resources may contain at least one instruction register.","The computation resource array may further contain at least one instruction fetching resource.","The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.","Certain embodiments of the invention include circuits generated by methods of this invention.","They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).","They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.","Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.","Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.","The shared bus wire bundle state is determined in part by the configuration register.","Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator."],["This application claims priority from the following provisional applications filed with the United States Patent and Trademark Office: Ser.","No.","60/204,113, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed May 15, 2000 by inventor, docket number ARITH001PR; Ser.","No.","60/215,894, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.","5, 2000 by inventor, docket number ARITH002PR; Ser.","No.","60/217,353, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.","11, 2000 by inventor, docket number ARITH003PR; Ser.","No.","60/231,873, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Sep. 12, 2000 by inventor, docket number ARITH004PR; Ser.","No.","60/261,066, entitled “Method and apparatus of a DSP resource circuit”, filed Jan. 11, 2001 by inventor, docket number ARITH005PR; and Ser.","No.","60/282,093, entitled “Method and apparatus of a DSP resource circuit”, filed Apr.","6, 2001 by inventor, docket number ARITH006PR.","TECHNICAL FIELD This invention relates to digital signal processing engines, instruction processing mechanisms, arithmetic operational units of same, as well as circuitry generated based upon use of some or all of these elements.","BACKGROUND OF INVENTION Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.","These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.","Most of these response and transform functions are linear in nature.","Many of these functions operate on a finite grid of data, frequently known as a data window.","While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.","Many application systems need non-linear functions.","Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.","These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.","Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.","The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.","What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.","The above mentioned finite grids of data are often sampled in real-time by sensor devices.","Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.","Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.","Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.","The consequence of this is that bits go unused.","By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.","Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.","What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.","Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.","There is a central problem associated with these representations, the accumulation of arithmetic errors.","This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.","Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.","The consequence of this is that as calculations progress, additional precision is required, but not available.","What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.","By way of example, many real-time DSP applications possess the following common constraints.","High speed input sample rates, often on the order of 100K to 100 Million samples per second.","High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.","Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.","Many of these applications are developed against a further constraint of short time-to-market requirement.","Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.","Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.","Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.","Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.","Commercial examples of MIMD products include the high-end products of the ADSP product line.","Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.","There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.","The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.","Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.","This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.","Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.","What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.","The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.","The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.","While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.","The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.","What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.","Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.","Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.","These computer have been largely constructed from arrays of FPGA's.","They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.","Such computer also tend to have problems with multiplication.","While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.","Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.","Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.","The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.","What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.","What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.","What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.","What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.","There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.","By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.","However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.","When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.","What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.","Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).","However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.","What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.","There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.","The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.","Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.","Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.","Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.","Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.","Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.","To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.","Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.","What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.","What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.","What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.","What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.","What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.","What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.","What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.","What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.","What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.","SUMMARY OF INVENTION Certain embodiments of the invention solve all the above mentioned problems found in the prior art.","Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.","The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.","Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.","These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.","Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.","The logarithmic conversion of a zero yields a represented number with a minus-infinity.","The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.","Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.","Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.","Special-plus added to special-plus results in a special-plus.","When a special-minus number and a special-plus number are added, the result is a special-minus result.","This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.","In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.","A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.","Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.","This supports selecting input-output resources for optimal data bandwidth throughput.","They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.","The computation resource array may contain at least one instruction propagating resource.","The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.","The computation resource array may further contain at least one instruction processing resource.","Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.","The instruction processing resources may contain at least one instruction register.","The computation resource array may further contain at least one instruction fetching resource.","The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.","Certain embodiments of the invention include circuits generated by methods of this invention.","They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).","They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.","Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.","Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.","The shared bus wire bundle state is determined in part by the configuration register.","Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator.","BRIEF DESCRIPTION OF DRAWINGS FIG.","1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention; FIG.","2 shows a refinement of FIG.","1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400; FIG.","3 shows a refinement of FIG.","2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.","4 shows a refinement of FIG.","3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.","5 shows a refinement of FIGS.","1, 2, 3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400; FIG.","6 shows a refinement of FIG.","5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100; FIG.","7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.","8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.","9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result; FIG.","10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300; FIG.","11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIG.","11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIGS.","12 to 16 show a configuration of instruction memories and MSRI[K] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples; FIG.","17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.","Note that in other embodiments of the invention, i could range over a different range; FIG.","17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.","FIGS.","18 to 20 show various configurations of PISRI's and their neighboring instruction memories; FIG.","21 shows a high level data flow diagram of a two dimensional array block 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported; FIG.","22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource and at least one datapath operational resource; FIG.","22B depicts a detail flowchart of user operation 2000 of FIG.","22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration; FIG.","23A depicts a detail flowchart of user operation 2000 of FIG.","22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration; FIG.","23B depicts a detail flowchart of user operation 2000 of FIG.","22A further performing the method of using/operating the array of computational resources; FIG.","24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500; FIG.","24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.","24A further comprised of instruction fetch mechanism 3800; FIG.","25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720; and FIG.","26 shows a detail block diagram of a branch processor 3700 extending the block diagram of FIG.","25 further comprised of cache manager 3740; FIG.","27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention; FIG.","28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.","27; FIG.","29 depicts a preferred version of ALU 1400, as used in FIG.","28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank; FIG.","30 depicts a flowchart of a method of processing numeric data, which may be variously embodied; FIG.","31 depicts a detail flowchart of operation 2222 of FIG.","30 further performing the arithmetic operation collection; FIG.","32A depicts a detail flowchart of operation 2272 of FIG.","31 further adding; FIG.","32B depicts a detail flowchart of operation 2282 of FIG.","31 further subtracting; FIG.","33A depicts a detail flowchart of operation 2292 of FIG.","31 further exponentiating; FIG.","33B depicts a detail flowchart of operation 2302 of FIG.","31 further logarithming; FIG.","34A depicts a detail flowchart of operation 2442 of FIG.","33B further setting the special part of the log-result; FIG.","34B depicts a detail flowchart of operation 2442 of FIG.","33B further setting the special part of the log-result; FIG.","35A depicts a detail flowchart of operation 2292 of FIG.","31 further exponentiating; FIG.","35B depicts a detail flowchart of operation 2302 of FIG.","31 further logarithming; FIG.","36A depicts a detail flowchart of operation 2532 of FIG.","35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.","36B depicts a detail flowchart of operation 2532 of FIG.","35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.","37A depicts a detail flowchart of operation 2232 of FIG.","30 further log-converting the input number collection member; FIG.","37B depicts a detail flowchart of operation 2232 of FIG.","30 further log-converting the input number collection member; FIG.","38A depicts a detail flowchart of operation 2242 of FIG.","30 further exp-converting; and FIG.","38B depicts a detail flowchart of operation 2242 of FIG.","30 further exp-converting.","DETAILED DESCRIPTION OF DRAWINGS As used herein a wire refers to a path connecting nodes of a circuit which carries a state between the connected nodes and/or refers to a resonant cavity propagating information in terms of state between the connected nodes.","A wire may be made out of metal, an optical chamber, or a tunnel path through a molecular substrate.","A wire bundle is a collection of at least one wire.","State as used herein refers to an element of a finite alphabet, which contains at least two symbols.","These two minimal symbols relate to ‘0’ and ‘1’ as used in Boolean logic.","FIG.","1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention.","The basic arithmetic unit 1400 includes a partitioning wire bundle 1002 presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1008 is presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1010 is presented only to first memory circuit 1200.First memory instruction wire bundle 1202 is presented to first memory circuit 1200.First memory circuit 1200 generates signals for first memory output wire bundle 1016 presented to first ALU 1300.First ALU instruction wire bundle 1302 is presented to first ALU circuit 1300.First ALU 1300 receives a carry-in wire bundle 1150, first ALU instruction wire bundle 1302 and generates a carry-out wire bundle 1152 and further generates signals for first ALU output wire bundle 1018.The basic arithmetic processing unit operates by receiving the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018, as well as first memory instruction wire bundle 1202, first ALU instruction wire bundle 1302 and carry-input wire bundle 1150.The received signal state of the partitioning wire bundle 1.002, further determines the operational partitioning of first memory circuit 1200 and first ALU circuit 1300 with regards to the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018.First memory circuit 1200 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010 and first memory instruction wire bundle 1202.Partitioning wire bundle 1002 signal state is used by first memory circuit 1200 to determine from the signal state of first memory instruction wire bundle 1202 at least one of first memory local instructions to be executed.","The first memory local instructions are executed by the first memory circuit 1200.First memory circuit 1200 asserts the signal state of first memory output wire bundle 1016.First ALU 1300 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, first memory output wire bundle 1016, first ALU instruction wire bundle 1302 and the effect of the signal state of carry-input wire bundle 1150.Partitioning wire bundle 1002 signal state is used by first ALU circuit 1300 to determine from the signal state of first ALU instruction wire bundle 1302 of at least one of first first ALU local instructions to be executed.","The first ALU local instructions are executed by the first ALU circuit 1300.First ALU circuit 1200 asserts the signal state of first ALU output wire bundle 1018.FIG.","2 shows a refinement of FIG.","1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400.Note that FIG.","2 operates in much the same fashion as FIG.","1, excepting Input-output circuit 1100 acts as an interface between at least three external data bus wire bundles, 1004 and 1006 controlled by instruction wire bundle 1102 as well as partitioning wire bundle 1002 and creating the state on wire bundles 1008, 1010 and 1012.FIG.","3 shows a refinement of FIG.","2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.","3 operates in a similar fashion to FIGS.","1 and 2 with regards to elements of similar reference numbers.","Second memory circuit 1250 may receive output 1018 from ALU 1300 as well as wire bundle 1014 from input-output circuit 1100 based upon partitioning wire bundle 1002.Second memory circuit 1250 may drive the state of wire bundle 1014.Second memory circuit 1250 may receive wire bundle 1012 from input-output circuit 1100.FIG.","4 shows a refinement of FIG.","3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.","4 operates in a similar fashion to FIG.","1, 2, and 3 with regards to elements of similar reference numbers.","FIG.","5 shows a refinement of FIGS.","1, 2,3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.","2, 3 and 4.FIG.","6 shows a refinement of FIG.","5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.","2, 3 and 4.FIG.","7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.FIG.","8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.In FIGS.","8 and 9, each ALU circuit 1310 may support a consistent datapath bit width.","Each ALU circuit 1310 may further support a constant datapath bit width.","The constant datapath bit width may be at least one of 3, 4, and 5.In FIGS.","8 and 9, ALU circuit 1300 may contain instances of ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.The number of instances of ALU circuit 1310 is a power of two not necessarily shown in these Figures.","The number of instances of ALU circuit 1310 may not be a power of two.","The number of instances of ALU circuit 1310 may be 12.The constant bit width of ALU circuit 1310 may be 4 and the number of instances of ALU circuit 1310 belongs to a collection comprising 8, 12, 16, 24, 32, 48 and 64.The constant bit width of ALU circuit 1310 is 3 and the number of instances of ALU circuit 1310 belongs to a collection comprising 12, 16, 24, 32, 48 and 64.FIG.","9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result.","Each shifter (S1, S2, S2, and S4)is controlled by add-instruction-components belonging to a collection comprising at least values representing same-sign, reverse-sign, do-not-use, shift-up and shift-down.","The effects of these values acting on the add-input are as follows: The add-input acted upon by the corresponding add-inst-component having same-sign generates the add-input.","The add-input acted upon by the corresponding add-inst-component having reverse-sign generates the negative of the add-input.","The add-input acted upon by the corresponding add-inst-component having do-not-use generates a zero.","The add-input acted upon by the corresponding add-inst-component having shift-up generates a positive power of two times the add-input.","The add-input acted upon by the corresponding add-inst-component having shift-down generates a negative power of two times the add-input.","The shift-instruction collection may further include shift-up-by-m and shift-down-by-m, where m is at least two.","Partitioning wire bundle 1002 is used to control carry propagation and shift bit propagation, but are not shown in this Figure to simplify the discussion.","The omission of partitioning wire bundle 1002 is not meant to limit nor require its presence for all embodiments of the invention.","Note that the logarithm of 0 will be negative-infinity, and that the square and square root of 0 are each 0.To preserve these facts in the logarithmic domain, shifting negative-infinity should produce negative infinity.","The add-inst controls for each shifter S1-S4 may further include shift-up-2 and shift-down-2, supporting shifting by 2 bits, as well as shift-up-3 and shift-down-3 supporting shifting by 3 bits.","Certain preferred embodiments of the invention employ a subset of all these add-inst controls including same-sign, reverse-sign, do-not-use, shift-up, shift-down, shift-up-2, shift-up-3, which may be coded as 2 bits designating same-sign, reverse-sign, do-not-use, shift-down; and 2 additional bits coding pass-through, shift-up, and shift-up-2.Note that as shown hereafter, when do-not-use is asserted, it does not matter what the other two bit field contains.","In general, when do-not-use is asserted, the contents of the other two bit field will be chosen to optimize at least one system characteristic, such as testability and/or logic complexity and/or signal propagation through the circuit, also known as the circuit's critical path delay.","The add-inst control signals may originate from the datapath instruction being presented for execution, or may alternatively be generated based upon part of a numeric data to perform a limited range of multiplications.","Consider the following table Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 do-not-use same-sign, shift-up-1 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => 4 same-sign, shift-up-2 do-not-use 101 => 5 = 4 + 1 same-sign, shift-up-2 same-sign, pass-through 110 => 6 = 4 + 2 same-sign, shift-up-2 same-sign, shift-up-1 111 => 7 = 8 − 1 reverse-sign, pass-through same-sign shift-up-3 Table One: a three bit multiplication based upon controlling a pair of shifter inputs.","Note that this further supports the circuitry shown in FIG.","9 as supporting the multiplication of 6 bits of an operand acting upon a second number, which may be further provided by a local memory.","Using this circuitry in such a fashion provides an interpolation capability supporting successive approximations, which may involve linear, multi-linear and through the feedback-cascading of partial results based upon this circuit, some forms of non-linear approximations to various non-linear functions including but not limited to various exponential functions, logarithms, trigonometric functions, among others.","An alternative approach to approximation interprets a three bit number as a signed number, leading to the following table: Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 same-sign, pass-through same-sign, pass-through 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => −4 reverse-sign, shift-up-1 reverse-sign, shift-up-1 101 => −3 = −4 + 1 reverse-sign, pass-through reverse-sign, shift-up-1 110 => −2 reverse-sign, pass-through reverse-sign, pass-through 111 => −1 reverse-sign, pass-through do-no-use Table Two: a signed three bit multiplication based upon controlling a pair of shifter inputs.","Note that these two tables may be concurrently employed in certain situations where a signed 6 bit numeric multiplication is desired.","The most significant 3 bits affect multiplication as shown in Table Two.","When the sign of the 6 bit quantity is negative, the least significant 3 bits may affect the multiplication as shown in Table One, with every instance of reverse-sign changed to same-sign, and every instance of same-sign changed to reverse-sign.","When the sign of the 6 bit quantity is positive, Table One may be used as shown.","These tables and discussions have been provided by way of example and are not meant to limit the scope of the claims.","As one of skill in the art will readily recognize, there are many alternative notations for the various operations presented herein which are essentially equivalent to the examples presented herein.","FIG.","10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300, in accordance with certain embodiments of the invention.","In certain embodiments of the invention, only one instruction memory 3200 is coupled to Arithmetic Processor Array 3000, feeding one instruction to each Arithmetic Processor 3002.FIGS.","11A to 16 refer to such embodiments of the invention.","Note that these embodiments of the invention do not require the partitioning wire bundle 3102, nor the partitioning wire bundle 1002.FIG.","11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.","Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.","Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.","The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIG.","11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.","Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.","Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.","The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIGS.","12 to 16 show a configuration of instruction memories and MSRI[k] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples.","Assume the horizontal datapath width of the ALU2 and memories in each MSRI is at least 3, preferably 4, bits.","When the horizontal datapath width of the ALU2 and memories in each MSRI is 4 bits, 10 bit samples could also be accepted.","The data stream enters MSRI[4] in FIG.","12 flowing to MSRI[5], then MSRI[6], MSRI[7], then MSRI[8] of FIG.","13 eventually leaving MSRI[231 having performed as a 12 pass arithmetic stream processor.","If each MSRI component performs a radix 4 FFT, then the system as a whole performs a 1K FFT by the time the data leaves MSRI[21], and a 4K FFT upon leaving MSRI[23].","The increase in depth of the MSRI's is to consistently extend the precision of the calculations to account for the accumulation of rounding errors.","In certain other embodiments of the invention, two instruction memories 3200 and 3300 are coupled to Arithmetic Processor Array 3000, feeding two instructions to each Arithmetic Processor 3002, the selection of which instruction is executed determined by the partitioning wire bundle 3102, which in turn drives the partitioning wire bundle 1002.FIGS.","17A to 20 refer to such embodiments of the invention.","FIG.","17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.","Note that in other embodiments of the invention, i could range over a different range.","Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the firstmemories 1210 as second memories.","Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.","The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[k] cell array where i ranges from 1 to k. This circuit and the circuit of FIG.","17B use the partitioning wire bundle 1002 to control carry propagation and determination of which of the two instructions are executed in each horizontal strip of the PISRI.","FIG.","17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.","Note that in other embodiments of the invention, i could range over a different range.","Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.","Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.","The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[K] cell array where i ranges from 1 to k. FIGS.","18 to 20 show various configurations of PISRI's and their neighboring instruction memories.","In FIG.","18, PSRI[11] can be configured to act as an MSRI[4] and MSRI[7], or as an MSRI[5] and MSRI[6].","The net effect can then be the processing flow of MSRI[4]==>MSRI[5]==>MSRI[6]==>MSRI[7].","Similarly, the PSRI[19]'s can be configured as ==>MSRI[9]==>MSRI[10]==>MSRI[11].","FIG.","19 shows a configuration of same size PISRI[8]'s which can be support dataflows as discussed above, and which provide a 32 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 4 bits.","Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.","FIG.","20 shows a configuration of same size PISRI[12]'s which can be support dataflows as discussed above, and which provide a 36 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 3 bits.","Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.","FIG.","21 shows a high level data flow diagram of a two dimensional array blocks 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported.","Data flow as depicted in FIG.","21 supports both nearest neighbor and global communication of array blocks in each of the two dimensions of the array.","Note that the vertical communications lines shown in FIG.","21 may be used to transfer data via an external data memory interface which is not shown.","Such an external data memory interface may receive addressing signal either from datapath element(s), and/or from one or more instruction processors.","FIG.","22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource in accordance with certain embodiments of the invention.","User operation 2000 starts the usage of this flowchart.","Arrow 2002 directs the usage flow from user operation 2000 to user operation 2004.User operation 2004 performs selecting the input-output resources to create an input-output access collection comprised of at least one input-output access parameter Arrow 2006 directs usage from user operation 2004 to user operation 2008.User operation 2008 performs selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.","Arrow 2010 directs usage from user operation 2008 to user operation 2012.User operation 2012 terminates the usage of this flowchart.","Note that in other embodiments of the invention, the flowchart of FIG.","22A may be used to depict a method of operating an array of computational resources containing at least one input-output resource, at least one datapath operational resource.","The array of computational resources may be implemented using one or more programmable logic devices, which may include or more programmable logic arrays and/or one or more Filed Programmable Gate Arrays (FPGAs).","Certain further embodiments of the invention include the array of computation resources further containing at least one instruction propagating resource.","FIG.","22B depicts a detail flowchart of user operation 2000 of FIG.","22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration, in accordance with certain embodiments of the invention.","Arrow 2030 directs the usage flow from starting user operation 2000 to user operation 2032.User operation 2032 performs selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.","Arrow 2034 directs usage from user operation 2032 to user operation 2036.User operation 2036 terminates the usage of this flowchart.","FIG.","23A depicts a detail flowchart of user operation 2000 of FIG.","22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration in accordance with certain embodiments of the invention.","Arrow 2050 directs the usage flow from starting user operation 2000 to user operation 2052.User operation 2052 performs selecting the instruction processing resources based upon the input-output access collection and based upon the datapath operational resource allocation collection and based upon the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration.","Arrow 2054 directs usage from user operation 2052 to user operation 2056.User operation 2056 terminates the usage of this flowchart.","Certain further embodiments of the invention include the array of computation resources further containing at least one instruction fetching resource and the instruction processing resources containing at least one instruction register.","FIG.","23B depicts a detail flowchart of user operation 2000 of FIG.","22A further performing the method of using/operating the array of computational resources in accordance with certain embodiments of the invention.","Arrow 2070 directs the usage flow from starting user operation 2000 to user operation 2072.User operation 2072 performs fetching using the instruction fetching resource to create a fetched instruction.","Arrow 2074 directs usage from user operation 2072 to user operation 2076.User operation 2076 performs loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.","Arrow 2078 directs usage from user operation 2076 to user operation 2080.User operation 2080 terminates the usage of this flowchart.","FIG.","24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500, in accordance with certain embodiments of the invention.","Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.","In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.","24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.","24A further comprised of instruction fetch mechanism 3800, in accordance with certain embodiments of the invention.","Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.","In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.","25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720, in accordance with certain embodiments of the invention.","Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.","In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.","Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.","FIG.","26 shows a detail block diagram of an branch processor 3700 extending the block diagram of FIG.","25 further comprised of cache manager 3740, in accordance with certain embodiments of the invention.","Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.","In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.","Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.","FIG.","27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention.","The DSP Resource Circuit is comprised a Datapath Resource Array 5000.The Datapath Resource Array 5000 is coupled to at least one of the following: the Digital Device Interface 5300 and the System and Control Interface 5400.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5312,5314 and/or 5316 with Digital Device Interface 5300.In certain applications, coupling 5312 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.","Coupling 5314 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5316 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.","Note that the couplings 5302, 5304, and 5306 preferably respectively relate to the external communications associated with couplings 5312, 5314, and 5316 in such applications.","In other applications, one input-output processor may strictly receive data via coupling 5316 which is generated based upon an external input stream via coupling 5306.Additionally, an input-output processor may strictly output data via coupling 5312 which is used to generate an external data stream presented via coupling 5302.In certain applications, each of these coupling may be preferably split into two such couplings, each under the control of a separate input-output processor.","The Datapath Resource Array 5000 may also be coupled to a Local Memory Interface 5500.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5512,5514 and/or 5516 to Local Memory Interface 5500.Preferably, coupling 5512 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.","Coupling 5514 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5516 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.","Note that preferably, couplings 5502, 5504, and 5506 respectively relate to the external communications associated with couplings 5512, 5514, and 5516.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5402 and/or 5404 to System and Control Interface 5400.Each or either of couplings 5402 and 5404 may be comprised of a collection of couplings such as discussed above in FIG.","27 with unidirectional couplings and possibly strictly bi-directional couplings.","In certain applications, coupling 5402 may preferably convey system control and status information related via 5406 with an external system environment.","In certain applications, coupling 5404 may convey data communicated via 5406 with the external system environment.","Such data may be provided during system initialization time for conveyance not only into internal memory within the Datapath Resource Array 5000.Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Local Memory Interface 5500 for storage in local memory.","Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Digital Device Interface 5300 for use elsewhere.","FIG.","28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.","27.Datapath Resource Array 5000 is comprised of at least one instruction processor 3800 and an array of DSP resources 1400.By way of example, FIG.","28 depicts an array of 8 rows and 8 columns of DSP resource 1400 instances.","Instruction processor 3800 may be closely coupled with an instruction memory array 3500.In certain applications, Datapath Resource Array 5000 is preferably comprised of two instruction processors 3800-1 and 3800-2.At this level of abstraction the partition control wire bundle is not visible.","However, it is assumed to support partitioning the array of DSP resources into two horizontal regions.","By way of example, the top three rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 1 3800-1.The remaining bottom 5 rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 2 3800-2.In certain applications a further partitioning of instruction processing resources may be preferred.","An instruction processor 3800 may be partitioned into two instruction processing streams, each with independent branch mechanism.","The first instruction processing stream may be partitioned to control the instruction state asserted for the three left-most columns of the array of DSP resources.","The second instruction processing stream would control the remaining 5 columns of the array of DSP resources.","Note that in certain applications, partitioning may support more than two instruction processing streams being sent from an instruction processor.","For simplicity of discussion, no more than two instruction streams will be discussed hereafter.","This is not meant to limit the scope of the claims.","By way of example, the applications and configurations of FIGS.","12 to 20 may be implemented by circuitry illustrated in FIG.","28.Digital Device Interface 5300 is comprised of at least one at least one input-output instruction processor 1130-1 controlling an input-output processor 1120-1.Digital Device Interface 5300 may be further comprised of a second input-output instruction processor 1130-2 controlling input-output processor 1120-2.In certain applications, at least one input-output instruction processor 1130 may be coupled to an input-output instruction memory 1140.Input-output processors preferably possess couplings to all the rows of their associated array of DSP resources 1400.Input-output processors preferably configure communication to the array of DSP resources based upon the partition state information which configures the rows of the array of DSP resources to communicate together.","By way of example, the first input-output processor 1120-1 may communicate with the top three rows of the array of DSP resources, when they are so partitioned.","The second input-output processor 1120-2 may then preferably communicate with the remaining 5 bottom rows.","Note that the rows of DSP resources may be partitioned into more than two communicating components.","Similarly, the Digital Device Interface 5300 may comprise more than two input-output instruction processors controlling more than two input-output processors.","Each input-output processor may include at least one of the following: a data memory, ALU, and specialized logical functions such as bit packing-unpacking circuits.","Note that the circuitry of FIGS.","27 and 28 may further reside in a package where at least one of the interfaces couples to analog circuitry including but not limited to at least one of the following: A/D converters, D/A converters, frequency synthesizers, threshold detectors, and amplifiers.","Note that implementations of multiple instances of the circuitry of FIG.","28 may include situations where one input-output processor of a first instance may not couple to elements of another array of DSP resources 1400 within another instance.","System and Control Interface 5400 may be comprised of at least one input-output processor 1120-3 controlled by input-output instruction processor 1130-3.The preceding discussion regarding the Digital Device Interface is applicable to System and Control Interface 5400, and will not be repeated for reasons of brevity.","However, this is not meant limit the scope of the claims.","A common branching mechanism is preferably employed through the instruction processors discussed in FIG.","28.This branching mechanism can be embodied to support caching and accessing of memory through a local memory interface 5500, or may be embodied without support of external memory.","The overall instruction processing principles embodied in this invention include the following design/architectural goals: Input-output and datapath configurations dominate the embodied architectures, not the other way around.","The hardware supports software debugging and test.","The embodied architectures can support multiple systems levels of instruction fetching.","They can support both SIMD and MIMD, as well as SISD and MISD processing applications.","Implementations support separate references to data and instructions.","There are several consequences to separate data and instruction references.","The runtime environments of procedural languages such C, C++, PASCAL, FORTRAN and JAVA.","By employing an invariant instruction set across a variety of datapath size ranges, a single program can handle a wide range of input data sizes with the arithmetical precision preserved by construction.","Another consequence is the requirement of parameter passing to functions and subroutines by reference only.","Consider for a moment the runtime stack frame requirements of the C programming language.","C's runtime stack frame contains the following components: Branch related pointers, loop counters, data address references and data values, all of which may have differing data widths.","By partitioning the stack frame into multiple stack frame into separate stack frames to handle differing data widths, the communication and manipulation of data to “fit” onto a single stack frame, or “fit” back into the processing element where it is needed is minimized.","There are further architectural preferences which ease the task of compilation of procedural language programs as well as improve the reliability of these translations.","FIG.","29 depicts a preferred version of ALU 1400, as used in FIG.","28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank.","Arithmetic processor 1400 preferably contains 3 ALUs, 1300-1, 1300-2 and 1300-3, with two memories 1200-1 and 1200-2 respectively feeding 1016 and 1018 the first two ALUs 1300-1 and 1300-2.The third ALU 1300-3 is feed by at least ALU 1300-2 and wire bundle 1002.System input 1002 preferably contains representations of normal numbers and numbers in a logarithmic domain.","The logarithmic domain will be discussed in detail shortly.","The normal number representation be stored in memory 1200-1.ALU 1300-2 may further be implemented in a fashion as shown in FIG.","9, with shift controls being provided from part of the numeric signaling received from wire bundle 1018, as well as at least part of the addressing being provided by parts of the numeric signals received from wire bundle 1018.The results of the two ALU 1300-1 and 1300-2 operations are sent 1018 and 1036 to ALU 1300-3, where the results are combined to form an efficient and very accurate approximation of a sum of numerical products.","As mentioned earlier, there are some fundamental problems with multipliers.","Most importantly, they tend to grow with the product of the input precision.","While this may be somewhat constrained by output precision requirements, it remains a fundamental problem.","An approach that solve this problem will now be described.","Consider the action of representing each member of a number collection by an integer part and a special part, where the special part contains exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity.","FIG.","30 depicts a flowchart of a method of processing numeric data, which may be variously embodied.","Arrow 2210 directs the flow of execution from starting operation 2200 to operation 2212.Operation 2212 performs representing each member of a number collection by an integerpart and a special part.","Arrow 2214 directs execution from operation 2212 to operation 226.Operation 226 terminates the operations of this flowchart.","Arrow 2220 directs the flow of execution from starting operation 2200 to operation 2222.Operation 2222 performs performing at least one member of the arithmetic operation collection upon at least one of the members of the number collection.","Arrow 2224 directs execution from operation 2222 to operation 226.Operation 226 terminates the operations of this flowchart.","Certain embodiments of the invention may perform all members of the arithmetic operation collection upon the relevant member of the number collection.","Certain embodiments of the invention may further include one or both of the following operational steps.","Arrow 2230 directs the flow of execution from starting operation 2200 to operation 2232.Operation 2232 performs log-converting a member of an input number collection to create a member of the number collection.","Arrow 2234 directs execution from operation 2232 to operation 226.Operation 226 terminates the operations of this flowchart.","Arrow 2240 directs the flow of execution from starting operation 2200 to operation 2242.Operation 2242 performs exp-converting a member of the number collection to create a member of an output number collection.","Arrow 2244 directs execution from operation 2242 to operation 2216.Operation 2216 terminates the operations of this flowchart.","FIG.","31 depicts a detail flowchart of operation 2222 of FIG.","30 further performing the arithmetic operation collection.","Arrow 2270 directs the flow of execution from starting operation 2222 to operation 2272.Operation 2272 performs adding the first number to the second number to create an add-result.","Arrow 2274 directs execution from operation 2272 to operation 2276.Operation 2276 terminates the operations of this flowchart.","Arrow 2280 directs the flow of execution from starting operation 2222 to operation 2282.Operation 2282 performs subtracting the first number by the second number to create a subtract-result.","Arrow 2284 directs execution from operation 2282 to operation 2276.Operation 2276 terminates the operations of this flowchart.","Arrow 2290 directs the flow of execution from starting operation 2222 to operation 2292.Operation 2292 performs exponentiating the first number to create an exp-result.","Arrow 2294 directs execution from operation 2292 to operation 2276.Operation 2276 terminates the operations of this flowchart.","Arrow 2300 directs the flow of execution from starting operation 2222 to operation 2302.Operation 2302 performs logarithming the first number to create a log-result.","Arrow 2304 directs execution from operation 2302 to operation 2276.Operation 2276 terminates the operations of this flowchart.","Note that the number collection is further comprised of the add-result, the subtract-result, the exp-result and the log-result.","FIG.","32A depicts a detail flowchart of operation 2272 of FIG.","31 further adding.","Arrow 2330 directs the flow of execution from starting operation 2272 to operation 2332.Operation 2332 performs determining whether the special part of the first number contains the negative-infinity.","Arrow 2334 directs execution from operation 2332 to operation 2336.Operation 2336 terminates the operations of this flowchart.","Arrow 2340 directs the flow of execution from starting operation 2272 to operation 2342.Operation 2342 performs determining whether the special part of the second number contains the negative-infinity.","Arrow 2344 directs execution from operation 2342 to operation 2336.Operation 2336 terminates the operations of this flowchart.","Arrow 2350 directs the flow of execution from starting operation 2272 to operation 2352.Operation 2352 performs setting the special part of the add-result to contain the negative-infinity whenever the special part of at least one member of the collection the first number and the second number contains the negative-infinity.","Arrow 2354 directs execution from operation 2352 to operation 2336.Operation 2336 terminates the operations of this flowchart.","FIG.","32B depicts a detail flowchart of operation 2282 of FIG.","31 further subtracting.","Arrow 2370 directs the flow of execution from starting operation 2282 to operation 2372.Operation 2372 performs determining whether the special part of the first number contains the negative-infinity.","Arrow 2374 directs execution from operation 2372 to operation 2376.Operation 2376 terminates the operations of this flowchart.","Arrow 2380 directs the flow of execution from starting operation 2282 to operation 2382.Operation 2382 performs setting the special part of the subtract-result to contain the negative-infinity whenever the special part of the first number contains the negative-infinity.","Arrow 2384 directs execution from operation 2382 to operation 2376.Operation 2376 terminates the operations of this flowchart.","FIG.","33A depicts a detail flowchart of operation 2292 of FIG.","31 further exponentiating.","Arrow 2410 directs the flow of execution from starting operation 292 to operation 2412.Operation 2412 performs determining whether the special part of the first number contains the negative-infinity.","Arrow 2414 directs execution from operation 2412 to operation 2416.Operation 2416 terminates the operations of this flowchart.","Arrow 2420 directs the flow of execution from starting operation 292 to operation 2422.Operation 2422 performs setting the special part of the exp-result to contain the not-negative-infinity and setting the integer part to a zero-representation whenever the special part of the first number contains the negative-infinity.","Arrow 2424 directs execution from operation 2422 to operation 2416.Operation 2416 terminates the operations of this flowchart.","FIG.","33B depicts a detail flowchart of operation 2302 of FIG.","31 further logarithming.","Arrow 2430 directs the flow of execution from starting operation 2302 to operation 2432.Operation 2432 performs determining whether the integer part of the first number is essentially equal to the zero-representation.","Arrow 2434 directs execution from operation 2432 to operation 2436.Operation 2436 terminates the operations of this flowchart.","Arrow 2440 directs the flow of execution from starting operation 2302 to operation 2442.Operation 2442 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number essentially equals the zero-representation.","Arrow 2444 directs execution from operation 2442 to operation 2436.Operation 2436 terminates the operations of this flowchart.","Note that the integer part of each of the number collection members may be in a non-redundant numeric notation.","FIG.","34A depicts a detail flowchart of operation 2442 of FIG.","33B further setting the special part of the log-result.","Arrow 2470 directs the flow of execution from starting operation 2442 to operation 2472.Operation 2472 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number equals the zero-representation.","Arrow 2474 directs execution from operation 2472 to operation 2476.Operation 2476 terminates the operations of this flowchart.","Alternatively, the integer part of each of the number collection members may be in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances.","FIG.","34B depicts a detail flowchart of operation 2442 of FIG.","33B further setting the special part of the log-result.","Arrow 2490 directs the flow of execution from starting operation 2442 to operation 2492.Operation 2492 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number is a member of the zero-representation collection.","Arrow 2494 directs execution from operation 2492 to operation 2496.Operation 2496 terminates the operations of this flowchart.","Note that the integer part of each member of the number collection may contains a sign and a magnitude.","Further, for each member of the number collection, the sign may be a member of a sign collection consisting essentially of a positive-sign and a negative-sign.","The special part of each member of the number collection may further contain exactly one member of a second special value collection comprising a special-minus and a special-plus.","FIG.","35A depicts a detail flowchart of operation 2292 of FIG.","31 further exponentiating.","Arrow 2510 directs the flow of execution from starting operation 2292 to operation 2512.Operation 2512 performs setting the sign of the exp-result to essentially the negative-sign whenever the special part of the first number contains the special-minus.","Arrow 2514 directs execution from operation 2512 to operation 2516.Operation 2516 terminates the operations of this flowchart.","FIG.","35B depicts a detail flowchart of operation 2302 of FIG.","31 further logarithming.","Arrow 2530 directs the flow of execution from starting operation 2302 to operation 2532.Operation 2532 performs determining whether the sign part of the first number is essentially equal to the negative-sign.","Arrow 2534 directs execution from operation 2532 to operation 2536.Operation 2536 terminates the operations of this flowchart.","Arrow 2540 directs the flow of execution from starting operation 2302 to operation 2542.Operation 2542 performs setting the special part of the log-result to contain the special-minus whenever the sign part of the first number is essentially the negative-sign.","Arrow 2544 directs execution from operation 2542 to operation 2536.Operation 2536 terminates the operations of this flowchart.","The integer part of each of the number collection members may be in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps.","FIG.","36A depicts a detail flowchart of operation 2532 of FIG.","35B further determining whether the sign part of the first number is essentially equal to the negative-sign.","Arrow 2570 directs the flow of execution from starting operation 2532 to operation 2572.Operation 2572 performs determining whether the sign part of the first number is equal to the negative-sign based upon performing at least one of the members of the negative-test collection.","Arrow 2574 directs execution from operation 2572 to operation 2576.Operation 2576 terminates the operations of this flowchart.","Alternatively, the integer part of each of the number collection members is in a non-redundant numeric notation possessing exactly one negative-test step.","FIG.","36B depicts a detail flowchart of operation 2532 of FIG.","35B further determining whether the sign part of the first number is essentially equal to the negative-sign.","Arrow 2590 directs the flow of execution from starting operation 2532 to operation 2592.Operation 2592 performs performing the exactly one negative-test step based upon the first number.","Arrow 2594 directs execution from operation 2592 to operation 2596.Operation 2596 terminates the operations of this flowchart.","Note that each member of the input number collection may be comprised of an integer part.","FIG.","37A depicts a detail flowchart of operation 2232 of FIG.","30 further log-converting the input number collection member.","Arrow 2600 directs the flow of execution from starting operation 2232 to operation 2602.Operation 2602 performs determining whether the integer part of the input number collection member is essentially equal to the zero-representation.","Arrow 2604 directs execution from operation 2602 to operation 2606.Operation 2606 terminates the operations of this flowchart.","Arrow 2610 directs the flow of execution from starting operation 2232 to operation 2612.Operation 2612 performs setting the special part of the number collection member to contain the negative-infinity whenever the integer part of the input number collection member essentially equals the zero-representation.","Arrow 2614 directs execution from operation 2612 to operation 2606.Operation 2606 terminates the operations of this flowchart.","Note that the integer part of each of the input number collection members may contain a sign belonging to the sign collection and a magnitude.","FIG.","37B depicts a detail flowchart of operation 2232 of FIG.","30 further log-converting the input number collection member.","Arrow 2630 directs the flow of execution from starting operation 2232 to operation 2632.Operation 2632 performs determining whether the sign part of the input number collection member is essentially equal to the negative-sign.","Arrow 2634 directs execution from operation 2632 to operation 2636.Operation 2636 terminates the operations of this flowchart.","Arrow 2640 directs the flow of execution from starting operation 2232 to operation 2642.Operation 2642 performs setting the special part of the number collection member to contain the special-minus whenever the sign part of the input number collection member is essentially the negative-sign.","Arrow 2644 directs execution from operation 2642 to operation 2636.Operation 2636 terminates the operations of this flowchart.","Note that each of the output number collection members may include a magnitude.","FIG.","38A depicts a detail flowchart of operation 2242 of FIG.","30 further exp-converting.","Arrow 2650 directs the flow of execution from starting operation 2242 to operation 2652.Operation 2652 performs setting the magnitude of the output number collection member to the zero-representation whenever the special part of the number collection member contains the negative-infinity.","Arrow 2654 directs execution from operation 2652 to operation 2656.Operation 2656 terminates the operations of this flowchart.","Also, each of the output number collection members may include a sign belonging to the sign collection.","FIG.","38B depicts a detail flowchart of operation 2242 of FIG.","30 further exp-converting.","Arrow 2670 directs the flow of execution from starting operation 2242 to operation 2672.Operation 2672 performs setting the sign of the output number collection member to the negative-sign whenever the special part of the number collection member contains the special-minus.","Arrow 2674 directs execution from operation 2672 to operation 2676.Operation 2676 terminates the operations of this flowchart.","Arrow 2680 directs the flow of execution from starting operation 2242 to operation 2682.Operation 2682 performs setting the sign of the output number collection member to the positive-sign whenever the special part of the number collection member contains the special-plus.","Arrow 2684 directs execution from operation 2682 to operation 2686.Operation 2686 terminates the operations of this flowchart.","Note that each of the input number collection members may be encoded as an N1 bit code; wherein the N1 is at least three.","The integer part of each of the number collection members may be encoded as an N2 bit code; wherein N2 is greater than N1.This method of numeric processing may be implemented as a program system comprised of program steps implementing the steps of the method.","The program steps may reside in a memory accessibly coupled to a computer, which executes these program steps.","The program system may further be implemented as program steps in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon the computer.","The invention includes circuitry generated from those program steps.","The invention also includes circuitry implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection.","Where the programmable logic device collection refers to all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array.","The fixed architecture device collection refers to all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.","The preceding embodiments of the invention have been provided by way of example and are not meant to constrain the scope of the following claims."]],"string":"[\n [\n \"Method and apparatus of dsp resource allocation and use\",\n \"Certain embodiments of the invention support the distribution and control of multiple instruction streams going to multiple steps of modules supporting arithmetic and memory, as well as method for numeric data processing optimizing non-additive functions and operations through a novel numeric representation and the use of novel ALU components to support numeric conversions and evaluations of non-additive functions.\"\n ],\n [\n \"1.A circuit supporting partitioned operation of multiple concurrently presented instructions comprising: a first instruction wire bundle; a second instruction wire bundle; a clock wire bundle possessing a capture state; a partition control wire bundle possessing a partition control state; an arithmetic module circuit coupled to said partition control wire bundle and further comprised of a first memory circuit coupled to said partition control wire bundle; and a first arithmetic logic unit coupled to said partition control wire bundle; wherein said module circuit samples said first instruction wire bundle and said second instruction wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and wherein said arithmetic module circuit responds to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state; wherein said arithmetic module circuit response is further comprised of: said first memory circuit responds to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responds to at least one member of said sample instruction collection based upon said partition control state; wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein said first memory circuit is further comprised of at least two memory sub-circuits, each memory sub-circuit corresponding to a member of said ALU part collection; wherein each member of said part ALU collection and said corresponding memory sub-circuit are coupled to said partition control wire bundle; and wherein each member of said part ALU collection and said corresponding memory sub-circuit both respond to exactly one and the same member of said sample instruction collection based upon said partition control state.\",\n \"2.The circuit of claim 1, wherein said first part ALU presents a first carry signal to said second part ALUs; wherein said second part ALU ignores said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.\",\n \"3.The circuit of claim 1, further comprising: an input-output circuit coupled to said arithmetic module circuit, to said partition control wire bundle; wherein said input-output circuit responds to at least one member of said sample instruction collection based upon said partition control state.\",\n \"4.The circuit of claim 3, wherein said input-output circuit presents a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; wherein said input-output circuit presents a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.\",\n \"5.The circuit of claim 4, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein each of said second sub-circuits corresponding to said member of said ALU responds to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, said second sub-circuit further responds by approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.\",\n \"6.The circuit of claim 5, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input; 7.The circuit of claim 6, wherein each member of said ALU part collection presents a result to said corresponding said second sub-circuit.\",\n \"8.The circuit of claim 7, further comprising a second of said arithmetic module circuits coupled to said input-output circuit; wherein said input-output circuit presents said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein said input-output circuit presents said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"9.The circuit of claim 8, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"10.The circuit of claim 1, further comprising a first datapath instruction processor coupled to said first datapath instruction wire bundle presented to said module circuit; wherein said first datapath instruction processor asserts said first datapath instruction wire bundle; and a second datapath instruction processor coupled to said second datapath instruction wire bundle presented to said module circuit; wherein said second datapath instruction processor asserts said second datapath instruction wire bundle.\",\n \"11.The circuit of claim 10, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said datapath instruction queue pointer responds to said clock wire bundle by asserting an asserted datapath instruction address; wherein said datapath instruction register responds to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and wherein said datapath instruction register further asserts said associated datapath instruction wire bundle based upon said datapath instruction capture state.\",\n \"12.The circuit of claim 11, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of: an instruction store coupled to said datapath instruction address wire bundle and to said datapath instruction wire bundle; wherein said instruction store responds to said asserted datapath instruction address via said datapath instruction address wire bundle to asserting said datapath instruction wire bundle based upon said asserted datapath instruction address.\",\n \"13.The circuit of claim 12, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.\",\n \"14.A circuit implementation of claim 1, wherein a component list comprises said arithmetic module circuit, said first memory circuit coupled to said partition control wire bundle, and said first arithmetic logic unit; wherein each member of said component list is implemented with at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"15.The circuit implementation of claim 14, wherein the implementation of all members of said component list further includes only circuitry responding to said exactly one and the same member of said sample instruction collection based upon said partition control state.\",\n \"16.A method of operating a module circuit containing at least an arithmetic module circuit further containing a first memory circuit and a first arithmetic logic unit using a clock wire bundle possessing a capture state and using a partition control wire bundle possessing a partition control state, said method comprising the steps of: said module circuit sampling a first instruction pair wire bundle and a second instruction pair wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and said arithmetic module circuit responding to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state; wherein the step of said arithmetic module circuit responding is further comprised of the steps of: said first memory circuit responding to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responding to at least one member of said sample instruction collection based upon said partition control state; wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein the step of said first arithmetic logic unit responding is further comprised of the steps of said first part ALU responding to exactly one member of said sample instruction collection based upon said partition control; and said second part ALU responding to exactly one member of said sample instruction collection based upon said partition control state; wherein said first memory circuit is comprised of at least two memory sub-circuits, each of said memory sub-circuits corresponding to a member of said ALU part collection; wherein the step of said first memory circuit responding is further comprised of, for each of said memory sub-circuits, the step of said memory sub-circuit responding to exactly one and the same member of said sample instruction collection as said corresponding member of said ALU part collection.\",\n \"17.The method of claim 16, further comprising the step of said first part ALU presenting a first carry signal to said second part ALU; wherein the step of said second ALU responding is comprised of the steps of: said second part ALU ignoring said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.\",\n \"18.The method of claim 16, further comprising the step of: an input-output circuit responding to at least one member of said sample instruction collection based upon said partition control state.\",\n \"19.The method of claim 18, further comprising the steps of: said input-output circuit presenting a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; said input-output circuit presenting a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.\",\n \"20.The method of claim 19, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein said method is further comprised, for each of said second sub-circuits corresponding to said member of said ALU part collection, of the step of: said second sub-circuit responding to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, the step of said second sub-circuit responding is further comprised of the step of: said second sub-circuit approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.\",\n \"21.The method of claim 20, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input.\",\n \"22.The method of claim 21, further comprising the steps of: for each member of said ALU part collection, said member presenting a result to said corresponding said second sub-circuit.\",\n \"23.The method of claim 22, wherein said module circuit is further comprised of a second of said arithmetic module circuits coupled to said input-output circuit; wherein said method is further comprised of the step of: said input-output circuit presenting said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said input-output circuit presenting said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"24.The method of claim 23, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein said method is further comprised, for each member of said second circuit collection coupled to said internal wire bundle of the step of: said member asserting a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said member asserting a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"25.The method of claim 16, further comprising the steps of a first datapath instruction processor asserting said first datapath instruction wire bundle presented to said module circuit; and a second datapath instruction processor asserting said second datapath instruction wire bundle presented to said module circuit.\",\n \"26.The method of claim 25, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said method is further comprised of the steps of said datapath instruction queue pointer responding to said clock wire bundle by asserting an asserted datapath instruction address; said datapath instruction register responding to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and said datapath instruction register asserting said associated datapath instruction wire bundle based upon said datapath instruction capture state.\",\n \"27.The method of claim 26, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of an instruction store; wherein said method is further comprised of the steps of said instruction store responding to said asserted datapath instruction address to assert said datapath instruction wire bundle based upon said asserted datapath instruction address.\",\n \"28.The method of claim 27, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.\",\n \"29.A circuit implementation of the method of claim 16, wherein the steps of the method are each implemented within at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"30.A method of operating a module circuit containing at least an arithmetic module circuit further containing a first memory circuit and a first arithmetic logic unit using a clock wire bundle possessing a capture state and using a partition control wire bundle possessing a partition control state, said method comprising the steps of: said module circuit sampling a first instruction pair wire bundle and a second instruction pair wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and said arithmetic module circuit responding to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state.\",\n \"31.The method of claim 30, wherein the step of said arithmetic module circuit responding is further comprised of the steps of: said first memory circuit responding to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responding to at least one member of said sample instruction collection based upon said partition control state.\",\n \"32.The method of claim 31, wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein the step of said first arithmetic logic unit responding is further comprised of the steps of said first part ALU responding to exactly one member of said sample instruction collection based upon said partition control; and said second part ALU responding to exactly one member of said sample instruction collection based upon said partition control state; wherein said first memory circuit is comprised of at least two memory sub-circuits, each of said memory sub-circuits corresponding to a member of said ALU part collection; wherein the step of said first memory circuit responding is further comprised of for each of said memory sub-circuits, the step of said memory sub-circuit responding to exactly one and the same member of said sample instruction collection as said corresponding member of said ALU part collection.\",\n \"33.The method of claim 32, further comprising the step of said first part ALU presenting a first carry signal to said second part ALU; wherein the step of said second ALU responding is comprised of the steps of: said second part ALU ignoring said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.\",\n \"34.The method of claim 32, further comprising the step of: an input-output circuit responding to at least one member of said sample instruction collection based upon said partition control state.\",\n \"35.The method of claim 34, further comprising the steps of: said input-output circuit presenting a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; said input-output circuit presenting a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.\",\n \"36.The method of claim 35, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein said method is further comprised, for each of said second sub-circuits corresponding to said member of said ALU part collection, of the step of: said second sub-circuit responding to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, the step of said second sub-circuit responding is further comprised of the step of: said second sub-circuit approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.\",\n \"37.The method of claim 36, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input.\",\n \"38.The method of claim 37, further comprising the steps of: for each member of said ALU part collection, said member presenting a result to said corresponding said second sub-circuit.\",\n \"39.The method of claim 38, wherein said module circuit is further comprised of a second of said arithmetic module circuits coupled to said input-output circuit; wherein said method is further comprised of the step of: said input-output circuit presenting said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said input-output circuit presenting said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"40.The method of claim 39, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein said method is further comprised, for each member of said second circuit collection coupled to said internal wire bundle of the step of: said member asserting a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said member asserting a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"41.The method of claim 30, further comprising the steps of a first datapath instruction processor asserting said first datapath instruction wire bundle presented to said module circuit; and a second datapath instruction processor asserting said second datapath instruction wire bundle presented to said module circuit.\",\n \"42.The method of claim 41, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said method is further comprised of the steps of said datapath instruction queue pointer responding to said clock wire bundle by asserting an asserted datapath instruction address; said datapath instruction register responding to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and said datapath instruction register asserting said associated datapath instruction wire bundle based upon said datapath instruction capture state.\",\n \"43.The method of claim 42, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of an instruction store; wherein said method is further comprised of the steps of said instruction store responding to said asserted datapath instruction address to assert said datapath instruction wire bundle based upon said asserted datapath instruction address.\",\n \"44.The method of claim 43, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle, and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.\",\n \"45.A circuit implementation of the method of claim 30, wherein the steps of the method are each implemented within at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"46.A circuit supporting partitioned operation of multiple concurrently presented instructions comprising: a first instruction wire bundle; a second instruction wire bundle; a clock wire bundle possessing a capture state; a partition control wire bundle possessing a partition control state; an arithmetic module circuit coupled to said partition control wire bundle and further comprised of a first memory circuit coupled to said partition control wire bundle; and a first arithmetic logic unit coupled to said partition control wire bundle; wherein said module circuit samples said first instruction wire bundle and said second instruction wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and wherein said arithmetic module circuit responds to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state.\",\n \"47.The circuit of claim 46, wherein said arithmetic module circuit response is further comprised of: said first memory circuit responds to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responds to at least one member of said sample instruction collection based upon said partition control state.\",\n \"48.The circuit of claim 47, wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein said first memory circuit is further comprised of at least two memory sub-circuits, each memory sub-circuit corresponding to a member of said ALU part collection; wherein each member of said part ALU collection and said corresponding memory sub-circuit are coupled to said partition control wire bundle; and wherein each member of said part ALU collection and said corresponding memory sub-circuit both respond to exactly one and the same member of said sample instruction collection based upon said partition control state.\",\n \"49.The circuit of claim 48, wherein said partition control state includes to a carry partition control belonging to a partition control state collection comprising first carry control state and a second carry control state; wherein said first part ALU presents a first carry signal to said second part ALUs; wherein said second part ALU ignores said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.\",\n \"50.The circuit of claim 48, further comprising: an input-output circuit coupled to said arithmetic module circuit, to said partition control wire bundle; wherein said input-output circuit responds to at least one member of said sample instruction collection based upon said partition control state.\",\n \"51.The circuit of claim 50, wherein said input-output circuit presents a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; wherein said input-output circuit presents a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.\",\n \"52.The circuit of claim 51, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein each of said second sub-circuits corresponding to said member of said ALU responds to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, said second sub-circuit further responds by approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.\",\n \"53.The circuit of claim 52, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input; 54.The circuit of claim 53, wherein each member of said ALU part collection presents a result to said corresponding said second sub-circuit.\",\n \"55.The circuit of claim 54, further comprising a second of said arithmetic module circuits coupled to said input-output circuit; wherein said input-output circuit presents said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein said input-output circuit presents said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"56.The circuit of claim 55, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.\",\n \"57.The circuit of claim 46, further comprising a first datapath instruction processor coupled to said first datapath instruction wire bundle presented to said module circuit; wherein said first datapath instruction processor asserts said first datapath instruction wire bundle; and a second datapath instruction processor coupled to said second datapath instruction wire bundle presented to said module circuit; wherein said second datapath instruction processor asserts said second datapath instruction wire bundle.\",\n \"58.The circuit of claim 57, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said datapath instruction queue pointer responds to said clock wire bundle by asserting an asserted datapath instruction address; wherein said datapath instruction register responds to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and wherein said datapath instruction register further asserts said associated datapath instruction wire bundle based upon said datapath instruction capture state.\",\n \"59.The circuit of claim 58, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of: an instruction store coupled to said datapath instruction address wire bundle and to said datapath instruction wire bundle; wherein said instruction store responds to said asserted datapath instruction address via said datapath instruction address wire bundle to asserting said datapath instruction wire bundle based upon said asserted datapath instruction address.\",\n \"60.The circuit of claim 59, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.\",\n \"61.A circuit implementation of claim 46, wherein a component list comprises said arithmetic module circuit, said first memory circuit coupled to said partition control wire bundle, and said first arithmetic logic unit; wherein each member of said component list is implemented with at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"62.The circuit implementation of claim 61,: wherein the implementation of all members of said component list further includes only circuitry responding to exactly one and the same member of said sample instruction collection based upon said partition control state.\",\n \"63.A method of processing numeric data, comprising the step of: representing each member of a number collection by an integer part and a special part; log-converting a member of an input number collection to create a member of said number collection; and exp-converting a member of said number collection to create a member of an output number collection; wherein said special part representing said member of said number collection contains of exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity; wherein said special part of each member of said number collection further contains exactly one member of a second special value collection comprising a special-minus and a special-plus; wherein said integer part of each member of said number collection contains a sign and a magnitude; wherein, for each member of said number collection, said sign is a member of a sign collection consisting essentially of a positive-sign and a negative-sign; wherein said number collection comprises at least a first number and a second number; said method further comprising the steps of: performing all members of the arithmetic operation collection upon at least one of said members of said number collection; wherein said arithmetic operation collection is comprised of the steps of: adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result; wherein said number collection is further comprised of said add-result, said subtract-result, said exp-result and said log-result; wherein the step of adding is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; determining whether said special part of said second number contains said negative-infinity; and setting said special part of said add-result to contain said negative-infinity whenever said special part of at least one member of the collection said first number and said second number contains said negative-infinity; wherein the step of subtracting is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said subtract-result to contain said negative-infinity whenever said special part of said first number contains said negative-infinity; wherein the step of exponentiating is further comprised of the step of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said exp-result to contain said not-negative-infinity and setting said integer part to a zero-representation whenever said special part of said first number contains said negative-infinity; and setting said sign of said exp-result to essentially said negative-sign whenever said special part of said first number contains said special-minus; wherein the step of logarithming is further comprised of the steps of: determining whether said integer part of said first number is essentially equal to said zero-representation; setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number essentially equals said zero-representation; determining whether said sign part of said first number is essentially equal to said negative-sign; and setting said special part of said log-result to contain said special-minus whenever said sign part of said first number is essentially said negative-sign; wherein said input number collection is comprised of a first input number and a second input number.\",\n \"wherein each member of said input number collection is comprised of an integer part; wherein the step log-converting said input number collection member is further comprised of the steps of: determining whether said integer part of said input number collection member is essentially equal to said zero-representation; and setting said special part of said number collection member to contain said negative-infinity whenever said integer part of said input number collection member essentially equals said zero-representation.\",\n \"wherein said output number collection is comprised of a first output number and a second output number; wherein each of said output number collection members include a magnitude; wherein the step exp-converting is further comprised of the steps of: setting said magnitude of said output number collection member to said zero-representation whenever said special part of said number collection member contains said negative-infinity; and setting said sign of said output number collection member to said positive-sign whenever said special part of said number collection member contains said special-plus.\",\n \"64.The method of claim 63, wherein said integer part of each of said number collection members is in a non-redundant numeric notation; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number equals said zero-representation.\",\n \"65.The method of claim 63, wherein said integer part of each of said number collection members is in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number is a member of said zero-representation collection.\",\n \"66.The method of claim 63, wherein said integer part of each of said number collection members is in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps; where the step determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: determining whether said sign part of said first number is equal to said negative-sign based upon performing at least one of the members of said negative-test collection.\",\n \"67.The method of claim 63, wherein said integer part of each of said number collection members is in a non-redundant numeric notation possessing exactly one negative-test step; wherein the step of determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: performing said exactly one negative-test step based upon said first number.\",\n \"68.The method of claim 63, wherein said integer part of each of said input number collection members contains a sign belonging to said sign collection and a magnitude; wherein the step of log-converting is further comprised of the steps of: determining whether said sign part of said input number collection member is essentially equal to said negative-sign; and setting said special part of said number collection member to contain said special-minus whenever said sign part of said input number collection member is essentially said negative-sign.\",\n \"69.The method of claim 63, wherein each of said output number collection members include a sign belong to said sign collection; wherein the step exp-converting is further comprised of the step of: setting said sign of said output number collection member to said negative-sign whenever said special part of said number collection member contains said special-minus.\",\n \"70.The method of claim 69, wherein each of said input number collection members is encoded as an N1 bit code; wherein said N1 is at least three; wherein said integer part of each of said number collection members is encoded as an N2 bit code; wherein N2 is greater than N1.71.A program system for processing numeric data, implementing the steps of claim 63, comprising program steps residing in a memory accessibly coupled to a computer, said program system comprising the program steps of: representing each member of a number collection by an integer part and a special part; log-converting a member of an input number collection to create a member of said number collection; exp-converting a member of said number collection to create a member of an output number collection; determining whether said special part of said first number contains said negative-infinity; adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result.\",\n \"72.The program system of claim 71, wherein the program steps implementing the method are embodied in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon said computer.\",\n \"73.A digital circuit generated from the program steps of claim 72.74.A circuit for processing numeric data, implementing the steps of claim 63, comprising: means for representing each member of a number collection by an integer part and a special part; means for determining whether said special part of said first number contains said negative-infinity; means for adding said first number to said second number to create an add-result; means for subtracting said first number by said second number to create a subtract-result; means for exponentiating said first number to create an exp-result; and means for logarithming said first number to create a log-result.\",\n \"75.The circuit of claim 74, wherein at least one of the means of claim 74 is implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array; and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"76.The circuit of claim 74, wherein at least one of said input number collection members are implemented as a wire state collection received from a wire bundle coupled to said circuit.\",\n \"77.The circuit of claim 74, wherein at least one of said output number collection members are implemented as a wire state asserted by said circuit onto a wire bundle.\",\n \"78.A method of processing numeric data, comprising the step of: representing each member of a number collection by an integer part and a special part; wherein said special part representing said member of said number collection contains of exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity; wherein said number collection comprises at least a first number and a second number; said method further comprising the steps of: performing at least one member of the arithmetic operation collection upon at least one of said members of said number collection; wherein said arithmetic operation collection is comprised of the steps of: adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result; wherein said number collection is further comprised of said add-result, said subtract-result, said exp-result and said log-result; wherein the step of adding is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; determining whether said special part of said second number contains said negative-infinity; and setting said special part of said add-result to contain said negative-infinity whenever said special part of at least one member of the collection said first number and said second number contains said negative-infinity; wherein the step of subtracting is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said subtract-result to contain said negative-infinity whenever said special part of said first number contains said negative-infinity; wherein the step of exponentiating is further comprised of the step of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said exp-result to contain said not-negative-infinity and setting said integer part to a zero-representation whenever said special part of said first number contains said negative-infinity; wherein the step of logarithming is further comprised of the steps of: determining whether said integer part of said first number is essentially equal to said zero-representation; and setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number essentially equals said zero-representation.\",\n \"79.The method of claim 78, wherein said integer part of each of said number collection members is in a non-redundant numeric notation; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number equals said zero-representation.\",\n \"80.The method of claim 78, wherein said integer part of each of said number collection members is in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number is a member of said zero-representation collection.\",\n \"81.The method of claim 78, wherein said integer part of each member of said number collection contains a sign and a magnitude; wherein, for each member of said number collection, said sign is a member of a sign collection consisting essentially of a positive-sign and a negative-sign; wherein said special part of each member of said number collection further contains exactly one member of a second special value collection comprising a special-minus and a special-plus; wherein the step of exponentiating is further comprised of the steps of: setting said sign of said exp-result to essentially said negative-sign whenever said special part of said first number contains said special-minus; wherein the step of logarithming is further comprised of the steps of: determining whether said sign part of said first number is essentially equal to said negative-sign; and setting said special part of said log-result to contain said special-minus whenever said sign part of said first number is essentially said negative-sign.\",\n \"82.The method of claim 81, wherein said integer part of each of said number collection members is in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps; where the step determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: determining whether said sign part of said first number is equal to said negative-sign based upon performing at least one of the members of said negative-test collection.\",\n \"83.The method of claim 81, wherein said integer part of each of said number collection members is in a non-redundant numeric notation possessing exactly one negative-test step; wherein the step of determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: performing said exactly one negative-test step based upon said first number.\",\n \"84.The method of claim 81, further comprising the step of: log-converting a member of an input number collection to create a member of said number collection; wherein said input number collection is comprised of a first input number and a second input number.\",\n \"85.The method of claim 84, wherein each member of said input number collection is comprised of an integer part; wherein the step log-converting said input number collection member is further comprised of the steps of: determining whether said integer part of said input number collection member is essentially equal to said zero-representation; and setting said special part of said number collection member to contain said negative-infinity whenever said integer part of said input number collection member essentially equals said zero-representation.\",\n \"86.The method of claim 85, wherein said integer part of each of said input number collection members contains a sign belonging to said sign collection and a magnitude; wherein the step of log-converting is further comprised of the steps of: determining whether said sign part of said input number collection member is essentially equal to said negative-sign; and setting said special part of said number collection member to contain said special-minus whenever said sign part of said input number collection member is essentially said negative-sign.\",\n \"87.The method of claim 86, further comprising the step of: exp-converting a member of said number collection to create a member of an output number collection; wherein said output number collection is comprised of a first output number and a second output number.\",\n \"88.The method of claim 87, wherein each of said output number collection members include a magnitude; wherein the step exp-converting is further comprised of the step of: setting said magnitude of said output number collection member to said zero-representation whenever said special part of said number collection member contains said negative-infinity.\",\n \"89.The method of claim 88, wherein each of said output number collection members include a sign belong to said sign collection; wherein the step exp-converting is further comprised of the steps of: setting said sign of said output number collection member to said negative-sign whenever said special part of said number collection member contains said special-minus; and setting said sign of said output number collection member to said positive-sign whenever said special part of said number collection member contains said special-plus.\",\n \"90.The method of claim 89, wherein each of said input number collection members is encoded as an N1 bit code; wherein said N1 is at least three; wherein said integer part of each of said number collection members is encoded as an N2 bit code; wherein N2 is greater than N1.91.A program system for processing numeric data, implementing the steps of claim 78, comprising program steps residing in a memory accessibly coupled to a computer, said program system comprising the program steps of: representing each member of a number collection by an integer part and a special part; adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result.\",\n \"92.The program system of claim 78, wherein the program steps implementing the method are embodied in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon said computer.\",\n \"93.A circuit generated from the program steps of claim 92.94.A circuit for processing numeric data, implementing the steps of claim 78, comprising: means for representing each member of a number collection by an integer part and a special part; means for adding said first number to said second number to create an add-result; means for subtracting said first number by said second number to create a subtract-result; means for exponentiating said first number to create an exp-result; and means for logarithming said first number to create a log-result.\",\n \"95.The circuit of claim 94, wherein at least one of the means of claim 94 is implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array; and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"96.The circuit of claim 94, wherein at least one of said input number collection members are implemented as a wire state collection received from a wire bundle coupled to said circuit.\",\n \"97.The circuit of claim 94, wherein at least one of said output number collection members are implemented as a wire state asserted by said circuit onto a wire bundle.\"\n ],\n [\n \" BACKGROUND OF INVENTION Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.\",\n \"These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.\",\n \"Most of these response and transform functions are linear in nature.\",\n \"Many of these functions operate on a finite grid of data, frequently known as a data window.\",\n \"While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.\",\n \"Many application systems need non-linear functions.\",\n \"Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.\",\n \"These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.\",\n \"Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.\",\n \"The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.\",\n \"What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.\",\n \"The above mentioned finite grids of data are often sampled in real-time by sensor devices.\",\n \"Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.\",\n \"Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.\",\n \"Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.\",\n \"The consequence of this is that bits go unused.\",\n \"By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.\",\n \"Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.\",\n \"What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.\",\n \"Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.\",\n \"There is a central problem associated with these representations, the accumulation of arithmetic errors.\",\n \"This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.\",\n \"Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.\",\n \"The consequence of this is that as calculations progress, additional precision is required, but not available.\",\n \"What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.\",\n \"By way of example, many real-time DSP applications possess the following common constraints.\",\n \"High speed input sample rates, often on the order of 100K to 100 Million samples per second.\",\n \"High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.\",\n \"Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.\",\n \"Many of these applications are developed against a further constraint of short time-to-market requirement.\",\n \"Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.\",\n \"Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.\",\n \"Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.\",\n \"Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.\",\n \"Commercial examples of MIMD products include the high-end products of the ADSP product line.\",\n \"Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.\",\n \"There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.\",\n \"The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.\",\n \"Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.\",\n \"This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.\",\n \"Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.\",\n \"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.\",\n \"The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.\",\n \"The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.\",\n \"While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.\",\n \"The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.\",\n \"What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.\",\n \"Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.\",\n \"Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.\",\n \"These computer have been largely constructed from arrays of FPGA's.\",\n \"They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.\",\n \"Such computer also tend to have problems with multiplication.\",\n \"While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.\",\n \"Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.\",\n \"Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.\",\n \"The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.\",\n \"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.\",\n \"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.\",\n \"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.\",\n \"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.\",\n \"There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.\",\n \"By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.\",\n \"However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.\",\n \"When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.\",\n \"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.\",\n \"Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).\",\n \"However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.\",\n \"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.\",\n \"There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.\",\n \"The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.\",\n \"Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.\",\n \"Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.\",\n \"Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.\",\n \"Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.\",\n \"Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.\",\n \"To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.\",\n \"Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.\",\n \"What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.\",\n \"What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.\",\n \"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.\",\n \"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.\",\n \"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.\",\n \"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.\",\n \"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.\",\n \"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.\",\n \"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.\"\n ],\n [\n \" SUMMARY OF INVENTION Certain embodiments of the invention solve all the above mentioned problems found in the prior art.\",\n \"Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.\",\n \"The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.\",\n \"Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.\",\n \"These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.\",\n \"Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.\",\n \"The logarithmic conversion of a zero yields a represented number with a minus-infinity.\",\n \"The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.\",\n \"Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.\",\n \"Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.\",\n \"Special-plus added to special-plus results in a special-plus.\",\n \"When a special-minus number and a special-plus number are added, the result is a special-minus result.\",\n \"This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.\",\n \"In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.\",\n \"A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.\",\n \"Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.\",\n \"This supports selecting input-output resources for optimal data bandwidth throughput.\",\n \"They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.\",\n \"The computation resource array may contain at least one instruction propagating resource.\",\n \"The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.\",\n \"The computation resource array may further contain at least one instruction processing resource.\",\n \"Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.\",\n \"The instruction processing resources may contain at least one instruction register.\",\n \"The computation resource array may further contain at least one instruction fetching resource.\",\n \"The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.\",\n \"Certain embodiments of the invention include circuits generated by methods of this invention.\",\n \"They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).\",\n \"They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.\",\n \"Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.\",\n \"Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.\",\n \"The shared bus wire bundle state is determined in part by the configuration register.\",\n \"Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator.\"\n ],\n [\n \"This application claims priority from the following provisional applications filed with the United States Patent and Trademark Office: Ser.\",\n \"No.\",\n \"60/204,113, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed May 15, 2000 by inventor, docket number ARITH001PR; Ser.\",\n \"No.\",\n \"60/215,894, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.\",\n \"5, 2000 by inventor, docket number ARITH002PR; Ser.\",\n \"No.\",\n \"60/217,353, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.\",\n \"11, 2000 by inventor, docket number ARITH003PR; Ser.\",\n \"No.\",\n \"60/231,873, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Sep. 12, 2000 by inventor, docket number ARITH004PR; Ser.\",\n \"No.\",\n \"60/261,066, entitled “Method and apparatus of a DSP resource circuit”, filed Jan. 11, 2001 by inventor, docket number ARITH005PR; and Ser.\",\n \"No.\",\n \"60/282,093, entitled “Method and apparatus of a DSP resource circuit”, filed Apr.\",\n \"6, 2001 by inventor, docket number ARITH006PR.\",\n \"TECHNICAL FIELD This invention relates to digital signal processing engines, instruction processing mechanisms, arithmetic operational units of same, as well as circuitry generated based upon use of some or all of these elements.\",\n \"BACKGROUND OF INVENTION Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.\",\n \"These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.\",\n \"Most of these response and transform functions are linear in nature.\",\n \"Many of these functions operate on a finite grid of data, frequently known as a data window.\",\n \"While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.\",\n \"Many application systems need non-linear functions.\",\n \"Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.\",\n \"These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.\",\n \"Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.\",\n \"The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.\",\n \"What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.\",\n \"The above mentioned finite grids of data are often sampled in real-time by sensor devices.\",\n \"Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.\",\n \"Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.\",\n \"Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.\",\n \"The consequence of this is that bits go unused.\",\n \"By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.\",\n \"Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.\",\n \"What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.\",\n \"Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.\",\n \"There is a central problem associated with these representations, the accumulation of arithmetic errors.\",\n \"This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.\",\n \"Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.\",\n \"The consequence of this is that as calculations progress, additional precision is required, but not available.\",\n \"What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.\",\n \"By way of example, many real-time DSP applications possess the following common constraints.\",\n \"High speed input sample rates, often on the order of 100K to 100 Million samples per second.\",\n \"High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.\",\n \"Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.\",\n \"Many of these applications are developed against a further constraint of short time-to-market requirement.\",\n \"Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.\",\n \"Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.\",\n \"Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.\",\n \"Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.\",\n \"Commercial examples of MIMD products include the high-end products of the ADSP product line.\",\n \"Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.\",\n \"There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.\",\n \"The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.\",\n \"Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.\",\n \"This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.\",\n \"Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.\",\n \"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.\",\n \"The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.\",\n \"The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.\",\n \"While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.\",\n \"The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.\",\n \"What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.\",\n \"Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.\",\n \"Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.\",\n \"These computer have been largely constructed from arrays of FPGA's.\",\n \"They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.\",\n \"Such computer also tend to have problems with multiplication.\",\n \"While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.\",\n \"Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.\",\n \"Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.\",\n \"The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.\",\n \"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.\",\n \"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.\",\n \"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.\",\n \"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.\",\n \"There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.\",\n \"By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.\",\n \"However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.\",\n \"When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.\",\n \"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.\",\n \"Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).\",\n \"However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.\",\n \"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.\",\n \"There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.\",\n \"The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.\",\n \"Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.\",\n \"Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.\",\n \"Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.\",\n \"Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.\",\n \"Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.\",\n \"To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.\",\n \"Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.\",\n \"What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.\",\n \"What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.\",\n \"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.\",\n \"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.\",\n \"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.\",\n \"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.\",\n \"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.\",\n \"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.\",\n \"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.\",\n \"SUMMARY OF INVENTION Certain embodiments of the invention solve all the above mentioned problems found in the prior art.\",\n \"Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.\",\n \"The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.\",\n \"Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.\",\n \"These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.\",\n \"Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.\",\n \"The logarithmic conversion of a zero yields a represented number with a minus-infinity.\",\n \"The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.\",\n \"Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.\",\n \"Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.\",\n \"Special-plus added to special-plus results in a special-plus.\",\n \"When a special-minus number and a special-plus number are added, the result is a special-minus result.\",\n \"This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.\",\n \"In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.\",\n \"A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.\",\n \"Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.\",\n \"This supports selecting input-output resources for optimal data bandwidth throughput.\",\n \"They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.\",\n \"The computation resource array may contain at least one instruction propagating resource.\",\n \"The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.\",\n \"The computation resource array may further contain at least one instruction processing resource.\",\n \"Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.\",\n \"The instruction processing resources may contain at least one instruction register.\",\n \"The computation resource array may further contain at least one instruction fetching resource.\",\n \"The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.\",\n \"Certain embodiments of the invention include circuits generated by methods of this invention.\",\n \"They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).\",\n \"They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.\",\n \"Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.\",\n \"Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.\",\n \"The shared bus wire bundle state is determined in part by the configuration register.\",\n \"Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator.\",\n \"BRIEF DESCRIPTION OF DRAWINGS FIG.\",\n \"1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention; FIG.\",\n \"2 shows a refinement of FIG.\",\n \"1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400; FIG.\",\n \"3 shows a refinement of FIG.\",\n \"2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.\",\n \"4 shows a refinement of FIG.\",\n \"3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.\",\n \"5 shows a refinement of FIGS.\",\n \"1, 2, 3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400; FIG.\",\n \"6 shows a refinement of FIG.\",\n \"5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100; FIG.\",\n \"7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.\",\n \"8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.\",\n \"9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result; FIG.\",\n \"10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300; FIG.\",\n \"11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIG.\",\n \"11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIGS.\",\n \"12 to 16 show a configuration of instruction memories and MSRI[K] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples; FIG.\",\n \"17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.\",\n \"Note that in other embodiments of the invention, i could range over a different range; FIG.\",\n \"17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.\",\n \"FIGS.\",\n \"18 to 20 show various configurations of PISRI's and their neighboring instruction memories; FIG.\",\n \"21 shows a high level data flow diagram of a two dimensional array block 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported; FIG.\",\n \"22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource and at least one datapath operational resource; FIG.\",\n \"22B depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration; FIG.\",\n \"23A depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration; FIG.\",\n \"23B depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A further performing the method of using/operating the array of computational resources; FIG.\",\n \"24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500; FIG.\",\n \"24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.\",\n \"24A further comprised of instruction fetch mechanism 3800; FIG.\",\n \"25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720; and FIG.\",\n \"26 shows a detail block diagram of a branch processor 3700 extending the block diagram of FIG.\",\n \"25 further comprised of cache manager 3740; FIG.\",\n \"27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention; FIG.\",\n \"28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.\",\n \"27; FIG.\",\n \"29 depicts a preferred version of ALU 1400, as used in FIG.\",\n \"28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank; FIG.\",\n \"30 depicts a flowchart of a method of processing numeric data, which may be variously embodied; FIG.\",\n \"31 depicts a detail flowchart of operation 2222 of FIG.\",\n \"30 further performing the arithmetic operation collection; FIG.\",\n \"32A depicts a detail flowchart of operation 2272 of FIG.\",\n \"31 further adding; FIG.\",\n \"32B depicts a detail flowchart of operation 2282 of FIG.\",\n \"31 further subtracting; FIG.\",\n \"33A depicts a detail flowchart of operation 2292 of FIG.\",\n \"31 further exponentiating; FIG.\",\n \"33B depicts a detail flowchart of operation 2302 of FIG.\",\n \"31 further logarithming; FIG.\",\n \"34A depicts a detail flowchart of operation 2442 of FIG.\",\n \"33B further setting the special part of the log-result; FIG.\",\n \"34B depicts a detail flowchart of operation 2442 of FIG.\",\n \"33B further setting the special part of the log-result; FIG.\",\n \"35A depicts a detail flowchart of operation 2292 of FIG.\",\n \"31 further exponentiating; FIG.\",\n \"35B depicts a detail flowchart of operation 2302 of FIG.\",\n \"31 further logarithming; FIG.\",\n \"36A depicts a detail flowchart of operation 2532 of FIG.\",\n \"35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.\",\n \"36B depicts a detail flowchart of operation 2532 of FIG.\",\n \"35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.\",\n \"37A depicts a detail flowchart of operation 2232 of FIG.\",\n \"30 further log-converting the input number collection member; FIG.\",\n \"37B depicts a detail flowchart of operation 2232 of FIG.\",\n \"30 further log-converting the input number collection member; FIG.\",\n \"38A depicts a detail flowchart of operation 2242 of FIG.\",\n \"30 further exp-converting; and FIG.\",\n \"38B depicts a detail flowchart of operation 2242 of FIG.\",\n \"30 further exp-converting.\",\n \"DETAILED DESCRIPTION OF DRAWINGS As used herein a wire refers to a path connecting nodes of a circuit which carries a state between the connected nodes and/or refers to a resonant cavity propagating information in terms of state between the connected nodes.\",\n \"A wire may be made out of metal, an optical chamber, or a tunnel path through a molecular substrate.\",\n \"A wire bundle is a collection of at least one wire.\",\n \"State as used herein refers to an element of a finite alphabet, which contains at least two symbols.\",\n \"These two minimal symbols relate to ‘0’ and ‘1’ as used in Boolean logic.\",\n \"FIG.\",\n \"1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention.\",\n \"The basic arithmetic unit 1400 includes a partitioning wire bundle 1002 presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1008 is presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1010 is presented only to first memory circuit 1200.First memory instruction wire bundle 1202 is presented to first memory circuit 1200.First memory circuit 1200 generates signals for first memory output wire bundle 1016 presented to first ALU 1300.First ALU instruction wire bundle 1302 is presented to first ALU circuit 1300.First ALU 1300 receives a carry-in wire bundle 1150, first ALU instruction wire bundle 1302 and generates a carry-out wire bundle 1152 and further generates signals for first ALU output wire bundle 1018.The basic arithmetic processing unit operates by receiving the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018, as well as first memory instruction wire bundle 1202, first ALU instruction wire bundle 1302 and carry-input wire bundle 1150.The received signal state of the partitioning wire bundle 1.002, further determines the operational partitioning of first memory circuit 1200 and first ALU circuit 1300 with regards to the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018.First memory circuit 1200 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010 and first memory instruction wire bundle 1202.Partitioning wire bundle 1002 signal state is used by first memory circuit 1200 to determine from the signal state of first memory instruction wire bundle 1202 at least one of first memory local instructions to be executed.\",\n \"The first memory local instructions are executed by the first memory circuit 1200.First memory circuit 1200 asserts the signal state of first memory output wire bundle 1016.First ALU 1300 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, first memory output wire bundle 1016, first ALU instruction wire bundle 1302 and the effect of the signal state of carry-input wire bundle 1150.Partitioning wire bundle 1002 signal state is used by first ALU circuit 1300 to determine from the signal state of first ALU instruction wire bundle 1302 of at least one of first first ALU local instructions to be executed.\",\n \"The first ALU local instructions are executed by the first ALU circuit 1300.First ALU circuit 1200 asserts the signal state of first ALU output wire bundle 1018.FIG.\",\n \"2 shows a refinement of FIG.\",\n \"1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400.Note that FIG.\",\n \"2 operates in much the same fashion as FIG.\",\n \"1, excepting Input-output circuit 1100 acts as an interface between at least three external data bus wire bundles, 1004 and 1006 controlled by instruction wire bundle 1102 as well as partitioning wire bundle 1002 and creating the state on wire bundles 1008, 1010 and 1012.FIG.\",\n \"3 shows a refinement of FIG.\",\n \"2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.\",\n \"3 operates in a similar fashion to FIGS.\",\n \"1 and 2 with regards to elements of similar reference numbers.\",\n \"Second memory circuit 1250 may receive output 1018 from ALU 1300 as well as wire bundle 1014 from input-output circuit 1100 based upon partitioning wire bundle 1002.Second memory circuit 1250 may drive the state of wire bundle 1014.Second memory circuit 1250 may receive wire bundle 1012 from input-output circuit 1100.FIG.\",\n \"4 shows a refinement of FIG.\",\n \"3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.\",\n \"4 operates in a similar fashion to FIG.\",\n \"1, 2, and 3 with regards to elements of similar reference numbers.\",\n \"FIG.\",\n \"5 shows a refinement of FIGS.\",\n \"1, 2,3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.\",\n \"2, 3 and 4.FIG.\",\n \"6 shows a refinement of FIG.\",\n \"5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.\",\n \"2, 3 and 4.FIG.\",\n \"7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.FIG.\",\n \"8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.In FIGS.\",\n \"8 and 9, each ALU circuit 1310 may support a consistent datapath bit width.\",\n \"Each ALU circuit 1310 may further support a constant datapath bit width.\",\n \"The constant datapath bit width may be at least one of 3, 4, and 5.In FIGS.\",\n \"8 and 9, ALU circuit 1300 may contain instances of ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.The number of instances of ALU circuit 1310 is a power of two not necessarily shown in these Figures.\",\n \"The number of instances of ALU circuit 1310 may not be a power of two.\",\n \"The number of instances of ALU circuit 1310 may be 12.The constant bit width of ALU circuit 1310 may be 4 and the number of instances of ALU circuit 1310 belongs to a collection comprising 8, 12, 16, 24, 32, 48 and 64.The constant bit width of ALU circuit 1310 is 3 and the number of instances of ALU circuit 1310 belongs to a collection comprising 12, 16, 24, 32, 48 and 64.FIG.\",\n \"9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result.\",\n \"Each shifter (S1, S2, S2, and S4)is controlled by add-instruction-components belonging to a collection comprising at least values representing same-sign, reverse-sign, do-not-use, shift-up and shift-down.\",\n \"The effects of these values acting on the add-input are as follows: The add-input acted upon by the corresponding add-inst-component having same-sign generates the add-input.\",\n \"The add-input acted upon by the corresponding add-inst-component having reverse-sign generates the negative of the add-input.\",\n \"The add-input acted upon by the corresponding add-inst-component having do-not-use generates a zero.\",\n \"The add-input acted upon by the corresponding add-inst-component having shift-up generates a positive power of two times the add-input.\",\n \"The add-input acted upon by the corresponding add-inst-component having shift-down generates a negative power of two times the add-input.\",\n \"The shift-instruction collection may further include shift-up-by-m and shift-down-by-m, where m is at least two.\",\n \"Partitioning wire bundle 1002 is used to control carry propagation and shift bit propagation, but are not shown in this Figure to simplify the discussion.\",\n \"The omission of partitioning wire bundle 1002 is not meant to limit nor require its presence for all embodiments of the invention.\",\n \"Note that the logarithm of 0 will be negative-infinity, and that the square and square root of 0 are each 0.To preserve these facts in the logarithmic domain, shifting negative-infinity should produce negative infinity.\",\n \"The add-inst controls for each shifter S1-S4 may further include shift-up-2 and shift-down-2, supporting shifting by 2 bits, as well as shift-up-3 and shift-down-3 supporting shifting by 3 bits.\",\n \"Certain preferred embodiments of the invention employ a subset of all these add-inst controls including same-sign, reverse-sign, do-not-use, shift-up, shift-down, shift-up-2, shift-up-3, which may be coded as 2 bits designating same-sign, reverse-sign, do-not-use, shift-down; and 2 additional bits coding pass-through, shift-up, and shift-up-2.Note that as shown hereafter, when do-not-use is asserted, it does not matter what the other two bit field contains.\",\n \"In general, when do-not-use is asserted, the contents of the other two bit field will be chosen to optimize at least one system characteristic, such as testability and/or logic complexity and/or signal propagation through the circuit, also known as the circuit's critical path delay.\",\n \"The add-inst control signals may originate from the datapath instruction being presented for execution, or may alternatively be generated based upon part of a numeric data to perform a limited range of multiplications.\",\n \"Consider the following table Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 do-not-use same-sign, shift-up-1 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => 4 same-sign, shift-up-2 do-not-use 101 => 5 = 4 + 1 same-sign, shift-up-2 same-sign, pass-through 110 => 6 = 4 + 2 same-sign, shift-up-2 same-sign, shift-up-1 111 => 7 = 8 − 1 reverse-sign, pass-through same-sign shift-up-3 Table One: a three bit multiplication based upon controlling a pair of shifter inputs.\",\n \"Note that this further supports the circuitry shown in FIG.\",\n \"9 as supporting the multiplication of 6 bits of an operand acting upon a second number, which may be further provided by a local memory.\",\n \"Using this circuitry in such a fashion provides an interpolation capability supporting successive approximations, which may involve linear, multi-linear and through the feedback-cascading of partial results based upon this circuit, some forms of non-linear approximations to various non-linear functions including but not limited to various exponential functions, logarithms, trigonometric functions, among others.\",\n \"An alternative approach to approximation interprets a three bit number as a signed number, leading to the following table: Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 same-sign, pass-through same-sign, pass-through 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => −4 reverse-sign, shift-up-1 reverse-sign, shift-up-1 101 => −3 = −4 + 1 reverse-sign, pass-through reverse-sign, shift-up-1 110 => −2 reverse-sign, pass-through reverse-sign, pass-through 111 => −1 reverse-sign, pass-through do-no-use Table Two: a signed three bit multiplication based upon controlling a pair of shifter inputs.\",\n \"Note that these two tables may be concurrently employed in certain situations where a signed 6 bit numeric multiplication is desired.\",\n \"The most significant 3 bits affect multiplication as shown in Table Two.\",\n \"When the sign of the 6 bit quantity is negative, the least significant 3 bits may affect the multiplication as shown in Table One, with every instance of reverse-sign changed to same-sign, and every instance of same-sign changed to reverse-sign.\",\n \"When the sign of the 6 bit quantity is positive, Table One may be used as shown.\",\n \"These tables and discussions have been provided by way of example and are not meant to limit the scope of the claims.\",\n \"As one of skill in the art will readily recognize, there are many alternative notations for the various operations presented herein which are essentially equivalent to the examples presented herein.\",\n \"FIG.\",\n \"10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300, in accordance with certain embodiments of the invention.\",\n \"In certain embodiments of the invention, only one instruction memory 3200 is coupled to Arithmetic Processor Array 3000, feeding one instruction to each Arithmetic Processor 3002.FIGS.\",\n \"11A to 16 refer to such embodiments of the invention.\",\n \"Note that these embodiments of the invention do not require the partitioning wire bundle 3102, nor the partitioning wire bundle 1002.FIG.\",\n \"11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.\",\n \"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.\",\n \"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.\",\n \"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIG.\",\n \"11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.\",\n \"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.\",\n \"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.\",\n \"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIGS.\",\n \"12 to 16 show a configuration of instruction memories and MSRI[k] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples.\",\n \"Assume the horizontal datapath width of the ALU2 and memories in each MSRI is at least 3, preferably 4, bits.\",\n \"When the horizontal datapath width of the ALU2 and memories in each MSRI is 4 bits, 10 bit samples could also be accepted.\",\n \"The data stream enters MSRI[4] in FIG.\",\n \"12 flowing to MSRI[5], then MSRI[6], MSRI[7], then MSRI[8] of FIG.\",\n \"13 eventually leaving MSRI[231 having performed as a 12 pass arithmetic stream processor.\",\n \"If each MSRI component performs a radix 4 FFT, then the system as a whole performs a 1K FFT by the time the data leaves MSRI[21], and a 4K FFT upon leaving MSRI[23].\",\n \"The increase in depth of the MSRI's is to consistently extend the precision of the calculations to account for the accumulation of rounding errors.\",\n \"In certain other embodiments of the invention, two instruction memories 3200 and 3300 are coupled to Arithmetic Processor Array 3000, feeding two instructions to each Arithmetic Processor 3002, the selection of which instruction is executed determined by the partitioning wire bundle 3102, which in turn drives the partitioning wire bundle 1002.FIGS.\",\n \"17A to 20 refer to such embodiments of the invention.\",\n \"FIG.\",\n \"17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.\",\n \"Note that in other embodiments of the invention, i could range over a different range.\",\n \"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the firstmemories 1210 as second memories.\",\n \"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.\",\n \"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[k] cell array where i ranges from 1 to k. This circuit and the circuit of FIG.\",\n \"17B use the partitioning wire bundle 1002 to control carry propagation and determination of which of the two instructions are executed in each horizontal strip of the PISRI.\",\n \"FIG.\",\n \"17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.\",\n \"Note that in other embodiments of the invention, i could range over a different range.\",\n \"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.\",\n \"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.\",\n \"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[K] cell array where i ranges from 1 to k. FIGS.\",\n \"18 to 20 show various configurations of PISRI's and their neighboring instruction memories.\",\n \"In FIG.\",\n \"18, PSRI[11] can be configured to act as an MSRI[4] and MSRI[7], or as an MSRI[5] and MSRI[6].\",\n \"The net effect can then be the processing flow of MSRI[4]==>MSRI[5]==>MSRI[6]==>MSRI[7].\",\n \"Similarly, the PSRI[19]'s can be configured as ==>MSRI[9]==>MSRI[10]==>MSRI[11].\",\n \"FIG.\",\n \"19 shows a configuration of same size PISRI[8]'s which can be support dataflows as discussed above, and which provide a 32 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 4 bits.\",\n \"Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.\",\n \"FIG.\",\n \"20 shows a configuration of same size PISRI[12]'s which can be support dataflows as discussed above, and which provide a 36 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 3 bits.\",\n \"Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.\",\n \"FIG.\",\n \"21 shows a high level data flow diagram of a two dimensional array blocks 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported.\",\n \"Data flow as depicted in FIG.\",\n \"21 supports both nearest neighbor and global communication of array blocks in each of the two dimensions of the array.\",\n \"Note that the vertical communications lines shown in FIG.\",\n \"21 may be used to transfer data via an external data memory interface which is not shown.\",\n \"Such an external data memory interface may receive addressing signal either from datapath element(s), and/or from one or more instruction processors.\",\n \"FIG.\",\n \"22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource in accordance with certain embodiments of the invention.\",\n \"User operation 2000 starts the usage of this flowchart.\",\n \"Arrow 2002 directs the usage flow from user operation 2000 to user operation 2004.User operation 2004 performs selecting the input-output resources to create an input-output access collection comprised of at least one input-output access parameter Arrow 2006 directs usage from user operation 2004 to user operation 2008.User operation 2008 performs selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.\",\n \"Arrow 2010 directs usage from user operation 2008 to user operation 2012.User operation 2012 terminates the usage of this flowchart.\",\n \"Note that in other embodiments of the invention, the flowchart of FIG.\",\n \"22A may be used to depict a method of operating an array of computational resources containing at least one input-output resource, at least one datapath operational resource.\",\n \"The array of computational resources may be implemented using one or more programmable logic devices, which may include or more programmable logic arrays and/or one or more Filed Programmable Gate Arrays (FPGAs).\",\n \"Certain further embodiments of the invention include the array of computation resources further containing at least one instruction propagating resource.\",\n \"FIG.\",\n \"22B depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration, in accordance with certain embodiments of the invention.\",\n \"Arrow 2030 directs the usage flow from starting user operation 2000 to user operation 2032.User operation 2032 performs selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.\",\n \"Arrow 2034 directs usage from user operation 2032 to user operation 2036.User operation 2036 terminates the usage of this flowchart.\",\n \"FIG.\",\n \"23A depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration in accordance with certain embodiments of the invention.\",\n \"Arrow 2050 directs the usage flow from starting user operation 2000 to user operation 2052.User operation 2052 performs selecting the instruction processing resources based upon the input-output access collection and based upon the datapath operational resource allocation collection and based upon the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration.\",\n \"Arrow 2054 directs usage from user operation 2052 to user operation 2056.User operation 2056 terminates the usage of this flowchart.\",\n \"Certain further embodiments of the invention include the array of computation resources further containing at least one instruction fetching resource and the instruction processing resources containing at least one instruction register.\",\n \"FIG.\",\n \"23B depicts a detail flowchart of user operation 2000 of FIG.\",\n \"22A further performing the method of using/operating the array of computational resources in accordance with certain embodiments of the invention.\",\n \"Arrow 2070 directs the usage flow from starting user operation 2000 to user operation 2072.User operation 2072 performs fetching using the instruction fetching resource to create a fetched instruction.\",\n \"Arrow 2074 directs usage from user operation 2072 to user operation 2076.User operation 2076 performs loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.\",\n \"Arrow 2078 directs usage from user operation 2076 to user operation 2080.User operation 2080 terminates the usage of this flowchart.\",\n \"FIG.\",\n \"24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500, in accordance with certain embodiments of the invention.\",\n \"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.\",\n \"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.\",\n \"24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.\",\n \"24A further comprised of instruction fetch mechanism 3800, in accordance with certain embodiments of the invention.\",\n \"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.\",\n \"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.\",\n \"25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720, in accordance with certain embodiments of the invention.\",\n \"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.\",\n \"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.\",\n \"Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.\",\n \"FIG.\",\n \"26 shows a detail block diagram of an branch processor 3700 extending the block diagram of FIG.\",\n \"25 further comprised of cache manager 3740, in accordance with certain embodiments of the invention.\",\n \"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.\",\n \"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.\",\n \"Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.\",\n \"FIG.\",\n \"27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention.\",\n \"The DSP Resource Circuit is comprised a Datapath Resource Array 5000.The Datapath Resource Array 5000 is coupled to at least one of the following: the Digital Device Interface 5300 and the System and Control Interface 5400.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5312,5314 and/or 5316 with Digital Device Interface 5300.In certain applications, coupling 5312 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.\",\n \"Coupling 5314 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5316 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.\",\n \"Note that the couplings 5302, 5304, and 5306 preferably respectively relate to the external communications associated with couplings 5312, 5314, and 5316 in such applications.\",\n \"In other applications, one input-output processor may strictly receive data via coupling 5316 which is generated based upon an external input stream via coupling 5306.Additionally, an input-output processor may strictly output data via coupling 5312 which is used to generate an external data stream presented via coupling 5302.In certain applications, each of these coupling may be preferably split into two such couplings, each under the control of a separate input-output processor.\",\n \"The Datapath Resource Array 5000 may also be coupled to a Local Memory Interface 5500.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5512,5514 and/or 5516 to Local Memory Interface 5500.Preferably, coupling 5512 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.\",\n \"Coupling 5514 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5516 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.\",\n \"Note that preferably, couplings 5502, 5504, and 5506 respectively relate to the external communications associated with couplings 5512, 5514, and 5516.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5402 and/or 5404 to System and Control Interface 5400.Each or either of couplings 5402 and 5404 may be comprised of a collection of couplings such as discussed above in FIG.\",\n \"27 with unidirectional couplings and possibly strictly bi-directional couplings.\",\n \"In certain applications, coupling 5402 may preferably convey system control and status information related via 5406 with an external system environment.\",\n \"In certain applications, coupling 5404 may convey data communicated via 5406 with the external system environment.\",\n \"Such data may be provided during system initialization time for conveyance not only into internal memory within the Datapath Resource Array 5000.Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Local Memory Interface 5500 for storage in local memory.\",\n \"Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Digital Device Interface 5300 for use elsewhere.\",\n \"FIG.\",\n \"28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.\",\n \"27.Datapath Resource Array 5000 is comprised of at least one instruction processor 3800 and an array of DSP resources 1400.By way of example, FIG.\",\n \"28 depicts an array of 8 rows and 8 columns of DSP resource 1400 instances.\",\n \"Instruction processor 3800 may be closely coupled with an instruction memory array 3500.In certain applications, Datapath Resource Array 5000 is preferably comprised of two instruction processors 3800-1 and 3800-2.At this level of abstraction the partition control wire bundle is not visible.\",\n \"However, it is assumed to support partitioning the array of DSP resources into two horizontal regions.\",\n \"By way of example, the top three rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 1 3800-1.The remaining bottom 5 rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 2 3800-2.In certain applications a further partitioning of instruction processing resources may be preferred.\",\n \"An instruction processor 3800 may be partitioned into two instruction processing streams, each with independent branch mechanism.\",\n \"The first instruction processing stream may be partitioned to control the instruction state asserted for the three left-most columns of the array of DSP resources.\",\n \"The second instruction processing stream would control the remaining 5 columns of the array of DSP resources.\",\n \"Note that in certain applications, partitioning may support more than two instruction processing streams being sent from an instruction processor.\",\n \"For simplicity of discussion, no more than two instruction streams will be discussed hereafter.\",\n \"This is not meant to limit the scope of the claims.\",\n \"By way of example, the applications and configurations of FIGS.\",\n \"12 to 20 may be implemented by circuitry illustrated in FIG.\",\n \"28.Digital Device Interface 5300 is comprised of at least one at least one input-output instruction processor 1130-1 controlling an input-output processor 1120-1.Digital Device Interface 5300 may be further comprised of a second input-output instruction processor 1130-2 controlling input-output processor 1120-2.In certain applications, at least one input-output instruction processor 1130 may be coupled to an input-output instruction memory 1140.Input-output processors preferably possess couplings to all the rows of their associated array of DSP resources 1400.Input-output processors preferably configure communication to the array of DSP resources based upon the partition state information which configures the rows of the array of DSP resources to communicate together.\",\n \"By way of example, the first input-output processor 1120-1 may communicate with the top three rows of the array of DSP resources, when they are so partitioned.\",\n \"The second input-output processor 1120-2 may then preferably communicate with the remaining 5 bottom rows.\",\n \"Note that the rows of DSP resources may be partitioned into more than two communicating components.\",\n \"Similarly, the Digital Device Interface 5300 may comprise more than two input-output instruction processors controlling more than two input-output processors.\",\n \"Each input-output processor may include at least one of the following: a data memory, ALU, and specialized logical functions such as bit packing-unpacking circuits.\",\n \"Note that the circuitry of FIGS.\",\n \"27 and 28 may further reside in a package where at least one of the interfaces couples to analog circuitry including but not limited to at least one of the following: A/D converters, D/A converters, frequency synthesizers, threshold detectors, and amplifiers.\",\n \"Note that implementations of multiple instances of the circuitry of FIG.\",\n \"28 may include situations where one input-output processor of a first instance may not couple to elements of another array of DSP resources 1400 within another instance.\",\n \"System and Control Interface 5400 may be comprised of at least one input-output processor 1120-3 controlled by input-output instruction processor 1130-3.The preceding discussion regarding the Digital Device Interface is applicable to System and Control Interface 5400, and will not be repeated for reasons of brevity.\",\n \"However, this is not meant limit the scope of the claims.\",\n \"A common branching mechanism is preferably employed through the instruction processors discussed in FIG.\",\n \"28.This branching mechanism can be embodied to support caching and accessing of memory through a local memory interface 5500, or may be embodied without support of external memory.\",\n \"The overall instruction processing principles embodied in this invention include the following design/architectural goals: Input-output and datapath configurations dominate the embodied architectures, not the other way around.\",\n \"The hardware supports software debugging and test.\",\n \"The embodied architectures can support multiple systems levels of instruction fetching.\",\n \"They can support both SIMD and MIMD, as well as SISD and MISD processing applications.\",\n \"Implementations support separate references to data and instructions.\",\n \"There are several consequences to separate data and instruction references.\",\n \"The runtime environments of procedural languages such C, C++, PASCAL, FORTRAN and JAVA.\",\n \"By employing an invariant instruction set across a variety of datapath size ranges, a single program can handle a wide range of input data sizes with the arithmetical precision preserved by construction.\",\n \"Another consequence is the requirement of parameter passing to functions and subroutines by reference only.\",\n \"Consider for a moment the runtime stack frame requirements of the C programming language.\",\n \"C's runtime stack frame contains the following components: Branch related pointers, loop counters, data address references and data values, all of which may have differing data widths.\",\n \"By partitioning the stack frame into multiple stack frame into separate stack frames to handle differing data widths, the communication and manipulation of data to “fit” onto a single stack frame, or “fit” back into the processing element where it is needed is minimized.\",\n \"There are further architectural preferences which ease the task of compilation of procedural language programs as well as improve the reliability of these translations.\",\n \"FIG.\",\n \"29 depicts a preferred version of ALU 1400, as used in FIG.\",\n \"28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank.\",\n \"Arithmetic processor 1400 preferably contains 3 ALUs, 1300-1, 1300-2 and 1300-3, with two memories 1200-1 and 1200-2 respectively feeding 1016 and 1018 the first two ALUs 1300-1 and 1300-2.The third ALU 1300-3 is feed by at least ALU 1300-2 and wire bundle 1002.System input 1002 preferably contains representations of normal numbers and numbers in a logarithmic domain.\",\n \"The logarithmic domain will be discussed in detail shortly.\",\n \"The normal number representation be stored in memory 1200-1.ALU 1300-2 may further be implemented in a fashion as shown in FIG.\",\n \"9, with shift controls being provided from part of the numeric signaling received from wire bundle 1018, as well as at least part of the addressing being provided by parts of the numeric signals received from wire bundle 1018.The results of the two ALU 1300-1 and 1300-2 operations are sent 1018 and 1036 to ALU 1300-3, where the results are combined to form an efficient and very accurate approximation of a sum of numerical products.\",\n \"As mentioned earlier, there are some fundamental problems with multipliers.\",\n \"Most importantly, they tend to grow with the product of the input precision.\",\n \"While this may be somewhat constrained by output precision requirements, it remains a fundamental problem.\",\n \"An approach that solve this problem will now be described.\",\n \"Consider the action of representing each member of a number collection by an integer part and a special part, where the special part contains exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity.\",\n \"FIG.\",\n \"30 depicts a flowchart of a method of processing numeric data, which may be variously embodied.\",\n \"Arrow 2210 directs the flow of execution from starting operation 2200 to operation 2212.Operation 2212 performs representing each member of a number collection by an integerpart and a special part.\",\n \"Arrow 2214 directs execution from operation 2212 to operation 226.Operation 226 terminates the operations of this flowchart.\",\n \"Arrow 2220 directs the flow of execution from starting operation 2200 to operation 2222.Operation 2222 performs performing at least one member of the arithmetic operation collection upon at least one of the members of the number collection.\",\n \"Arrow 2224 directs execution from operation 2222 to operation 226.Operation 226 terminates the operations of this flowchart.\",\n \"Certain embodiments of the invention may perform all members of the arithmetic operation collection upon the relevant member of the number collection.\",\n \"Certain embodiments of the invention may further include one or both of the following operational steps.\",\n \"Arrow 2230 directs the flow of execution from starting operation 2200 to operation 2232.Operation 2232 performs log-converting a member of an input number collection to create a member of the number collection.\",\n \"Arrow 2234 directs execution from operation 2232 to operation 226.Operation 226 terminates the operations of this flowchart.\",\n \"Arrow 2240 directs the flow of execution from starting operation 2200 to operation 2242.Operation 2242 performs exp-converting a member of the number collection to create a member of an output number collection.\",\n \"Arrow 2244 directs execution from operation 2242 to operation 2216.Operation 2216 terminates the operations of this flowchart.\",\n \"FIG.\",\n \"31 depicts a detail flowchart of operation 2222 of FIG.\",\n \"30 further performing the arithmetic operation collection.\",\n \"Arrow 2270 directs the flow of execution from starting operation 2222 to operation 2272.Operation 2272 performs adding the first number to the second number to create an add-result.\",\n \"Arrow 2274 directs execution from operation 2272 to operation 2276.Operation 2276 terminates the operations of this flowchart.\",\n \"Arrow 2280 directs the flow of execution from starting operation 2222 to operation 2282.Operation 2282 performs subtracting the first number by the second number to create a subtract-result.\",\n \"Arrow 2284 directs execution from operation 2282 to operation 2276.Operation 2276 terminates the operations of this flowchart.\",\n \"Arrow 2290 directs the flow of execution from starting operation 2222 to operation 2292.Operation 2292 performs exponentiating the first number to create an exp-result.\",\n \"Arrow 2294 directs execution from operation 2292 to operation 2276.Operation 2276 terminates the operations of this flowchart.\",\n \"Arrow 2300 directs the flow of execution from starting operation 2222 to operation 2302.Operation 2302 performs logarithming the first number to create a log-result.\",\n \"Arrow 2304 directs execution from operation 2302 to operation 2276.Operation 2276 terminates the operations of this flowchart.\",\n \"Note that the number collection is further comprised of the add-result, the subtract-result, the exp-result and the log-result.\",\n \"FIG.\",\n \"32A depicts a detail flowchart of operation 2272 of FIG.\",\n \"31 further adding.\",\n \"Arrow 2330 directs the flow of execution from starting operation 2272 to operation 2332.Operation 2332 performs determining whether the special part of the first number contains the negative-infinity.\",\n \"Arrow 2334 directs execution from operation 2332 to operation 2336.Operation 2336 terminates the operations of this flowchart.\",\n \"Arrow 2340 directs the flow of execution from starting operation 2272 to operation 2342.Operation 2342 performs determining whether the special part of the second number contains the negative-infinity.\",\n \"Arrow 2344 directs execution from operation 2342 to operation 2336.Operation 2336 terminates the operations of this flowchart.\",\n \"Arrow 2350 directs the flow of execution from starting operation 2272 to operation 2352.Operation 2352 performs setting the special part of the add-result to contain the negative-infinity whenever the special part of at least one member of the collection the first number and the second number contains the negative-infinity.\",\n \"Arrow 2354 directs execution from operation 2352 to operation 2336.Operation 2336 terminates the operations of this flowchart.\",\n \"FIG.\",\n \"32B depicts a detail flowchart of operation 2282 of FIG.\",\n \"31 further subtracting.\",\n \"Arrow 2370 directs the flow of execution from starting operation 2282 to operation 2372.Operation 2372 performs determining whether the special part of the first number contains the negative-infinity.\",\n \"Arrow 2374 directs execution from operation 2372 to operation 2376.Operation 2376 terminates the operations of this flowchart.\",\n \"Arrow 2380 directs the flow of execution from starting operation 2282 to operation 2382.Operation 2382 performs setting the special part of the subtract-result to contain the negative-infinity whenever the special part of the first number contains the negative-infinity.\",\n \"Arrow 2384 directs execution from operation 2382 to operation 2376.Operation 2376 terminates the operations of this flowchart.\",\n \"FIG.\",\n \"33A depicts a detail flowchart of operation 2292 of FIG.\",\n \"31 further exponentiating.\",\n \"Arrow 2410 directs the flow of execution from starting operation 292 to operation 2412.Operation 2412 performs determining whether the special part of the first number contains the negative-infinity.\",\n \"Arrow 2414 directs execution from operation 2412 to operation 2416.Operation 2416 terminates the operations of this flowchart.\",\n \"Arrow 2420 directs the flow of execution from starting operation 292 to operation 2422.Operation 2422 performs setting the special part of the exp-result to contain the not-negative-infinity and setting the integer part to a zero-representation whenever the special part of the first number contains the negative-infinity.\",\n \"Arrow 2424 directs execution from operation 2422 to operation 2416.Operation 2416 terminates the operations of this flowchart.\",\n \"FIG.\",\n \"33B depicts a detail flowchart of operation 2302 of FIG.\",\n \"31 further logarithming.\",\n \"Arrow 2430 directs the flow of execution from starting operation 2302 to operation 2432.Operation 2432 performs determining whether the integer part of the first number is essentially equal to the zero-representation.\",\n \"Arrow 2434 directs execution from operation 2432 to operation 2436.Operation 2436 terminates the operations of this flowchart.\",\n \"Arrow 2440 directs the flow of execution from starting operation 2302 to operation 2442.Operation 2442 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number essentially equals the zero-representation.\",\n \"Arrow 2444 directs execution from operation 2442 to operation 2436.Operation 2436 terminates the operations of this flowchart.\",\n \"Note that the integer part of each of the number collection members may be in a non-redundant numeric notation.\",\n \"FIG.\",\n \"34A depicts a detail flowchart of operation 2442 of FIG.\",\n \"33B further setting the special part of the log-result.\",\n \"Arrow 2470 directs the flow of execution from starting operation 2442 to operation 2472.Operation 2472 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number equals the zero-representation.\",\n \"Arrow 2474 directs execution from operation 2472 to operation 2476.Operation 2476 terminates the operations of this flowchart.\",\n \"Alternatively, the integer part of each of the number collection members may be in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances.\",\n \"FIG.\",\n \"34B depicts a detail flowchart of operation 2442 of FIG.\",\n \"33B further setting the special part of the log-result.\",\n \"Arrow 2490 directs the flow of execution from starting operation 2442 to operation 2492.Operation 2492 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number is a member of the zero-representation collection.\",\n \"Arrow 2494 directs execution from operation 2492 to operation 2496.Operation 2496 terminates the operations of this flowchart.\",\n \"Note that the integer part of each member of the number collection may contains a sign and a magnitude.\",\n \"Further, for each member of the number collection, the sign may be a member of a sign collection consisting essentially of a positive-sign and a negative-sign.\",\n \"The special part of each member of the number collection may further contain exactly one member of a second special value collection comprising a special-minus and a special-plus.\",\n \"FIG.\",\n \"35A depicts a detail flowchart of operation 2292 of FIG.\",\n \"31 further exponentiating.\",\n \"Arrow 2510 directs the flow of execution from starting operation 2292 to operation 2512.Operation 2512 performs setting the sign of the exp-result to essentially the negative-sign whenever the special part of the first number contains the special-minus.\",\n \"Arrow 2514 directs execution from operation 2512 to operation 2516.Operation 2516 terminates the operations of this flowchart.\",\n \"FIG.\",\n \"35B depicts a detail flowchart of operation 2302 of FIG.\",\n \"31 further logarithming.\",\n \"Arrow 2530 directs the flow of execution from starting operation 2302 to operation 2532.Operation 2532 performs determining whether the sign part of the first number is essentially equal to the negative-sign.\",\n \"Arrow 2534 directs execution from operation 2532 to operation 2536.Operation 2536 terminates the operations of this flowchart.\",\n \"Arrow 2540 directs the flow of execution from starting operation 2302 to operation 2542.Operation 2542 performs setting the special part of the log-result to contain the special-minus whenever the sign part of the first number is essentially the negative-sign.\",\n \"Arrow 2544 directs execution from operation 2542 to operation 2536.Operation 2536 terminates the operations of this flowchart.\",\n \"The integer part of each of the number collection members may be in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps.\",\n \"FIG.\",\n \"36A depicts a detail flowchart of operation 2532 of FIG.\",\n \"35B further determining whether the sign part of the first number is essentially equal to the negative-sign.\",\n \"Arrow 2570 directs the flow of execution from starting operation 2532 to operation 2572.Operation 2572 performs determining whether the sign part of the first number is equal to the negative-sign based upon performing at least one of the members of the negative-test collection.\",\n \"Arrow 2574 directs execution from operation 2572 to operation 2576.Operation 2576 terminates the operations of this flowchart.\",\n \"Alternatively, the integer part of each of the number collection members is in a non-redundant numeric notation possessing exactly one negative-test step.\",\n \"FIG.\",\n \"36B depicts a detail flowchart of operation 2532 of FIG.\",\n \"35B further determining whether the sign part of the first number is essentially equal to the negative-sign.\",\n \"Arrow 2590 directs the flow of execution from starting operation 2532 to operation 2592.Operation 2592 performs performing the exactly one negative-test step based upon the first number.\",\n \"Arrow 2594 directs execution from operation 2592 to operation 2596.Operation 2596 terminates the operations of this flowchart.\",\n \"Note that each member of the input number collection may be comprised of an integer part.\",\n \"FIG.\",\n \"37A depicts a detail flowchart of operation 2232 of FIG.\",\n \"30 further log-converting the input number collection member.\",\n \"Arrow 2600 directs the flow of execution from starting operation 2232 to operation 2602.Operation 2602 performs determining whether the integer part of the input number collection member is essentially equal to the zero-representation.\",\n \"Arrow 2604 directs execution from operation 2602 to operation 2606.Operation 2606 terminates the operations of this flowchart.\",\n \"Arrow 2610 directs the flow of execution from starting operation 2232 to operation 2612.Operation 2612 performs setting the special part of the number collection member to contain the negative-infinity whenever the integer part of the input number collection member essentially equals the zero-representation.\",\n \"Arrow 2614 directs execution from operation 2612 to operation 2606.Operation 2606 terminates the operations of this flowchart.\",\n \"Note that the integer part of each of the input number collection members may contain a sign belonging to the sign collection and a magnitude.\",\n \"FIG.\",\n \"37B depicts a detail flowchart of operation 2232 of FIG.\",\n \"30 further log-converting the input number collection member.\",\n \"Arrow 2630 directs the flow of execution from starting operation 2232 to operation 2632.Operation 2632 performs determining whether the sign part of the input number collection member is essentially equal to the negative-sign.\",\n \"Arrow 2634 directs execution from operation 2632 to operation 2636.Operation 2636 terminates the operations of this flowchart.\",\n \"Arrow 2640 directs the flow of execution from starting operation 2232 to operation 2642.Operation 2642 performs setting the special part of the number collection member to contain the special-minus whenever the sign part of the input number collection member is essentially the negative-sign.\",\n \"Arrow 2644 directs execution from operation 2642 to operation 2636.Operation 2636 terminates the operations of this flowchart.\",\n \"Note that each of the output number collection members may include a magnitude.\",\n \"FIG.\",\n \"38A depicts a detail flowchart of operation 2242 of FIG.\",\n \"30 further exp-converting.\",\n \"Arrow 2650 directs the flow of execution from starting operation 2242 to operation 2652.Operation 2652 performs setting the magnitude of the output number collection member to the zero-representation whenever the special part of the number collection member contains the negative-infinity.\",\n \"Arrow 2654 directs execution from operation 2652 to operation 2656.Operation 2656 terminates the operations of this flowchart.\",\n \"Also, each of the output number collection members may include a sign belonging to the sign collection.\",\n \"FIG.\",\n \"38B depicts a detail flowchart of operation 2242 of FIG.\",\n \"30 further exp-converting.\",\n \"Arrow 2670 directs the flow of execution from starting operation 2242 to operation 2672.Operation 2672 performs setting the sign of the output number collection member to the negative-sign whenever the special part of the number collection member contains the special-minus.\",\n \"Arrow 2674 directs execution from operation 2672 to operation 2676.Operation 2676 terminates the operations of this flowchart.\",\n \"Arrow 2680 directs the flow of execution from starting operation 2242 to operation 2682.Operation 2682 performs setting the sign of the output number collection member to the positive-sign whenever the special part of the number collection member contains the special-plus.\",\n \"Arrow 2684 directs execution from operation 2682 to operation 2686.Operation 2686 terminates the operations of this flowchart.\",\n \"Note that each of the input number collection members may be encoded as an N1 bit code; wherein the N1 is at least three.\",\n \"The integer part of each of the number collection members may be encoded as an N2 bit code; wherein N2 is greater than N1.This method of numeric processing may be implemented as a program system comprised of program steps implementing the steps of the method.\",\n \"The program steps may reside in a memory accessibly coupled to a computer, which executes these program steps.\",\n \"The program system may further be implemented as program steps in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon the computer.\",\n \"The invention includes circuitry generated from those program steps.\",\n \"The invention also includes circuitry implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection.\",\n \"Where the programmable logic device collection refers to all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array.\",\n \"The fixed architecture device collection refers to all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.\",\n \"The preceding embodiments of the invention have been provided by way of example and are not meant to constrain the scope of the following claims.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10276414"}}},{"rowIdx":85,"cells":{"text":{"kind":"list like","value":[["Centrifugal pump, in particular a spirally-housed centrifugal pump for a heat exchange medium","In a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), parts of the casing cover (18) are a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction.","The bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a), and the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56)."],["1.A centrifugal pump, in particular a spiral-casing centrifugal pump, for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), characterised in that a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover (18).","2.A centrifugal pump as set forth in claim 1 characterised in that the bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a).","3.A centrifugal pump as set-forth in claim 1 or claim 2 characterised in that the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56).","4.A centrifugal pump as set forth in claim 3 characterised in that at their ends remote from the base region (20) of the casing cover (18) the support ribs (26) are connected by the edge ring (30) surrounding the longitudinal axis (A) of the pump.","5.A centrifugal pump as set forth in one of claims 1 through 4 characterised in that the support ribs (26) are inclined at an angle (w) to the longitudinal axis (A) of the pump from the base region (20) of the casing cover (18) towards the shaft seal (56).","6.A centrifugal pump as set forth in claim 5 characterised by an angle (w) of between 30° and 60°.","7.A centrifugal pump as set forth in one of claims 1 through 6 characterised in that the cross-sectional height (h) of the support ribs (26) which preferably extend in axial planes (E) passing radially through the longitudinal axis (A) of the pump increases towards the sliding ring seal (56).","8.A centrifugal pump as set forth in one of claims 1 through 7 characterised in that the sealing chamber (48) adjoins the shaft bearing (76, 76a).","9.A centrifugal pump as set forth in one of claims 1 through 8 characterised in that at least one abutment step (28) directed away from the spiral casing (24) for a further pump portion (96) is arranged at the casing cover (18).","10.A centrifugal pump as set forth in claim 9 characterised in that the abutment step is a front ring (22) formed on the casing cover (18).","11.A centrifugal pump as set forth in claim 9 or claim 10 characterised in that the abutment step or the front ring (22) projects from an annular flange (21) which surrounds the support ribs (26) and which is disposed at the transition thereof to a base region (20) of the casing cover (18).","12.A centrifugal pump comprising a sealing ring (59) of the shaft seal which is in the form of a sliding ring seal (56), the sealing ring extending peripherally at a counterpart ring (60) provided at the housing side, as set forth in one of claims 1 through 11, characterised in that the counterpart ring (60) for the sealing ring (59) is arranged in a shaped stub portion (64) of the casing attachment portion (34), the stub portion projecting axially into the sealing chamber (48), and associated with the stub portion is at least one passage (58) inclined with respect to the longitudinal axis (A) of the pump, with a closure screw (52) closing the passage.","13.A centrifugal pump as set forth in claim 12 characterised by an angle (z) between the longitudinal axis (A) of the pump and the longitudinal axis (B) of the passage (53) of about 45°.","14.A centrifugal pump as set forth in claim 12 or claim 13 characterised by an axial length (f) of the stub portion (64) which corresponds approximately to half the axial length (e1) of the sealing chamber (48).","15.A centrifugal pump as set forth in one of claims 1 through 14 characterised in that the shaft bearing is in the form of a plain bearing (76) with a ratio of the length (g) of the bearing bush (77) to the diameter (d) of the pump shaft (35, 36, 36a) of between about 1.5 and 2.0, preferably about 1.8.16.A centrifugal pump as set forth in claim 15 characterised in that the bearing bush (77) of the plain bearing (76, 76a) is adapted to be dismantleable.","17.A centrifugal pump as set forth in claim 15 or claim 16 characterised in that the plain bearing (76, 76a) or its bearing bush (77) is externally embraced approximately at its longitudinal center by a clamping or tolerance ring (80) which engages into the bearing cup (28).","18.A centrifugal pump as set forth in claim 17 characterised in that the width (n) of the clamping or tolerance ring (80) corresponds to a fraction of the length (g) of the bearing bush (77).","19.A centrifugal pump as set forth in claim 18 characterised in that the width (n) of the external clamping or tolerance ring (80) corresponds approximately to between a fifth and a sixth of the length (g) of the bearing bush (77).","20.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76a) includes an inner bearing sleeve (82) surrounded by a bearing bush (77) at the bearing cup side (FIGS.","6 and 7).","21.A centrifugal pump as set forth in one of claims 15 through 20 characterised in that within the plain bearing (76a) the pump shaft (35, 36a) is embraced by at least one tolerance ring (80i) which engages into the pump shaft.","22.A centrifugal pump as set forth in one of claims 17 through 21 characterised in that provided on respective sides of the outer tolerance ring (80), viewed in longitudinal section of the centrifugal pump (10a), is a respective inward tolerance ring (80i) which engages into the pump shaft (35, 36a).","23.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76) includes a bearing bush (77) at the bearing cup side, with a slidable internal peripheral surface portion (78) embracing the pump shaft (36).","24.A centrifugal pump as set forth in claim 23 characterised by an internal peripheral surface portion (28) of the plain bearing (76) comprising carbon (FIGS.","4 and 5).","25.A centrifugal pump as set forth in claim 23 or claim 24 characterised by a hardened pump shaft (36) or a corresponding shaft portion which is associated with the bearing bush (77).","26.A centrifugal pump as set forth in one of claims 1 through 25 characterised in that the sliding ring seal (56) embraces a shaft sleeve (89) at the shaft side, wherein at least one O-ring (88) is provided between the shaft sleeve (89) and the pump shaft (36, 36a).","27.A centrifugal pump as set forth in one of claims 1 through 26 characterised in that a rearward shaft bearing (68) for the pump shaft (36, 36a) is arranged in the casing attachment portion (34) at an axial spacing relative to the sliding ring seal (56) outside the sealing chamber (48).","28.A centrifugal pump as set forth in claim 27 characterised in that at least one hollow space (66) is arranged between the sealing chamber (48) and the shaft bearing (68) in the casing attachment portion (34).","29.A centrifugal pump as set forth in one of claims 1 through 28 characterised in that the pump shaft (36a) is in the form of a plug-in stub shaft with clamping ring (92).","30.A centrifugal pump as set forth in one of claims 8 through 29 characterised in that the spiral casing (14) and the bearing carrier casing (16) are embraced by a spacer (96) which butts against the abutment step or steps or the front ring (22) respectively and it is attached to a motor (100)."],["The invention concerns a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing having an impeller, a casing cover and an associated casing attachment portion for a pump shaft provided with a shaft bearing near the spiral casing and a shaft seal disposed at a spacing in relation to the shaft bearing in a sealing chamber.","Such centrifugal pumps are described by way of example in a prospectus from the present applicants in relation to the range NHT for heat carrier purposes up to 350° C. A spiral casing for an impeller is connected to a bearing casing by means of a throttle section having external ribs.","The impeller is fixed at one end of a pump shaft which extends in the throttle section.","Arranged between the throttle section and the impeller is a plain bearing of silicon carbide and arranged downstream of the throttle section is a sliding ring seal which is followed by a rolling bearing.","This arrangement involves a horizontal spiral-casing centrifugal pump of a process type of structure which is single-stage in terms of intake flow.","The pump-side silicon carbide plain bearing is lubricated by the fluid being delivered, while used at the drive side is a grease-lubricated grooved-type ball bearing.","In consideration of that state of the art, the inventor set himself the aim of providing a centrifugal pump of the kind set forth in the opening part of this specification, which is simple to manufacture and which can be inexpensively and particularly well used.","That object is attained by the teaching of the independent claim; the appendant claims set forth advantageous developments.","In addition the scope of the invention embraces all combinations comprising at least two of the features disclosed in the description, the drawing and/or the claims.","In accordance with the invention a bearing cup accommodating the shaft bearing, a tubular portion surrounding the pump shaft as a heat barrier and so-called support ribs or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover.","Those features permit the pump to be of a simple structure with only two pressure-bearing parts for the bearing carrier casing—comprising the casing cover and the casing attachment portion—as well as a particularly high level of stability for the bearing carrier casing.","In addition the bearing cup is to be held at an axial spacing towards the impeller by the tubular portion which surrounds the pump shaft.","The sealing chamber for the shaft seal is advantageously delimited by the casing attachment portion which is preferably joined to the casing cover or an edge ring which is carried by the support ribs thereof and which surrounds the longitudinal axis of the pump; the edge ring is connected to and preferably integrally formed on the support ribs at their ends remote from the base region of the casing cover.","Disposed between the spiral casing and the above-mentioned sealing chamber is a latticework-like or trussing-like rib arrangement comprising those support ribs which are to be inclined towards the longitudinal axis from the spiral casing at a preferred angle of between 40° and 45° and—at a radial spacing relative to each other—surround the bearing cup and the heat barrier and form jointly therewith the casing cover.","For that purpose it has proven to be desirable for the cross-sectional height of the support ribs which preferably extend in axial planes which pass radially through the longitudinal axis of the pump to be caused to increase towards the sliding ring seal in order to enhance the level of stability and minimise heat losses.","In accordance with a further feature of the invention the sealing chamber is to adjoin the shaft bearing.","Inter alia that ensures an adequate supply of lubricating medium to the bearing.","In addition the bearing contributes to circulation of the medium in the sealing chamber for flushing and cooling the seal.","In order to enlarge the use of the centrifugal pump according to the invention and the combination options thereof, it is possible to arrange on the casing cover at least one abutment stage or step directed away from the spiral casing, for a further part of the pump; that abutment step should preferably be a front ring which is formed on the casing cover.","An associated pump part can moreover be a spacer which extends around the bearing carrier casing and which abuts against the abutment step or steps or the front ring and to which a motor is attached.","It has proven to be desirable for the abutment step or the front ring to be arranged to project from an annular flange which extends around the support ribs and which is mounted to the transition thereof to a base region of the casing cover.","The scope of the invention includes a centrifugal pump with a sealing ring of the shaft seal—in the form of a sliding ring seal—, the sealing ring extending therearound on a counterpart ring provided at the casing side; the counterpart ring is arranged for the sealing ring in a shaped stub portion of the casing attachment portion, the stub portion projecting axially into the sealing chamber.","At last one opening or passage which is inclined with respect to the longitudinal axis of the pump and with a closure screw for closing it is to be associated with the stub portion—which is of an axial length approximately corresponding to half the axial length of the sealing chamber—; the longitudinal axis of the passage advantageously delimits an angle of about 45° with the longitudinal axis of the pump.","Thus the sliding ring seal is advanced within the sealing chamber in order to make available sufficient space for collecting air or outgassings of the delivery medium—in particular when the arrangement is positioned vertically.","The described arrangement of the passage with the closure screw easily permits complete venting of the sealing chamber both when the pump is positioned horizontally and also vertically.","The frictional heat of the sliding ring seal is desirably dissipated to the surroundings by virtue of a large surface area of the surrounding sealing chamber and a large volume of fluid in the sealing chamber.","In accordance with the invention the shaft bearing is in the form of a plain bearing with a ratio of the length of the bearing bush to the diameter of the pump shaft of between about 1.5 and 2.0, preferably about 1.8.In addition the bearing bush of the plain bearing should be designed to be dismantled.","Advantageously the plain bearing, as viewed from the impeller, is disposed behind the heat barrier, which contributes to simplicity of possible fixing and also contributes to the heat barrier being of a desirable design configuration.","In order to achieve a low level of heat conduction from the spiral casing to the sealing chamber, a small cross-section and a long length of the tubular portion, which is in the form of the heat barrier, of the casing cover has proven to be desirable.","That can be implemented particularly well with the specified bearing arrangement.","The positioning of the impeller-side bearing behind the heat barrier means that the delivery medium which serves as a lubricating agent is at a lower temperature at that location than in the pump casing.","As a result it is of higher viscosity, which is advantageous for the lubricating capability.","In addition the vapor pressure of the medium at that location is markedly lower than in the pump casing, thereby minimising the risk of the bearing running dry.","It has proven to be desirable for the plain bearing or the bearing bush thereof, for fixing purposes, to be surrounded approximately at the longitudinal center thereof on the outside by a clamping or tolerance ring which engages into the bearing cup.","The width of the clamping or tolerance ring should correspond to a fraction of the length of the bearing bush, for example between a fifth and a sixth of the length of the bearing bush.","That provides for easy (dis-)mantling as well as compensation for alignment errors by virtue of tilting mobility.","There are a number of design configurations of the plain bearing, in accordance with the invention.","In one case the plain bearing includes an inner bearing sleeve which is surrounded by a bearing bush at the bearing cup side.","In that case the pump shaft is surrounded within the plain bearing by at least one tolerance ring which engages into the pump shaft.","In particular an inward tolerance ring should be provided on each of the two sides of the outer tolerance ring which engages into the bearing cup—as viewed in longitudinal section of the centrifugal pump—, the inward tolerance ring engaging into the pump shaft.","The bearing sleeve and the bearing bush are preferably made from silicon carbide.","Another design configuration in accordance with the invention involves a plain bearing with a bearing bush at the bearing cup side, the bearing bush including a slidable internal peripheral surface portion which extends around the pump shaft.","In that case the dismantleable plain bearing bush is preferably provided with an inner carbon coating.","The bush runs on a hardened shaft or a hardened shaft portion.","Fixing is effected by a centrally disposed outer tolerance ring, as already mentioned.","In accordance with a further feature of the invention the sliding ring seal extends around a shaft sleeve at the shaft side, wherein at least one O-ring is provided between the shaft sleeve and the pump shaft.","That arrangement, in a construction which is relieved of load, permits assembly of the sliding ring seal from the impeller side, which is desirable in particular when the shaft is in the form of a plug-in shaft.","In accordance with another feature of the invention arranged in the casing attachment portion at an axial spacing relative to the sliding ring seal outside the sealing chamber is a rearward shaft bearing for the pump shaft; at least one hollow space can also be provided in the casing attachment portion between the sealing chamber and the shaft bearing.","In a particular configuration the pump shaft is in the form of a plug-in shaft with clamping ring.","By virtue of the described different design configurations of the plain bearing, in combination with the three types of structure: pump with main plate and coupling; pump of a block-type structure; and pump of an in-line type of structure there are at least six structural variants for the centrifugal pump according to the invention.","Further advantages, features and details of the invention will be apparent from the description hereinafter of preferred embodiments and with reference to the drawing in which: FIGS.","1 and 8 each show a perspective view of different embodiments of a centrifugal pump according to the invention for heat carrier media, FIG.","2 shows an end view of the FIG.","1 structure, FIG.","3 shows a partial longitudinal section through FIG.","2 along line III-III therein, FIG.","4 shows a view in longitudinal section on an enlarged scale through the pump in FIG.","2 taken along line IV-IV therein, FIG.","5 shows a view on an enlarged scale of a part of the FIG.","4 structure, FIG.","6 shows a longitudinal section corresponding to FIG.","4 through another embodiment of the pump shown in FIG.","1, FIG.","7 shows a view on an enlarged scale of a portion of the FIG.","6 structure, FIG.","9 shows an end view of the FIG.","8 structure, FIG.","10 shows a perspective view of a casing cover of the pump, FIG.","11 shows a view on an enlarged scale through the pump of FIGS.","8 and 9 taken along line XI-XI in FIG.","9, FIG.","12 shows a partial longitudinal section on an enlarged scale in relation to FIG.","11 taken along line XII-XII in FIG.","9, FIGS.","13 and 14 show different perspective views of a part of a further embodiment of a centrifugal pump according to the invention, FIG.","15 shows an end view of the structure shown in FIGS.","13 and 14, FIG.","16 shows a partial longitudinal section through the pump of FIGS.","13 through 15 taken along line XVI-XVI in FIG.","15, and FIG.","17 shows a partial section on an enlarged scale in relation to FIG.","16 through that pump taken along line XVII-XVII in FIG.","15.A spiral-casing centrifugal pump 10, on a volute or spiral casing 14 provided with connecting flanges 12, 12a,—with the interposition of seals (not shown)—has a bearing carrier casing 16 which includes a casing cover 18 with a casing attachment portion 34 fitted thereto.","The casing cover 18 is provided with an annular flange 21 which projects from a dish-like base region 20 and through which pass connecting screws 24, on a front ring 22 which is formed on the annular flange 21 and which forms an abutment step; the connecting screws 24 are fitted into sleeve ribs 15 which are formed in axis-parallel relationship externally on a peripheral wall 13 of the spiral casing 14, the peripheral wall engaging over the dish wall 19, and the screws 24 centeringly connect the casing cover 18 to the spiral casing 14.Formed on the casing cover 18 or the base region 20 thereof respectively are support ribs 26 of substantially constant thickness b, which are determined by axial planes E—passing radially through the longitudinal axis A of the pump—and which are inclined at an angle w of about 40° towards the longitudinal axis A of the pump.","The cross-sectional height h of the support ribs 26 steadily increases in the longitudinal extent or in the axial direction away from the casing cover 18.The support ribs 26 are cast at the other end to a bearing cup 28 axially associated with the longitudinal axis A of the pump and an edge ring 30 which surrounds the bearing cup.","An axial tubular portion 32 which is integral with the casing cover 18 connects the bearing cup 28 thereto, in centered relationship.","The bearing cup 28, with its edge ring 30, and the support rings 26—of which there are four here—are also part of the casing cover 18 whose length a, approximately corresponds to a third of the pump length a.","Passing through the casing cover 18 and the rearward casing attachment portion 34—that is to say the bearing carrier casing 16—is a pump shaft 36 of a diameter d, while an impeller 40 is held on the front stepped end 37 thereof by a nut 38.The impeller 40 rotates in the spiral casing 14 which includes an intake or suction chamber 42, whose inlet in the region of the axial connecting flange 12 is identified by reference 43 and whose radial outlet in the connecting flange 12a is identified by reference 44.Disposed near the impeller 40 in the casing cover 18 is a stuffing-box packing 46 at the periphery of the cylindrical tubular portion 32.The internal space 29 of the bearing cup 28 is connected to the cylindrical tubular portion 32 which extends around the pump shaft 36 to form a heat barrier.","If for example a flow agent of low viscosity at about 350° C. passes through the inlet 43, flow agent of higher viscosity at 130° C. is disposed in the internal space of the bearing cup.","The internal space 29 of the bearing cup 28, which adjoins the cylindrical tube portion 32, communicates with a sealing chamber 48 which is arranged in the bearing carrier casing 16 or in the casing attachment portion 34 and which is surrounded by an annular wall 50 and which is provided with at least one closure screw 52 together with a screwthreaded opening or passage 53 which accommodates the screw and which is inclined at an angle z of 45° relative to the longitudinal axis A of the pump, and with blade-like surface elements 55 which are inclined relative to each other and which each have a respective edge 54 parallel to the axis, the axial length e1 thereof approximately corresponding to half the length e of the casing attachment portion 34.An outlet screw 52b towards the bottom is arranged in a radial opening or passage in the region of the chamber which is towards the bottom.","In addition FIG.","2 shows a lateral closure screw 52a which is required for venting when the pump 10 is used in a vertical position.","A rear axial sliding ring seal 56—which is not relieved of load—can be seen in the sealing chamber 48.Projecting from the annular wall 50 are four radial lugs 51 which are connected to corresponding counterpart lugs 51a of the edge ring 30 by screws 24a.","In embodiments which are not shown here—in particular of large dimensioning—an annular flange can also be provided, instead of the above-mentioned radial lugs 51.In order to increase the surface area of the outside of the annular wall, axially extending surface portions can be arranged on the outside of the annular wall.","The sliding ring seal 56 includes a coil spring 58—which is supported against a ring step or a ring 38 which is pushed on to the pump shaft 36—and which loads a rotating sliding ring 59 which with a stationary counterpart ring 60 forms a sealing gap.","The counterpart ring 60 is disposed in the tubular space 62 of an axial shaped stub portion 64 of an axial length f which here corresponds approximately to half the sealing chamber length e1.Arranged subsequently to the annular end part 65 of the stub portion 64 in the casing attachment portion 34 is a dish-like hollow space 66.Between the latter and the adjacent stepped shaft end 37e the pump shaft 36 is surrounded by a grooved-type ball bearing 68 with spacer disks 69 and a securing ring 70; that unit is covered by a bearing cover 72 of the casing attachment portion 34.Projecting radially beneath the latter is a plate-like support leg 74 which is fixed by screws 24b, comprising an angle plate member, in opposite relationship to which, in the region of the spiral casing 14, are two supports 74a which project in parallel in eccentric relationship.","The supports 74a and the support leg 74 are connected at the other end to a base plate (not shown).","Arranged in the internal space 29 in the bearing cup 28 in FIGS.","3 through 5 is a front hydrodynamic plain bearing 76 comprising a bearing bush 77 of a length g with an internal peripheral surface portion 78 of carbon material; the plain bearing 78, at its longitudinal center, is surrounded by a clamping or tolerance ring 80 of a width n, the ring 80 being provided in the bearing cup 28—that is to say at the housing side—; the ring 80 is of an elastic nature and force-lockingly connects the plain bearing 76 to the bearing cup.","The plain bearing 76a of the pump 10a as shown in FIGS.","6 and 7 has within the bearing bush 77 a coaxial bearing sleeve 82 and—besides the above-described outer tolerance ring 80 at the longitudinal center—two inner tolerance rings 80i of a width i which are disposed at the shaft side and which—viewed in longitudinal section—flank the outer tolerance ring 80 at the pump shaft 36 at a radial spacing on both sides.","The width n of the outer tolerance ring 80 and the width i of the inner tolerance rings 80i are relatively short in relation to the length g of the bearing bush, for example the latter corresponds approximately to between five and six times the width i and n respectively.","Reference 84 in FIGS.","6 and 7 shows a nut which is disposed between the front plain bearing 76a and the rear sliding ring seal 56—which is here relieved of load—for the purposes of fixing the bearing sleeve 82.The sliding ring components for carrying the radial forces which occur preferably comprise silicon carbide in the illustrated embodiment.","The two above-discussed bearing types 76, 76a can also be used in the centrifugal pump 11 shown in FIGS.","8 through 12 in which the pump shaft 35 is in the form of a plug-in or stub shaft and is provided with an O-ring 88 within a shaft sleeve 86 of the sliding ring seal 56a, the shaft sleeve 86 surrounding the pump shaft.","The O-ring 88 is disposed between the pump shaft 35 and a shaft sleeve 89 embracing it.","At the rearward end of the bearing carrier casing 16 the pump shaft 35 includes a clamping ring 92 which carries a fan wheel 90 and which is surrounded towards the front by a protective grid 93 as protection to prevent articles from being poked in.","The rearward shaft bearing—which is disposed in the motor—cannot be seen in the drawing.","The impeller 90 is surrounded by a wall ring 94—with apertured plate members 95—of a spacer 96 with endward flange rings 97, 97a; they are connected by longitudinal ribs 98 which are parallel to the axis.","The flange ring 97 which is at the right in the drawing is screwed to a parallel flange ring 99 of a motor 100, the flange ring 99 bearing against the spiral casing 14; the motor 100 is fixed by means of the spacer 96 to the pump 11.In the configuration of a centrifugal pump 11a with a vertical shaft 35 as shown in FIG.","13 ff, the inlet 43 and the outlet 44 of the spiral casing 14 are on a straight diametral line Q.","This pump 11a can also be provided with both of the described bearing forms 76, 76a."]],"string":"[\n [\n \"Centrifugal pump, in particular a spirally-housed centrifugal pump for a heat exchange medium\",\n \"In a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), parts of the casing cover (18) are a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction.\",\n \"The bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a), and the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56).\"\n ],\n [\n \"1.A centrifugal pump, in particular a spiral-casing centrifugal pump, for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), characterised in that a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover (18).\",\n \"2.A centrifugal pump as set forth in claim 1 characterised in that the bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a).\",\n \"3.A centrifugal pump as set-forth in claim 1 or claim 2 characterised in that the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56).\",\n \"4.A centrifugal pump as set forth in claim 3 characterised in that at their ends remote from the base region (20) of the casing cover (18) the support ribs (26) are connected by the edge ring (30) surrounding the longitudinal axis (A) of the pump.\",\n \"5.A centrifugal pump as set forth in one of claims 1 through 4 characterised in that the support ribs (26) are inclined at an angle (w) to the longitudinal axis (A) of the pump from the base region (20) of the casing cover (18) towards the shaft seal (56).\",\n \"6.A centrifugal pump as set forth in claim 5 characterised by an angle (w) of between 30° and 60°.\",\n \"7.A centrifugal pump as set forth in one of claims 1 through 6 characterised in that the cross-sectional height (h) of the support ribs (26) which preferably extend in axial planes (E) passing radially through the longitudinal axis (A) of the pump increases towards the sliding ring seal (56).\",\n \"8.A centrifugal pump as set forth in one of claims 1 through 7 characterised in that the sealing chamber (48) adjoins the shaft bearing (76, 76a).\",\n \"9.A centrifugal pump as set forth in one of claims 1 through 8 characterised in that at least one abutment step (28) directed away from the spiral casing (24) for a further pump portion (96) is arranged at the casing cover (18).\",\n \"10.A centrifugal pump as set forth in claim 9 characterised in that the abutment step is a front ring (22) formed on the casing cover (18).\",\n \"11.A centrifugal pump as set forth in claim 9 or claim 10 characterised in that the abutment step or the front ring (22) projects from an annular flange (21) which surrounds the support ribs (26) and which is disposed at the transition thereof to a base region (20) of the casing cover (18).\",\n \"12.A centrifugal pump comprising a sealing ring (59) of the shaft seal which is in the form of a sliding ring seal (56), the sealing ring extending peripherally at a counterpart ring (60) provided at the housing side, as set forth in one of claims 1 through 11, characterised in that the counterpart ring (60) for the sealing ring (59) is arranged in a shaped stub portion (64) of the casing attachment portion (34), the stub portion projecting axially into the sealing chamber (48), and associated with the stub portion is at least one passage (58) inclined with respect to the longitudinal axis (A) of the pump, with a closure screw (52) closing the passage.\",\n \"13.A centrifugal pump as set forth in claim 12 characterised by an angle (z) between the longitudinal axis (A) of the pump and the longitudinal axis (B) of the passage (53) of about 45°.\",\n \"14.A centrifugal pump as set forth in claim 12 or claim 13 characterised by an axial length (f) of the stub portion (64) which corresponds approximately to half the axial length (e1) of the sealing chamber (48).\",\n \"15.A centrifugal pump as set forth in one of claims 1 through 14 characterised in that the shaft bearing is in the form of a plain bearing (76) with a ratio of the length (g) of the bearing bush (77) to the diameter (d) of the pump shaft (35, 36, 36a) of between about 1.5 and 2.0, preferably about 1.8.16.A centrifugal pump as set forth in claim 15 characterised in that the bearing bush (77) of the plain bearing (76, 76a) is adapted to be dismantleable.\",\n \"17.A centrifugal pump as set forth in claim 15 or claim 16 characterised in that the plain bearing (76, 76a) or its bearing bush (77) is externally embraced approximately at its longitudinal center by a clamping or tolerance ring (80) which engages into the bearing cup (28).\",\n \"18.A centrifugal pump as set forth in claim 17 characterised in that the width (n) of the clamping or tolerance ring (80) corresponds to a fraction of the length (g) of the bearing bush (77).\",\n \"19.A centrifugal pump as set forth in claim 18 characterised in that the width (n) of the external clamping or tolerance ring (80) corresponds approximately to between a fifth and a sixth of the length (g) of the bearing bush (77).\",\n \"20.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76a) includes an inner bearing sleeve (82) surrounded by a bearing bush (77) at the bearing cup side (FIGS.\",\n \"6 and 7).\",\n \"21.A centrifugal pump as set forth in one of claims 15 through 20 characterised in that within the plain bearing (76a) the pump shaft (35, 36a) is embraced by at least one tolerance ring (80i) which engages into the pump shaft.\",\n \"22.A centrifugal pump as set forth in one of claims 17 through 21 characterised in that provided on respective sides of the outer tolerance ring (80), viewed in longitudinal section of the centrifugal pump (10a), is a respective inward tolerance ring (80i) which engages into the pump shaft (35, 36a).\",\n \"23.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76) includes a bearing bush (77) at the bearing cup side, with a slidable internal peripheral surface portion (78) embracing the pump shaft (36).\",\n \"24.A centrifugal pump as set forth in claim 23 characterised by an internal peripheral surface portion (28) of the plain bearing (76) comprising carbon (FIGS.\",\n \"4 and 5).\",\n \"25.A centrifugal pump as set forth in claim 23 or claim 24 characterised by a hardened pump shaft (36) or a corresponding shaft portion which is associated with the bearing bush (77).\",\n \"26.A centrifugal pump as set forth in one of claims 1 through 25 characterised in that the sliding ring seal (56) embraces a shaft sleeve (89) at the shaft side, wherein at least one O-ring (88) is provided between the shaft sleeve (89) and the pump shaft (36, 36a).\",\n \"27.A centrifugal pump as set forth in one of claims 1 through 26 characterised in that a rearward shaft bearing (68) for the pump shaft (36, 36a) is arranged in the casing attachment portion (34) at an axial spacing relative to the sliding ring seal (56) outside the sealing chamber (48).\",\n \"28.A centrifugal pump as set forth in claim 27 characterised in that at least one hollow space (66) is arranged between the sealing chamber (48) and the shaft bearing (68) in the casing attachment portion (34).\",\n \"29.A centrifugal pump as set forth in one of claims 1 through 28 characterised in that the pump shaft (36a) is in the form of a plug-in stub shaft with clamping ring (92).\",\n \"30.A centrifugal pump as set forth in one of claims 8 through 29 characterised in that the spiral casing (14) and the bearing carrier casing (16) are embraced by a spacer (96) which butts against the abutment step or steps or the front ring (22) respectively and it is attached to a motor (100).\"\n ],\n [\n \"The invention concerns a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing having an impeller, a casing cover and an associated casing attachment portion for a pump shaft provided with a shaft bearing near the spiral casing and a shaft seal disposed at a spacing in relation to the shaft bearing in a sealing chamber.\",\n \"Such centrifugal pumps are described by way of example in a prospectus from the present applicants in relation to the range NHT for heat carrier purposes up to 350° C. A spiral casing for an impeller is connected to a bearing casing by means of a throttle section having external ribs.\",\n \"The impeller is fixed at one end of a pump shaft which extends in the throttle section.\",\n \"Arranged between the throttle section and the impeller is a plain bearing of silicon carbide and arranged downstream of the throttle section is a sliding ring seal which is followed by a rolling bearing.\",\n \"This arrangement involves a horizontal spiral-casing centrifugal pump of a process type of structure which is single-stage in terms of intake flow.\",\n \"The pump-side silicon carbide plain bearing is lubricated by the fluid being delivered, while used at the drive side is a grease-lubricated grooved-type ball bearing.\",\n \"In consideration of that state of the art, the inventor set himself the aim of providing a centrifugal pump of the kind set forth in the opening part of this specification, which is simple to manufacture and which can be inexpensively and particularly well used.\",\n \"That object is attained by the teaching of the independent claim; the appendant claims set forth advantageous developments.\",\n \"In addition the scope of the invention embraces all combinations comprising at least two of the features disclosed in the description, the drawing and/or the claims.\",\n \"In accordance with the invention a bearing cup accommodating the shaft bearing, a tubular portion surrounding the pump shaft as a heat barrier and so-called support ribs or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover.\",\n \"Those features permit the pump to be of a simple structure with only two pressure-bearing parts for the bearing carrier casing—comprising the casing cover and the casing attachment portion—as well as a particularly high level of stability for the bearing carrier casing.\",\n \"In addition the bearing cup is to be held at an axial spacing towards the impeller by the tubular portion which surrounds the pump shaft.\",\n \"The sealing chamber for the shaft seal is advantageously delimited by the casing attachment portion which is preferably joined to the casing cover or an edge ring which is carried by the support ribs thereof and which surrounds the longitudinal axis of the pump; the edge ring is connected to and preferably integrally formed on the support ribs at their ends remote from the base region of the casing cover.\",\n \"Disposed between the spiral casing and the above-mentioned sealing chamber is a latticework-like or trussing-like rib arrangement comprising those support ribs which are to be inclined towards the longitudinal axis from the spiral casing at a preferred angle of between 40° and 45° and—at a radial spacing relative to each other—surround the bearing cup and the heat barrier and form jointly therewith the casing cover.\",\n \"For that purpose it has proven to be desirable for the cross-sectional height of the support ribs which preferably extend in axial planes which pass radially through the longitudinal axis of the pump to be caused to increase towards the sliding ring seal in order to enhance the level of stability and minimise heat losses.\",\n \"In accordance with a further feature of the invention the sealing chamber is to adjoin the shaft bearing.\",\n \"Inter alia that ensures an adequate supply of lubricating medium to the bearing.\",\n \"In addition the bearing contributes to circulation of the medium in the sealing chamber for flushing and cooling the seal.\",\n \"In order to enlarge the use of the centrifugal pump according to the invention and the combination options thereof, it is possible to arrange on the casing cover at least one abutment stage or step directed away from the spiral casing, for a further part of the pump; that abutment step should preferably be a front ring which is formed on the casing cover.\",\n \"An associated pump part can moreover be a spacer which extends around the bearing carrier casing and which abuts against the abutment step or steps or the front ring and to which a motor is attached.\",\n \"It has proven to be desirable for the abutment step or the front ring to be arranged to project from an annular flange which extends around the support ribs and which is mounted to the transition thereof to a base region of the casing cover.\",\n \"The scope of the invention includes a centrifugal pump with a sealing ring of the shaft seal—in the form of a sliding ring seal—, the sealing ring extending therearound on a counterpart ring provided at the casing side; the counterpart ring is arranged for the sealing ring in a shaped stub portion of the casing attachment portion, the stub portion projecting axially into the sealing chamber.\",\n \"At last one opening or passage which is inclined with respect to the longitudinal axis of the pump and with a closure screw for closing it is to be associated with the stub portion—which is of an axial length approximately corresponding to half the axial length of the sealing chamber—; the longitudinal axis of the passage advantageously delimits an angle of about 45° with the longitudinal axis of the pump.\",\n \"Thus the sliding ring seal is advanced within the sealing chamber in order to make available sufficient space for collecting air or outgassings of the delivery medium—in particular when the arrangement is positioned vertically.\",\n \"The described arrangement of the passage with the closure screw easily permits complete venting of the sealing chamber both when the pump is positioned horizontally and also vertically.\",\n \"The frictional heat of the sliding ring seal is desirably dissipated to the surroundings by virtue of a large surface area of the surrounding sealing chamber and a large volume of fluid in the sealing chamber.\",\n \"In accordance with the invention the shaft bearing is in the form of a plain bearing with a ratio of the length of the bearing bush to the diameter of the pump shaft of between about 1.5 and 2.0, preferably about 1.8.In addition the bearing bush of the plain bearing should be designed to be dismantled.\",\n \"Advantageously the plain bearing, as viewed from the impeller, is disposed behind the heat barrier, which contributes to simplicity of possible fixing and also contributes to the heat barrier being of a desirable design configuration.\",\n \"In order to achieve a low level of heat conduction from the spiral casing to the sealing chamber, a small cross-section and a long length of the tubular portion, which is in the form of the heat barrier, of the casing cover has proven to be desirable.\",\n \"That can be implemented particularly well with the specified bearing arrangement.\",\n \"The positioning of the impeller-side bearing behind the heat barrier means that the delivery medium which serves as a lubricating agent is at a lower temperature at that location than in the pump casing.\",\n \"As a result it is of higher viscosity, which is advantageous for the lubricating capability.\",\n \"In addition the vapor pressure of the medium at that location is markedly lower than in the pump casing, thereby minimising the risk of the bearing running dry.\",\n \"It has proven to be desirable for the plain bearing or the bearing bush thereof, for fixing purposes, to be surrounded approximately at the longitudinal center thereof on the outside by a clamping or tolerance ring which engages into the bearing cup.\",\n \"The width of the clamping or tolerance ring should correspond to a fraction of the length of the bearing bush, for example between a fifth and a sixth of the length of the bearing bush.\",\n \"That provides for easy (dis-)mantling as well as compensation for alignment errors by virtue of tilting mobility.\",\n \"There are a number of design configurations of the plain bearing, in accordance with the invention.\",\n \"In one case the plain bearing includes an inner bearing sleeve which is surrounded by a bearing bush at the bearing cup side.\",\n \"In that case the pump shaft is surrounded within the plain bearing by at least one tolerance ring which engages into the pump shaft.\",\n \"In particular an inward tolerance ring should be provided on each of the two sides of the outer tolerance ring which engages into the bearing cup—as viewed in longitudinal section of the centrifugal pump—, the inward tolerance ring engaging into the pump shaft.\",\n \"The bearing sleeve and the bearing bush are preferably made from silicon carbide.\",\n \"Another design configuration in accordance with the invention involves a plain bearing with a bearing bush at the bearing cup side, the bearing bush including a slidable internal peripheral surface portion which extends around the pump shaft.\",\n \"In that case the dismantleable plain bearing bush is preferably provided with an inner carbon coating.\",\n \"The bush runs on a hardened shaft or a hardened shaft portion.\",\n \"Fixing is effected by a centrally disposed outer tolerance ring, as already mentioned.\",\n \"In accordance with a further feature of the invention the sliding ring seal extends around a shaft sleeve at the shaft side, wherein at least one O-ring is provided between the shaft sleeve and the pump shaft.\",\n \"That arrangement, in a construction which is relieved of load, permits assembly of the sliding ring seal from the impeller side, which is desirable in particular when the shaft is in the form of a plug-in shaft.\",\n \"In accordance with another feature of the invention arranged in the casing attachment portion at an axial spacing relative to the sliding ring seal outside the sealing chamber is a rearward shaft bearing for the pump shaft; at least one hollow space can also be provided in the casing attachment portion between the sealing chamber and the shaft bearing.\",\n \"In a particular configuration the pump shaft is in the form of a plug-in shaft with clamping ring.\",\n \"By virtue of the described different design configurations of the plain bearing, in combination with the three types of structure: pump with main plate and coupling; pump of a block-type structure; and pump of an in-line type of structure there are at least six structural variants for the centrifugal pump according to the invention.\",\n \"Further advantages, features and details of the invention will be apparent from the description hereinafter of preferred embodiments and with reference to the drawing in which: FIGS.\",\n \"1 and 8 each show a perspective view of different embodiments of a centrifugal pump according to the invention for heat carrier media, FIG.\",\n \"2 shows an end view of the FIG.\",\n \"1 structure, FIG.\",\n \"3 shows a partial longitudinal section through FIG.\",\n \"2 along line III-III therein, FIG.\",\n \"4 shows a view in longitudinal section on an enlarged scale through the pump in FIG.\",\n \"2 taken along line IV-IV therein, FIG.\",\n \"5 shows a view on an enlarged scale of a part of the FIG.\",\n \"4 structure, FIG.\",\n \"6 shows a longitudinal section corresponding to FIG.\",\n \"4 through another embodiment of the pump shown in FIG.\",\n \"1, FIG.\",\n \"7 shows a view on an enlarged scale of a portion of the FIG.\",\n \"6 structure, FIG.\",\n \"9 shows an end view of the FIG.\",\n \"8 structure, FIG.\",\n \"10 shows a perspective view of a casing cover of the pump, FIG.\",\n \"11 shows a view on an enlarged scale through the pump of FIGS.\",\n \"8 and 9 taken along line XI-XI in FIG.\",\n \"9, FIG.\",\n \"12 shows a partial longitudinal section on an enlarged scale in relation to FIG.\",\n \"11 taken along line XII-XII in FIG.\",\n \"9, FIGS.\",\n \"13 and 14 show different perspective views of a part of a further embodiment of a centrifugal pump according to the invention, FIG.\",\n \"15 shows an end view of the structure shown in FIGS.\",\n \"13 and 14, FIG.\",\n \"16 shows a partial longitudinal section through the pump of FIGS.\",\n \"13 through 15 taken along line XVI-XVI in FIG.\",\n \"15, and FIG.\",\n \"17 shows a partial section on an enlarged scale in relation to FIG.\",\n \"16 through that pump taken along line XVII-XVII in FIG.\",\n \"15.A spiral-casing centrifugal pump 10, on a volute or spiral casing 14 provided with connecting flanges 12, 12a,—with the interposition of seals (not shown)—has a bearing carrier casing 16 which includes a casing cover 18 with a casing attachment portion 34 fitted thereto.\",\n \"The casing cover 18 is provided with an annular flange 21 which projects from a dish-like base region 20 and through which pass connecting screws 24, on a front ring 22 which is formed on the annular flange 21 and which forms an abutment step; the connecting screws 24 are fitted into sleeve ribs 15 which are formed in axis-parallel relationship externally on a peripheral wall 13 of the spiral casing 14, the peripheral wall engaging over the dish wall 19, and the screws 24 centeringly connect the casing cover 18 to the spiral casing 14.Formed on the casing cover 18 or the base region 20 thereof respectively are support ribs 26 of substantially constant thickness b, which are determined by axial planes E—passing radially through the longitudinal axis A of the pump—and which are inclined at an angle w of about 40° towards the longitudinal axis A of the pump.\",\n \"The cross-sectional height h of the support ribs 26 steadily increases in the longitudinal extent or in the axial direction away from the casing cover 18.The support ribs 26 are cast at the other end to a bearing cup 28 axially associated with the longitudinal axis A of the pump and an edge ring 30 which surrounds the bearing cup.\",\n \"An axial tubular portion 32 which is integral with the casing cover 18 connects the bearing cup 28 thereto, in centered relationship.\",\n \"The bearing cup 28, with its edge ring 30, and the support rings 26—of which there are four here—are also part of the casing cover 18 whose length a, approximately corresponds to a third of the pump length a.\",\n \"Passing through the casing cover 18 and the rearward casing attachment portion 34—that is to say the bearing carrier casing 16—is a pump shaft 36 of a diameter d, while an impeller 40 is held on the front stepped end 37 thereof by a nut 38.The impeller 40 rotates in the spiral casing 14 which includes an intake or suction chamber 42, whose inlet in the region of the axial connecting flange 12 is identified by reference 43 and whose radial outlet in the connecting flange 12a is identified by reference 44.Disposed near the impeller 40 in the casing cover 18 is a stuffing-box packing 46 at the periphery of the cylindrical tubular portion 32.The internal space 29 of the bearing cup 28 is connected to the cylindrical tubular portion 32 which extends around the pump shaft 36 to form a heat barrier.\",\n \"If for example a flow agent of low viscosity at about 350° C. passes through the inlet 43, flow agent of higher viscosity at 130° C. is disposed in the internal space of the bearing cup.\",\n \"The internal space 29 of the bearing cup 28, which adjoins the cylindrical tube portion 32, communicates with a sealing chamber 48 which is arranged in the bearing carrier casing 16 or in the casing attachment portion 34 and which is surrounded by an annular wall 50 and which is provided with at least one closure screw 52 together with a screwthreaded opening or passage 53 which accommodates the screw and which is inclined at an angle z of 45° relative to the longitudinal axis A of the pump, and with blade-like surface elements 55 which are inclined relative to each other and which each have a respective edge 54 parallel to the axis, the axial length e1 thereof approximately corresponding to half the length e of the casing attachment portion 34.An outlet screw 52b towards the bottom is arranged in a radial opening or passage in the region of the chamber which is towards the bottom.\",\n \"In addition FIG.\",\n \"2 shows a lateral closure screw 52a which is required for venting when the pump 10 is used in a vertical position.\",\n \"A rear axial sliding ring seal 56—which is not relieved of load—can be seen in the sealing chamber 48.Projecting from the annular wall 50 are four radial lugs 51 which are connected to corresponding counterpart lugs 51a of the edge ring 30 by screws 24a.\",\n \"In embodiments which are not shown here—in particular of large dimensioning—an annular flange can also be provided, instead of the above-mentioned radial lugs 51.In order to increase the surface area of the outside of the annular wall, axially extending surface portions can be arranged on the outside of the annular wall.\",\n \"The sliding ring seal 56 includes a coil spring 58—which is supported against a ring step or a ring 38 which is pushed on to the pump shaft 36—and which loads a rotating sliding ring 59 which with a stationary counterpart ring 60 forms a sealing gap.\",\n \"The counterpart ring 60 is disposed in the tubular space 62 of an axial shaped stub portion 64 of an axial length f which here corresponds approximately to half the sealing chamber length e1.Arranged subsequently to the annular end part 65 of the stub portion 64 in the casing attachment portion 34 is a dish-like hollow space 66.Between the latter and the adjacent stepped shaft end 37e the pump shaft 36 is surrounded by a grooved-type ball bearing 68 with spacer disks 69 and a securing ring 70; that unit is covered by a bearing cover 72 of the casing attachment portion 34.Projecting radially beneath the latter is a plate-like support leg 74 which is fixed by screws 24b, comprising an angle plate member, in opposite relationship to which, in the region of the spiral casing 14, are two supports 74a which project in parallel in eccentric relationship.\",\n \"The supports 74a and the support leg 74 are connected at the other end to a base plate (not shown).\",\n \"Arranged in the internal space 29 in the bearing cup 28 in FIGS.\",\n \"3 through 5 is a front hydrodynamic plain bearing 76 comprising a bearing bush 77 of a length g with an internal peripheral surface portion 78 of carbon material; the plain bearing 78, at its longitudinal center, is surrounded by a clamping or tolerance ring 80 of a width n, the ring 80 being provided in the bearing cup 28—that is to say at the housing side—; the ring 80 is of an elastic nature and force-lockingly connects the plain bearing 76 to the bearing cup.\",\n \"The plain bearing 76a of the pump 10a as shown in FIGS.\",\n \"6 and 7 has within the bearing bush 77 a coaxial bearing sleeve 82 and—besides the above-described outer tolerance ring 80 at the longitudinal center—two inner tolerance rings 80i of a width i which are disposed at the shaft side and which—viewed in longitudinal section—flank the outer tolerance ring 80 at the pump shaft 36 at a radial spacing on both sides.\",\n \"The width n of the outer tolerance ring 80 and the width i of the inner tolerance rings 80i are relatively short in relation to the length g of the bearing bush, for example the latter corresponds approximately to between five and six times the width i and n respectively.\",\n \"Reference 84 in FIGS.\",\n \"6 and 7 shows a nut which is disposed between the front plain bearing 76a and the rear sliding ring seal 56—which is here relieved of load—for the purposes of fixing the bearing sleeve 82.The sliding ring components for carrying the radial forces which occur preferably comprise silicon carbide in the illustrated embodiment.\",\n \"The two above-discussed bearing types 76, 76a can also be used in the centrifugal pump 11 shown in FIGS.\",\n \"8 through 12 in which the pump shaft 35 is in the form of a plug-in or stub shaft and is provided with an O-ring 88 within a shaft sleeve 86 of the sliding ring seal 56a, the shaft sleeve 86 surrounding the pump shaft.\",\n \"The O-ring 88 is disposed between the pump shaft 35 and a shaft sleeve 89 embracing it.\",\n \"At the rearward end of the bearing carrier casing 16 the pump shaft 35 includes a clamping ring 92 which carries a fan wheel 90 and which is surrounded towards the front by a protective grid 93 as protection to prevent articles from being poked in.\",\n \"The rearward shaft bearing—which is disposed in the motor—cannot be seen in the drawing.\",\n \"The impeller 90 is surrounded by a wall ring 94—with apertured plate members 95—of a spacer 96 with endward flange rings 97, 97a; they are connected by longitudinal ribs 98 which are parallel to the axis.\",\n \"The flange ring 97 which is at the right in the drawing is screwed to a parallel flange ring 99 of a motor 100, the flange ring 99 bearing against the spiral casing 14; the motor 100 is fixed by means of the spacer 96 to the pump 11.In the configuration of a centrifugal pump 11a with a vertical shaft 35 as shown in FIG.\",\n \"13 ff, the inlet 43 and the outlet 44 of the spiral casing 14 are on a straight diametral line Q.\",\n \"This pump 11a can also be provided with both of the described bearing forms 76, 76a.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10276581"}}},{"rowIdx":86,"cells":{"text":{"kind":"list like","value":[["Allocating access across shared communication medium","A method of providing network access across a shared communications medium between competing users (86) includes the step of allocating network access for each user for a future time interval (102).","Features include forecasting network access (1100) of the users in a future time interval, and prioritizing the users for allocating network access to the users.","The network access allocations represent network access allowances available to the users during the future time interval, and further may represent network access usage.","Classes of users can be allocated network access first (1114), and then each user allocated network access from the class allocation."],["1.A method of providing network access across a shared communications medium between at least two competing users, comprising the steps of: (a) prior to a first time interval, determining for each user a respective first network access allowance representing a respective first maximum level of network access available to the user during the first time interval, but not necessarily representing the level of network access that will be utilized by the user during the first time interval; (b) during the first time interval, providing network access to each user such that the respective first network access allowance for each user is not exceeded; (c) prior to a second consecutive time interval, determining for each user a respective second network access allowance representing a respective second maximum level of network access available to the user during the second time interval, but not necessarily representing the level of network access that will be utilized by the user during the second time interval, at least one respective second network access allowance for a user differing from such user's respective first network access allowance; and (d) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.","2.The method of claim 1, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","3.The method of claim 1, wherein the first and second time intervals are consecutive time intervals.","4.The method of claim 1, wherein each of the first time interval and the second time interval has a period of between one minute and sixty minutes.","5.The method of claim 1, wherein the second network access allowance for each respective user differs from the first network access allowance for such user.","6.The method of claim 1, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.","7.The method of claim 6, wherein the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.","8.The method of claim 7, wherein the first allocation policy differs from the second allocation policy.","9.The method of claim 7, further comprising requesting a minimum level of network access for the first time interval for a user, and wherein said allocating network access to such user for the first time interval comprises setting the level of network access allocated to such user to a level equal to or greater than the requested minimum level.","10.The method of claim 9, wherein said allocating network access to such user for the second time interval comprises setting the level of network access allocated to such user to a level less than the requested minimum level for the first time interval.","11.The method of claim 1, further comprising the step of monitoring network access usage by each user.","12.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted from and to each user during a past time interval.","13.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a past time interval.","14.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.","15.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a past time interval.","16.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a time interval.","17.The method of claim 1, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.","18.The method of claim 1, wherein the Shared Access Carrier Network comprises a wireless network.","19.The method of claim 1, further comprising prioritizing the users for determining network access allowances.","20.The method of claim 19, wherein said prioritizing is based on fairness considerations.","21.The method of claim 20, wherein the users are prioritized based on user throughput during a past time interval, with a user with lesser throughput receiving priority over a user with greater throughput.","22.The method of claim 20, wherein the users are prioritized based on data loss for each user during a past time interval, with a user with greater data loss having priority over a user with lesser data loss.","23.The method of claim 20, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","24.The method of claim 20, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.","25.The method of claim 20, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","26.The method of claim 19, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.","27.The method of claim 26, further comprising the step of monitoring network access usage by each user.","28.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","29.The method of claim 27, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","30.The method of claim 27, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.","31.The method of claim 27, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","32.The method of claim 27, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","33.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises setting the respective levels of network access equal to each user's specified respective minimum level of network access.","34.The method of claim 11, further comprising the step of forecasting network access usage by each user during a future time interval based on said step of monitoring network access usage by each user.","35.The method of claim 34, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","36.The method of claim 34, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.","37.The method of claim 34, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.","38.The method of claim 37, wherein said step of allocating network access comprises setting the respective levels of network access of the users proportional to each user's forecasted network access usage.","39.The method of claim 37, further comprising the step of prioritizing the users for allocating network access.","40.The method of claim 39, wherein said prioritizing is based on each user's forecasted network access usage.","41.The method of claim 40, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","42.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","43.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","44.A method of providing during two consecutive time intervals network access across a shared communications medium between two competing users, comprising the step of varying between the two time intervals a respective network access allowance for each user, each respective network access allowance representing a maximum level of network access available to the respective user during a particular time interval, but not necessarily representing the level of network access that is utilized by the user during the particular time interval.","45.A method of providing network access across a shared communications medium in a downstream direction towards competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; and (b) based on said monitored network access usage, allocating network access to each user on a per user basis for a future time interval.","46.The method of claim 45, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","47.The method of claim 45, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.","48.The method of claim 47, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.","49.The method of claim 45, wherein said step of monitoring comprises monitoring bandwidth that is consumed by each user in a downstream direction at time intervals of fifteen minutes to sixty minutes.","50.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","51.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","52.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted to each user during a time interval.","53.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted to each user during a time interval.","54.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","55.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","56.The method of claim 45, wherein said step of allocating network access comprises allocating network access equally to the users.","57.The method of claim 45, wherein the shared communications medium is part of a Shared Access Carrier Network.","58.The method of claim 57, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","59.The method of claim 57, wherein the Shared Access Carrier Network comprises a wireless network.","60.The method of claim 45, further comprising prioritizing the users for allocating network access.","61.The method of claim 60, wherein said prioritizing is based on fairness considerations.","62.The method of claim 61, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput receiving priority over a user with greater throughput.","63.The method of claim 61, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss having priority over a user with lesser data loss.","64.The method of claim 61, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","65.The method of claim 61, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.","66.The method of claim 61, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","67.The method of claim 60, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.","68.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","69.The method of claim 67, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","70.The method of claim 67, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.","71.The method of claim 67, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","72.The method of claim 67, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","73.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.","74.The method of claim 45, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.","75.The method of claim 74, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","76.The method of claim 74, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.","77.The method of claim 74, wherein said step of allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.","78.The method of claim 74, further comprising the step of prioritizing the users for allocating network access.","79.The method of claim 78, wherein said prioritizing is based on each user's forecasted network access usage.","80.The method of claim 78, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","81.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","82.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","83.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) forecasting network access usage by each user during a future time interval based on said monitored network access usage by each user; and (c) based on said forecasted network access usage, allocating network access to each user for the future time interval.","84.The method of claim 83, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","85.The method of claim 83, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.","86.The method of claim 83, wherein said step of allocating network access comprises allocating network access to the users proportionally based on each user's forecasted network access usage.","87.The method of claim 83, further comprising the step of prioritizing the users for allocating network access.","88.The method of claim 87, wherein said prioritizing is based on each user's forecasted network access usage.","89.The method of claim 87, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","90.The method of claim 87, wherein said prioritizing is based on fairness considerations.","91.The method of claim 87, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.","92.The method of claim 87, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.","93.The method of claim 87, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","94.The method of claim 87, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.","95.The method of claim 87, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","96.The method of claim 87, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.","97.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","98.The method of claim 96, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","99.The method of claim 96, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usages both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access would have been utilized for such users.","100.The method of claim 96, wherein SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","101.The method of claim 96, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","102.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.","103.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","104.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","105.The method of claim 83, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.","106.The method of claim 83, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.","107.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) of each user that will be transmitted over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.","108.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage requested by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) that will be requested by each user over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.","109.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","110.The method of claim 109, wherein said step of allocating further comprises allocating any surplus network access remaining equally to the users.","111.The method of claim 109, wherein said step of allocating further comprises allocating in decreasing user priority any surplus network access to the users proportionally to the user's forecasted network access usage.","112.The method of claim 109, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","113.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.","114.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.","115.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","116.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","117.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","118.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","119.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","120.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","121.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.","122.The method of claim 109, wherein the shared communications medium is part of a Shared Access Carrier Network.","123.The method of claim 122, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","124.The method of claim 122, wherein the Shared Access Carrier Network comprises a wireless network.","125.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total available network access to the total forecasted network access usage of all the users, and subject to a respective, predetermined maximum value and subject to availability.","126.The method of claim 125, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","127.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.","128.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.","129.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","130.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","131.The method of claim 125, wherein said step of monitoring network access includes collecting data representative of the logical data units transmitted from and to each user during a time interval.","132.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","133.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","134.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","135.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.","136.The method of claim 125, wherein the shared communications medium is part of a Shared Access Carrier Network.","137.The method of claim 126, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","138.The method of claim 126, wherein the Shared Access Carrier Network comprises a wireless network.","139.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","140.The method of claim 139, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","141.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.","142.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.","143.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","144.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","145.The method of claim 139, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","146.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","147.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","148.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","149.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.","150.The method of claim 139, wherein the shared communications medium is part of a Shared Access Carrier Network.","151.The method of claim 150, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","152.The method of claim 150, wherein the Shared Access Carrier Network comprises a wireless network.","153.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum specified value and subject to availability.","154.The method of claim 153, wherein any additional network access remaining after said allocation is earmarked for the users such that each user's allocation equals the user's forecasted network access usage, subject to a respective, predetermined maximum allowed level of network access and subject to availability.","155.The method of claim 154, wherein any surplus network access remaining after said distribution is further earmarked in equal amounts for the users, subject to a respective, predetermined maximum allowed level of network access.","156.The method of claim 154, further comprising allocating to the users network access only in an amount equal to the forecasted network access usage multiplied by the ratio of total network access available for allocation to the total forecasted network access usage of the users, but only when the total forecasted network access usage of the users exceeds the total network access available for allocation.","157.The method of claim 153, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","158.The method of claim 153, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.","159.The method of claim 153, wherein said step of monitoring comprises monitoring network access by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.","160.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","161.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","162.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","163.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","164.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are dropped during a time interval.","165.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of each user that are dropped during a time interval.","166.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are requested to be transmitted in the upstream direction during a time interval.","167.The method of claim 153, wherein the shared communications medium is part of a Shared Access Carrier Network.","168.The method of claim 167, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","169.The method of claim 167, wherein the Shared Access Carrier Network comprises a wireless network.","170.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing said monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.","171.The method of claim 170, wherein the threshold value represents a respective maximum level of network access for each user.","172.The method of claim 170, wherein the threshold value represents a respective maximum burstable level of network access with target probability for each user.","173.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via email.","174.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via instant messaging.","175.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.","176.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.","177.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.","178.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent basis.","179.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user with a maximum burstable level of network access with target probability.","180.The method of claim 170, further comprising charging the user a fee for the modification of the SLA.","181.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a temporary basis.","182.The method of claim 170, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","183.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in an upstream direction of communication across the shared communications medium at time intervals of one minute to fifteen minutes.","184.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in a downstream direction of communication across the shared communications medium at time intervals of fifteen minutes to sixty minutes.","185.The method of claim 170, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","186.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","187.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","188.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","189.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.","190.The method of claim 170, wherein the shared communications medium is part of a Shared Access Carrier Network.","191.The method of claim 190, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.","192.The method of claim 190, wherein the Shared Access Carrier Network comprises a wireless network.","193.The method of claim 170, further comprising, based on said monitored network access usage, allocating network access to each user for a future time interval.","194.The method of claim 193, wherein said step of allocating network access comprises allocating network access equally to the users.","195.The method of claim 193, further comprising prioritizing the users for allocating network access.","196.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for the users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","197.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","198.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.","199.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","200.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","201.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.","202.The method of claim 195, wherein said prioritizing is based on fairness considerations.","203.The method of claim 202, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.","204.The method of claim 202, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.","205.The method of claim 202, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","206.The method of claim 202, wherein the users are prioritized based on both user throughput and data loss during a time interval.","207.The method of claim 202, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","208.The method of claim 193, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.","209.The method of claim 208, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","210.The method of claim 208, wherein said step of forecasting comprises applying an adaptive-response-rate signal exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.","211.The method of claim 208, wherein said step of allocating comprises allocating network access to the users proportionally based on each user's forecasted network access usage.","212.The method of claim 208, further comprising the step of prioritizing the users for allocating network access.","213.The method of claim 212, wherein said prioritizing is based on each user's forecasted network access usage.","214.The method of claim 212, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","215.The method of claim 212, wherein said step of allocating comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","216.The method of claim 212, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","217.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user for respective predetermined past time intervals; (b) identifying a recurrent period of high network access usage of a user based on said monitoring; and (c) soliciting a user to modify the user's SLA to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.","218.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via email.","219.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via instant messaging.","220.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.","221.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.","222.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.","223.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for all future recurrent periods of high network access usage.","224.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for a predetermined number of future recurrent periods of high network access usage.","225.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user with a maximum burstable level of network access with target probability for the future period of high network access usage.","226.The method of claim 217, wherein the recurrent time period of high network access usage comprises a particular time of day.","227.The method of claim 217, further comprising charging the user a fee for modification of the user's SLA.","228.A method of providing network access across a DOCSIS 1.0 compliant cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user a respective first bandwidth allowance for a first future time interval; (b) generating a first set of cable modem configuration files, each of which limits bandwidth consumption by a cable modem (CM) of a respective user to that user's first bandwidth allowance; (c) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; (d) sending a command either to each user's CM or to a cable modem termination system (CMTS) to which each user's CM is connected to cause each CM to acquire its new respective configuration file of the first set for the first time interval; (e) determining for each user a respective second bandwidth allowance for a second future time interval, a second bandwidth allowance of at least one of the users differing from that user's first bandwidth allowance; and (f) for each user having a second bandwidth allowance different from that user's first bandwidth allowance, (i) generating a second cable modem configuration file which limits bandwidth consumption of that user's CM to a value representative of that user's second bandwidth allowance; (ii) sending the second configuration file for that user to the TFTP Server of the DOC Network; and (iii) sending a command either to that user's CM or to the CMTS to which that user's CM is connected to cause the CM to acquire the second configuration file for that user for the second time interval.","229.The method of claim 228, further comprising the steps of monitoring bandwidth of each user for a time interval and, based on said monitored bandwidth, determining each user's second bandwidth allowance.","230.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes prioritizing each user and allocating bandwidth to each user according to such priority.","231.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth with respective values specified by service level agreements (SLAs) of the users.","232.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth of each user with an established minimum quality of service value.","233.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes forecasting each user's bandwidth for the second future time interval.","234.The method of claim 233, wherein said step of determining each user's bandwidth allowance includes prioritizing the users for allocation of bandwidth based on said forecasting.","235.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the upstream direction during time intervals of one minute to fifteen minutes.","236.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is requested by each CM in the upstream direction during time intervals of one minute to fifteen minutes.","237.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the downstream direction during time intervals of fifteen minutes to sixty minutes.","238.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","239.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","240.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","241.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","242.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.","243.The method of claim 228, wherein the first time interval and the second time interval are consecutive time intervals.","244.The method of claim 228, wherein each of the first time interval and the second time interval has a period of one to sixty minutes.","245.The method of claim 228, wherein for each user, the first bandwidth allowance of that user differs from the second bandwidth allowance of that user.","246.The method of claim 228, wherein the first bandwidth allowance for each user differs from the first bandwidth allowance of all other users.","247.The method of claim 228, wherein the second bandwidth allowance for each user differs from the second bandwidth allowance of all other users.","248.The method of claim 228, wherein the first bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.","249.The method of claim 248, wherein the second bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.","250.The method of claim 249, wherein the first and second allocation policies differ.","251.The method of claim 249, wherein the first and second allocation policies are the same.","252.A method of continuously providing network access across a cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user during present time intervals bandwidth allowances of such user for respective future time intervals, each bandwidth allowance of such user for a future time interval representing a maximum level of bandwidth consumption for such user during the future time interval, but not necessarily representing the amount of bandwidth that will be consumed by such user during such future time interval, the bandwidth allowances of each user varying as between time intervals; and (b) for each user, limiting during each respective future time interval bandwidth consumption of that user's cable modem (CM) to that user's respective bandwidth allowance for such time interval by implementing a new cable modem configuration file in the CM.","253.The method of claim 252, wherein the present and future time intervals have a uniform period of one minute to sixty minutes.","254.The method of claim 252, wherein each user's bandwidth allowance for each future time interval is determined by allocating bandwidth to the users on a per user basis for such future time interval in accordance with a selected allocation.","255.A Carrier Network for providing communication between multiple users and a Service Provider, comprising: (a) computer network equipment defining an Intermediate Network and a shared communications medium, said Intermediate Network and said shared communications medium extending between the users and the Service Provider for conveying data therebetween, said shared communications medium joining the users with said Intermediate Network such that data of each user is conveyed over said shared communications medium to and from the Intermediate Network whereby the users compete for network access; and (b) software for managing network access of the users across said shared communications medium, said software including computer-executable instructions that performs the steps of: (i) monitoring the network access usage by each user for a time interval; and (ii) based on the monitored network access usage, setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.","256.The Carrier Network of claim 255, wherein said network equipment futher defines a Cable Network and said shared communications medium comprises a coaxial cable of said Cable Network.","257.The Carrier Network of claim 256, wherein said software comprises software modules distributed within computer readable media of said network equipment.","258.The Carrier Network of claim 256, wherein said software comprises software modules stored in a computer readable medium of a hardware component.","259.The Carrier Network of claim 258, wherein said hardware component is physically located with said network equipment.","260.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a regional data center of said DOC Network.","261.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a headend of said DOC Network.","262.The Carrier Network of claim 258, wherein said hardware component is physically located remotely from said network equipment.","263.The Carrier Network of claim 262, wherein said hardware component is directly connected with said network equipment for communication therebetween.","264.The Carrier Network of claim 262, wherein said hardware component is indirectly connected with said network equipment through the Internet for communication therebetween.","265.The Carrier Network of claim 256, wherein said software modules include: (i) a first software component that manages a database for storage and retrieval of data; (ii) a second software component that collects data representative of network access usage from said network equipment, and that sends data representative of network access usage to said first component for storage thereof; and (iii) a third component that retrieves data representative of network access usage stored in said first component and sets, based at least partially thereon, network access allowances for the users for the future time interval.","266.The Carrier Network of claim 265, further includes a fourth component having a graphical user interface for sending data to said first component for storage and for retrieval of data stored by said first component.","267.The Carrier Network of claim 255, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","268.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in an upstream direction across said shared communications medium in time intervals of one minute to fifteen minutes.","269.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in the downstream direction across said shared communications medium in time intervals of fifteen minutes to sixty minutes.","270.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.","271.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.","272.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.","273.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.","274.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.","275.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.","276.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction across said shared communications medium during a time interval.","277.The Carrier Network of claim 255, wherein said step of setting a network access allowance for each user comprises allocating network access equally to the users and setting the network access allowance for each user equal to that user's allocated network access.","278.The Carrier Network of claim 277, wherein said software further includes computer-executable instructions that perform the additional step of prioritizing the users for allocating network access.","279.The Carrier Network of claim 278, wherein the prioritizing is based on fairness considerations.","280.The Carrier Network of claim 279, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.","281.The Carrier Network of claim 279, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.","282.The Carrier Network of claim 279, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","283.The Carrier Network of claim 279, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.","284.The Carrier Network of claim 279, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","285.The Carrier Network of claim 278, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.","286.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing each user's monitored network access usage with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","287.The Carrier Network of claim 285, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","288.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.","289.The Carrier Network of claim 285, wherein SLAs provide respective fees for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","290.The Carrier Network of claim 285, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","291.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.","292.The Carrier Network of claim 285, wherein said software further includes computer-executable instructions that perform the step of forecasting network access usage by each user during the future time interval based on the monitoring of network access usage by each user.","293.The Carrier Network of claim 292, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","294.The Carrier Network of claim 292, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.","295.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to users proportional to each user's forecasted network access usage.","296.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions that perform the step of prioritizing the users for allocating network access.","297.The Carrier Network of claim 296, wherein prioritizing is based on each user's forecasted network access usage.","298.The Carrier Network of claim 296, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","299.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","300.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","301.A computer-readable medium having computer-executable instructions that manage network access across a shared communications medium between competing users of a Carrier Network, said instructions performing the steps of: (a) monitoring the network access usage by each user for a time interval; and (b) setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.","302.The computer-readable medium of claim 301, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions performing the step of prioritizing the users for allocating network access.","303.The computer-readable medium of claim 302, wherein the prioritizing is based on fairness considerations.","304.The computer-readable medium of claim 302, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.","305.The computer-readable medium of claim 302, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.","306.The computer-readable medium of claim 302, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.","307.The computer-readable medium of claim 302, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.","308.The computer-readable medium of claim 302, wherein users are prioritized based on an established minimum quality of service (QoS) standard.","309.The computer-readable medium of claim 302, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.","310.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when the respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.","311.The computer-readable medium of claim 309, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.","312.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.","313.The computer-readable medium of claim 309, wherein SLAs provide a respective fee for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.","314.The computer-readable medium of claim 309, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.","315.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.","316.The computer-readable medium of claim 309, further comprising computer-executable instructions performing the step of forecasting network access usage by each user during the future time interval based the monitoring of network access usage by each user.","317.The computer-readable medium of claim 316, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.","318.The computer-readable medium of claim 316, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.","319.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.","320.The computer-readable medium of claim 316, further comprising computer-executable instructions performing the step of prioritizing the users for allocating network access.","321.The computer-readable medium of claim 320, wherein prioritizing is based on each user's forecasted network access usage.","322.The computer-readable medium of claim 320, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.","323.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.","324.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.","325.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users; and (d) allocating bandwidth to each user sequentially in decreasing order of user priority.","326.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth requested for each user.","327.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth consumption of each user.","328.The computerized method of claim 327, wherein the bandwidth that is forecasted is the bandwidth consumption of each user.","329.The computerized method of claim 328, wherein users are prioritized based on each user's forecasted future bandwidth consumption in increasing order, whereby a user with a lesser forecasted bandwidth consumption receives a higher priority than a user with a greater forecasted bandwidth consumption.","330.The computerized method of claim 328, wherein each user's allocation of bandwidth for the future time interval is set to equal each user's forecasted bandwidth consumption subject to a respective, predetermined maximum value and subject to bandwidth availability 331.The computerized method of claim 330, further comprising distributing equally to the users any unallocated bandwidth.","332.The computerized method of claim 330, further comprising distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidth consumptions.","333.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise the number of logical data units transmitted from and to each user during the past time interval.","334.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes transmitted from and to each user during the past time interval.","335.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of data packets transmitted from and to each user during the past time interval.","336.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.","337.The computerized method of claim 336, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.","338.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.","339.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.","340.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee charged to each user in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.","341.The computerized method of claim 340, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.","342.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.","343.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.","344.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective bandwidth levels specified to the users; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.","345.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.","346.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.","347.The computerized method of claim 344, further comprising setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.","348.The computerized method of claim 344, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.","349.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.","350.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.","351.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.","352.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing equally to the users any unallocated bandwidth.","353.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidths.","354.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.","355.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth by each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.","356.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective levels of network access specified to the users; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.","357.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.","358.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.","359.The computer-readable medium of claim 356, wherein said instructions further perform the step of setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.","360.A method of providing network access across a shared communications medium between at least four competing users, with at least a first pair of users being grouped within a first class and at least a second pair of different users being grouped within a second class, comprising the steps of: (a) determining class and user allowances of network access for a first time interval by allocating network access to each user class for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval, (b) providing network access to each user during the first time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' network allowance; (c) determining class and user allowances of network access for a second time interval by allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval, the allocated network access for the second time interval for at least one user differing from that user's allocated network access for the first time interval; and (d) providing network access to each user during the second time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' allowance.","361.The method of claim 360, wherein for at least one class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.","362.The method of claim 360, wherein for each class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.","363.The method of claim 360, wherein the collective network access allocated to each class for the first time interval differs from the collective network access allocated to each class for the second time interval.","364.The method of claim 360, further comprising requesting a minimum level of network access for a user for utilization during the first future time interval, and wherein said allocating network access to such user for the first future time interval comprises setting the level of network access allocated to such user to an amount equal to or greater than the requested minimum level.","365.The method of claim 364, wherein said allocating network access to such user for the second future time interval comprises setting the level of network access allocated to such user to an amount less than the requested minimum level for the first time interval.","366.The method of claim 360, wherein each of the first future time interval and the second future time interval has a period of between one minute and sixty minutes.","367.The method of claim 360, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.","368.The method of claim 360, further comprising the step of monitoring network access usage by each user.","369.The method of claim 368, further comprising the step of tracking network access usage for each user class.","370.The method of claim 368, wherein said step of monitoring network access usage by each user includes collecting data representative of logical data units transmitted from and to each user during a past time interval.","371.The method of claim 368, further comprising tracking logical data units transmitted from and to each user class during a past time interval.","372.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.","373.The method of claim 368, further comprising tracking the number of logical data units that are dropped for each user class during a past time interval.","374.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a past time interval.","375.The method of claim 368, further comprising tracking the number of logical data units of each user class that are requested to be transmitted in an upstream direction during a past time interval.","376.The method of claim 360, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.","377.The method of claim 360, wherein the shared communications medium is part of a Shared Access Carrier Network comprises a wireless network.","378.The method of claim 360, further comprising prioritizing the user classes for allocating network access.","379.The method of claim 378, further comprising prioritizing the users for allocating, network access.","380.The method of claim 378, wherein said prioritizing of the user classes is based on fairness considerations.","381.The method of claim 380, wherein the user classes are prioritized based on collective user throughput during a past time interval, with a user class with lesser collective user throughput receiving priority over a user class with greater collective user throughput.","382.The method of claim 380, wherein the user classes are prioritized based on collective data loss for each user class during a past time interval, with a user class with greater collective data loss having priority over a user class with lesser collective data loss.","383.The method of claim 380, wherein the user classes are prioritized based on collective network access usage for a particular time of day, with a user class with lesser collective network access usage for the particular time of day receiving priority over a user class with greater collective network access usage for the particular time of day.","384.The method of claim 380, wherein the user classes are prioritized based on both collective user throughput for the class and collective data loss of the users for the class during a time interval.","385.The method of claim 380, wherein user classes are prioritized based on an established minimum quality of service (QoS) standard.","386.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) for at least two user classes regarding the provision of network access to each respective class.","387.The method of claim 386, further comprising the step of tracking network access usage by each user class.","388.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for the user classes with the specified respective minimum levels of collective network access therefor, and awarding priority to a user class when said monitored network access usage for such user class falls below the specified respective minimum level of collective network access for such class.","389.The method of claim 387, wherein each CSLA specifies a respective time-of-day (TOD) minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for such user classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a user class when said monitored network access usage during the specified respective TOD for such user class falls below the specified respective TOD minimum level of collective network access of such user class.","390.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access up to a maximum burstable level with target probability for the respective users therein, and said step of prioritizing includes comparing for each user class said monitored network access usage both with the respective minimum level of collective network access and with the respective maximum burstable level of collective network access, and comparing the instances the respective maximum level of network access were obtained out of all instances the respective maximum level of network access could have been utilized.","391.The method of claim 387, wherein each CSLA provides a respective fee for collective network access usage, and said step of prioritizing comprises sorting such user classes based on each class' respective fee in decreasing order, with a user class with a higher fee receiving priority over a user class with a lesser fee.","392.The method of claim 387, wherein each CSLA provides a respective credit for a level of collective network access falling below a respective guaranteed level for the user class, and said step of prioritizing comprises sorting such user classes based on each class' respective credit in decreasing order, with a user class with a higher credit receiving priority over a user class with a lower credit.","393.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the user class, and said step of allocating network access comprises setting the respective level of collective network access of the user class equal to the class' specified respective minimum level of collective network access.","394.The method of claim 368, further comprising the step of forecasting collective network access usage by each user class during a future time interval based on said step of monitoring network access usage by each user.","395.The method of claim 394, wherein said step of forecasting comprises summing within each class a forecasted network access usage of each user.","396.The method of claim 394, wherein said step of forecasting network access usage of each user comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of each user.","397.The method of claim 395, wherein said step of allocating network access to each user class comprises setting the respective levels of network access of the user classes proportional to each class' forecasted network access usage.","398.The method of claim 395, further comprising the step of prioritizing the user classes for allocating network access.","399.The method of claim 398, wherein said prioritizing is based on each class' forecasted network access usage.","400.The method of claim 398, wherein said user classes are prioritized in increasing order of each class' forecasted network access usage, with a user class with a lesser forecasted network access usage receiving priority over a user class with a greater forecasted network access usage.","401.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportional to the number of users within each class.","402.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportionally based on each class' forecasted network access usage.","403.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) regarding the provision of collective network access.","404.The method of claim 403, further comprising the step of monitoring collective network access usage of each class.","405.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes with the specified respective minimum levels of collective network access, and awarding priority to a class when said respective monitored network access usage for such class falls below the class' specified respective minimum level of collective network access.","406.The method of claim 404, wherein CSLAs specify respective time-of-day (TOD) minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a class when said monitored network access usage during the specified respective TOD for such class falls below the class' specified respective TOD minimum level of collective network access.","407.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access up to a maximum burstable levels with target probability for classes, and said step of prioritizing includes comparing said monitored network access usage for each such class both with the respective minimum levels of collective network access and with the respective maximum burstable levels of collective network access, and comparing the instances the respective maximum levels of collective network access were obtained for each such class out of all instances the respective maximum levels of collective network access could have been utilized.","408.The method of claim 404, wherein CSLAs provide respective fees for collective network access usage of classes, and said step of prioritizing comprises sorting such classes based on each class' respective fee in decreasing order, with a class with a higher fee receiving priority over a class with a lower fee.","409.The method of claim 404, wherein CSLAs provide respective credits for levels of collective network access below respective guaranteed levels for classes, and said step of prioritizing comprises sorting such classes based on each class' respective credit in decreasing order, with a class with a higher credit receiving priority over a class with a lower credit.","410.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of allocating network access comprises setting the allocations of collective network access for such classes equal to each class' specified respective minimum level of collective network access"],[" BACKGROUND OF THE PRESENT INVENTION As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.","The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).","The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.","The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.","For example, a Carrier or a related entity may serve as an Internet service provider (ISP).","Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.","One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.","Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).","For example, with regard to DOC Networks, and with reference to FIG.","1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30 , which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.","All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34 .","Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.","Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.","The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.","Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.","This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.","1 .","The DOC Network 40 also includes multiple headends 36 , 64 , 66 .","In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.","Examples of a dedicated connection are shown for comparison in FIG.","1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76 .","Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.","Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.","A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.","A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.","Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.","Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.","It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.","Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.","For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.","Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.","Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.","A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.","Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.","Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.","Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.","The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.","In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.","In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.","Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.","Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.","Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.","A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.","A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.","More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.","At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.","While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.","Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.","Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.","Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.","Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.","Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium."],[" SUMMARY OF THE PRESENT INVENTION Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.","First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.","In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.","Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.","The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.","The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.","Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.","Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.","In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.","Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.","Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.","With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.","Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.","In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.","In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.","Another additional feature of the present invention includes the prioritizing of the users for allocating network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.","Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.","Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.","In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.","Another feature of the present invention includes the prioritizing of the users for allocating network access.","A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.","A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.","Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.","The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.","Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.","Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.","The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.","The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.","The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.","The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.","A charge preferably is applied to the user for the modification.","A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.","Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.","A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.","Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.","Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.","The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.","The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.","The Carrier Network also includes software that manages the network access of the users.","In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.","In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.","The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.","One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.","Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.","The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.","A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.","Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.","In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.","The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.","Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.","A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.","Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.","The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.","Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.","In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.","The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.","Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.","The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.","As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.","As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.","The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.","The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.","Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.","In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.","Accordingly, all users of a shared communications network can be classified.","In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.","Another feature includes the step of prioritizing the users for the allocation of network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.","Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted."],["FIELD OF THE PRESENT INVENTION The present invention generally relates to allocating access across a shared communications medium and, in particular, to allocating bandwidth used to convey data of competing users across a shared communications medium of a Carrier Network.","BACKGROUND OF THE PRESENT INVENTION As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.","The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).","The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.","The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.","For example, a Carrier or a related entity may serve as an Internet service provider (ISP).","Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.","One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.","Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).","For example, with regard to DOC Networks, and with reference to FIG.","1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30, which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.","All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34.Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.","Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.","The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.","Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.","This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.","1.The DOC Network 40 also includes multiple headends 36,64,66.In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.","Examples of a dedicated connection are shown for comparison in FIG.","1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76.Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.","Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.","A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.","A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.","Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.","Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.","It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.","Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.","For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.","Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.","Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.","A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.","Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.","Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.","Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.","The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.","In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.","In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.","Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.","Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.","Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.","A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.","A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.","More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.","At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.","While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.","Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.","Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.","Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.","Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.","Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium.","SUMMARY OF THE PRESENT INVENTION Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.","First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.","In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.","Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.","The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.","The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.","Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.","Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.","In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.","Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.","Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.","With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.","Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.","In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.","In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.","Another additional feature of the present invention includes the prioritizing of the users for allocating network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.","Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.","Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.","In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.","Another feature of the present invention includes the prioritizing of the users for allocating network access.","A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.","A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.","Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.","Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.","The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.","Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.","Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.","The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.","The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.","The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.","The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.","A charge preferably is applied to the user for the modification.","A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.","Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.","A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.","Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.","Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.","The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.","The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.","The Carrier Network also includes software that manages the network access of the users.","In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.","In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.","The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.","One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.","Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.","The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.","A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.","Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.","In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.","The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.","Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.","A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.","Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.","The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.","Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.","In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.","The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.","Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.","The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.","As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.","As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.","The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.","The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.","Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.","In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.","Accordingly, all users of a shared communications network can be classified.","In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.","Another feature includes the step of prioritizing the users for the allocation of network access.","One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.","Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.","Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.","In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.","BRIEF DESCRIPTION OF THE DRAWINGS Further features and benefits of the present invention will be apparent from a detailed description of preferred embodiments thereof taken in conjunction with the following drawings, wherein like elements are referred to with like reference numbers, and wherein: FIG.","1 illustrates a conventional DOC Network; FIG.","2 illustrates a first DOC Network of the present invention; FIG.","3 illustrates a second DOC Network of the present invention; FIG.","4 illustrates a third DOC Network of the present invention; FIG.","5 illustrates a fourth DOC Network of the present invention; FIG.","6 illustrates a system architecture of software components that perform preferred methods of the present invention in the DOC Networks of FIGS.","2-5; FIG.","7 illustrates a flowchart of the steps of a preferred routine for forecasting bandwidth of each user for a future time interval; FIG.","8 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the ARRSES Function of the preferred routine of FIG.","7; FIG.","9 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the HW Function of the preferred routine of FIG.","7; FIG.","10 illustrates a graph of user throughput rates versus user data loss rates for two users relative to a target minimum QoS standard; FIG.","11 illustrates a flowchart of a first preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.","12 illustrates a flowchart of a second preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.","13 illustrates a flowchart of a third preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.","14 illustrates a flowchart of a fourth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.","15a and 15b illustrate a flowchart of a fifth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.","16a and 16b illustrate a flowchart of a sixth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.","17 illustrates a flowchart of a first preferred method of prioritizing users and allocating bandwidth to each user within a class; FIG.","18 illustrates a flowchart of a second preferred method of prioritizing users and allocating bandwidth within a class; FIG.","19 illustrates a flowchart of a third preferred method of prioritizing users and allocating bandwidth within a class; FIG.","20 illustrates a flowchart of a fourth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.","21a and 21b illustrate a flowchart of a fifth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.","22a and 22b illustrate a flowchart of a sixth preferred method of prioritizing users and allocating bandwidth within a class; FIG.","23 illustrates a flowchart of a preferred method of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network; FIG.","24 illustrates the allocation of bandwidth to users within a class during a first time interval; FIG.","25 illustrates the allocation of bandwidth to the users of FIG.","24 during a second time interval; and FIG.","26 illustrates a flowchart of a preferred method of soliciting a user to modify the user's SLA based on monitored network access usage of the user.","DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In the following detailed description, numerous specific details are set forth with regard to preferred embodiments of the present invention in order to provide a thorough understanding of the present invention; however, it will be apparent to ordinary artisans that the present invention may be practiced without all of these specific details.","Well-known structures and devices also are shown in block diagram form, the specific details of which are not considered a necessary part of the present invention.","Furthermore, as will become apparent to ordinary artisans, the present invention may be embodied in or performed by hardware, firmware, or software, or various combinations thereof.","As described above, a conventional DOC Network 40 is shown in FIG.","1 and includes a plurality of Cable Networks 38, with a particular Cable Network 38 being illustrated in an expanded view and comprising a group of CMs 34, each connected to a computer 44 representing a user.","Additionally, as used herein, “user” includes not only a person who interacts with a computer 44, but any additional persons who also interact with the same computer 44, as well as any group of persons all of whom interact with computers attached either to the same CM 34 or to the same computer 44 which, itself, is attached to a CM 34.While not shown, such additional arrangements are well known in the art.","The CMs 34 are connected by a coaxial cable 32 with a CMTS 30 and, specifically, to a card 31 mounted within the CMTS 30.Each of the CMTSs of the DOC Network 40 preferably includes a plurality of cards, with each card supporting a group of CMs connected thereto in an inverted tree configuration to define a Cable Network 38.Furthermore, each CMTS conventionally supports up to 1,500 users, although recent CMTSs have been introduced that support up to 15,000 users.","Each Cable Network 38 defines a Shared Access Carrier Network, wherein data of respective users in each are conveyed together through a shared coaxial cable.","For instance, data packets (or frames) addressed to at least one of the computers 44 are transmitted by the CMTS 30 downstream over the coaxial cable 32 to all of the CMs 34 within a 6 MHz data channel.","Conversely, data packets intended for delivery to the CMTS 30 and beyond are transmitted by a CM 34 upstream to the CMTS 30 over the coaxial cable 32 within a 2 MHz channel.","The Cable Network 38 shown in expanded view in FIG.","1 is a traditional all coaxial cable network.","The other Cable Networks 38 collectively include both traditional all coaxial cable networks as well as HFC networks.","The CMTS 30 transmits and receives data packets between the Cable Networks 38 and an Intermediate Network 46, which begins with a router 48 in the headend 36, and includes switched and routed network equipment at a Regional Data Center 50 that provides connectivity to service providers 52,54,56,58, either directly or through the Internet 60.In this regard, during user communications the router 48 conveys data packets from the CMTS 30 to the Regional Data Center 50 of the DOC Network 40 and, conversely, routes data packets received from the Regional Data Center 50 to the appropriate CMTS for delivery to a particular user.","Data packets that are conveyed to the Regional Data Center 50, in turn, are directed on to an appropriate service provider 52,54 directly connected to the Regional Data Center 50, or to an appropriate service provider 56,58 indirectly connected to the Regional Data Center 50 via the Internet 60.Alternatively, data packets from users are conveyed to a server of an application server group 62 of the Regional Data Center 50, which includes, for example, servers supporting Web hosting, news, chat, SMTP, POP3, Proxy, cache and content replication, and streaming media.","The Cable Networks 38 stemming from headend 36 are maintained by a Carrier which also may maintain the Regional Data Center 50 as well as serve as a service provider.","Moreover, the Carrier may maintain the Cable Networks of additional headends 64,66, or of only one or more of the headends 64,66.In any event, the Cable Networks that are maintained by the Carrier are administered on a daily basis through an element management system (EMS) 68.The EMS 68 comprises an operations system designed specifically to configure and manage CMTSs and associated CMs, and includes a CM database 70.Operational tasks performed by the EMS 68 include provisioning, day-to-day administration, and testing of various components of each CMTS.","The EMS 68 typically is located at a central network operations center of the Carrier, but may be collocated at the headend 36 of the Carrier as shown in FIG.","1.The DOC Network 40 is managed through a control plane server group 72 typically located at the Regional Data Center 50.The control plane server group 72 includes the usual servers necessary to run the DOC Network 40, such as user authorization and accounting servers, log control servers (Syslog), IP address assignment and administration servers (DHCP, TFTP), domain name servers (DNS), and DOCSIS control servers.","For purposes of comparison, two dedicated connections also are shown in FIG.","1, wherein a telephony modem 74 and an ISDN modem 76 are connected directly to the Intermediate Network 46 at the Regional Data Center 50.As will be immediately apparent, data conveyed over each dedicated connection is between a single user and the Intermediate Network 46, and is not combined with data of other users over a shared communications medium as in each Cable Network 38.As is common in conventional Cable Networks 38 such as those shown in the DOC Network 40 of FIG.","1, when a CM comes online the CM is assigned a configuration file which, inter alia, sets a constant limit on the bandwidth that can be utilized in the downstream direction by the CM during any particular interval of time, and sets a constant limit on the bandwidth that can be utilized in the upstream direction by the CM during any particular interval of time.","The configuration file also includes other parameters, such as the IP address for the CM.","The configuration file for each CM conventionally is obtained by the CM when first brought online, or when the CM is reset.","The upstream and downstream bandwidth limits are predetermined by the Carrier or other appropriate entity, the determination of which is based on the expected number of users to be serviced by the particular Cable Network 38 to which the CM belongs.","With particular regard to data transmissions in the downstream direction, when the bandwidth limit is reached in receiving data within a particular time interval, the CM transmits a signal to the router 48 to cease further data forwarding for the remainder of the time interval.","Thereafter, whereas any data received by a CMTS is relayed on to the CM as the data is received, any additional data received by the router 48 during the remainder of this time interval is stored for later transmission in a buffer up to a threshold limit and, thereafter, any further data received within the time interval is dropped.","With regard to data transmissions in the upstream direction, when the CM registers with the CMTS following receipt by the CM of its configuration file, the CM informs the CMTS of the constant bandwidth limit to be applied to upstream transmissions from the CM.","Then, actual requests for bandwidth (i.e., requests for timeslots) for transmission of data in the upstream direction are submitted regularly by each CM to the CMTS.","In response to the submissions, the CMTS schedules timeslots in a particular time interval to the CMs for exclusive transmission of data within each timeslot by a respective CM.","However, the CMTS does not grant an amount of bandwidth (by assigning too many timeslots) to a particular CM that would exceed the constant bandwidth limit for the particular CM.","The timeslots are assigned to requesting CMs based on an established assignment policy.","For example, timeslots may be assigned by the CMTS on a first-in-first-out basis, or timeslots may be assigned equally to the CMs that request bandwidth within a particular window of time.","The requesting CMs also may be prioritized by the CMTS for assignment of the timeslots.","Preferred embodiments 78,80,82,84 of a DOC Network in accordance with the present invention are shown, respectively, in FIGS.","2-5, wherein each includes a “network access manager” 86 in accordance with the present invention.","In FIG.","2 the network access manager 86 is located in the headend 36 of the DOC Network 78, in FIG.","3 the network access manager 86 is located at the Regional Data Center 50 of the DOC Network 80, and in FIGS.","4-5 the network access manager 86 is remotely located, but is disposed for communication with the respective DOC Network 82,84, either directly as shown in the DOC Network 82 of FIG.","4, or indirectly via the Internet 60 as shown in the DOC Network 84 of FIG.","5.The network access manager 86 preferably comprises a hardware component having software modules for performing methods in accordance with the present invention.","For commercial purposes, especially in enhancing existing DOC Networks, preferably the network access manager 86 is self-contained and need only be connected in communication with the DOC Network to operate correctly.","In a DOC Network that is being upgraded or established, preferably the software modules are distributed within the DOC Network itself and may or may not include any additional hardware components such as the network access manager 86.For example, the software modules may be incorporated into the EMS, CMTS, and control plane server group of a DOC Network, thereby avoiding the expense of additional computer hardware components.","In order to accommodate deployment and implementation of the present invention, the software modules preferably are designed as peers within a messaging infrastructure and, in particular, within a CORBA infrastructure 87, the system architecture of which is shown in FIG.","6.Due to the interoperability of the peers to the CORBA infrastructure 87, the separate modules readily call upon each other as described in detail below without regard to differences in location between the modules.","Nevertheless, for ease of deployment, the network access manager 86 is best suited for deployment and implementation of the present invention in established DOC Networks, whether situated within the Intermediate Network as in FIGS.","2-3, or remotely situated as in FIGS.","4-5.The software modules include a Data Collector 88, a Database Manager 90, Bandwidth Allocator 92, and GUI & Report Generating Engine 94.The Data Collector 88 and Bandwidth Allocator 92 each includes an external system interface layer 96,98, respectively, that enables it to communicate with network equipment of a DOC Network.","In the system architecture of preferred embodiments, the Data Collector 88 communicates with each CMTS and CMs of each Cable Network for which network access is managed by the network access manager 86, and the Bandwidth Allocator 92 communicates with the control plane server group 72 of the DOC Network as well as with the CMTS and CMs.","If a DOC Network is DOCSIS 1.0 compliant, then each external system interface layer 96,98 is a DOCSIS external system interface layer.","If a DOC Network uses proprietary interface specifications, then each external system interface layer 96,98 is designed based on the proprietary interface specifications.","In either case, however, the Data Collector 88 and Bandwidth Allocator 92 generally need not be modified; only the external systems interface layers 96,98 thereof need be changed based on the particularities of the DOC Network.","Each of the Data Collector 88 and Bandwidth Allocator 92 also includes a scheduling element 100,102, respectively, that schedules the timing of actions and communications thereof with the network equipment of a DOC Network.","The GUI & Report Generating Engine 94 communicates with an Administrator 106 of the network access manager 86, preferably through a web server, whereby the Administrator 106 sets up and configures the network access manager 86 and accesses reports generated by the network access manager 86, such as graphs of bandwidth consumption and bandwidth requested per time interval for a user.","The Administrator 106 may be the Carrier, a service provider, or some other entity, such as the entity managing the Regional Data Center 50 or a third-party responsible for maintenance of the network access manager 86.The Database Manager 90 stores configuration and setup information received from the GUI & Report Generating Engine 94, as well as information processed by the Data Collector 88.The Database Manager 90 also provides information to the Bandwidth Allocator 92 and GUI & Report Generating Engine 94 as requested via the CORBA infrastructure 87.Having now described in detail the structure of preferred DOC Networks 78,80,82,84, preferred methods of the present invention will be described with reference thereto.","In accordance with preferred methods of the present invention, network access usages of each user in the upstream and downstream directions are monitored through the Data Collector 88.Specifically, the Data Collector 88 issues queries to the CMTS and CM to which counter values of logical data units (LDUs) are returned for a user.","Preferably, counter values are returned for the number of bytes and the number of data packets that are transmitted in both the upstream and downstream directions, the number of bytes and the number of data packets that are dropped in both the upstream and downstream directions, the number of bytes and the number of packets that are requested to be transmitted in the upstream direction, and the time for which the counter values are returned.","Accordingly, as used herein the phrase “monitoring network access usage” is intended to refer to the collection of data representative of at least one of: (i) the number of LDUs that are transmitted in a particular direction across a shared communications medium; (ii) the number of LDUs that are dropped in transmitting in a particular direction across a shared communications medium; and (iii) the number of LDUs that are requested to be transmitted in a particular direction across a shared communications medium.","In a DOCSIS compliant DOC Network, the information is collected from the CMTS and CMs of a Cable Network via the simple network management protocol (SNMP).","The counter values for bytes and data packets that are transmitted and that are dropped in the upstream direction from each CM, and the number of bytes and data packets that are requested to be transmitted in the upstream direction from each CM, are recorded by the CMTS in accordance with a management information base (MIB) of a DOCSIS compliant CMTS.","Likewise, the counter values for bytes and data packets that are transmitted and that are dropped in the downstream direction from the CMTS to a CM are recorded by the CM in accordance with a MIB of a DOCSIS compliant CM.","Both bytes and data packets are monitored since each data packet may vary in the number of bytes it contains.","The scheduling element 100 of the Data Collector 88 initiates the data collection from each CMTS and from the CMs connected thereto, preferably at different predetermined time intervals.","For example, the data collection from a CMTS preferably occurs at five minute intervals and data collection from the CMs connected thereto preferably occurs at thirty minute intervals.","The data collection from the CMs preferably is less often than the data collection from the CMTS in order to minimize consumption of bandwidth across the Cable Network that otherwise would be allocated to users.","When the counter values and time thereof are returned to the Data Collector 88, the Data Collector 88 calculates the change over time for each counter value to arrive at the average rates of bytes and data packets that are successfully transmitted, the average rates of bytes and data packets that are requested to be transmitted, and the average rates of bytes and data packets that are dropped.","The respective rates and time intervals for the rates (as well as the counter values and time stamp data) are then communicated to the Database Manager 90, which stores the information in a user statistics table (“stats”) for later use by the Bandwidth Allocator 92 and GUI & Report Generating Engine 94.The Bandwidth Allocator 92 continually determines the network access—or bandwidth in a Cable Network—that may be utilized by each user class, and by each user within each class, over succeeding time intervals.","Each allowance is determined by first allocating bandwidth to the user classes, and then allocating bandwidth to the users in each class, in accordance with one or more selected allocation policies.","Furthermore, as set forth above, each allowance is an amount of bandwidth up to which a user class or user may consume, but is not necessarily the amount of bandwidth that a user class or user will consume; it is an upper limit on such amount.","For example, with reference to FIG.","24, a selected allocation policy has resulted in the allocation of bandwidth to the users of the shared communications medium 2450 for a time interval extending from t0 to (t0+dt), User 2 and User K each is allocated a single bandwidth unit (b/w unit 3 and b/w unit X, respectively), while User 1 and User 3 each is allocated two bandwidth units (b/w unit 1 and b/w unit 2 to User 1, and b/w unit 4 and b/w unit 5 to User 3).","As shown in FIG.","25, in the next time interval extending from (t0+dt) to (t0+2dt), User 1, User 3, and User K each is allocated a single bandwidth unit (b/w unit 1, b/w unit 5, and b/w unit X, respectively), while User 2 is allocated three bandwidth units (b/w unit 2, b/w unit 3, and b/w unit 4).","In this example, all users are grouped within the same class, and the bandwidth units in this example broadly represent network access to the communication member 2400 that is shared between the users across the shared communications medium 2450.In accordance with the present invention, respective user bandwidth allowances for each time interval are equated with these user allocations of bandwidth, whereby no user receives more bandwidth in a time interval than that user's respective bandwidth allowance for that time interval.","Furthermore, it is important to distinguish what a user actually may be “allocated” in the context of the bandwidth that is actually utilized or consumed by such user, as opposed to bandwidth allocations to a user in accordance with the present invention.","The bandwidth allocation in accordance with the present invention represents a limit on the amount of bandwidth that can be allocated to a user for a time interval—and hence is equated with a bandwidth allowance; it does not represent per se the amount of bandwidth that the user actually will utilize in the time interval.","In determining network access allocations (and thus allowances) in the preferred embodiments herein described, the Bandwidth Allocator 92 preferably performs three routines, including: the prediction of bandwidth of each user class, and each user within each class, in a predetermined future interval of time (“First Routine”); the prioritization of user classes, and users within each class, for allocation of bandwidth (“Second Routine”); and the actual allocation of bandwidth for each user class, and each user within each class, for determining the bandwidth allowances for the future time interval (“Third Routine”).","The First Routine preferably is performed utilizing statistical analysis of past bandwidth consumption of each user or, alternatively, past bandwidth requested for each user, and the forecasted bandwidth includes the bandwidth expected to be consumed by each user or, alternatively, the bandwidth expected to be requested by each user.","Any function, method, or algorithm that generates an estimate of a future sample based on previously encountered samples may be used and many are well known in the art of statistical analysis as is evident from SPYROS MAKRIDAKIS ET AL., FORECASTING METHODS AND APPLICATIONS (3d.","Ed.","John Wiley & Sons 1998), which is hereby incorporated by reference.","With regard to user classes, preferably a collective forecasted bandwidth for each class is determined by summing the forecasted bandwidth of all users within the class.","The preferred algorithm for predicting each user's forecasted bandwidth includes the combined use of an adaptive-response-rate single exponential smoothing function (ARRSES Function) and a Holt-Winters' seasonal exponential smoothing function (HW Function).","These two functions are utilized according to the forecast generation flowchart of FIG.","7.The input includes a list of active users and the applicable time intervals for bandwidth allocation.","The First Routine 700 begins by identification (Step 702) of the users of the Cable Network to which bandwidth is to be allocated in the Third Routine.","Then, for each user, bandwidth for a succeeding time interval is predicted according to either the ARRSES Function or HW Function by first determining (Step 704) whether the user previously has been assigned a forecast function.","If not, then in Step 706 the ARRSES Function is assigned to the user and the ARRSES Function is used to generate and record the forecasted bandwidth for the succeeding time interval.","On the other hand, if it is determined in Step 704 that a forecast function is assigned, but it is determined in Step 707 that the forecast function is not the HW Function, then a determination is made (Step 708) whether to check for a seasonal cycle of the user.","This determination in Step 708 is made by checking the elapsed time since the last seasonal check was made, with a seasonal check being made after a predetermined period of time elapses.","If the determination in Step 708 is affirmative, then a seasonal identifier algorithm is executed (Step 710), in which an autocorrelation function and a seasonal identifier function are performed.","The autocorrelation function is well known in the art of statistical analysis, and is used to identify elements in a time series which are influential on a current observation of that same series.","Based on the output of the autocorrelation function, the seasonal identifier function identifies possible seasonal cycles of the time series by identifying local maxima of the results of the autocorrelation function.","Based on the results of the seasonal identifier function, a determination is made (Step 712) whether an actual seasonal pattern exists.","If a seasonal pattern is not found, or if it is not yet time to check for a seasonal cycle, then a forecast is generated and recorded (Step 714) using the ARRSES Function.","If a seasonal pattern is found, then the HW Function is assigned (Step 716) to the user, the HW Function is initialized (Step 718), and the first forecast is generated and recorded (Step 720) using the HW Function.","If it is determined in Step 707 that the current function assigned to the user already is the HW Function, then the determination is made (Step 722) whether the last forecasted bandwidth was acceptable.","This determination is made by comparing whether the forecasted bandwidth was within 10% of the actual bandwidth consumed or requested.","If this determination in Step 722 is negative, then the ARRSES Function is assigned to the user and the new forecast is generated and recorded in accordance with the ARRSES Function (Step 706).","If the last forecast is determined (Step 722) to have been acceptable, then a determination is made (Step 724) whether the seasonal cycle has ended.","If the seasonal cycle has ended, then the HW Function is reinitialized (Step 726), and the first forecast of the next seasonal cycle is generated and recorded (Step 728) via the HW Function.","If the seasonal cycle has not expired, then the next forecast is generated and recorded (Step 730) in accordance with the HW Function.","Following each of Step 706, Step 714, Step 728, and Step 730, the Bandwidth Allocator 92 determines (Step 732) whether the forecasting has been completed for all users and, if not, then repeats (Step 738) a forecast loop for a remaining user.","If it is determined in Step 732 that all users have been evaluated, then the forecasts are communicated (Step 736) to the Database Manager 90 and the forecasting routine ends.","A forecast of bandwidth for a user in a future time interval is generated in accordance with the ARRSES Function via the following formulas: FN+1=FN+αN(BN−FN) αN+1=|SEN/SAEN| SEN+1=SEN+β(BN+1−FN+1−SEN) SAEN=β|(BN−FN)|+(1−β)SAEN−1 wherein, F is the bandwidth that is expected to be consumed by a user for a time interval (or the bandwidth that is expected to be requested by a user); B is the bandwidth that is actually consumed by a user for the time interval (or the bandwidth that is actually requested by a user); N is the present time interval; N−1 is the previous (immediate past) time interval; N+1 is the next (immediate future) time interval; and β is a selected parameter affecting the responsiveness to change of the ARRSES Function when the bandwidth of a user changes between time intervals.","Bandwidth is predicted both for the 6 MHz channel in the downstream direction as well as the 2 MHz channel in the upstream direction.","Preferably each time interval is thirty minutes in length, but preferably may range from fifteen minutes to sixty minutes in length when bandwidth is forecast in the downstream direction.","Preferably each time interval is five minutes in length, but preferably may range from one minute to fifteen minutes in length when bandwidth is forecast in the upstream direction.","The steps in generating a forecast in accordance with the ARRSES Function are set forth in FIG.","8, and include the calculation (Step 802) of a forecast error, the calculation (Step 804) of a smoothed error, the calculation (Step 806) of a smoothed absolute error, the calculation (Step 808) of alpha, and the calculation (Step 810) of the new forecast.","A forecast of bandwidth of a user for a future time interval is generated in accordance the HW Function via the following formulas: Ls=1/s(Y1+Y2+ .",".",".","+Ys) bs=1/S[(Ys+1−Y1)/s+(Ys+2−Y2)/s+ .",".",".","+(Y2s−Ys)/s] S1=Y1/Ls,S2=Y2/Ls, .",".",".","Ss=Ys/Ls Lt=α(Yt/St−s)+(1−α)(Lt−1+bt−1) bt=β(Lt−Lt−1)+(1−β)bt−1 St=γYt/Lt+(1−γ)St−s Ft+m=(Lt+btm)St−s+m wherein, Li=an average level of bandwidth after time interval i, bi=the trend after time interval i, si=the seasonal influence at time interval i, s=length of seasonal cycle (in number of time intervals), Yi=monitored bandwidth consumed or requested in time interval i t=time of initialization, m=the number of time intervals into the future for which a forecast is made, and α, β, and γ are parameters of the forecast method whose values are determined by doing a grid search over the domain of possible values of these parameters in an attempt to minimize the mean-squared-error of the forecast method, each of α, β, and γ falling between 0 and 1.The steps in generating a forecast in accordance with the HW Function are set forth in FIG.","9, and include the initialization of the HW Function by determining Ls, bs, and S1, S2 .",".",".",", Ss in Step 902, if appropriate; the determination of the intermediate values of Lt, bt, and St in Step 904; and the determination of the forecast in Step 906, all in accordance with the above formulas.","The Second Routine performed by the Bandwidth Allocator 92 comprises the prioritizing of user classes, and of users within each class, to determine respective orders of allocations.","Prioritization is performed in accordance with one or more of various possible prioritization policies for users and for user classes.","With regard to users within each class, the prioritization policies may depend upon, for example, (i) each user's SLA, (ii) each user's forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.","User SLAs that at least partially affect prioritization policies include those that specify, for example: (i) a guaranteed minimum level of bandwidth; (ii) a time-of-day (TOD) minimum level of bandwidth; or (iii) a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability.","Equivalently, such provisions also may be found in a CSLA for a class of which the user is a member.","Under a SLA or CSLA providing for a guaranteed minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for use at all times.","Accordingly, if the available bandwidth to such a user otherwise would fall below the minimum guaranteed level, then such a user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.","Similarly, under a SLA or CSLA providing for a TOD minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for a particular TOD.","If the available bandwidth to such a user otherwise would fall below the minimum guaranteed level during the particular TOD, then such user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.","Finally, under a SLA or CSLA providing for a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability for a user, a user will have a guaranteed minimum level of bandwidth at all times and, in addition thereto, probably will have additional bandwidth up to a maximum level at any given time in accordance with the target probability.","Accordingly, if the bandwidth available to such user otherwise would fall below the minimum guaranteed level, then the user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.","The user also is given priority over such other users in allocating additional bandwidth as needed up to the maximum level in accordance with the target probability.","Other SLA or CSLA provisions not relating to guaranteed levels of bandwidth also may affect a prioritization policy for users.","Thus, for example, a SLA or CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon bandwidth consumption by a user for a particular amount of time, and the fee may be different as between users.","Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid.","Similarly, a SLA or CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier to an account based upon a bandwidth shortfall to a user for a particular amount of time when a guaranteed level of bandwidth for the user is not met.","Moreover, the credit may be different as between users.","Under these circumstances, prioritization may be determined so as to minimize the collective credits that a Carrier must apply.","An example of prioritization based upon the forecasted bandwidth of each user includes giving priority to a first user over all other users, each of whom have a forecasted bandwidth that is greater than that of the first user.","Prioritization may also be performed based on unilateral fairness considerations, especially when SLAs or CSLAs do not guarantee minimum levels of bandwidth for individual users, or when users otherwise would share equally in priority.","Thus, users may be prioritized based on, for example: (i) the throughput of each of the users for a given time interval, with priority going to the user with the lesser throughput; (ii) data packets dropped over a given time interval, with priority going to the user with the greater data loss; and (iii) throughput experienced during a particular time of day or day of the week with priority going to the user with the lesser throughput for the particular time of day or day of the week.","An example of fairness considerations that may be utilized in determining priority is illustrated in FIG.","10, wherein user throughput for a time interval is graphed against user data packets dropped in the time interval for Users A and B.","A target QoS standard for minimum throughput and maximum packet loss rates are established by the Carrier, whereby in the illustrated example each user is prioritized based on the user's absolute distance from the target QoS standard.","Thus, under this policy, User A experiencing higher throughput rate and a lower packet loss rate, and thus having a shorter distance from the standard, is prioritized lower than User B having a lower throughput rate and higher data loss rate.","With regard to user classes, prioritization policies are similar to those of the users and include, for example, (i) each CSLA, (ii) each class' collective forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.","CSLAs that at least partially affect prioritization policies for user classes include those that specify, for example: (i) a guaranteed minimum level of collective bandwidth for the user class; (ii) a time-of-day (TOD) minimum level of collective bandwidth for the user class; or (iii) a guaranteed minimum level of collective bandwidth up to a maximum burstable level of collective bandwidth with target probability for the user class.","Other CSLA provisions not relating to guaranteed levels of collective bandwidth also may affect a prioritization policy.","Thus, for example, each CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon collective bandwidth consumption by the users of a class for a particular amount of time, and the fee may be different as between different classes of users.","Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid to a Carrier.","Similarly, each CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier based upon a collective bandwidth shortfall to the users of the class for a particular amount of time when a guaranteed level of collective bandwidth is not met.","Moreover, the credit may be different as between user classes.","Under these circumstances, prioritization may be determined so as to minimize the total credits that a Carrier may have to apply.","An example of prioritization based upon the collective forecasted bandwidth of each user class includes giving priority to a first user class over all other user classes, each of which has a respective collective forecasted bandwidth that is greater than that of the first user class.","Prioritization may also be performed based on unilateral fairness considerations, especially when CSLAs do not guarantee minimum levels of collective bandwidth, or when classes otherwise would share equally in priority.","Thus, user classes may be prioritized based on, for example: (i) the collective throughput of the users of a class for a given time interval, with priority going to the class with the lesser collective throughput; (ii) the collective data packets of a user class that are dropped over a given time interval, with priority going to the user class with the greater collective data loss; and (iii) the collective throughput of the users of a class experienced during a particular time of day or day of the week, with priority going to the user class with the lesser collective throughput for the particular time of day or day of the week.","The Third Routine performed by the Bandwidth Allocator 92 is the allocation of bandwidth to the user classes, and then to the users within each class, in accordance with one or more allocation policies as desired.","Examples of allocation policies for users include: (i) the equal distribution of all available bandwidth to all users; (ii) the distribution of all available bandwidth to all users proportional to each user's respective forecasted bandwidth; (iii) the distribution of bandwidth to each user equal to the user's respective forecasted bandwidth, with any surplus bandwidth being distributed to the users either equally or proportionally based upon the user's respective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user based upon the minimum of the user's guaranteed bandwidth or the forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users.","Likewise, examples of allocation policies for user classes include: (i) the distribution of all available bandwidth by the Bandwidth Allocator 92 to all user classes proportional to the number of active users in each class; (ii) the distribution of all available bandwidth to all user classes proportional to each class' respective collective forecasted bandwidth; (iii) the distribution of bandwidth to each user class equal to the class' respective collective forecasted bandwidth, with any surplus bandwidth being distributed to the user classes either equally or proportionally based upon the class' respective collective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user class based upon the minimum of the class' guaranteed collective bandwidth or the collective forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users classes.","Examples of alternate preferred methods of prioritizing user classes, and then allocating bandwidth to the classes, will now be described in detail, each of which utilizes one or more of the aforementioned user class prioritization and allocation policies.","Alternative preferred methods of prioritizing users within each class, and then allocating bandwidth to the users in each class, are set forth thereafter.","In either case, the preferred methods of prioritizing and allocating are initiated pursuant to the scheduling module 102 of the Bandwidth Allocator 92, which operates independently of the scheduling module 100 of the Data Collector 88.With regard to prioritization of and allocation to user classes, a first preferred method 1100 is illustrated in FIG.","11 and begins with the retrieval (Step 1102) of the collective forecasted bandwidth from the Database Manager 90 for all active user classes.","Whether a user class is active is determined by past collective bandwidth consumption of the class (or, alternatively, collective requested bandwidth for the users of the class), as revealed by the user stats maintained by the Database Manager 90.All user classes are then prioritized (Step 1104) based on each class' collective forecast in increasing order, whereby a class having a lesser collective forecasted bandwidth will be prioritized over a class having larger collective forecasted bandwidth.","A “surplus” is then set (Step 1106) to the total bandwidth available for allocation to the classes in the particular direction of communication over the shared communications medium at issue, and the total bandwidth available is then allocated (Step 1108) to each user class in an amount equaling the collective forecasted bandwidth subject to a respective maximum collective bandwidth value of the user class.","Preferably the maximum collective bandwidth value is determined either in the appropriate CSLA or by the Carrier, Administrator 106, or other entity.","Allocation of bandwidth to a user class additionally is subject to the actual availability of bandwidth following previous allocations thereof to user classes with equal or higher priority.","Following allocations to all user classes, any bandwidth determined (Step 1110) to be remaining is then allocated (Step 1112) to the classes in amount proportional to the number of active users in each class, subject of course to the respective maximum collective bandwidth value of the class.","The resulting class allocations are then recorded in the Database Manager 90 (Step 1114) as the bandwidth allowances for the classes.","The method 1200 illustrated in FIG.","12 is the same as that of FIG.","11, except that surplus bandwidth, if any, is allocated (Step 1102) proportional to the collective forecasted bandwidths of the user classes, again subject to the respective maximum collective bandwidth value of each user class.","The preferred method 1300 illustrated in FIG.","13 does not prioritize the user classes for purposes of allocation but, instead, treats all classes equally.","The method 1300 begins with the retrieval (Step 1302) of the collective forecasted bandwidth of each user class from the Database Manager 90.The surplus is then set to the total bandwidth available in the particular direction of communication, and the sum of all the collective forecasts is calculated (Step 1304).","The available bandwidth then is allocated (Step 1306) to all classes proportional to the class' collective forecasted bandwidth, again subject to the respective maximum collective bandwidth value for each class.","The resulting class allocations then are recorded in the Database Manage 90 (Step 1308) as the bandwidth allowances for the classes.","The preferred method 1400 illustrated in FIG.","14 seeks to maximize revenues from fees (F) that are paid for class bandwidth consumption.","The method 1400 begins with the retrieval (Step 1402) of the collective forecast for each user class as well as a fee that is paid for the collective bandwidth of the class.","The classes are then sorted (Step 1404) based on these fees in decreasing order, with the class with the highest fee receiving the highest priority.","Next the surplus is set (Step 1406) to the total bandwidth available for allocation to the classes in the particular direction of communication.","Bandwidth then is allocated (Step 1408) to the classes as available from highest to lowest priority in an amount equal to the class' collective forecasted bandwidth, subject to the respective maximum collective bandwidth value for the class.","Both preferred method 1500 of FIGS.","15a and 15b, and preferred method 1600 of FIGS.","16a and 16b differ from the other methods 1100,1200,1300,1400 in that these two methods allocate bandwidth to the user classes in multiple allocation rounds.","Method 1500 begins in FIG.","15a with the retrieval (Step 1502) of the collective forecasted bandwidths of the classes as well as a credit (C) that applies if a respective class does not receive up to a guaranteed maximum level of collective bandwidth.","The classes are then prioritized (Step 1504) based on each class' respective credit in decreasing order, with those classes having higher credits being given priority over classes with lesser credits.","Next, the surplus is set (Step 1506) to the total bandwidth available to the classes in the particular direction of communication.","Bandwidth then is allocated (Step 1508) as available in a first round to the classes from highest to lowest priority.","The allocation for each class in the first round is equal to the minimum of the collective forecasted bandwidth or the maximum collective bandwidth that is guaranteed, subject to the respective maximum collective bandwidth value for the class.","If any additional bandwidth is determined (Step 1510) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 1514).","Bandwidth then is allocated (Step 1516) as available to each class in the same class order.","Assuming sufficient bandwidth remains available, the allocation in the second round brings each class' allocation up to the class' collective forecasted bandwidth subject to the class' respective maximum collective bandwidth value.","Following the second allocation round, a determination is made (Step 1518) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 1522) to the classes proportional to each class' collective forecasted bandwidth, and subject to each class' respective maximum collective bandwidth value.","The resulting class allocations are then recorded (Step 1524) in the Database Manager 90 as the bandwidth allowances of the classes.","If it is determined that no bandwidth remains available in either of Step 1510 or Step 1518, then the class allocations are completed and are recorded in the Database Manager 90 in Steps 1512,1524, respectively.","Method 1600 of FIGS.","16a and 16b differs from that of FIGS.","15a and 15b only in that the sum of the collective forecasted bandwidths for all classes is calculated (Step 1602) and a determination is made (Step 1604) whether the sum exceeds the total bandwidth available for allocation to the classes.","If the sum exceeds the total available bandwidth, then bandwidth is allocated (Step 1606) to each class in an amount equal to the collective forecasted bandwidth of the class, subject to the class' maximum guaranteed collective bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.","Thus, for example, if the sum of all collective forecasted bandwidths exceeds the total available bandwidth for allocation in an amount equal to 20% of all collective forecasted bandwidths, then each class is allocated bandwidth in an amount equal to the class' collective forecasted bandwidth (subject to the class' maximum guaranteed collective bandwidth), then less 20% thereof.","The information including fees, credits, guaranteed collective bandwidths, and respective maximum collective bandwidth values in the aforementioned preferred methods, is obtained from each CSLA and/or is predetermined by the Administrator 106, Carrier, or other entity.","Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a CSLA table for each class as well as information regarding users associated therewith, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of class and user records in a timely fashion.","Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once class allocations have been determined, the Database Manager 90 is updated with the new class allocations.","Then, for each class, allocations of bandwidth are made to the users in the class.","Furthermore, allocations within each class may be made by different methods.","A first preferred method 1700 of prioritizing users and allocating bandwidth (whether upstream or downstream) by the Bandwidth Allocator 92 is illustrated in FIG.","17 and begins with the retrieval (Step 1702) of the forecasted bandwidth from the Database Manager 90 for all active users.","Whether a user is active is determined by past bandwidth consumption of the user (or, alternatively, requested bandwidth for the user), as revealed by the user stats maintained by the Database Manager 90.All users are then prioritized (Step 1704) based on each user's forecast in increasing order, whereby users having lesser forecasted bandwidths will be prioritized over users having larger forecasted bandwidths.","The “surplus” is then set (Step 1706) to the total allocated bandwidth of the class (i.e., the class' collective bandwidth allowance) in the particular direction of communication, and the entire bandwidth allowance of the class is then allocated (Step 1708) to each user in an amount equaling the forecasted bandwidth of the user subject to a respective maximum bandwidth value of the user.","Preferably the respective maximum bandwidth value is determined either in the user's SLA, the respective CSLA of the class, or by the Carrier, Administrator 106, or other entity.","Allocation of bandwidth to a user additionally is subject to the actual availability of bandwidth following previous allocations thereof to users with equal or higher priority.","Following allocations to all users, any bandwidth determined (Step 1710) to be remaining out of the total class allowance is then allocated equally (Step 1712) to the users subject to the respective maximum bandwidth value for each user.","The new user allocations are then incorporated (Step 1714) into the DOC Network as the bandwidth allowances of the users.","The method 1800 illustrated in FIG.","18 is the same as that of FIG.","17, except that surplus bandwidth in the class, if any, is allocated (Step 1802) proportional to the forecasted bandwidths of the users in the class, again subject to each user's respective maximum bandwidth value.","The preferred method 1900 illustrated in FIG.","19 does not prioritize the users for purposes of allocation but, instead, treats all users equally.","The method 1900 begins with the retrieval (Step 1902) of the forecasted bandwidth for each user in the class from the Database Manager 90.The surplus is then set to the total allocated bandwidth of the class in the particular direction of communication, and the sum of all forecasts of the users in the class is calculated (Step 1904).","The total allocated bandwidth of the class then is allocated (Step 1906) to all users in the class proportional to the user's forecasted bandwidth, again subject to each user's respective maximum bandwidth value.","The user allocations then are incorporated into the DOC Network (Step 1908) as the bandwidth allowances of the users.","The preferred method 2000 illustrated in FIG.","20 seeks to maximize revenues from fees (F) that are paid for bandwidth consumption by the users.","The method 2000 begins with the retrieval (Step 2002) of the forecast for each user as well as a fee that is paid for bandwidth by each user.","The users are then sorted (Step 2004) based on user fees in decreasing order, with the user paying the most for bandwidth receiving the highest priority.","Next, the surplus is set (Step 2006) to the total allocated bandwidth of the class in the particular direction of communication.","Bandwidth then is allocated (Step 2008) to the users in the class as available from highest to lowest priority in an amount equal to each user's forecasted bandwidth, and subject to the user's respective maximum bandwidth value.","Both preferred method 2100 of FIGS.","21a and 21b, and preferred method 2200 of FIGS.","22a and 22b differ from the other methods 1700,1800,1900,2000 in that these two methods allocate bandwidth to the users in multiple allocation rounds.","Method 2100 begins in FIG.","21a with the retrieval (Step 2102) of the forecasted bandwidths of the users as well as a credit (C) that applies if a respective user does not receive up to a guaranteed maximum level of bandwidth.","The users are then prioritized (Step 2104) based on each user's respective credit in decreasing order, with those users having higher credits being given priority over users with lesser credits.","Next, the surplus is set (Step 2106) to the total allocated bandwidth of the class in the particular direction of communication.","Bandwidth then is allocated (Step 2108) as available in a first round to the users from highest to lowest priority.","The allocation in the first round for each user is equal to the minimum of the forecasted bandwidth or the maximum bandwidth that is guaranteed, subject to the user's respective maximum bandwidth value.","If any additional bandwidth is determined (Step 2110) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 2114).","Bandwidth then is allocated (Step 2116) as available to each user in the same user order.","Assuming sufficient bandwidth remains available, the allocation in the second round brings the user's allocation up to the user's forecasted bandwidth subject to the user's respective maximum bandwidth value.","Following the second allocation round, a determination is made (Step 2118) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 2122) equally to the users, subject to each user's respective maximum bandwidth value.","The user allocations are then incorporated (Step 2124) into the DOC Network as the users' bandwidth allowances.","If it is determined that no bandwidth remains available in either of Step 2110 or Step 2118, then the user allocations are completed and are incorporated into DOC Network in Steps 2112,2124, respectively, as the users' bandwidth allowances.","Method 2200 of FIGS.","22a and 22b differs from that of FIGS.","21a and 21b only in that the sum of the forecasted bandwidths for all users is calculated (Step 2202) and a determination is made (Step 2204) whether the sum exceeds the total allocated bandwidth of the class.","If the sum exceeds the total allocated bandwidth of the class, then the bandwidth is allocated (Step 2206) to each user in an amount equal to the forecasted bandwidth, subject to the user's maximum guaranteed bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.","Thus, for example, if the sum of all forecasted bandwidths exceeds the total allocated bandwidth of the class in an amount equal to 20% of the sum of all the forecasted bandwidths, then each user is allocated bandwidth in an amount equal to the user's forecasted bandwidth (subject to the user's maximum guaranteed bandwidth), then less 20% thereof.","The applicable class bandwidth allowances used in the aforementioned methods are obtained from the Database Manager 90.The information, including fees, credits, guaranteed user bandwidths, and maximum bandwidth values in the aforementioned methods, is obtained from each user's SLA or from any applicable CSLA, and/or is predetermined by the Administrator 106, Carrier, or other entity.","Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a user SLA table as well as a user billing table, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred not only for the CSLAs, but also for the SLAs and user billing tables, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of user records in a timely fashion.","Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once user allocations have been determined under the aforementioned allocation policies, the respective DOC Network is updated with the resulting user allocations as the bandwidth allowances for the users for a particular time interval.","Each user is then allocated bandwidth during the particular time interval in an amount that is less than, or equal to, that user's bandwidth allowance.","Similarly, the collective bandwidth consumptions of a class by users therein is limited by that class' bandwidth allowance.","Preferably, the DOC Network is updated at periodic intervals of between one to fifteen minutes and, preferably every five minutes.","Furthermore, the periodic interval preferably corresponds to the scheduling of the Bandwidth Allocator 92 with regard to upstream transmissions.","With particular reference to FIG.","23, a preferred method 2300 of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network with the user allowances is illustrated.","The DOC Network is updated by incorporating (Step 2302) the user allocations as bandwidth allowances (i.e., bandwidth limits) into CM configuration files (MD-5 files) for the CMs of the respective users.","As set forth above, each CM configuration file contains instructions for a respective CM that limits the actual bandwidth consumed by the CM in the upstream direction and in the downstream direction.","The CM configuration files are then sent (Step 2304) by the Bandwidth Allocator 92 to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network, which maintains CM configuration files for the CMs of the Cable Network.","A command is also sent (Step 2306) to either of the CMs or the CMTS of the respective Cable Network causing the CMs to acquire and implement the CM configuration files maintained on the TFTP Server.","In addition to maintaining information regarding CSLAs, class allocations, SLAs, and user billing data in the Database Manager 90, the GUI & Report Generating Engine 94 further enables the Administrator 106 to analyze the user stats updated by the Data Collector 88, including the generation of reports and graphs regarding, for example, network access usage of the users over time as well as user throughput rates vs. data loss rates similar to that shown in FIG.","10.It additionally should be noted that a user may or may not be permitted to be grouped in one or more classes in accordance with the present invention.","If it is desired that classes be mutually exclusive, then some policy should be established for determining which class with which a user is associated as between competing classes.","If it is desired that classes not be mutually exclusive, then users falling within two or more classes will be allocated bandwidth within each class to the extent that no conflict arises as between the classes, and subject to any maximum allowed aggregated user bandwidth for all classes that may be established.","As now will readily be seen, the preferred methods and preferred networks of the present invention described in detail herein enable a Carrier to accommodate bandwidth concerns of service providers competing for the business of users of a shared communications medium in a Shared Access Carrier Network.","In particular, CSLAs now can be constructed in accordance with the present invention whereby a service provider is guaranteed some collective level of network access for the users of the shared communications medium that are customers of the service provider.","Furthermore, the provision of bandwidth to users who are customers of competing service providers can now be based on fairness considerations, even if one of the service providers is related to the Carrier.","In addition thereto, the differing demands for instantaneous throughput by users competing for access across the shared communications medium now can be accommodated in accordance with the present invention.","Indeed, a Carrier now is able to continuously vary bandwidth consumption limits for each user on an individual basis and for small time intervals, either in accordance with fairness considerations, forecasted network access usage of the users, or under contractual provisions governing network access.","It also will now be evident that the present invention gives rise to new business models that may be implemented by service providers for providing network access to users thereof and, in particular, to new ways of selling network access, which is also considered part of the present invention.","For example, in accordance with the present invention, network access now can be “wholesaled” to service providers by considering the users of the service provider a class and allocating bulk network access to such class pursuant to a CSLA between the Carrier and the service provider.","Through a CSLA, a Carrier can offer to the service provider a guaranteed minimum level of network access for the class that is constant throughout the day or week, or a guaranteed minimum level of network access that varies depending upon considerations such as the time of day or the day of week.","A Carrier also now can offer a guaranteed minimum level of network access to the class with a guaranteed maximum level of network access provided as needed in accordance with a target probability.","The service providers, in turn, then can offer different SLAs to the users that are its customers, essentially selling network access at the retail level.","Accordingly, service providers can be assured of levels of network access for the users that are their customers, and users can be assured of appropriate levels of network access to meet their individual demands.","Moreover, Carriers and/or service providers now can differentiate between users in charging for network access, thereby allowing Carriers and/or service providers to differentiate revenue streams for maximization of revenues.","The present invention also enables Carriers and/or service providers to offer “dynamic SLAs” to users.","The term “dynamic SLA” refers to a SLA that can be modified by a user as the user's demand for network access significantly changes, whether such modification is permanent or temporary.","In this regard, and in accordance with a preferred method 2600 of the present invention as illustrated in FIG.","26, the “network access retailer” (the entity selling the network access to the user) monitors (Step 2602) network access usage by users of a Shared Access Carrier Network and determines (Step 2604), for each user based on network access usage, whether a SLA provision other than those found in the user's current SLA would better meet the user's needs.","This determination is made by comparing the user's throughput, bandwidth consumption, and/or bandwidth requested for a predetermined period of time against a set of threshold values, including any guaranteed level of network access provided for in the user's SLA as well as any minimum QoS standard that are deemed necessary for user satisfaction by the network access retailer or other appropriate entity.","Thus, if the user's level of throughput, bandwidth consumption, and/or bandwidth requested for the predetermined time interval differs by a predetermined tolerance from a minimum threshold value, then the user is identified (Step 2606) as a “candidate” for modifying the SLA.","A similar process alternatively is used, wherein the user's forecasted bandwidth is compared to the threshold values and, if the difference exceeds a predetermined tolerance, then the user is deemed a candidate for modifying the user's SLA.","Once users have been identified as candidates, the candidates are filtered by screening (Step 2608) the candidates against a list of users for which solicitations are not to be made.","Those candidates passing the screening are then invited (Step 2610) to modify their respective SLAs.","The solicitation of the user preferably is performed via email, instant messaging, redirection of the user's web browser to a solicitation web page, generation and mailing of solicitation literature via U.S. mail, telemarketing, or other means of communication.","The solicitation includes an invitation for the user to modify the user's SLA by increasing for a fee the minimum level of network access guaranteed to the user.","The solicitation preferably also includes an invitation to make the modification permanent, or to make the modification only temporary and for a specific period of time.","Thus, for example, if a user is identified as having a high usage pattern at recurrent periods of time (such as every Saturday night when a particular webcast is viewed, or when an Internet game is played), then the user automatically is solicited with an invitation via instant messaging on the following Saturday night to increase the user's guaranteed network access for that night, for a predetermined number of following Saturday nights, and/or for every Saturday night.","Acceptance of the invitation by each user results in the modification (Step 2612) of the user's SLA for the appropriate period of time by increasing the level of network access the user is guaranteed (and/or the user's respective maximum bandwidth value, depending upon the policies used).","The solicited modification to the user's SLA is updated in the SLA database, which is then used during user prioritization and allocation of bandwidth by the Bandwidth Allocator 92.The resulting higher bandwidth allowance should enhance the user's experience and overall satisfaction with the Carrier Network.","In particular, the higher bandwidth (greater network access) should enhance the viewing of the webcast or the playing of the Internet game.","On the other hand, SLAs for which users decline solicitations are not modified.","Furthermore, if deemed appropriate, users declining a solicitation are recorded in the list against which candidates are screened.","Preferably, the Bandwidth Allocator 92 analyzes the user stats maintained by the Database Manager 90, identifies those users that are candidates for SLA modification, and initiates the solicitation of such candidates.","Information for each user's SLA for comparison with the user's stats automatically is obtained either from the Database Manager 90, or from an external database maintained by the network access retailer or other appropriate entity.","Furthermore, the Bandwidth Allocator 92 preferably performs this analysis for solicitation on a regularly scheduled basis.","In addition to such solicitations, a user of course may request a change in the level of network access guaranteed without having to receive first a solicitation.","Furthermore, the user may request that the change be for a temporary period of time such that, for example, the change is reversed after only a few hours, which would cover a viewing of a particular webcast or the playing of a particular Internet game beginning at the time of the request.","In view of the foregoing detailed description of the preferred embodiments and methods of the present invention, it readily will be understood by those persons skilled in the art that the present invention is susceptible of broad utility and application.","Many embodiments and adaptations of the present invention other than those herein described, as well as many variations, modifications, and equivalent arrangements, will be apparent from or reasonably suggested by the present invention and the foregoing description thereof, without departing from the substance or scope of the present invention.","Accordingly, while the present invention has been described herein in detail in relation to preferred embodiments, it is to be understood that this disclosure only is illustrative and exemplary of the present invention and is made merely for purposes of providing a full and enabling disclosure of the invention.","The foregoing disclosure is not intended nor is to be construed to limit the present invention or otherwise to exclude any such other embodiments, adaptations, variations, modifications and equivalent arrangements, the present invention being limited only by the claims appended hereto and the equivalents thereof.","Thus, for example, it will be apparent that, while preferred embodiments of the present invention have been described in the context of DOC Networks (including either a network of all coaxial cable, or a HFC network), the present invention nevertheless relates to any other network (whether wireline or wireless) wherein competing users share access across a shared communications medium including, for example, home networks and small networks in mass transit vehicles."]],"string":"[\n [\n \"Allocating access across shared communication medium\",\n \"A method of providing network access across a shared communications medium between competing users (86) includes the step of allocating network access for each user for a future time interval (102).\",\n \"Features include forecasting network access (1100) of the users in a future time interval, and prioritizing the users for allocating network access to the users.\",\n \"The network access allocations represent network access allowances available to the users during the future time interval, and further may represent network access usage.\",\n \"Classes of users can be allocated network access first (1114), and then each user allocated network access from the class allocation.\"\n ],\n [\n \"1.A method of providing network access across a shared communications medium between at least two competing users, comprising the steps of: (a) prior to a first time interval, determining for each user a respective first network access allowance representing a respective first maximum level of network access available to the user during the first time interval, but not necessarily representing the level of network access that will be utilized by the user during the first time interval; (b) during the first time interval, providing network access to each user such that the respective first network access allowance for each user is not exceeded; (c) prior to a second consecutive time interval, determining for each user a respective second network access allowance representing a respective second maximum level of network access available to the user during the second time interval, but not necessarily representing the level of network access that will be utilized by the user during the second time interval, at least one respective second network access allowance for a user differing from such user's respective first network access allowance; and (d) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.\",\n \"2.The method of claim 1, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"3.The method of claim 1, wherein the first and second time intervals are consecutive time intervals.\",\n \"4.The method of claim 1, wherein each of the first time interval and the second time interval has a period of between one minute and sixty minutes.\",\n \"5.The method of claim 1, wherein the second network access allowance for each respective user differs from the first network access allowance for such user.\",\n \"6.The method of claim 1, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.\",\n \"7.The method of claim 6, wherein the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.\",\n \"8.The method of claim 7, wherein the first allocation policy differs from the second allocation policy.\",\n \"9.The method of claim 7, further comprising requesting a minimum level of network access for the first time interval for a user, and wherein said allocating network access to such user for the first time interval comprises setting the level of network access allocated to such user to a level equal to or greater than the requested minimum level.\",\n \"10.The method of claim 9, wherein said allocating network access to such user for the second time interval comprises setting the level of network access allocated to such user to a level less than the requested minimum level for the first time interval.\",\n \"11.The method of claim 1, further comprising the step of monitoring network access usage by each user.\",\n \"12.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted from and to each user during a past time interval.\",\n \"13.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a past time interval.\",\n \"14.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.\",\n \"15.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a past time interval.\",\n \"16.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a time interval.\",\n \"17.The method of claim 1, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"18.The method of claim 1, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"19.The method of claim 1, further comprising prioritizing the users for determining network access allowances.\",\n \"20.The method of claim 19, wherein said prioritizing is based on fairness considerations.\",\n \"21.The method of claim 20, wherein the users are prioritized based on user throughput during a past time interval, with a user with lesser throughput receiving priority over a user with greater throughput.\",\n \"22.The method of claim 20, wherein the users are prioritized based on data loss for each user during a past time interval, with a user with greater data loss having priority over a user with lesser data loss.\",\n \"23.The method of claim 20, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"24.The method of claim 20, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.\",\n \"25.The method of claim 20, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"26.The method of claim 19, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.\",\n \"27.The method of claim 26, further comprising the step of monitoring network access usage by each user.\",\n \"28.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"29.The method of claim 27, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"30.The method of claim 27, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.\",\n \"31.The method of claim 27, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"32.The method of claim 27, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"33.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises setting the respective levels of network access equal to each user's specified respective minimum level of network access.\",\n \"34.The method of claim 11, further comprising the step of forecasting network access usage by each user during a future time interval based on said step of monitoring network access usage by each user.\",\n \"35.The method of claim 34, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"36.The method of claim 34, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.\",\n \"37.The method of claim 34, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.\",\n \"38.The method of claim 37, wherein said step of allocating network access comprises setting the respective levels of network access of the users proportional to each user's forecasted network access usage.\",\n \"39.The method of claim 37, further comprising the step of prioritizing the users for allocating network access.\",\n \"40.The method of claim 39, wherein said prioritizing is based on each user's forecasted network access usage.\",\n \"41.The method of claim 40, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"42.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"43.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"44.A method of providing during two consecutive time intervals network access across a shared communications medium between two competing users, comprising the step of varying between the two time intervals a respective network access allowance for each user, each respective network access allowance representing a maximum level of network access available to the respective user during a particular time interval, but not necessarily representing the level of network access that is utilized by the user during the particular time interval.\",\n \"45.A method of providing network access across a shared communications medium in a downstream direction towards competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; and (b) based on said monitored network access usage, allocating network access to each user on a per user basis for a future time interval.\",\n \"46.The method of claim 45, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"47.The method of claim 45, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.\",\n \"48.The method of claim 47, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.\",\n \"49.The method of claim 45, wherein said step of monitoring comprises monitoring bandwidth that is consumed by each user in a downstream direction at time intervals of fifteen minutes to sixty minutes.\",\n \"50.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"51.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"52.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted to each user during a time interval.\",\n \"53.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted to each user during a time interval.\",\n \"54.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"55.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"56.The method of claim 45, wherein said step of allocating network access comprises allocating network access equally to the users.\",\n \"57.The method of claim 45, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"58.The method of claim 57, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"59.The method of claim 57, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"60.The method of claim 45, further comprising prioritizing the users for allocating network access.\",\n \"61.The method of claim 60, wherein said prioritizing is based on fairness considerations.\",\n \"62.The method of claim 61, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput receiving priority over a user with greater throughput.\",\n \"63.The method of claim 61, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss having priority over a user with lesser data loss.\",\n \"64.The method of claim 61, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"65.The method of claim 61, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.\",\n \"66.The method of claim 61, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"67.The method of claim 60, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.\",\n \"68.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"69.The method of claim 67, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"70.The method of claim 67, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.\",\n \"71.The method of claim 67, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"72.The method of claim 67, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"73.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.\",\n \"74.The method of claim 45, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.\",\n \"75.The method of claim 74, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"76.The method of claim 74, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.\",\n \"77.The method of claim 74, wherein said step of allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.\",\n \"78.The method of claim 74, further comprising the step of prioritizing the users for allocating network access.\",\n \"79.The method of claim 78, wherein said prioritizing is based on each user's forecasted network access usage.\",\n \"80.The method of claim 78, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"81.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"82.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"83.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) forecasting network access usage by each user during a future time interval based on said monitored network access usage by each user; and (c) based on said forecasted network access usage, allocating network access to each user for the future time interval.\",\n \"84.The method of claim 83, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"85.The method of claim 83, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.\",\n \"86.The method of claim 83, wherein said step of allocating network access comprises allocating network access to the users proportionally based on each user's forecasted network access usage.\",\n \"87.The method of claim 83, further comprising the step of prioritizing the users for allocating network access.\",\n \"88.The method of claim 87, wherein said prioritizing is based on each user's forecasted network access usage.\",\n \"89.The method of claim 87, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"90.The method of claim 87, wherein said prioritizing is based on fairness considerations.\",\n \"91.The method of claim 87, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.\",\n \"92.The method of claim 87, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.\",\n \"93.The method of claim 87, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"94.The method of claim 87, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.\",\n \"95.The method of claim 87, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"96.The method of claim 87, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.\",\n \"97.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"98.The method of claim 96, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"99.The method of claim 96, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usages both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access would have been utilized for such users.\",\n \"100.The method of claim 96, wherein SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"101.The method of claim 96, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"102.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.\",\n \"103.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"104.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"105.The method of claim 83, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.\",\n \"106.The method of claim 83, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.\",\n \"107.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) of each user that will be transmitted over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.\",\n \"108.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage requested by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) that will be requested by each user over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.\",\n \"109.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"110.The method of claim 109, wherein said step of allocating further comprises allocating any surplus network access remaining equally to the users.\",\n \"111.The method of claim 109, wherein said step of allocating further comprises allocating in decreasing user priority any surplus network access to the users proportionally to the user's forecasted network access usage.\",\n \"112.The method of claim 109, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"113.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.\",\n \"114.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.\",\n \"115.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"116.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"117.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"118.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"119.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"120.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"121.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"122.The method of claim 109, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"123.The method of claim 122, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"124.The method of claim 122, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"125.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total available network access to the total forecasted network access usage of all the users, and subject to a respective, predetermined maximum value and subject to availability.\",\n \"126.The method of claim 125, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"127.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.\",\n \"128.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.\",\n \"129.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"130.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"131.The method of claim 125, wherein said step of monitoring network access includes collecting data representative of the logical data units transmitted from and to each user during a time interval.\",\n \"132.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"133.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"134.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"135.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"136.The method of claim 125, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"137.The method of claim 126, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"138.The method of claim 126, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"139.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"140.The method of claim 139, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"141.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.\",\n \"142.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.\",\n \"143.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"144.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"145.The method of claim 139, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"146.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"147.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"148.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"149.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"150.The method of claim 139, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"151.The method of claim 150, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"152.The method of claim 150, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"153.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum specified value and subject to availability.\",\n \"154.The method of claim 153, wherein any additional network access remaining after said allocation is earmarked for the users such that each user's allocation equals the user's forecasted network access usage, subject to a respective, predetermined maximum allowed level of network access and subject to availability.\",\n \"155.The method of claim 154, wherein any surplus network access remaining after said distribution is further earmarked in equal amounts for the users, subject to a respective, predetermined maximum allowed level of network access.\",\n \"156.The method of claim 154, further comprising allocating to the users network access only in an amount equal to the forecasted network access usage multiplied by the ratio of total network access available for allocation to the total forecasted network access usage of the users, but only when the total forecasted network access usage of the users exceeds the total network access available for allocation.\",\n \"157.The method of claim 153, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"158.The method of claim 153, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.\",\n \"159.The method of claim 153, wherein said step of monitoring comprises monitoring network access by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.\",\n \"160.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"161.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"162.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"163.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"164.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are dropped during a time interval.\",\n \"165.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of each user that are dropped during a time interval.\",\n \"166.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"167.The method of claim 153, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"168.The method of claim 167, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"169.The method of claim 167, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"170.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing said monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.\",\n \"171.The method of claim 170, wherein the threshold value represents a respective maximum level of network access for each user.\",\n \"172.The method of claim 170, wherein the threshold value represents a respective maximum burstable level of network access with target probability for each user.\",\n \"173.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via email.\",\n \"174.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via instant messaging.\",\n \"175.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.\",\n \"176.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.\",\n \"177.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.\",\n \"178.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent basis.\",\n \"179.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user with a maximum burstable level of network access with target probability.\",\n \"180.The method of claim 170, further comprising charging the user a fee for the modification of the SLA.\",\n \"181.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a temporary basis.\",\n \"182.The method of claim 170, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"183.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in an upstream direction of communication across the shared communications medium at time intervals of one minute to fifteen minutes.\",\n \"184.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in a downstream direction of communication across the shared communications medium at time intervals of fifteen minutes to sixty minutes.\",\n \"185.The method of claim 170, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"186.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"187.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"188.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"189.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"190.The method of claim 170, wherein the shared communications medium is part of a Shared Access Carrier Network.\",\n \"191.The method of claim 190, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"192.The method of claim 190, wherein the Shared Access Carrier Network comprises a wireless network.\",\n \"193.The method of claim 170, further comprising, based on said monitored network access usage, allocating network access to each user for a future time interval.\",\n \"194.The method of claim 193, wherein said step of allocating network access comprises allocating network access equally to the users.\",\n \"195.The method of claim 193, further comprising prioritizing the users for allocating network access.\",\n \"196.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for the users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"197.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"198.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.\",\n \"199.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"200.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"201.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.\",\n \"202.The method of claim 195, wherein said prioritizing is based on fairness considerations.\",\n \"203.The method of claim 202, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.\",\n \"204.The method of claim 202, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.\",\n \"205.The method of claim 202, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"206.The method of claim 202, wherein the users are prioritized based on both user throughput and data loss during a time interval.\",\n \"207.The method of claim 202, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"208.The method of claim 193, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.\",\n \"209.The method of claim 208, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"210.The method of claim 208, wherein said step of forecasting comprises applying an adaptive-response-rate signal exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.\",\n \"211.The method of claim 208, wherein said step of allocating comprises allocating network access to the users proportionally based on each user's forecasted network access usage.\",\n \"212.The method of claim 208, further comprising the step of prioritizing the users for allocating network access.\",\n \"213.The method of claim 212, wherein said prioritizing is based on each user's forecasted network access usage.\",\n \"214.The method of claim 212, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"215.The method of claim 212, wherein said step of allocating comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"216.The method of claim 212, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"217.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user for respective predetermined past time intervals; (b) identifying a recurrent period of high network access usage of a user based on said monitoring; and (c) soliciting a user to modify the user's SLA to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.\",\n \"218.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via email.\",\n \"219.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via instant messaging.\",\n \"220.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.\",\n \"221.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.\",\n \"222.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.\",\n \"223.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for all future recurrent periods of high network access usage.\",\n \"224.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for a predetermined number of future recurrent periods of high network access usage.\",\n \"225.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user with a maximum burstable level of network access with target probability for the future period of high network access usage.\",\n \"226.The method of claim 217, wherein the recurrent time period of high network access usage comprises a particular time of day.\",\n \"227.The method of claim 217, further comprising charging the user a fee for modification of the user's SLA.\",\n \"228.A method of providing network access across a DOCSIS 1.0 compliant cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user a respective first bandwidth allowance for a first future time interval; (b) generating a first set of cable modem configuration files, each of which limits bandwidth consumption by a cable modem (CM) of a respective user to that user's first bandwidth allowance; (c) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; (d) sending a command either to each user's CM or to a cable modem termination system (CMTS) to which each user's CM is connected to cause each CM to acquire its new respective configuration file of the first set for the first time interval; (e) determining for each user a respective second bandwidth allowance for a second future time interval, a second bandwidth allowance of at least one of the users differing from that user's first bandwidth allowance; and (f) for each user having a second bandwidth allowance different from that user's first bandwidth allowance, (i) generating a second cable modem configuration file which limits bandwidth consumption of that user's CM to a value representative of that user's second bandwidth allowance; (ii) sending the second configuration file for that user to the TFTP Server of the DOC Network; and (iii) sending a command either to that user's CM or to the CMTS to which that user's CM is connected to cause the CM to acquire the second configuration file for that user for the second time interval.\",\n \"229.The method of claim 228, further comprising the steps of monitoring bandwidth of each user for a time interval and, based on said monitored bandwidth, determining each user's second bandwidth allowance.\",\n \"230.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes prioritizing each user and allocating bandwidth to each user according to such priority.\",\n \"231.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth with respective values specified by service level agreements (SLAs) of the users.\",\n \"232.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth of each user with an established minimum quality of service value.\",\n \"233.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes forecasting each user's bandwidth for the second future time interval.\",\n \"234.The method of claim 233, wherein said step of determining each user's bandwidth allowance includes prioritizing the users for allocation of bandwidth based on said forecasting.\",\n \"235.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the upstream direction during time intervals of one minute to fifteen minutes.\",\n \"236.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is requested by each CM in the upstream direction during time intervals of one minute to fifteen minutes.\",\n \"237.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the downstream direction during time intervals of fifteen minutes to sixty minutes.\",\n \"238.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"239.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"240.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"241.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"242.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.\",\n \"243.The method of claim 228, wherein the first time interval and the second time interval are consecutive time intervals.\",\n \"244.The method of claim 228, wherein each of the first time interval and the second time interval has a period of one to sixty minutes.\",\n \"245.The method of claim 228, wherein for each user, the first bandwidth allowance of that user differs from the second bandwidth allowance of that user.\",\n \"246.The method of claim 228, wherein the first bandwidth allowance for each user differs from the first bandwidth allowance of all other users.\",\n \"247.The method of claim 228, wherein the second bandwidth allowance for each user differs from the second bandwidth allowance of all other users.\",\n \"248.The method of claim 228, wherein the first bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.\",\n \"249.The method of claim 248, wherein the second bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.\",\n \"250.The method of claim 249, wherein the first and second allocation policies differ.\",\n \"251.The method of claim 249, wherein the first and second allocation policies are the same.\",\n \"252.A method of continuously providing network access across a cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user during present time intervals bandwidth allowances of such user for respective future time intervals, each bandwidth allowance of such user for a future time interval representing a maximum level of bandwidth consumption for such user during the future time interval, but not necessarily representing the amount of bandwidth that will be consumed by such user during such future time interval, the bandwidth allowances of each user varying as between time intervals; and (b) for each user, limiting during each respective future time interval bandwidth consumption of that user's cable modem (CM) to that user's respective bandwidth allowance for such time interval by implementing a new cable modem configuration file in the CM.\",\n \"253.The method of claim 252, wherein the present and future time intervals have a uniform period of one minute to sixty minutes.\",\n \"254.The method of claim 252, wherein each user's bandwidth allowance for each future time interval is determined by allocating bandwidth to the users on a per user basis for such future time interval in accordance with a selected allocation.\",\n \"255.A Carrier Network for providing communication between multiple users and a Service Provider, comprising: (a) computer network equipment defining an Intermediate Network and a shared communications medium, said Intermediate Network and said shared communications medium extending between the users and the Service Provider for conveying data therebetween, said shared communications medium joining the users with said Intermediate Network such that data of each user is conveyed over said shared communications medium to and from the Intermediate Network whereby the users compete for network access; and (b) software for managing network access of the users across said shared communications medium, said software including computer-executable instructions that performs the steps of: (i) monitoring the network access usage by each user for a time interval; and (ii) based on the monitored network access usage, setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.\",\n \"256.The Carrier Network of claim 255, wherein said network equipment futher defines a Cable Network and said shared communications medium comprises a coaxial cable of said Cable Network.\",\n \"257.The Carrier Network of claim 256, wherein said software comprises software modules distributed within computer readable media of said network equipment.\",\n \"258.The Carrier Network of claim 256, wherein said software comprises software modules stored in a computer readable medium of a hardware component.\",\n \"259.The Carrier Network of claim 258, wherein said hardware component is physically located with said network equipment.\",\n \"260.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a regional data center of said DOC Network.\",\n \"261.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a headend of said DOC Network.\",\n \"262.The Carrier Network of claim 258, wherein said hardware component is physically located remotely from said network equipment.\",\n \"263.The Carrier Network of claim 262, wherein said hardware component is directly connected with said network equipment for communication therebetween.\",\n \"264.The Carrier Network of claim 262, wherein said hardware component is indirectly connected with said network equipment through the Internet for communication therebetween.\",\n \"265.The Carrier Network of claim 256, wherein said software modules include: (i) a first software component that manages a database for storage and retrieval of data; (ii) a second software component that collects data representative of network access usage from said network equipment, and that sends data representative of network access usage to said first component for storage thereof; and (iii) a third component that retrieves data representative of network access usage stored in said first component and sets, based at least partially thereon, network access allowances for the users for the future time interval.\",\n \"266.The Carrier Network of claim 265, further includes a fourth component having a graphical user interface for sending data to said first component for storage and for retrieval of data stored by said first component.\",\n \"267.The Carrier Network of claim 255, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"268.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in an upstream direction across said shared communications medium in time intervals of one minute to fifteen minutes.\",\n \"269.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in the downstream direction across said shared communications medium in time intervals of fifteen minutes to sixty minutes.\",\n \"270.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.\",\n \"271.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.\",\n \"272.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.\",\n \"273.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.\",\n \"274.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.\",\n \"275.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.\",\n \"276.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction across said shared communications medium during a time interval.\",\n \"277.The Carrier Network of claim 255, wherein said step of setting a network access allowance for each user comprises allocating network access equally to the users and setting the network access allowance for each user equal to that user's allocated network access.\",\n \"278.The Carrier Network of claim 277, wherein said software further includes computer-executable instructions that perform the additional step of prioritizing the users for allocating network access.\",\n \"279.The Carrier Network of claim 278, wherein the prioritizing is based on fairness considerations.\",\n \"280.The Carrier Network of claim 279, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.\",\n \"281.The Carrier Network of claim 279, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.\",\n \"282.The Carrier Network of claim 279, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"283.The Carrier Network of claim 279, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.\",\n \"284.The Carrier Network of claim 279, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"285.The Carrier Network of claim 278, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.\",\n \"286.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing each user's monitored network access usage with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"287.The Carrier Network of claim 285, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"288.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.\",\n \"289.The Carrier Network of claim 285, wherein SLAs provide respective fees for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"290.The Carrier Network of claim 285, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"291.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.\",\n \"292.The Carrier Network of claim 285, wherein said software further includes computer-executable instructions that perform the step of forecasting network access usage by each user during the future time interval based on the monitoring of network access usage by each user.\",\n \"293.The Carrier Network of claim 292, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"294.The Carrier Network of claim 292, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.\",\n \"295.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to users proportional to each user's forecasted network access usage.\",\n \"296.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions that perform the step of prioritizing the users for allocating network access.\",\n \"297.The Carrier Network of claim 296, wherein prioritizing is based on each user's forecasted network access usage.\",\n \"298.The Carrier Network of claim 296, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"299.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"300.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"301.A computer-readable medium having computer-executable instructions that manage network access across a shared communications medium between competing users of a Carrier Network, said instructions performing the steps of: (a) monitoring the network access usage by each user for a time interval; and (b) setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.\",\n \"302.The computer-readable medium of claim 301, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions performing the step of prioritizing the users for allocating network access.\",\n \"303.The computer-readable medium of claim 302, wherein the prioritizing is based on fairness considerations.\",\n \"304.The computer-readable medium of claim 302, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.\",\n \"305.The computer-readable medium of claim 302, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.\",\n \"306.The computer-readable medium of claim 302, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.\",\n \"307.The computer-readable medium of claim 302, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.\",\n \"308.The computer-readable medium of claim 302, wherein users are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"309.The computer-readable medium of claim 302, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.\",\n \"310.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when the respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.\",\n \"311.The computer-readable medium of claim 309, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.\",\n \"312.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.\",\n \"313.The computer-readable medium of claim 309, wherein SLAs provide a respective fee for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.\",\n \"314.The computer-readable medium of claim 309, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.\",\n \"315.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.\",\n \"316.The computer-readable medium of claim 309, further comprising computer-executable instructions performing the step of forecasting network access usage by each user during the future time interval based the monitoring of network access usage by each user.\",\n \"317.The computer-readable medium of claim 316, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.\",\n \"318.The computer-readable medium of claim 316, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.\",\n \"319.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.\",\n \"320.The computer-readable medium of claim 316, further comprising computer-executable instructions performing the step of prioritizing the users for allocating network access.\",\n \"321.The computer-readable medium of claim 320, wherein prioritizing is based on each user's forecasted network access usage.\",\n \"322.The computer-readable medium of claim 320, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.\",\n \"323.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.\",\n \"324.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.\",\n \"325.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users; and (d) allocating bandwidth to each user sequentially in decreasing order of user priority.\",\n \"326.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth requested for each user.\",\n \"327.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth consumption of each user.\",\n \"328.The computerized method of claim 327, wherein the bandwidth that is forecasted is the bandwidth consumption of each user.\",\n \"329.The computerized method of claim 328, wherein users are prioritized based on each user's forecasted future bandwidth consumption in increasing order, whereby a user with a lesser forecasted bandwidth consumption receives a higher priority than a user with a greater forecasted bandwidth consumption.\",\n \"330.The computerized method of claim 328, wherein each user's allocation of bandwidth for the future time interval is set to equal each user's forecasted bandwidth consumption subject to a respective, predetermined maximum value and subject to bandwidth availability 331.The computerized method of claim 330, further comprising distributing equally to the users any unallocated bandwidth.\",\n \"332.The computerized method of claim 330, further comprising distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidth consumptions.\",\n \"333.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise the number of logical data units transmitted from and to each user during the past time interval.\",\n \"334.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes transmitted from and to each user during the past time interval.\",\n \"335.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of data packets transmitted from and to each user during the past time interval.\",\n \"336.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.\",\n \"337.The computerized method of claim 336, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.\",\n \"338.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.\",\n \"339.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.\",\n \"340.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee charged to each user in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.\",\n \"341.The computerized method of claim 340, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.\",\n \"342.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.\",\n \"343.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.\",\n \"344.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective bandwidth levels specified to the users; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.\",\n \"345.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.\",\n \"346.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.\",\n \"347.The computerized method of claim 344, further comprising setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.\",\n \"348.The computerized method of claim 344, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.\",\n \"349.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.\",\n \"350.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.\",\n \"351.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.\",\n \"352.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing equally to the users any unallocated bandwidth.\",\n \"353.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidths.\",\n \"354.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.\",\n \"355.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth by each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.\",\n \"356.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective levels of network access specified to the users; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.\",\n \"357.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.\",\n \"358.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.\",\n \"359.The computer-readable medium of claim 356, wherein said instructions further perform the step of setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.\",\n \"360.A method of providing network access across a shared communications medium between at least four competing users, with at least a first pair of users being grouped within a first class and at least a second pair of different users being grouped within a second class, comprising the steps of: (a) determining class and user allowances of network access for a first time interval by allocating network access to each user class for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval, (b) providing network access to each user during the first time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' network allowance; (c) determining class and user allowances of network access for a second time interval by allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval, the allocated network access for the second time interval for at least one user differing from that user's allocated network access for the first time interval; and (d) providing network access to each user during the second time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' allowance.\",\n \"361.The method of claim 360, wherein for at least one class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.\",\n \"362.The method of claim 360, wherein for each class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.\",\n \"363.The method of claim 360, wherein the collective network access allocated to each class for the first time interval differs from the collective network access allocated to each class for the second time interval.\",\n \"364.The method of claim 360, further comprising requesting a minimum level of network access for a user for utilization during the first future time interval, and wherein said allocating network access to such user for the first future time interval comprises setting the level of network access allocated to such user to an amount equal to or greater than the requested minimum level.\",\n \"365.The method of claim 364, wherein said allocating network access to such user for the second future time interval comprises setting the level of network access allocated to such user to an amount less than the requested minimum level for the first time interval.\",\n \"366.The method of claim 360, wherein each of the first future time interval and the second future time interval has a period of between one minute and sixty minutes.\",\n \"367.The method of claim 360, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.\",\n \"368.The method of claim 360, further comprising the step of monitoring network access usage by each user.\",\n \"369.The method of claim 368, further comprising the step of tracking network access usage for each user class.\",\n \"370.The method of claim 368, wherein said step of monitoring network access usage by each user includes collecting data representative of logical data units transmitted from and to each user during a past time interval.\",\n \"371.The method of claim 368, further comprising tracking logical data units transmitted from and to each user class during a past time interval.\",\n \"372.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.\",\n \"373.The method of claim 368, further comprising tracking the number of logical data units that are dropped for each user class during a past time interval.\",\n \"374.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a past time interval.\",\n \"375.The method of claim 368, further comprising tracking the number of logical data units of each user class that are requested to be transmitted in an upstream direction during a past time interval.\",\n \"376.The method of claim 360, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.\",\n \"377.The method of claim 360, wherein the shared communications medium is part of a Shared Access Carrier Network comprises a wireless network.\",\n \"378.The method of claim 360, further comprising prioritizing the user classes for allocating network access.\",\n \"379.The method of claim 378, further comprising prioritizing the users for allocating, network access.\",\n \"380.The method of claim 378, wherein said prioritizing of the user classes is based on fairness considerations.\",\n \"381.The method of claim 380, wherein the user classes are prioritized based on collective user throughput during a past time interval, with a user class with lesser collective user throughput receiving priority over a user class with greater collective user throughput.\",\n \"382.The method of claim 380, wherein the user classes are prioritized based on collective data loss for each user class during a past time interval, with a user class with greater collective data loss having priority over a user class with lesser collective data loss.\",\n \"383.The method of claim 380, wherein the user classes are prioritized based on collective network access usage for a particular time of day, with a user class with lesser collective network access usage for the particular time of day receiving priority over a user class with greater collective network access usage for the particular time of day.\",\n \"384.The method of claim 380, wherein the user classes are prioritized based on both collective user throughput for the class and collective data loss of the users for the class during a time interval.\",\n \"385.The method of claim 380, wherein user classes are prioritized based on an established minimum quality of service (QoS) standard.\",\n \"386.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) for at least two user classes regarding the provision of network access to each respective class.\",\n \"387.The method of claim 386, further comprising the step of tracking network access usage by each user class.\",\n \"388.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for the user classes with the specified respective minimum levels of collective network access therefor, and awarding priority to a user class when said monitored network access usage for such user class falls below the specified respective minimum level of collective network access for such class.\",\n \"389.The method of claim 387, wherein each CSLA specifies a respective time-of-day (TOD) minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for such user classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a user class when said monitored network access usage during the specified respective TOD for such user class falls below the specified respective TOD minimum level of collective network access of such user class.\",\n \"390.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access up to a maximum burstable level with target probability for the respective users therein, and said step of prioritizing includes comparing for each user class said monitored network access usage both with the respective minimum level of collective network access and with the respective maximum burstable level of collective network access, and comparing the instances the respective maximum level of network access were obtained out of all instances the respective maximum level of network access could have been utilized.\",\n \"391.The method of claim 387, wherein each CSLA provides a respective fee for collective network access usage, and said step of prioritizing comprises sorting such user classes based on each class' respective fee in decreasing order, with a user class with a higher fee receiving priority over a user class with a lesser fee.\",\n \"392.The method of claim 387, wherein each CSLA provides a respective credit for a level of collective network access falling below a respective guaranteed level for the user class, and said step of prioritizing comprises sorting such user classes based on each class' respective credit in decreasing order, with a user class with a higher credit receiving priority over a user class with a lower credit.\",\n \"393.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the user class, and said step of allocating network access comprises setting the respective level of collective network access of the user class equal to the class' specified respective minimum level of collective network access.\",\n \"394.The method of claim 368, further comprising the step of forecasting collective network access usage by each user class during a future time interval based on said step of monitoring network access usage by each user.\",\n \"395.The method of claim 394, wherein said step of forecasting comprises summing within each class a forecasted network access usage of each user.\",\n \"396.The method of claim 394, wherein said step of forecasting network access usage of each user comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of each user.\",\n \"397.The method of claim 395, wherein said step of allocating network access to each user class comprises setting the respective levels of network access of the user classes proportional to each class' forecasted network access usage.\",\n \"398.The method of claim 395, further comprising the step of prioritizing the user classes for allocating network access.\",\n \"399.The method of claim 398, wherein said prioritizing is based on each class' forecasted network access usage.\",\n \"400.The method of claim 398, wherein said user classes are prioritized in increasing order of each class' forecasted network access usage, with a user class with a lesser forecasted network access usage receiving priority over a user class with a greater forecasted network access usage.\",\n \"401.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportional to the number of users within each class.\",\n \"402.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportionally based on each class' forecasted network access usage.\",\n \"403.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) regarding the provision of collective network access.\",\n \"404.The method of claim 403, further comprising the step of monitoring collective network access usage of each class.\",\n \"405.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes with the specified respective minimum levels of collective network access, and awarding priority to a class when said respective monitored network access usage for such class falls below the class' specified respective minimum level of collective network access.\",\n \"406.The method of claim 404, wherein CSLAs specify respective time-of-day (TOD) minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a class when said monitored network access usage during the specified respective TOD for such class falls below the class' specified respective TOD minimum level of collective network access.\",\n \"407.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access up to a maximum burstable levels with target probability for classes, and said step of prioritizing includes comparing said monitored network access usage for each such class both with the respective minimum levels of collective network access and with the respective maximum burstable levels of collective network access, and comparing the instances the respective maximum levels of collective network access were obtained for each such class out of all instances the respective maximum levels of collective network access could have been utilized.\",\n \"408.The method of claim 404, wherein CSLAs provide respective fees for collective network access usage of classes, and said step of prioritizing comprises sorting such classes based on each class' respective fee in decreasing order, with a class with a higher fee receiving priority over a class with a lower fee.\",\n \"409.The method of claim 404, wherein CSLAs provide respective credits for levels of collective network access below respective guaranteed levels for classes, and said step of prioritizing comprises sorting such classes based on each class' respective credit in decreasing order, with a class with a higher credit receiving priority over a class with a lower credit.\",\n \"410.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of allocating network access comprises setting the allocations of collective network access for such classes equal to each class' specified respective minimum level of collective network access\"\n ],\n [\n \" BACKGROUND OF THE PRESENT INVENTION As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.\",\n \"The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).\",\n \"The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.\",\n \"The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.\",\n \"For example, a Carrier or a related entity may serve as an Internet service provider (ISP).\",\n \"Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.\",\n \"One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.\",\n \"Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).\",\n \"For example, with regard to DOC Networks, and with reference to FIG.\",\n \"1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30 , which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.\",\n \"All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34 .\",\n \"Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.\",\n \"Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.\",\n \"The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.\",\n \"Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.\",\n \"This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.\",\n \"1 .\",\n \"The DOC Network 40 also includes multiple headends 36 , 64 , 66 .\",\n \"In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.\",\n \"Examples of a dedicated connection are shown for comparison in FIG.\",\n \"1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76 .\",\n \"Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.\",\n \"Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.\",\n \"A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.\",\n \"A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.\",\n \"Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.\",\n \"Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.\",\n \"It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.\",\n \"Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.\",\n \"For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.\",\n \"Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.\",\n \"Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.\",\n \"A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.\",\n \"Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.\",\n \"Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.\",\n \"Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.\",\n \"The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.\",\n \"In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.\",\n \"In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.\",\n \"Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.\",\n \"Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.\",\n \"Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.\",\n \"A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.\",\n \"A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.\",\n \"More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.\",\n \"At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.\",\n \"While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.\",\n \"Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.\",\n \"Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.\",\n \"Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.\",\n \"Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.\",\n \"Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium.\"\n ],\n [\n \" SUMMARY OF THE PRESENT INVENTION Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.\",\n \"First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.\",\n \"In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.\",\n \"Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.\",\n \"The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.\",\n \"The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.\",\n \"Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.\",\n \"Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.\",\n \"In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.\",\n \"Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.\",\n \"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\",\n \"With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.\",\n \"Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.\",\n \"In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.\",\n \"In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.\",\n \"Another additional feature of the present invention includes the prioritizing of the users for allocating network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.\",\n \"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\",\n \"Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.\",\n \"In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.\",\n \"Another feature of the present invention includes the prioritizing of the users for allocating network access.\",\n \"A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.\",\n \"A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.\",\n \"Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.\",\n \"The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.\",\n \"Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.\",\n \"Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.\",\n \"The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.\",\n \"The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.\",\n \"The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.\",\n \"The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.\",\n \"A charge preferably is applied to the user for the modification.\",\n \"A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.\",\n \"Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.\",\n \"A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.\",\n \"Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.\",\n \"Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.\",\n \"The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.\",\n \"The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.\",\n \"The Carrier Network also includes software that manages the network access of the users.\",\n \"In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.\",\n \"In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.\",\n \"The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.\",\n \"One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.\",\n \"Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.\",\n \"The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.\",\n \"A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.\",\n \"Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.\",\n \"In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.\",\n \"The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.\",\n \"Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.\",\n \"A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.\",\n \"Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.\",\n \"The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.\",\n \"Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.\",\n \"In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.\",\n \"The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.\",\n \"Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.\",\n \"The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.\",\n \"As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.\",\n \"As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.\",\n \"The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.\",\n \"The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.\",\n \"Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.\",\n \"In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.\",\n \"Accordingly, all users of a shared communications network can be classified.\",\n \"In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.\",\n \"Another feature includes the step of prioritizing the users for the allocation of network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.\",\n \"Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\"\n ],\n [\n \"FIELD OF THE PRESENT INVENTION The present invention generally relates to allocating access across a shared communications medium and, in particular, to allocating bandwidth used to convey data of competing users across a shared communications medium of a Carrier Network.\",\n \"BACKGROUND OF THE PRESENT INVENTION As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.\",\n \"The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).\",\n \"The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.\",\n \"The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.\",\n \"For example, a Carrier or a related entity may serve as an Internet service provider (ISP).\",\n \"Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.\",\n \"One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.\",\n \"Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).\",\n \"For example, with regard to DOC Networks, and with reference to FIG.\",\n \"1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30, which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.\",\n \"All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34.Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.\",\n \"Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.\",\n \"The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.\",\n \"Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.\",\n \"This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.\",\n \"1.The DOC Network 40 also includes multiple headends 36,64,66.In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.\",\n \"Examples of a dedicated connection are shown for comparison in FIG.\",\n \"1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76.Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.\",\n \"Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.\",\n \"A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.\",\n \"A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.\",\n \"Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.\",\n \"Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.\",\n \"It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.\",\n \"Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.\",\n \"For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.\",\n \"Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.\",\n \"Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.\",\n \"A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.\",\n \"Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.\",\n \"Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.\",\n \"Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.\",\n \"The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.\",\n \"In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.\",\n \"In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.\",\n \"Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.\",\n \"Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.\",\n \"Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.\",\n \"A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.\",\n \"A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.\",\n \"More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.\",\n \"At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.\",\n \"While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.\",\n \"Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.\",\n \"Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.\",\n \"Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.\",\n \"Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.\",\n \"Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium.\",\n \"SUMMARY OF THE PRESENT INVENTION Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.\",\n \"First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.\",\n \"In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.\",\n \"Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.\",\n \"The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.\",\n \"The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.\",\n \"Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.\",\n \"Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.\",\n \"In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.\",\n \"Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.\",\n \"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\",\n \"With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.\",\n \"Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.\",\n \"In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.\",\n \"In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.\",\n \"Another additional feature of the present invention includes the prioritizing of the users for allocating network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.\",\n \"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\",\n \"Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.\",\n \"In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.\",\n \"Another feature of the present invention includes the prioritizing of the users for allocating network access.\",\n \"A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.\",\n \"A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.\",\n \"Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.\",\n \"Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.\",\n \"The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.\",\n \"Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.\",\n \"Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.\",\n \"The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.\",\n \"The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.\",\n \"The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.\",\n \"The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.\",\n \"A charge preferably is applied to the user for the modification.\",\n \"A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.\",\n \"Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.\",\n \"A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.\",\n \"Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.\",\n \"Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.\",\n \"The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.\",\n \"The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.\",\n \"The Carrier Network also includes software that manages the network access of the users.\",\n \"In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.\",\n \"In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.\",\n \"The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.\",\n \"One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.\",\n \"Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.\",\n \"The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.\",\n \"A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.\",\n \"Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.\",\n \"In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.\",\n \"The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.\",\n \"Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.\",\n \"A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.\",\n \"Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.\",\n \"The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.\",\n \"Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.\",\n \"In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.\",\n \"The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.\",\n \"Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.\",\n \"The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.\",\n \"As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.\",\n \"As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.\",\n \"The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.\",\n \"The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.\",\n \"Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.\",\n \"In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.\",\n \"Accordingly, all users of a shared communications network can be classified.\",\n \"In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.\",\n \"Another feature includes the step of prioritizing the users for the allocation of network access.\",\n \"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.\",\n \"Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.\",\n \"Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.\",\n \"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS Further features and benefits of the present invention will be apparent from a detailed description of preferred embodiments thereof taken in conjunction with the following drawings, wherein like elements are referred to with like reference numbers, and wherein: FIG.\",\n \"1 illustrates a conventional DOC Network; FIG.\",\n \"2 illustrates a first DOC Network of the present invention; FIG.\",\n \"3 illustrates a second DOC Network of the present invention; FIG.\",\n \"4 illustrates a third DOC Network of the present invention; FIG.\",\n \"5 illustrates a fourth DOC Network of the present invention; FIG.\",\n \"6 illustrates a system architecture of software components that perform preferred methods of the present invention in the DOC Networks of FIGS.\",\n \"2-5; FIG.\",\n \"7 illustrates a flowchart of the steps of a preferred routine for forecasting bandwidth of each user for a future time interval; FIG.\",\n \"8 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the ARRSES Function of the preferred routine of FIG.\",\n \"7; FIG.\",\n \"9 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the HW Function of the preferred routine of FIG.\",\n \"7; FIG.\",\n \"10 illustrates a graph of user throughput rates versus user data loss rates for two users relative to a target minimum QoS standard; FIG.\",\n \"11 illustrates a flowchart of a first preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.\",\n \"12 illustrates a flowchart of a second preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.\",\n \"13 illustrates a flowchart of a third preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.\",\n \"14 illustrates a flowchart of a fourth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.\",\n \"15a and 15b illustrate a flowchart of a fifth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.\",\n \"16a and 16b illustrate a flowchart of a sixth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.\",\n \"17 illustrates a flowchart of a first preferred method of prioritizing users and allocating bandwidth to each user within a class; FIG.\",\n \"18 illustrates a flowchart of a second preferred method of prioritizing users and allocating bandwidth within a class; FIG.\",\n \"19 illustrates a flowchart of a third preferred method of prioritizing users and allocating bandwidth within a class; FIG.\",\n \"20 illustrates a flowchart of a fourth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.\",\n \"21a and 21b illustrate a flowchart of a fifth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.\",\n \"22a and 22b illustrate a flowchart of a sixth preferred method of prioritizing users and allocating bandwidth within a class; FIG.\",\n \"23 illustrates a flowchart of a preferred method of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network; FIG.\",\n \"24 illustrates the allocation of bandwidth to users within a class during a first time interval; FIG.\",\n \"25 illustrates the allocation of bandwidth to the users of FIG.\",\n \"24 during a second time interval; and FIG.\",\n \"26 illustrates a flowchart of a preferred method of soliciting a user to modify the user's SLA based on monitored network access usage of the user.\",\n \"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In the following detailed description, numerous specific details are set forth with regard to preferred embodiments of the present invention in order to provide a thorough understanding of the present invention; however, it will be apparent to ordinary artisans that the present invention may be practiced without all of these specific details.\",\n \"Well-known structures and devices also are shown in block diagram form, the specific details of which are not considered a necessary part of the present invention.\",\n \"Furthermore, as will become apparent to ordinary artisans, the present invention may be embodied in or performed by hardware, firmware, or software, or various combinations thereof.\",\n \"As described above, a conventional DOC Network 40 is shown in FIG.\",\n \"1 and includes a plurality of Cable Networks 38, with a particular Cable Network 38 being illustrated in an expanded view and comprising a group of CMs 34, each connected to a computer 44 representing a user.\",\n \"Additionally, as used herein, “user” includes not only a person who interacts with a computer 44, but any additional persons who also interact with the same computer 44, as well as any group of persons all of whom interact with computers attached either to the same CM 34 or to the same computer 44 which, itself, is attached to a CM 34.While not shown, such additional arrangements are well known in the art.\",\n \"The CMs 34 are connected by a coaxial cable 32 with a CMTS 30 and, specifically, to a card 31 mounted within the CMTS 30.Each of the CMTSs of the DOC Network 40 preferably includes a plurality of cards, with each card supporting a group of CMs connected thereto in an inverted tree configuration to define a Cable Network 38.Furthermore, each CMTS conventionally supports up to 1,500 users, although recent CMTSs have been introduced that support up to 15,000 users.\",\n \"Each Cable Network 38 defines a Shared Access Carrier Network, wherein data of respective users in each are conveyed together through a shared coaxial cable.\",\n \"For instance, data packets (or frames) addressed to at least one of the computers 44 are transmitted by the CMTS 30 downstream over the coaxial cable 32 to all of the CMs 34 within a 6 MHz data channel.\",\n \"Conversely, data packets intended for delivery to the CMTS 30 and beyond are transmitted by a CM 34 upstream to the CMTS 30 over the coaxial cable 32 within a 2 MHz channel.\",\n \"The Cable Network 38 shown in expanded view in FIG.\",\n \"1 is a traditional all coaxial cable network.\",\n \"The other Cable Networks 38 collectively include both traditional all coaxial cable networks as well as HFC networks.\",\n \"The CMTS 30 transmits and receives data packets between the Cable Networks 38 and an Intermediate Network 46, which begins with a router 48 in the headend 36, and includes switched and routed network equipment at a Regional Data Center 50 that provides connectivity to service providers 52,54,56,58, either directly or through the Internet 60.In this regard, during user communications the router 48 conveys data packets from the CMTS 30 to the Regional Data Center 50 of the DOC Network 40 and, conversely, routes data packets received from the Regional Data Center 50 to the appropriate CMTS for delivery to a particular user.\",\n \"Data packets that are conveyed to the Regional Data Center 50, in turn, are directed on to an appropriate service provider 52,54 directly connected to the Regional Data Center 50, or to an appropriate service provider 56,58 indirectly connected to the Regional Data Center 50 via the Internet 60.Alternatively, data packets from users are conveyed to a server of an application server group 62 of the Regional Data Center 50, which includes, for example, servers supporting Web hosting, news, chat, SMTP, POP3, Proxy, cache and content replication, and streaming media.\",\n \"The Cable Networks 38 stemming from headend 36 are maintained by a Carrier which also may maintain the Regional Data Center 50 as well as serve as a service provider.\",\n \"Moreover, the Carrier may maintain the Cable Networks of additional headends 64,66, or of only one or more of the headends 64,66.In any event, the Cable Networks that are maintained by the Carrier are administered on a daily basis through an element management system (EMS) 68.The EMS 68 comprises an operations system designed specifically to configure and manage CMTSs and associated CMs, and includes a CM database 70.Operational tasks performed by the EMS 68 include provisioning, day-to-day administration, and testing of various components of each CMTS.\",\n \"The EMS 68 typically is located at a central network operations center of the Carrier, but may be collocated at the headend 36 of the Carrier as shown in FIG.\",\n \"1.The DOC Network 40 is managed through a control plane server group 72 typically located at the Regional Data Center 50.The control plane server group 72 includes the usual servers necessary to run the DOC Network 40, such as user authorization and accounting servers, log control servers (Syslog), IP address assignment and administration servers (DHCP, TFTP), domain name servers (DNS), and DOCSIS control servers.\",\n \"For purposes of comparison, two dedicated connections also are shown in FIG.\",\n \"1, wherein a telephony modem 74 and an ISDN modem 76 are connected directly to the Intermediate Network 46 at the Regional Data Center 50.As will be immediately apparent, data conveyed over each dedicated connection is between a single user and the Intermediate Network 46, and is not combined with data of other users over a shared communications medium as in each Cable Network 38.As is common in conventional Cable Networks 38 such as those shown in the DOC Network 40 of FIG.\",\n \"1, when a CM comes online the CM is assigned a configuration file which, inter alia, sets a constant limit on the bandwidth that can be utilized in the downstream direction by the CM during any particular interval of time, and sets a constant limit on the bandwidth that can be utilized in the upstream direction by the CM during any particular interval of time.\",\n \"The configuration file also includes other parameters, such as the IP address for the CM.\",\n \"The configuration file for each CM conventionally is obtained by the CM when first brought online, or when the CM is reset.\",\n \"The upstream and downstream bandwidth limits are predetermined by the Carrier or other appropriate entity, the determination of which is based on the expected number of users to be serviced by the particular Cable Network 38 to which the CM belongs.\",\n \"With particular regard to data transmissions in the downstream direction, when the bandwidth limit is reached in receiving data within a particular time interval, the CM transmits a signal to the router 48 to cease further data forwarding for the remainder of the time interval.\",\n \"Thereafter, whereas any data received by a CMTS is relayed on to the CM as the data is received, any additional data received by the router 48 during the remainder of this time interval is stored for later transmission in a buffer up to a threshold limit and, thereafter, any further data received within the time interval is dropped.\",\n \"With regard to data transmissions in the upstream direction, when the CM registers with the CMTS following receipt by the CM of its configuration file, the CM informs the CMTS of the constant bandwidth limit to be applied to upstream transmissions from the CM.\",\n \"Then, actual requests for bandwidth (i.e., requests for timeslots) for transmission of data in the upstream direction are submitted regularly by each CM to the CMTS.\",\n \"In response to the submissions, the CMTS schedules timeslots in a particular time interval to the CMs for exclusive transmission of data within each timeslot by a respective CM.\",\n \"However, the CMTS does not grant an amount of bandwidth (by assigning too many timeslots) to a particular CM that would exceed the constant bandwidth limit for the particular CM.\",\n \"The timeslots are assigned to requesting CMs based on an established assignment policy.\",\n \"For example, timeslots may be assigned by the CMTS on a first-in-first-out basis, or timeslots may be assigned equally to the CMs that request bandwidth within a particular window of time.\",\n \"The requesting CMs also may be prioritized by the CMTS for assignment of the timeslots.\",\n \"Preferred embodiments 78,80,82,84 of a DOC Network in accordance with the present invention are shown, respectively, in FIGS.\",\n \"2-5, wherein each includes a “network access manager” 86 in accordance with the present invention.\",\n \"In FIG.\",\n \"2 the network access manager 86 is located in the headend 36 of the DOC Network 78, in FIG.\",\n \"3 the network access manager 86 is located at the Regional Data Center 50 of the DOC Network 80, and in FIGS.\",\n \"4-5 the network access manager 86 is remotely located, but is disposed for communication with the respective DOC Network 82,84, either directly as shown in the DOC Network 82 of FIG.\",\n \"4, or indirectly via the Internet 60 as shown in the DOC Network 84 of FIG.\",\n \"5.The network access manager 86 preferably comprises a hardware component having software modules for performing methods in accordance with the present invention.\",\n \"For commercial purposes, especially in enhancing existing DOC Networks, preferably the network access manager 86 is self-contained and need only be connected in communication with the DOC Network to operate correctly.\",\n \"In a DOC Network that is being upgraded or established, preferably the software modules are distributed within the DOC Network itself and may or may not include any additional hardware components such as the network access manager 86.For example, the software modules may be incorporated into the EMS, CMTS, and control plane server group of a DOC Network, thereby avoiding the expense of additional computer hardware components.\",\n \"In order to accommodate deployment and implementation of the present invention, the software modules preferably are designed as peers within a messaging infrastructure and, in particular, within a CORBA infrastructure 87, the system architecture of which is shown in FIG.\",\n \"6.Due to the interoperability of the peers to the CORBA infrastructure 87, the separate modules readily call upon each other as described in detail below without regard to differences in location between the modules.\",\n \"Nevertheless, for ease of deployment, the network access manager 86 is best suited for deployment and implementation of the present invention in established DOC Networks, whether situated within the Intermediate Network as in FIGS.\",\n \"2-3, or remotely situated as in FIGS.\",\n \"4-5.The software modules include a Data Collector 88, a Database Manager 90, Bandwidth Allocator 92, and GUI & Report Generating Engine 94.The Data Collector 88 and Bandwidth Allocator 92 each includes an external system interface layer 96,98, respectively, that enables it to communicate with network equipment of a DOC Network.\",\n \"In the system architecture of preferred embodiments, the Data Collector 88 communicates with each CMTS and CMs of each Cable Network for which network access is managed by the network access manager 86, and the Bandwidth Allocator 92 communicates with the control plane server group 72 of the DOC Network as well as with the CMTS and CMs.\",\n \"If a DOC Network is DOCSIS 1.0 compliant, then each external system interface layer 96,98 is a DOCSIS external system interface layer.\",\n \"If a DOC Network uses proprietary interface specifications, then each external system interface layer 96,98 is designed based on the proprietary interface specifications.\",\n \"In either case, however, the Data Collector 88 and Bandwidth Allocator 92 generally need not be modified; only the external systems interface layers 96,98 thereof need be changed based on the particularities of the DOC Network.\",\n \"Each of the Data Collector 88 and Bandwidth Allocator 92 also includes a scheduling element 100,102, respectively, that schedules the timing of actions and communications thereof with the network equipment of a DOC Network.\",\n \"The GUI & Report Generating Engine 94 communicates with an Administrator 106 of the network access manager 86, preferably through a web server, whereby the Administrator 106 sets up and configures the network access manager 86 and accesses reports generated by the network access manager 86, such as graphs of bandwidth consumption and bandwidth requested per time interval for a user.\",\n \"The Administrator 106 may be the Carrier, a service provider, or some other entity, such as the entity managing the Regional Data Center 50 or a third-party responsible for maintenance of the network access manager 86.The Database Manager 90 stores configuration and setup information received from the GUI & Report Generating Engine 94, as well as information processed by the Data Collector 88.The Database Manager 90 also provides information to the Bandwidth Allocator 92 and GUI & Report Generating Engine 94 as requested via the CORBA infrastructure 87.Having now described in detail the structure of preferred DOC Networks 78,80,82,84, preferred methods of the present invention will be described with reference thereto.\",\n \"In accordance with preferred methods of the present invention, network access usages of each user in the upstream and downstream directions are monitored through the Data Collector 88.Specifically, the Data Collector 88 issues queries to the CMTS and CM to which counter values of logical data units (LDUs) are returned for a user.\",\n \"Preferably, counter values are returned for the number of bytes and the number of data packets that are transmitted in both the upstream and downstream directions, the number of bytes and the number of data packets that are dropped in both the upstream and downstream directions, the number of bytes and the number of packets that are requested to be transmitted in the upstream direction, and the time for which the counter values are returned.\",\n \"Accordingly, as used herein the phrase “monitoring network access usage” is intended to refer to the collection of data representative of at least one of: (i) the number of LDUs that are transmitted in a particular direction across a shared communications medium; (ii) the number of LDUs that are dropped in transmitting in a particular direction across a shared communications medium; and (iii) the number of LDUs that are requested to be transmitted in a particular direction across a shared communications medium.\",\n \"In a DOCSIS compliant DOC Network, the information is collected from the CMTS and CMs of a Cable Network via the simple network management protocol (SNMP).\",\n \"The counter values for bytes and data packets that are transmitted and that are dropped in the upstream direction from each CM, and the number of bytes and data packets that are requested to be transmitted in the upstream direction from each CM, are recorded by the CMTS in accordance with a management information base (MIB) of a DOCSIS compliant CMTS.\",\n \"Likewise, the counter values for bytes and data packets that are transmitted and that are dropped in the downstream direction from the CMTS to a CM are recorded by the CM in accordance with a MIB of a DOCSIS compliant CM.\",\n \"Both bytes and data packets are monitored since each data packet may vary in the number of bytes it contains.\",\n \"The scheduling element 100 of the Data Collector 88 initiates the data collection from each CMTS and from the CMs connected thereto, preferably at different predetermined time intervals.\",\n \"For example, the data collection from a CMTS preferably occurs at five minute intervals and data collection from the CMs connected thereto preferably occurs at thirty minute intervals.\",\n \"The data collection from the CMs preferably is less often than the data collection from the CMTS in order to minimize consumption of bandwidth across the Cable Network that otherwise would be allocated to users.\",\n \"When the counter values and time thereof are returned to the Data Collector 88, the Data Collector 88 calculates the change over time for each counter value to arrive at the average rates of bytes and data packets that are successfully transmitted, the average rates of bytes and data packets that are requested to be transmitted, and the average rates of bytes and data packets that are dropped.\",\n \"The respective rates and time intervals for the rates (as well as the counter values and time stamp data) are then communicated to the Database Manager 90, which stores the information in a user statistics table (“stats”) for later use by the Bandwidth Allocator 92 and GUI & Report Generating Engine 94.The Bandwidth Allocator 92 continually determines the network access—or bandwidth in a Cable Network—that may be utilized by each user class, and by each user within each class, over succeeding time intervals.\",\n \"Each allowance is determined by first allocating bandwidth to the user classes, and then allocating bandwidth to the users in each class, in accordance with one or more selected allocation policies.\",\n \"Furthermore, as set forth above, each allowance is an amount of bandwidth up to which a user class or user may consume, but is not necessarily the amount of bandwidth that a user class or user will consume; it is an upper limit on such amount.\",\n \"For example, with reference to FIG.\",\n \"24, a selected allocation policy has resulted in the allocation of bandwidth to the users of the shared communications medium 2450 for a time interval extending from t0 to (t0+dt), User 2 and User K each is allocated a single bandwidth unit (b/w unit 3 and b/w unit X, respectively), while User 1 and User 3 each is allocated two bandwidth units (b/w unit 1 and b/w unit 2 to User 1, and b/w unit 4 and b/w unit 5 to User 3).\",\n \"As shown in FIG.\",\n \"25, in the next time interval extending from (t0+dt) to (t0+2dt), User 1, User 3, and User K each is allocated a single bandwidth unit (b/w unit 1, b/w unit 5, and b/w unit X, respectively), while User 2 is allocated three bandwidth units (b/w unit 2, b/w unit 3, and b/w unit 4).\",\n \"In this example, all users are grouped within the same class, and the bandwidth units in this example broadly represent network access to the communication member 2400 that is shared between the users across the shared communications medium 2450.In accordance with the present invention, respective user bandwidth allowances for each time interval are equated with these user allocations of bandwidth, whereby no user receives more bandwidth in a time interval than that user's respective bandwidth allowance for that time interval.\",\n \"Furthermore, it is important to distinguish what a user actually may be “allocated” in the context of the bandwidth that is actually utilized or consumed by such user, as opposed to bandwidth allocations to a user in accordance with the present invention.\",\n \"The bandwidth allocation in accordance with the present invention represents a limit on the amount of bandwidth that can be allocated to a user for a time interval—and hence is equated with a bandwidth allowance; it does not represent per se the amount of bandwidth that the user actually will utilize in the time interval.\",\n \"In determining network access allocations (and thus allowances) in the preferred embodiments herein described, the Bandwidth Allocator 92 preferably performs three routines, including: the prediction of bandwidth of each user class, and each user within each class, in a predetermined future interval of time (“First Routine”); the prioritization of user classes, and users within each class, for allocation of bandwidth (“Second Routine”); and the actual allocation of bandwidth for each user class, and each user within each class, for determining the bandwidth allowances for the future time interval (“Third Routine”).\",\n \"The First Routine preferably is performed utilizing statistical analysis of past bandwidth consumption of each user or, alternatively, past bandwidth requested for each user, and the forecasted bandwidth includes the bandwidth expected to be consumed by each user or, alternatively, the bandwidth expected to be requested by each user.\",\n \"Any function, method, or algorithm that generates an estimate of a future sample based on previously encountered samples may be used and many are well known in the art of statistical analysis as is evident from SPYROS MAKRIDAKIS ET AL., FORECASTING METHODS AND APPLICATIONS (3d.\",\n \"Ed.\",\n \"John Wiley & Sons 1998), which is hereby incorporated by reference.\",\n \"With regard to user classes, preferably a collective forecasted bandwidth for each class is determined by summing the forecasted bandwidth of all users within the class.\",\n \"The preferred algorithm for predicting each user's forecasted bandwidth includes the combined use of an adaptive-response-rate single exponential smoothing function (ARRSES Function) and a Holt-Winters' seasonal exponential smoothing function (HW Function).\",\n \"These two functions are utilized according to the forecast generation flowchart of FIG.\",\n \"7.The input includes a list of active users and the applicable time intervals for bandwidth allocation.\",\n \"The First Routine 700 begins by identification (Step 702) of the users of the Cable Network to which bandwidth is to be allocated in the Third Routine.\",\n \"Then, for each user, bandwidth for a succeeding time interval is predicted according to either the ARRSES Function or HW Function by first determining (Step 704) whether the user previously has been assigned a forecast function.\",\n \"If not, then in Step 706 the ARRSES Function is assigned to the user and the ARRSES Function is used to generate and record the forecasted bandwidth for the succeeding time interval.\",\n \"On the other hand, if it is determined in Step 704 that a forecast function is assigned, but it is determined in Step 707 that the forecast function is not the HW Function, then a determination is made (Step 708) whether to check for a seasonal cycle of the user.\",\n \"This determination in Step 708 is made by checking the elapsed time since the last seasonal check was made, with a seasonal check being made after a predetermined period of time elapses.\",\n \"If the determination in Step 708 is affirmative, then a seasonal identifier algorithm is executed (Step 710), in which an autocorrelation function and a seasonal identifier function are performed.\",\n \"The autocorrelation function is well known in the art of statistical analysis, and is used to identify elements in a time series which are influential on a current observation of that same series.\",\n \"Based on the output of the autocorrelation function, the seasonal identifier function identifies possible seasonal cycles of the time series by identifying local maxima of the results of the autocorrelation function.\",\n \"Based on the results of the seasonal identifier function, a determination is made (Step 712) whether an actual seasonal pattern exists.\",\n \"If a seasonal pattern is not found, or if it is not yet time to check for a seasonal cycle, then a forecast is generated and recorded (Step 714) using the ARRSES Function.\",\n \"If a seasonal pattern is found, then the HW Function is assigned (Step 716) to the user, the HW Function is initialized (Step 718), and the first forecast is generated and recorded (Step 720) using the HW Function.\",\n \"If it is determined in Step 707 that the current function assigned to the user already is the HW Function, then the determination is made (Step 722) whether the last forecasted bandwidth was acceptable.\",\n \"This determination is made by comparing whether the forecasted bandwidth was within 10% of the actual bandwidth consumed or requested.\",\n \"If this determination in Step 722 is negative, then the ARRSES Function is assigned to the user and the new forecast is generated and recorded in accordance with the ARRSES Function (Step 706).\",\n \"If the last forecast is determined (Step 722) to have been acceptable, then a determination is made (Step 724) whether the seasonal cycle has ended.\",\n \"If the seasonal cycle has ended, then the HW Function is reinitialized (Step 726), and the first forecast of the next seasonal cycle is generated and recorded (Step 728) via the HW Function.\",\n \"If the seasonal cycle has not expired, then the next forecast is generated and recorded (Step 730) in accordance with the HW Function.\",\n \"Following each of Step 706, Step 714, Step 728, and Step 730, the Bandwidth Allocator 92 determines (Step 732) whether the forecasting has been completed for all users and, if not, then repeats (Step 738) a forecast loop for a remaining user.\",\n \"If it is determined in Step 732 that all users have been evaluated, then the forecasts are communicated (Step 736) to the Database Manager 90 and the forecasting routine ends.\",\n \"A forecast of bandwidth for a user in a future time interval is generated in accordance with the ARRSES Function via the following formulas: FN+1=FN+αN(BN−FN) αN+1=|SEN/SAEN| SEN+1=SEN+β(BN+1−FN+1−SEN) SAEN=β|(BN−FN)|+(1−β)SAEN−1 wherein, F is the bandwidth that is expected to be consumed by a user for a time interval (or the bandwidth that is expected to be requested by a user); B is the bandwidth that is actually consumed by a user for the time interval (or the bandwidth that is actually requested by a user); N is the present time interval; N−1 is the previous (immediate past) time interval; N+1 is the next (immediate future) time interval; and β is a selected parameter affecting the responsiveness to change of the ARRSES Function when the bandwidth of a user changes between time intervals.\",\n \"Bandwidth is predicted both for the 6 MHz channel in the downstream direction as well as the 2 MHz channel in the upstream direction.\",\n \"Preferably each time interval is thirty minutes in length, but preferably may range from fifteen minutes to sixty minutes in length when bandwidth is forecast in the downstream direction.\",\n \"Preferably each time interval is five minutes in length, but preferably may range from one minute to fifteen minutes in length when bandwidth is forecast in the upstream direction.\",\n \"The steps in generating a forecast in accordance with the ARRSES Function are set forth in FIG.\",\n \"8, and include the calculation (Step 802) of a forecast error, the calculation (Step 804) of a smoothed error, the calculation (Step 806) of a smoothed absolute error, the calculation (Step 808) of alpha, and the calculation (Step 810) of the new forecast.\",\n \"A forecast of bandwidth of a user for a future time interval is generated in accordance the HW Function via the following formulas: Ls=1/s(Y1+Y2+ .\",\n \".\",\n \".\",\n \"+Ys) bs=1/S[(Ys+1−Y1)/s+(Ys+2−Y2)/s+ .\",\n \".\",\n \".\",\n \"+(Y2s−Ys)/s] S1=Y1/Ls,S2=Y2/Ls, .\",\n \".\",\n \".\",\n \"Ss=Ys/Ls Lt=α(Yt/St−s)+(1−α)(Lt−1+bt−1) bt=β(Lt−Lt−1)+(1−β)bt−1 St=γYt/Lt+(1−γ)St−s Ft+m=(Lt+btm)St−s+m wherein, Li=an average level of bandwidth after time interval i, bi=the trend after time interval i, si=the seasonal influence at time interval i, s=length of seasonal cycle (in number of time intervals), Yi=monitored bandwidth consumed or requested in time interval i t=time of initialization, m=the number of time intervals into the future for which a forecast is made, and α, β, and γ are parameters of the forecast method whose values are determined by doing a grid search over the domain of possible values of these parameters in an attempt to minimize the mean-squared-error of the forecast method, each of α, β, and γ falling between 0 and 1.The steps in generating a forecast in accordance with the HW Function are set forth in FIG.\",\n \"9, and include the initialization of the HW Function by determining Ls, bs, and S1, S2 .\",\n \".\",\n \".\",\n \", Ss in Step 902, if appropriate; the determination of the intermediate values of Lt, bt, and St in Step 904; and the determination of the forecast in Step 906, all in accordance with the above formulas.\",\n \"The Second Routine performed by the Bandwidth Allocator 92 comprises the prioritizing of user classes, and of users within each class, to determine respective orders of allocations.\",\n \"Prioritization is performed in accordance with one or more of various possible prioritization policies for users and for user classes.\",\n \"With regard to users within each class, the prioritization policies may depend upon, for example, (i) each user's SLA, (ii) each user's forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.\",\n \"User SLAs that at least partially affect prioritization policies include those that specify, for example: (i) a guaranteed minimum level of bandwidth; (ii) a time-of-day (TOD) minimum level of bandwidth; or (iii) a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability.\",\n \"Equivalently, such provisions also may be found in a CSLA for a class of which the user is a member.\",\n \"Under a SLA or CSLA providing for a guaranteed minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for use at all times.\",\n \"Accordingly, if the available bandwidth to such a user otherwise would fall below the minimum guaranteed level, then such a user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.\",\n \"Similarly, under a SLA or CSLA providing for a TOD minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for a particular TOD.\",\n \"If the available bandwidth to such a user otherwise would fall below the minimum guaranteed level during the particular TOD, then such user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.\",\n \"Finally, under a SLA or CSLA providing for a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability for a user, a user will have a guaranteed minimum level of bandwidth at all times and, in addition thereto, probably will have additional bandwidth up to a maximum level at any given time in accordance with the target probability.\",\n \"Accordingly, if the bandwidth available to such user otherwise would fall below the minimum guaranteed level, then the user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.\",\n \"The user also is given priority over such other users in allocating additional bandwidth as needed up to the maximum level in accordance with the target probability.\",\n \"Other SLA or CSLA provisions not relating to guaranteed levels of bandwidth also may affect a prioritization policy for users.\",\n \"Thus, for example, a SLA or CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon bandwidth consumption by a user for a particular amount of time, and the fee may be different as between users.\",\n \"Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid.\",\n \"Similarly, a SLA or CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier to an account based upon a bandwidth shortfall to a user for a particular amount of time when a guaranteed level of bandwidth for the user is not met.\",\n \"Moreover, the credit may be different as between users.\",\n \"Under these circumstances, prioritization may be determined so as to minimize the collective credits that a Carrier must apply.\",\n \"An example of prioritization based upon the forecasted bandwidth of each user includes giving priority to a first user over all other users, each of whom have a forecasted bandwidth that is greater than that of the first user.\",\n \"Prioritization may also be performed based on unilateral fairness considerations, especially when SLAs or CSLAs do not guarantee minimum levels of bandwidth for individual users, or when users otherwise would share equally in priority.\",\n \"Thus, users may be prioritized based on, for example: (i) the throughput of each of the users for a given time interval, with priority going to the user with the lesser throughput; (ii) data packets dropped over a given time interval, with priority going to the user with the greater data loss; and (iii) throughput experienced during a particular time of day or day of the week with priority going to the user with the lesser throughput for the particular time of day or day of the week.\",\n \"An example of fairness considerations that may be utilized in determining priority is illustrated in FIG.\",\n \"10, wherein user throughput for a time interval is graphed against user data packets dropped in the time interval for Users A and B.\",\n \"A target QoS standard for minimum throughput and maximum packet loss rates are established by the Carrier, whereby in the illustrated example each user is prioritized based on the user's absolute distance from the target QoS standard.\",\n \"Thus, under this policy, User A experiencing higher throughput rate and a lower packet loss rate, and thus having a shorter distance from the standard, is prioritized lower than User B having a lower throughput rate and higher data loss rate.\",\n \"With regard to user classes, prioritization policies are similar to those of the users and include, for example, (i) each CSLA, (ii) each class' collective forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.\",\n \"CSLAs that at least partially affect prioritization policies for user classes include those that specify, for example: (i) a guaranteed minimum level of collective bandwidth for the user class; (ii) a time-of-day (TOD) minimum level of collective bandwidth for the user class; or (iii) a guaranteed minimum level of collective bandwidth up to a maximum burstable level of collective bandwidth with target probability for the user class.\",\n \"Other CSLA provisions not relating to guaranteed levels of collective bandwidth also may affect a prioritization policy.\",\n \"Thus, for example, each CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon collective bandwidth consumption by the users of a class for a particular amount of time, and the fee may be different as between different classes of users.\",\n \"Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid to a Carrier.\",\n \"Similarly, each CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier based upon a collective bandwidth shortfall to the users of the class for a particular amount of time when a guaranteed level of collective bandwidth is not met.\",\n \"Moreover, the credit may be different as between user classes.\",\n \"Under these circumstances, prioritization may be determined so as to minimize the total credits that a Carrier may have to apply.\",\n \"An example of prioritization based upon the collective forecasted bandwidth of each user class includes giving priority to a first user class over all other user classes, each of which has a respective collective forecasted bandwidth that is greater than that of the first user class.\",\n \"Prioritization may also be performed based on unilateral fairness considerations, especially when CSLAs do not guarantee minimum levels of collective bandwidth, or when classes otherwise would share equally in priority.\",\n \"Thus, user classes may be prioritized based on, for example: (i) the collective throughput of the users of a class for a given time interval, with priority going to the class with the lesser collective throughput; (ii) the collective data packets of a user class that are dropped over a given time interval, with priority going to the user class with the greater collective data loss; and (iii) the collective throughput of the users of a class experienced during a particular time of day or day of the week, with priority going to the user class with the lesser collective throughput for the particular time of day or day of the week.\",\n \"The Third Routine performed by the Bandwidth Allocator 92 is the allocation of bandwidth to the user classes, and then to the users within each class, in accordance with one or more allocation policies as desired.\",\n \"Examples of allocation policies for users include: (i) the equal distribution of all available bandwidth to all users; (ii) the distribution of all available bandwidth to all users proportional to each user's respective forecasted bandwidth; (iii) the distribution of bandwidth to each user equal to the user's respective forecasted bandwidth, with any surplus bandwidth being distributed to the users either equally or proportionally based upon the user's respective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user based upon the minimum of the user's guaranteed bandwidth or the forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users.\",\n \"Likewise, examples of allocation policies for user classes include: (i) the distribution of all available bandwidth by the Bandwidth Allocator 92 to all user classes proportional to the number of active users in each class; (ii) the distribution of all available bandwidth to all user classes proportional to each class' respective collective forecasted bandwidth; (iii) the distribution of bandwidth to each user class equal to the class' respective collective forecasted bandwidth, with any surplus bandwidth being distributed to the user classes either equally or proportionally based upon the class' respective collective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user class based upon the minimum of the class' guaranteed collective bandwidth or the collective forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users classes.\",\n \"Examples of alternate preferred methods of prioritizing user classes, and then allocating bandwidth to the classes, will now be described in detail, each of which utilizes one or more of the aforementioned user class prioritization and allocation policies.\",\n \"Alternative preferred methods of prioritizing users within each class, and then allocating bandwidth to the users in each class, are set forth thereafter.\",\n \"In either case, the preferred methods of prioritizing and allocating are initiated pursuant to the scheduling module 102 of the Bandwidth Allocator 92, which operates independently of the scheduling module 100 of the Data Collector 88.With regard to prioritization of and allocation to user classes, a first preferred method 1100 is illustrated in FIG.\",\n \"11 and begins with the retrieval (Step 1102) of the collective forecasted bandwidth from the Database Manager 90 for all active user classes.\",\n \"Whether a user class is active is determined by past collective bandwidth consumption of the class (or, alternatively, collective requested bandwidth for the users of the class), as revealed by the user stats maintained by the Database Manager 90.All user classes are then prioritized (Step 1104) based on each class' collective forecast in increasing order, whereby a class having a lesser collective forecasted bandwidth will be prioritized over a class having larger collective forecasted bandwidth.\",\n \"A “surplus” is then set (Step 1106) to the total bandwidth available for allocation to the classes in the particular direction of communication over the shared communications medium at issue, and the total bandwidth available is then allocated (Step 1108) to each user class in an amount equaling the collective forecasted bandwidth subject to a respective maximum collective bandwidth value of the user class.\",\n \"Preferably the maximum collective bandwidth value is determined either in the appropriate CSLA or by the Carrier, Administrator 106, or other entity.\",\n \"Allocation of bandwidth to a user class additionally is subject to the actual availability of bandwidth following previous allocations thereof to user classes with equal or higher priority.\",\n \"Following allocations to all user classes, any bandwidth determined (Step 1110) to be remaining is then allocated (Step 1112) to the classes in amount proportional to the number of active users in each class, subject of course to the respective maximum collective bandwidth value of the class.\",\n \"The resulting class allocations are then recorded in the Database Manager 90 (Step 1114) as the bandwidth allowances for the classes.\",\n \"The method 1200 illustrated in FIG.\",\n \"12 is the same as that of FIG.\",\n \"11, except that surplus bandwidth, if any, is allocated (Step 1102) proportional to the collective forecasted bandwidths of the user classes, again subject to the respective maximum collective bandwidth value of each user class.\",\n \"The preferred method 1300 illustrated in FIG.\",\n \"13 does not prioritize the user classes for purposes of allocation but, instead, treats all classes equally.\",\n \"The method 1300 begins with the retrieval (Step 1302) of the collective forecasted bandwidth of each user class from the Database Manager 90.The surplus is then set to the total bandwidth available in the particular direction of communication, and the sum of all the collective forecasts is calculated (Step 1304).\",\n \"The available bandwidth then is allocated (Step 1306) to all classes proportional to the class' collective forecasted bandwidth, again subject to the respective maximum collective bandwidth value for each class.\",\n \"The resulting class allocations then are recorded in the Database Manage 90 (Step 1308) as the bandwidth allowances for the classes.\",\n \"The preferred method 1400 illustrated in FIG.\",\n \"14 seeks to maximize revenues from fees (F) that are paid for class bandwidth consumption.\",\n \"The method 1400 begins with the retrieval (Step 1402) of the collective forecast for each user class as well as a fee that is paid for the collective bandwidth of the class.\",\n \"The classes are then sorted (Step 1404) based on these fees in decreasing order, with the class with the highest fee receiving the highest priority.\",\n \"Next the surplus is set (Step 1406) to the total bandwidth available for allocation to the classes in the particular direction of communication.\",\n \"Bandwidth then is allocated (Step 1408) to the classes as available from highest to lowest priority in an amount equal to the class' collective forecasted bandwidth, subject to the respective maximum collective bandwidth value for the class.\",\n \"Both preferred method 1500 of FIGS.\",\n \"15a and 15b, and preferred method 1600 of FIGS.\",\n \"16a and 16b differ from the other methods 1100,1200,1300,1400 in that these two methods allocate bandwidth to the user classes in multiple allocation rounds.\",\n \"Method 1500 begins in FIG.\",\n \"15a with the retrieval (Step 1502) of the collective forecasted bandwidths of the classes as well as a credit (C) that applies if a respective class does not receive up to a guaranteed maximum level of collective bandwidth.\",\n \"The classes are then prioritized (Step 1504) based on each class' respective credit in decreasing order, with those classes having higher credits being given priority over classes with lesser credits.\",\n \"Next, the surplus is set (Step 1506) to the total bandwidth available to the classes in the particular direction of communication.\",\n \"Bandwidth then is allocated (Step 1508) as available in a first round to the classes from highest to lowest priority.\",\n \"The allocation for each class in the first round is equal to the minimum of the collective forecasted bandwidth or the maximum collective bandwidth that is guaranteed, subject to the respective maximum collective bandwidth value for the class.\",\n \"If any additional bandwidth is determined (Step 1510) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 1514).\",\n \"Bandwidth then is allocated (Step 1516) as available to each class in the same class order.\",\n \"Assuming sufficient bandwidth remains available, the allocation in the second round brings each class' allocation up to the class' collective forecasted bandwidth subject to the class' respective maximum collective bandwidth value.\",\n \"Following the second allocation round, a determination is made (Step 1518) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 1522) to the classes proportional to each class' collective forecasted bandwidth, and subject to each class' respective maximum collective bandwidth value.\",\n \"The resulting class allocations are then recorded (Step 1524) in the Database Manager 90 as the bandwidth allowances of the classes.\",\n \"If it is determined that no bandwidth remains available in either of Step 1510 or Step 1518, then the class allocations are completed and are recorded in the Database Manager 90 in Steps 1512,1524, respectively.\",\n \"Method 1600 of FIGS.\",\n \"16a and 16b differs from that of FIGS.\",\n \"15a and 15b only in that the sum of the collective forecasted bandwidths for all classes is calculated (Step 1602) and a determination is made (Step 1604) whether the sum exceeds the total bandwidth available for allocation to the classes.\",\n \"If the sum exceeds the total available bandwidth, then bandwidth is allocated (Step 1606) to each class in an amount equal to the collective forecasted bandwidth of the class, subject to the class' maximum guaranteed collective bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.\",\n \"Thus, for example, if the sum of all collective forecasted bandwidths exceeds the total available bandwidth for allocation in an amount equal to 20% of all collective forecasted bandwidths, then each class is allocated bandwidth in an amount equal to the class' collective forecasted bandwidth (subject to the class' maximum guaranteed collective bandwidth), then less 20% thereof.\",\n \"The information including fees, credits, guaranteed collective bandwidths, and respective maximum collective bandwidth values in the aforementioned preferred methods, is obtained from each CSLA and/or is predetermined by the Administrator 106, Carrier, or other entity.\",\n \"Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a CSLA table for each class as well as information regarding users associated therewith, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of class and user records in a timely fashion.\",\n \"Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once class allocations have been determined, the Database Manager 90 is updated with the new class allocations.\",\n \"Then, for each class, allocations of bandwidth are made to the users in the class.\",\n \"Furthermore, allocations within each class may be made by different methods.\",\n \"A first preferred method 1700 of prioritizing users and allocating bandwidth (whether upstream or downstream) by the Bandwidth Allocator 92 is illustrated in FIG.\",\n \"17 and begins with the retrieval (Step 1702) of the forecasted bandwidth from the Database Manager 90 for all active users.\",\n \"Whether a user is active is determined by past bandwidth consumption of the user (or, alternatively, requested bandwidth for the user), as revealed by the user stats maintained by the Database Manager 90.All users are then prioritized (Step 1704) based on each user's forecast in increasing order, whereby users having lesser forecasted bandwidths will be prioritized over users having larger forecasted bandwidths.\",\n \"The “surplus” is then set (Step 1706) to the total allocated bandwidth of the class (i.e., the class' collective bandwidth allowance) in the particular direction of communication, and the entire bandwidth allowance of the class is then allocated (Step 1708) to each user in an amount equaling the forecasted bandwidth of the user subject to a respective maximum bandwidth value of the user.\",\n \"Preferably the respective maximum bandwidth value is determined either in the user's SLA, the respective CSLA of the class, or by the Carrier, Administrator 106, or other entity.\",\n \"Allocation of bandwidth to a user additionally is subject to the actual availability of bandwidth following previous allocations thereof to users with equal or higher priority.\",\n \"Following allocations to all users, any bandwidth determined (Step 1710) to be remaining out of the total class allowance is then allocated equally (Step 1712) to the users subject to the respective maximum bandwidth value for each user.\",\n \"The new user allocations are then incorporated (Step 1714) into the DOC Network as the bandwidth allowances of the users.\",\n \"The method 1800 illustrated in FIG.\",\n \"18 is the same as that of FIG.\",\n \"17, except that surplus bandwidth in the class, if any, is allocated (Step 1802) proportional to the forecasted bandwidths of the users in the class, again subject to each user's respective maximum bandwidth value.\",\n \"The preferred method 1900 illustrated in FIG.\",\n \"19 does not prioritize the users for purposes of allocation but, instead, treats all users equally.\",\n \"The method 1900 begins with the retrieval (Step 1902) of the forecasted bandwidth for each user in the class from the Database Manager 90.The surplus is then set to the total allocated bandwidth of the class in the particular direction of communication, and the sum of all forecasts of the users in the class is calculated (Step 1904).\",\n \"The total allocated bandwidth of the class then is allocated (Step 1906) to all users in the class proportional to the user's forecasted bandwidth, again subject to each user's respective maximum bandwidth value.\",\n \"The user allocations then are incorporated into the DOC Network (Step 1908) as the bandwidth allowances of the users.\",\n \"The preferred method 2000 illustrated in FIG.\",\n \"20 seeks to maximize revenues from fees (F) that are paid for bandwidth consumption by the users.\",\n \"The method 2000 begins with the retrieval (Step 2002) of the forecast for each user as well as a fee that is paid for bandwidth by each user.\",\n \"The users are then sorted (Step 2004) based on user fees in decreasing order, with the user paying the most for bandwidth receiving the highest priority.\",\n \"Next, the surplus is set (Step 2006) to the total allocated bandwidth of the class in the particular direction of communication.\",\n \"Bandwidth then is allocated (Step 2008) to the users in the class as available from highest to lowest priority in an amount equal to each user's forecasted bandwidth, and subject to the user's respective maximum bandwidth value.\",\n \"Both preferred method 2100 of FIGS.\",\n \"21a and 21b, and preferred method 2200 of FIGS.\",\n \"22a and 22b differ from the other methods 1700,1800,1900,2000 in that these two methods allocate bandwidth to the users in multiple allocation rounds.\",\n \"Method 2100 begins in FIG.\",\n \"21a with the retrieval (Step 2102) of the forecasted bandwidths of the users as well as a credit (C) that applies if a respective user does not receive up to a guaranteed maximum level of bandwidth.\",\n \"The users are then prioritized (Step 2104) based on each user's respective credit in decreasing order, with those users having higher credits being given priority over users with lesser credits.\",\n \"Next, the surplus is set (Step 2106) to the total allocated bandwidth of the class in the particular direction of communication.\",\n \"Bandwidth then is allocated (Step 2108) as available in a first round to the users from highest to lowest priority.\",\n \"The allocation in the first round for each user is equal to the minimum of the forecasted bandwidth or the maximum bandwidth that is guaranteed, subject to the user's respective maximum bandwidth value.\",\n \"If any additional bandwidth is determined (Step 2110) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 2114).\",\n \"Bandwidth then is allocated (Step 2116) as available to each user in the same user order.\",\n \"Assuming sufficient bandwidth remains available, the allocation in the second round brings the user's allocation up to the user's forecasted bandwidth subject to the user's respective maximum bandwidth value.\",\n \"Following the second allocation round, a determination is made (Step 2118) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 2122) equally to the users, subject to each user's respective maximum bandwidth value.\",\n \"The user allocations are then incorporated (Step 2124) into the DOC Network as the users' bandwidth allowances.\",\n \"If it is determined that no bandwidth remains available in either of Step 2110 or Step 2118, then the user allocations are completed and are incorporated into DOC Network in Steps 2112,2124, respectively, as the users' bandwidth allowances.\",\n \"Method 2200 of FIGS.\",\n \"22a and 22b differs from that of FIGS.\",\n \"21a and 21b only in that the sum of the forecasted bandwidths for all users is calculated (Step 2202) and a determination is made (Step 2204) whether the sum exceeds the total allocated bandwidth of the class.\",\n \"If the sum exceeds the total allocated bandwidth of the class, then the bandwidth is allocated (Step 2206) to each user in an amount equal to the forecasted bandwidth, subject to the user's maximum guaranteed bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.\",\n \"Thus, for example, if the sum of all forecasted bandwidths exceeds the total allocated bandwidth of the class in an amount equal to 20% of the sum of all the forecasted bandwidths, then each user is allocated bandwidth in an amount equal to the user's forecasted bandwidth (subject to the user's maximum guaranteed bandwidth), then less 20% thereof.\",\n \"The applicable class bandwidth allowances used in the aforementioned methods are obtained from the Database Manager 90.The information, including fees, credits, guaranteed user bandwidths, and maximum bandwidth values in the aforementioned methods, is obtained from each user's SLA or from any applicable CSLA, and/or is predetermined by the Administrator 106, Carrier, or other entity.\",\n \"Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a user SLA table as well as a user billing table, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred not only for the CSLAs, but also for the SLAs and user billing tables, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of user records in a timely fashion.\",\n \"Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once user allocations have been determined under the aforementioned allocation policies, the respective DOC Network is updated with the resulting user allocations as the bandwidth allowances for the users for a particular time interval.\",\n \"Each user is then allocated bandwidth during the particular time interval in an amount that is less than, or equal to, that user's bandwidth allowance.\",\n \"Similarly, the collective bandwidth consumptions of a class by users therein is limited by that class' bandwidth allowance.\",\n \"Preferably, the DOC Network is updated at periodic intervals of between one to fifteen minutes and, preferably every five minutes.\",\n \"Furthermore, the periodic interval preferably corresponds to the scheduling of the Bandwidth Allocator 92 with regard to upstream transmissions.\",\n \"With particular reference to FIG.\",\n \"23, a preferred method 2300 of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network with the user allowances is illustrated.\",\n \"The DOC Network is updated by incorporating (Step 2302) the user allocations as bandwidth allowances (i.e., bandwidth limits) into CM configuration files (MD-5 files) for the CMs of the respective users.\",\n \"As set forth above, each CM configuration file contains instructions for a respective CM that limits the actual bandwidth consumed by the CM in the upstream direction and in the downstream direction.\",\n \"The CM configuration files are then sent (Step 2304) by the Bandwidth Allocator 92 to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network, which maintains CM configuration files for the CMs of the Cable Network.\",\n \"A command is also sent (Step 2306) to either of the CMs or the CMTS of the respective Cable Network causing the CMs to acquire and implement the CM configuration files maintained on the TFTP Server.\",\n \"In addition to maintaining information regarding CSLAs, class allocations, SLAs, and user billing data in the Database Manager 90, the GUI & Report Generating Engine 94 further enables the Administrator 106 to analyze the user stats updated by the Data Collector 88, including the generation of reports and graphs regarding, for example, network access usage of the users over time as well as user throughput rates vs. data loss rates similar to that shown in FIG.\",\n \"10.It additionally should be noted that a user may or may not be permitted to be grouped in one or more classes in accordance with the present invention.\",\n \"If it is desired that classes be mutually exclusive, then some policy should be established for determining which class with which a user is associated as between competing classes.\",\n \"If it is desired that classes not be mutually exclusive, then users falling within two or more classes will be allocated bandwidth within each class to the extent that no conflict arises as between the classes, and subject to any maximum allowed aggregated user bandwidth for all classes that may be established.\",\n \"As now will readily be seen, the preferred methods and preferred networks of the present invention described in detail herein enable a Carrier to accommodate bandwidth concerns of service providers competing for the business of users of a shared communications medium in a Shared Access Carrier Network.\",\n \"In particular, CSLAs now can be constructed in accordance with the present invention whereby a service provider is guaranteed some collective level of network access for the users of the shared communications medium that are customers of the service provider.\",\n \"Furthermore, the provision of bandwidth to users who are customers of competing service providers can now be based on fairness considerations, even if one of the service providers is related to the Carrier.\",\n \"In addition thereto, the differing demands for instantaneous throughput by users competing for access across the shared communications medium now can be accommodated in accordance with the present invention.\",\n \"Indeed, a Carrier now is able to continuously vary bandwidth consumption limits for each user on an individual basis and for small time intervals, either in accordance with fairness considerations, forecasted network access usage of the users, or under contractual provisions governing network access.\",\n \"It also will now be evident that the present invention gives rise to new business models that may be implemented by service providers for providing network access to users thereof and, in particular, to new ways of selling network access, which is also considered part of the present invention.\",\n \"For example, in accordance with the present invention, network access now can be “wholesaled” to service providers by considering the users of the service provider a class and allocating bulk network access to such class pursuant to a CSLA between the Carrier and the service provider.\",\n \"Through a CSLA, a Carrier can offer to the service provider a guaranteed minimum level of network access for the class that is constant throughout the day or week, or a guaranteed minimum level of network access that varies depending upon considerations such as the time of day or the day of week.\",\n \"A Carrier also now can offer a guaranteed minimum level of network access to the class with a guaranteed maximum level of network access provided as needed in accordance with a target probability.\",\n \"The service providers, in turn, then can offer different SLAs to the users that are its customers, essentially selling network access at the retail level.\",\n \"Accordingly, service providers can be assured of levels of network access for the users that are their customers, and users can be assured of appropriate levels of network access to meet their individual demands.\",\n \"Moreover, Carriers and/or service providers now can differentiate between users in charging for network access, thereby allowing Carriers and/or service providers to differentiate revenue streams for maximization of revenues.\",\n \"The present invention also enables Carriers and/or service providers to offer “dynamic SLAs” to users.\",\n \"The term “dynamic SLA” refers to a SLA that can be modified by a user as the user's demand for network access significantly changes, whether such modification is permanent or temporary.\",\n \"In this regard, and in accordance with a preferred method 2600 of the present invention as illustrated in FIG.\",\n \"26, the “network access retailer” (the entity selling the network access to the user) monitors (Step 2602) network access usage by users of a Shared Access Carrier Network and determines (Step 2604), for each user based on network access usage, whether a SLA provision other than those found in the user's current SLA would better meet the user's needs.\",\n \"This determination is made by comparing the user's throughput, bandwidth consumption, and/or bandwidth requested for a predetermined period of time against a set of threshold values, including any guaranteed level of network access provided for in the user's SLA as well as any minimum QoS standard that are deemed necessary for user satisfaction by the network access retailer or other appropriate entity.\",\n \"Thus, if the user's level of throughput, bandwidth consumption, and/or bandwidth requested for the predetermined time interval differs by a predetermined tolerance from a minimum threshold value, then the user is identified (Step 2606) as a “candidate” for modifying the SLA.\",\n \"A similar process alternatively is used, wherein the user's forecasted bandwidth is compared to the threshold values and, if the difference exceeds a predetermined tolerance, then the user is deemed a candidate for modifying the user's SLA.\",\n \"Once users have been identified as candidates, the candidates are filtered by screening (Step 2608) the candidates against a list of users for which solicitations are not to be made.\",\n \"Those candidates passing the screening are then invited (Step 2610) to modify their respective SLAs.\",\n \"The solicitation of the user preferably is performed via email, instant messaging, redirection of the user's web browser to a solicitation web page, generation and mailing of solicitation literature via U.S. mail, telemarketing, or other means of communication.\",\n \"The solicitation includes an invitation for the user to modify the user's SLA by increasing for a fee the minimum level of network access guaranteed to the user.\",\n \"The solicitation preferably also includes an invitation to make the modification permanent, or to make the modification only temporary and for a specific period of time.\",\n \"Thus, for example, if a user is identified as having a high usage pattern at recurrent periods of time (such as every Saturday night when a particular webcast is viewed, or when an Internet game is played), then the user automatically is solicited with an invitation via instant messaging on the following Saturday night to increase the user's guaranteed network access for that night, for a predetermined number of following Saturday nights, and/or for every Saturday night.\",\n \"Acceptance of the invitation by each user results in the modification (Step 2612) of the user's SLA for the appropriate period of time by increasing the level of network access the user is guaranteed (and/or the user's respective maximum bandwidth value, depending upon the policies used).\",\n \"The solicited modification to the user's SLA is updated in the SLA database, which is then used during user prioritization and allocation of bandwidth by the Bandwidth Allocator 92.The resulting higher bandwidth allowance should enhance the user's experience and overall satisfaction with the Carrier Network.\",\n \"In particular, the higher bandwidth (greater network access) should enhance the viewing of the webcast or the playing of the Internet game.\",\n \"On the other hand, SLAs for which users decline solicitations are not modified.\",\n \"Furthermore, if deemed appropriate, users declining a solicitation are recorded in the list against which candidates are screened.\",\n \"Preferably, the Bandwidth Allocator 92 analyzes the user stats maintained by the Database Manager 90, identifies those users that are candidates for SLA modification, and initiates the solicitation of such candidates.\",\n \"Information for each user's SLA for comparison with the user's stats automatically is obtained either from the Database Manager 90, or from an external database maintained by the network access retailer or other appropriate entity.\",\n \"Furthermore, the Bandwidth Allocator 92 preferably performs this analysis for solicitation on a regularly scheduled basis.\",\n \"In addition to such solicitations, a user of course may request a change in the level of network access guaranteed without having to receive first a solicitation.\",\n \"Furthermore, the user may request that the change be for a temporary period of time such that, for example, the change is reversed after only a few hours, which would cover a viewing of a particular webcast or the playing of a particular Internet game beginning at the time of the request.\",\n \"In view of the foregoing detailed description of the preferred embodiments and methods of the present invention, it readily will be understood by those persons skilled in the art that the present invention is susceptible of broad utility and application.\",\n \"Many embodiments and adaptations of the present invention other than those herein described, as well as many variations, modifications, and equivalent arrangements, will be apparent from or reasonably suggested by the present invention and the foregoing description thereof, without departing from the substance or scope of the present invention.\",\n \"Accordingly, while the present invention has been described herein in detail in relation to preferred embodiments, it is to be understood that this disclosure only is illustrative and exemplary of the present invention and is made merely for purposes of providing a full and enabling disclosure of the invention.\",\n \"The foregoing disclosure is not intended nor is to be construed to limit the present invention or otherwise to exclude any such other embodiments, adaptations, variations, modifications and equivalent arrangements, the present invention being limited only by the claims appended hereto and the equivalents thereof.\",\n \"Thus, for example, it will be apparent that, while preferred embodiments of the present invention have been described in the context of DOC Networks (including either a network of all coaxial cable, or a HFC network), the present invention nevertheless relates to any other network (whether wireline or wireless) wherein competing users share access across a shared communications medium including, for example, home networks and small networks in mass transit vehicles.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10276654"}}},{"rowIdx":87,"cells":{"text":{"kind":"list like","value":[["Novel nucleic acids and polypeptides","The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."],["1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, and complementary sequences thereof.","2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.","3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.","5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.","6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.","10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-8051.11.A composition comprising the polypeptide of claim 10 and a carrier.","12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.","14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.","15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.","16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.","17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.","19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-8051, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).","20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8052-16102, the mature protein portion thereof, or the active domain thereof.","21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.","22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-8051.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.","24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.","25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.","26.The collection of claim 22, wherein the collection is provided in a computer-readable format.","27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.","28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier."],[" 2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences."],[" 3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.","In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.","2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.","The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).","More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.","Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.","3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.","The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.","The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.","The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.","These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.","In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.","In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.","The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.","The identifying sequence can be 100 base pairs in length.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.","The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.","In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.","The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.","The collection can also be provided in a computer-readable format.","This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.","Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.","In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.","In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.","orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.","The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.","Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.","The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.","host cells) of the invention.","The invention also provides compositions comprising a polypeptide of the invention.","Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The invention also provides host cells transformed or transfected with a polynucleotide of the invention.","The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.","Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.","Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.","These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.","For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.","In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.","The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.","For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.","Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.","The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.","Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.","In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.","The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.","Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.","The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.","The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.","The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.","Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.","The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.","Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.","Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.","The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.","The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.","In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.","Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.","The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).","If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.","4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.","The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.","According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.","Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.","The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.","The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.","For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.","Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.","The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.","The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.","The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.","The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.","PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.","Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.","The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.","As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.","EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).","One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.","The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.","These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.","In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).","It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).","Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.","The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.","The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.","Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.","Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.","A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.","Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-8051.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.","(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).","They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.","Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.","The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-8051.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.","Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.","Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.","The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.","Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.","The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).","The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.","The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.","The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.","The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.","For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.","While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.","repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.","The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.","A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.","The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.","A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.","The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.","Preferably the peptide is from about 5 to about 200 amino acids.","To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.","The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.","The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.","The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.","The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.","The peptide may have been produced by processing in the cell which removes any leader/signal sequence.","The mature protein portion may or may not include an initial methionine residue.","The methionine residue may be removed from the protein during processing in the cell.","The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.","The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.","The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.","Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.","Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.","Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.","Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.","“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.","For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.","“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.","The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.","Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.","Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.","For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.","Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.","For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.","The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.","In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).","The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.","In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.","The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.","The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.","“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.","As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.","Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.","The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.","An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.","Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.","Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.","This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.","The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.","Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.","This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.","Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.","The cells can be prokaryotic or eukaryotic.","The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.","“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.","“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.","“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.","Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.","Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.","(1998) Annu.","Rev.","Immunol.","16:27-55) Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.","Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.","The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.","Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).","Other exemplary hybridization conditions are described herein in the examples.","In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).","As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.","Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).","Such a sequence is said to have 65% sequence identity to the listed sequence.","In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).","Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.","Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.","Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.","For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.","For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.","Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.","(1990) Methods Enzymol.","183:626-645).","Identity between sequences can also be determined by other methods known in the art, e.g.","by varying hybridization conditions.","The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.","The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.","The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.","The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.","As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.","UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.","The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.","The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.","As described above, a UMF will increase the frequency of uptake of a linked marker sequence.","Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.","4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.","The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-8051; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 8052-16102; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 8052-16102.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-8051; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 8052-16102.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.","The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.","The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.","The present invention also provides genes corresponding to the cDNA sequences disclosed herein.","The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.","Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.","Further 5′ and 3′ sequence can be obtained using methods known in the art.","For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-8051 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-8051 or a portion thereof as a probe.","Alternatively, the polynucleotides of SEQ ID NO: 1-8051 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.","The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.","The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.","The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.","Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.","Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-8051, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.","Fragments of, e.g.","15, 17, or 20 nucleotides or more that are selective for (i.e.","specifically hybridize to any one of the polynucleotides of the invention) are contemplated.","Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.","The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.","Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-8051, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-8051 with a sequence from another isolate of the same species.","Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.","In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.","The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-8051 can be obtained by searching a database using an algorithm or a program.","Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.","Evol.","36 290-300 (1993) and Altschul S. F. et al.","J. Mol.","Biol.","21:403-410 (1990)).","Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.","Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.","Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.","The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.","The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.","These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.","There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.","Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.","These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).","Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.","Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.","Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.","Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.","Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.","In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.","This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.","In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).","A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.","10:6487-6500 (1982).","PCR may also be used to create amino acid sequence variants of the novel nucleic acids.","When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.","PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.","The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.","A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.","Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.","Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.","Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.","The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.","The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.","Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.","In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-8051, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.","Also included are the cDNA inserts of any of the clones identified herein.","A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.","(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).","Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.","Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.","In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.","Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.","A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.","The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof or any other polynucleotides of the invention.","In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof is inserted, in a forward or reverse orientation.","In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.","Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.","The following vectors are provided by way of example.","Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).","Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).","The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.","19, 4485-4490 (1991), in order to produce the protein recombinantly.","Many suitable expression control sequences are known in the art.","General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).","As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.","Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.","Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.","Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.","Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.","Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.","Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.","The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.","Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.","Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.","The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.","Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.","As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).","Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).","These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.","Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.","Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.","Polynucleotides of the invention can also be used to induce immune responses.","For example, as described in Fan et al., Nat.","Biotech.","17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.","The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.","4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-8051, or fragments, analogs or derivatives thereof.","An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.","In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.","Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 8052-16102 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-8051 are additionally provided.","In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.","The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.","In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.","The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).","Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-8051), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.","The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.","For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.","An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.","An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.","For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.","Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.","Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).","The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.","The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.","An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.","Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.","For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.","The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.","To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.","In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule.","An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al.","(1987) Nucleic Acids Res 15: 6625-6641).","The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.","(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.","(1987) FEBS Lett 215: 327-330).","4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.","Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.","Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.","A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-8051).","For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-8051 (see, e.g., Cech et al.","U.S. Pat.","No.","4,987,071; and Cech et al.","U.S. Pat.","No.","5,116,742).","Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.","See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.","See generally, Helene.","(1991) Anticancer Drug Des.","6: 569-84; Helene.","et al.","(1992) Ann.","N.Y. Acad.","Sci.","660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.","For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.","(1996) Bioorg Med Chem 4: 5-23).","As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.","DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.","The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.","The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.","(1996) above; Perry-O'Keefe et al.","(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.","For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.","PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.","(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.","(1996), above; Perry-O'Keefe (1996), above).","In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.","For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.","Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.","PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).","The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.","(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.","(1989) Nucl Acid Res 17: 5973-88).","PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.","(1996) above).","Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.","See, Petersen et al.","(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.","Natl.","Acad.","Sci.","U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.","Natl.","Acad.","Sci.","84:648-652; PCT Publication No.","W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.","W089/10134).","In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.","Res.","5:539-549).","To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.","4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.","For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.","The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.","See, for example, PCT International Publication No.","WO94/12650, PCT International Publication No.","WO92/20808, and PCT International Publication No.","WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.","The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.","Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).","The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.","Any host/vector system can be used to express one or more of the ORFs of the present invention.","These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.","The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.","Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.","Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.","Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.","Various mammalian cell culture systems can also be employed to express recombinant protein.","Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).","Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.","Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.","DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.","Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.","Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.","Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.","Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.","Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.","Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.","Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.","If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.","Such covalent attachments may be accomplished using known chemical or enzymatic methods.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 8052-16102 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-8051 or the corresponding full length or mature protein.","Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.","The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.","Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 8052-16102.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.","Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.","Chem.","Soc.","114, 9245-9253 (1992), both of which are incorporated herein by reference.","Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.","The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.","The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.","The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.","The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.","Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.","In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.","Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.","The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.","By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.","Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.","A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.","At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.","The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.","This technique is particularly useful in producing small peptides and fragments of larger polypeptides.","Fragments are useful, for example, in generating antibodies against the native polypeptide.","Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.","The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.","As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.","One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.","The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.","For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.","The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.","Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.","In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.","One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.","These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.","See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.","Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.","The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.","These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 8052-16102.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.","The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.","For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.","Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.","For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.","Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.","No.","4,518,584).","Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.","Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.","This type of analysis determines the importance of the substituted amino acid(s) in biological activity.","Regions of the protein that are important for protein function may be determined by the eMATRIX program.","Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.","Such modifications are encompassed by the present invention.","The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.","Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.","1555 (1987), incorporated herein by reference.","As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.","The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.","The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.","Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.","For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His-tag.","Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.","), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.","The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.","One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).","Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.","Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.","The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).","This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.","Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.","Such analogs may exhibit improved properties such as activity and/or stability.","Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.","Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.","Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.","4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.","Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.","Biol.","215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.","vol.","25, pp.","3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.","Biol., Vol.","6, pp.","219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.","4, pp.","202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.","26 (1), pp.","320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.","Biol, 157, pp.","105-31 (1982), incorporated herein by reference).","The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.","NCB NLM NIH Bethesda, Md.","20894; Altschul, S., et al., J. Mol.","Biol.","215:403-410 (1990).","4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.","As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.","Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.","In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.","In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.","Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.","The polypeptide can be fused to the N-terminus or C-terminus.","For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.","In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.","In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.","The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.","The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.","Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival.","Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.","A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.","For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.","In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.","Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.","(eds.)","CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).","Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).","A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.","4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.","The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.","Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).","See, for example, Anderson, Nature, supplement to vol.","392, no.","6679, pp.","25-20 (1998).","For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).","Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.","It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.","Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.","Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.","The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.","These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.","Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.","Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.","The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.","See, for example, PCT International Publication No.","WO 94/12650, PCT International Publication No.","WO 92/20808, and PCT International Publication No.","WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.","If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.","In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.","As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.","Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.","Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.","These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.","The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.","Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.","Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.","Here, the naturally occurring sequences are deleted and new sequences are added.","In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.","The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.","Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.","The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.","No.","5,272,071 to Chappel; U.S. Pat.","No.","5,578,461 to Sherwin et al.","; International Application No.","PCT/US92/09627 (WO93/09222) by Selden et al.","; and International Application No.","PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.","4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.","Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.","In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].","Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.","Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.","Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.","No.","5,557,032, incorporated herein by reference.","Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.","Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.","Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.","No.","5,489,743 and PCT Publication No.","WO94/28122, incorporated herein by reference.","Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.","Inactivation can be carried out using homologous recombination methods described above.","Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.","The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.","4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.","Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).","The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.","Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.","Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.","The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.","4.10.1 Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.","The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.","Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.","The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.","Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.","Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.","Methods for performing the uses listed above are well known to those skilled in the art.","References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.","Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.","In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.","In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.","4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.","The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.","Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Bertagnolli et al., J. Immunol.","145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.","149:3778-3783, 1992; Bowman et al., I. Immunol.","152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.12.1-3.12.14, John Wiley and Sons, Toronto.","1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","6.8.1-6.8.8, John Wiley and Sons, Toronto.","1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","6.3.1-6.3.12, John Wiley and Sons, Toronto.","1991; deVries et al., J. Exp.","Med.","173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.","Natl.","Acad.","Sci.","U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.6.1-6.6.5, John Wiley and Sons, Toronto.","1991; Smith et al., Proc.","Natl.","Aced.","Sci.","U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.15.1 John Wiley and Sons, Toronto.","1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.","In Current Protocols in Immunology.","J. E. Coligan eds.","Vol 1 pp.","6.13.1, John Wiley and Sons, Toronto.","1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.","Natl.","Acad.","Sci.","USA 77:6091-6095, 1980; Weinberger et al., Eur.","J. Immuni.","11:405-411, 1981; Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.","Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.","The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.","It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein I-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).","Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.","Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.","This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.","Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.","Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.","No.","5,690,926).","Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.","This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.","These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.","These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.","Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.","For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.","The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.","In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.","Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.","A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.","The selectable marker allows only cells of the desired type to survive.","For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.","Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.","Lanza et al., Academic Press (1997)).","Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.","In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.","Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.","Proc.","Natl.","Acad.","Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.","The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.","as described by Bernstein et al., Blood, 77: 2316-2321 (1991).","4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.","Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.","in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.","Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.","Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.","Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.","Natl.","Acad.","Sci.","USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.","In Culture of Hematopoietic Cells.","R. I. Freshney, et al.","eds.","Vol pp.","139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.","A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.","Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.","De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.","A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.","Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)","mediated by inflammatory processes may also be possible using the composition of the invention.","Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.","Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.","Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.","De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.","The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.","The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.","The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.","The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.","for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.","More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.","Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.","Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.","Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.","Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.","Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.","A polypeptide of the present invention may also exhibit angiogenic activity.","A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.","A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.","Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.","WO95/16035 (bone, cartilage, tendon); International Patent Publication No.","WO95/05846 (nerve, neuronal); International Patent Publication No.","WO91/07491 (skin, endothelium).","Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.","71-112 (Maibach, H. I. and Rovee, D. T., eds.","), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz J.","Invest.","Dermatol 71:382-84 (1978).","4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.","A polynucleotide of the invention can encode a polypeptide exhibiting such activities.","A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.","These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.","More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.","and various fungal infections such as candidiasis.","Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.","include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.","Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.","Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.","The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch.","Toxocol.","73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.","Environ.","Health 53: 563-79).","Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.","Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.","The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.","Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.","Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.","Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.","Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).","For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.","Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.","The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.","Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.","Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.","To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.","The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.","Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.","Natl.","Acad.","Sci USA, 89:11102-11105 (1992).","In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.","Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.","Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.","Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.","Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.","Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.","The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.","Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.","840-856).","Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.","Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.","For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.","Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.","Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.","The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.","A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.","In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.","Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.","Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.","Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.","The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.","Natl.","Acad.","Sci.","USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.","128:1968-1974, 1982; Handa et al., J. Immunol.","135:1564-1572, 1985; Takai et al., I. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.","153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.","144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.","J. E. e.a.","Coligan eds.","Vol 1 pp.","3.8.1-3.8.16, John Wiley and Sons, Toronto.","1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.","137:3494-3500, 1986; Takai et al., J. Immunol.","140:508-512, 1988; Bertagnolli et al., J. Immunol.","149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.","134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.","Nat.","Acad.","Sci.","USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.","A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.","Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).","Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.","Administration of sufficient amounts of other inhibins can induce infertility in these mammals.","Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.","See, for example, U.S. Pat.","No.","4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods.","Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.","Natl.","Acad.","Sci.","USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.","Chemotactic or chemokinetic compositions (e.g.","proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.","For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.","A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.","Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.","Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.","Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.","Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.","J. Clin.","Invest.","95:1370-1376, 1995; Lind et al.","APMIS 103:140-146, 1995; Muller et al Eur.","J. Immunol.","25:1744-1748; Gruber et al.","J. of Immunol.","152:5860-5867, 1994; Johnston et al.","J. of Immunol.","153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.","A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.","Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.","A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).","Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.","Pharmacol.","26:131-140, 1986; Burdick et al., Thrombosis Res.","45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.","Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.","For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.","Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.","Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.","Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.","Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.","Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.","Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.","reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.","The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.","An anticancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.","The use of anti-cancer cocktails as a cancer treatment is routine.","Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.","In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.","There are hereditary conditions and/or environmental situations (e.g.","exposure to carcinogens) known in the art that predispose an individual to developing cancers.","Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.","In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.","These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.","Can.","Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.","J. Dev.","Biol., 40: 1189-97 (1999) and Li et al., Clin.","Exp.","Metastasis, 17:423-9 (1999), respectively.","Suitable tumor cells lines are available, e.g.","from American Type Tissue Culture Collection catalogs.","4.10.12 Receptor/Ligand Activity A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.","A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.","Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.","Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.","A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.","The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology; Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.","Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.","Natl.","Acad.","Sci.","USA 84:6864-6868, 1987; Bierer et al., J. Exp.","Med.","168:1145-1156, 1988; Rosenstein et al., J. Exp.","Med.","169:149-160 1989; Stoltenborg et al., J. Immunol.","Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).","Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.","Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.","The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.","(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.","182 (1990) Academic Press, Inc. San Diego).","Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.","Examples of toxins include, but are not limited, to ricin.","4.10.13 Drug Screening This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.","The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.","One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.","Drugs are screened against such transformed cells in competitive binding assays.","Such cells, either in viable or fixed form, can be used for standard binding assays.","One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.","Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.","Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.","The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.","Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.","For a review, see Science 282:63-68 (1998).","Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.","Of particular interest are peptide and oligonucleotide combinatorial libraries.","Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.","For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.","Opin.","Biotechnol.","8:701-707 (1997).","For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.","Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1 (1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).","Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.","The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.","In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.","The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.","The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.","Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.","4.10.14 Assay for Receptor Activity The invention also provides methods to detect specific binding of a polypeptide e.g.","a ligand or a receptor.","The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.","For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.","As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.","There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.","Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.","The responses of the two cell populations to the addition of ligands(s) are then compared.","Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).","As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.","The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.","For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.","The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.","Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.","phosphorylation.","Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.","4.10.15 Anti-Inflammatory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.","The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.","Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.","Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.","4.10.16 Leukemias Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.","Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).","4.10.17 Nervous System Disorders Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.","Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.","Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.","For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.","Such effects may be measured by any method known in the art.","In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.","(1990, J. Neurosci.","10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.","(1980, Exp.","Neurol.","70:65-82) or Brown et al.","(1981, Ann.","Rev.","Neurosci.","4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.","In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).","4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.","4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.","Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.","For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.","Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.","For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.","Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).","In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.","Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.","The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.","In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.","Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.","4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.","The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.","Arch.","Allergy Appl.","Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).","The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.","The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.","The control consists of administering PBS only.","The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.","An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.","4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.","Examples of therapeutic applications include, but are not limited to, those exemplified herein.","4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.","While the mode of administration is not particularly important, parenteral administration is preferred.","An exemplary mode of administration is to deliver an intravenous bolus.","The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.","It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.","Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.","For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.","Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.","The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.","The preparation of such solutions is within the skill of the art.","4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.","Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.","The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).","The characteristics of the carrier will depend on the route of administration.","The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.","In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.","The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.","Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.","Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).","A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.","As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.","As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).","Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.","A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.","When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.","When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.","In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.","Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.","When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.","If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.","4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.","Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.","Intravenous administration to the patient is preferred.","Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.","In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.","Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.","The liposomes will be targeted to and taken up selectively by the afflicted tissue.","The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.","The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.","Preferably for wound treatment, one administers the therapeutic compound directly to the site.","Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.","Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.","4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.","These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.","Proper formulation is dependent upon the route of administration chosen.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.","When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.","The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.","When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.","The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.","When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active-ingredient of the present invention.","When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.","The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.","A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.","The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.","For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.","For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.","Such penetrants are generally known in the art.","For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.","Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.","Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.","Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).","If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.","Dragee cores are provided with suitable coatings.","For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.","Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.","Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.","The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.","In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.","In addition, stabilizers may be added.","All formulations for oral administration should be in dosages suitable for such administration.","For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.","For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.","In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.","Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.","The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.","Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.","The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.","Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.","Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.","Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.","Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.","Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.","Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.","The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.","In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.","Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.","Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.","A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.","The co-solvent system may be the VPD co-solvent system.","VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.","The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.","This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.","Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.","Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.","polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.","Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.","Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.","Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.","Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.","Various types of sustained-release materials have been established and are well known by those skilled in the art.","Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.","Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.","The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.","Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.","Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.","Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.","The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.","The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.","B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.","T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.","MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.","The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.","Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.","The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.","Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.","Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.","Nos.","4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.","The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.","Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.","Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.","Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.","It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.","For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.","When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.","Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.","Topical administration may be suitable for wound healing and tissue repair.","Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.","Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.","Such matrices may be formed of materials presently in use for other implanted medical applications.","The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.","The particular application of the compositions will define the appropriate formulation.","Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.","Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.","Further matrices are comprised of pure proteins or extracellular matrix components.","Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.","Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.","The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.","Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.","In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.","A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).","Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).","The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.","In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.","These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).","The therapeutic compositions are also presently valuable for veterinary applications.","Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.","The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.","The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.","For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.","Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.","Polynucleotides of the present invention can also be used for gene therapy.","Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.","Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).","Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.","Treated cells can then be introduced in vivo for therapeutic purposes.","4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.","More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.","Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.","For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.","For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.","Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).","The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.","The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.","The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.","The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.","The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.","See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.","1 p. 1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).","The MEC will vary for each compound but can be estimated from in vitro data.","Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.","However, HPLC assays or bioassays can be used to determine plasma concentrations.","Dosage intervals can also be determined using MEC value.","Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.","In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.","An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.","Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.","The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.","4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.","The pack may, for example, comprise metal or plastic foil, such as a blister pack.","The pack or dispenser device may be accompanied by instructions for administration.","Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.","4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.","The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.","Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library.","In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.","Certain classes have subclasses as well, such as IgG1, IgG2, and others.","Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.","Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.","An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.","The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.","An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of any of the full length proteins of the invention, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.","Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.","Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.","In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention, e.g., a hydrophilic region.","A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.","As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.","See, e.g., Hopp and Woods, 1981, Proc.","Nat.","Acad.","Sci USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.","Biol.","157: 105-142, each of which is incorporated herein by reference in its entirety.","Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.","A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.","Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).","Some of these antibodies are discussed below.","5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.","An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.","Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.","Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.","The preparation can further include an adjuvant.","Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.","), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.","Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).","The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.","Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.","Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.","14, No.","8 (Apr.","17, 2000), pp.","25-28).","5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.","In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.","MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.","Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).","In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.","Alternatively, the lymphocytes can be immunized in vitro.","The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.","Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.","The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.","59-103).","Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.","Usually, rat or mouse myeloma cell lines are employed.","The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.","For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.","Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.","More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.","Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.","51-63).","The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.","Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).","Such techniques and assays are known in the art.","The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.","Biochem., 107:220 (1980).","Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.","After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.","Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.","Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.","The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.","The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.","No.","4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).","The hybridoma cells of the invention serve as a preferred source of such DNA.","Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.","The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.","No.","4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.","Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.","5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.","These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.","Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.","Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.","(See also U.S. Pat.","No.","5,225,539.)","In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.","Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.","In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.","The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.","Op.","Struct.","Biol., 2:593-596 (1992)).","5.133 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.","Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.","Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.","77-96).","In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.","Biol., 227:381 (1991); Marks et al., J. Mol.","Biol., 222:581 (1991)).","Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.","Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.","This approach is described, for example, in U.S. Pat.","Nos.","5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et at.","(Bio/Technology 10, 779-783 (1992)); Lonberg et al.","(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.","Rev.","Immunol.","13 65-93 (1995)).","Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.","(See PCT publication WO94/02602).","The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.","The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.","An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.","The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.","The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.","Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.","An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.","No.","5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.","A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.","No.","5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.","The hybrid cell expresses an antibody containing the heavy chain and the light chain.","In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.","No.","4,946,778).","In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.","Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.","5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.","In the present case, one of the binding specificities is for an antigenic protein of the invention.","The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.","Methods for making bispecific antibodies are known in the art.","Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).","Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.","The purification of the correct molecule is usually accomplished by affinity chromatography steps.","Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.","The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.","It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.","DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.","For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).","According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.","The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.","In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g tyrosine or tryptophan).","Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.","alanine or threonine).","This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.","Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.","F(ab′)2 bispecific antibodies).","Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.","For example, bispecific antibodies can be prepared using chemical linkage.","Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.","These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.","The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.","One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.","The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.","Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.","Shalaby et al., J. Exp.","Med.","175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.","Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.","The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.","Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.","For example, bispecific antibodies have been produced using leucine zippers.","Kostelny et al., J. Immunol.","148(5):1547-1553 (1992).","The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.","The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.","This method can also be utilized for the production of antibody homodimers.","The “diabody” technology described by Hollinger et al., Proc.","Natl.","Acad.","Sci.","USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.","The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.","Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.","Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.","See, Gruber et al., J. Immunol.","152:5368 (1994).","Antibodies with more than two valencies are contemplated.","For example, trispecific antibodies can be prepared.","Tutt et al., J. Immunol.","147:60 (1991).","Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.","Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.","CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.","Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.","These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.","Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).","5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.","Heteroconjugate antibodies are composed of two covalently joined antibodies.","Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.","No.","4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).","It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.","For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.","Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.","No.","4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.","For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.","The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).","See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.","Immunol., 148: 2918-2922 (1992).","Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.","Cancer Research, 53: 2560-2565 (1993).","Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.","See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).","5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).","Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.","Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.","A variety of radionuclides are available for the production of radioconjugated antibodies.","Examples include 212Bi, 131I, 131In, 90Y, and 186Re.","Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).","For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).","Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.","See WO94/1 1026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.","4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.","As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.","Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.","A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.","As used herein, “recorded” refers to a process for storing information on computer readable medium.","A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.","A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.","The choice of the data storage structure will generally be based on the means chosen to access the stored information.","In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.","The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.","A skilled artisan can readily adapt any number of data processor structuring formats (e.g.","text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.","By providing any of the nucleotide sequences SEQ ID NO: 1-8051 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-8051 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.","Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.","The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.","Biol.","215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.","Chem.","17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.","Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.","As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.","The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.","A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.","As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.","As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.","As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.","Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.","A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.","Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).","A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.","As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.","A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.","The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.","However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.","As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.","There are a variety of target motifs known in the art.","Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.","Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).","4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.","Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem.","56:560, (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.","4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.","In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.","Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.","In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.","In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.","Conditions for incubating a nucleic acid probe or antibody with a test sample vary.","Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.","One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.","Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.","1 (1982), Vol.","2 (1983), Vol.","3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).","The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.","The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.","Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.","In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.","Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.","In detail, a compartment kit includes any kit in which reagents are contained in separate containers.","Such containers include small glass containers, plastic containers or strips of plastic or paper.","Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.","Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.","), and containers which contain the reagents used to detect the bound antibody or probe.","Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.","One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.","4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).","See, e.g., Kunkel et al., U.S. Pat.","No.","5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.","4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-8051, or bind to a specific domain of the polypeptide encoded by the nucleic acid.","In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.","In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.","Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.","Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).","Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).","Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.","The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.","The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.","For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.","Alternatively, agents may be rationally selected or designed.","As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.","For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.","Freeman, NY (1992), pp.","289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.","In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.","As described above, such agents can be randomly screened or rationally designed/selected.","Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.","One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.","Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.","Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.","Acids Res.","6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.","56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).","Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.","Both techniques have been demonstrated to be effective in model systems.","Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.","Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.","Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.","4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.","The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-8051.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-8051 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.","Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.","PCR as described in U.S. Pat.","Nos.","4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.","Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.","The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.","Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.","Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.","The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.","The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.","These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.","The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.","Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981 f).","Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.","The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.","4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.","Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.","One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.","Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.","Microbiol.","28 (6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol.","Cell Probes 3 (2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.","Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.","For example, Broude et al.","(1994) Proc.","Natl.","Acad.","Sci.","USA 91 (8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.","Streptavidin-coated beads may be purchased from Dynal, Oslo.","Of course, this same linking chemistry is applicable to coating any surface with streptavidin.","Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).","Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.","Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.","CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.","CovaLink Modules may be purchased from Nunc Laboratories.","DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.","Biochem.","198 (1) 13842).","The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).","In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.","11 (8) 6513-29).","This is beneficial as immobilization using only a single covalent bond is preferred.","The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.","To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.","It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.","More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.","at 95° C. and cooling on ice for 10 min.","Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIM7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.","Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIM7, is made fresh and 25 ul added per well.","The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).","It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.","This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.","The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.","Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.","An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.","For example, addressable laser-activated photodeprotection may be Fodor et al.","(1991) Science 251 (4995) 767-73, incorporated herein by reference.","Probes may also be immobilized on nylon supports as described by Van Ness et at (1991) Nucleic Acids Res.","19 (12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.","Biochem.","169 (1) 104-8; all references being specifically incorporated herein.","To link an oligonucleotide to a nylon support, as described by Van Ness et al.","(1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.","One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91 (11) 5022-6, incorporated herein by reference).","These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).","These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.","A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.","4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.","For example, Sambrook et al.","(1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).","DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.","Samples may be prepared or dispensed in multiwell plates.","About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.","The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.","(1989), shearing by ultrasound and NaOH treatment.","Low pressure shearing is also appropriate, as described by Schriefer et al.","(1990) Nucleic Acids Res.","18 (24) 7455-6, incorporated herein by reference).","In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.","A lever device allows controlled application of low to intermediate pressures to the cell.","The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.","One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.","(1992) Nucleic Acids Res.","20 (14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.","The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.","Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).","Fitzgerald et al.","(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.","Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.","As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.","Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.","This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.","Phosphate groups must also be removed from genomic DNA by methods known in the art.","4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.","Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.","By offset printing, a density of dots higher than the density of the wells is achieved.","One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.","By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.","Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.","Each of the subarrays may represent replica spotting of the same samples.","In one example, a selected gene segment may be amplified from 64 patients.","For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).","A plate for each of the 64 patients is prepared.","By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.","Subarrays may contain 64 samples, one from each patient.","Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.","Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.","a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.","A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.","The present invention is illustrated in the following examples.","Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.","Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.","The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.","Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.","Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.","All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.","5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.","The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.","Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.","The clones were clustered into groups of similar or identical sequences.","Representative clones were selected for sequencing.","In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.","PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.","Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.","In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.","5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.","The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-8051 and are provided in the attached Sequence Listing.","The contigs were assembled using an EST sequence as a seed.","Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.","The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.","Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.","The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-8051) of the present invention are incorporated in the attached Sequence Listing.","A subset of the predicted polypeptide sequences contain an unknown amino acid; a stop codon; a possible nucleotide deletion; or a possible nucleotide insertion.","These sequences have also been shown in their entirety in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-8051.Table 2 also indicates the method by which the polypeptide was predicted.","Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fasta.bioch.virginia,edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).","Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.","Biol., 268:78-94 (1997), incorporated herein by reference).","Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.","The nearest neighbor results for SEQ ID NO: 1-8051 were obtained by a BLASTX version 2.0al 19 MP-WashU search against Genpept release 123 and Geneseq release 200110 (Derwent), using BLAST algorithm.","The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-8051.The nearest neighbor results for SEQ ID NO: 1-8051, having identifiable function(s) are incorporated in the attached Sequence Listing.","Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.","Biol., Vol.","6 pp.","219-235 (1999) herein incorporated by reference), all the polypeptide sequences were examined to determine whether they had identifiable signature regions.","The attached Sequence Listing provides the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.","Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.","26 (1) pp.","320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.","The attached Sequence Listing provides the results obtained by pFam analysis for each polypeptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.","Tables 1 and 2 follow.","Table 1 shows the various tissue sources of SEQ ID NO: 1-8051.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.","Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.","No.","09/577,408.LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00001 Please refer to the end of the specification for access instructions.","LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00002 Please refer to the end of the specification for access instructions.","LENGTHY TABLE The patent application contains a lengthy table section.","A copy of the table is available in electronic form from the USPTO web site ().","An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3)."]],"string":"[\n [\n \"Novel nucleic acids and polypeptides\",\n \"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.\"\n ],\n [\n \"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, and complementary sequences thereof.\",\n \"2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.\",\n \"3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.\",\n \"5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.\",\n \"6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.\",\n \"10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-8051.11.A composition comprising the polypeptide of claim 10 and a carrier.\",\n \"12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.\",\n \"14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.\",\n \"15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.\",\n \"16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.\",\n \"17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.\",\n \"19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-8051, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).\",\n \"20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8052-16102, the mature protein portion thereof, or the active domain thereof.\",\n \"21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.\",\n \"22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-8051.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.\",\n \"24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.\",\n \"25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.\",\n \"26.The collection of claim 22, wherein the collection is provided in a computer-readable format.\",\n \"27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\",\n \"28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.\"\n ],\n [\n \" 2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\"\n ],\n [\n \" 3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.\",\n \"In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.\",\n \"2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.\",\n \"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).\",\n \"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.\",\n \"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.\",\n \"3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.\",\n \"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.\",\n \"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.\",\n \"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.\",\n \"These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.\",\n \"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.\",\n \"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.\",\n \"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.\",\n \"The identifying sequence can be 100 base pairs in length.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.\",\n \"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.\",\n \"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.\",\n \"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.\",\n \"The collection can also be provided in a computer-readable format.\",\n \"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.\",\n \"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.\",\n \"In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.\",\n \"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.\",\n \"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.\",\n \"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.\",\n \"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.\",\n \"host cells) of the invention.\",\n \"The invention also provides compositions comprising a polypeptide of the invention.\",\n \"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.\",\n \"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.\",\n \"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.\",\n \"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.\",\n \"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.\",\n \"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.\",\n \"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.\",\n \"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.\",\n \"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.\",\n \"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.\",\n \"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.\",\n \"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.\",\n \"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.\",\n \"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.\",\n \"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.\",\n \"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.\",\n \"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.\",\n \"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.\",\n \"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.\",\n \"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.\",\n \"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.\",\n \"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.\",\n \"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.\",\n \"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.\",\n \"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.\",\n \"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.\",\n \"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).\",\n \"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.\",\n \"4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.\",\n \"The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.\",\n \"According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.\",\n \"Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.\",\n \"The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.\",\n \"The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.\",\n \"For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.\",\n \"Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.\",\n \"The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.\",\n \"The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.\",\n \"The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.\",\n \"The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.\",\n \"PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.\",\n \"Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.\",\n \"The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.\",\n \"As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.\",\n \"EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).\",\n \"One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.\",\n \"The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.\",\n \"These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.\",\n \"In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).\",\n \"It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).\",\n \"Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.\",\n \"The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.\",\n \"The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.\",\n \"Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.\",\n \"Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.\",\n \"A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.\",\n \"Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-8051.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.\",\n \"(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).\",\n \"They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.\",\n \"Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.\",\n \"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-8051.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.\",\n \"Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.\",\n \"Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.\",\n \"The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.\",\n \"Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.\",\n \"The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).\",\n \"The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.\",\n \"The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.\",\n \"The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.\",\n \"The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.\",\n \"For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.\",\n \"While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.\",\n \"repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.\",\n \"The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.\",\n \"A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.\",\n \"The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.\",\n \"A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.\",\n \"The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.\",\n \"Preferably the peptide is from about 5 to about 200 amino acids.\",\n \"To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.\",\n \"The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.\",\n \"The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.\",\n \"The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.\",\n \"The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.\",\n \"The peptide may have been produced by processing in the cell which removes any leader/signal sequence.\",\n \"The mature protein portion may or may not include an initial methionine residue.\",\n \"The methionine residue may be removed from the protein during processing in the cell.\",\n \"The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.\",\n \"The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.\",\n \"The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.\",\n \"Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.\",\n \"Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.\",\n \"Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.\",\n \"Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.\",\n \"“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.\",\n \"For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.\",\n \"“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.\",\n \"The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.\",\n \"Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.\",\n \"Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.\",\n \"For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.\",\n \"Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.\",\n \"For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.\",\n \"The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.\",\n \"In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).\",\n \"The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.\",\n \"In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.\",\n \"The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.\",\n \"The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.\",\n \"“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.\",\n \"As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.\",\n \"Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.\",\n \"The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.\",\n \"An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.\",\n \"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.\",\n \"Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.\",\n \"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.\",\n \"The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.\",\n \"Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.\",\n \"This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.\",\n \"Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.\",\n \"The cells can be prokaryotic or eukaryotic.\",\n \"The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.\",\n \"“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.\",\n \"“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.\",\n \"“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.\",\n \"Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.\",\n \"Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.\",\n \"(1998) Annu.\",\n \"Rev.\",\n \"Immunol.\",\n \"16:27-55) Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.\",\n \"Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.\",\n \"The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.\",\n \"Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).\",\n \"Other exemplary hybridization conditions are described herein in the examples.\",\n \"In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).\",\n \"As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.\",\n \"Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).\",\n \"Such a sequence is said to have 65% sequence identity to the listed sequence.\",\n \"In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).\",\n \"Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.\",\n \"Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.\",\n \"Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.\",\n \"For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.\",\n \"For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.\",\n \"Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.\",\n \"(1990) Methods Enzymol.\",\n \"183:626-645).\",\n \"Identity between sequences can also be determined by other methods known in the art, e.g.\",\n \"by varying hybridization conditions.\",\n \"The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.\",\n \"The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.\",\n \"The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.\",\n \"The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.\",\n \"As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.\",\n \"UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.\",\n \"The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.\",\n \"The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.\",\n \"As described above, a UMF will increase the frequency of uptake of a linked marker sequence.\",\n \"Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.\",\n \"4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.\",\n \"The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-8051; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 8052-16102; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 8052-16102.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-8051; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 8052-16102.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.\",\n \"The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.\",\n \"The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.\",\n \"The present invention also provides genes corresponding to the cDNA sequences disclosed herein.\",\n \"The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.\",\n \"Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.\",\n \"Further 5′ and 3′ sequence can be obtained using methods known in the art.\",\n \"For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-8051 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-8051 or a portion thereof as a probe.\",\n \"Alternatively, the polynucleotides of SEQ ID NO: 1-8051 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.\",\n \"The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.\",\n \"The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.\",\n \"The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.\",\n \"Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.\",\n \"Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-8051, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.\",\n \"Fragments of, e.g.\",\n \"15, 17, or 20 nucleotides or more that are selective for (i.e.\",\n \"specifically hybridize to any one of the polynucleotides of the invention) are contemplated.\",\n \"Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.\",\n \"The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.\",\n \"Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-8051, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-8051 with a sequence from another isolate of the same species.\",\n \"Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.\",\n \"In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.\",\n \"The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-8051 can be obtained by searching a database using an algorithm or a program.\",\n \"Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.\",\n \"Evol.\",\n \"36 290-300 (1993) and Altschul S. F. et al.\",\n \"J. Mol.\",\n \"Biol.\",\n \"21:403-410 (1990)).\",\n \"Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.\",\n \"Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.\",\n \"Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.\",\n \"The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.\",\n \"The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.\",\n \"These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.\",\n \"There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.\",\n \"Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.\",\n \"These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).\",\n \"Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.\",\n \"Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.\",\n \"Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.\",\n \"Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.\",\n \"Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.\",\n \"In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.\",\n \"This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.\",\n \"In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).\",\n \"A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.\",\n \"10:6487-6500 (1982).\",\n \"PCR may also be used to create amino acid sequence variants of the novel nucleic acids.\",\n \"When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.\",\n \"PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.\",\n \"The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.\",\n \"A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.\",\n \"Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.\",\n \"Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.\",\n \"Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.\",\n \"The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.\",\n \"The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.\",\n \"Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.\",\n \"In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-8051, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.\",\n \"Also included are the cDNA inserts of any of the clones identified herein.\",\n \"A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.\",\n \"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).\",\n \"Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.\",\n \"Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.\",\n \"In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.\",\n \"Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.\",\n \"A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.\",\n \"The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof or any other polynucleotides of the invention.\",\n \"In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof is inserted, in a forward or reverse orientation.\",\n \"In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.\",\n \"Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.\",\n \"The following vectors are provided by way of example.\",\n \"Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).\",\n \"Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).\",\n \"The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.\",\n \"19, 4485-4490 (1991), in order to produce the protein recombinantly.\",\n \"Many suitable expression control sequences are known in the art.\",\n \"General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).\",\n \"As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.\",\n \"Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.\",\n \"Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.\",\n \"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.\",\n \"Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.\",\n \"Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.\",\n \"Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.\",\n \"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.\",\n \"Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.\",\n \"Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.\",\n \"The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.\",\n \"Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.\",\n \"As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).\",\n \"Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).\",\n \"These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.\",\n \"Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.\",\n \"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.\",\n \"Polynucleotides of the invention can also be used to induce immune responses.\",\n \"For example, as described in Fan et al., Nat.\",\n \"Biotech.\",\n \"17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.\",\n \"The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.\",\n \"4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-8051, or fragments, analogs or derivatives thereof.\",\n \"An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.\",\n \"In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.\",\n \"Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 8052-16102 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-8051 are additionally provided.\",\n \"In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.\",\n \"In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.\",\n \"The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).\",\n \"Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-8051), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.\",\n \"The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.\",\n \"For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.\",\n \"An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.\",\n \"An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.\",\n \"For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.\",\n \"Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.\",\n \"Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).\",\n \"The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.\",\n \"The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.\",\n \"An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.\",\n \"Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.\",\n \"For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.\",\n \"The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.\",\n \"To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.\",\n \"In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule.\",\n \"An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al.\",\n \"(1987) Nucleic Acids Res 15: 6625-6641).\",\n \"The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.\",\n \"(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.\",\n \"(1987) FEBS Lett 215: 327-330).\",\n \"4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.\",\n \"Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.\",\n \"Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.\",\n \"A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-8051).\",\n \"For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-8051 (see, e.g., Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,987,071; and Cech et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,116,742).\",\n \"Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.\",\n \"See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.\",\n \"See generally, Helene.\",\n \"(1991) Anticancer Drug Des.\",\n \"6: 569-84; Helene.\",\n \"et al.\",\n \"(1992) Ann.\",\n \"N.Y. Acad.\",\n \"Sci.\",\n \"660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.\",\n \"For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.\",\n \"(1996) Bioorg Med Chem 4: 5-23).\",\n \"As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.\",\n \"DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.\",\n \"The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.\",\n \"The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.\",\n \"(1996) above; Perry-O'Keefe et al.\",\n \"(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.\",\n \"For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.\",\n \"PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.\",\n \"(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.\",\n \"(1996), above; Perry-O'Keefe (1996), above).\",\n \"In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.\",\n \"For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.\",\n \"Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.\",\n \"PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).\",\n \"The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.\",\n \"(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.\",\n \"(1989) Nucl Acid Res 17: 5973-88).\",\n \"PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.\",\n \"(1996) above).\",\n \"Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.\",\n \"See, Petersen et al.\",\n \"(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"84:648-652; PCT Publication No.\",\n \"W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.\",\n \"W089/10134).\",\n \"In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.\",\n \"Res.\",\n \"5:539-549).\",\n \"To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.\",\n \"4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.\",\n \"For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.\",\n \"The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO94/12650, PCT International Publication No.\",\n \"WO92/20808, and PCT International Publication No.\",\n \"WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.\",\n \"The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.\",\n \"Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).\",\n \"The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.\",\n \"Any host/vector system can be used to express one or more of the ORFs of the present invention.\",\n \"These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.\",\n \"The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.\",\n \"Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.\",\n \"Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.\",\n \"Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.\",\n \"Various mammalian cell culture systems can also be employed to express recombinant protein.\",\n \"Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).\",\n \"Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.\",\n \"Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.\",\n \"DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.\",\n \"Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.\",\n \"Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.\",\n \"Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.\",\n \"Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.\",\n \"Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.\",\n \"Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.\",\n \"Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.\",\n \"If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.\",\n \"Such covalent attachments may be accomplished using known chemical or enzymatic methods.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 8052-16102 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-8051 or the corresponding full length or mature protein.\",\n \"Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.\",\n \"The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.\",\n \"Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 8052-16102.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.\",\n \"Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.\",\n \"Chem.\",\n \"Soc.\",\n \"114, 9245-9253 (1992), both of which are incorporated herein by reference.\",\n \"Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.\",\n \"The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.\",\n \"The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.\",\n \"The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.\",\n \"The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.\",\n \"Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.\",\n \"In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.\",\n \"Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.\",\n \"The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.\",\n \"By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.\",\n \"Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.\",\n \"A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.\",\n \"At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.\",\n \"The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.\",\n \"This technique is particularly useful in producing small peptides and fragments of larger polypeptides.\",\n \"Fragments are useful, for example, in generating antibodies against the native polypeptide.\",\n \"Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.\",\n \"The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.\",\n \"As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.\",\n \"One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.\",\n \"The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.\",\n \"For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.\",\n \"The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.\",\n \"Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.\",\n \"In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.\",\n \"One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.\",\n \"These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.\",\n \"See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.\",\n \"Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.\",\n \"The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.\",\n \"These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 8052-16102.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.\",\n \"The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.\",\n \"For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.\",\n \"Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.\",\n \"For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.\",\n \"Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.\",\n \"No.\",\n \"4,518,584).\",\n \"Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.\",\n \"Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.\",\n \"This type of analysis determines the importance of the substituted amino acid(s) in biological activity.\",\n \"Regions of the protein that are important for protein function may be determined by the eMATRIX program.\",\n \"Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.\",\n \"Such modifications are encompassed by the present invention.\",\n \"The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.\",\n \"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.\",\n \"1555 (1987), incorporated herein by reference.\",\n \"As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.\",\n \"The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.\",\n \"The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.\",\n \"Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.\",\n \"For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His-tag.\",\n \"Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.\",\n \"), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.\",\n \"The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.\",\n \"One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).\",\n \"Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.\",\n \"Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.\",\n \"The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).\",\n \"This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.\",\n \"Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.\",\n \"Such analogs may exhibit improved properties such as activity and/or stability.\",\n \"Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.\",\n \"Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.\",\n \"Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.\",\n \"4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.\",\n \"Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.\",\n \"Biol.\",\n \"215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.\",\n \"vol.\",\n \"25, pp.\",\n \"3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6, pp.\",\n \"219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.\",\n \"4, pp.\",\n \"202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26 (1), pp.\",\n \"320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.\",\n \"Biol, 157, pp.\",\n \"105-31 (1982), incorporated herein by reference).\",\n \"The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.\",\n \"NCB NLM NIH Bethesda, Md.\",\n \"20894; Altschul, S., et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990).\",\n \"4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.\",\n \"As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.\",\n \"Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.\",\n \"In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.\",\n \"In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.\",\n \"Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.\",\n \"The polypeptide can be fused to the N-terminus or C-terminus.\",\n \"For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.\",\n \"In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.\",\n \"In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.\",\n \"The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.\",\n \"The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.\",\n \"Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival.\",\n \"Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.\",\n \"A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.\",\n \"For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.\",\n \"In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.\",\n \"Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.\",\n \"(eds.)\",\n \"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).\",\n \"Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).\",\n \"A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.\",\n \"4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.\",\n \"The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.\",\n \"Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).\",\n \"See, for example, Anderson, Nature, supplement to vol.\",\n \"392, no.\",\n \"6679, pp.\",\n \"25-20 (1998).\",\n \"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).\",\n \"Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.\",\n \"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.\",\n \"Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.\",\n \"Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.\",\n \"The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.\",\n \"These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.\",\n \"Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.\",\n \"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.\",\n \"The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.\",\n \"See, for example, PCT International Publication No.\",\n \"WO 94/12650, PCT International Publication No.\",\n \"WO 92/20808, and PCT International Publication No.\",\n \"WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.\",\n \"If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.\",\n \"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.\",\n \"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.\",\n \"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.\",\n \"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.\",\n \"These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.\",\n \"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.\",\n \"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.\",\n \"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.\",\n \"Here, the naturally occurring sequences are deleted and new sequences are added.\",\n \"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.\",\n \"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.\",\n \"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.\",\n \"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.\",\n \"No.\",\n \"5,272,071 to Chappel; U.S. Pat.\",\n \"No.\",\n \"5,578,461 to Sherwin et al.\",\n \"; International Application No.\",\n \"PCT/US92/09627 (WO93/09222) by Selden et al.\",\n \"; and International Application No.\",\n \"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.\",\n \"4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.\",\n \"Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.\",\n \"In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].\",\n \"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.\",\n \"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.\",\n \"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.\",\n \"No.\",\n \"5,557,032, incorporated herein by reference.\",\n \"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.\",\n \"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.\",\n \"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.\",\n \"No.\",\n \"5,489,743 and PCT Publication No.\",\n \"WO94/28122, incorporated herein by reference.\",\n \"Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.\",\n \"Inactivation can be carried out using homologous recombination methods described above.\",\n \"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.\",\n \"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.\",\n \"4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.\",\n \"Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).\",\n \"The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.\",\n \"Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.\",\n \"Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.\",\n \"The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.\",\n \"4.10.1 Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.\",\n \"The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.\",\n \"Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.\",\n \"The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.\",\n \"Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.\",\n \"Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.\",\n \"Methods for performing the uses listed above are well known to those skilled in the art.\",\n \"References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.\",\n \"Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.\",\n \"In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.\",\n \"In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.\",\n \"4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.\",\n \"The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Bertagnolli et al., J. Immunol.\",\n \"145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.\",\n \"149:3778-3783, 1992; Bowman et al., I. Immunol.\",\n \"152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.12.1-3.12.14, John Wiley and Sons, Toronto.\",\n \"1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.8.1-6.8.8, John Wiley and Sons, Toronto.\",\n \"1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.3.1-6.3.12, John Wiley and Sons, Toronto.\",\n \"1991; deVries et al., J. Exp.\",\n \"Med.\",\n \"173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.6.1-6.6.5, John Wiley and Sons, Toronto.\",\n \"1991; Smith et al., Proc.\",\n \"Natl.\",\n \"Aced.\",\n \"Sci.\",\n \"U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.15.1 John Wiley and Sons, Toronto.\",\n \"1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.\",\n \"In Current Protocols in Immunology.\",\n \"J. E. Coligan eds.\",\n \"Vol 1 pp.\",\n \"6.13.1, John Wiley and Sons, Toronto.\",\n \"1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 77:6091-6095, 1980; Weinberger et al., Eur.\",\n \"J. Immuni.\",\n \"11:405-411, 1981; Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.\",\n \"Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.\",\n \"The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.\",\n \"It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein I-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).\",\n \"Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.\",\n \"Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.\",\n \"This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.\",\n \"Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.\",\n \"Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.\",\n \"No.\",\n \"5,690,926).\",\n \"Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.\",\n \"This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.\",\n \"These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.\",\n \"These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.\",\n \"Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.\",\n \"For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.\",\n \"The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.\",\n \"Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.\",\n \"A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.\",\n \"The selectable marker allows only cells of the desired type to survive.\",\n \"For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.\",\n \"Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.\",\n \"Lanza et al., Academic Press (1997)).\",\n \"Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.\",\n \"In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.\",\n \"Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.\",\n \"Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.\",\n \"The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.\",\n \"as described by Bernstein et al., Blood, 77: 2316-2321 (1991).\",\n \"4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.\",\n \"Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.\",\n \"in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.\",\n \"Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.\",\n \"Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.\",\n \"Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.\",\n \"In Culture of Hematopoietic Cells.\",\n \"R. I. Freshney, et al.\",\n \"eds.\",\n \"Vol pp.\",\n \"139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.\",\n \"A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.\",\n \"Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.\",\n \"De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.\",\n \"A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.\",\n \"Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)\",\n \"mediated by inflammatory processes may also be possible using the composition of the invention.\",\n \"Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.\",\n \"Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.\",\n \"Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.\",\n \"De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.\",\n \"The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.\",\n \"The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.\",\n \"The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.\",\n \"The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.\",\n \"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.\",\n \"More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.\",\n \"Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.\",\n \"Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.\",\n \"Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.\",\n \"Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.\",\n \"Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.\",\n \"A polypeptide of the present invention may also exhibit angiogenic activity.\",\n \"A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.\",\n \"A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.\",\n \"WO95/16035 (bone, cartilage, tendon); International Patent Publication No.\",\n \"WO95/05846 (nerve, neuronal); International Patent Publication No.\",\n \"WO91/07491 (skin, endothelium).\",\n \"Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.\",\n \"71-112 (Maibach, H. I. and Rovee, D. T., eds.\",\n \"), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz J.\",\n \"Invest.\",\n \"Dermatol 71:382-84 (1978).\",\n \"4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such activities.\",\n \"A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.\",\n \"These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.\",\n \"More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.\",\n \"and various fungal infections such as candidiasis.\",\n \"Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.\",\n \"include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.\",\n \"Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.\",\n \"Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.\",\n \"The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch.\",\n \"Toxocol.\",\n \"73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.\",\n \"Environ.\",\n \"Health 53: 563-79).\",\n \"Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.\",\n \"Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.\",\n \"The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.\",\n \"Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.\",\n \"Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.\",\n \"Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.\",\n \"Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).\",\n \"For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.\",\n \"Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.\",\n \"The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.\",\n \"Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.\",\n \"Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.\",\n \"To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.\",\n \"The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.\",\n \"Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci USA, 89:11102-11105 (1992).\",\n \"In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.\",\n \"Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.\",\n \"Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.\",\n \"Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.\",\n \"Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.\",\n \"Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.\",\n \"The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.\",\n \"Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.\",\n \"840-856).\",\n \"Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.\",\n \"Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.\",\n \"For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.\",\n \"Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.\",\n \"Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.\",\n \"The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.\",\n \"A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.\",\n \"In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.\",\n \"Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.\",\n \"Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.\",\n \"Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.\",\n \"The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.\",\n \"128:1968-1974, 1982; Handa et al., J. Immunol.\",\n \"135:1564-1572, 1985; Takai et al., I. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.\",\n \"153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.\",\n \"144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.\",\n \"J. E. e.a.\",\n \"Coligan eds.\",\n \"Vol 1 pp.\",\n \"3.8.1-3.8.16, John Wiley and Sons, Toronto.\",\n \"1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.\",\n \"137:3494-3500, 1986; Takai et al., J. Immunol.\",\n \"140:508-512, 1988; Bertagnolli et al., J. Immunol.\",\n \"149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.\",\n \"134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.\",\n \"A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.\",\n \"Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).\",\n \"Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.\",\n \"Administration of sufficient amounts of other inhibins can induce infertility in these mammals.\",\n \"Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.\",\n \"See, for example, U.S. Pat.\",\n \"No.\",\n \"4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods.\",\n \"Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.\",\n \"Chemotactic or chemokinetic compositions (e.g.\",\n \"proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.\",\n \"For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.\",\n \"A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.\",\n \"Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.\",\n \"Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.\",\n \"Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.\",\n \"Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.\",\n \"J. Clin.\",\n \"Invest.\",\n \"95:1370-1376, 1995; Lind et al.\",\n \"APMIS 103:140-146, 1995; Muller et al Eur.\",\n \"J. Immunol.\",\n \"25:1744-1748; Gruber et al.\",\n \"J. of Immunol.\",\n \"152:5860-5867, 1994; Johnston et al.\",\n \"J. of Immunol.\",\n \"153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.\",\n \"Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.\",\n \"A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).\",\n \"Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.\",\n \"Pharmacol.\",\n \"26:131-140, 1986; Burdick et al., Thrombosis Res.\",\n \"45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.\",\n \"Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.\",\n \"For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.\",\n \"Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.\",\n \"Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.\",\n \"Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.\",\n \"Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.\",\n \"Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.\",\n \"Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.\",\n \"reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.\",\n \"The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.\",\n \"An anticancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.\",\n \"The use of anti-cancer cocktails as a cancer treatment is routine.\",\n \"Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.\",\n \"In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.\",\n \"There are hereditary conditions and/or environmental situations (e.g.\",\n \"exposure to carcinogens) known in the art that predispose an individual to developing cancers.\",\n \"Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.\",\n \"In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.\",\n \"These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.\",\n \"Can.\",\n \"Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.\",\n \"J. Dev.\",\n \"Biol., 40: 1189-97 (1999) and Li et al., Clin.\",\n \"Exp.\",\n \"Metastasis, 17:423-9 (1999), respectively.\",\n \"Suitable tumor cells lines are available, e.g.\",\n \"from American Type Tissue Culture Collection catalogs.\",\n \"4.10.12 Receptor/Ligand Activity A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.\",\n \"A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.\",\n \"Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.\",\n \"Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.\",\n \"A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.\",\n \"The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology; Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.\",\n \"Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 84:6864-6868, 1987; Bierer et al., J. Exp.\",\n \"Med.\",\n \"168:1145-1156, 1988; Rosenstein et al., J. Exp.\",\n \"Med.\",\n \"169:149-160 1989; Stoltenborg et al., J. Immunol.\",\n \"Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).\",\n \"Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.\",\n \"Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.\",\n \"The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.\",\n \"(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.\",\n \"182 (1990) Academic Press, Inc. San Diego).\",\n \"Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.\",\n \"Examples of toxins include, but are not limited, to ricin.\",\n \"4.10.13 Drug Screening This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.\",\n \"The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.\",\n \"One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.\",\n \"Drugs are screened against such transformed cells in competitive binding assays.\",\n \"Such cells, either in viable or fixed form, can be used for standard binding assays.\",\n \"One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.\",\n \"Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.\",\n \"Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.\",\n \"The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.\",\n \"Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.\",\n \"For a review, see Science 282:63-68 (1998).\",\n \"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.\",\n \"Of particular interest are peptide and oligonucleotide combinatorial libraries.\",\n \"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.\",\n \"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.\",\n \"Opin.\",\n \"Biotechnol.\",\n \"8:701-707 (1997).\",\n \"For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.\",\n \"Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1 (1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).\",\n \"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.\",\n \"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.\",\n \"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.\",\n \"The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.\",\n \"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.\",\n \"Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.\",\n \"4.10.14 Assay for Receptor Activity The invention also provides methods to detect specific binding of a polypeptide e.g.\",\n \"a ligand or a receptor.\",\n \"The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.\",\n \"For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.\",\n \"As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.\",\n \"There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.\",\n \"Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.\",\n \"The responses of the two cell populations to the addition of ligands(s) are then compared.\",\n \"Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).\",\n \"As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.\",\n \"The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.\",\n \"For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.\",\n \"The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.\",\n \"Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.\",\n \"phosphorylation.\",\n \"Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.\",\n \"4.10.15 Anti-Inflammatory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.\",\n \"The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.\",\n \"Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.\",\n \"Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.\",\n \"4.10.16 Leukemias Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.\",\n \"Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).\",\n \"4.10.17 Nervous System Disorders Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.\",\n \"Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.\",\n \"Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.\",\n \"For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.\",\n \"Such effects may be measured by any method known in the art.\",\n \"In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.\",\n \"(1990, J. Neurosci.\",\n \"10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.\",\n \"(1980, Exp.\",\n \"Neurol.\",\n \"70:65-82) or Brown et al.\",\n \"(1981, Ann.\",\n \"Rev.\",\n \"Neurosci.\",\n \"4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.\",\n \"In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).\",\n \"4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.\",\n \"4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.\",\n \"Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.\",\n \"For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.\",\n \"Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.\",\n \"For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.\",\n \"Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).\",\n \"In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.\",\n \"Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.\",\n \"The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.\",\n \"In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.\",\n \"Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.\",\n \"4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.\",\n \"The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.\",\n \"Arch.\",\n \"Allergy Appl.\",\n \"Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).\",\n \"The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.\",\n \"The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.\",\n \"The control consists of administering PBS only.\",\n \"The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.\",\n \"An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.\",\n \"4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.\",\n \"Examples of therapeutic applications include, but are not limited to, those exemplified herein.\",\n \"4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.\",\n \"While the mode of administration is not particularly important, parenteral administration is preferred.\",\n \"An exemplary mode of administration is to deliver an intravenous bolus.\",\n \"The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.\",\n \"It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.\",\n \"Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.\",\n \"For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.\",\n \"Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.\",\n \"The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.\",\n \"The preparation of such solutions is within the skill of the art.\",\n \"4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.\",\n \"Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.\",\n \"The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).\",\n \"The characteristics of the carrier will depend on the route of administration.\",\n \"The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.\",\n \"In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.\",\n \"The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.\",\n \"Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.\",\n \"Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).\",\n \"A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.\",\n \"As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.\",\n \"As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).\",\n \"Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.\",\n \"A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.\",\n \"When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.\",\n \"When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.\",\n \"In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.\",\n \"Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.\",\n \"When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.\",\n \"If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.\",\n \"4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.\",\n \"Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.\",\n \"Intravenous administration to the patient is preferred.\",\n \"Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.\",\n \"In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.\",\n \"Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.\",\n \"The liposomes will be targeted to and taken up selectively by the afflicted tissue.\",\n \"The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.\",\n \"The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.\",\n \"Preferably for wound treatment, one administers the therapeutic compound directly to the site.\",\n \"Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.\",\n \"Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.\",\n \"4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.\",\n \"These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.\",\n \"Proper formulation is dependent upon the route of administration chosen.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.\",\n \"When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.\",\n \"The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.\",\n \"When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.\",\n \"The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.\",\n \"When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active-ingredient of the present invention.\",\n \"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.\",\n \"The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.\",\n \"A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.\",\n \"The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.\",\n \"For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.\",\n \"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.\",\n \"Such penetrants are generally known in the art.\",\n \"For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.\",\n \"Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.\",\n \"Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.\",\n \"Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).\",\n \"If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.\",\n \"Dragee cores are provided with suitable coatings.\",\n \"For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.\",\n \"Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.\",\n \"Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.\",\n \"The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.\",\n \"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.\",\n \"In addition, stabilizers may be added.\",\n \"All formulations for oral administration should be in dosages suitable for such administration.\",\n \"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.\",\n \"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.\",\n \"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.\",\n \"Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.\",\n \"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.\",\n \"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.\",\n \"The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.\",\n \"Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.\",\n \"Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.\",\n \"Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.\",\n \"Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.\",\n \"Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.\",\n \"Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.\",\n \"The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.\",\n \"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.\",\n \"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.\",\n \"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.\",\n \"A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.\",\n \"The co-solvent system may be the VPD co-solvent system.\",\n \"VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.\",\n \"The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.\",\n \"This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.\",\n \"Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.\",\n \"Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.\",\n \"polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.\",\n \"Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.\",\n \"Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.\",\n \"Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.\",\n \"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.\",\n \"Various types of sustained-release materials have been established and are well known by those skilled in the art.\",\n \"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.\",\n \"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.\",\n \"The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.\",\n \"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.\",\n \"Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.\",\n \"Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.\",\n \"The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.\",\n \"The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.\",\n \"B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.\",\n \"T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.\",\n \"MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.\",\n \"The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.\",\n \"Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.\",\n \"The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.\",\n \"Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.\",\n \"Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.\",\n \"Nos.\",\n \"4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.\",\n \"The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.\",\n \"Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.\",\n \"Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.\",\n \"Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.\",\n \"It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.\",\n \"For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.\",\n \"When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.\",\n \"Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.\",\n \"Topical administration may be suitable for wound healing and tissue repair.\",\n \"Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.\",\n \"Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.\",\n \"Such matrices may be formed of materials presently in use for other implanted medical applications.\",\n \"The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.\",\n \"The particular application of the compositions will define the appropriate formulation.\",\n \"Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.\",\n \"Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.\",\n \"Further matrices are comprised of pure proteins or extracellular matrix components.\",\n \"Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.\",\n \"Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.\",\n \"The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.\",\n \"Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.\",\n \"In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.\",\n \"A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).\",\n \"Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).\",\n \"The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.\",\n \"In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.\",\n \"These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).\",\n \"The therapeutic compositions are also presently valuable for veterinary applications.\",\n \"Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.\",\n \"The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.\",\n \"The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.\",\n \"For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.\",\n \"Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.\",\n \"Polynucleotides of the present invention can also be used for gene therapy.\",\n \"Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.\",\n \"Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).\",\n \"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.\",\n \"Treated cells can then be introduced in vivo for therapeutic purposes.\",\n \"4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.\",\n \"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.\",\n \"Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.\",\n \"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.\",\n \"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.\",\n \"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).\",\n \"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.\",\n \"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.\",\n \"The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.\",\n \"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.\",\n \"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.\",\n \"See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.\",\n \"1 p. 1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).\",\n \"The MEC will vary for each compound but can be estimated from in vitro data.\",\n \"Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.\",\n \"However, HPLC assays or bioassays can be used to determine plasma concentrations.\",\n \"Dosage intervals can also be determined using MEC value.\",\n \"Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.\",\n \"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.\",\n \"An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.\",\n \"Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.\",\n \"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.\",\n \"4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.\",\n \"The pack may, for example, comprise metal or plastic foil, such as a blister pack.\",\n \"The pack or dispenser device may be accompanied by instructions for administration.\",\n \"Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.\",\n \"4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.\",\n \"The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.\",\n \"Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library.\",\n \"In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.\",\n \"Certain classes have subclasses as well, such as IgG1, IgG2, and others.\",\n \"Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.\",\n \"Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.\",\n \"An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.\",\n \"The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.\",\n \"An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of any of the full length proteins of the invention, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.\",\n \"Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.\",\n \"Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.\",\n \"In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention, e.g., a hydrophilic region.\",\n \"A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.\",\n \"As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.\",\n \"See, e.g., Hopp and Woods, 1981, Proc.\",\n \"Nat.\",\n \"Acad.\",\n \"Sci USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.\",\n \"Biol.\",\n \"157: 105-142, each of which is incorporated herein by reference in its entirety.\",\n \"Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.\",\n \"A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.\",\n \"Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).\",\n \"Some of these antibodies are discussed below.\",\n \"5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.\",\n \"An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.\",\n \"Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.\",\n \"Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.\",\n \"The preparation can further include an adjuvant.\",\n \"Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.\",\n \"), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.\",\n \"Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).\",\n \"The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.\",\n \"Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.\",\n \"Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.\",\n \"14, No.\",\n \"8 (Apr.\",\n \"17, 2000), pp.\",\n \"25-28).\",\n \"5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.\",\n \"In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.\",\n \"MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.\",\n \"Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).\",\n \"In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.\",\n \"Alternatively, the lymphocytes can be immunized in vitro.\",\n \"The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.\",\n \"Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.\",\n \"The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.\",\n \"59-103).\",\n \"Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.\",\n \"Usually, rat or mouse myeloma cell lines are employed.\",\n \"The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.\",\n \"For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.\",\n \"Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.\",\n \"More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.\",\n \"Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.\",\n \"51-63).\",\n \"The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.\",\n \"Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).\",\n \"Such techniques and assays are known in the art.\",\n \"The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.\",\n \"Biochem., 107:220 (1980).\",\n \"Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.\",\n \"After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.\",\n \"Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.\",\n \"Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.\",\n \"The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.\",\n \"The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.\",\n \"No.\",\n \"4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).\",\n \"The hybridoma cells of the invention serve as a preferred source of such DNA.\",\n \"Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.\",\n \"The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.\",\n \"No.\",\n \"4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.\",\n \"Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.\",\n \"5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.\",\n \"These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.\",\n \"Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.\",\n \"Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.\",\n \"(See also U.S. Pat.\",\n \"No.\",\n \"5,225,539.)\",\n \"In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.\",\n \"Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.\",\n \"In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.\",\n \"The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.\",\n \"Op.\",\n \"Struct.\",\n \"Biol., 2:593-596 (1992)).\",\n \"5.133 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.\",\n \"Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.\",\n \"Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.\",\n \"77-96).\",\n \"In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.\",\n \"Biol., 227:381 (1991); Marks et al., J. Mol.\",\n \"Biol., 222:581 (1991)).\",\n \"Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.\",\n \"Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.\",\n \"This approach is described, for example, in U.S. Pat.\",\n \"Nos.\",\n \"5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et at.\",\n \"(Bio/Technology 10, 779-783 (1992)); Lonberg et al.\",\n \"(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.\",\n \"Rev.\",\n \"Immunol.\",\n \"13 65-93 (1995)).\",\n \"Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.\",\n \"(See PCT publication WO94/02602).\",\n \"The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.\",\n \"The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.\",\n \"An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.\",\n \"The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.\",\n \"The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.\",\n \"Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.\",\n \"An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.\",\n \"A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.\",\n \"No.\",\n \"5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.\",\n \"The hybrid cell expresses an antibody containing the heavy chain and the light chain.\",\n \"In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.\",\n \"No.\",\n \"4,946,778).\",\n \"In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.\",\n \"Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.\",\n \"5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.\",\n \"In the present case, one of the binding specificities is for an antigenic protein of the invention.\",\n \"The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.\",\n \"Methods for making bispecific antibodies are known in the art.\",\n \"Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).\",\n \"Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.\",\n \"The purification of the correct molecule is usually accomplished by affinity chromatography steps.\",\n \"Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.\",\n \"The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.\",\n \"It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.\",\n \"DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.\",\n \"For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).\",\n \"According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.\",\n \"The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.\",\n \"In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g tyrosine or tryptophan).\",\n \"Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.\",\n \"alanine or threonine).\",\n \"This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.\",\n \"Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.\",\n \"F(ab′)2 bispecific antibodies).\",\n \"Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.\",\n \"For example, bispecific antibodies can be prepared using chemical linkage.\",\n \"Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.\",\n \"These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.\",\n \"The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.\",\n \"One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.\",\n \"The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.\",\n \"Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.\",\n \"Shalaby et al., J. Exp.\",\n \"Med.\",\n \"175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.\",\n \"Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.\",\n \"The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.\",\n \"Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.\",\n \"For example, bispecific antibodies have been produced using leucine zippers.\",\n \"Kostelny et al., J. Immunol.\",\n \"148(5):1547-1553 (1992).\",\n \"The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.\",\n \"The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.\",\n \"This method can also be utilized for the production of antibody homodimers.\",\n \"The “diabody” technology described by Hollinger et al., Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.\",\n \"The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.\",\n \"Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.\",\n \"Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.\",\n \"See, Gruber et al., J. Immunol.\",\n \"152:5368 (1994).\",\n \"Antibodies with more than two valencies are contemplated.\",\n \"For example, trispecific antibodies can be prepared.\",\n \"Tutt et al., J. Immunol.\",\n \"147:60 (1991).\",\n \"Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.\",\n \"Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.\",\n \"CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.\",\n \"Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.\",\n \"These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.\",\n \"Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).\",\n \"5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.\",\n \"Heteroconjugate antibodies are composed of two covalently joined antibodies.\",\n \"Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.\",\n \"No.\",\n \"4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).\",\n \"It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.\",\n \"For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.\",\n \"Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.\",\n \"No.\",\n \"4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.\",\n \"For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.\",\n \"The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).\",\n \"See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.\",\n \"Immunol., 148: 2918-2922 (1992).\",\n \"Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.\",\n \"Cancer Research, 53: 2560-2565 (1993).\",\n \"Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.\",\n \"See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).\",\n \"5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).\",\n \"Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.\",\n \"Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.\",\n \"A variety of radionuclides are available for the production of radioconjugated antibodies.\",\n \"Examples include 212Bi, 131I, 131In, 90Y, and 186Re.\",\n \"Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).\",\n \"For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).\",\n \"Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.\",\n \"See WO94/1 1026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.\",\n \"4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.\",\n \"As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.\",\n \"Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.\",\n \"A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"As used herein, “recorded” refers to a process for storing information on computer readable medium.\",\n \"A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.\",\n \"A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.\",\n \"The choice of the data storage structure will generally be based on the means chosen to access the stored information.\",\n \"In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.\",\n \"The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.\",\n \"A skilled artisan can readily adapt any number of data processor structuring formats (e.g.\",\n \"text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.\",\n \"By providing any of the nucleotide sequences SEQ ID NO: 1-8051 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-8051 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.\",\n \"Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.\",\n \"The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.\",\n \"Biol.\",\n \"215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.\",\n \"Chem.\",\n \"17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.\",\n \"Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.\",\n \"As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.\",\n \"The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.\",\n \"A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.\",\n \"As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.\",\n \"As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.\",\n \"As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.\",\n \"Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.\",\n \"A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.\",\n \"Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).\",\n \"A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.\",\n \"As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.\",\n \"A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.\",\n \"The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.\",\n \"However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.\",\n \"As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.\",\n \"There are a variety of target motifs known in the art.\",\n \"Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.\",\n \"Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).\",\n \"4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.\",\n \"Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem.\",\n \"56:560, (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.\",\n \"4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.\",\n \"In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.\",\n \"Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.\",\n \"In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.\",\n \"In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.\",\n \"Conditions for incubating a nucleic acid probe or antibody with a test sample vary.\",\n \"Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.\",\n \"One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.\",\n \"Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.\",\n \"1 (1982), Vol.\",\n \"2 (1983), Vol.\",\n \"3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).\",\n \"The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.\",\n \"The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.\",\n \"Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.\",\n \"In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.\",\n \"Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.\",\n \"In detail, a compartment kit includes any kit in which reagents are contained in separate containers.\",\n \"Such containers include small glass containers, plastic containers or strips of plastic or paper.\",\n \"Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.\",\n \"Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.\",\n \"), and containers which contain the reagents used to detect the bound antibody or probe.\",\n \"Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.\",\n \"One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.\",\n \"4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).\",\n \"See, e.g., Kunkel et al., U.S. Pat.\",\n \"No.\",\n \"5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.\",\n \"4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-8051, or bind to a specific domain of the polypeptide encoded by the nucleic acid.\",\n \"In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.\",\n \"In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.\",\n \"Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.\",\n \"Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).\",\n \"Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).\",\n \"Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.\",\n \"The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.\",\n \"The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.\",\n \"For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.\",\n \"Alternatively, agents may be rationally selected or designed.\",\n \"As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.\",\n \"For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.\",\n \"Freeman, NY (1992), pp.\",\n \"289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.\",\n \"In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.\",\n \"As described above, such agents can be randomly screened or rationally designed/selected.\",\n \"Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.\",\n \"One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.\",\n \"Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.\",\n \"Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.\",\n \"Acids Res.\",\n \"6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.\",\n \"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).\",\n \"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.\",\n \"Both techniques have been demonstrated to be effective in model systems.\",\n \"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.\",\n \"Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.\",\n \"Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.\",\n \"4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.\",\n \"The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-8051.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-8051 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.\",\n \"Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.\",\n \"PCR as described in U.S. Pat.\",\n \"Nos.\",\n \"4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.\",\n \"Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.\",\n \"The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.\",\n \"Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.\",\n \"Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.\",\n \"The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.\",\n \"The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.\",\n \"These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.\",\n \"The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.\",\n \"Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981 f).\",\n \"Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.\",\n \"The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.\",\n \"4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.\",\n \"Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.\",\n \"One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.\",\n \"Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.\",\n \"Microbiol.\",\n \"28 (6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol.\",\n \"Cell Probes 3 (2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.\",\n \"Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.\",\n \"For example, Broude et al.\",\n \"(1994) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA 91 (8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.\",\n \"Streptavidin-coated beads may be purchased from Dynal, Oslo.\",\n \"Of course, this same linking chemistry is applicable to coating any surface with streptavidin.\",\n \"Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).\",\n \"Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.\",\n \"Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.\",\n \"CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.\",\n \"CovaLink Modules may be purchased from Nunc Laboratories.\",\n \"DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.\",\n \"Biochem.\",\n \"198 (1) 13842).\",\n \"The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).\",\n \"In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.\",\n \"11 (8) 6513-29).\",\n \"This is beneficial as immobilization using only a single covalent bond is preferred.\",\n \"The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.\",\n \"To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.\",\n \"It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.\",\n \"More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.\",\n \"at 95° C. and cooling on ice for 10 min.\",\n \"Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIM7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.\",\n \"Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIM7, is made fresh and 25 ul added per well.\",\n \"The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).\",\n \"It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.\",\n \"This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.\",\n \"The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.\",\n \"Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.\",\n \"An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.\",\n \"For example, addressable laser-activated photodeprotection may be Fodor et al.\",\n \"(1991) Science 251 (4995) 767-73, incorporated herein by reference.\",\n \"Probes may also be immobilized on nylon supports as described by Van Ness et at (1991) Nucleic Acids Res.\",\n \"19 (12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.\",\n \"Biochem.\",\n \"169 (1) 104-8; all references being specifically incorporated herein.\",\n \"To link an oligonucleotide to a nylon support, as described by Van Ness et al.\",\n \"(1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.\",\n \"One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91 (11) 5022-6, incorporated herein by reference).\",\n \"These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).\",\n \"These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.\",\n \"A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.\",\n \"4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.\",\n \"For example, Sambrook et al.\",\n \"(1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).\",\n \"DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.\",\n \"Samples may be prepared or dispensed in multiwell plates.\",\n \"About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.\",\n \"The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.\",\n \"(1989), shearing by ultrasound and NaOH treatment.\",\n \"Low pressure shearing is also appropriate, as described by Schriefer et al.\",\n \"(1990) Nucleic Acids Res.\",\n \"18 (24) 7455-6, incorporated herein by reference).\",\n \"In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.\",\n \"A lever device allows controlled application of low to intermediate pressures to the cell.\",\n \"The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.\",\n \"One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.\",\n \"(1992) Nucleic Acids Res.\",\n \"20 (14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.\",\n \"The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.\",\n \"Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).\",\n \"Fitzgerald et al.\",\n \"(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.\",\n \"Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.\",\n \"As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.\",\n \"Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.\",\n \"This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.\",\n \"Phosphate groups must also be removed from genomic DNA by methods known in the art.\",\n \"4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.\",\n \"Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.\",\n \"By offset printing, a density of dots higher than the density of the wells is achieved.\",\n \"One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.\",\n \"By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.\",\n \"Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.\",\n \"Each of the subarrays may represent replica spotting of the same samples.\",\n \"In one example, a selected gene segment may be amplified from 64 patients.\",\n \"For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).\",\n \"A plate for each of the 64 patients is prepared.\",\n \"By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.\",\n \"Subarrays may contain 64 samples, one from each patient.\",\n \"Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.\",\n \"Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.\",\n \"a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.\",\n \"A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.\",\n \"The present invention is illustrated in the following examples.\",\n \"Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.\",\n \"Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.\",\n \"The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.\",\n \"Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.\",\n \"Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.\",\n \"All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.\",\n \"5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.\",\n \"The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.\",\n \"Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.\",\n \"The clones were clustered into groups of similar or identical sequences.\",\n \"Representative clones were selected for sequencing.\",\n \"In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.\",\n \"PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.\",\n \"Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.\",\n \"In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.\",\n \"5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.\",\n \"The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-8051 and are provided in the attached Sequence Listing.\",\n \"The contigs were assembled using an EST sequence as a seed.\",\n \"Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.\",\n \"The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.\",\n \"Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.\",\n \"The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-8051) of the present invention are incorporated in the attached Sequence Listing.\",\n \"A subset of the predicted polypeptide sequences contain an unknown amino acid; a stop codon; a possible nucleotide deletion; or a possible nucleotide insertion.\",\n \"These sequences have also been shown in their entirety in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-8051.Table 2 also indicates the method by which the polypeptide was predicted.\",\n \"Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fasta.bioch.virginia,edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).\",\n \"Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.\",\n \"Biol., 268:78-94 (1997), incorporated herein by reference).\",\n \"Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.\",\n \"The nearest neighbor results for SEQ ID NO: 1-8051 were obtained by a BLASTX version 2.0al 19 MP-WashU search against Genpept release 123 and Geneseq release 200110 (Derwent), using BLAST algorithm.\",\n \"The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-8051.The nearest neighbor results for SEQ ID NO: 1-8051, having identifiable function(s) are incorporated in the attached Sequence Listing.\",\n \"Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.\",\n \"Biol., Vol.\",\n \"6 pp.\",\n \"219-235 (1999) herein incorporated by reference), all the polypeptide sequences were examined to determine whether they had identifiable signature regions.\",\n \"The attached Sequence Listing provides the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.\",\n \"Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.\",\n \"26 (1) pp.\",\n \"320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.\",\n \"The attached Sequence Listing provides the results obtained by pFam analysis for each polypeptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.\",\n \"Tables 1 and 2 follow.\",\n \"Table 1 shows the various tissue sources of SEQ ID NO: 1-8051.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.\",\n \"Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.\",\n \"No.\",\n \"09/577,408.LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00001 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00002 Please refer to the end of the specification for access instructions.\",\n \"LENGTHY TABLE The patent application contains a lengthy table section.\",\n \"A copy of the table is available in electronic form from the USPTO web site ().\",\n \"An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10276817"}}},{"rowIdx":88,"cells":{"text":{"kind":"list like","value":[["Automatic scanning system, shoppers scanpass system","The checkout system described herein is to show a system how to eliminate the lines at the regiters, to eliminate the removal of items from shopping cart in order to “ring” them at the checkout counters.","As clients lowering their goods into shopping carts, an auto scanner mounted at the top of the cart shall trigger a beam to scan the items which are lower into the cart.","Once client completes his shopping he than read the total amount of sale which can be verified at the top of the shopping cart.","The cart with all the intended purchased items is wheeled to a checkout areas, in which the total amount is paid either by cash or credit cards."],["1.Checkout system eliminating the lines at the register, so to eliminate the removal of items from shopping carts in order to “ring” them at the checkout counters As clients lowering their goods into shopping carts, an automatic scanner mounted at the top of the carts shall trigger a beam to scan the items which are lowered into the carts."],["Systematic design for most retail establishments which “ring” their items thru a checkout counter, where clients/shoppers can conduct the process of shopping without the standard waiting on lines in front of cash registers.","DESCRIPTION Items meanning any commodity which can be handled mostly by hand, as a self service method of purchase, within retail establishment such as supermarkets, retail chains stores, retail chain outlets of any type of marketing in a self-service system, can benefit greatly by using this system.","Any items which are for the most part are picked up from the selling offering area and are lower into shopping wagons by clients who wheel the wanted goods to the cash registers counters.","Shopping cart.","Specifically designed as carriers and for an automatic instant scanning for all items which are lowered into them.","An automatically scanning devise installed at the top brim, frame of shopping cart that electronically sensetive to the coded pricing tags attached to every item.","Once an item which carried the coded price tag is passed thru the top frame of the shopping cart and lower onto cart will instantally trigger the pricing scanner installed at their top Wagons O Matic Pricing Scanner.","Wagons, Carts are made of a type of materials, components and designed specifically to be used as a carriers and scanners.","The designed wagons/carts will described in a separate chapter.","Shoppers will have the ability to monitor each item by the unit pricing as well as the price itself thru a scanner window installed at the top of each shopping cart.","In the event of a reversed scanning such as a removal of an item from cart, the scanner installed at the top frame of cart will automatically deduct the amount from the total desired purchase.","(By pressing a botton which says CANCEL).","A processing checkout booths shall be constructed at the location designated to receive all carts filled with goods which are wheeled into specifically designed Cashiers scanning booths.","Each booth equipped with a remote scanner shall beam directly into the “total price” window located at the top of each shopping cart.","A total amount of sale shall be displayed and announced for each individual shopping cart.","Scanning booths shall be equipped with slots machines mounted on their walls to recieve payment for each purchase by cash, credit cards or store cards.","Customers shall be instructed to insert cards at appropriate slot machines.","Once payments accepted a gate/door will be opened for each shopping cart to be wheeled out into a packing booths or a similar packing counter.","At each gate/door a push button shall activate a trigger for the total sale reciept.","Each time door/gate is opened a cc tv system will monitor that individual shopping cart.","All Check—points of entries and exits shall be monitored by a cc tv systems.","An optional checkout cashier counters, “the old system” will still be operational for those who wish to be informed about various store applications as well as the privilege to converse with cashier in general.","The advantage use of the SCANPASS system is to reduce the long lines in front of each cash registers.","Shoppers will not leave unwanted items by registers.","There will be a dramatic reduction in shoplifting, probably not at all.","If store employees are engaged in theft, than any items not accounted for, unscanned will have to exit the back doors, an area which can be easily monitored at all hours.","Produce and related loose food items shall be packed and wrapped accordingly meaning by wanted desired volume, with a protected see thru wrap, bag which eliminate the direct contact of skin.","Each wrapped produced, food bag will carry a scanning tag.","and be protected against an outside contact of foreign bacterias, as well as human breath, speach, cough, hair, etc.","No item could be carried out of the store undetected unpaid or unaccounted for, since the scanned tag attached will always trigger the alarm systematic devise on the way out.","And if any attempt shall be made to carry the item out of the store thru the SCANPASS exit (gate), then simply the gate will not open.","Type of “labels” attached to all products will be of a design that applicable to the size and shape of the item, and be constructed as a wrap around ribbon with a thin cord inserted or attached to it, visible from all directions for scanning detections and if item relatively large in size than 2 or three scanning labels shall be attached.","Once all shopping carts are moved into the bagging counters which shall be supported by store employees or upon shoppers choice to bag their items themselves, A conveyor belt shall be constructed at that area for all carts to be wheeled on and eventually be carried to the the front store entrance.","SCANPASS System An Automatic SCANPASS System.","Specifically designed/Systematic design for retail establishments which “ring” their items thru a checkout counter, where clients/shoppers conduct the process of shopping without the standard of waiting on lines in front of the cash registers.","and use the specifically special designed built shopping cart/wagon equipped with an automatic scanner at the top of its frame to scan/trigger all items which are entering, lowering into wagons/carts.","SCANPASS To be registered as a trade mark.","And said title shall be recognized as a title for the system described herein on page 5 and on exhibit E. Wagon O Matic System Wagon O Matic Pricing Scanner.","Specifically designed shopping wagon equipped with an automatic scanner/trigger, mounted at the top of its frame to scan/trigger all item which are lowered into the wagon.","Wagon O Matic To be registered as a trade mark.","and said title shall be recognized as a title for the system described herein on page 6 and on exhibit F."]],"string":"[\n [\n \"Automatic scanning system, shoppers scanpass system\",\n \"The checkout system described herein is to show a system how to eliminate the lines at the regiters, to eliminate the removal of items from shopping cart in order to “ring” them at the checkout counters.\",\n \"As clients lowering their goods into shopping carts, an auto scanner mounted at the top of the cart shall trigger a beam to scan the items which are lower into the cart.\",\n \"Once client completes his shopping he than read the total amount of sale which can be verified at the top of the shopping cart.\",\n \"The cart with all the intended purchased items is wheeled to a checkout areas, in which the total amount is paid either by cash or credit cards.\"\n ],\n [\n \"1.Checkout system eliminating the lines at the register, so to eliminate the removal of items from shopping carts in order to “ring” them at the checkout counters As clients lowering their goods into shopping carts, an automatic scanner mounted at the top of the carts shall trigger a beam to scan the items which are lowered into the carts.\"\n ],\n [\n \"Systematic design for most retail establishments which “ring” their items thru a checkout counter, where clients/shoppers can conduct the process of shopping without the standard waiting on lines in front of cash registers.\",\n \"DESCRIPTION Items meanning any commodity which can be handled mostly by hand, as a self service method of purchase, within retail establishment such as supermarkets, retail chains stores, retail chain outlets of any type of marketing in a self-service system, can benefit greatly by using this system.\",\n \"Any items which are for the most part are picked up from the selling offering area and are lower into shopping wagons by clients who wheel the wanted goods to the cash registers counters.\",\n \"Shopping cart.\",\n \"Specifically designed as carriers and for an automatic instant scanning for all items which are lowered into them.\",\n \"An automatically scanning devise installed at the top brim, frame of shopping cart that electronically sensetive to the coded pricing tags attached to every item.\",\n \"Once an item which carried the coded price tag is passed thru the top frame of the shopping cart and lower onto cart will instantally trigger the pricing scanner installed at their top Wagons O Matic Pricing Scanner.\",\n \"Wagons, Carts are made of a type of materials, components and designed specifically to be used as a carriers and scanners.\",\n \"The designed wagons/carts will described in a separate chapter.\",\n \"Shoppers will have the ability to monitor each item by the unit pricing as well as the price itself thru a scanner window installed at the top of each shopping cart.\",\n \"In the event of a reversed scanning such as a removal of an item from cart, the scanner installed at the top frame of cart will automatically deduct the amount from the total desired purchase.\",\n \"(By pressing a botton which says CANCEL).\",\n \"A processing checkout booths shall be constructed at the location designated to receive all carts filled with goods which are wheeled into specifically designed Cashiers scanning booths.\",\n \"Each booth equipped with a remote scanner shall beam directly into the “total price” window located at the top of each shopping cart.\",\n \"A total amount of sale shall be displayed and announced for each individual shopping cart.\",\n \"Scanning booths shall be equipped with slots machines mounted on their walls to recieve payment for each purchase by cash, credit cards or store cards.\",\n \"Customers shall be instructed to insert cards at appropriate slot machines.\",\n \"Once payments accepted a gate/door will be opened for each shopping cart to be wheeled out into a packing booths or a similar packing counter.\",\n \"At each gate/door a push button shall activate a trigger for the total sale reciept.\",\n \"Each time door/gate is opened a cc tv system will monitor that individual shopping cart.\",\n \"All Check—points of entries and exits shall be monitored by a cc tv systems.\",\n \"An optional checkout cashier counters, “the old system” will still be operational for those who wish to be informed about various store applications as well as the privilege to converse with cashier in general.\",\n \"The advantage use of the SCANPASS system is to reduce the long lines in front of each cash registers.\",\n \"Shoppers will not leave unwanted items by registers.\",\n \"There will be a dramatic reduction in shoplifting, probably not at all.\",\n \"If store employees are engaged in theft, than any items not accounted for, unscanned will have to exit the back doors, an area which can be easily monitored at all hours.\",\n \"Produce and related loose food items shall be packed and wrapped accordingly meaning by wanted desired volume, with a protected see thru wrap, bag which eliminate the direct contact of skin.\",\n \"Each wrapped produced, food bag will carry a scanning tag.\",\n \"and be protected against an outside contact of foreign bacterias, as well as human breath, speach, cough, hair, etc.\",\n \"No item could be carried out of the store undetected unpaid or unaccounted for, since the scanned tag attached will always trigger the alarm systematic devise on the way out.\",\n \"And if any attempt shall be made to carry the item out of the store thru the SCANPASS exit (gate), then simply the gate will not open.\",\n \"Type of “labels” attached to all products will be of a design that applicable to the size and shape of the item, and be constructed as a wrap around ribbon with a thin cord inserted or attached to it, visible from all directions for scanning detections and if item relatively large in size than 2 or three scanning labels shall be attached.\",\n \"Once all shopping carts are moved into the bagging counters which shall be supported by store employees or upon shoppers choice to bag their items themselves, A conveyor belt shall be constructed at that area for all carts to be wheeled on and eventually be carried to the the front store entrance.\",\n \"SCANPASS System An Automatic SCANPASS System.\",\n \"Specifically designed/Systematic design for retail establishments which “ring” their items thru a checkout counter, where clients/shoppers conduct the process of shopping without the standard of waiting on lines in front of the cash registers.\",\n \"and use the specifically special designed built shopping cart/wagon equipped with an automatic scanner at the top of its frame to scan/trigger all items which are entering, lowering into wagons/carts.\",\n \"SCANPASS To be registered as a trade mark.\",\n \"And said title shall be recognized as a title for the system described herein on page 5 and on exhibit E. Wagon O Matic System Wagon O Matic Pricing Scanner.\",\n \"Specifically designed shopping wagon equipped with an automatic scanner/trigger, mounted at the top of its frame to scan/trigger all item which are lowered into the wagon.\",\n \"Wagon O Matic To be registered as a trade mark.\",\n \"and said title shall be recognized as a title for the system described herein on page 6 and on exhibit F.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10292688"}}},{"rowIdx":89,"cells":{"text":{"kind":"list like","value":[["Thermoplastic Hydraulic Composition, Formed Article Prepared From The Composition By Hydration- Hardening And Method For Preparing The Hydration- Hardened Former Article","The object of the present invention is to produce general machine parts, OA machine part, and the like which can be produced by injection molding and which have excellent mechanical properties, thermal resistance, dimension stability and processability.","The thermoplastic hydraulic composition of the present invention comprises 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.","It is characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.","It is characterized in that the hydraulic composition preferably is a mixed powder which comprises from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less."],["1.A thermoplastic hydraulic composition that is formed into a given shape and then is hardened by removal of at least a part of a thermoplastic resin from the inside, followed by the introduction of moisture, which is characterized by comprising 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.","2.The thermoplastic hydraulic composition according to claim 1 further comprising at least one of from 1 to 200 parts by weight of a fiber and from 0.5 to 10.0 parts by weight of a releasing agent added.","3.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic composition is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.","4.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic powder is at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium alumino-ferrite, calcium phosphate, hemihydrate or anhydride gypsum and a powder of calcium oxide possessing a self-hardening property.","5.The thermoplastic hydraulic composition according to claim 1, wherein the non-hydraulic powder is at least one powder selected from a potassium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a flyash powder, a silica powder, a clay powder and a silica fume powder.","6.The thermoplastic hydraulic composition according to claim 1, wherein the thermoplastic resin is constituted of from 50 to 90 wt % of a thermoplastic low molecular compound and 50 to 10 wt % of a thermoplastic high molecular compound.","7.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic low molecular compound is selected from low melting point compounds of paraffin wax, montan wax, carnauba wax, a fatty acid ester, glycerite and modified wax.","8.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic high molecular compound is a single substance or a copolymer of two or more substances selected from polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.","9.A hydration-hardened formed article characterized by being obtained by forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape, subsequently removing the thermoplastic resin from the inside of the resulting formed article, and further introducing moisture into the formed article to harden it.","10.A hydration-hardened formed article characterized by being obtained by forming the hydraulic composition according to claim 1 into a given shape, subsequently removing the thermoplastic resin from the resulting formed article completely or removing only a thermoplastic low molecular compound, and further introducing moisture into the inside of the formed article to harden it.","11.A method for producing a hydrogen-hardened formed article, the method being characterized in that it comprises forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.","12.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic resin from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic high molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.","13.The method for producing a hydration-hardened formed article according to claim 11, wherein the degreasing step is carried out at a temperature of from 400 to 500° C. for a period of 12 hours or longer.","14.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.","15.The method for producing a hydration-hardened formed article according to claim 14, wherein the degreasing step is carried out at a temperature of 270° C. or lower for a period of 12 hours or shorter.","16.The method for producing a hydration-hardened formed article according to claim 11, wherein said curing step is carried out at a temperature of 80° C. or higher by any one of normal pressure steam curing, high pressure steam curing and hot water curing, or in combination of the normal pressure steam curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing."],[" BACKGROUND ART As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.","However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.","Hei-10-177099, Hei-10-177100 and Hei-11-233636.)","However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.","The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions."],["TECHNICAL FIELD The present invention relates to a thermoplastic hydraulic composition that allows an injection molding technique mainly employed for production of plastic molded articles and ceramic molded articles to be applicable even for hydraulic compositions, and to formed articles prepared from the hydraulic compositions by hydration-hardening.","The present invention relates more particularly to provide thermoplastic hydraulic compositions, formed articles thereof having excellent mechanical properties, thermal resistance, dimensional stability and processability, as well as being capable of being applied for, e.g., general machine parts and OA machine parts.","BACKGROUND ART As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.","However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.","Hei-10-177099, Hei-10-177100 and Hei-11-233636.)","However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.","The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions.","DISCLOSURE OF THE INVENTION The object of the present invention is to provide a thermoplastic hydraulic composition and a formed article prepared from the composition by hydration-hardening that make it possible to produce machine parts with a shape more complex than those obtained by conventional pressing or extrusion forming by allowing the above-mentioned hydraulic composition for machine parts to have a composition such that the resulting hydraulic composition can be injection molded and forming it.","The present inventors made intensive studies to achieve the above object and found that a thermoplastic hydraulic composition containing a thermoplastic resin, a hydraulic powder, a fiber and a releasing agent respectively in given amounts is of excellent injection moldability and that a molded article obtained by injection molding the thermoplastic hydraulic composition and hardening it by curing is excellent in mechanical properties, thermal properties and formability, thus achieving reproduction of a complex shape.","Specifically, the thermoplastic hydraulic composition of the present invention is characterized by containing 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition, and is preferably characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.","The foregoing hydraulic composition is preferably a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.","BEST MODE FOR CARRYING OUT OF THE INVENTION A more detailed description on the thermoplastic hydraulic composition of the present invention will be made below.","(1) Hydraulic Composition (1-1) In the thermoplastic hydraulic composition of the present invention, it is preferable that the hydraulic composition is used in an amount of from 500 to 1000 parts by weight, particularly from 650 to 750 parts by weight, based on 100 parts by weight of the two types of thermoplastic resins differing in melting point.","If less than 500 parts by weight, the strength of formed articles will be reduced, whereas when exceeding 1000 parts by weight, the amount of the thermoplastic resin incorporated will be reduced, resulting in deterioration of formability to cause a problem.","By the hydraulic composition is meant a powder that is hardened by water and preferably comprises at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium aluminoferrite, calcium phosphate, hemihydrate or anhydride gypsum, and a powder of calcium oxide possessing a self-hardening property.","The hydraulic powder preferably has an average particle diameter of from about 10 to about 40 μm, and preferably has a specific surface area by blaine of 2500 cm2/g or more from the viewpoint of securing a high strength of formed articles.","(1-2) The aforementioned hydraulic composition preferably is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less.","The aforementioned non-hydraulic powder indicates a powder that is incapable of hardening by itself even by its contact with water and includes a powder such that some of the components thereof are eluted in an alkaline or acid state or under a high-pressure steam atmosphere and then react with other eluted components to form a product.","Preferred as the non-hydraulic powder is at least one powder selected from a calcium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a fly ash powder, a silica powder, a clay powder and a silica fume powder.","The non-hydraulic powder has an average particle diameter at least one order, preferably at least two order, smaller than that of the hydraulic powder, but no lower limit is set unless the effect of the present invention is damaged.","(2) The thermoplastic resin is a resin which can obtain a plasticity to a degree such that it can be formed by heating and is a resin which can be used for extrusion forming and injection molding.","In the present invention, a hydraulic composition is melt kneaded together with the thermoplastic resin at a temperature not lower than the softening point of the thermoplastic resin to be formed into a pellet form, which is used as a raw material for the subsequent injection molding.","The pellet-formed raw material is melted and kneaded again within a heating cylinder mounted inside an injection molding machine and is filled into a mold by the injection device.","The mixture of the hydraulic composition and the thermoplastic resin filled into the mold is formed into a formed article through cooling and hardening of the thermoplastic resin, which can be removed from the mold.","Hydraulic compositions are generally provided with a flowability by water, but it takes a long time for them to be released from a mold.","Therefore, forming methods such as injection molding can not be applied for them.","Moreover, if the hydraulic compositions come into contact with water, a hydration reaction will proceed and therefore articles defectively formed can not be recycled.","However, a mixture of the aforementioned thermoplastic resin and the hydraulic composition can afford a shape to the hydraulic composition without use of water and can realize releasing from a mold in a short time.","In addition, since no water is used in a stage of forming, no hydration reaction of the hydraulic composition is started and therefore the mixture can be recycled multiple times if it has not been cured yet.","Since the hydraulic composition of the present invention uses no water during its forming, it is necessary to supply moisture after the forming.","The spaces between particles of the hydraulic composition in a formed article after injection molding are filled with a thermoplastic resin.","Therefore, if the formed article is left in such a condition, the supply of water to the inside of the formed article will be inhibited, resulting in an insufficient hydration reaction of the formed article.","For this reason, it is necessary to remove a part or the whole of the resin from the inside of the formed article to form a channel for water supply.","When removing the resin from the inside of a formed article, it is desirable to use two types of resins differing in melting point when considering minimizing the dimensional change of the formed article.","Use of two types of resins differing in melting point makes it possible to injection mold the thermoplastic hydraulic composition and to obtain a formed article with a complex shape because a low molecular resin melts at a relatively low temperature and enables the hydraulic composition to fluidize easily.","Such a low molecular resin starts to decompose at a relatively low temperature (about 100° C. in the case of some resins), but a high molecular resin starts to decompose at a relatively high temperature (about 200° C. in the case of some resins).","Accordingly, if the removal of resin components at an intermediate temperature, only the low molecular resin is removed from the inside of the formed article and the vacant spaces resulting from the removal of the low molecular resin becomes channels for moisture supply, which facilitates a hydration reaction in the inside of the formed article.","On the other hand, the high molecular resin remaining in the inside of the formed article instead of being decomposed exists in the inside of the formed article to inhibit, e.g., dimensional change of the formed article.","In addition, when the resin components are removed at a temperature not lower than the melting point of the high molecular resin, all of the low molecular resin and the high molecular resin are removed and a completely inorganic hardened article will be obtained.","In such a case, since the low molecular resin has a melting point different than that of the high molecular resin, the low molecular resin is removed first and then the high molecular resin is removed.","Such removal of the resin components with a time lag can inhibit dimensional change of formed articles.","The thermoplastic low molecular compound with a lower melting point preferably has a molecular weight of 200 to several thousands.","The thermoplastic high molecular compound with a higher melting point preferably has a molecular weight of 10000 or more.","It is desirable to set upper limits based on appropriate selection from the viewpoint of kneadability and the like, because the increase of molecular weight have a great influence to kneadability.","Regarding blending of the thermoplastic resin, it is preferable to blend a low molecular compound in an amount of from 50 to 90 wt %, as described above, and most preferably from 55 to 65 wt %.","The amount of the thermoplastic high molecular compound is preferably from 50 to 10 wt %, and more preferably from 45 to 35 wt %.","If the amount of the thermoplastic low molecular compound is less than 50 wt %, the channel for moisture supply is reduced and hydration proceeds insufficiently.","On the other hand, if it is more than 90 wt %, the dimensional change during removal from a mold will adversely become large.","(2-1) As the aforementioned thermoplastic low molecular compound, it is preferable to use a single substance or two or more substances selected from low melting point compounds including paraffin wax, montan wax, carnauba wax, fatty acid esters, glycerite, and modified wax.","(2-2) As the thermoplastic high molecular compound, used are a single substance or two or more substances of polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.","(3) The fiber is preferably added in an amount of from 1 to 200 parts by weight, and more preferably from 100 to 170 parts by weight based on 100 parts by weight of the thermoplastic resin.","When no fiber is added or when the amount of fiber added is less than 40 parts by weight, it may be impossible to improve the low impact strength and low tensile strength, which are inherent to hydraulic compositions.","If more than 200 parts by weight, it will have much influence to flowability to result in poor formability.","As the fiber, fibers of glass, carbon, aramid, polyamide, boron and the like and potassium titanate whisker can be used.","The length of the fiber is preferably from 0.1 to 6 mm, and more preferably from 3 to 6 mm.","The diameter of the fiber is preferably from 5 to 30 μm.","(4) The releasing agent is added preferably in an amount of from 0.5 to 10.0 parts by weight, and more preferably in an amount of from 2.0 to 3.0 parts by weight.","If less than 0.5 part by weight, the releasability from a mold will be poor.","If exceeding 10.0 parts by weight, the hydration reaction of the hydraulic composition may be inhibited.","As the releasing agent, stearic acid, stearyl alcohol, ethylene bisstearic acid amide, glycerol triester, glycerol monoester and the like can be used.","As other additives, alkylphenols, 2.6-tertialbutylparacresol, bisphenol A and the like for inhibiting oxidation of the thermoplastic resin can be used.","In addition, salicylic acid esters, benzene acid esters or the like may optionally be added as an ultraviolet absorber.","(5) Method for Producing Hardened Articles The hydration-hardened formed article of the present invention is a product produced by forming a thermoplastic hydraulic composition comprising a thermoplastic resin and a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently heating it at a temperature not lower than the melting point of the thermoplastic resin to remove the thermoplastic resin from the inside of the formed article (this operation is also called degreasing), and curing the resulting formed article to introduce moisture, thereby hydration-hardening the formed article.","The thermoplastic resin may be made up of one compound, but it is preferable to use one made up of two types of substances including a thermoplastic low molecular compound and a thermoplastic high molecular compound as described previously.","When the thermoplastic resin is constituted of such two types of compounds, preferred is degreasing at a temperature between the melting point of the thermoplastic low molecular compound having a lower melting point and the melting point of the thermoplastic high molecular compound having a higher melting point (270° C. or less).","This will remove only the thermoplastic low molecular compound from the inside of a formed article.","Introduction of moisture using the vacant spaces resulting from the degreasing as channels for moisture supply will increase the hydration reaction rate to cause hydration hardening.","Specifically, injection molding, extrusion forming, pressure forming and the like can be employed as a forming method.","Furthermore, regarding a degreasing method, when DEP and an ethylene-vinyl acetate copolymer are used as a low molecular compound and a high molecular compound, respectively, decomposition of DEP starts at 100° C. and ends at 190° C.; whereas the ethylene-vinyl acetate copolymer starts to decompose at 210° C. Accordingly, it is possible to selectively remove only DEP by heating formed articles at 200° C. When curing at a temperature of from 400 to 500° C., it is possible to remove these thermoplastic resins completely.","The time required for resin removal can be set optionally depending, for example, on the kind, incorporation amount, pressure and temperature of the resin.","When the combination of the resins described in the above example is degreased at 200° C. under atmospheric pressure, the degreasing ratio will reach about 70% in 3 hours and about 100% in 12 hours.","Further, curing may be carried out by a curing method such as a normal pressure steam curing, a high pressure steam curing and a hot water curing conducted alone at a temperature not lower than 80° C., or in combination of the normal pressure curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing.","As a degreasing method, methods other than heating can be applied and are exemplified by solvent extraction and a removal method using reduced pressure.","The following is a description on embodiments of the present invention.","EXPERIMENTAL EXAMPLE 1 A thermoplastic hydraulic composition was prepared by blending an ethylene-vinyl acetate copolymer resin (EVA, molecular weight: 30000 to 50000) as a thermoplastic high molecular compound, carnauba wax (molecular weight: 300 to 500) as a thermoplastic low molecular compound and stearic acid as a releasing agent, respectively in the ratios given in Table 1, to a powder resulting from mixing of portland cement (average particle diameter 20 μm) as a hydraulic powder and fly ash and silica powder as non-hydraulic powders, and then was kneaded with a hot roll at 140° C. for 45 minutes to yield pellets.","Subsequently, an injection molded article 120 mm long, 10 mm wide and 3 mm thick was obtained by use of the pellets.","The resulting unhardened molded article was degreased through a degreasing step by heating (1): at 200° C. for 12 hours, 2) at 500° C. for 12 hours) and then a molded article was produced by autoclave curing (175° C., 7 hours, 8.8 atm).","The molded article was tested for its flexural strength, deflection temperature under load, and coefficient of linear expansion and was compared.","The results are shown in Table 1. HDT Test (Test of Deflection Temperature under Load) .",".",".","According to JIS K 7191-2, Method A A specimen 120 mm long, 10 mm wide and 3 mm thick was prepared.","The specimen was held between supporting points with an interval of 100 mm and the temperature thereof was raised at a constant rate while a flexural stress of 1.8 MPa was applied downward to its center.","The temperature at which the deflection reached a standard deflection was used as the deflection temperature under load.","Coefficient of linear expansion .",".",".","According to ASTM D-648 A φ3×20 mm specimen was prepared and the coefficient of linear expansion was measured in the temperature range of from 30 to 80° C. using a push-rod type measuring device.","TABLE 1 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 1 2 3 4 5 1 2 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 37.0 40.4 105 110 22.5 ** 8.6 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 80 80 38 39 150 ** 250 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.","The results shown above reveal that all the molded articles of the present invention are superior in mechanical strength in terms of flexural strength to those of the Comparative Examples.","In addition, it is shown that the molded articles of the present invention have higher deflection temperatures under load and therefore are superior also in thermal resistance.","Moreover, the fact that the molded articles of the present invention have smaller coefficients of linear expansion shows that those molded articles are superior also in dimension stability.","On the other hand, the molded articles of Comparative Examples are inferior in all of these properties to those of the present invention.","In Comparative Example 2, cracks were formed during curing.","EXPERIMENTAL EXAMPLE 2 Using the same materials as those used in Experimental Example 1, a thermoplastic hydraulic composition with the composition shown in Table 2 was kneaded while being heated to 150° C. with a hot roll and formed into a pellet form.","Subsequently, the resultant was formed into a 120 mm long, 10 mm wide, 3 mm thick specimen using an injection machine and then the unhardened molded article was degreased through a degreasing step by heating ((1): at 200° C. for 12 hours, (2) at 500° C. for 12 hours).","A molded article thereafter was produced by normal pressure steam curing (100° C., 7 hours) and then compared its flexural strength, deflection temperature under load, and coefficient of linear expansion in the same way as Experimental Example 1.The results are shown in Table 2.TABLE 2 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 6 7 8 9 10 3 4 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 31.2 32.3 70.5 80.4 19.6 ** 6.0 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 100 100 50 48 179 ** 254 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.","The results shown above reveal that all the formed articles of the present invention are superior in mechanical strength measured as flexural strength to those of Comparative Examples.","Further, it is also shown that the formed articles of the present invention have higher deflection temperature under load and are superior in thermal resistance.","Furthermore, the fact that those formed article have small coefficients of linear expansion shows that those formed articles are also superior in dimensional stability.","On the other hand, the formed articles of the Comparative Examples are inferior in all of such properties to the formed articles of the present invention.","In Comparative Example 3, cracks were formed during curing.","As described above, the thermoplastic hydraulic composition of the present invention can be injection molded without addition of water.","When this composition is cured, general machine parts, OA machine parts and the like with excellent mechanical properties, thermal resistance, dimensional stability and processability can be produced.","By blending two types of thermoplastic resins differing in melting point and removing only a thermoplastic low molecular compound having a low melting point followed by curing, it is possible to promote the hydration reaction to the inside of formed articles and simultaneously to harden the formed articles while maintaining their shapes by the action of the remaining high molecular compounds having high melting points.","In such a manner, machine parts excellent in mechanical characteristics, thermal resistance, dimensional stability or the like can be produced.","Furthermore, curing after complete removal of the thermoplastic resin at higher temperatures makes it possible to further improve such properties of formed articles."]],"string":"[\n [\n \"Thermoplastic Hydraulic Composition, Formed Article Prepared From The Composition By Hydration- Hardening And Method For Preparing The Hydration- Hardened Former Article\",\n \"The object of the present invention is to produce general machine parts, OA machine part, and the like which can be produced by injection molding and which have excellent mechanical properties, thermal resistance, dimension stability and processability.\",\n \"The thermoplastic hydraulic composition of the present invention comprises 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.\",\n \"It is characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.\",\n \"It is characterized in that the hydraulic composition preferably is a mixed powder which comprises from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less.\"\n ],\n [\n \"1.A thermoplastic hydraulic composition that is formed into a given shape and then is hardened by removal of at least a part of a thermoplastic resin from the inside, followed by the introduction of moisture, which is characterized by comprising 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.\",\n \"2.The thermoplastic hydraulic composition according to claim 1 further comprising at least one of from 1 to 200 parts by weight of a fiber and from 0.5 to 10.0 parts by weight of a releasing agent added.\",\n \"3.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic composition is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.\",\n \"4.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic powder is at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium alumino-ferrite, calcium phosphate, hemihydrate or anhydride gypsum and a powder of calcium oxide possessing a self-hardening property.\",\n \"5.The thermoplastic hydraulic composition according to claim 1, wherein the non-hydraulic powder is at least one powder selected from a potassium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a flyash powder, a silica powder, a clay powder and a silica fume powder.\",\n \"6.The thermoplastic hydraulic composition according to claim 1, wherein the thermoplastic resin is constituted of from 50 to 90 wt % of a thermoplastic low molecular compound and 50 to 10 wt % of a thermoplastic high molecular compound.\",\n \"7.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic low molecular compound is selected from low melting point compounds of paraffin wax, montan wax, carnauba wax, a fatty acid ester, glycerite and modified wax.\",\n \"8.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic high molecular compound is a single substance or a copolymer of two or more substances selected from polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.\",\n \"9.A hydration-hardened formed article characterized by being obtained by forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape, subsequently removing the thermoplastic resin from the inside of the resulting formed article, and further introducing moisture into the formed article to harden it.\",\n \"10.A hydration-hardened formed article characterized by being obtained by forming the hydraulic composition according to claim 1 into a given shape, subsequently removing the thermoplastic resin from the resulting formed article completely or removing only a thermoplastic low molecular compound, and further introducing moisture into the inside of the formed article to harden it.\",\n \"11.A method for producing a hydrogen-hardened formed article, the method being characterized in that it comprises forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.\",\n \"12.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic resin from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic high molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.\",\n \"13.The method for producing a hydration-hardened formed article according to claim 11, wherein the degreasing step is carried out at a temperature of from 400 to 500° C. for a period of 12 hours or longer.\",\n \"14.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.\",\n \"15.The method for producing a hydration-hardened formed article according to claim 14, wherein the degreasing step is carried out at a temperature of 270° C. or lower for a period of 12 hours or shorter.\",\n \"16.The method for producing a hydration-hardened formed article according to claim 11, wherein said curing step is carried out at a temperature of 80° C. or higher by any one of normal pressure steam curing, high pressure steam curing and hot water curing, or in combination of the normal pressure steam curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing.\"\n ],\n [\n \" BACKGROUND ART As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.\",\n \"However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.\",\n \"Hei-10-177099, Hei-10-177100 and Hei-11-233636.)\",\n \"However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.\",\n \"The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions.\"\n ],\n [\n \"TECHNICAL FIELD The present invention relates to a thermoplastic hydraulic composition that allows an injection molding technique mainly employed for production of plastic molded articles and ceramic molded articles to be applicable even for hydraulic compositions, and to formed articles prepared from the hydraulic compositions by hydration-hardening.\",\n \"The present invention relates more particularly to provide thermoplastic hydraulic compositions, formed articles thereof having excellent mechanical properties, thermal resistance, dimensional stability and processability, as well as being capable of being applied for, e.g., general machine parts and OA machine parts.\",\n \"BACKGROUND ART As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.\",\n \"However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.\",\n \"Hei-10-177099, Hei-10-177100 and Hei-11-233636.)\",\n \"However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.\",\n \"The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions.\",\n \"DISCLOSURE OF THE INVENTION The object of the present invention is to provide a thermoplastic hydraulic composition and a formed article prepared from the composition by hydration-hardening that make it possible to produce machine parts with a shape more complex than those obtained by conventional pressing or extrusion forming by allowing the above-mentioned hydraulic composition for machine parts to have a composition such that the resulting hydraulic composition can be injection molded and forming it.\",\n \"The present inventors made intensive studies to achieve the above object and found that a thermoplastic hydraulic composition containing a thermoplastic resin, a hydraulic powder, a fiber and a releasing agent respectively in given amounts is of excellent injection moldability and that a molded article obtained by injection molding the thermoplastic hydraulic composition and hardening it by curing is excellent in mechanical properties, thermal properties and formability, thus achieving reproduction of a complex shape.\",\n \"Specifically, the thermoplastic hydraulic composition of the present invention is characterized by containing 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition, and is preferably characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.\",\n \"The foregoing hydraulic composition is preferably a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.\",\n \"BEST MODE FOR CARRYING OUT OF THE INVENTION A more detailed description on the thermoplastic hydraulic composition of the present invention will be made below.\",\n \"(1) Hydraulic Composition (1-1) In the thermoplastic hydraulic composition of the present invention, it is preferable that the hydraulic composition is used in an amount of from 500 to 1000 parts by weight, particularly from 650 to 750 parts by weight, based on 100 parts by weight of the two types of thermoplastic resins differing in melting point.\",\n \"If less than 500 parts by weight, the strength of formed articles will be reduced, whereas when exceeding 1000 parts by weight, the amount of the thermoplastic resin incorporated will be reduced, resulting in deterioration of formability to cause a problem.\",\n \"By the hydraulic composition is meant a powder that is hardened by water and preferably comprises at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium aluminoferrite, calcium phosphate, hemihydrate or anhydride gypsum, and a powder of calcium oxide possessing a self-hardening property.\",\n \"The hydraulic powder preferably has an average particle diameter of from about 10 to about 40 μm, and preferably has a specific surface area by blaine of 2500 cm2/g or more from the viewpoint of securing a high strength of formed articles.\",\n \"(1-2) The aforementioned hydraulic composition preferably is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less.\",\n \"The aforementioned non-hydraulic powder indicates a powder that is incapable of hardening by itself even by its contact with water and includes a powder such that some of the components thereof are eluted in an alkaline or acid state or under a high-pressure steam atmosphere and then react with other eluted components to form a product.\",\n \"Preferred as the non-hydraulic powder is at least one powder selected from a calcium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a fly ash powder, a silica powder, a clay powder and a silica fume powder.\",\n \"The non-hydraulic powder has an average particle diameter at least one order, preferably at least two order, smaller than that of the hydraulic powder, but no lower limit is set unless the effect of the present invention is damaged.\",\n \"(2) The thermoplastic resin is a resin which can obtain a plasticity to a degree such that it can be formed by heating and is a resin which can be used for extrusion forming and injection molding.\",\n \"In the present invention, a hydraulic composition is melt kneaded together with the thermoplastic resin at a temperature not lower than the softening point of the thermoplastic resin to be formed into a pellet form, which is used as a raw material for the subsequent injection molding.\",\n \"The pellet-formed raw material is melted and kneaded again within a heating cylinder mounted inside an injection molding machine and is filled into a mold by the injection device.\",\n \"The mixture of the hydraulic composition and the thermoplastic resin filled into the mold is formed into a formed article through cooling and hardening of the thermoplastic resin, which can be removed from the mold.\",\n \"Hydraulic compositions are generally provided with a flowability by water, but it takes a long time for them to be released from a mold.\",\n \"Therefore, forming methods such as injection molding can not be applied for them.\",\n \"Moreover, if the hydraulic compositions come into contact with water, a hydration reaction will proceed and therefore articles defectively formed can not be recycled.\",\n \"However, a mixture of the aforementioned thermoplastic resin and the hydraulic composition can afford a shape to the hydraulic composition without use of water and can realize releasing from a mold in a short time.\",\n \"In addition, since no water is used in a stage of forming, no hydration reaction of the hydraulic composition is started and therefore the mixture can be recycled multiple times if it has not been cured yet.\",\n \"Since the hydraulic composition of the present invention uses no water during its forming, it is necessary to supply moisture after the forming.\",\n \"The spaces between particles of the hydraulic composition in a formed article after injection molding are filled with a thermoplastic resin.\",\n \"Therefore, if the formed article is left in such a condition, the supply of water to the inside of the formed article will be inhibited, resulting in an insufficient hydration reaction of the formed article.\",\n \"For this reason, it is necessary to remove a part or the whole of the resin from the inside of the formed article to form a channel for water supply.\",\n \"When removing the resin from the inside of a formed article, it is desirable to use two types of resins differing in melting point when considering minimizing the dimensional change of the formed article.\",\n \"Use of two types of resins differing in melting point makes it possible to injection mold the thermoplastic hydraulic composition and to obtain a formed article with a complex shape because a low molecular resin melts at a relatively low temperature and enables the hydraulic composition to fluidize easily.\",\n \"Such a low molecular resin starts to decompose at a relatively low temperature (about 100° C. in the case of some resins), but a high molecular resin starts to decompose at a relatively high temperature (about 200° C. in the case of some resins).\",\n \"Accordingly, if the removal of resin components at an intermediate temperature, only the low molecular resin is removed from the inside of the formed article and the vacant spaces resulting from the removal of the low molecular resin becomes channels for moisture supply, which facilitates a hydration reaction in the inside of the formed article.\",\n \"On the other hand, the high molecular resin remaining in the inside of the formed article instead of being decomposed exists in the inside of the formed article to inhibit, e.g., dimensional change of the formed article.\",\n \"In addition, when the resin components are removed at a temperature not lower than the melting point of the high molecular resin, all of the low molecular resin and the high molecular resin are removed and a completely inorganic hardened article will be obtained.\",\n \"In such a case, since the low molecular resin has a melting point different than that of the high molecular resin, the low molecular resin is removed first and then the high molecular resin is removed.\",\n \"Such removal of the resin components with a time lag can inhibit dimensional change of formed articles.\",\n \"The thermoplastic low molecular compound with a lower melting point preferably has a molecular weight of 200 to several thousands.\",\n \"The thermoplastic high molecular compound with a higher melting point preferably has a molecular weight of 10000 or more.\",\n \"It is desirable to set upper limits based on appropriate selection from the viewpoint of kneadability and the like, because the increase of molecular weight have a great influence to kneadability.\",\n \"Regarding blending of the thermoplastic resin, it is preferable to blend a low molecular compound in an amount of from 50 to 90 wt %, as described above, and most preferably from 55 to 65 wt %.\",\n \"The amount of the thermoplastic high molecular compound is preferably from 50 to 10 wt %, and more preferably from 45 to 35 wt %.\",\n \"If the amount of the thermoplastic low molecular compound is less than 50 wt %, the channel for moisture supply is reduced and hydration proceeds insufficiently.\",\n \"On the other hand, if it is more than 90 wt %, the dimensional change during removal from a mold will adversely become large.\",\n \"(2-1) As the aforementioned thermoplastic low molecular compound, it is preferable to use a single substance or two or more substances selected from low melting point compounds including paraffin wax, montan wax, carnauba wax, fatty acid esters, glycerite, and modified wax.\",\n \"(2-2) As the thermoplastic high molecular compound, used are a single substance or two or more substances of polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.\",\n \"(3) The fiber is preferably added in an amount of from 1 to 200 parts by weight, and more preferably from 100 to 170 parts by weight based on 100 parts by weight of the thermoplastic resin.\",\n \"When no fiber is added or when the amount of fiber added is less than 40 parts by weight, it may be impossible to improve the low impact strength and low tensile strength, which are inherent to hydraulic compositions.\",\n \"If more than 200 parts by weight, it will have much influence to flowability to result in poor formability.\",\n \"As the fiber, fibers of glass, carbon, aramid, polyamide, boron and the like and potassium titanate whisker can be used.\",\n \"The length of the fiber is preferably from 0.1 to 6 mm, and more preferably from 3 to 6 mm.\",\n \"The diameter of the fiber is preferably from 5 to 30 μm.\",\n \"(4) The releasing agent is added preferably in an amount of from 0.5 to 10.0 parts by weight, and more preferably in an amount of from 2.0 to 3.0 parts by weight.\",\n \"If less than 0.5 part by weight, the releasability from a mold will be poor.\",\n \"If exceeding 10.0 parts by weight, the hydration reaction of the hydraulic composition may be inhibited.\",\n \"As the releasing agent, stearic acid, stearyl alcohol, ethylene bisstearic acid amide, glycerol triester, glycerol monoester and the like can be used.\",\n \"As other additives, alkylphenols, 2.6-tertialbutylparacresol, bisphenol A and the like for inhibiting oxidation of the thermoplastic resin can be used.\",\n \"In addition, salicylic acid esters, benzene acid esters or the like may optionally be added as an ultraviolet absorber.\",\n \"(5) Method for Producing Hardened Articles The hydration-hardened formed article of the present invention is a product produced by forming a thermoplastic hydraulic composition comprising a thermoplastic resin and a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently heating it at a temperature not lower than the melting point of the thermoplastic resin to remove the thermoplastic resin from the inside of the formed article (this operation is also called degreasing), and curing the resulting formed article to introduce moisture, thereby hydration-hardening the formed article.\",\n \"The thermoplastic resin may be made up of one compound, but it is preferable to use one made up of two types of substances including a thermoplastic low molecular compound and a thermoplastic high molecular compound as described previously.\",\n \"When the thermoplastic resin is constituted of such two types of compounds, preferred is degreasing at a temperature between the melting point of the thermoplastic low molecular compound having a lower melting point and the melting point of the thermoplastic high molecular compound having a higher melting point (270° C. or less).\",\n \"This will remove only the thermoplastic low molecular compound from the inside of a formed article.\",\n \"Introduction of moisture using the vacant spaces resulting from the degreasing as channels for moisture supply will increase the hydration reaction rate to cause hydration hardening.\",\n \"Specifically, injection molding, extrusion forming, pressure forming and the like can be employed as a forming method.\",\n \"Furthermore, regarding a degreasing method, when DEP and an ethylene-vinyl acetate copolymer are used as a low molecular compound and a high molecular compound, respectively, decomposition of DEP starts at 100° C. and ends at 190° C.; whereas the ethylene-vinyl acetate copolymer starts to decompose at 210° C. Accordingly, it is possible to selectively remove only DEP by heating formed articles at 200° C. When curing at a temperature of from 400 to 500° C., it is possible to remove these thermoplastic resins completely.\",\n \"The time required for resin removal can be set optionally depending, for example, on the kind, incorporation amount, pressure and temperature of the resin.\",\n \"When the combination of the resins described in the above example is degreased at 200° C. under atmospheric pressure, the degreasing ratio will reach about 70% in 3 hours and about 100% in 12 hours.\",\n \"Further, curing may be carried out by a curing method such as a normal pressure steam curing, a high pressure steam curing and a hot water curing conducted alone at a temperature not lower than 80° C., or in combination of the normal pressure curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing.\",\n \"As a degreasing method, methods other than heating can be applied and are exemplified by solvent extraction and a removal method using reduced pressure.\",\n \"The following is a description on embodiments of the present invention.\",\n \"EXPERIMENTAL EXAMPLE 1 A thermoplastic hydraulic composition was prepared by blending an ethylene-vinyl acetate copolymer resin (EVA, molecular weight: 30000 to 50000) as a thermoplastic high molecular compound, carnauba wax (molecular weight: 300 to 500) as a thermoplastic low molecular compound and stearic acid as a releasing agent, respectively in the ratios given in Table 1, to a powder resulting from mixing of portland cement (average particle diameter 20 μm) as a hydraulic powder and fly ash and silica powder as non-hydraulic powders, and then was kneaded with a hot roll at 140° C. for 45 minutes to yield pellets.\",\n \"Subsequently, an injection molded article 120 mm long, 10 mm wide and 3 mm thick was obtained by use of the pellets.\",\n \"The resulting unhardened molded article was degreased through a degreasing step by heating (1): at 200° C. for 12 hours, 2) at 500° C. for 12 hours) and then a molded article was produced by autoclave curing (175° C., 7 hours, 8.8 atm).\",\n \"The molded article was tested for its flexural strength, deflection temperature under load, and coefficient of linear expansion and was compared.\",\n \"The results are shown in Table 1. HDT Test (Test of Deflection Temperature under Load) .\",\n \".\",\n \".\",\n \"According to JIS K 7191-2, Method A A specimen 120 mm long, 10 mm wide and 3 mm thick was prepared.\",\n \"The specimen was held between supporting points with an interval of 100 mm and the temperature thereof was raised at a constant rate while a flexural stress of 1.8 MPa was applied downward to its center.\",\n \"The temperature at which the deflection reached a standard deflection was used as the deflection temperature under load.\",\n \"Coefficient of linear expansion .\",\n \".\",\n \".\",\n \"According to ASTM D-648 A φ3×20 mm specimen was prepared and the coefficient of linear expansion was measured in the temperature range of from 30 to 80° C. using a push-rod type measuring device.\",\n \"TABLE 1 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 1 2 3 4 5 1 2 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 37.0 40.4 105 110 22.5 ** 8.6 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 80 80 38 39 150 ** 250 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.\",\n \"The results shown above reveal that all the molded articles of the present invention are superior in mechanical strength in terms of flexural strength to those of the Comparative Examples.\",\n \"In addition, it is shown that the molded articles of the present invention have higher deflection temperatures under load and therefore are superior also in thermal resistance.\",\n \"Moreover, the fact that the molded articles of the present invention have smaller coefficients of linear expansion shows that those molded articles are superior also in dimension stability.\",\n \"On the other hand, the molded articles of Comparative Examples are inferior in all of these properties to those of the present invention.\",\n \"In Comparative Example 2, cracks were formed during curing.\",\n \"EXPERIMENTAL EXAMPLE 2 Using the same materials as those used in Experimental Example 1, a thermoplastic hydraulic composition with the composition shown in Table 2 was kneaded while being heated to 150° C. with a hot roll and formed into a pellet form.\",\n \"Subsequently, the resultant was formed into a 120 mm long, 10 mm wide, 3 mm thick specimen using an injection machine and then the unhardened molded article was degreased through a degreasing step by heating ((1): at 200° C. for 12 hours, (2) at 500° C. for 12 hours).\",\n \"A molded article thereafter was produced by normal pressure steam curing (100° C., 7 hours) and then compared its flexural strength, deflection temperature under load, and coefficient of linear expansion in the same way as Experimental Example 1.The results are shown in Table 2.TABLE 2 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 6 7 8 9 10 3 4 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 31.2 32.3 70.5 80.4 19.6 ** 6.0 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 100 100 50 48 179 ** 254 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.\",\n \"The results shown above reveal that all the formed articles of the present invention are superior in mechanical strength measured as flexural strength to those of Comparative Examples.\",\n \"Further, it is also shown that the formed articles of the present invention have higher deflection temperature under load and are superior in thermal resistance.\",\n \"Furthermore, the fact that those formed article have small coefficients of linear expansion shows that those formed articles are also superior in dimensional stability.\",\n \"On the other hand, the formed articles of the Comparative Examples are inferior in all of such properties to the formed articles of the present invention.\",\n \"In Comparative Example 3, cracks were formed during curing.\",\n \"As described above, the thermoplastic hydraulic composition of the present invention can be injection molded without addition of water.\",\n \"When this composition is cured, general machine parts, OA machine parts and the like with excellent mechanical properties, thermal resistance, dimensional stability and processability can be produced.\",\n \"By blending two types of thermoplastic resins differing in melting point and removing only a thermoplastic low molecular compound having a low melting point followed by curing, it is possible to promote the hydration reaction to the inside of formed articles and simultaneously to harden the formed articles while maintaining their shapes by the action of the remaining high molecular compounds having high melting points.\",\n \"In such a manner, machine parts excellent in mechanical characteristics, thermal resistance, dimensional stability or the like can be produced.\",\n \"Furthermore, curing after complete removal of the thermoplastic resin at higher temperatures makes it possible to further improve such properties of formed articles.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10296092"}}},{"rowIdx":90,"cells":{"text":{"kind":"list like","value":[["Combinations of enzyme inhibitor-containing preparations and the use thereof","The invention comprises a process wherein the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ) and of T cells as well is inhibited by the simultaneous and combined inhibition of the enzyme activity (I) of alanyl aminopeptidase and of dipeptidyl peptidase IV; (II) of dipeptidyl peptidase IV and of the angiotensin-converting enzyme; (III) of dipeptidyl peptidase IV and of prolyl oligopeptidase; and (IV) of dipeptidyl peptidase IV and of X-Pro-aminopeptidase to an extent which cannot be achieved by an application of a single one of said enzyme inhibitors, even at a higher dosage.","Although the said inhibitors exercise an influence on the very same process finally, i.e.","the DNA synthesis and, thus, the proliferation of immune cells, this effect is not complete and is not long lasting when a single inhibitor is applied.","From the functional overlap of enzymatic activities results an additive/superadditive inhibitory effect on the DNA synthesis and proliferation by the simultaneous inhibition of more than one of the above enzymes, as our data show.","Our invention shows that the simultaneous application of substances inhibiting the above enzymes or of corresponding preparations and administration forms, respectively, is well suitable for a therapy of autoimmune diseases and chronic diseases with an inflammatory genesis as well as for a treatment of rejection episodes after a transplantation."],["1.Use of inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having the same substrate specificity (DP IV-analogous enzyme activity) in combination with inhibitors of alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro-aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and/or of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) for a superadditive inhibition of an activation, DNA synthesis and proliferation of human T lymphocytes and mononuclear cells.","2.Use according to claim 1, wherein the inhibitors of DP IV are preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivative), corresponding derivatives, preferably dipeptide phosphonic acid diaryl estates, dipeptide boronic acids (e.g.","Pro-boro-Pro) and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably NE-4-nitrobenzyloxycarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure.","3.Use according to claim 1, wherein amino acid amides, e.g., NE-4-nitrobenzyloxy-carbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative, are preferably employed as the DP IV inhibitor.","4.Use according to claim 1, wherein Actinonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phosphinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, preferably act as inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-L-proline, the corresponding L-proline methyl esters, (2S,3R)-HAMH/(2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof preferably act as inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof preferably act as inhibitors of ACE; Postatin, Eurystatin A or B, Na-protected peptide aldehydes, preferably benzyloxy-carbonyl-L-prolyl-L-prolinal or benzyloxycarbonyl-L-thioprolyl-L-thioprolinal, Na-protected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyano-pyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof preferably act as inhibitors of POP (PEP).","5.Use of inhibitor combinations of claim 1 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.","6.Use of inhibitor combinations of claim 1 for a suppression of transplant rejection and for a therapy of tumor diseases.","7.Pharmaceutical preparations, comprising inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having DP IV-analogous enzyme activity in combination with inhibitors of any of the enzymes alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) and in combination with per se known carrier, additive and/or auxiliary substances.","8.Pharmaceutical preparation according to claim 7, comprising preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivatives) corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably Ne-4-nitrobenzyloxcarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure, as the inhibitors of DP IV.","9.Pharmaceutical preparation according to claim 7, comprising preferably amino acid amides, e.g.","Ne-4-nitrobenzyloxcarbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative as the inhibitors of DP IV.","10.Pharmaceutical preparation according to claim 7, comprising, as the inhibitors of APN, APP, ACE and POP (PEP), preferably Actnonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phoshinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, as the inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof as the inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof as inhibitors of ACE; Postatin, Eurystatin A or B, Naprotected peptide aldehydes, preferably benzyloxycarbonyl-L-prolyl-L-prolinal or benzyloxcarbonyl-L-thioprolyl-L-thioprolinal, Naprotected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyanopyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof as inhibitors of POP (PEP).","11.Pharmaceutical preparation according to claim 7 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.","12.Pharmaceutical preparation according to claim 7 for a systemic use of the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramusclar, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.","13.Pharmaceutical preparation according to claim 7 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.","14.Use of inhibitor combinations of claim 2 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.","15.Use of inhibitor combinations of claim 2 for a suppression of transplant rejection and for a therapy of tumor diseases.","16.Use of inhibitor combinations of claim 3 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.","17.Use of inhibitor combinations of claim 3 for a suppression of transplant rejection and for a therapy of tumor diseases.","18.Use of inhibitor combinations of claim 4 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.","19.Use of inhibitor combinations of claim 4 for a suppression of transplant rejection and for a therapy of tumor diseases.","20.Pharmaceutical preparation according to claim 8 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.","21.Pharmaceutical preparation according to claim 8 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.","22.Pharmaceutical preparation according to claim 8 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.","23.Pharmaceutical preparation according to claim 9 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.","24.Pharmaceutical preparation according to claim 9 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.","25.Pharmaceutical preparation according to claim 9 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.","26.Pharmaceutical preparation according to claim 10 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.","27.Pharmaceutical preparation according to claim 10 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.","28.Pharmaceutical preparation according to claim 10 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.","29.Pharmaceutical preparation according to claim 11 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, virginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.","30.Pharmaceutical preparation according to claim 11 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications."],["The present invention describes the combined inhibition of the activity of the enzymes Aminopeptidase N (APN; E.C.","3.4.11.2.; CD13), Dipeptidyl peptidase IV (DP IV; E.C.","3.4.14.5; CD26), Prolyl oligopeptidase (POP; Prolyl endopeptidase; PEP; E.C.","3.4.21.26), the membrane-adherent Aminopeptidase P (X-Pro-Aminopeptidase; APP; XPNPEP2; E.C.","3.4.11.9) and Angiotensin-converting Enzyme (Angiotensin-konvertierendes Enzym; ACE; CD156; E.C.","3.4.15.1;CD156) by a simultaneous application of respective specific inhibitors of the above-referenced enzymes on the basis of amino acid derivatives, peptides or peptide derivatives, by which the activation, the DNA synthesis and, thus, the proliferation of immune cells is suppressed.","It is applicable to all diseases showing autoimmune pathogenesis that the disease is based on, or consists of, an activation and proliferation of immune cells, in particular of autoreactive T cells.","The very same mechanisms effect the acute or chronic rejection episodes after a transplantation of organs.","It was shown that membrane-adherent peptidases as, for example, DP IV or APN play a key role in the process of activation and clonal expansion of immune cells, particularly of T lymphocytes [B. Fleischer: “CD26, a surface protease involved in T cell activation”, Immunology Today 1994, 15, 180-184; U. Lendeckel et al.",": “Role of alanyl peptidase in growth and function of human T cells”, International Journal of Molecular Medicine 1999, 4, 17-27; D. Riemann et al.",": “CD13—not just a marker in leukemia typing”, Immunology Today 1999, 20, 83-88].","Several functions of mitogene-stimulated mononuclear cells (mitogen-stimulierte mononukleäre Zellen; MNZ) or of enriched T lymphocytes as, for example, the DNA synthesis, production and secretion of immune-stimulating cytokines (IL2, IL6, IL12, IFN-γ) and helper functions for B cells (synthesis of IgG and IgM) may be inhibited in the presence of specific inhibitors DP IV and APN [E. Schön et al,: “The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes”, Biomed.","Biochim Acta 1985, 2, K9-K15; E. Schön et al.",": “The role of dipeptidyl peptidase IV in humane T lymphocyte activation, inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro”, Eur.","J. Immunol.","1987, 17, 1821-1826; D. Reinhold et al.",": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360; U. Lendeckel et al.",": “Induction of the membrane alanyl peptidase gene and surface expression in human T cells by mitogenic activation”, Biochem.","J.","1996, 319, 817-823; T. Kähne et al.",": “Dipeptidyl peptidase IV: A cell surface peptidase involved in regulating T cell growth (Review)”, Int.","J. Mol.","Med.","1999, 4, 3-15; U. Lendeckel et al.",": “Role of alanyl aminopeptidase in growth and function of human T cells (Review)”, Int.","J. Mol.","Med.","1999, 4, 17-27].","It is already known that the treatment of autoimmune diseases and rejection reactions after transplantations can be effected by an inhibition of dipeptidyl peptidase IV localized on immune cells by means of synthetic inhibitors.","Reference is made, for example, to EP 764 151 A1, WO 09529691, EP 731 789 A1, EP 528 858).","With respect to the enzymes aminopeptidase N, angiotensin-converting enzyme, X-Pro-aminopeptidase and prolyl oligopeptidase, such effects are not known up to now.","The invention is based on the surprising finding that the simultaneous inhibition of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, as well as (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase inhibits the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ), and of T cells as well, to an extent which cannot be achieved by the application of a single one of these enzyme inhibitors, even at a higher dosage.","Although the above-mentioned inhibitors finally exhibit an effect to the same process, i.e.","the DNA synthesis and, thus, the proliferation of immune cells, said effect is marked to a weaker extent and is not long lasting in the case of an application of single inhibitors.","Due to the functional overlap of the enzymatic activities of said enzymes, there can be observed a superadditive inhibitory effect on the DNA synthesis and proliferation resulting from the simultaneous inhibition of two or more of these enzymes, as can be concluded from our data.","Our invention shows that the simultaneous application of inhibitory substances of the above-mentioned enzymes or, respectively, of corresponding preparations and administration forms is well suitable for the therapy of autoimmune diseases and inflammatory diseases as well as for the treatment of rejection reactions after a transplantation.","In detail, the invention is based on the finding that the DNA synthesis of mononuclear cells (MNZ) and T cells is inhibited, in a superadditive way, by the simultaneous administration of inhibitors of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase.","The application of enzyme inhibitors is a new method and complementary therapy form for the treatment of the above-mentioned diseases.","The inhibitors of dipeptidyl peptidase IV, of aminopeptidase N, of prolyl oligopeptidase, of the angiotensin-converting enzyme and of X-Pro-aminopeptidase applied in accordance with the invention may be employed in pharmaceutically applicable formulation complexes as inhibitors, substrates, pseudo-substrates, peptides and peptide derivatives having inhibitory effect and as antibodies of said enzymes as well.","Preferred effectors for DP IV are, for example, Xaa-Pro-dipeptides, their corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and their salts, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (n=0-10), their corresponding derivatives and their salts and amino acid (Xaa)-amides, their corresponding derivatives and salts, wherein Xaa is an α-amino acid/imino acid or an α-amino acid/imino acid derivative, respectively, preferably Nε-4-nitrobenzylcarbonyl-L-lysin, -L-prolin, -L-tryptophan, -L-isoleucin, -L-valin, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine, and their derivatives serve as the amide structure.","Such compounds and their preparation were described in a prior patent (K. Neubert et al., DD 296 075 A5).","The inhibitors are administered simultaneously with known carrier substances.","The administration is conducted, on one hand, as a topical application in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles, respectively, including instillative applications and, on the other hand, systemic applications for an oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular administration in suitable recipes and in a suitable galenic application form, respectively.","WORKING EXAMPLES Example 1 Inhibition of the DNA Synthesis of Human T Lymphocytes by Incubation with Synthetic Inhibitors of DP IV and of APN Our investigations showed that the DNA synthesis of human peripheric T lymphocytes is inhibited in a superadditive manner by the simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (Actinonin).","The T cells were incubated for 72 h in the presence of said inhibitors and subsequently, the DNA synthesis rate was determined via a measurement of the 3[H]-thymidin incorporation, as was described by Reinhold et al.","[D. Reinhold et al,: “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","1 shows the dose-dependent superadditive inhibition of the DNA synthesis.","FIG.","1 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and aminopeptidase N (actinonine) on the DNA synthesis of human T lymphocytes.","Human peripheric T cells were incubated over 3 days with the concentrations of the inhibitors shown.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine inserted into the DNA was measured after further 6 hours.","Example 2 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of APN Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (actinonine).","The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.","[D. Reinhold et al.",": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","2 shows the dose-dependent, superadditive inhibition of the DNA synthesis.","FIG.","2 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and APN (actinonine) on the DNA synthesis of human mononuclear cells (MNZ).","Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.","Example 3 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-thiazolidid).","The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.","[D. Reinhold et al.",": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","3 shows the dose-dependent, superadditive inhibition of the DNA synthesis.","FIG.","3 show a the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human peripheric T lymphocytes.","Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.","Example 4 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-Thiazolidid).","The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.","[D. Reinhold et al.",": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","4 shows the dose-dependent, superadditive inhibition of the DNA synthesis.","FIG.","4 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human mononuclear cells (MNZ).","Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.","Example 5 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).","The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.","[D. Reinhold et al.",": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","5 shows the dose-dependent, superadditive inhibition of the DNA synthesis.","FIG.","5 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) X and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human peripheric T lymphocytes.","Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.","Example 6 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).","The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.","[D. Reinhold et al.","; “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].","FIG.","6 shows the dose-dependent, superadditive inhibition of the DNA synthesis.","FIG.","6 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human mononuclear cells (MNZ).","Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.","Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.","Example 7 Inhibition of the Proliferation of Human Peripheric Mononuclear Cells (MNZ) by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin) FIG.","7: The MNZ were incubated for a time period of 72 h without addition (control), with an addition of the mitogenic lectin phytohemagglutinin (PHA), and with the addition of PHA and the inhibitors mentioned above, respectively.","Subsequently, the number amount of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.","Example 8 Inhibition of the Proliferation of the Human T Cell Line KARPAS-299 by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of APN (Actinonin and Probestin) FIG.","8: The KARPAS-299 cells were incubated without addition (control) and in the presence of single inhibitors mentioned above as well as in the presence of combinations thereof, respectively.","Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.","Example 9 Inhibition of the Proliferation of Activated Human Peripheric T Cells by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin and Probestin) FIG.","9: The T cells, with the exception of the untreated control, were activated by an addition of phytohemagglutinine and phorbol-12-myristate-13-acetate to the culture medium and were incubated for a time period of 72 h in the presence of the above-mentioned inhibitors single and in combination.","Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.","Example 10 Inhibition of the Proliferation of PHA-Activated Human Mononuclear Cells (MNZ) by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of X-Pro-Aminopeptidase (APP) (Apstatin) FIG.","10: The mononuclear cells (MNZ) were incubated for a time period of 72 h in the presence of the inhibitors mentioned above single and in combination.","Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer."]],"string":"[\n [\n \"Combinations of enzyme inhibitor-containing preparations and the use thereof\",\n \"The invention comprises a process wherein the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ) and of T cells as well is inhibited by the simultaneous and combined inhibition of the enzyme activity (I) of alanyl aminopeptidase and of dipeptidyl peptidase IV; (II) of dipeptidyl peptidase IV and of the angiotensin-converting enzyme; (III) of dipeptidyl peptidase IV and of prolyl oligopeptidase; and (IV) of dipeptidyl peptidase IV and of X-Pro-aminopeptidase to an extent which cannot be achieved by an application of a single one of said enzyme inhibitors, even at a higher dosage.\",\n \"Although the said inhibitors exercise an influence on the very same process finally, i.e.\",\n \"the DNA synthesis and, thus, the proliferation of immune cells, this effect is not complete and is not long lasting when a single inhibitor is applied.\",\n \"From the functional overlap of enzymatic activities results an additive/superadditive inhibitory effect on the DNA synthesis and proliferation by the simultaneous inhibition of more than one of the above enzymes, as our data show.\",\n \"Our invention shows that the simultaneous application of substances inhibiting the above enzymes or of corresponding preparations and administration forms, respectively, is well suitable for a therapy of autoimmune diseases and chronic diseases with an inflammatory genesis as well as for a treatment of rejection episodes after a transplantation.\"\n ],\n [\n \"1.Use of inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having the same substrate specificity (DP IV-analogous enzyme activity) in combination with inhibitors of alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro-aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and/or of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) for a superadditive inhibition of an activation, DNA synthesis and proliferation of human T lymphocytes and mononuclear cells.\",\n \"2.Use according to claim 1, wherein the inhibitors of DP IV are preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivative), corresponding derivatives, preferably dipeptide phosphonic acid diaryl estates, dipeptide boronic acids (e.g.\",\n \"Pro-boro-Pro) and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably NE-4-nitrobenzyloxycarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure.\",\n \"3.Use according to claim 1, wherein amino acid amides, e.g., NE-4-nitrobenzyloxy-carbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative, are preferably employed as the DP IV inhibitor.\",\n \"4.Use according to claim 1, wherein Actinonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phosphinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, preferably act as inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-L-proline, the corresponding L-proline methyl esters, (2S,3R)-HAMH/(2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof preferably act as inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof preferably act as inhibitors of ACE; Postatin, Eurystatin A or B, Na-protected peptide aldehydes, preferably benzyloxy-carbonyl-L-prolyl-L-prolinal or benzyloxycarbonyl-L-thioprolyl-L-thioprolinal, Na-protected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyano-pyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof preferably act as inhibitors of POP (PEP).\",\n \"5.Use of inhibitor combinations of claim 1 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.\",\n \"6.Use of inhibitor combinations of claim 1 for a suppression of transplant rejection and for a therapy of tumor diseases.\",\n \"7.Pharmaceutical preparations, comprising inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having DP IV-analogous enzyme activity in combination with inhibitors of any of the enzymes alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) and in combination with per se known carrier, additive and/or auxiliary substances.\",\n \"8.Pharmaceutical preparation according to claim 7, comprising preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivatives) corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably Ne-4-nitrobenzyloxcarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure, as the inhibitors of DP IV.\",\n \"9.Pharmaceutical preparation according to claim 7, comprising preferably amino acid amides, e.g.\",\n \"Ne-4-nitrobenzyloxcarbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative as the inhibitors of DP IV.\",\n \"10.Pharmaceutical preparation according to claim 7, comprising, as the inhibitors of APN, APP, ACE and POP (PEP), preferably Actnonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phoshinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, as the inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof as the inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof as inhibitors of ACE; Postatin, Eurystatin A or B, Naprotected peptide aldehydes, preferably benzyloxycarbonyl-L-prolyl-L-prolinal or benzyloxcarbonyl-L-thioprolyl-L-thioprolinal, Naprotected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyanopyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof as inhibitors of POP (PEP).\",\n \"11.Pharmaceutical preparation according to claim 7 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.\",\n \"12.Pharmaceutical preparation according to claim 7 for a systemic use of the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramusclar, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.\",\n \"13.Pharmaceutical preparation according to claim 7 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.\",\n \"14.Use of inhibitor combinations of claim 2 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.\",\n \"15.Use of inhibitor combinations of claim 2 for a suppression of transplant rejection and for a therapy of tumor diseases.\",\n \"16.Use of inhibitor combinations of claim 3 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.\",\n \"17.Use of inhibitor combinations of claim 3 for a suppression of transplant rejection and for a therapy of tumor diseases.\",\n \"18.Use of inhibitor combinations of claim 4 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.\",\n \"19.Use of inhibitor combinations of claim 4 for a suppression of transplant rejection and for a therapy of tumor diseases.\",\n \"20.Pharmaceutical preparation according to claim 8 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.\",\n \"21.Pharmaceutical preparation according to claim 8 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.\",\n \"22.Pharmaceutical preparation according to claim 8 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.\",\n \"23.Pharmaceutical preparation according to claim 9 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.\",\n \"24.Pharmaceutical preparation according to claim 9 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.\",\n \"25.Pharmaceutical preparation according to claim 9 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.\",\n \"26.Pharmaceutical preparation according to claim 10 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.\",\n \"27.Pharmaceutical preparation according to claim 10 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.\",\n \"28.Pharmaceutical preparation according to claim 10 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.\",\n \"29.Pharmaceutical preparation according to claim 11 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, virginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.\",\n \"30.Pharmaceutical preparation according to claim 11 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.\"\n ],\n [\n \"The present invention describes the combined inhibition of the activity of the enzymes Aminopeptidase N (APN; E.C.\",\n \"3.4.11.2.; CD13), Dipeptidyl peptidase IV (DP IV; E.C.\",\n \"3.4.14.5; CD26), Prolyl oligopeptidase (POP; Prolyl endopeptidase; PEP; E.C.\",\n \"3.4.21.26), the membrane-adherent Aminopeptidase P (X-Pro-Aminopeptidase; APP; XPNPEP2; E.C.\",\n \"3.4.11.9) and Angiotensin-converting Enzyme (Angiotensin-konvertierendes Enzym; ACE; CD156; E.C.\",\n \"3.4.15.1;CD156) by a simultaneous application of respective specific inhibitors of the above-referenced enzymes on the basis of amino acid derivatives, peptides or peptide derivatives, by which the activation, the DNA synthesis and, thus, the proliferation of immune cells is suppressed.\",\n \"It is applicable to all diseases showing autoimmune pathogenesis that the disease is based on, or consists of, an activation and proliferation of immune cells, in particular of autoreactive T cells.\",\n \"The very same mechanisms effect the acute or chronic rejection episodes after a transplantation of organs.\",\n \"It was shown that membrane-adherent peptidases as, for example, DP IV or APN play a key role in the process of activation and clonal expansion of immune cells, particularly of T lymphocytes [B. Fleischer: “CD26, a surface protease involved in T cell activation”, Immunology Today 1994, 15, 180-184; U. Lendeckel et al.\",\n \": “Role of alanyl peptidase in growth and function of human T cells”, International Journal of Molecular Medicine 1999, 4, 17-27; D. Riemann et al.\",\n \": “CD13—not just a marker in leukemia typing”, Immunology Today 1999, 20, 83-88].\",\n \"Several functions of mitogene-stimulated mononuclear cells (mitogen-stimulierte mononukleäre Zellen; MNZ) or of enriched T lymphocytes as, for example, the DNA synthesis, production and secretion of immune-stimulating cytokines (IL2, IL6, IL12, IFN-γ) and helper functions for B cells (synthesis of IgG and IgM) may be inhibited in the presence of specific inhibitors DP IV and APN [E. Schön et al,: “The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes”, Biomed.\",\n \"Biochim Acta 1985, 2, K9-K15; E. Schön et al.\",\n \": “The role of dipeptidyl peptidase IV in humane T lymphocyte activation, inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro”, Eur.\",\n \"J. Immunol.\",\n \"1987, 17, 1821-1826; D. Reinhold et al.\",\n \": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360; U. Lendeckel et al.\",\n \": “Induction of the membrane alanyl peptidase gene and surface expression in human T cells by mitogenic activation”, Biochem.\",\n \"J.\",\n \"1996, 319, 817-823; T. Kähne et al.\",\n \": “Dipeptidyl peptidase IV: A cell surface peptidase involved in regulating T cell growth (Review)”, Int.\",\n \"J. Mol.\",\n \"Med.\",\n \"1999, 4, 3-15; U. Lendeckel et al.\",\n \": “Role of alanyl aminopeptidase in growth and function of human T cells (Review)”, Int.\",\n \"J. Mol.\",\n \"Med.\",\n \"1999, 4, 17-27].\",\n \"It is already known that the treatment of autoimmune diseases and rejection reactions after transplantations can be effected by an inhibition of dipeptidyl peptidase IV localized on immune cells by means of synthetic inhibitors.\",\n \"Reference is made, for example, to EP 764 151 A1, WO 09529691, EP 731 789 A1, EP 528 858).\",\n \"With respect to the enzymes aminopeptidase N, angiotensin-converting enzyme, X-Pro-aminopeptidase and prolyl oligopeptidase, such effects are not known up to now.\",\n \"The invention is based on the surprising finding that the simultaneous inhibition of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, as well as (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase inhibits the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ), and of T cells as well, to an extent which cannot be achieved by the application of a single one of these enzyme inhibitors, even at a higher dosage.\",\n \"Although the above-mentioned inhibitors finally exhibit an effect to the same process, i.e.\",\n \"the DNA synthesis and, thus, the proliferation of immune cells, said effect is marked to a weaker extent and is not long lasting in the case of an application of single inhibitors.\",\n \"Due to the functional overlap of the enzymatic activities of said enzymes, there can be observed a superadditive inhibitory effect on the DNA synthesis and proliferation resulting from the simultaneous inhibition of two or more of these enzymes, as can be concluded from our data.\",\n \"Our invention shows that the simultaneous application of inhibitory substances of the above-mentioned enzymes or, respectively, of corresponding preparations and administration forms is well suitable for the therapy of autoimmune diseases and inflammatory diseases as well as for the treatment of rejection reactions after a transplantation.\",\n \"In detail, the invention is based on the finding that the DNA synthesis of mononuclear cells (MNZ) and T cells is inhibited, in a superadditive way, by the simultaneous administration of inhibitors of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase.\",\n \"The application of enzyme inhibitors is a new method and complementary therapy form for the treatment of the above-mentioned diseases.\",\n \"The inhibitors of dipeptidyl peptidase IV, of aminopeptidase N, of prolyl oligopeptidase, of the angiotensin-converting enzyme and of X-Pro-aminopeptidase applied in accordance with the invention may be employed in pharmaceutically applicable formulation complexes as inhibitors, substrates, pseudo-substrates, peptides and peptide derivatives having inhibitory effect and as antibodies of said enzymes as well.\",\n \"Preferred effectors for DP IV are, for example, Xaa-Pro-dipeptides, their corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and their salts, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (n=0-10), their corresponding derivatives and their salts and amino acid (Xaa)-amides, their corresponding derivatives and salts, wherein Xaa is an α-amino acid/imino acid or an α-amino acid/imino acid derivative, respectively, preferably Nε-4-nitrobenzylcarbonyl-L-lysin, -L-prolin, -L-tryptophan, -L-isoleucin, -L-valin, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine, and their derivatives serve as the amide structure.\",\n \"Such compounds and their preparation were described in a prior patent (K. Neubert et al., DD 296 075 A5).\",\n \"The inhibitors are administered simultaneously with known carrier substances.\",\n \"The administration is conducted, on one hand, as a topical application in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles, respectively, including instillative applications and, on the other hand, systemic applications for an oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular administration in suitable recipes and in a suitable galenic application form, respectively.\",\n \"WORKING EXAMPLES Example 1 Inhibition of the DNA Synthesis of Human T Lymphocytes by Incubation with Synthetic Inhibitors of DP IV and of APN Our investigations showed that the DNA synthesis of human peripheric T lymphocytes is inhibited in a superadditive manner by the simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (Actinonin).\",\n \"The T cells were incubated for 72 h in the presence of said inhibitors and subsequently, the DNA synthesis rate was determined via a measurement of the 3[H]-thymidin incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al,: “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"1 shows the dose-dependent superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"1 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and aminopeptidase N (actinonine) on the DNA synthesis of human T lymphocytes.\",\n \"Human peripheric T cells were incubated over 3 days with the concentrations of the inhibitors shown.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine inserted into the DNA was measured after further 6 hours.\",\n \"Example 2 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of APN Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (actinonine).\",\n \"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al.\",\n \": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"2 shows the dose-dependent, superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"2 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and APN (actinonine) on the DNA synthesis of human mononuclear cells (MNZ).\",\n \"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.\",\n \"Example 3 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-thiazolidid).\",\n \"The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al.\",\n \": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"3 shows the dose-dependent, superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"3 show a the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human peripheric T lymphocytes.\",\n \"Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.\",\n \"Example 4 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-Thiazolidid).\",\n \"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al.\",\n \": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"4 shows the dose-dependent, superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"4 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human mononuclear cells (MNZ).\",\n \"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.\",\n \"Example 5 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).\",\n \"The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al.\",\n \": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"5 shows the dose-dependent, superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"5 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) X and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human peripheric T lymphocytes.\",\n \"Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.\",\n \"Example 6 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).\",\n \"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.\",\n \"[D. Reinhold et al.\",\n \"; “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].\",\n \"FIG.\",\n \"6 shows the dose-dependent, superadditive inhibition of the DNA synthesis.\",\n \"FIG.\",\n \"6 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human mononuclear cells (MNZ).\",\n \"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.\",\n \"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.\",\n \"Example 7 Inhibition of the Proliferation of Human Peripheric Mononuclear Cells (MNZ) by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin) FIG.\",\n \"7: The MNZ were incubated for a time period of 72 h without addition (control), with an addition of the mitogenic lectin phytohemagglutinin (PHA), and with the addition of PHA and the inhibitors mentioned above, respectively.\",\n \"Subsequently, the number amount of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.\",\n \"Example 8 Inhibition of the Proliferation of the Human T Cell Line KARPAS-299 by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of APN (Actinonin and Probestin) FIG.\",\n \"8: The KARPAS-299 cells were incubated without addition (control) and in the presence of single inhibitors mentioned above as well as in the presence of combinations thereof, respectively.\",\n \"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.\",\n \"Example 9 Inhibition of the Proliferation of Activated Human Peripheric T Cells by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin and Probestin) FIG.\",\n \"9: The T cells, with the exception of the untreated control, were activated by an addition of phytohemagglutinine and phorbol-12-myristate-13-acetate to the culture medium and were incubated for a time period of 72 h in the presence of the above-mentioned inhibitors single and in combination.\",\n \"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.\",\n \"Example 10 Inhibition of the Proliferation of PHA-Activated Human Mononuclear Cells (MNZ) by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of X-Pro-Aminopeptidase (APP) (Apstatin) FIG.\",\n \"10: The mononuclear cells (MNZ) were incubated for a time period of 72 h in the presence of the inhibitors mentioned above single and in combination.\",\n \"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10296102"}}},{"rowIdx":91,"cells":{"text":{"kind":"list like","value":[["Induction heating of rail welds","An apparatus (10) and method for preheating welds uses a centered induction plate (12) having preferably a plurality of induction coils (30, 32) to impart the generation of heat in the materials to be welded (14), being interactively controlled by at least a temperature sensor (90, 92) and power supply control loop (16) so that even preheating can be obtained for a selected length of time given the parameters of the weld desired."],["1.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating element affixed to the first side of the tool and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensor positioned proximate the first workpiece and the first heating element; a second induction heating element affixed to the second side of the tool and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensor positioned proximate the first workpiece and the second heating element; a controller coupled to the first and second temperature sensors, the controller activating the first and second heating elements independently to achieve a predetermined welding temperature in both first and second heating zones.","2.The induction heating system of claim 1, further comprising a first power supply coupled to the first heating element, and a second power supply coupled to the second heating element, wherein both first and second power supplies are coupled to the controller.","3.The induction heating system of claim 1, wherein the first and second induction heating elements are ferrite core heating elements.","4.The induction heating system of claim 1, wherein the temperature sensors comprise thermocouple temperature sensors.","5.The induction heating system of claim 1, wherein the temperature sensors comprise pyrometer temperature sensors.","6.The induction heating system of claim 1, wherein the controller provides a discernible indication when the workpieces have reached the predetermined welding temperature.","7.The induction heating system of claim 6, wherein the discernible indication is a visual indication.","8.The induction heating system of claim 1, wherein the controller provides positioning information to a positioning robot that adjusts relative position of the tool until the predetermined welding temperature is achieved.","9.The induction heating system of claim 1, wherein cooling fluid is provided proximate each of the heating elements.","10.The induction heating system of claim 9, wherein the controller controls cooling fluid activation.","11.The induction heating system of claim 1, wherein there are three or more heating elements.","12.The induction heating system of claim 1, wherein there are three or more heating zones.","13.The induction heating system of claim 1, wherein the first and second workpieces are railroad rails.","14.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the work-pieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; an induction heating element affixed to the tool and disposed proximate the first and second workpieces; a heating zone defined by a temperature sensor positioned proximate the first workpiece and the heating element; a controller coupled to the temperature sensor, the controller activating the heating element to achieve a predetermined welding temperature in the heating zone.","15.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a plurality of induction heating elements affixed to the tool and disposed proximate the first and second workpieces; as plurality of heating zones defined by multiple temperature sensors positioned proximate the first workpiece and the heating elements; a controller coupled to the temperature sensors, the controller activating the heating elements to achieve a predetermined welding temperature in the heating zones.","16.An induction heating system for preheating first and second workpieces to be welded together comprising: mounting means having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, wherein the mounting means further further includes mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating means affixed to the first side of the mounting means and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensing means positioned proximate the first workpiece and the first heating means; a second induction heating means affixed to the second side of the mounting means and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensing means positioned proximate the first workpiece and the second heating means; a controller means coupled to the first and second temperature sensing means, the controller means activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones.","17.The induction heating system of claim 16, wherein the mounting means comprises a tool having first and second opposing sides.","18.The induction heating system of claim 16, further comprising a first power supply means coupled to the first heating means, and a second power supply means coupled to the second heating means, wherein both first and second power supplies means are coupled to the controller means.","19.The induction heating system of claim 16, wherein the first and second induction heating means comprise ferrite core heating elements.","20.The induction heating system of claim 16, wherein the temperature sensing means comprise thermocouple temperature sensors.","21.The induction heating system of claim 16, wherein the temperature sensing means comprise pyrometer temperature sensors.","22.The induction heating system of claim 16, wherein the controller means provides a discernible indication when the workpieces have reached the predetermined welding temperature.","23.The induction heating system of claim 22, wherein the discernible indication is a visual indication.","24.The induction heating system of claim 16, wherein the controller means provides positioning information to a positioning means that adjusts relative position of the tool until the predetermined welding temperature is achieved.","25.The induction heating system of claim 16, wherein cooling fluid is provided proximate each of the heating means.","26.The induction heating system of claim 25, wherein the controller means controls cooling fluid activation.","27.The induction heating system of claim 16, wherein there are three or more heating means.","28.The induction heating system of claim 16, wherein there are three or more heating zones.","29.The induction heating system of claim 16, wherein the first and second workpieces are railroad rails.","30.A method for preheating first and second workpieces to be welded together, the method comprising the steps of: (a) disposing, within a gap defined by facing ends of the workpieces, mounting means having first and second opposing sides; (b) providing mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; (c) disposing a first induction heating means affixed to the first side of the mounting means proximate the first and second workpieces; (d) defining a first heating zone by positioning a first temperature sensing means proximate the first workpiece and the first heating means; (e) disposing a second induction heating means affixed to the second side of the mounting means proximate the first and second workpieces; (f) defining a second heating zone by positioning a second temperature sensing means proximate the first workpiece and the second heating means; and (g) activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones."],[" BACKGROUND OF THE INVENTION 1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.","The descriptions herein of rail welds should be considered with this more expansive use in mind.","Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.","This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.","This eliminates the human element, resulting in an automated high quality weld preheat method.","The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.","One advantage in this regard is the ability to manipulate the heat gradient.","Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.","The invention provides consistent heat through a wide range of ambient temperatures.","Another advantage is that of use in different rail geometry, rail chemistries and welding methods.","In addition to heating, an analogous plate or array can be used to control cooling after welding.","In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.","Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.","An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.","While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.","2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.","The use of items such as gas or other torches, gas burners or electrically powered devices.","Field welding in the past commonly preheated with torches and gas burners.","Such methods introduce human intervention positioning, timing or estimating heat input and temperature.","Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.","Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control."],[" BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a schematic view showing the layout of the components of the invention.","FIG.","2 is an elevational view showing the induction plate of the invention.","FIG.","2A is an elevational view showing an induction coil with ferrite core.","FIG.","3 is a wiring diagram showing the control wiring of the invention.","FIG.","4 is a wiring diagram showing the power supply wiring to control separate heating zones.","FIG.","5 is a side elevational view showing the induction plate of the invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["CLAIM OF PRIORITY Priority is claimed based upon Provisional Patent Application Ser.","No.","60/209,030, filed Jun.","2, 2000, which is incorporated by reference as if fully set forth herein.","STATEMENT OF INVENTORSHIP Assignee, Holland Company, believes that the subject matter was the invention of Richard F. Miller.","FIELD OF THE INVENTION Some welding techniques require precise consistent and controlled heating, which is difficult or impossible to obtain with torches, gas burners or other electric devices.","Instead, this is achievable through the use of induction heating wherein a plate or other locating and holding device is placed in a gap between work pieces to be welded, the plate containing an array of induction heating elements which, when energized, produce shaped, varying electromagnetic fields which link with and induce a voltage in the work pieces which in turn results in eddy current flows and subsequent power losses, as well as hysteresis losses, so that the workpiece temperature is raised to a desired, uniform level prior to welding.","BACKGROUND OF THE INVENTION 1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.","The descriptions herein of rail welds should be considered with this more expansive use in mind.","Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.","This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.","This eliminates the human element, resulting in an automated high quality weld preheat method.","The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.","One advantage in this regard is the ability to manipulate the heat gradient.","Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.","The invention provides consistent heat through a wide range of ambient temperatures.","Another advantage is that of use in different rail geometry, rail chemistries and welding methods.","In addition to heating, an analogous plate or array can be used to control cooling after welding.","In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.","Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.","An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.","While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.","2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.","The use of items such as gas or other torches, gas burners or electrically powered devices.","Field welding in the past commonly preheated with torches and gas burners.","Such methods introduce human intervention positioning, timing or estimating heat input and temperature.","Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.","Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control.","BRIEF DESCRIPTION OF THE DRAWINGS FIG.","1 is a schematic view showing the layout of the components of the invention.","FIG.","2 is an elevational view showing the induction plate of the invention.","FIG.","2A is an elevational view showing an induction coil with ferrite core.","FIG.","3 is a wiring diagram showing the control wiring of the invention.","FIG.","4 is a wiring diagram showing the power supply wiring to control separate heating zones.","FIG.","5 is a side elevational view showing the induction plate of the invention.","DETAILED DESCRIPTION OF THE INVENTION Weld quality and consistency can be improved with precise control of heating (temperatures, location zones and heat gradients).","Heating becomes more critical when dealing with certain alloying, geometry or ambient temperatures.","This is more of an art gained through experience or a routine that must be followed carefully to attempt to produce consistent, quality welds.","An induction heating system 10 uses a tool or plate 12 to heat a railroad rail 14.Power control 16 is operatively connected to a pair of power supplies 18,20.Power supplies 18,20 are in turn operatively connected to a heat zone control unit 22 using control output connections 24, 26.Cables (not shown) interconnect power supplies 18,20 to plate 12, generally, and to heating elements 30, 32 specifically through connectors 34, 36.FIG.","2A shows the heating element 32 in more detail.","Each element 30, 32 is generally broken out into a separate heating module 38, 40 using the elements 30, 32 and other features more fully described below to provide a unitary heating module 38, 40.The heating system 10 is a 5 Kw, 25 Khz induction heating system broken out in the form of two 2.5 Kw modules 38, 40.While this is the preferred capacity of the system, different welding operations could advantageously be accommodated through the use of a heating system with a different heating capacity.","These are capable of independent heating of two heat zones, 42, 44.Each module 38, 40 is connected to a heating element 30, 32 with ferrite cores 46, 48 in a common housing in plate 12 capable of being inserted in an approximate one inch gap between rails 14.It will be understood that the rails 14 are shown in section and in the field there will be to rails 14 with a gap between them intended to be welded.","For preheating of normal section railroad rails 14 for gas shielded arc welding, the preferred heat zones 42, 44 consist of two heating locations 50, 52 generally near the bottom flanges 54, 56.The preferred embodiment will be further described below after general description of the field that requires induction preheating.","Gas shielded arc welding without the preheating taught by the invention in this application has been practiced under controlled laboratory and/or workshop conditions but is believed unsuitable for use in the field.","One method used under controlled conditions is generally taught by U.S. Pat.","Nos.","5,773,779 and 5,877,468, which are incorporated by reference as if fully set forth herein.","It is believed that one reason the method described in these two patents is inoperative in field conditions is inadequate control of preheating.","Delivery of gas shielded arc welding equipment and the alignment and restraint of railroad rails and the deployment of a weld containment unit for application of weld beads is taught in published International Application No.","WO 99/31322 published 16 Dec. 1998 entitled “Mail Welding Apparatus Incorporating Rail Restraining Device, Weld Containment Device and Weld Delivery Unit.” The teachings of this application are also incorporated by reference.","It is believed that the apparatus and method taught herein are essential in effective practice of gas shielded arc welding of rails.","Additionally, other welding methods are believed to be capable of enhancement through the use of the invention taught here.","Other weld methods, such as thermite, on site foundry and even certain flux-based arc welding may prove suitable for high strength welds of complex shapes with adequate and well controlled preheating.","While the preferred embodiment of induction heating for rail welding using gas shielded arc welding anticipates using two heating modules 38, 40, other uses could use fewer, more or differently arranged heating modules.","Thus, for certain rail welding or joining methods it may prove advantageous to heat the entire rail section simultaneously.","The invention is not limited to rail welding using two heating modules.","Plate 12 is formed to fully support and contain heating elements 30, 32.Accordingly plate 12 has a body portion 60 ending in a protective ceramic cover 62 which fully covers ferrite cores 46, 48 and the corresponding conductors of elements 30, 32.Side edges 64, 66 of plate 12 are fitted with centering bar assemblies 68, 70.Assemblies 68, 70 use centering adjustment mechanism 72 to adjust bars 74, 76 outwardly or inwardly to fit the rail gap.","It will be understood that assemblies 68, 70 are symmetric and accordingly only one assembly 70 is shown and illustrated in FIG.","5.Precise cutting of rails in the field is quite difficult, thus there is often variation in the size of gaps and the orientation of their faces.","The adjustable and expandable centering bar assembles enabling side to side and top to bottom centering are important in aligning plate 12 as close to the center of the gap as practicable to maximize the uniform heating of the rail ends.","In this manner the assemblies 68, 70 are aligned for maximum effectiveness and uniformity, being centered between faces that may themselves be non-parallel due to the difficulty of cutting in the field.","Each heating element 30, 32 is fitted with a respective pair of water/power connections 80, 82 illustrated in FIG.","2A.","These enable both the electrical power connection necessary to energize the ferrite core 46, 48 and provide conduits for the transmission of cooling fluid to dissipate the heat radiantly transmitted from the preheated rails 14 to the plate 12.Spring clips 84, 86 are used to retain elements 30, 32 in place in plate 12.Temperature measuring devices such as spring loaded thermocouples 90, 92 are used as an integral part of both plate 12 and heat zone control unit 22.Thermocouples 90, 92 are operatively connected to heat zone control unit 22 which is in turn used to control power supplies 18, 20.Power supplies 18, 20 receive signals from unit 22 interconnected through connections 24, 26 which provide power on or power off control signals depending on the heat measured at thermocouples 90, 92.It will be observed, particularly from FIG.","1 and 2 that thermocouples 90 and 92 are positioned proximate ferrite cores 48, thereby providing an accurate temperature reading from rails 14 which are heated as a result of the energizing of elements 30, 32 and particularly cores 46, 48.The general principles of induction heating will be recognized, namely the providing of a sufficiently large energy output at elements 30, 32 and cores 46, 48 will create heat in adjacent steel rails 14.In this manner heat zones 42, 44 are interactively controlled so that a controlled heat results although a variety of factors, whether an imprecise gap, power, magnetic fluctuations, unequal temperature differential or even environmental factors such as a crosswind, impact the actual temperature and heat distribution in the rails.","As noted above while locating heat zones 42, 44 near the rail flanges is advantageous for gas shielded arc welding other types of welding may require the use of different heat zones such as heating the entire rail section including flanges, web and head, or for the welding of different shapes such as I-beams and the like.","In these other uses and applications, the number of heat zones and their orientation can be controlled consistent with the principles of this invention.","The use of one or more coil elements 30, 32 gives flexibility in applying the two preferred heat zones 46, 48.In other, particularly non-rail welding applications, a single heat zone or multiple heat zones could be used.","A plurality of coil elements, two or more, also provide flexibility in temperature differentials that may be required by particular metallurgical or welding considerations.","The invention enables precision controlling of the heat gradient in the pieces to be welded.","The interactive control between elements 30, 32 and thermocouples 90, 92 in zones 42, 44 enables the ability to manipulate the effects of applied heat to the particular metallurgy of the rails 14 and weld material and method used.","Control unit 22 can also be interconnected to corresponding additional fixtures or controls.","The invention contemplates a feedback system that enables, but is not limited to input from a robot or positioner or a controller/computer that calculates heat soak/rail temperature for particular conditions.","With this data compared to the induction heat inputs directly supplied by unit 22 and temperature measurement enabled by thermocouples 90, 92, control unit can be modified for particular time and energy parameters, given known metallurgical and welding requirements.","This full feedback system maximizes the quality control of the welding process so that it will be repeatable and monitored.","The full feedback system also records actual temperatures and adjusts automatically.","The fill feedback system is further programmable for various materials, conditions and methods.","With greater and better data regarding when the weld pieces reach correct temperature(s) in singular and/or multiple zones fully integrated with weld delivery controls, the user is provided a seamless system with no additional mechanisms, components required, such as the prior art burners or torches, and welding materials requiring multiple unrelated and uncontrolled steps.","Finally, the fully integrated system can be manipulated by robot for deployment, movements during heating process and retractment.","In operation, the steps of the invention are premised on the step in which each heat zone is monitored by a temperature measuring device which checks temperature on one side of the zone.","Even heating is achieved on either side of the zone because the inductor is centered between the rails with a mechanical centering device, which independently and exactly centers the inductor relative to side one and side two.","By so doing each side is brought up to a preferably preheat temperature even though the rails may have a variable gap.","It will be noted that the temperature may, of course, vary based on the materials welded and the method of welding used.","Rail ends are preheated by induction heating for preparation of welding.","The ideal temperature and heat gradient is controlled by a feedback system.","The feedback system uses temperature-measuring devices like thermocouple's, pyrometers, and other heat sensors with or without a controlling device.","During the pre-heating, different zone(s) of rail ends can be simultaneously heated independently of each other.","Frequency, proximity and number of cycles allow for control of the heat gradient.","This is complemented though coil designs in the tool and/or power inputs.","This tool fits between the rail ends in the gap and can be manipulated by a robotic arm or manually.","The gaps between rails are approximately ¼″ and up.","Material and mechanical designs of this tool enhance durability and efficiencies.","Process requirements are monitored and recorded for quality control.","Parameter measurements give a go/no go signal to proceed with welding or intervene with corrections to meet parameters.","In addition post-heating enjoys many of the same benefits.","The complete system is mobile and portable.","In operation, each zone begins heating simultaneously.","Should one zone reach temperature prior to the other the heater output is reduced so as to maintain at temperature until the second zone also achieves required temperature.","Only at this time does the controller send a signal to the weld controller indicating that welding can commence."]],"string":"[\n [\n \"Induction heating of rail welds\",\n \"An apparatus (10) and method for preheating welds uses a centered induction plate (12) having preferably a plurality of induction coils (30, 32) to impart the generation of heat in the materials to be welded (14), being interactively controlled by at least a temperature sensor (90, 92) and power supply control loop (16) so that even preheating can be obtained for a selected length of time given the parameters of the weld desired.\"\n ],\n [\n \"1.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating element affixed to the first side of the tool and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensor positioned proximate the first workpiece and the first heating element; a second induction heating element affixed to the second side of the tool and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensor positioned proximate the first workpiece and the second heating element; a controller coupled to the first and second temperature sensors, the controller activating the first and second heating elements independently to achieve a predetermined welding temperature in both first and second heating zones.\",\n \"2.The induction heating system of claim 1, further comprising a first power supply coupled to the first heating element, and a second power supply coupled to the second heating element, wherein both first and second power supplies are coupled to the controller.\",\n \"3.The induction heating system of claim 1, wherein the first and second induction heating elements are ferrite core heating elements.\",\n \"4.The induction heating system of claim 1, wherein the temperature sensors comprise thermocouple temperature sensors.\",\n \"5.The induction heating system of claim 1, wherein the temperature sensors comprise pyrometer temperature sensors.\",\n \"6.The induction heating system of claim 1, wherein the controller provides a discernible indication when the workpieces have reached the predetermined welding temperature.\",\n \"7.The induction heating system of claim 6, wherein the discernible indication is a visual indication.\",\n \"8.The induction heating system of claim 1, wherein the controller provides positioning information to a positioning robot that adjusts relative position of the tool until the predetermined welding temperature is achieved.\",\n \"9.The induction heating system of claim 1, wherein cooling fluid is provided proximate each of the heating elements.\",\n \"10.The induction heating system of claim 9, wherein the controller controls cooling fluid activation.\",\n \"11.The induction heating system of claim 1, wherein there are three or more heating elements.\",\n \"12.The induction heating system of claim 1, wherein there are three or more heating zones.\",\n \"13.The induction heating system of claim 1, wherein the first and second workpieces are railroad rails.\",\n \"14.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the work-pieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; an induction heating element affixed to the tool and disposed proximate the first and second workpieces; a heating zone defined by a temperature sensor positioned proximate the first workpiece and the heating element; a controller coupled to the temperature sensor, the controller activating the heating element to achieve a predetermined welding temperature in the heating zone.\",\n \"15.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a plurality of induction heating elements affixed to the tool and disposed proximate the first and second workpieces; as plurality of heating zones defined by multiple temperature sensors positioned proximate the first workpiece and the heating elements; a controller coupled to the temperature sensors, the controller activating the heating elements to achieve a predetermined welding temperature in the heating zones.\",\n \"16.An induction heating system for preheating first and second workpieces to be welded together comprising: mounting means having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, wherein the mounting means further further includes mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating means affixed to the first side of the mounting means and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensing means positioned proximate the first workpiece and the first heating means; a second induction heating means affixed to the second side of the mounting means and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensing means positioned proximate the first workpiece and the second heating means; a controller means coupled to the first and second temperature sensing means, the controller means activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones.\",\n \"17.The induction heating system of claim 16, wherein the mounting means comprises a tool having first and second opposing sides.\",\n \"18.The induction heating system of claim 16, further comprising a first power supply means coupled to the first heating means, and a second power supply means coupled to the second heating means, wherein both first and second power supplies means are coupled to the controller means.\",\n \"19.The induction heating system of claim 16, wherein the first and second induction heating means comprise ferrite core heating elements.\",\n \"20.The induction heating system of claim 16, wherein the temperature sensing means comprise thermocouple temperature sensors.\",\n \"21.The induction heating system of claim 16, wherein the temperature sensing means comprise pyrometer temperature sensors.\",\n \"22.The induction heating system of claim 16, wherein the controller means provides a discernible indication when the workpieces have reached the predetermined welding temperature.\",\n \"23.The induction heating system of claim 22, wherein the discernible indication is a visual indication.\",\n \"24.The induction heating system of claim 16, wherein the controller means provides positioning information to a positioning means that adjusts relative position of the tool until the predetermined welding temperature is achieved.\",\n \"25.The induction heating system of claim 16, wherein cooling fluid is provided proximate each of the heating means.\",\n \"26.The induction heating system of claim 25, wherein the controller means controls cooling fluid activation.\",\n \"27.The induction heating system of claim 16, wherein there are three or more heating means.\",\n \"28.The induction heating system of claim 16, wherein there are three or more heating zones.\",\n \"29.The induction heating system of claim 16, wherein the first and second workpieces are railroad rails.\",\n \"30.A method for preheating first and second workpieces to be welded together, the method comprising the steps of: (a) disposing, within a gap defined by facing ends of the workpieces, mounting means having first and second opposing sides; (b) providing mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; (c) disposing a first induction heating means affixed to the first side of the mounting means proximate the first and second workpieces; (d) defining a first heating zone by positioning a first temperature sensing means proximate the first workpiece and the first heating means; (e) disposing a second induction heating means affixed to the second side of the mounting means proximate the first and second workpieces; (f) defining a second heating zone by positioning a second temperature sensing means proximate the first workpiece and the second heating means; and (g) activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION 1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.\",\n \"The descriptions herein of rail welds should be considered with this more expansive use in mind.\",\n \"Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.\",\n \"This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.\",\n \"This eliminates the human element, resulting in an automated high quality weld preheat method.\",\n \"The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.\",\n \"One advantage in this regard is the ability to manipulate the heat gradient.\",\n \"Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.\",\n \"The invention provides consistent heat through a wide range of ambient temperatures.\",\n \"Another advantage is that of use in different rail geometry, rail chemistries and welding methods.\",\n \"In addition to heating, an analogous plate or array can be used to control cooling after welding.\",\n \"In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.\",\n \"Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.\",\n \"An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.\",\n \"While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.\",\n \"2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.\",\n \"The use of items such as gas or other torches, gas burners or electrically powered devices.\",\n \"Field welding in the past commonly preheated with torches and gas burners.\",\n \"Such methods introduce human intervention positioning, timing or estimating heat input and temperature.\",\n \"Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.\",\n \"Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control.\"\n ],\n [\n \" BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a schematic view showing the layout of the components of the invention.\",\n \"FIG.\",\n \"2 is an elevational view showing the induction plate of the invention.\",\n \"FIG.\",\n \"2A is an elevational view showing an induction coil with ferrite core.\",\n \"FIG.\",\n \"3 is a wiring diagram showing the control wiring of the invention.\",\n \"FIG.\",\n \"4 is a wiring diagram showing the power supply wiring to control separate heating zones.\",\n \"FIG.\",\n \"5 is a side elevational view showing the induction plate of the invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"CLAIM OF PRIORITY Priority is claimed based upon Provisional Patent Application Ser.\",\n \"No.\",\n \"60/209,030, filed Jun.\",\n \"2, 2000, which is incorporated by reference as if fully set forth herein.\",\n \"STATEMENT OF INVENTORSHIP Assignee, Holland Company, believes that the subject matter was the invention of Richard F. Miller.\",\n \"FIELD OF THE INVENTION Some welding techniques require precise consistent and controlled heating, which is difficult or impossible to obtain with torches, gas burners or other electric devices.\",\n \"Instead, this is achievable through the use of induction heating wherein a plate or other locating and holding device is placed in a gap between work pieces to be welded, the plate containing an array of induction heating elements which, when energized, produce shaped, varying electromagnetic fields which link with and induce a voltage in the work pieces which in turn results in eddy current flows and subsequent power losses, as well as hysteresis losses, so that the workpiece temperature is raised to a desired, uniform level prior to welding.\",\n \"BACKGROUND OF THE INVENTION 1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.\",\n \"The descriptions herein of rail welds should be considered with this more expansive use in mind.\",\n \"Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.\",\n \"This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.\",\n \"This eliminates the human element, resulting in an automated high quality weld preheat method.\",\n \"The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.\",\n \"One advantage in this regard is the ability to manipulate the heat gradient.\",\n \"Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.\",\n \"The invention provides consistent heat through a wide range of ambient temperatures.\",\n \"Another advantage is that of use in different rail geometry, rail chemistries and welding methods.\",\n \"In addition to heating, an analogous plate or array can be used to control cooling after welding.\",\n \"In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.\",\n \"Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.\",\n \"An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.\",\n \"While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.\",\n \"2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.\",\n \"The use of items such as gas or other torches, gas burners or electrically powered devices.\",\n \"Field welding in the past commonly preheated with torches and gas burners.\",\n \"Such methods introduce human intervention positioning, timing or estimating heat input and temperature.\",\n \"Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.\",\n \"Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 is a schematic view showing the layout of the components of the invention.\",\n \"FIG.\",\n \"2 is an elevational view showing the induction plate of the invention.\",\n \"FIG.\",\n \"2A is an elevational view showing an induction coil with ferrite core.\",\n \"FIG.\",\n \"3 is a wiring diagram showing the control wiring of the invention.\",\n \"FIG.\",\n \"4 is a wiring diagram showing the power supply wiring to control separate heating zones.\",\n \"FIG.\",\n \"5 is a side elevational view showing the induction plate of the invention.\",\n \"DETAILED DESCRIPTION OF THE INVENTION Weld quality and consistency can be improved with precise control of heating (temperatures, location zones and heat gradients).\",\n \"Heating becomes more critical when dealing with certain alloying, geometry or ambient temperatures.\",\n \"This is more of an art gained through experience or a routine that must be followed carefully to attempt to produce consistent, quality welds.\",\n \"An induction heating system 10 uses a tool or plate 12 to heat a railroad rail 14.Power control 16 is operatively connected to a pair of power supplies 18,20.Power supplies 18,20 are in turn operatively connected to a heat zone control unit 22 using control output connections 24, 26.Cables (not shown) interconnect power supplies 18,20 to plate 12, generally, and to heating elements 30, 32 specifically through connectors 34, 36.FIG.\",\n \"2A shows the heating element 32 in more detail.\",\n \"Each element 30, 32 is generally broken out into a separate heating module 38, 40 using the elements 30, 32 and other features more fully described below to provide a unitary heating module 38, 40.The heating system 10 is a 5 Kw, 25 Khz induction heating system broken out in the form of two 2.5 Kw modules 38, 40.While this is the preferred capacity of the system, different welding operations could advantageously be accommodated through the use of a heating system with a different heating capacity.\",\n \"These are capable of independent heating of two heat zones, 42, 44.Each module 38, 40 is connected to a heating element 30, 32 with ferrite cores 46, 48 in a common housing in plate 12 capable of being inserted in an approximate one inch gap between rails 14.It will be understood that the rails 14 are shown in section and in the field there will be to rails 14 with a gap between them intended to be welded.\",\n \"For preheating of normal section railroad rails 14 for gas shielded arc welding, the preferred heat zones 42, 44 consist of two heating locations 50, 52 generally near the bottom flanges 54, 56.The preferred embodiment will be further described below after general description of the field that requires induction preheating.\",\n \"Gas shielded arc welding without the preheating taught by the invention in this application has been practiced under controlled laboratory and/or workshop conditions but is believed unsuitable for use in the field.\",\n \"One method used under controlled conditions is generally taught by U.S. Pat.\",\n \"Nos.\",\n \"5,773,779 and 5,877,468, which are incorporated by reference as if fully set forth herein.\",\n \"It is believed that one reason the method described in these two patents is inoperative in field conditions is inadequate control of preheating.\",\n \"Delivery of gas shielded arc welding equipment and the alignment and restraint of railroad rails and the deployment of a weld containment unit for application of weld beads is taught in published International Application No.\",\n \"WO 99/31322 published 16 Dec. 1998 entitled “Mail Welding Apparatus Incorporating Rail Restraining Device, Weld Containment Device and Weld Delivery Unit.” The teachings of this application are also incorporated by reference.\",\n \"It is believed that the apparatus and method taught herein are essential in effective practice of gas shielded arc welding of rails.\",\n \"Additionally, other welding methods are believed to be capable of enhancement through the use of the invention taught here.\",\n \"Other weld methods, such as thermite, on site foundry and even certain flux-based arc welding may prove suitable for high strength welds of complex shapes with adequate and well controlled preheating.\",\n \"While the preferred embodiment of induction heating for rail welding using gas shielded arc welding anticipates using two heating modules 38, 40, other uses could use fewer, more or differently arranged heating modules.\",\n \"Thus, for certain rail welding or joining methods it may prove advantageous to heat the entire rail section simultaneously.\",\n \"The invention is not limited to rail welding using two heating modules.\",\n \"Plate 12 is formed to fully support and contain heating elements 30, 32.Accordingly plate 12 has a body portion 60 ending in a protective ceramic cover 62 which fully covers ferrite cores 46, 48 and the corresponding conductors of elements 30, 32.Side edges 64, 66 of plate 12 are fitted with centering bar assemblies 68, 70.Assemblies 68, 70 use centering adjustment mechanism 72 to adjust bars 74, 76 outwardly or inwardly to fit the rail gap.\",\n \"It will be understood that assemblies 68, 70 are symmetric and accordingly only one assembly 70 is shown and illustrated in FIG.\",\n \"5.Precise cutting of rails in the field is quite difficult, thus there is often variation in the size of gaps and the orientation of their faces.\",\n \"The adjustable and expandable centering bar assembles enabling side to side and top to bottom centering are important in aligning plate 12 as close to the center of the gap as practicable to maximize the uniform heating of the rail ends.\",\n \"In this manner the assemblies 68, 70 are aligned for maximum effectiveness and uniformity, being centered between faces that may themselves be non-parallel due to the difficulty of cutting in the field.\",\n \"Each heating element 30, 32 is fitted with a respective pair of water/power connections 80, 82 illustrated in FIG.\",\n \"2A.\",\n \"These enable both the electrical power connection necessary to energize the ferrite core 46, 48 and provide conduits for the transmission of cooling fluid to dissipate the heat radiantly transmitted from the preheated rails 14 to the plate 12.Spring clips 84, 86 are used to retain elements 30, 32 in place in plate 12.Temperature measuring devices such as spring loaded thermocouples 90, 92 are used as an integral part of both plate 12 and heat zone control unit 22.Thermocouples 90, 92 are operatively connected to heat zone control unit 22 which is in turn used to control power supplies 18, 20.Power supplies 18, 20 receive signals from unit 22 interconnected through connections 24, 26 which provide power on or power off control signals depending on the heat measured at thermocouples 90, 92.It will be observed, particularly from FIG.\",\n \"1 and 2 that thermocouples 90 and 92 are positioned proximate ferrite cores 48, thereby providing an accurate temperature reading from rails 14 which are heated as a result of the energizing of elements 30, 32 and particularly cores 46, 48.The general principles of induction heating will be recognized, namely the providing of a sufficiently large energy output at elements 30, 32 and cores 46, 48 will create heat in adjacent steel rails 14.In this manner heat zones 42, 44 are interactively controlled so that a controlled heat results although a variety of factors, whether an imprecise gap, power, magnetic fluctuations, unequal temperature differential or even environmental factors such as a crosswind, impact the actual temperature and heat distribution in the rails.\",\n \"As noted above while locating heat zones 42, 44 near the rail flanges is advantageous for gas shielded arc welding other types of welding may require the use of different heat zones such as heating the entire rail section including flanges, web and head, or for the welding of different shapes such as I-beams and the like.\",\n \"In these other uses and applications, the number of heat zones and their orientation can be controlled consistent with the principles of this invention.\",\n \"The use of one or more coil elements 30, 32 gives flexibility in applying the two preferred heat zones 46, 48.In other, particularly non-rail welding applications, a single heat zone or multiple heat zones could be used.\",\n \"A plurality of coil elements, two or more, also provide flexibility in temperature differentials that may be required by particular metallurgical or welding considerations.\",\n \"The invention enables precision controlling of the heat gradient in the pieces to be welded.\",\n \"The interactive control between elements 30, 32 and thermocouples 90, 92 in zones 42, 44 enables the ability to manipulate the effects of applied heat to the particular metallurgy of the rails 14 and weld material and method used.\",\n \"Control unit 22 can also be interconnected to corresponding additional fixtures or controls.\",\n \"The invention contemplates a feedback system that enables, but is not limited to input from a robot or positioner or a controller/computer that calculates heat soak/rail temperature for particular conditions.\",\n \"With this data compared to the induction heat inputs directly supplied by unit 22 and temperature measurement enabled by thermocouples 90, 92, control unit can be modified for particular time and energy parameters, given known metallurgical and welding requirements.\",\n \"This full feedback system maximizes the quality control of the welding process so that it will be repeatable and monitored.\",\n \"The full feedback system also records actual temperatures and adjusts automatically.\",\n \"The fill feedback system is further programmable for various materials, conditions and methods.\",\n \"With greater and better data regarding when the weld pieces reach correct temperature(s) in singular and/or multiple zones fully integrated with weld delivery controls, the user is provided a seamless system with no additional mechanisms, components required, such as the prior art burners or torches, and welding materials requiring multiple unrelated and uncontrolled steps.\",\n \"Finally, the fully integrated system can be manipulated by robot for deployment, movements during heating process and retractment.\",\n \"In operation, the steps of the invention are premised on the step in which each heat zone is monitored by a temperature measuring device which checks temperature on one side of the zone.\",\n \"Even heating is achieved on either side of the zone because the inductor is centered between the rails with a mechanical centering device, which independently and exactly centers the inductor relative to side one and side two.\",\n \"By so doing each side is brought up to a preferably preheat temperature even though the rails may have a variable gap.\",\n \"It will be noted that the temperature may, of course, vary based on the materials welded and the method of welding used.\",\n \"Rail ends are preheated by induction heating for preparation of welding.\",\n \"The ideal temperature and heat gradient is controlled by a feedback system.\",\n \"The feedback system uses temperature-measuring devices like thermocouple's, pyrometers, and other heat sensors with or without a controlling device.\",\n \"During the pre-heating, different zone(s) of rail ends can be simultaneously heated independently of each other.\",\n \"Frequency, proximity and number of cycles allow for control of the heat gradient.\",\n \"This is complemented though coil designs in the tool and/or power inputs.\",\n \"This tool fits between the rail ends in the gap and can be manipulated by a robotic arm or manually.\",\n \"The gaps between rails are approximately ¼″ and up.\",\n \"Material and mechanical designs of this tool enhance durability and efficiencies.\",\n \"Process requirements are monitored and recorded for quality control.\",\n \"Parameter measurements give a go/no go signal to proceed with welding or intervene with corrections to meet parameters.\",\n \"In addition post-heating enjoys many of the same benefits.\",\n \"The complete system is mobile and portable.\",\n \"In operation, each zone begins heating simultaneously.\",\n \"Should one zone reach temperature prior to the other the heater output is reduced so as to maintain at temperature until the second zone also achieves required temperature.\",\n \"Only at this time does the controller send a signal to the weld controller indicating that welding can commence.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10296635"}}},{"rowIdx":92,"cells":{"text":{"kind":"list like","value":[["Carboxylate salts in heat-storage applications","The use of an alkali metal salt or alkaline earth metal salt or of a bring solution of a C1-C16 carboxylic acid, or a mixture thereof, as a medium for the storage and use of thermal energy.","The salts or solution can be used in a heat exchange fluid or a lubricant or hydraulic fluid or soap."],["1-13.","(canceled) 14.A method for the storage and use of thermal energy comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.","15.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C3-C18 carboxylic acid or hydrated salts of a C3 to C7 carboxylic acid with a source of thermal energy.","16.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C2 carboxylic acids and a salt of one or more C3-C5 carboxylic acids.","17.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C5 carboxylic acids and one or more C6-C18 carboxylic acids.","18.The method of claim 4 wherein said mixture comprises a salt of one or more C3-C5 carboxylic acids and one or more C6-C16 carboxylic acids.","19.The method of claim 1 wherein the temperature range of the thermal energy source is 20 to 180 degrees C. 20.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.","21.The method of claim 1 further comprising contacting one or a mixture of hydrated alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.","22.A method for improving the thermal properties of a fluid comprising dispersing in said fluid one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.","23.The method of claim 9 wherein the fluid is a water soluble alcohol freezing point depressant selected from the group consisting of ethylene glycol, propylene glycol, ethanol and methanol.","24.The method of claim 9 wherein the fluid is a lubricant or hydraulic fluid selected from the group consisting of mineral oil, synthetic oil, mineral soap, synthetic soap, mineral grease and synthetic grease."],[" BACKGROUND OF THE INVENTION Thermal energy originating from any energy source is reusable if it can be stored.","Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.","in refineries and steam generation plants).","Energy generated in peak load time can be managed and stored for later use.","Examples are solar heating and electrical heating on low tariff hours.","The problem of cold car engine start in wintertime is well known.","Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.","Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.","Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.","However these solutions put an extra burden on the vehicle's electrical power system.","Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.","Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.","Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.","An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.","The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.","The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates."],["This invention relates to the application of carboxylate salts for storing thermal energy.","Melted salts are used for heat-storage because salts absorb heat during the transition from the solid to the liquid phase.","This heat is stored in latent form as long as the liquid state persists and released again during the transition from the liquid to the solid phase when the liquid salt solidifies.","BACKGROUND OF THE INVENTION Thermal energy originating from any energy source is reusable if it can be stored.","Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.","in refineries and steam generation plants).","Energy generated in peak load time can be managed and stored for later use.","Examples are solar heating and electrical heating on low tariff hours.","The problem of cold car engine start in wintertime is well known.","Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.","Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.","Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.","However these solutions put an extra burden on the vehicle's electrical power system.","Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.","Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.","Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.","An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.","The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.","The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates.","PRIOR ART Hydrated fluoride-, chloride-, sulfate- and nitrate salts or salt combinations have been described as heat-storage media.","U.S. Pat.","No.","4,104,185 describes a heat accumulator in which the heat-energy storage medium consists essentially of a potassium fluoride/water solution having a fluoride content between 44 and 48% by weight.","U.S. Pat.","No.","5,567,346 relates to a latent heat storage material composition comprising 65 to 85 wt % of sodium sulfate decahydrate, 1 to 20 wt % of ammonium chloride and 1 to 20 wt % of sodium bromide, and optionally 1 to 20 wt % of ammonium sulfate.","U.S. Pat.","No.","5,728,316 relates to heat-storage salt mixtures composed of magnesium nitrate hexahydrate and lithium nitrate in mass ratio 86-81:14-19.U.S.","Pat.","No.","5,755,988 relates to a process for moderating the thermal energy content of closed container comprising mixtures of organic acids.","Co-assigned EP 0,229,440, EP 0,251,480, EP 0,308,037 and EP 0,564,721 describe the use of carboxylate salts as corrosion inhibitors in aqueous heat exchange fluids or corrosion-inhibited antifreeze formulations.","EPA No.","99930566.1 describes aqueous solutions of carboxylates that provide frost and corrosion protection.","Aqueous solutions of low carbon (C1-C2) carboxylic acid salts, in combination with higher carbon (C3-C5) carboxylic acid salts, were found to provide eutectic freezing protection.","Improved corrosion protection was found by adding one or more than one C6-C16 carboxylic acids.","The advantage of these carboxylic salts based cooling fluids over ethylene glycol- or propylene glycol cooling fluids is improved heat-transfer due to a higher specific heat and improved fluidity resulting from the higher water content at the same frost protection.","It is another objective of this invention to add heat-storage capacity to the above heat-exchange fluids and other functional fluids and soaps like lubricants and greases.","It is an object of the present invention to provide heat storage salt combinations that are less toxic and less burdensome to the environment than the fluoride-, chloride-, sulfate- and nitrate salts or the acidic salt combinations used in prior art Another object of the invention is to provide heat storage salt combinations that are less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.","FIELD OF THE INVENTION One aspect of the invention relates to the application of alkali metal salts or alkali earth metal salts of carboxylic acids, and combinations of such salts as latent heat-storage media The carboxylate heat-storage salts of this invention are less toxic and more environmentally friendly than the fluoride-, chloride-, sulfate- and nitrate salts or salt combinations used in prior art.","They are also less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.","They are similar to the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.","They are also compatible with the carboxylates (formates and/or acetates) used as freezing point depressant in aqueous heat-exchange fluids.","In heat storage applications it is important to find media with melting temperatures that are in line with the temperature operating range of the heat source and which have high latent heat capacity.","It is another aspect of this invention that mixtures of carboxylic salts can be tuned to provide melting temperatures that fit the application temperatures.","Similarly, combinations with high heat capacity can be selected to optimize storage capacity.","This can be done by mixing different salts of the same carboxylate (for instance the potassium, lithium and/or sodium salt of the same carboxylate) or by mixing the salts of different carboxylates.","In heat storage applications it is also important that the heat storage salts can withstand unchanged and unlimited cycles of heat storage and heat release.","Hydrated heat-storage salts are particularly susceptible.","Loss of water from hydrolyzed crystals will introduce anhydrous crystalline structures with different melting temperatures and different latent heat capacities that may no longer be suitable for the application.","Dehydration at temperatures above the melting temperature of a hydrated salt can be avoided by using hermetically sealed containers and limiting the free space where water can condense without contact with the heat-storage salts.","These measures limit to some extent the use of hydrated salts in heat-storage applications.","It is another aspect of this invention to disperse carboxylate salts with heat-storage capacity in the heat-transfer fluid.","Heat-storage salts can be selected that have limited solubility in the heat-transfer fluid of choice.","The total amount of heat-storage salts added to the solution can be tuned to the heat capacity required in a particular system.","As the melting temperature of the dispersed heat-storage salts is reached, the salts will start to melt and extract heat from the fluid by phase transmission.","The fluid temperature can only rise again when all the heat-storage salts are in molten state.","In the case where hydrated heat-storage salts are used, the use of an aqueous heat-exchange fluid in which the salts are dispersed ensures hydration.","Heat-storage salts can be selected that have densities that are close in solid and liquid phase so that there is no risk of damage to the container or system due to expansion upon phase transition.","In many heat-exchange applications, however, a fluid phase will be preferred to allow easy transport of heat.","Dual heat-exchange systems can of course be used, in which the primary system contains the heat-storage salts, and the secondary system contains the heat-transport fluid.","It is another aspect of the invention to improve the heat capacity of a heat-exchange fluid by dispersion of heat-storage particles in existing heat exchange fluids or other functional fluids or soaps.","Examples are: 1 Heat-exchange fluids based on water soluble alcohol freezing point depressants such as ethylene glycol, propylene glycol, ethanol or methanol.","2 Heat-exchange fluids based on aqueous solutions of low carbon (C1-C2) carboxylic acid salts (formates, acetates) or mixtures thereof.","3 Heat-exchange fluids, lubricants or hydraulic fluids based on mineral- or synthetic oil, mineral and synthetic soaps or greases.","Suspended particles provide heat-storage capacity in the bulk of the existing exchange medium, lubricant or grease.","The alkali metal salts of carboxylic acids have low toxicity, are biodegradable and are not corrosive towards many materials.","An additional advantage of alkali metal carboxylates is that they are similar and/or compatible with the carboxylates used as freezing point depressant and with the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.","EXAMPLES The invention will be more specifically described by way of reference to the following examples.","A number of formulations were evaluated, by subjecting known quantities of salts to controlled heating and cooling cycles between 20° C. and 180° C. EXAMPLE COMPOSITION Comparative A Magnesium Chloride Hexahydrate Comparative B Magnesium Nitrate Hexahydrate Invention 1 Potassium Octanoate Invention 2 Potassium Heptanoate Invention 3 Potassium Octanoate (90%)/Potassium Heptanoate (10%) Invention 4 Potassium Propionate Invention 5 Sodium Propionate (30%)/Potassium Formate (70%) Invention 6 Potassium Octanoate (70%)/Potassium Heptanoate (30%) Invention 7 Brine solution of 80 w/w % Potassium Propionate Invention 8 Sodium Propionate (20%)/Potassium Formate (20%)/ Potassium Heptanoate (10%)/Water (50%) FIGURES The Figures show heat transformation curves for the formulations of the examples.","They are more specifically described hereinafter.","DISCLOSURE OF THE INVENTION Application of Carboxylate Salts in Heat-Storage Applications.","One aspect of the invention is that alkali metal salts and alkali earth metal salts of carboxylic acids have been found to have heat-storage capacities which allow these salts to be used in heat-storage applications.","To evaluate the heat-storage capacities, salts were subjected to controlled heating and cooling cycles over a preset temperature range.","For instance, to evaluate possible automotive applications, known quantities of the salts were subjected to controlled heating and cooling cycles between 20° C. and 180° C. When, upon heating, the melting point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is melted.","By measuring the temperature differential between salt and a reference recipient subjected to the same temperature cycles, the melting point can be determined.","By integrating the temperature differential over time, the latent heat capacity of the sample can be measured.","Similarly, when, upon cooling the solidification point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is solidified.","Again, by integrating the temperature differential overtime, the latent heat capacity of the sample can be estimated (differential scanning calorimetric technique).","By repeating the temperature cycles, the stability of the heat-storage salt can be evaluated.","Literature provides information on the melting point and heat capacity of some known heat-storage salts.","For instance, magnesium chloride hexahydrate (comparative example A) has been reported to have a melting point of 117° C. and a latent heat capacity of 165 KJ/kg.","FIG.","1 shows experimental curves for magnesium chloride hexahydrate.","The temperature cycle has been repeated five times.","FIG.","2 shows the temperature differentials versus time.","In FIG.","3, the temperature differentials are plotted in function of temperature.","From these curves it can be derived that the melting point is indeed 117° C. Under-cooling upon solidification is shown.","Repeatability of the melting point in successive temperature cycles or series is good.","Some reduction in heat capacity is however noted, likely the result of partial dehydration of the salt.","More extreme shifts in melting point(s) and heat-storage capacity are shown in FIG.","4 for magnesium nitrate hexahydrate (comparative example B).","In this experiment heat-storage capacity is lost in the second and third temperature cycle (series 2 and 3).","Additional temperature cycles for the sample of magnesium nitrate hexahydrate are shown in FIG.","5.Dehydration of the salt apparently causes further changes and shifts in melting and solidification points towards higher temperatures.","Carboxylate Salts Provide Stable Heat-Storage Properties.","Surprisingly, a much more stable behavior is found for alkali metal salts of carboxylic acids that are also employed as corrosion inhibitors.","For example, FIG.","6 shows five successive temperature cycles for potassium octanoate (invention example 1).","The melting point of the salt is 57° C. An additional example using potassium heptanoate is shown in FIG.","7 (invention example 2).","The melting point for potassium heptanoate is 61° C. The Melting Point of Carboxylate Salts can be Tuned for a Specific Heat-Storage Application.","The melting point can be tuned for a specific application by the selection and mixing ratio of the alkali metal carboxylates.","For example, a mixture of potassium octanoate (90%) and potassium heptanoate (10%) (invention example 3 shown in FIG.","8) was found to have a melting temperature of about 48° C., particularly suited for heat-storage at lower temperatures.","In aqueous solutes these carboxylate salts or salt combinations show excellent corrosion protection properties.","In addition, they are similar and thus fully compatible with the carboxylates used as corrosion inhibitors in ethylene glycol and propylene glycol heat-exchange fluids and water treatment chemicals.","The low carbon (C1-C2) carboxylic acid alkali metal salts and the medium carbon (C3-C5) carboxylic acid alkali metal salts, or combinations of the two can be used as heat-storage salts.","For example, FIG.","9 (invention example 4) shows consecutive heating and cooling cycles for potassium propionate, with melting temperature of 79° C. A mixture of 30% sodium propionate and 70% potassium formate—FIG.","10 (invention example 5)—was found to have a melting temperature of 167° C. Heat-Storage Properties of Wetted or Hydrated Carboxylate Salts are Easily Restored.","It was found that, starting from hydrated or wetted carboxylate salts, stable heat-storage properties can easily be obtained by one or more temperature cycles in which the water is evaporated.","This is illustrated in FIG.","11 (invention example 6) for a mixture of the potassium octanoate (70%) and potassium heptanoate (30%), with a melting temperature of about 42° C.—water is boiled off from a wet sample in the first heatig cycle and latent heat can already be recovered in the first cooling cycle at a solidification temperature of about 45° C. This allows heat-exchange applications in which water is evaporated or added to the heat-storage salts, for instance to remove excess heat efficiently.","Brine Solutions of Carboxylate Salts have Heat-Storage Capacity.","Brine solutions of carboxylate salts can also be used as heat-storage medium.","For example, FIGS.","12 to 14 (invention example 7) shows the different curves for consecutive heating and cooling cycles for a brine solution of 80 w/w % of potassium propionate.","Contrary to the salts, the aqueous brine solution was contained in a closed container, not allowing evaporation of water.","In the experiment, phase transition on the lower temperature range was apparently not completed when the heating cycle was re-started, due to the high heat-storage capacity of the medium.","Silicone oil was used as reference fluid.","Dispersed Carboxylates Salts Provide Heat-Storage Capacity to Fluids or Soaps.","It is another aspect of this invention to disperse the hydrated salts with heat-storage capacity in the heat-transfer fluid.","For example, FIG.","15 (invention example 8) shows the consecutive heating and cooling cycles for a mixture of 20% sodium propionate and 20% potassium formate and 10% potassium heptanoate with 50% water in comparison with a brine solution without the addition of the potassium heptanoate.","The effect of the heptanoate additions is clearly seen.","This is even more evident from the curves in FIG.","16, showing the differential temperatures in function of time.","FIG.","17 shows the effect of the potassium heptanoate.","The effect of solidification at about 73° C. which is also observed for pure potassium heptanoate (FIG.","7) is clearly seen.","Dispersion of carboxylate heat-storage salts in other fluids will have similar effects.","This will particularly be the case for glycol based heat exchange fluids.","Many carboxylate salts have limited solubility in glycol and water and can thus be dispersed in such fluids to add heat-storage capacity.","Similarly, this is possible in other functional products such as lubricants or hydraulic fluids based on mineral- or synthetic oil, and in mineral-and synthetic soaps or greases."]],"string":"[\n [\n \"Carboxylate salts in heat-storage applications\",\n \"The use of an alkali metal salt or alkaline earth metal salt or of a bring solution of a C1-C16 carboxylic acid, or a mixture thereof, as a medium for the storage and use of thermal energy.\",\n \"The salts or solution can be used in a heat exchange fluid or a lubricant or hydraulic fluid or soap.\"\n ],\n [\n \"1-13.\",\n \"(canceled) 14.A method for the storage and use of thermal energy comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.\",\n \"15.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C3-C18 carboxylic acid or hydrated salts of a C3 to C7 carboxylic acid with a source of thermal energy.\",\n \"16.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C2 carboxylic acids and a salt of one or more C3-C5 carboxylic acids.\",\n \"17.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C5 carboxylic acids and one or more C6-C18 carboxylic acids.\",\n \"18.The method of claim 4 wherein said mixture comprises a salt of one or more C3-C5 carboxylic acids and one or more C6-C16 carboxylic acids.\",\n \"19.The method of claim 1 wherein the temperature range of the thermal energy source is 20 to 180 degrees C. 20.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.\",\n \"21.The method of claim 1 further comprising contacting one or a mixture of hydrated alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.\",\n \"22.A method for improving the thermal properties of a fluid comprising dispersing in said fluid one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.\",\n \"23.The method of claim 9 wherein the fluid is a water soluble alcohol freezing point depressant selected from the group consisting of ethylene glycol, propylene glycol, ethanol and methanol.\",\n \"24.The method of claim 9 wherein the fluid is a lubricant or hydraulic fluid selected from the group consisting of mineral oil, synthetic oil, mineral soap, synthetic soap, mineral grease and synthetic grease.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION Thermal energy originating from any energy source is reusable if it can be stored.\",\n \"Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.\",\n \"in refineries and steam generation plants).\",\n \"Energy generated in peak load time can be managed and stored for later use.\",\n \"Examples are solar heating and electrical heating on low tariff hours.\",\n \"The problem of cold car engine start in wintertime is well known.\",\n \"Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.\",\n \"Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.\",\n \"Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.\",\n \"However these solutions put an extra burden on the vehicle's electrical power system.\",\n \"Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.\",\n \"Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.\",\n \"Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.\",\n \"An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.\",\n \"The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.\",\n \"The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates.\"\n ],\n [\n \"This invention relates to the application of carboxylate salts for storing thermal energy.\",\n \"Melted salts are used for heat-storage because salts absorb heat during the transition from the solid to the liquid phase.\",\n \"This heat is stored in latent form as long as the liquid state persists and released again during the transition from the liquid to the solid phase when the liquid salt solidifies.\",\n \"BACKGROUND OF THE INVENTION Thermal energy originating from any energy source is reusable if it can be stored.\",\n \"Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.\",\n \"in refineries and steam generation plants).\",\n \"Energy generated in peak load time can be managed and stored for later use.\",\n \"Examples are solar heating and electrical heating on low tariff hours.\",\n \"The problem of cold car engine start in wintertime is well known.\",\n \"Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.\",\n \"Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.\",\n \"Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.\",\n \"However these solutions put an extra burden on the vehicle's electrical power system.\",\n \"Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.\",\n \"Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.\",\n \"Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.\",\n \"An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.\",\n \"The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.\",\n \"The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates.\",\n \"PRIOR ART Hydrated fluoride-, chloride-, sulfate- and nitrate salts or salt combinations have been described as heat-storage media.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,104,185 describes a heat accumulator in which the heat-energy storage medium consists essentially of a potassium fluoride/water solution having a fluoride content between 44 and 48% by weight.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,567,346 relates to a latent heat storage material composition comprising 65 to 85 wt % of sodium sulfate decahydrate, 1 to 20 wt % of ammonium chloride and 1 to 20 wt % of sodium bromide, and optionally 1 to 20 wt % of ammonium sulfate.\",\n \"U.S. Pat.\",\n \"No.\",\n \"5,728,316 relates to heat-storage salt mixtures composed of magnesium nitrate hexahydrate and lithium nitrate in mass ratio 86-81:14-19.U.S.\",\n \"Pat.\",\n \"No.\",\n \"5,755,988 relates to a process for moderating the thermal energy content of closed container comprising mixtures of organic acids.\",\n \"Co-assigned EP 0,229,440, EP 0,251,480, EP 0,308,037 and EP 0,564,721 describe the use of carboxylate salts as corrosion inhibitors in aqueous heat exchange fluids or corrosion-inhibited antifreeze formulations.\",\n \"EPA No.\",\n \"99930566.1 describes aqueous solutions of carboxylates that provide frost and corrosion protection.\",\n \"Aqueous solutions of low carbon (C1-C2) carboxylic acid salts, in combination with higher carbon (C3-C5) carboxylic acid salts, were found to provide eutectic freezing protection.\",\n \"Improved corrosion protection was found by adding one or more than one C6-C16 carboxylic acids.\",\n \"The advantage of these carboxylic salts based cooling fluids over ethylene glycol- or propylene glycol cooling fluids is improved heat-transfer due to a higher specific heat and improved fluidity resulting from the higher water content at the same frost protection.\",\n \"It is another objective of this invention to add heat-storage capacity to the above heat-exchange fluids and other functional fluids and soaps like lubricants and greases.\",\n \"It is an object of the present invention to provide heat storage salt combinations that are less toxic and less burdensome to the environment than the fluoride-, chloride-, sulfate- and nitrate salts or the acidic salt combinations used in prior art Another object of the invention is to provide heat storage salt combinations that are less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.\",\n \"FIELD OF THE INVENTION One aspect of the invention relates to the application of alkali metal salts or alkali earth metal salts of carboxylic acids, and combinations of such salts as latent heat-storage media The carboxylate heat-storage salts of this invention are less toxic and more environmentally friendly than the fluoride-, chloride-, sulfate- and nitrate salts or salt combinations used in prior art.\",\n \"They are also less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.\",\n \"They are similar to the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.\",\n \"They are also compatible with the carboxylates (formates and/or acetates) used as freezing point depressant in aqueous heat-exchange fluids.\",\n \"In heat storage applications it is important to find media with melting temperatures that are in line with the temperature operating range of the heat source and which have high latent heat capacity.\",\n \"It is another aspect of this invention that mixtures of carboxylic salts can be tuned to provide melting temperatures that fit the application temperatures.\",\n \"Similarly, combinations with high heat capacity can be selected to optimize storage capacity.\",\n \"This can be done by mixing different salts of the same carboxylate (for instance the potassium, lithium and/or sodium salt of the same carboxylate) or by mixing the salts of different carboxylates.\",\n \"In heat storage applications it is also important that the heat storage salts can withstand unchanged and unlimited cycles of heat storage and heat release.\",\n \"Hydrated heat-storage salts are particularly susceptible.\",\n \"Loss of water from hydrolyzed crystals will introduce anhydrous crystalline structures with different melting temperatures and different latent heat capacities that may no longer be suitable for the application.\",\n \"Dehydration at temperatures above the melting temperature of a hydrated salt can be avoided by using hermetically sealed containers and limiting the free space where water can condense without contact with the heat-storage salts.\",\n \"These measures limit to some extent the use of hydrated salts in heat-storage applications.\",\n \"It is another aspect of this invention to disperse carboxylate salts with heat-storage capacity in the heat-transfer fluid.\",\n \"Heat-storage salts can be selected that have limited solubility in the heat-transfer fluid of choice.\",\n \"The total amount of heat-storage salts added to the solution can be tuned to the heat capacity required in a particular system.\",\n \"As the melting temperature of the dispersed heat-storage salts is reached, the salts will start to melt and extract heat from the fluid by phase transmission.\",\n \"The fluid temperature can only rise again when all the heat-storage salts are in molten state.\",\n \"In the case where hydrated heat-storage salts are used, the use of an aqueous heat-exchange fluid in which the salts are dispersed ensures hydration.\",\n \"Heat-storage salts can be selected that have densities that are close in solid and liquid phase so that there is no risk of damage to the container or system due to expansion upon phase transition.\",\n \"In many heat-exchange applications, however, a fluid phase will be preferred to allow easy transport of heat.\",\n \"Dual heat-exchange systems can of course be used, in which the primary system contains the heat-storage salts, and the secondary system contains the heat-transport fluid.\",\n \"It is another aspect of the invention to improve the heat capacity of a heat-exchange fluid by dispersion of heat-storage particles in existing heat exchange fluids or other functional fluids or soaps.\",\n \"Examples are: 1 Heat-exchange fluids based on water soluble alcohol freezing point depressants such as ethylene glycol, propylene glycol, ethanol or methanol.\",\n \"2 Heat-exchange fluids based on aqueous solutions of low carbon (C1-C2) carboxylic acid salts (formates, acetates) or mixtures thereof.\",\n \"3 Heat-exchange fluids, lubricants or hydraulic fluids based on mineral- or synthetic oil, mineral and synthetic soaps or greases.\",\n \"Suspended particles provide heat-storage capacity in the bulk of the existing exchange medium, lubricant or grease.\",\n \"The alkali metal salts of carboxylic acids have low toxicity, are biodegradable and are not corrosive towards many materials.\",\n \"An additional advantage of alkali metal carboxylates is that they are similar and/or compatible with the carboxylates used as freezing point depressant and with the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.\",\n \"EXAMPLES The invention will be more specifically described by way of reference to the following examples.\",\n \"A number of formulations were evaluated, by subjecting known quantities of salts to controlled heating and cooling cycles between 20° C. and 180° C. EXAMPLE COMPOSITION Comparative A Magnesium Chloride Hexahydrate Comparative B Magnesium Nitrate Hexahydrate Invention 1 Potassium Octanoate Invention 2 Potassium Heptanoate Invention 3 Potassium Octanoate (90%)/Potassium Heptanoate (10%) Invention 4 Potassium Propionate Invention 5 Sodium Propionate (30%)/Potassium Formate (70%) Invention 6 Potassium Octanoate (70%)/Potassium Heptanoate (30%) Invention 7 Brine solution of 80 w/w % Potassium Propionate Invention 8 Sodium Propionate (20%)/Potassium Formate (20%)/ Potassium Heptanoate (10%)/Water (50%) FIGURES The Figures show heat transformation curves for the formulations of the examples.\",\n \"They are more specifically described hereinafter.\",\n \"DISCLOSURE OF THE INVENTION Application of Carboxylate Salts in Heat-Storage Applications.\",\n \"One aspect of the invention is that alkali metal salts and alkali earth metal salts of carboxylic acids have been found to have heat-storage capacities which allow these salts to be used in heat-storage applications.\",\n \"To evaluate the heat-storage capacities, salts were subjected to controlled heating and cooling cycles over a preset temperature range.\",\n \"For instance, to evaluate possible automotive applications, known quantities of the salts were subjected to controlled heating and cooling cycles between 20° C. and 180° C. When, upon heating, the melting point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is melted.\",\n \"By measuring the temperature differential between salt and a reference recipient subjected to the same temperature cycles, the melting point can be determined.\",\n \"By integrating the temperature differential over time, the latent heat capacity of the sample can be measured.\",\n \"Similarly, when, upon cooling the solidification point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is solidified.\",\n \"Again, by integrating the temperature differential overtime, the latent heat capacity of the sample can be estimated (differential scanning calorimetric technique).\",\n \"By repeating the temperature cycles, the stability of the heat-storage salt can be evaluated.\",\n \"Literature provides information on the melting point and heat capacity of some known heat-storage salts.\",\n \"For instance, magnesium chloride hexahydrate (comparative example A) has been reported to have a melting point of 117° C. and a latent heat capacity of 165 KJ/kg.\",\n \"FIG.\",\n \"1 shows experimental curves for magnesium chloride hexahydrate.\",\n \"The temperature cycle has been repeated five times.\",\n \"FIG.\",\n \"2 shows the temperature differentials versus time.\",\n \"In FIG.\",\n \"3, the temperature differentials are plotted in function of temperature.\",\n \"From these curves it can be derived that the melting point is indeed 117° C. Under-cooling upon solidification is shown.\",\n \"Repeatability of the melting point in successive temperature cycles or series is good.\",\n \"Some reduction in heat capacity is however noted, likely the result of partial dehydration of the salt.\",\n \"More extreme shifts in melting point(s) and heat-storage capacity are shown in FIG.\",\n \"4 for magnesium nitrate hexahydrate (comparative example B).\",\n \"In this experiment heat-storage capacity is lost in the second and third temperature cycle (series 2 and 3).\",\n \"Additional temperature cycles for the sample of magnesium nitrate hexahydrate are shown in FIG.\",\n \"5.Dehydration of the salt apparently causes further changes and shifts in melting and solidification points towards higher temperatures.\",\n \"Carboxylate Salts Provide Stable Heat-Storage Properties.\",\n \"Surprisingly, a much more stable behavior is found for alkali metal salts of carboxylic acids that are also employed as corrosion inhibitors.\",\n \"For example, FIG.\",\n \"6 shows five successive temperature cycles for potassium octanoate (invention example 1).\",\n \"The melting point of the salt is 57° C. An additional example using potassium heptanoate is shown in FIG.\",\n \"7 (invention example 2).\",\n \"The melting point for potassium heptanoate is 61° C. The Melting Point of Carboxylate Salts can be Tuned for a Specific Heat-Storage Application.\",\n \"The melting point can be tuned for a specific application by the selection and mixing ratio of the alkali metal carboxylates.\",\n \"For example, a mixture of potassium octanoate (90%) and potassium heptanoate (10%) (invention example 3 shown in FIG.\",\n \"8) was found to have a melting temperature of about 48° C., particularly suited for heat-storage at lower temperatures.\",\n \"In aqueous solutes these carboxylate salts or salt combinations show excellent corrosion protection properties.\",\n \"In addition, they are similar and thus fully compatible with the carboxylates used as corrosion inhibitors in ethylene glycol and propylene glycol heat-exchange fluids and water treatment chemicals.\",\n \"The low carbon (C1-C2) carboxylic acid alkali metal salts and the medium carbon (C3-C5) carboxylic acid alkali metal salts, or combinations of the two can be used as heat-storage salts.\",\n \"For example, FIG.\",\n \"9 (invention example 4) shows consecutive heating and cooling cycles for potassium propionate, with melting temperature of 79° C. A mixture of 30% sodium propionate and 70% potassium formate—FIG.\",\n \"10 (invention example 5)—was found to have a melting temperature of 167° C. Heat-Storage Properties of Wetted or Hydrated Carboxylate Salts are Easily Restored.\",\n \"It was found that, starting from hydrated or wetted carboxylate salts, stable heat-storage properties can easily be obtained by one or more temperature cycles in which the water is evaporated.\",\n \"This is illustrated in FIG.\",\n \"11 (invention example 6) for a mixture of the potassium octanoate (70%) and potassium heptanoate (30%), with a melting temperature of about 42° C.—water is boiled off from a wet sample in the first heatig cycle and latent heat can already be recovered in the first cooling cycle at a solidification temperature of about 45° C. This allows heat-exchange applications in which water is evaporated or added to the heat-storage salts, for instance to remove excess heat efficiently.\",\n \"Brine Solutions of Carboxylate Salts have Heat-Storage Capacity.\",\n \"Brine solutions of carboxylate salts can also be used as heat-storage medium.\",\n \"For example, FIGS.\",\n \"12 to 14 (invention example 7) shows the different curves for consecutive heating and cooling cycles for a brine solution of 80 w/w % of potassium propionate.\",\n \"Contrary to the salts, the aqueous brine solution was contained in a closed container, not allowing evaporation of water.\",\n \"In the experiment, phase transition on the lower temperature range was apparently not completed when the heating cycle was re-started, due to the high heat-storage capacity of the medium.\",\n \"Silicone oil was used as reference fluid.\",\n \"Dispersed Carboxylates Salts Provide Heat-Storage Capacity to Fluids or Soaps.\",\n \"It is another aspect of this invention to disperse the hydrated salts with heat-storage capacity in the heat-transfer fluid.\",\n \"For example, FIG.\",\n \"15 (invention example 8) shows the consecutive heating and cooling cycles for a mixture of 20% sodium propionate and 20% potassium formate and 10% potassium heptanoate with 50% water in comparison with a brine solution without the addition of the potassium heptanoate.\",\n \"The effect of the heptanoate additions is clearly seen.\",\n \"This is even more evident from the curves in FIG.\",\n \"16, showing the differential temperatures in function of time.\",\n \"FIG.\",\n \"17 shows the effect of the potassium heptanoate.\",\n \"The effect of solidification at about 73° C. which is also observed for pure potassium heptanoate (FIG.\",\n \"7) is clearly seen.\",\n \"Dispersion of carboxylate heat-storage salts in other fluids will have similar effects.\",\n \"This will particularly be the case for glycol based heat exchange fluids.\",\n \"Many carboxylate salts have limited solubility in glycol and water and can thus be dispersed in such fluids to add heat-storage capacity.\",\n \"Similarly, this is possible in other functional products such as lubricants or hydraulic fluids based on mineral- or synthetic oil, and in mineral-and synthetic soaps or greases.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10296826"}}},{"rowIdx":93,"cells":{"text":{"kind":"list like","value":[["Stabiliser for radiopharmaceuticals","The present invention provides an improved stabiliser for radiopharmaceuticals which inhibits impurities from being produced by two kinds of decomposition mechanisms and exhibits such an effect that the shelf life of a radiopharmaceutical after its preparation is prolonged as compared with conventional ones.","The improvement comprises a combination of an amino-substituted aromatic carboxylic acid or its salt, ester or amide in combination with a diphosphonic acid or its salt."],["1.A stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical which is susceptible to either reductive degradation or radiolysis; (ii) a first stabiliser for said radiopharmaceutical which includes an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a second stabiliser for said radiopharmaceutical which includes a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.","2.The composition of claim 1 wherein the amino-ubstituted aromatic carboxylic acid or its salt, ester or amide is selected from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 3, 5-diaminobenzoic acid, 4-amino-salicylic acid, and salts, esters and amides thereof.","3.The composition of claim 1, wherein the diphosphonic acid or its salt is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof.","4.The composition of claim 1, wherein the amino-substituted aromatic carboxylic acid or salt, ester or amide thereof is sodium 4-aminobenzoate, and the diphosphonic acid is methylenediphosphonic acid.","5.The composition of claim 1, wherein the radiopharmaceutical includes a γ-ray emitter or a β-ray emitter.","6.The composition of claim 5, wherein the radiopharmaceutical includes Tc-99m, Re-186 or Re-188.7.The composition of claim 1, wherein the radiopharmaceutical includes a metal complex of a radionuclide with a ligand.","8.The composition of claim 7, wherein the ligand is an amine oxime.","9.The composition of claim 8, wherein the amine oxime is selected from the group consisting of d,l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.","10.A non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition of claim 1 which comprises: (i) a first stabiliser which comprises an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; and (ii) a second stabiliser which comprises a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.","11.The kit of claim 10, which includes 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.","12.The kit of claim 10, further comprising a tin (II) reductant, wherein the molar ratio of the diphosphonic acid or its salt to tin (II) is in the range 1 to 10.13.The kit of claim 10, further comprising an amine oxime ligand.","14.The kit of claim 13 wherein the amine oxime is selected from the group consisting of d, l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.","15.The kit of claim 10, wherein the first and second stabilzers are lyophilised.","16.In a method of preparing a radiopharmaceutical composition which includes adding stabilizers to a radiopharmaceutical, wherein the radiopharmaceutical is not a metal complex of diphosphonic acid, the improvement comprising using a diphosphonic acid or a salt thereof as a stabiliser.","17.Cancelled 18.The method of claim 16, wherein the radiopharmaceutical comprises Tc-99m.","19.The method of claim 16, wherein the diphosphonic acid is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof."],[" BACKGROUND TO THE INVENTION Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.","Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.","In the case of Tc-99m, the latter is referred to as post-reconstitution instability.","U.S. Pat.","No.","4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99m Tc-complexes of diphosphonic acids.","Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99m Tc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.","99m Tc-HMPAO is particularly unstable with respect to post-reconstitution stability.","99m Tc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.","The function of the stannous ion is to reduce the 99m Tc-pertechnetate ( 99m TcO 4 − ), ie.","technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99m Tc-HMPAO metal complex.","The radiochemical purity (Rcp) of 99m Tc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99m Tc labelling.","After Tc-99m radiolabelling, the Rcp of 99m Tc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99m Tc complex of unknown structure derived from 99m Tc-HMPAO, 99m Tc-pertechnetate ( 99m TcO 4 − ) and reduced-hydrolysed-technetium [ 99m Tc].","Of these impurities, both the secondary complex and 99m Tc-pertechnetate are decomposition products of 99m Tc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.","Med.","29, 1568-1576, 1988).","The secondary complex is believed to be produced when the lipophilic 99m Tc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.","stannous) remaining from the pertechnetate reduction step.","On the other hand, the 99m Tc-pertechnetate impurity is produced when 99m Tc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.","radiolysis of the solvent.","Accordingly, in order to inhibit the production of both the 99m Tc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.","Thus, Nucl.","Med.","Biol.","7, 675-680 (1989); Eur.","J. Nucl.","Med.","16, 541 (1990); Eur.","J. Nucl.","Med.","20, 661-666 (1993) and Eur.","J. Nucl.","Med.","22, 1163-1172 (1995) all report attempts to stabilise 99m Tc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.","In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99m Tc-HMPAO to at least 80% at 4 hours post reconstitution.","Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99m Tc-HMPAO at 6 hours post reconstitution to at least 80%.","The stabilisation mechanisms of 99m Tc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.","Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99m Tc-HMPAO.","However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99m Tc-blue or cobalt chloride is used as a stabiliser for 99m Tc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99m Tc-pertechnetate.","Accordingly, any kit for the preparation of 99m Tc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).","One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.","The other is a vial containing the stabiliser (methylene blue or cobalt chloride).","Thus, the most successful prior art methods of stabilising 99m Tc-HMPAO to date all require the use of 2-vial kits.","When the kit for preparing 99m Tc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99m Tc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.","methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.","It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.","Too short a time, and 99m Tc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.","Too long a time, and the stabilising effect is delayed.","In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.","The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.","In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.","Moreover, when methylene blue is added to 99m Tc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.","There is therefore a need for a single vial kit for the preparation of 99m Tc-HMPAO which has both shelf-life and post-radiolabelling stability.","The present invention provides a kit which solves this problem, and is straightforward to use.","The Present Invention.","The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid."],["FIELD OF THE INVENTION The present invention relates to a stabilised radiopharmaceutical composition, together with non-radioactive kits for the preparation of the stabilised radiopharmaceutical composition.","BACKGROUND TO THE INVENTION Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.","Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.","In the case of Tc-99m, the latter is referred to as post-reconstitution instability.","U.S. Pat.","No.","4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99mTc-complexes of diphosphonic acids.","Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99mTc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.","99mTc-HMPAO is particularly unstable with respect to post-reconstitution stability.","99mTc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.","The function of the stannous ion is to reduce the 99mTc-pertechnetate (99mTcO4−), ie.","technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99mTc-HMPAO metal complex.","The radiochemical purity (Rcp) of 99mTc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99mTc labelling.","After Tc-99m radiolabelling, the Rcp of 99mTc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99mTc complex of unknown structure derived from 99mTc-HMPAO, 99mTc-pertechnetate (99mTcO4−) and reduced-hydrolysed-technetium [99mTc].","Of these impurities, both the secondary complex and 99mTc-pertechnetate are decomposition products of 99mTc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.","Med.","29, 1568-1576, 1988).","The secondary complex is believed to be produced when the lipophilic 99mTc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.","stannous) remaining from the pertechnetate reduction step.","On the other hand, the 99mTc-pertechnetate impurity is produced when 99mTc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.","radiolysis of the solvent.","Accordingly, in order to inhibit the production of both the 99mTc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.","Thus, Nucl.","Med.","Biol.","7, 675-680 (1989); Eur.","J. Nucl.","Med.","16, 541 (1990); Eur.","J. Nucl.","Med.","20, 661-666 (1993) and Eur.","J. Nucl.","Med.","22, 1163-1172 (1995) all report attempts to stabilise 99mTc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.","In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99mTc-HMPAO to at least 80% at 4 hours post reconstitution.","Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99mTc-HMPAO at 6 hours post reconstitution to at least 80%.","The stabilisation mechanisms of 99mTc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.","Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99mTc-HMPAO.","However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99mTc-blue or cobalt chloride is used as a stabiliser for 99mTc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99mTc-pertechnetate.","Accordingly, any kit for the preparation of 99mTc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).","One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.","The other is a vial containing the stabiliser (methylene blue or cobalt chloride).","Thus, the most successful prior art methods of stabilising 99mTc-HMPAO to date all require the use of 2-vial kits.","When the kit for preparing 99mTc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99mTc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.","methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.","It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.","Too short a time, and 99mTc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.","Too long a time, and the stabilising effect is delayed.","In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.","The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.","In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.","Moreover, when methylene blue is added to 99mTc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.","There is therefore a need for a single vial kit for the preparation of 99mTc-HMPAO which has both shelf-life and post-radiolabelling stability.","The present invention provides a kit which solves this problem, and is straightforward to use.","The Present Invention.","The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.","DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides: a stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical; (ii) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a diphosphonic acid or salt thereof, is with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.","By the term ‘amino-substituted aromatic carboxylic acid’ is meant a compound in which at least one hydrogen atom on the aromatic ring of an aromatic carboxylic acid is substituted with at least one amino group.","The aromatic group is preferably benzene.","Preferred amino-substituted aromatic carboxylic acids are: 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid (pABA), 3,5-diaminobenzoic acid and 4-aminosalicylic acid.","4-aminobenzoic acid (pABA) is especially preferred.","The salt of the amino-substituted aromatic carboxylic acid is suitably a salt with a biocompatible cation.","By the term “biocompatible cation” is meant a positively charged counterion which forms a salt with an ionised, negatively charged group (here a carboxylate group), where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.","Examples of suitable biocompatible cations include: the alkali metals (eg.","sodium or potassium); the alkaline earth metals (eg.","calcium, magnesium and barium); and the ammonium ion.","A preferred biocompatible cation is sodium.","Preferred salts of the present invention include: sodium 2-aminobenzoate, sodium 3-aminobenzoate, sodium 4-aminobenzoate (NapABA), sodium 3,5-diaminobenzoate, sodium 4-aminosalicylate, potassium 2-aminobenzoate, potassium 3-aminobenzoate, potassium 4-aminobenzoate, potassium 3,5-diaminobenzoate and potassium 4-aminosalicylate.","Sodium 4-aminobenzoate (NapABA) is especially preferred.","Suitable esters of the aromatic carboxylic acid include methyl, ethyl or propyl esters.","Preferred esters are: methyl 2-aminobenzoate, methyl 3-aminobenzoate, methyl 4-aminobenzoate, methyl 3,5-diaminobenzoate, methyl 4-aminosalicylate, ethyl 2-aminobenzoate, ethyl 3-aminobenzoate, ethyl 4-aminobenzoate, ethyl 3,5-diaminobenzoate, ethyl 4-aminosalicylate, propyl 2-aminobenzoate, propyl 3-aminobenzoate, propyl 4-aminobenzoate, propyl 3,5-diaminobenzoate and propyl 4-aminosalicylate.","Suitable amides of the amino-substituted aromatic carboxylic acid are amides formed by derivatising the carboxyl group of the aromatic carboxylic acid with either ammonia or a compound having at least one amino group, and include such compounds as 2-aminobenzamide, 3-aminobenzamide and 4-aminobenzamide.","The diphosphonic acid of the present invention is suitably a 1,1- or a 1,2-diphosphonic acid, or a diphosphonic acid derivative of an amine such as ethylenediaminetetraphosphonic acid (EDTMP).","1,1-diphosphonic acids are preferred, such as methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP).","Methylenediphosphonic acid (MDP) and hydroxymethanediphosphonic acid (HMDP) are especially preferred.","Suitable salts of the diphosphonic acid are with a ‘biocompatible cation’ as defined above.","Preferred such diphosphonate salts include: sodium methylenediphosphonate, sodium hydroxymethanediphosphonate, sodium hydroxyethanediphosphonate, sodium ethylenediamine-tetraphosphonate, ammonium methylenediphosphonate, ammonium hydroxymethanediphosphonate, ammonium hydroxyethanediphosphonate and ammonium ethylenediamine-tetraphosphonate.","The combination of compounds in the stabiliser for radiopharmaceuticals of the present invention is preferably a combination of sodium 4-aminobenzoate (NapABA) with methylenediphosphonic acid (MDP) or hydroxymethanediphosphonic acid (HMDP).","Without wishing to be bound by theory, it is believed that the stabilisers of the present invention act as follows: The amino-substituted aromatic carboxylic acid or its salt, ester or amide has a reducing ability and removes any oxidising free radicals which result from the radiolysis of the solution, and hence inhibits the oxidative destruction of the radiopharmaceutical by free radical attack.","On the other hand, when excess tin(II) reductant remains after completion of the radiolabelling, the diphosphonic acid or its salt inhibits the excess stannous ion from reductively degrading the radiopharmaceutical.","It is a surprising finding of the present invention that such stabilisers can be present in a single step radiopharmaceutical preparation without either: (i) preventing or hindering the stannous reduction of the radiopharmaceutical precursor, with consequent adverse effect on Rcp; (ii) complexing with the radiometal to form undesirable radioactive impurities (eg.","diphosphonic acid complexes of the radionuclide).","Accordingly, the amount of the diphosphonic acid or its salt to be added to the radiopharmaceutical depends upon the amount of the reducing tin(II) contained in the radiopharmaceutical preparation or kit.","The amount of the diphosphonic acid or its salt effective to stabilise the radiopharmaceutical is 1 to 10 moles per mole of stannous, preferably 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.","The radionuclide of the radiopharmaceutical of the present invention is a γ-ray or β-ray emitter, preferably Tc-99m, Re-186 or Re-188, most preferably Tc-99m.","γ-ray emitters are mainly used for radiodiagnosis and β-ray emitters are mainly used for radiotherapy.","When a radiodiagnostic radiopharmaceutical of this invention is intracorporeally administered to a human being, the level of radioactivity used is in the range of from 370 to 1,480 MBq, preferably from 370 to 1,110 MBq.","When a radiotherapeutic agent of the radiopharmaceuticals of this invention is intracorporeally administered to a human being, the radioactivity level is in the range of from 37 to 18,500 MBq, preferably from 370 to 7,400 MBq.","The radiopharmaceutical comprises an active ingredient which is susceptible to either degradation by the reducing action of the reductant (present to help effect labelling of the radionuclide), or radiolysis.","By using the stabiliser composition of the present invention to stabilise such active ingredients, it is possible to prolong the useful lifetime post-radiolabelling to at least twice that of the prior art.","The stabilisers of the present invention are particularly useful when the active ingredient comprises a ligand having a tetradentate diaminedioxime donor set, especially d,l-hexamethylpropyleneamine oxime or HMPAO (exametazime), 4,9-diaza-3,3,10,10-tetramethyldodecane-2,11-dione dioxime (PnAO), and similar compounds.","In a second aspect, the present invention provides a non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition described above which comprises: (i) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (ii) a diphosphonic acid or salt thereof, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.","Thus, the stabiliser for radiopharmaceuticals of the present invention can be pre-mixed with the ligand prior to radiolabelling, so that the formulation of a single vial, non-radioactive kit for the preparation of a stabilised radiopharmaceutical (which is not a radionuclide complex of the diphosphonic acid stabilizer) is possible.","This simplifies the labelling procedure of the prior art 2-vial kit, shortens the labelling time and diminishes the risk of operator exposure to harmful radiation.","The single vial kit of the present invention also has an extended stability post radioactive preparation, which in turn extends the useful lifetime for the user of the kit, eg.","a clinician.","Suitable non-radioactive kits of the present invention comprise 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.","When the kit contains stannous, the amount of the diphosphonic acid or its salt effective to stabilise is 1 to 10 moles per mole of stannous.","Preferably, the ratio of diphosphonic acid or its salt is 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.","In a third aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid suitable diphosphonic acid salts are those with a biocompatible cation as described above.","Preferably the radiopharmaceutical also comprises stannous in the formulation.","Most preferably, the radiopharmaceutical comprises 99mTc.","Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.","In a fourth aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for non-radioactive kits for the preparation of radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.","Suitable diphosphonic acid salts are those with a biocompatible cation as described above.","Preferably the non-radioactive kit further comprises stannous in the formulation.","Most preferably, the non-radioactive kit is for the preparation of a 99mTc radiopharmaceutical.","Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.","DESCRIPTION OF THE DRAWINGS FIG.","1 compares the change in Rcp of 99mTc-HMPAO prepared from Composition III, Composition III+lactose and a commercially available kit for preparing 99mTc-HMPAO, each of which had been stored for 6 months prior to reconstitution.","The present invention is explained in more detail with reference to the following Examples: Example 1 is a comparative example for a two step process (including prior art stabilisers such as cobalt chloride).","An Rcp of 99mTc-HMPAO of at least 80% 3 hours after the addition is judged as effective stabilisation.","However, as is clear from Table 1, the only single stabiliser which meets this criterion is cobalt chloride.","Example 2 shows that 99mTc-HMPAO can be effectively stabilised in a single-step process, using a mixture of two compounds as stabilisers, and that the stability is significantly increased over that achieved using the 2-step process of Example 1.Example 3 provides further data on optimising the combinations of MDP/NapABA and HMDP/NapABA in a single step process.","The molar ratio of Sn2+ to MDP/HMDP was kept constant (1:5), and the amounts added were varied.","Example 4 shows that lyophilised non-radioactive kits for the preparation of 99mTc radiopharmaceuticals can be prepared using the compositions of the present invention.","Lactose (a known cryoprotectant excipient in freeze-dried formulations), is shown to be useful to preserve stannous levels for extended shelf-life periods.","Example 5 shows that the combination HMDP/NapABA is also effective as a stabiliser for 99mTc-HMPAO.","Example 6 provides data on the stannous or tin(II) content of the freeze-dried kits, as a function of shelf-life storage in the dark at 4° C., and shows that the amount of reducing tin(II) in Composition III+lactose is higher than that in Composition III.","Moreover, the amount of the reducing tin(II) in Composition III shows some tendency to decrease with increasing storage time.","Accordingly, when the freeze-dried kit is stored for a prolonged period of time, lactose may be used in the kit composition to maintain the amount of the tin(II) reductant constant.","Example 7 compares the Rcp after 6-months storage of the freeze-dried kits of the present invention, with the Rcp for 99mTc-HMPAO prepared from a commercially available kit.","The Rcp decreases as a function of time post-reconstitution for the commercial kit, but for Composition III and Composition III+lactose, the Rcp's at 6 hours post-reconstitution were at least 80%.","This shows that, whilst the stannous levels without lactose do show some modest decrease (Example 5), the decrease does not impair the performance of the kit.","Example 8 shows that freeze-dried kits containing the stabiliser composition HMDP/NapABA have an effective shelf-life.","Example 9 compares the biodistribution in rats of 99mTc-HMPAO prepared using kit Composition III and kit Composition III+lactose of the present invention, with a commercial 99mTc-HMPAO kit preparation.","No significant difference in uptake in target organs was found.","This includes the brain, which is the diagnostic imaging application of the present commercial 99mTc-HMPAO agent.","These results show that the added compounds (MDP, NapABA and lactose), which are not present in the commercially available kit, do not adversely affect the biological behaviour of the 99mTc radiopharmaceutical.","Example 10 describes various lyophilisation methods suitable for freeze-dried kit preparation of formulations of the present invention.","Example 11 compares the Rcp of the various freeze-dried kits of Example 10.Example 1 The Rcp of 99mTc-HMPAO Using a Single Stabiliser: Two Step Process (Comparative Example) The radiochemical purity (Rcp) of 99mTc-HMPAO prepared using a commercially available kit (CERETEC™) for preparing 99mTc-HMPAO is 80% at one hour post-reconstitution, and the Rcp decreases thereafter with time.","On the other hand, in the case of 99mTc-HMPAO stabilised with cobalt chloride, the Rcp 6 hours after radiolabelling is 80%.","The following known medically acceptable compounds and additives were studied: ascorbic acid, sodium ascorbate, gentisic acid, gentisic acid ethanolamide, methylenediphosphonic acid (MDP), succinic acid, 4-aminobenzoic acid (pABA) and sodium 4-aminobenzoate (NapABA).","To each of ten vials each containing 0.5 mg of HMPAO and 4.0 μg of Sn2+ (stannous), was added sodium pertechnetate (1.48 GBq in 5 ml of saline) to carry out the radiolabelling.","To each of 9 of the ten reconstituted vials was individually added the compound shown in Table 1 at a time of 2 minutes post-radiolabelling.","Nothing was added to the remaining vial.","At 1 minute and 3 hours after the addition, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).","The results are given in Table 1: TABLE 1 Radiochemical purity (Rcp) of 99mTc-HMPAO after the addition of a single stabiliser (%, n = 3).","Name of Additive Rcp 1 min from Rcp 3 hours from (amount) addition (%) addition (%) No additive 87 ± 4 38 ± 15 Cobalt chloride 94 ± 3 89 ± 2 (200 μg) Ascorbic acid 83* 41* (4.5 μg) Sodium ascorbate 90* 51* (10 mg) Gentisic acid 87 ± 2 77 ± 3 30 μg) Gentisic acid ethanolamide 61 ± 3 29 ± 13 (30 μg) Methylene diphosphonic acid 79* 30* (10 μg) 4-Aminobenzoic acid 80 ± 8 67 ± 5 (30 μg) Sodium4-amino-benzoate 69 ± 5 39 ± 11 (30 μg) Succinic acid 51* 14* (30 μg) Note: *n = 1 Example 2 Examination of Stabilisation of 99mTc-HMPAO by a Combination of Two Compounds: Single Step Process The combinations studied were: (i) ascorbic acid and hydroxymethanediphosphonic acid (HMDP); (ii) sodium 4-aminobenzoate (NapABA) and methylenediphosphonic acid (MDP).","A composition comprising 0.5 mg of HMPAO, 4.0 μg of Sn2+, 1.0 μg of HMDP and 0.5 μg of ascorbic acid (Composition A), and a composition comprising 0.5 mg of HMPAO, 5.4 μg of Sn2+, 40.5 μg of MDP and 0.5 mg of NapABA (Composition B) were prepared.","To each of A and B was added sodium pertechnetate (1.48 GBq in 5 ml) to carry out the labelling.","After 3 hours, an aliquot was taken from each, and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).","The Rcp of 99mTc-HMPAO 3 hours after Tc-99m labelling was about 62% in the case of Composition A and about 80% in the case of Composition B.","Example 3 Optimisation of Stabiliser Composition: Single Step Process As shown in Table 2, six different sample compositions were prepared and stored in the dark at 4° C. One vial of each formulation was taken out at each time point (1, 7, 31 and 32-days storage), and sodium pertechnetate (1.48 GBq in 5 ml) was added.","At 6 hours post addition of pertechnetate, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).","The results are shown in Table 2: TABLE 2 Sample Compositions and Rcp of 99mTc-HMPAO 6 hours post-radiolabelling (n = 1, *n = 3).","Composition I II III IV V VI Preparation HMPAO 0.5 0.5 0.5 0.5 0.5 0.5 Conditions (mg) NapABA 0.5 0.1 0.1 0.1 0.5 — Sn2+ 5.4 5.4 8.1 8.1 8.1 8.1 (μg) MDP 40.5 40.5 61.0 61.0 — — (μg) HMPD — — — — 66.0 66.0 (μg) Rcp (%) - 1 day 86.4 79.5 82.2 73.8 84.8 ± 2.7* 77.3 ± 5.5* after storage 7 days 80.8 80.9 82.1 81.1 — — time 32 days 79.0 81.8 84.3 66.5 — — 31 days — — — — 83.5 ± 2.2* 72.0 ± 0.2* Note: *n = 3.Compositions II, III and V show that, even in the case of samples stored for 32 days in the dark at 4° C., the Rcp at 6 hours post-reconstitution was at least 80%.","Compositions III and V, which exhibited a higher Rcp, 5 were therefore used as the basis for subsequent tests.","Example 4 Stabilisation of 99mTc-HMPAO by a Combination of MDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition III of Example 3, and a composition prepared by adding lactose to Composition III (referred to hereinafter as Composition III+lactose) were used to prepare four lots of freeze-dried kit in each case.","The amounts of compounds shown below correspond to the scale for a production batch of about 100 freeze-dried kit vials.","First, anhydrous stannous chloride (25.8 mg) and MDP (122 mg) were dissolved in 0.1 M hydrochloric acid (1,000 ml).","The resulting solution is referred to hereinafter as Solution 1.d,l-HMPAO (100 mg) and NapABA (100 mg) were dissolved in Solution 1 (100 ml), to give a solution referred to hereinafter as Solution 2.Solution 2 was divided into two 50 ml portions, one of which was adjusted to a pH of 8.5-9.0 with sodium hydroxide and thereafter the total volume was adjusted to 10.0 ml with water by use of a measuring cylinder.","In the other 50 ml portion was dissolved 300 mg of lactose monohydrate, the resulting solution was adjusted to a pH of 8.5 to 9.0 with sodium hydroxide, and thereafter, the total volume was adjusted to 100 ml with water by use of a measuring cylinder.","1.0 ml of each of the solutions obtained was placed in each vial, which was then frozen at −50 to −78° C. and then freeze-dried for about 24 hours.","After completion of the freeze-drying, the vials were sealed and the freeze-dried kit was stored in the dark at 4° C. The same procedure was repeated four times to prepare four separate lots of each of, Composition III and Composition III+lactose.","Lot Nos.","ID-01 to ID-04 were of Composition III and Lot Nos.","ID-05 to ID-08 were of Composition III+lactose.","The production conditions and results of each lot are shown in Table 3.TABLE 3 Production conditions and results of each lot Freezing Number of pH before temperature Vials Lot No.","freezing (° C.) Produced Property# ID-01 9.0 −50 99 Fair ID-02 9.0 −78 90 Bad ID-03 8.5 −78 39 Bad ID-04 9.0 −50 99 Fair ID-05 9.0 −50 91 Good ID-06 9.0 −78 92 Good ID-07 8.5 −78 94 Good ID-08 8.5 −50 91 Good #where ’property' refers to the behaviour during lyophilisation: Good = the cake in the freeze-dried kits kept well-formed; Fair = the cake in the freeze-dried kits was partly broken, but there was no loss of powder from the vials during lyophilisation; Bad = the cake in the freeze-dried kits was completely broken, and some of the powder was lost from the vials during lyophilisation.","In all the lots other than ID-03 (which is of Composition III), at least 90 vials were produced.","Among the four lots of Composition III, only ID-03 was adjusted to a pH of 8.5 before freezing.","Moreover, in the case of Composition III+lactose, even when the pH was adjusted to 8.5 before freezing (ID-07, ID-08), at least 90 vials could be produced.","Accordingly, when a freeze-dried kit is produced, it is desirable to adjust the pH to 9.0 in the case of lactose-free Composition III.","Example 5 Stabilisation of 99mTc-HMPAO by a Combination of HMDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition V of Example 3, and a composition prepared by adding lactose to Composition V (referred to hereinafter as Composition V+lactose) were used to prepare one lot of freeze-dried kits in each case.","The freeze-dried kits were prepared in much the same manner as that of Example 4, except that Solution 1 was slightly different—in this case anhydrous stannous chloride (25.8 mg) and HMDP-2Na (132.0 mg) were dissolved in 0.1M hydrochloric acid (1,000 mL).","Lot Nos.","ID-09 was of Composition V and Lot Nos.","ID-10 was of Composition V+lactose.","The production conditions and results of each lot are shown in Table 4.TABLE 4 Production conditions and results of each lot.","Freezing Number of pH before Temperature Vials freezing (° C.) Produced Property# ID-09 8.8 −50° C. 49 Fair ID-10 8.8 −50° C. 45 Good #defined in Example 4.Example 6 Determination of the Amount of tin(II) in the Freeze-Dried Kit as a Function of Storage Time Vials of Composition III (ID-01, O2, 03 and 04), Composition III+lactose (ID-05, 06, 07 and 08), Composition V (ID-09) and Composition V+lactose (ID-10) which had been stored in the dark at 4° C. for periods of 1 day, 3-months and 6-months, were allowed to warm to room temperature.","The amount of tin(II) was then determined by an absorbance method.","The results obtained are shown in Table 5.TABLE 5 Amounts of reducing tin(II) determined (μg/vial, n = 2).","1-day storage 3-month storage 6-month storage Composition III 9.5 ± 0.2 9.1 ± 0.5 8.7 ± 0.5 Composition III + 10.3 ± 0.5 10.5 ± 0.5 10.8 ± 0.4 lactose Composition V 10.1 ± 0.0 — — Composition V + 10.3 ± 0.3 — — lactose Note Each value was an average value of 4 lots.","Example 7 Radiochemical Purity of Freeze-Dried Kits Containing MDP and NapABA Studied over a 6-Month Shelf-Life Storage Period Four lots of each of Composition III and Composition III+lactose produced in Example 4 were subjected to examination of radiochemical purity after 6 -months storage in the dark at 4° C. Vials of freeze-dried Composition III (ID-01, O2, 03 and 04) and Composition III+lactose (ID-OS, 06, 07 and 08) were stored for 6 months in the dark at 4° C. After a storage period of 1 day, 1, 3 and 6 months, the vials were allowed to warm to room temperature, and Tc-99m labelling was carried out.","Thus, to one vial of each lot was added sodium pertechnetate (1.48 GBq in 5 ml).","Aliquots were taken after times of 2 minutes, one hour, 3 hours and 6 hours and the Rcp was measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline and filter paper/50% aqueous acetonitrile).","The results obtained at 6 hours are shown in Table 6.When Composition III and Composition III+lactose were radiolabelled after 6-month shelf-life storage, simultaneously, sodium pertechnetate (1.48 GBq/5 ml) was added to a commercially available kit for preparing 99mTc-HMPAO (containing 0.5 mg of HMPAO and 4.0 μg of Sn2+) as a control.","An aliquot thereof was taken after times of one minute, one hour, 3 hours and 6 hours, and then the Rcp was measured in the same manner.","The results obtained are shown in FIG.","1.TABLE 6 Rcp of 99mTc-HMPAO at 6 hr post reconstitution by a combination of MDP and NapABA as a function of storage time.","STORAGE PERIOD 1-day 1-month 3-months 6-months Rcp Composition III 86.4 ± 2.2 87.4 ± 1.5 86.5± 2.7 88.4 ± 3.4 (%) Composition III + 86.2 ± 2.3 86.2 ± 2.4 87.9 ± 2.6 85.6 ± 2.7 Lactose Example 8 Radiochemical Purity of Freeze-Dried Kits Containing HMDP and NapABA for 1-Month Shelf-Life Storage One lot of each of Composition V and Composition V+lactose produced in Example 5 were subjected to examination of radiochemical purity for 1-month storage in the dark at 4° C. The Rcp was measured in the same manner.","The results obtained are shown in Table 7.TABLE 7 Rcp of 99mTc-HMPAO at 6 hr post-reconstitution stabilised by a combination of HMDP and NapABA as a function of storage time.","Storage Time 1-day 1-month Rcp Composition V 84.8 ± 2.7 83.5 ± 2.2 (%) Composition V + 87.3 ± 2.8 86.0 ± 1.0 Lactose Example 9 Biodistribution in the Rat To one vial of each of Composition III (ID-O2) and Composition III+lactose (ID-06) obtained in Example 4 was added sodium pertechnetate in a proportion of 1.48 GBq/5 ml to carry out the radiolabelling.","After 2 hours, 3.0 to 3.7 MBq of the reconstituted solution was administered to a female Sprague-Dawley strain rat (body weight 140-170 g) which had been previously anaesthetised with sodium thiopentobarbital in the tail vein.","At one hour post-administration, the animal was sacrificed and the radioactivity in each organ was measured using a NaI single channel analyser.","Separately, sodium pertechnetate was added in a proportion of 1.48 GBq/5 ml to the commercially available kit for preparing 99mTc-HMPAO.","In the same manner, at 15 minutes post-reconstitution, 3.0-3.7 MBq of the solution was administered to the same kind of rat, and the biodistribution of radioactivity in each organ measured.","The results obtained are shown in Table 8: TABLE 8 Biodistribution of Commercial 99mTc-HMPAO, Composition III (ID-02) and Composition III + lactose (ID-06) in the female rat at one hour post administration (upper row: % ID/organ, lower row: % ID/g, n = 5).","Composition III + Commercial Composition lactose 99mTc-HMPAO III (ID-02) (ID-06) Brain 1.66 ± 0.13 1.65 ± 0.10 1.73 ± 0.15 0.96 ± 0.07 0.96 ± 0.15 1.01 ± 0.07 Blood 15.38 ± 0.72 14.41 ± 1.68 14.07 ± 1.51 1.56 ± 0.04 1.64 ± 0.06 1.45 ± 0.18 Heart 0.81 ± 0.04 0.85 ± 0.03 0.90 ± 0.10 1.61 ± 0.08 1.75 ± 0.10 1.72 ± 0.03 Lung 3.41 ± 0.20 3.15 ± 0.18 3.39 ± 0.38 4.05 ± 0.28 4.26 ± 0.29 4.14 ± 0.36 Liver 5.91 ± 0.20 6.49 ± 0.48 7.68 ± 0.38 1.10 ± 0.07 1.36 ± 0.13 1.44 ± 0.14 Spleen 0.78 ± 0.10 0.79 ± 0.18 0.86 ± 0.12 2.17 ± 0.12 2.24 ± 0.12 2.25 ± 0.21 Kidney 4.27 ± 0.27 5.04 ± 0.48 4.81 ± 0.31 4.39 ± 0.16 4.78 ± 0.64 4.46 ± 0.21 Stomach 1.41 ± 0.54 2.15 ± 0.46 1.70 ± 0.79 0.49 ± 0.17 1.24 ± 0.43 0.93 ± 0.54 Small 17.27 ± 0.85 17.61 ± 0.87 20.23 ± 1.50 Intestine 2.86 ± 0.17 3.47 ± 0.33 3.94 ± 0.46 Large 2.08 ± 0.25 2.14 ± 0.34 2.05 ± 0.24 Intestine 0.35 ± 0.03 0.40 ± 0.04 0.34 ± 0.03 Skin 0.78 ± 0.61 0.68 ± 0.55 0.51 ± 0.34 0.39 ± 0.04 0.37 ± 0.09 0.31 ± 0.09 Muscle 0.91 ± 0.49 0.67 ± 0.41 0.49 ± 0.21 0.28 ± 0.03 0.29 ± 0.03 0.16 ± 0.02 Urine 12.36 ± 1.18 11.78 ± 2.18 12.07 ± 1.11 Carcass 36.96 ± 0.83 37.76 ± 2.18 33.95 ± 1.37 0.34 ± 0.02 0.37 ± 0.02 0.31 ± 0.01 Example 10 Methods of Freeze-Dried Kit Preparation The freeze-dried kit (Composition V+lactose of Example 5) was prepared by two methods.","The first method (ID-11) was similar to that of Example 5, but with a modified Solution 2—thus, d,l-HMPAO (250 mg),NapABA (250 mg) and lactose monohydrate (1.5 g) were dissolved in Solution 1 (250 mL).","The solution was added to water (250 mL) and adjusted to pH 8.9.The production conditions and results for ID-11 are shown in Table 9.The second method (ID-12) is described below.","Thus, lactose monohydrate (3.03 g), NapABA (505 mg), d,l-HMPAO (505 mg), HMDP (66.6 mg) and anhydrous stannous chloride (13.1 mg) were dissolved in 0.1M hydrochloric acid (1000 mL) in the order given.","The solution was then adjusted to pH 9.4.The production conditions and results for ID-12 are shown in Table 9.TABLE 9 Production conditions and results of each lot Freezing Number pH before Temperature of Vials freezing ° ( C.) Produced Property# ID-11 8.9 −40° C. 290 Good ID-12 9.4 −40° C. 240 Good #as defined in Example 4.Example 11 RCP of Freeze-Dried Kits Prepared by Different Methods The freeze-dried kits in Example 10 were used to examine the RCP.","The RCP was measured in the same manner as in Example 7.The results obtained are shown in Table 10.TABLE 10 Rcp of 99mTc-HMPAO at the time after reconstitution 2 min 1 hour 3 hours 6 hours ID-11 90.2 ± 0.4 89.3 ± 1.8 85.9 ± 1.7 85.7 ± 1.7 ID-12 87.1 ± 2.1 81.4 ± 2.4 70.6 ± 1.7 60.7 ± 4.0"]],"string":"[\n [\n \"Stabiliser for radiopharmaceuticals\",\n \"The present invention provides an improved stabiliser for radiopharmaceuticals which inhibits impurities from being produced by two kinds of decomposition mechanisms and exhibits such an effect that the shelf life of a radiopharmaceutical after its preparation is prolonged as compared with conventional ones.\",\n \"The improvement comprises a combination of an amino-substituted aromatic carboxylic acid or its salt, ester or amide in combination with a diphosphonic acid or its salt.\"\n ],\n [\n \"1.A stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical which is susceptible to either reductive degradation or radiolysis; (ii) a first stabiliser for said radiopharmaceutical which includes an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a second stabiliser for said radiopharmaceutical which includes a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"2.The composition of claim 1 wherein the amino-ubstituted aromatic carboxylic acid or its salt, ester or amide is selected from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 3, 5-diaminobenzoic acid, 4-amino-salicylic acid, and salts, esters and amides thereof.\",\n \"3.The composition of claim 1, wherein the diphosphonic acid or its salt is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof.\",\n \"4.The composition of claim 1, wherein the amino-substituted aromatic carboxylic acid or salt, ester or amide thereof is sodium 4-aminobenzoate, and the diphosphonic acid is methylenediphosphonic acid.\",\n \"5.The composition of claim 1, wherein the radiopharmaceutical includes a γ-ray emitter or a β-ray emitter.\",\n \"6.The composition of claim 5, wherein the radiopharmaceutical includes Tc-99m, Re-186 or Re-188.7.The composition of claim 1, wherein the radiopharmaceutical includes a metal complex of a radionuclide with a ligand.\",\n \"8.The composition of claim 7, wherein the ligand is an amine oxime.\",\n \"9.The composition of claim 8, wherein the amine oxime is selected from the group consisting of d,l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.\",\n \"10.A non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition of claim 1 which comprises: (i) a first stabiliser which comprises an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; and (ii) a second stabiliser which comprises a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"11.The kit of claim 10, which includes 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.\",\n \"12.The kit of claim 10, further comprising a tin (II) reductant, wherein the molar ratio of the diphosphonic acid or its salt to tin (II) is in the range 1 to 10.13.The kit of claim 10, further comprising an amine oxime ligand.\",\n \"14.The kit of claim 13 wherein the amine oxime is selected from the group consisting of d, l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.\",\n \"15.The kit of claim 10, wherein the first and second stabilzers are lyophilised.\",\n \"16.In a method of preparing a radiopharmaceutical composition which includes adding stabilizers to a radiopharmaceutical, wherein the radiopharmaceutical is not a metal complex of diphosphonic acid, the improvement comprising using a diphosphonic acid or a salt thereof as a stabiliser.\",\n \"17.Cancelled 18.The method of claim 16, wherein the radiopharmaceutical comprises Tc-99m.\",\n \"19.The method of claim 16, wherein the diphosphonic acid is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof.\"\n ],\n [\n \" BACKGROUND TO THE INVENTION Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.\",\n \"Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.\",\n \"In the case of Tc-99m, the latter is referred to as post-reconstitution instability.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99m Tc-complexes of diphosphonic acids.\",\n \"Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99m Tc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.\",\n \"99m Tc-HMPAO is particularly unstable with respect to post-reconstitution stability.\",\n \"99m Tc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.\",\n \"The function of the stannous ion is to reduce the 99m Tc-pertechnetate ( 99m TcO 4 − ), ie.\",\n \"technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99m Tc-HMPAO metal complex.\",\n \"The radiochemical purity (Rcp) of 99m Tc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99m Tc labelling.\",\n \"After Tc-99m radiolabelling, the Rcp of 99m Tc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99m Tc complex of unknown structure derived from 99m Tc-HMPAO, 99m Tc-pertechnetate ( 99m TcO 4 − ) and reduced-hydrolysed-technetium [ 99m Tc].\",\n \"Of these impurities, both the secondary complex and 99m Tc-pertechnetate are decomposition products of 99m Tc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.\",\n \"Med.\",\n \"29, 1568-1576, 1988).\",\n \"The secondary complex is believed to be produced when the lipophilic 99m Tc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.\",\n \"stannous) remaining from the pertechnetate reduction step.\",\n \"On the other hand, the 99m Tc-pertechnetate impurity is produced when 99m Tc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.\",\n \"radiolysis of the solvent.\",\n \"Accordingly, in order to inhibit the production of both the 99m Tc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.\",\n \"Thus, Nucl.\",\n \"Med.\",\n \"Biol.\",\n \"7, 675-680 (1989); Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"16, 541 (1990); Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"20, 661-666 (1993) and Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"22, 1163-1172 (1995) all report attempts to stabilise 99m Tc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.\",\n \"In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99m Tc-HMPAO to at least 80% at 4 hours post reconstitution.\",\n \"Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99m Tc-HMPAO at 6 hours post reconstitution to at least 80%.\",\n \"The stabilisation mechanisms of 99m Tc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.\",\n \"Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99m Tc-HMPAO.\",\n \"However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99m Tc-blue or cobalt chloride is used as a stabiliser for 99m Tc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99m Tc-pertechnetate.\",\n \"Accordingly, any kit for the preparation of 99m Tc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).\",\n \"One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.\",\n \"The other is a vial containing the stabiliser (methylene blue or cobalt chloride).\",\n \"Thus, the most successful prior art methods of stabilising 99m Tc-HMPAO to date all require the use of 2-vial kits.\",\n \"When the kit for preparing 99m Tc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99m Tc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.\",\n \"methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.\",\n \"It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.\",\n \"Too short a time, and 99m Tc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.\",\n \"Too long a time, and the stabilising effect is delayed.\",\n \"In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.\",\n \"The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.\",\n \"In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.\",\n \"Moreover, when methylene blue is added to 99m Tc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.\",\n \"There is therefore a need for a single vial kit for the preparation of 99m Tc-HMPAO which has both shelf-life and post-radiolabelling stability.\",\n \"The present invention provides a kit which solves this problem, and is straightforward to use.\",\n \"The Present Invention.\",\n \"The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to a stabilised radiopharmaceutical composition, together with non-radioactive kits for the preparation of the stabilised radiopharmaceutical composition.\",\n \"BACKGROUND TO THE INVENTION Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.\",\n \"Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.\",\n \"In the case of Tc-99m, the latter is referred to as post-reconstitution instability.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99mTc-complexes of diphosphonic acids.\",\n \"Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99mTc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.\",\n \"99mTc-HMPAO is particularly unstable with respect to post-reconstitution stability.\",\n \"99mTc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.\",\n \"The function of the stannous ion is to reduce the 99mTc-pertechnetate (99mTcO4−), ie.\",\n \"technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99mTc-HMPAO metal complex.\",\n \"The radiochemical purity (Rcp) of 99mTc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99mTc labelling.\",\n \"After Tc-99m radiolabelling, the Rcp of 99mTc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99mTc complex of unknown structure derived from 99mTc-HMPAO, 99mTc-pertechnetate (99mTcO4−) and reduced-hydrolysed-technetium [99mTc].\",\n \"Of these impurities, both the secondary complex and 99mTc-pertechnetate are decomposition products of 99mTc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.\",\n \"Med.\",\n \"29, 1568-1576, 1988).\",\n \"The secondary complex is believed to be produced when the lipophilic 99mTc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.\",\n \"stannous) remaining from the pertechnetate reduction step.\",\n \"On the other hand, the 99mTc-pertechnetate impurity is produced when 99mTc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.\",\n \"radiolysis of the solvent.\",\n \"Accordingly, in order to inhibit the production of both the 99mTc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.\",\n \"Thus, Nucl.\",\n \"Med.\",\n \"Biol.\",\n \"7, 675-680 (1989); Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"16, 541 (1990); Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"20, 661-666 (1993) and Eur.\",\n \"J. Nucl.\",\n \"Med.\",\n \"22, 1163-1172 (1995) all report attempts to stabilise 99mTc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.\",\n \"In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99mTc-HMPAO to at least 80% at 4 hours post reconstitution.\",\n \"Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99mTc-HMPAO at 6 hours post reconstitution to at least 80%.\",\n \"The stabilisation mechanisms of 99mTc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.\",\n \"Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99mTc-HMPAO.\",\n \"However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99mTc-blue or cobalt chloride is used as a stabiliser for 99mTc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99mTc-pertechnetate.\",\n \"Accordingly, any kit for the preparation of 99mTc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).\",\n \"One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.\",\n \"The other is a vial containing the stabiliser (methylene blue or cobalt chloride).\",\n \"Thus, the most successful prior art methods of stabilising 99mTc-HMPAO to date all require the use of 2-vial kits.\",\n \"When the kit for preparing 99mTc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99mTc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.\",\n \"methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.\",\n \"It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.\",\n \"Too short a time, and 99mTc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.\",\n \"Too long a time, and the stabilising effect is delayed.\",\n \"In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.\",\n \"The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.\",\n \"In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.\",\n \"Moreover, when methylene blue is added to 99mTc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.\",\n \"There is therefore a need for a single vial kit for the preparation of 99mTc-HMPAO which has both shelf-life and post-radiolabelling stability.\",\n \"The present invention provides a kit which solves this problem, and is straightforward to use.\",\n \"The Present Invention.\",\n \"The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides: a stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical; (ii) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a diphosphonic acid or salt thereof, is with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"By the term ‘amino-substituted aromatic carboxylic acid’ is meant a compound in which at least one hydrogen atom on the aromatic ring of an aromatic carboxylic acid is substituted with at least one amino group.\",\n \"The aromatic group is preferably benzene.\",\n \"Preferred amino-substituted aromatic carboxylic acids are: 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid (pABA), 3,5-diaminobenzoic acid and 4-aminosalicylic acid.\",\n \"4-aminobenzoic acid (pABA) is especially preferred.\",\n \"The salt of the amino-substituted aromatic carboxylic acid is suitably a salt with a biocompatible cation.\",\n \"By the term “biocompatible cation” is meant a positively charged counterion which forms a salt with an ionised, negatively charged group (here a carboxylate group), where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.\",\n \"Examples of suitable biocompatible cations include: the alkali metals (eg.\",\n \"sodium or potassium); the alkaline earth metals (eg.\",\n \"calcium, magnesium and barium); and the ammonium ion.\",\n \"A preferred biocompatible cation is sodium.\",\n \"Preferred salts of the present invention include: sodium 2-aminobenzoate, sodium 3-aminobenzoate, sodium 4-aminobenzoate (NapABA), sodium 3,5-diaminobenzoate, sodium 4-aminosalicylate, potassium 2-aminobenzoate, potassium 3-aminobenzoate, potassium 4-aminobenzoate, potassium 3,5-diaminobenzoate and potassium 4-aminosalicylate.\",\n \"Sodium 4-aminobenzoate (NapABA) is especially preferred.\",\n \"Suitable esters of the aromatic carboxylic acid include methyl, ethyl or propyl esters.\",\n \"Preferred esters are: methyl 2-aminobenzoate, methyl 3-aminobenzoate, methyl 4-aminobenzoate, methyl 3,5-diaminobenzoate, methyl 4-aminosalicylate, ethyl 2-aminobenzoate, ethyl 3-aminobenzoate, ethyl 4-aminobenzoate, ethyl 3,5-diaminobenzoate, ethyl 4-aminosalicylate, propyl 2-aminobenzoate, propyl 3-aminobenzoate, propyl 4-aminobenzoate, propyl 3,5-diaminobenzoate and propyl 4-aminosalicylate.\",\n \"Suitable amides of the amino-substituted aromatic carboxylic acid are amides formed by derivatising the carboxyl group of the aromatic carboxylic acid with either ammonia or a compound having at least one amino group, and include such compounds as 2-aminobenzamide, 3-aminobenzamide and 4-aminobenzamide.\",\n \"The diphosphonic acid of the present invention is suitably a 1,1- or a 1,2-diphosphonic acid, or a diphosphonic acid derivative of an amine such as ethylenediaminetetraphosphonic acid (EDTMP).\",\n \"1,1-diphosphonic acids are preferred, such as methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP).\",\n \"Methylenediphosphonic acid (MDP) and hydroxymethanediphosphonic acid (HMDP) are especially preferred.\",\n \"Suitable salts of the diphosphonic acid are with a ‘biocompatible cation’ as defined above.\",\n \"Preferred such diphosphonate salts include: sodium methylenediphosphonate, sodium hydroxymethanediphosphonate, sodium hydroxyethanediphosphonate, sodium ethylenediamine-tetraphosphonate, ammonium methylenediphosphonate, ammonium hydroxymethanediphosphonate, ammonium hydroxyethanediphosphonate and ammonium ethylenediamine-tetraphosphonate.\",\n \"The combination of compounds in the stabiliser for radiopharmaceuticals of the present invention is preferably a combination of sodium 4-aminobenzoate (NapABA) with methylenediphosphonic acid (MDP) or hydroxymethanediphosphonic acid (HMDP).\",\n \"Without wishing to be bound by theory, it is believed that the stabilisers of the present invention act as follows: The amino-substituted aromatic carboxylic acid or its salt, ester or amide has a reducing ability and removes any oxidising free radicals which result from the radiolysis of the solution, and hence inhibits the oxidative destruction of the radiopharmaceutical by free radical attack.\",\n \"On the other hand, when excess tin(II) reductant remains after completion of the radiolabelling, the diphosphonic acid or its salt inhibits the excess stannous ion from reductively degrading the radiopharmaceutical.\",\n \"It is a surprising finding of the present invention that such stabilisers can be present in a single step radiopharmaceutical preparation without either: (i) preventing or hindering the stannous reduction of the radiopharmaceutical precursor, with consequent adverse effect on Rcp; (ii) complexing with the radiometal to form undesirable radioactive impurities (eg.\",\n \"diphosphonic acid complexes of the radionuclide).\",\n \"Accordingly, the amount of the diphosphonic acid or its salt to be added to the radiopharmaceutical depends upon the amount of the reducing tin(II) contained in the radiopharmaceutical preparation or kit.\",\n \"The amount of the diphosphonic acid or its salt effective to stabilise the radiopharmaceutical is 1 to 10 moles per mole of stannous, preferably 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.\",\n \"The radionuclide of the radiopharmaceutical of the present invention is a γ-ray or β-ray emitter, preferably Tc-99m, Re-186 or Re-188, most preferably Tc-99m.\",\n \"γ-ray emitters are mainly used for radiodiagnosis and β-ray emitters are mainly used for radiotherapy.\",\n \"When a radiodiagnostic radiopharmaceutical of this invention is intracorporeally administered to a human being, the level of radioactivity used is in the range of from 370 to 1,480 MBq, preferably from 370 to 1,110 MBq.\",\n \"When a radiotherapeutic agent of the radiopharmaceuticals of this invention is intracorporeally administered to a human being, the radioactivity level is in the range of from 37 to 18,500 MBq, preferably from 370 to 7,400 MBq.\",\n \"The radiopharmaceutical comprises an active ingredient which is susceptible to either degradation by the reducing action of the reductant (present to help effect labelling of the radionuclide), or radiolysis.\",\n \"By using the stabiliser composition of the present invention to stabilise such active ingredients, it is possible to prolong the useful lifetime post-radiolabelling to at least twice that of the prior art.\",\n \"The stabilisers of the present invention are particularly useful when the active ingredient comprises a ligand having a tetradentate diaminedioxime donor set, especially d,l-hexamethylpropyleneamine oxime or HMPAO (exametazime), 4,9-diaza-3,3,10,10-tetramethyldodecane-2,11-dione dioxime (PnAO), and similar compounds.\",\n \"In a second aspect, the present invention provides a non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition described above which comprises: (i) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (ii) a diphosphonic acid or salt thereof, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"Thus, the stabiliser for radiopharmaceuticals of the present invention can be pre-mixed with the ligand prior to radiolabelling, so that the formulation of a single vial, non-radioactive kit for the preparation of a stabilised radiopharmaceutical (which is not a radionuclide complex of the diphosphonic acid stabilizer) is possible.\",\n \"This simplifies the labelling procedure of the prior art 2-vial kit, shortens the labelling time and diminishes the risk of operator exposure to harmful radiation.\",\n \"The single vial kit of the present invention also has an extended stability post radioactive preparation, which in turn extends the useful lifetime for the user of the kit, eg.\",\n \"a clinician.\",\n \"Suitable non-radioactive kits of the present invention comprise 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.\",\n \"When the kit contains stannous, the amount of the diphosphonic acid or its salt effective to stabilise is 1 to 10 moles per mole of stannous.\",\n \"Preferably, the ratio of diphosphonic acid or its salt is 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.\",\n \"In a third aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid suitable diphosphonic acid salts are those with a biocompatible cation as described above.\",\n \"Preferably the radiopharmaceutical also comprises stannous in the formulation.\",\n \"Most preferably, the radiopharmaceutical comprises 99mTc.\",\n \"Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.\",\n \"In a fourth aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for non-radioactive kits for the preparation of radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.\",\n \"Suitable diphosphonic acid salts are those with a biocompatible cation as described above.\",\n \"Preferably the non-radioactive kit further comprises stannous in the formulation.\",\n \"Most preferably, the non-radioactive kit is for the preparation of a 99mTc radiopharmaceutical.\",\n \"Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.\",\n \"DESCRIPTION OF THE DRAWINGS FIG.\",\n \"1 compares the change in Rcp of 99mTc-HMPAO prepared from Composition III, Composition III+lactose and a commercially available kit for preparing 99mTc-HMPAO, each of which had been stored for 6 months prior to reconstitution.\",\n \"The present invention is explained in more detail with reference to the following Examples: Example 1 is a comparative example for a two step process (including prior art stabilisers such as cobalt chloride).\",\n \"An Rcp of 99mTc-HMPAO of at least 80% 3 hours after the addition is judged as effective stabilisation.\",\n \"However, as is clear from Table 1, the only single stabiliser which meets this criterion is cobalt chloride.\",\n \"Example 2 shows that 99mTc-HMPAO can be effectively stabilised in a single-step process, using a mixture of two compounds as stabilisers, and that the stability is significantly increased over that achieved using the 2-step process of Example 1.Example 3 provides further data on optimising the combinations of MDP/NapABA and HMDP/NapABA in a single step process.\",\n \"The molar ratio of Sn2+ to MDP/HMDP was kept constant (1:5), and the amounts added were varied.\",\n \"Example 4 shows that lyophilised non-radioactive kits for the preparation of 99mTc radiopharmaceuticals can be prepared using the compositions of the present invention.\",\n \"Lactose (a known cryoprotectant excipient in freeze-dried formulations), is shown to be useful to preserve stannous levels for extended shelf-life periods.\",\n \"Example 5 shows that the combination HMDP/NapABA is also effective as a stabiliser for 99mTc-HMPAO.\",\n \"Example 6 provides data on the stannous or tin(II) content of the freeze-dried kits, as a function of shelf-life storage in the dark at 4° C., and shows that the amount of reducing tin(II) in Composition III+lactose is higher than that in Composition III.\",\n \"Moreover, the amount of the reducing tin(II) in Composition III shows some tendency to decrease with increasing storage time.\",\n \"Accordingly, when the freeze-dried kit is stored for a prolonged period of time, lactose may be used in the kit composition to maintain the amount of the tin(II) reductant constant.\",\n \"Example 7 compares the Rcp after 6-months storage of the freeze-dried kits of the present invention, with the Rcp for 99mTc-HMPAO prepared from a commercially available kit.\",\n \"The Rcp decreases as a function of time post-reconstitution for the commercial kit, but for Composition III and Composition III+lactose, the Rcp's at 6 hours post-reconstitution were at least 80%.\",\n \"This shows that, whilst the stannous levels without lactose do show some modest decrease (Example 5), the decrease does not impair the performance of the kit.\",\n \"Example 8 shows that freeze-dried kits containing the stabiliser composition HMDP/NapABA have an effective shelf-life.\",\n \"Example 9 compares the biodistribution in rats of 99mTc-HMPAO prepared using kit Composition III and kit Composition III+lactose of the present invention, with a commercial 99mTc-HMPAO kit preparation.\",\n \"No significant difference in uptake in target organs was found.\",\n \"This includes the brain, which is the diagnostic imaging application of the present commercial 99mTc-HMPAO agent.\",\n \"These results show that the added compounds (MDP, NapABA and lactose), which are not present in the commercially available kit, do not adversely affect the biological behaviour of the 99mTc radiopharmaceutical.\",\n \"Example 10 describes various lyophilisation methods suitable for freeze-dried kit preparation of formulations of the present invention.\",\n \"Example 11 compares the Rcp of the various freeze-dried kits of Example 10.Example 1 The Rcp of 99mTc-HMPAO Using a Single Stabiliser: Two Step Process (Comparative Example) The radiochemical purity (Rcp) of 99mTc-HMPAO prepared using a commercially available kit (CERETEC™) for preparing 99mTc-HMPAO is 80% at one hour post-reconstitution, and the Rcp decreases thereafter with time.\",\n \"On the other hand, in the case of 99mTc-HMPAO stabilised with cobalt chloride, the Rcp 6 hours after radiolabelling is 80%.\",\n \"The following known medically acceptable compounds and additives were studied: ascorbic acid, sodium ascorbate, gentisic acid, gentisic acid ethanolamide, methylenediphosphonic acid (MDP), succinic acid, 4-aminobenzoic acid (pABA) and sodium 4-aminobenzoate (NapABA).\",\n \"To each of ten vials each containing 0.5 mg of HMPAO and 4.0 μg of Sn2+ (stannous), was added sodium pertechnetate (1.48 GBq in 5 ml of saline) to carry out the radiolabelling.\",\n \"To each of 9 of the ten reconstituted vials was individually added the compound shown in Table 1 at a time of 2 minutes post-radiolabelling.\",\n \"Nothing was added to the remaining vial.\",\n \"At 1 minute and 3 hours after the addition, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).\",\n \"The results are given in Table 1: TABLE 1 Radiochemical purity (Rcp) of 99mTc-HMPAO after the addition of a single stabiliser (%, n = 3).\",\n \"Name of Additive Rcp 1 min from Rcp 3 hours from (amount) addition (%) addition (%) No additive 87 ± 4 38 ± 15 Cobalt chloride 94 ± 3 89 ± 2 (200 μg) Ascorbic acid 83* 41* (4.5 μg) Sodium ascorbate 90* 51* (10 mg) Gentisic acid 87 ± 2 77 ± 3 30 μg) Gentisic acid ethanolamide 61 ± 3 29 ± 13 (30 μg) Methylene diphosphonic acid 79* 30* (10 μg) 4-Aminobenzoic acid 80 ± 8 67 ± 5 (30 μg) Sodium4-amino-benzoate 69 ± 5 39 ± 11 (30 μg) Succinic acid 51* 14* (30 μg) Note: *n = 1 Example 2 Examination of Stabilisation of 99mTc-HMPAO by a Combination of Two Compounds: Single Step Process The combinations studied were: (i) ascorbic acid and hydroxymethanediphosphonic acid (HMDP); (ii) sodium 4-aminobenzoate (NapABA) and methylenediphosphonic acid (MDP).\",\n \"A composition comprising 0.5 mg of HMPAO, 4.0 μg of Sn2+, 1.0 μg of HMDP and 0.5 μg of ascorbic acid (Composition A), and a composition comprising 0.5 mg of HMPAO, 5.4 μg of Sn2+, 40.5 μg of MDP and 0.5 mg of NapABA (Composition B) were prepared.\",\n \"To each of A and B was added sodium pertechnetate (1.48 GBq in 5 ml) to carry out the labelling.\",\n \"After 3 hours, an aliquot was taken from each, and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).\",\n \"The Rcp of 99mTc-HMPAO 3 hours after Tc-99m labelling was about 62% in the case of Composition A and about 80% in the case of Composition B.\",\n \"Example 3 Optimisation of Stabiliser Composition: Single Step Process As shown in Table 2, six different sample compositions were prepared and stored in the dark at 4° C. One vial of each formulation was taken out at each time point (1, 7, 31 and 32-days storage), and sodium pertechnetate (1.48 GBq in 5 ml) was added.\",\n \"At 6 hours post addition of pertechnetate, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).\",\n \"The results are shown in Table 2: TABLE 2 Sample Compositions and Rcp of 99mTc-HMPAO 6 hours post-radiolabelling (n = 1, *n = 3).\",\n \"Composition I II III IV V VI Preparation HMPAO 0.5 0.5 0.5 0.5 0.5 0.5 Conditions (mg) NapABA 0.5 0.1 0.1 0.1 0.5 — Sn2+ 5.4 5.4 8.1 8.1 8.1 8.1 (μg) MDP 40.5 40.5 61.0 61.0 — — (μg) HMPD — — — — 66.0 66.0 (μg) Rcp (%) - 1 day 86.4 79.5 82.2 73.8 84.8 ± 2.7* 77.3 ± 5.5* after storage 7 days 80.8 80.9 82.1 81.1 — — time 32 days 79.0 81.8 84.3 66.5 — — 31 days — — — — 83.5 ± 2.2* 72.0 ± 0.2* Note: *n = 3.Compositions II, III and V show that, even in the case of samples stored for 32 days in the dark at 4° C., the Rcp at 6 hours post-reconstitution was at least 80%.\",\n \"Compositions III and V, which exhibited a higher Rcp, 5 were therefore used as the basis for subsequent tests.\",\n \"Example 4 Stabilisation of 99mTc-HMPAO by a Combination of MDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition III of Example 3, and a composition prepared by adding lactose to Composition III (referred to hereinafter as Composition III+lactose) were used to prepare four lots of freeze-dried kit in each case.\",\n \"The amounts of compounds shown below correspond to the scale for a production batch of about 100 freeze-dried kit vials.\",\n \"First, anhydrous stannous chloride (25.8 mg) and MDP (122 mg) were dissolved in 0.1 M hydrochloric acid (1,000 ml).\",\n \"The resulting solution is referred to hereinafter as Solution 1.d,l-HMPAO (100 mg) and NapABA (100 mg) were dissolved in Solution 1 (100 ml), to give a solution referred to hereinafter as Solution 2.Solution 2 was divided into two 50 ml portions, one of which was adjusted to a pH of 8.5-9.0 with sodium hydroxide and thereafter the total volume was adjusted to 10.0 ml with water by use of a measuring cylinder.\",\n \"In the other 50 ml portion was dissolved 300 mg of lactose monohydrate, the resulting solution was adjusted to a pH of 8.5 to 9.0 with sodium hydroxide, and thereafter, the total volume was adjusted to 100 ml with water by use of a measuring cylinder.\",\n \"1.0 ml of each of the solutions obtained was placed in each vial, which was then frozen at −50 to −78° C. and then freeze-dried for about 24 hours.\",\n \"After completion of the freeze-drying, the vials were sealed and the freeze-dried kit was stored in the dark at 4° C. The same procedure was repeated four times to prepare four separate lots of each of, Composition III and Composition III+lactose.\",\n \"Lot Nos.\",\n \"ID-01 to ID-04 were of Composition III and Lot Nos.\",\n \"ID-05 to ID-08 were of Composition III+lactose.\",\n \"The production conditions and results of each lot are shown in Table 3.TABLE 3 Production conditions and results of each lot Freezing Number of pH before temperature Vials Lot No.\",\n \"freezing (° C.) Produced Property# ID-01 9.0 −50 99 Fair ID-02 9.0 −78 90 Bad ID-03 8.5 −78 39 Bad ID-04 9.0 −50 99 Fair ID-05 9.0 −50 91 Good ID-06 9.0 −78 92 Good ID-07 8.5 −78 94 Good ID-08 8.5 −50 91 Good #where ’property' refers to the behaviour during lyophilisation: Good = the cake in the freeze-dried kits kept well-formed; Fair = the cake in the freeze-dried kits was partly broken, but there was no loss of powder from the vials during lyophilisation; Bad = the cake in the freeze-dried kits was completely broken, and some of the powder was lost from the vials during lyophilisation.\",\n \"In all the lots other than ID-03 (which is of Composition III), at least 90 vials were produced.\",\n \"Among the four lots of Composition III, only ID-03 was adjusted to a pH of 8.5 before freezing.\",\n \"Moreover, in the case of Composition III+lactose, even when the pH was adjusted to 8.5 before freezing (ID-07, ID-08), at least 90 vials could be produced.\",\n \"Accordingly, when a freeze-dried kit is produced, it is desirable to adjust the pH to 9.0 in the case of lactose-free Composition III.\",\n \"Example 5 Stabilisation of 99mTc-HMPAO by a Combination of HMDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition V of Example 3, and a composition prepared by adding lactose to Composition V (referred to hereinafter as Composition V+lactose) were used to prepare one lot of freeze-dried kits in each case.\",\n \"The freeze-dried kits were prepared in much the same manner as that of Example 4, except that Solution 1 was slightly different—in this case anhydrous stannous chloride (25.8 mg) and HMDP-2Na (132.0 mg) were dissolved in 0.1M hydrochloric acid (1,000 mL).\",\n \"Lot Nos.\",\n \"ID-09 was of Composition V and Lot Nos.\",\n \"ID-10 was of Composition V+lactose.\",\n \"The production conditions and results of each lot are shown in Table 4.TABLE 4 Production conditions and results of each lot.\",\n \"Freezing Number of pH before Temperature Vials freezing (° C.) Produced Property# ID-09 8.8 −50° C. 49 Fair ID-10 8.8 −50° C. 45 Good #defined in Example 4.Example 6 Determination of the Amount of tin(II) in the Freeze-Dried Kit as a Function of Storage Time Vials of Composition III (ID-01, O2, 03 and 04), Composition III+lactose (ID-05, 06, 07 and 08), Composition V (ID-09) and Composition V+lactose (ID-10) which had been stored in the dark at 4° C. for periods of 1 day, 3-months and 6-months, were allowed to warm to room temperature.\",\n \"The amount of tin(II) was then determined by an absorbance method.\",\n \"The results obtained are shown in Table 5.TABLE 5 Amounts of reducing tin(II) determined (μg/vial, n = 2).\",\n \"1-day storage 3-month storage 6-month storage Composition III 9.5 ± 0.2 9.1 ± 0.5 8.7 ± 0.5 Composition III + 10.3 ± 0.5 10.5 ± 0.5 10.8 ± 0.4 lactose Composition V 10.1 ± 0.0 — — Composition V + 10.3 ± 0.3 — — lactose Note Each value was an average value of 4 lots.\",\n \"Example 7 Radiochemical Purity of Freeze-Dried Kits Containing MDP and NapABA Studied over a 6-Month Shelf-Life Storage Period Four lots of each of Composition III and Composition III+lactose produced in Example 4 were subjected to examination of radiochemical purity after 6 -months storage in the dark at 4° C. Vials of freeze-dried Composition III (ID-01, O2, 03 and 04) and Composition III+lactose (ID-OS, 06, 07 and 08) were stored for 6 months in the dark at 4° C. After a storage period of 1 day, 1, 3 and 6 months, the vials were allowed to warm to room temperature, and Tc-99m labelling was carried out.\",\n \"Thus, to one vial of each lot was added sodium pertechnetate (1.48 GBq in 5 ml).\",\n \"Aliquots were taken after times of 2 minutes, one hour, 3 hours and 6 hours and the Rcp was measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline and filter paper/50% aqueous acetonitrile).\",\n \"The results obtained at 6 hours are shown in Table 6.When Composition III and Composition III+lactose were radiolabelled after 6-month shelf-life storage, simultaneously, sodium pertechnetate (1.48 GBq/5 ml) was added to a commercially available kit for preparing 99mTc-HMPAO (containing 0.5 mg of HMPAO and 4.0 μg of Sn2+) as a control.\",\n \"An aliquot thereof was taken after times of one minute, one hour, 3 hours and 6 hours, and then the Rcp was measured in the same manner.\",\n \"The results obtained are shown in FIG.\",\n \"1.TABLE 6 Rcp of 99mTc-HMPAO at 6 hr post reconstitution by a combination of MDP and NapABA as a function of storage time.\",\n \"STORAGE PERIOD 1-day 1-month 3-months 6-months Rcp Composition III 86.4 ± 2.2 87.4 ± 1.5 86.5± 2.7 88.4 ± 3.4 (%) Composition III + 86.2 ± 2.3 86.2 ± 2.4 87.9 ± 2.6 85.6 ± 2.7 Lactose Example 8 Radiochemical Purity of Freeze-Dried Kits Containing HMDP and NapABA for 1-Month Shelf-Life Storage One lot of each of Composition V and Composition V+lactose produced in Example 5 were subjected to examination of radiochemical purity for 1-month storage in the dark at 4° C. The Rcp was measured in the same manner.\",\n \"The results obtained are shown in Table 7.TABLE 7 Rcp of 99mTc-HMPAO at 6 hr post-reconstitution stabilised by a combination of HMDP and NapABA as a function of storage time.\",\n \"Storage Time 1-day 1-month Rcp Composition V 84.8 ± 2.7 83.5 ± 2.2 (%) Composition V + 87.3 ± 2.8 86.0 ± 1.0 Lactose Example 9 Biodistribution in the Rat To one vial of each of Composition III (ID-O2) and Composition III+lactose (ID-06) obtained in Example 4 was added sodium pertechnetate in a proportion of 1.48 GBq/5 ml to carry out the radiolabelling.\",\n \"After 2 hours, 3.0 to 3.7 MBq of the reconstituted solution was administered to a female Sprague-Dawley strain rat (body weight 140-170 g) which had been previously anaesthetised with sodium thiopentobarbital in the tail vein.\",\n \"At one hour post-administration, the animal was sacrificed and the radioactivity in each organ was measured using a NaI single channel analyser.\",\n \"Separately, sodium pertechnetate was added in a proportion of 1.48 GBq/5 ml to the commercially available kit for preparing 99mTc-HMPAO.\",\n \"In the same manner, at 15 minutes post-reconstitution, 3.0-3.7 MBq of the solution was administered to the same kind of rat, and the biodistribution of radioactivity in each organ measured.\",\n \"The results obtained are shown in Table 8: TABLE 8 Biodistribution of Commercial 99mTc-HMPAO, Composition III (ID-02) and Composition III + lactose (ID-06) in the female rat at one hour post administration (upper row: % ID/organ, lower row: % ID/g, n = 5).\",\n \"Composition III + Commercial Composition lactose 99mTc-HMPAO III (ID-02) (ID-06) Brain 1.66 ± 0.13 1.65 ± 0.10 1.73 ± 0.15 0.96 ± 0.07 0.96 ± 0.15 1.01 ± 0.07 Blood 15.38 ± 0.72 14.41 ± 1.68 14.07 ± 1.51 1.56 ± 0.04 1.64 ± 0.06 1.45 ± 0.18 Heart 0.81 ± 0.04 0.85 ± 0.03 0.90 ± 0.10 1.61 ± 0.08 1.75 ± 0.10 1.72 ± 0.03 Lung 3.41 ± 0.20 3.15 ± 0.18 3.39 ± 0.38 4.05 ± 0.28 4.26 ± 0.29 4.14 ± 0.36 Liver 5.91 ± 0.20 6.49 ± 0.48 7.68 ± 0.38 1.10 ± 0.07 1.36 ± 0.13 1.44 ± 0.14 Spleen 0.78 ± 0.10 0.79 ± 0.18 0.86 ± 0.12 2.17 ± 0.12 2.24 ± 0.12 2.25 ± 0.21 Kidney 4.27 ± 0.27 5.04 ± 0.48 4.81 ± 0.31 4.39 ± 0.16 4.78 ± 0.64 4.46 ± 0.21 Stomach 1.41 ± 0.54 2.15 ± 0.46 1.70 ± 0.79 0.49 ± 0.17 1.24 ± 0.43 0.93 ± 0.54 Small 17.27 ± 0.85 17.61 ± 0.87 20.23 ± 1.50 Intestine 2.86 ± 0.17 3.47 ± 0.33 3.94 ± 0.46 Large 2.08 ± 0.25 2.14 ± 0.34 2.05 ± 0.24 Intestine 0.35 ± 0.03 0.40 ± 0.04 0.34 ± 0.03 Skin 0.78 ± 0.61 0.68 ± 0.55 0.51 ± 0.34 0.39 ± 0.04 0.37 ± 0.09 0.31 ± 0.09 Muscle 0.91 ± 0.49 0.67 ± 0.41 0.49 ± 0.21 0.28 ± 0.03 0.29 ± 0.03 0.16 ± 0.02 Urine 12.36 ± 1.18 11.78 ± 2.18 12.07 ± 1.11 Carcass 36.96 ± 0.83 37.76 ± 2.18 33.95 ± 1.37 0.34 ± 0.02 0.37 ± 0.02 0.31 ± 0.01 Example 10 Methods of Freeze-Dried Kit Preparation The freeze-dried kit (Composition V+lactose of Example 5) was prepared by two methods.\",\n \"The first method (ID-11) was similar to that of Example 5, but with a modified Solution 2—thus, d,l-HMPAO (250 mg),NapABA (250 mg) and lactose monohydrate (1.5 g) were dissolved in Solution 1 (250 mL).\",\n \"The solution was added to water (250 mL) and adjusted to pH 8.9.The production conditions and results for ID-11 are shown in Table 9.The second method (ID-12) is described below.\",\n \"Thus, lactose monohydrate (3.03 g), NapABA (505 mg), d,l-HMPAO (505 mg), HMDP (66.6 mg) and anhydrous stannous chloride (13.1 mg) were dissolved in 0.1M hydrochloric acid (1000 mL) in the order given.\",\n \"The solution was then adjusted to pH 9.4.The production conditions and results for ID-12 are shown in Table 9.TABLE 9 Production conditions and results of each lot Freezing Number pH before Temperature of Vials freezing ° ( C.) Produced Property# ID-11 8.9 −40° C. 290 Good ID-12 9.4 −40° C. 240 Good #as defined in Example 4.Example 11 RCP of Freeze-Dried Kits Prepared by Different Methods The freeze-dried kits in Example 10 were used to examine the RCP.\",\n \"The RCP was measured in the same manner as in Example 7.The results obtained are shown in Table 10.TABLE 10 Rcp of 99mTc-HMPAO at the time after reconstitution 2 min 1 hour 3 hours 6 hours ID-11 90.2 ± 0.4 89.3 ± 1.8 85.9 ± 1.7 85.7 ± 1.7 ID-12 87.1 ± 2.1 81.4 ± 2.4 70.6 ± 1.7 60.7 ± 4.0\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10296952"}}},{"rowIdx":94,"cells":{"text":{"kind":"list like","value":[["METHODS FOR MEDIATING GENE SUPPRESSION","The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.","The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.","It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs."],["1.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","2.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","3.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","4.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","5.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","6.A method according to any one of claims 1 to 5, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.","7.A method according to any one of claims 1 to 6, wherein the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.","8.A method according to any one of claims 1 to 7, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.","9.A method according to any one of claims 1 to 8, wherein the factor is selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","10.A method according to any one of claims 1 to 9, wherein the RNAi factor is an res sequence.","11.A method according to claim 10, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","12.A method according to claim 11, wherein the res sequence is obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.13.A method according to claim 11, wherein the res sequence is represented by Seq ID Nos 1 to 4.14.A method according to any one of claims 1 to 13, wherein the cell is a eukaryotic cell.","15.A method according to 14, wherein the eukaryotic cell is a mammalian.","16.A method according to claim 1 or claim 2, wherein the cell is a Schizosaccharomyces pombe cell.","17.A method according to any one of claims 1 to 16, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.","18.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","19.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","20.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","21.A cell according to claim 20, wherein the cell is a eukaryotic cell.","22.A cell according to claim 20 or claim 21, wherein the cell is a Schizosaccharomyces pombe cell.","23.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.","24.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","25.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","26.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","27.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","28.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","29.A method according to any one of claims 23 to 28, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.","30.A method according to any one of claims 23 to 29, wherein the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.","31.A method according to any one of claims 23 to 30, wherein the cell is a eukaryotic cell.","32.A method according to 31, wherein the eukaryotic cell is a mammalian.","33.A method according to claim 23 or claim 24, wherein the cell is a Schizosaccharomyces pombe cell.","34.A method according to any one of claims 23 to 33, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.","35.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","36.A cell according to claim 35, wherein the cell is a eukaryotic cell.","37.A cell according to claim 35 or claim 36, wherein the cell is a Schizosaccharomyces pombe cell.","38.Method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.","39.A factor identified by the method of claim 38.40.A factor according to claim 39, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.","41.A factor according to claim 39 or claim 40, wherein the factor is selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","42.A factor according to any one of claims 39 to 41, wherein the factor is an res sequence.","43.A factor according to claim 42, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","44.An RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.45.An RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.46.A Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed."],[" BACKGROUND OF THE INVENTION The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.","Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.","It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.","One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.","The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.","Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.","The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.","These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.","The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.","The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.","Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.","The second strategy for finding key players in RNAi has involved the use of cell free assays.","These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.","However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.","Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.","This would enable the identification of factors which can enhance or reduce the gene silencing effect.","It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative."],[" SUMMARY OF THE INVENTION Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.","Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.","This is indicative of an RNAi-like mechanism of gene suppression.","It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.","This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.","The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.","The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.","According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.","Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.","It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.","Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.","More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","Even more preferred is an RNAi factor which is an res sequence.","Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.","In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.","According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.","As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.","According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.","Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.","Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.","Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.","However it will be understood that the entire native sequence of the target nucleic acid may be employed.","Conveniently, the target nucleic acid may be coupled to a selectable marker.","The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.","In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.","According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.","According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.","According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.","The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.","More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","Even more preferred is a factor having an res sequence.","Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.","The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.","The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.","This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).","The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).","It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.","The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.","It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.","The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.","The effective concentration range can be determined using routine methodology known in the art.","Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.","Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.","The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics."],["TECHNICAL FIELD The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.","The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.","It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs.","BACKGROUND OF THE INVENTION The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.","Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.","It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.","One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.","The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.","Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.","The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.","These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.","The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.","The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.","Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.","The second strategy for finding key players in RNAi has involved the use of cell free assays.","These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.","However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.","Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.","This would enable the identification of factors which can enhance or reduce the gene silencing effect.","It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative.","SUMMARY OF THE INVENTION Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.","Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.","This is indicative of an RNAi-like mechanism of gene suppression.","It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.","This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.","The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.","The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.","According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.","Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.","It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.","Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.","More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","Even more preferred is an RNAi factor which is an res sequence.","Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.","In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.","According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.","According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.","As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.","According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.","According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.","According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.","In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.","Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.","Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.","Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.","However it will be understood that the entire native sequence of the target nucleic acid may be employed.","Conveniently, the target nucleic acid may be coupled to a selectable marker.","The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.","In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.","Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.","According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.","The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.","According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.","According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.","The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.","More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.","Even more preferred is a factor having an res sequence.","Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.","According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.","The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.","The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.","This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).","The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).","It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.","The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.","It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.","The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.","The effective concentration range can be determined using routine methodology known in the art.","Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.","Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.","The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics.","BRIEF DESCRIPTION OF THE FIGURES FIG.","1.Target genes and antisense fragments.","a, The strain KC4-6 expresses low levels of lacZ RNA.","The lacZ expression cassette is integrated at the ura4 locus on chromosome III and is composed of the SV40 early promoter, the E. coli lacZ gene and the SV40 3′ processing signal.","The 5′ and 3′ flanking DNA sequences of the ura4 locus are as indicated.","b, The strain RB3-2, which expresses high levels of lacZ RNA, contains an expression cassette containing the S. pombe adh1 promoter, the lacZ coding region, and the S. pombe ura4 3′ processing signal.","c, The full-length 3.5 kb antisense lacZ fragment is shown.","The crippled sense fragment was generated by end-filling the ClaI cut lacZ vector and religating on itself.","d, The AML1 strain contains a c-myc-lacZ fusion cassette integrated at the ura4 locus.","Exons 2 and 3 of the human c-myc gene were employed as a target.","The relative position of the c-myc antisense fragment is shown.","Transcription initiation sites are indicated by the bent arrows.","The straight arrows represent the normal direction of transcription for a particular DNA fragment.","FIG.","2.dsRNA-mediated gene silencing in fission yeast.","a, A fission yeast strain containing the integrated lacZ gene under control of the strong adh1 promoter was transformed with lacZ antisense vectors.","The antisense gene was under control of the weak nmt41 promoter (low antisense), the strong nmt1 promoter in single copy (15) (medium antisense), or the nmt1 promoter in multi-copy (high antisense).","For the strain expressing maximum antisense RNA two episomal nmt1-driven antisense vectors containing different selectable markers were co-transformed into the target strain.","b, A fission yeast strain containing the integrated lacZ gene under control of the weak SV40 promoter (low target) or strong adh1 promoter (high target) was transformed with the antisense lacZ vector or antisense lacZ and sense lacZ vectors.","Strains were co-transformed with vectors containing appropriate selectable markers to complement auxotrophy.","c, The lacZ inverted repeat vector was expressed in the high-expressing lacZ target strain.","The strain expressing the antisense construct only is also indicated.","d, A fission yeast strain containing an integrated c-myc-lacZ fusion gene was transformed with the sense construct, the antisense construct, or both.","Antisense and sense constructs were also expressed in the high-expressing lacZ strain.","All strains were transformed with appropriate control plasmids to complement auxotrophy.","FIG.","3.Effect of co-expressing single copies of sense and antisense genes.","Strains containing target lacZ alone (RB3-2), the target and a single copy of the antisense gene (K40-7), and the target and both the antisense and frameshifted lacZ genes (M62-1) were assayed for β-galactosidase activity.","pREP2 and pREP4 are parental plasmids containing the LEU2 and ura4 selectable markers respectively.","Strains were transformed with the appropriate control plasmids to complement auxotrophy.","For each sample three independent colonies were assayed in triplicate.","FIG.","4.RNA expression profiles in fission yeast strains a, The target strain RB3-2 was transformed with the lacZ inverted repeat vector (pM53-1) or co-transformed with antisense (pGT2) and sense (pM54-3) vectors and grown to mid-log in the absence of thiamine.","Total RNA from each strain was separated on a 1% denaturing agarose gel, transferred to a nylon membrane and probed with the radioactively labeled 2.2 kb nmt1 fragment.","The episomal lacZ signal was normalized with the endogenous nmt1 transcript and quantitated by phosphorimage analysis.","The relative level of episomal lacZ expression is shown.","b, The strain M38-1 which contains a single copy stably integrated lacZ inverted repeat gene was grown to mid-log in the absence of thiamine, its RNA isolated and analyzed by Northern hybridization.","The relative steady-state level of the “panhandle” lacZ RNA is indicated in comparison to episomally expressed antisense lacZ RNA.","FIG.","5.In vivo dsRNA assay.","a, The constructs employed for assaying the ability of the lacZ inverted repeat to form an intramolecular RNA duplex are shown.","Each vector contains a functional lacZ fragment which gives the transformed strain a blue color phenotype when grown in the presence of X-GAL.","The predicted secondary structure of the pM81-2 generated transcript is indicated.","b, The strain 2037 was transformed with the vectors pM85-1, pM91-1 and pM81-2 in the absence of thiamine Single colonies were streaked on minimal media plates and overlayed with 0.5% agarose medium containing 500 μg/ml X-gal and 0.01% SDS.","FIG.","6.The effect of ded1 on dsRNA-mediated gene regulation.","a, The ded1 gene was co-expressed in a fission yeast strain containing the integrated lacZ gene.","β-galactosidase activity of the strain transformed with and without the antisense lacZ vector is shown.","b, The target strain was transformed with a vector expressing a short lacZ antisense gene or with both the short lacZ antisense vector and the ded1-expressing vector.","Strains were transformed with appropriate control plasmids to complement auxotrophy.","FIG.","7.Over-expression of thi1 in an antisense lacZ expressing strain.","β-galactosidase activity of the target strain RB3-2 transformed with and without the antisense lacZ vector is shown.","Over-expression of the thi1 gene in the antisense expressing strain is also indicated.","Strains were transformed with appropriate control plasmids to complement auxotrophy.","FIG.","8.Over-expression screening strategy for RNAi modulating factors.","A target strain containing the integrated lacZ gene under control of the adh1 promoter and the episomal vector containing the nmt1-driven lacZ antisense gene is transformed with an S. pombe cDNA library.","Library fragments are driven by the nmt1 promoter.","Transformants are individually screened for a change in the lacZ-encoded blue colony colour phenotype.","These transformants are then quantitatively assayed for β-galactosidase activity in the presence and absence of thiamine.","The antisense vector is then segregated out of transformants showing a cDNA dependent modification of lacZ suppression.","A tertiary β-galactosidase assay is performed to determine if the effect is dependent on the presence of dsRNA.","cDNA vectors are recovered from strains of interest, sequenced, and subjected to BLASTN analysis.","FIG.","9.Over-expression screen of a S. pombe cDNA library.","a, Transformants were grown on minimal media plates and over-layed with X-GAL-containing medium.","Those which showed a reduced blue colour-phenotype (black arrow) were analysed further.","Transformants demonstrating an enhanced blue-colour phenotype were also identified (white arrow).","b, Transformants which showed a visual reduction in the blue phenotype were assayed for β-galactosidase activity in liquid culture in the absence of thiamine.","Thiamine was added to the medium to demonstrate that the enhanced lacZ suppression was dependent on RNA expression.","Transformants were again assayed for β-galactosidase activity following antisense vector segregation.","c, Over-expression of unique res genes in dsRNA-expressing strains.","FIG.","10.lacZ panhandle-mediated gene silencing.","(A) The lacZ panhandle construct contains the full-length crippled lacZ gene which is followed by the inverted 5′ 2.5 kb lacZ fragment.","Intramolecular hybridization generates an RNA with 2.5 kb RNA duplex and a 1 kb loop.","(B) The relative steady-state level of the episomally expressed panhandle lacZ RNA (7.0 kb) is shown in comparison to episomally expressed antisense lacZ RNA (4.5 kb).","The lacZ signals were normalized to the endogenous nmt1 transcript (1.3 kb) and quantitated by phosphorimage analysis.","(C) The target strain was transformed with the episomally expressed lacZ panhandle and analyzed for b-galactosidase activity.","The appropriate plasmids were co-introduced to complement auxotrophy.","At least three independent colonies were assayed in triplicate for each strain.","Transformants were assayed in the presence of thiamine to abrogate expression of the panhandle RNA (hatched).","(D) The target lacZ strain was transformed with the panhandle vector only or both the panhandle and aes2 vector.","FIG.","11.Co-expression of ded1 and lacZ antisense genes.","b-galactosidase activity of the target strain co-transformed with the ded1 gene and the antisense lacZ vectors is shown.","Ded1 was expressed from the ura4-based plasmid pREP4 while the antisense genes were expressed from the LEU2-based plasmid pREP2.Strains expressing antisense RNAs or ded1 RNA alone are also indicated.","Strains were transformed with appropriate control plasmids to complement auxotrophy.","Three independent colonies were assayed in triplicate for each strain.","FIG.","12.DNA sequence for aes1 factor FIG.","13.DNA sequence for aes2 factor FIG.","14.DNA sequence for aes3 factor FIG.","15.DNA sequence for aes4 factor DESCRIPTION OF THE PREFERRED EMBODIMENTS Using the lacZ fission yeast model to investigate features of antisense-RNA technology in vivo (1-4), it has been shown that the degree of target gene suppression is dependent on the level of antisense RNA (FIG.","2A)(1).","During the course of developing this model it was surprisingly found that by increasing the steady-state levels of the lacZ target mRNA 20-fold in the presence of the same pool of antisense RNA that antisense-mediated suppression was not only maintained, but enhanced (FIG.","2B).","To verify that this effect was independent of the target mRNA a lacZ gene containing a frameshift mutation (leading to an inability to translate the β-galactosidase enzyme) was co-expressed in antisense-expressing strains.","Again, additional stimulation of lacZ inhibition was observed (FIG.","2B).","These observations are consistent with the potential formation of additional dsRNA and consequent stimulation of gene silencing.","They also indicate that the dsRNA-mediated gene silencing is dose-dependent in this system.","To further show that dsRNA was the key component in target gene inhibition in fission yeast dsRNA was generated by expressing a lacZ inverted repeat with 2.5 kb of internal complementarity in the presence of lacZ target mRNA.","Expression of this construct also reduced β-galactosidase activity in a lacZ expressing strain to a similar level to the full-length antisense RNA (FIG.","2C).","Overall, these data suggested that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, resulted in efficient interference of target gene expression in S. pombe.","To test the ability of dsRNA to specifically interfere with another target sequence in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (3).","A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene was previously found to suppress β-galactosidase activity within the c-myc-lacZ fusion target strain by 47% (3).","β-galactosidase assays demonstrated that co-expression of the antisense and sense c-myc constructs in the target strain enhanced c-myc suppression compared with the antisense c-myc vector alone (FIG.","2D).","Transformation of the strain expressing only the lacZ target with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is sequence-specific (FIG.","2D).","All of the above observations, as well as the reduction in target mRNA levels in response to additional dsRNA (data not shown), are consistent with features associated with RNAi.","This is the first example of dsRNA-mediated gene regulation in yeast and, unlike RNAi in all other organisms except zebrafish, appears to be dependent on the concentration of intracellular dsRNA.","The variation in RNAi efficacy between organisms may be due to the absence or under-expression of some components of the RNAi pathway or the presence of RNAi inhibitors.","We have now further developed this genetic model for identification of factors involved in RNAi.","Without wishing to be bound by any particular mechanism, it is probable that a multi-protein complex exists which mediates the post-transcriptional degradation of target mRNA in RNAi.","Several genes which are involved in the RNAi phenomenon have been identified through genetic screens in Neurospora and nematodes.","These genes include an RNA-dependent RNA polymerase (qde-1, ego-1) a RecQ DNA helicase (qde-3), an RNase D homologue (mut-7), and a putative translation initiation factor (rde-1, qde-2).","The genes mut-2, rde-2, rde-4, and rde-7 have also been identified as being involved in either the initiation or maintenance of RNAi.","Additionally, Drosophila cell free assays have shown that RNAi is mediated by nuclease degradation of the targeted mRNA while a 21-25 nt RNA species appears to be integral to specific post-transcriptional genetic interference.","RNAi has also been shown to be dependent on ATP which may be required for strand dissociation of dsRNA.","However, a missing component of the proposed multi-protein complex in the RNAi pathway is the implied RNA helicase.","The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.","In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.","Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.","The first gene that was tested in mediating RNAi activity in the present model was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.","As we have previously shown that antisense RNA-mediated gene suppression is dose dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.","The thi1 open reading frame was PCR amplified and subcloned into pREP4 as a BamHI fragment.","This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.","As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.","7).","This result indicated that host-encoded factors are present which affect the robustness of dsRNA-mediated gene suppression in fission yeast and also validated the overexpression strategy.","The second gene investigated has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.","According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.","It was therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.","Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.","6A).","When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.","6A).","The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (16).","Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.","6B).","It was thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.","By over-expressing a cDNA library in a dsRNA fission yeast model we have identified four novel, genes with potential roles in the RNAi pathway.","All of the known genes identified are essential and have natural associations with DNA, RNA or nucleic acid binding proteins, consistent with the expectation of RNAi cellular factors.","In addition, two of the three known cDNAs encoded proteins involved in the process of translation.","These findings along with our previous identification of an ATP-dependent RNA helicase (Raponi & Arndt, submitted) and reports of the involvement of a translation initiation factor in RNAi and the co-purification of ribonuclease activity with ribosomal fractions, suggests that this may be one site at which RNAi functions.","Alternatively, the identification of such varied proteins as DNA helicases and translational components as part of the RNAi machinery implies that particular cellular proteins may be recruited into more than one multiprotein complex.","Under the latter conditions, over-expression of these specific proteins may result in the generation of an RNAi complex that recognises dsRNA and mediates target gene suppression.","Certain of these proteins may be rate-limiting or rate-determining in RNAi and only through supplementation of these factors are their roles in RNAi uncovered.","It is proposed, without wishing to be bound by any particular mechanism, that in addition to the previously identified core proteins in the RNAi multiprotein complex(es), additional factors such as EF Tu, L7a, sna41 and res1 may be enlisted for a role in dsRNA-mediated gene suppression.","The over-expression strategy described overcomes some limitations associated with mutagenesis by identifying essential genes with a role in RNAi.","In addition, this strategy complements these other systems by allowing the isolation of cellular factors that modify the efficacy of RNAi in vivo.","The roles of these modulators of RNAi may be varied and include recognition and amplification of the dsRNA, delivery of the small 21-25 nt dsRNAs to the target mRNA, association between the antisense and target mRNA strands, and RNAi complex formation.","Alternatively, these modulators may control the rate of RNAi or the formation of different complexes within cell types or for different forms of post-transcriptional gene silencing.","The fact that over-expression of specific RNAi modulators enhanced dsRNA-mediated gene regulation in fission yeast indicates that a similar approach could be used to identify RNAi modulators in other organisms.","In addition, the co-expression of these factors with different forms of post-transcriptional gene silencing including co-suppression, quelling, and antisense RNA could be one way of enhancing the efficacy of these methods.","This may be especially important for application of RNAi to mammalian cells and tissues or to genes which have been somewhat recalcitrant to this form of regulation.","The present invention demonstrates for the first time the intrinsic involvement of an ATP-dependent RNA helicase as a key component in RNAi.","Further, it can be rate limiting, as the over-expression leads to increased RNAi activity in this system.","This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.","These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.","The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.","The present invention also provides for the first time a novel and quantitative genetic system based on S.pombe, for rapidly identifying essential cellular factors involved in RNAi.","The use of this model has enabled verification of RNA helicase activity as a critical contributor to efficient RNAi activity and isolated novel RNAi factors including EF Tu, L7a, sna41, and an unidentified gene res1.The present invention also demonstrates that gene silencing may be enhanced by concomitant expression of such RNAi factors.","The invention will now be described more particularly with reference to non-limiting examples of certain preferred embodiments of the invention.","Example 1 Materials and Methods Used to Exemplify the Invention S. pombe Media and Manipulations.","All yeast strains were maintained on standard YES or EMM media (6).","Repression of nmt1 transcription was achieved by the addition of thiamine to EMM media at a final concentration of 4 μM (7).","Yeast cells were transformed with plasmid DNA by electroporation (8) and stable integrants were identified as previously described (6).","A glass bead procedure (9) was used to isolate genomic DNA which was used for Southern analysis and PCR diagnosis.","Total RNA was extracted as previously described (10).","S. pombe Strain and Plasmid Construction.","Construction of the low expressing lacZ strain, KC4-6 (h−, ura4::SV40-lacZ, leu1-32), has been previously described (2).","The strain RB3-2 (h−, ura4::adh1-lacZ, leu1-32) which expresses higher levels β-galactosidase has also been previously described (1).","The target strain which contains the c-myc:lacZ fusion has previously been described (3).","The construction of the long lacZ antisense containing episomal plasmid, pGT2, and corresponding control plasmids have been described (2).","Plasmid pREP4-As was generated by subcloning the lacZ BamHI fragment contained in pGT2 into the plasmid pREP4 (11).","pREP4 is identical to pREP1 except that the S. cerevisiae LEU2 gene has been replaced with the S. pombe ura4 selectable marker.","To decrease the steady-state level of episomally expressed antisense lacZ, plasmids pREP42-As and pREP82-As were constructed by subcloning the lacZ BamHI fragment from pGT2 into pREP42 and pREP82, respectively.","These plasmids are derivatives of pREP4 with mutations in the TATA box of the nmt1 promoter (12).","The crippled lacZ vector, pGT62, was generated by end-filling the ClaI site of pGT2 and re-ligating (2).","This frameshifted fragment was then subcloned into the BamHI site of pREP4 to generate the plasmid pM54-3.The lacZ panhandle integration vector, pM30-8, was generated by first introducing a NotI site into the XmaI site of pRIP1/s (11) using the self-complementary linker 5′ CCG GGC GGC CGC 3′ to generate pL121-14.The 2.5 kb sequence of the 5′ end of the frameshifted lacZ gene (2) was then PCR-amplified to give it NotI ends using the forward primer 5′ ATGCGGCCGCAATTCCCGGGGATCGAAAGA 3′ and reverse primer 5′ ATGCGGCCGCAATGGGGTCGCTTCACTTA 3′.","This product was cloned using a TA cloning kit (TOPO: Invitrogen, San Diego, Calif., USA) and then subcloned into the NotI site of pL121-14 in the antisense orientation.","The integrating vector was introduced into target strains in single copy by using the sup3-5/ade6-704 complementation system (13).","The full-length frameshifted lacZ fragment was then introduced into the BamHI site of this vector in the sense orientation upstream of the 2.5 kb antisense fragment to generate pM30-8.The episomal version of this vector (pM53-1) was made by removing the Pst1 sup3-5 fragment and introducing the autonomous replicating sequence as an EcoRI fragment (11).","For testing the ability of the panhandle construct to form dsRNA in vivo, the frameshifted lacZ fragment was replaced with the functional lacZ fragment in the vectors pM54-3 and pM53-1, to generate the vectors pM85-1 and pM81-2, respectively.","The vector pM91-1, which is unable to form a lacZ panhandle transcript, was generated by removing the 2.5 kb NotI lacZ fragment from pM81-2 and reintroducing it in the sense orientation.","Generation of the c-myc antisense construct, pCM-17, has been described elsewhere (3).","The 792 by BglII c-myc fragment from pCM-17 was subcloned into the BamHI site of pREP4 in the sense orientation to generate pN12-1.The ded1, and thi1, open reading frames were amplified from fission yeast genomic DNA (strain 1913).","ded1 was amplified to give it BamHI ends using the forward primer 5′ ATGGGATCCCAACCAAACACTTCAACTCAG 3′ and the reverse primer 5′ ATGGGATCCTCAGAAGCCTGTGCATAACAC 3′.","thi1 was amplified to give it BglII ends using the forward primer 5′ ATGAGATCTGTGGTTGGTATTCTAGAGAGA 3′ and the reverse primer 5′ ATGAGATCTAACAAAGACCTGCAAAAAACC 3′.","PCR products were purified (Qiagen PCR purification kit), digested with either BamHI or BglII, gel-purified (Qiagen), and subcloned into the BamHI site of pREP4 in the sense orientation.","Southern and Northern Analysis.","Nucleic acid electrophoresis and membrane transfer was performed as described (14).","Southern and Northern blots were hybridized using ExpressHyb solution according to the manufacturer's instructions (Clontech Laboratories).","DNA probes were 32P-labelled using the Megaprime labelling kit (Amersham).","Probes included a 960 bp BamHI/ClaI lacZ fragment from pI2-1 (1), a 570 by HindIII/EcoRI ura4-3′ fragment from pGT113 (15), and a 2.2 kb PstI/SacI nmt1 fragment from pRIP1/s.","Radioactive signals were detected by autoradiography and quantitated by phosphorimage analysis (ImageQuant; Molecular Dynamics).","Plasmid Distribution Analysis.","Plasmid co-transformants of strain RB3-2 were grown under selective conditions to a cell density of 1-2×107 cells/ml.","A serial dilution of each culture was performed and cells plated for single colonies in triplicate onto each of YES, EMM, EMM+leucine, and EMM+uracil agar media.","The number of colonies grown on YES was taken as the total number of viable cells, while colonies growing on EMM represented the cells in the sampled population that contained both the ura4-containing (pREP4-based) and the LEU2-containing (pREP1-based) plasmids.","Cells containing either the ura4-containing plasmid or the LEU2-containing plasmid were identified from the EMM+leucine and EMM+uracil plates respectively.","The ratio of the number of colonies grown on selective media to the total number of viable colonies was used as the quantitative measure of the proportion of cells in the population, grown under selection, which contained plasmids.","β-galactosidase Assays.","The expression of the lacZ gene-encoded product, β-galactosidase, was quantitated using a cell permeabilization protocol as previously described (Raponi et al., 2000).","A semi-quantitative overlay assay was also employed for rapid screening of yeast transformants (3).","Isolation of S. pombe cDNA Clones That Alter Antisense RNA Efficacy.","The S. pombe cDNA library was originally constructed in pREP3Xho by Bruce Edgar and Chris Norbury (5).","The vector pREP3Xho is derived from pREP3 which contains the LEU2 marker and inserts are under control of the nmt1 promoter (11).","A total of 5 μg of library DNA was transformed into the strain RB3-2 containing the episomal antisense lacZ vector pREP4-lacZAS and grown in EMM liquid media to the mid-logarithmic phase.","Transformants were then plated on EMM solid media and grown at 30° C. for 3 days.","Colonies were over-layed with medium containing 0.5 M sodium phosphate, 0.5% agarose, 2% dimethylformamide, 0.01% SDS, and 500 μg/ml X-GAL (Progen, Australia).","Plates were then incubated at 37° C. for 3 hrs, colonies of interest recovered and assayed for β-galactosidase activity.","Plasmid Segregation.","Strains were plated on EMM containing limiting uracil and 1 mg/ml 5-fluoroorotic acid (6).","Strains were then replica plated on both selective and non-selective media.","Those colonies that did not grow on selective media were identified as having lost the ura4-containing antisense plasmid.","To determine the mitotic stability of pREP-based plasmids we used the plasmid segregation method previously described (16).","This method indicated that up to 30% of the cells grown in selective media did not contain the resident plasmid.","Example 2 Effect of Steady-State Level of Target mRNA and Antisense RNA on Antisense Efficacy To determine the role of target mRNA steady-state levels in antisense-mediated gene suppression, we investigated the ability of a long lacZ antisense RNA (2) to regulate lacZ target genes under control of both weak and strong constitutive promoters.","The low-level expressing strain, KC4-6, contained the lacZ gene driven by the SV40 early promoter integrated at the ura4 locus in chromosome III (FIG.","1A; (2)).","The high-level lacZ expressing strain, RB3-2, was constructed by placing the lacZ gene under control of the strong fission yeast adh1 promoter and the 3′ processing signal from the ura4 gene and integrating this expression cassette at the ura4 locus (FIG.","1B; (1)).","Characterization of both strains indicated that RB3-2 expressed 20-fold more lacZ mRNA than KC4-6 while an approximate 40-fold increase in β-galactosidase was also detected (data not shown).","Both strains were transformed with the lacZ antisense gene-containing plasmid (pGT2) and its corresponding sense control plasmid (pGT62).","β-galactosidase assays indicated that the antisense RNA suppressed β-galactosidase activity by 45% in KC4-6 while the same RNA reduced β-galactosidase activity by 52% in RB3-2 (FIG.","2B).","No reduction in β-galactosidase activity was seen in either strain when transformed with the control plasmid pGT62 which expresses a crippled version of lacZ.","These results indicated that despite a 20-fold increase in the steady state level of the lacZ target mRNA in RB3-2 the efficacy of antisense-mediated down regulation was not only maintained, but enhanced.","The increase in gene suppression demonstrated with strain RB3-2 was also seen when antisense molecules targeted to subregions of the lacZ target gene were used, but was not as pronounced.","These data suggested an increase in target mRNA can result in enhanced target gene suppression.","It has previously, been demonstrated that a stably integrated antisense lacZ gene acts in a dose dependent fashion with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number (1).","Here the role of the steady-state level of episomally expressed antisense RNA in both the low lacZ expressing target strain, KC4-6, and the high lacZ expressing target strain, RB3-2, was investigated.","The expression of lacZ antisense RNA was increased by co-transforming KC4-6 and RB3-2 with plasmids pGT2 and pREP4-As, each of which contains the lacZ antisense gene under control of the nmt1 promoter, but different selectable markers.","To decrease the steady-state level of antisense RNA, the plasmids pREP42-As and pREP82-AS were employed.","The nmt1 promoter in these vectors contain deletions in the TATA box sequence which affect the level of transcription, but have no impact on the site of transcription initiation or thiamine repressibility (12).","Each of the different antisense gene-containing plasmids was co-transformed with a control plasmid to complement auxotrophy where appropriate.","This resulted in a set of co-transformants of both RB3-2 and KC4-6 each containing the same lacZ antisense gene, but with different promoter capacities.","Each co-transformant was analyzed for antisense RNA steady-state levels and β-galactosidase activity.","Table 1 indicates that with both strains the degree of target suppression is enhanced with the increase of antisense lacZ RNA expression.","TABLE 1 Effects of varying the level of antisense RNA in strains expressing both low and high level target.","Relative β-gal.","activity antisense Strain Plasmids Descriptions (units) % suppressiona RNA levelb KC4-6 pREP2, pREP4 Vector 1.07 0 0 pREP2, pREP82-AS Low As 1.10 0 0.04 pREP2, pREP42-AS Medium As 1.07 0.1 0.14 pREP2, pREP4-AS Full As-ura4 0.75 30 1.0 pGT2, pREP4 Full As-LEU2 0.70 34.6 2.2 pGT2, pREP4-AS Double-As 0.54 49.2 5.6 RB3-2 pREP2, pREP4 Vector 48.5 0 0 pREP2, pREP82-AS Low As 44.0 9.4 0.05 pREP2, pREP42-AS Medium As 43.9 9.6 0.18 pREP2, pREP4-AS Full As-ura4 27.2 43.9 1.0 pGT2, pREP4 Full As-LEU2 24.9 48.7 1.9 pGT2, pREP4-AS Double-As 16.6 65.8 5.4 aThe % suppression of β-galactosidase activity was determined by expressing each β-galactosidase activity as a percentage of the activity found in the pREP2, pREP4 co-transformant.","bWithin each strain the steady-state level of long lacZ antisense RNA was normalized to nmt1 mRNA and then expressed relative to the level observed in the pREP2, pREP4-As co-transformant.","These data agree with the previous report that antisense RNA-mediated gene inhibition is dose dependent in S. pombe (1).","However, for each co-transformant the degree of β-galactosidase suppression was greater by 10-15% in the high lacZ expressing strain, RB3-2, than the low lacZ expressing strain, KC4-6, again demonstrating that low levels of target mRNA can limit antisense efficacy.","Example 3 Effect of Increasing Sense RNA on Antisense RNA-Mediated Target Gene Suppression To determine whether the increase in gene suppression was due to formation of an antisense RNA:target mRNA hybrid or an antisense RNA:sense RNA hybrid, a version of lacZ which is unable to be translated into functional β-galactosidase was co-expressed in strains expressing the lacZ antisense gene.","If antisense RNA was required to hybridize to target mRNA for inhibition of the gene expression pathway, then over-expression of the sense RNA would compete with the target for the available antisense molecules and a decrease in lacZ gene suppression would result.","Initially, both antisense and sense lacZ vectors were integrated in single copy into separate target strains and then crossed with each other.","In strains containing the antisense gene alone, β-galactosidase activity was reduced by approximately 40% (FIG.","3; (1)) while there was no reduction in the strain expressing sense lacZ alone (data not shown).","However, in the strain expressing both complementary transcripts (M62-1), β-galactosidase activity was reduced by 50% (FIG.","3).","These results suggested that increasing the intracellular concentration of sense RNA in the presence of antisense RNA, and therefore potentially generating more dsRNA, stimulates target gene suppression.","To further increase the potential formation of intracellular dsRNA an episomal sense lacZ plasmid (pM54-3; FIG.","2B) was co-transformed with the episomal antisense lacZ plasmid, pGT2, into the target strain R133-2.Both of these sequences were expressed by the strong conditional nmt1 promoter and RNA analysis showed that each transcript was being expressed at high levels when grown in the absence of thiamine (FIG.","4A).","When both RNAs were co-expressed in RB3-2, the target lacZ was suppressed by 65% compared to 50% in the strain expressing the antisense plasmid alone (FIG.","2B).","Northern analysis demonstrated that the total relative level of episomally expressed nmt1-driven transgenes was approximately 60% higher than when either vector was expressed alone (FIG.","4A).","Although expression of these complementary RNA transcripts is at a high level compared with single copy integrants only an additional 15% suppression was observed.","One explanation for the absence of higher levels of target gene suppression is plasmid segregation.","S. pombe undergoes asymmetric segregation, with the result that mitosis produces a daughter cell which lacks the segregating plasmid (17).","Therefore, when the 65% suppression level seen in the total population is corrected for only those cells containing the ura4-based and LEU2-based plasmids the level of suppression approaches approximately 100% (data not shown).","Overall these data suggest that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, is required for efficient interference of target gene expression in S. pombe.","However, unlike the phenomenon of RNAi seen in plants and nematodes, the dsRNA-mediated gene suppression demonstrated in fission yeast seems to be dependent on the concentration of intracellular dsRNA or a threshold level of dsRNA is required to invoke potent gene silencing.","Example 4 Effect of a lacZ Inverted Repeat on lacZ Gene Expression To further investigate the ability of dsRNA to inhibit gene expression in fission yeast we generated a vector containing the full-length 3.5 kb frameshifted lacZ sequence with a 2.5 kb inverted repeat.","This construct generates a transcript of approximately 7 kb in length with 2.5 kb of self-complementarity which, predictably, will form a strong intramolecular RNA duplex.","This gene was initially integrated into a fission yeast strain in single copy and then crossed with the strain RB3-2.The resulting strain which contained the single copy inverted repeat gene and target lacZ showed no reduction in β-galactosidase activity when transcription of the inverted repeat was activated.","Southern analysis confirmed that the cassette was intact (data not shown) while RNA analysis indicated that the 7 kb transcript was being generated but approximately 10-fold less than episomally expressed antisense lacZ (FIG.","4A&B).","To increase the steady-state level of the inverted repeat RNA we generated the episomal plasmid pM53-1 containing this cassette.","RNA analysis demonstrated that this vector is expressed at a similar level to episomally expressed antisense lacZ (FIG.","4A).","When expressed in RB3-2 pM53-1 inhibited β-galactosidase activity by 40% (FIG.","2C), while addition of thiamine to the culture medium returned β-galactosidase activity to control level.","To confirm that gene inhibition was due to this construct forming an RNA duplex, an in vivo assay for dsRNA was developed.","To this end, a series of vectors were generated which contained functional lacZ sequences including lacZ alone (pM85-1), a lacZ inverted repeat (pM81-2), and the lacZ repeat with both sequences in the sense orientation (pM91-1) (FIG.","5A).","These vectors were then introduced into a strain lacking the integrated target sequence (NCYC2037; h+, ura4-D18) and resulting transformants were overlayed with X-GAL-containing agarose.","Both strains containing control vectors generated high levels of β-galactosidase while the strain expressing the lacZ inverted repeat did not (FIG.","5B).","In each case Northern analysis demonstrated that strains were expressing the transgene.","These results indicate that the inverted repeat RNA could not be efficiently translated to produce β-galactosidase, most probably due to the formation of a strong intramolecular hairpin structure.","Taken together, these results show that a construct which has the ability to form dsRNA by intramolecular hybridization will interfere with target gene expression when expressed at high levels, again indicating that dsRNA mediates target gene suppression in a dose-dependent manner in fission yeast.","Example 5 Gene-Specificity of dsRNA-Meditated Gene Silencing in Fission Yeast To test the ability of dsRNA to specifically interfere with other target sequences in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (FIG.","1D; (3)).","A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene (named CM-17) was previously found to suppress β-galactosidase activity within a c-myc-lacZ fusion target strain (AML1) by 47% (3).","CM-17 was subsequently subcloned into pREP4 in the sense orientation to generate pN12-1.The antisense c-myc vector (pCM-17) and the sense c-myc vector were transformed into AML1 both independently and together.","β-galactosidase assays demonstrated that co-expression of the antisense and sense constructs enhanced c-myc suppression by an additional 13% compared with the antisense c-myc vector alone (FIG.","2D).","Transformation of RB3-2 with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is gene-specific (FIG.","2D).","These results demonstrate that dsRNA can specifically interfere with multiple targets in fission yeast including one of human origin.","Example 6 Over-Expression of a Translation Factor can Enhance RNAi in Fission Yeast The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.","In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.","Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.","The first gene that we have tested in mediating RNAi activity in the present model has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.","According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.","We therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.","Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.","6A).","When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.","6A).","The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (2).","Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.","6B).","We have thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.","Although an ATP-dependent RNA helicase has recently been suggested to be a key component in RNAi, this is the first demonstration of its intrinsic involvement.","Further, it can clearly be rate limiting, as the over-expression leads to increased RNAi activity in this system.","This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.","These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.","The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.","Example 7 Over-Expression of a Transcription Factor can Enhance RNAi in Fission Yeast The second gene investigated was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.","As we have previously shown that antisense RNA-mediated gene suppression is dose-dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.","The thi1 open reading frame was PCR-amplified and subcloned into pREP4 as a BamHI fragment.","This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.","As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.","7).","This result indicated that host-encoded factors are present which modulate the efficacy of dsRNA-mediated gene suppression in fission yeast and also validated the over-expression strategy.","Example 8 Screening for Novel RNAi Modulating Factors Over-expression of a fission yeast cDNA library (5) in an antisense lacZ expressing strain, revealed a series of transformants in which β-galactosidase activity was significantly reduced from that demonstrated in antisense RNA-expressing strains alone.","The screening strategy is shown in FIG.","8.Transformants were grown on selective plates and overlayed with X-GAL-containing media to generate a blue phenotype which could be analysed visually in a semi-quantitative manner (FIG.","9A)(3).","From 12,000 transformants screened, 48 were initially identified as having a reduced blue phenotype compared with background transformants.","Of these, 25 demonstrated a reproducible enhancement in antisense RNA-mediated lacZ suppression when quantified by a liquid β-galactosidase assay (FIG.","9B).","To demonstrate that the observed effects were due to transcription of the cDNA insert, transformants were grown in the presence of thiamine.","This repression of the nmt1 promoter resulted in a return to control levels of β-galactosidase activity in all transformants (FIG.","9B).","This indicated that the enhanced suppression was due to expression of the cDNA rather than other genetic events such as lacZ reporter mutations.","To determine if the effect was dependent on the presence of dsRNA, the antisense plasmid was segregated out of the transformants and the strains were again assayed for β-galactosidase activity.","Nine of the 25 transformants demonstrated a return to the level of β-galactosidase seen in the parental strain indicating that the effect was dsRNA-dependent (FIG.","9B).","The cDNAs being expressed in these transformants were named RNAi enhancing sequences (res).","All transformants displayed normal growth phenotypes indicating that over-expression of the resident cDNAs did not affect general biology of the cell.","In the absence of antisense RNA the remaining 16 transformants retained a reduced β-galactosidase activity indicating that the enhanced gene silencing was not dependent on the presence of dsRNA.","This suggests that the cDNA-encoded proteins in these strains could have been affecting lacZ at the level of transcription or the stability or function of the protein.","The library plasmids were recovered from the aes-containing strains (also referred to as res-containing strains) and their cDNA inserts sequenced.","BLASTN and BLASTP analysis identified clones W18, W20, and W30 (named aes2) as homologues of domains 2 and 3 of the mitochondrial elongation factor Tu (EF Tu).","EF Tu is an essential protein which plays a role in transporting tRNA to the A site in the ribosome for peptide elongation.","The cDNA in transformants W21, W23, and W32 (named aes3) was homologous to a putative protein that was identified in a screen for fission yeast ORFs.","Interestingly, the cDNA insert was also homologous to the antisense strand of the 3′ UTR of the fission yeast gene sna41 which has previously been shown to be involved in DNA replication.","It is thus possible that aes3 may operate through more than one mechanism in enhancing antisense RNA activity.","The cDNA in transformant W47 (named aes4) was homologous to the antisense strand of the ribosomal protein L7a, a component of the 60S ribosomal subunit.","aes4 also contained a small ORF of unknown biological function.","The inserts in transformants W27 and W28 (named aes1) shared homology with a putative protein from C. albicans that was identified in a screen for genes essential for cell growth.","A tertiary quantitative β-galactosidase assay was performed to obtain accurate levels of gene silencing augmentation in the antisense lacZ strains co-expressing these unique factors (FIG.","9C).","This analysis showed that these co-factors enhanced antisense suppression by up to 50%.","These results demonstrated that over-expression of a cDNA library was an effective way of identifying novel co-factors that magnify the suppressive effect mediated by antisense RNA.","aes1 shared 43% identity with amino acids 4 to 202 of a C. albicans hypothetical protein (AJ390519).","aes2 shared 99% identity with nucleotides 10452 to 9484 of the translation elongation factor EF Tu (AL049769).","aes3 shared 94% identity with nucleotides 776 to 1145 of D89239 S. pombe ORF (D89239) and 93% identity with nucleotides 3246 to 2876 of the antisense strand of the DNA replication factor sna41 (AB001379).","aes3 also contained a 220 nt stretch of a GA repeat sequence at its 3′ end.","aes4 shared 99% identity with nucleotides 9678 to 8897 of the antisense strand of ribosomal protein L7a (AJ001133) and 99% identity with nucleoStides 1365 to 584 of the antisense strand of ribosomal protein L4 (AB005750).","The reference numerals in brackets refer to accession numbers in the GeneBank database (the GeneBank database can be accessed from the following web site: http://www.ncbi.nlm.nih.gov/).","EF Tu is analogous to the eukaryotic EF1α and acts by transporting tRNA to the A site in the ribosome for peptide elongation.","EF1α is an essential protein which has also been implicated in a large array of cellular activities including actin binding, microtubule severing, cellular transformation, cell senescence, protein ubiquitination, and protein folding.","Detailed analysis of the EF Tu-expressing strain showed that it enhanced antisense RNA-mediated lacZ silencing by an additional 15% (FIG.","9C).","It has been proposed that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and dissociated by an ATP-dependent RNA helicase.","The small antisense fragments are then free to attack homologous mRNA by RNA nuclease-mediated degradation.","It has recently been suggested that an associative mechanism could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.","This may involve the transport of fragmented dsRNA to the ribosome where, in a competitive reaction with tRNA complexes, the complementary antisense strand binds to the mRNA.","This would permit the association of the antisense strand of the dsRNA with the target mRNA when the latter is in a structurally exposed state and both inhibit translation and target the mRNA for ribonuclease degradation.","Without wishing to be bound by any particular mechanism of action, EF Tu could act by binding to RNAi-dependent short dsRNA species and bring them to the site of action.","The protein encoded by sna41 has previously been shown to be involved in DNA replication.","sna41 has low homology with CDC45 and might have DNA helicase properties which could facilitate the expression of complementary sequences.","It is conceivable that the antisense plasmid and target DNA sequences may ectopically pair by intermolecular complementarity.","Such pairing may inhibit RNA expression and consequently reduce the level of intracellular dsRNA.","Similarly the intramolecular pairing of inverted repeat DNA sequences may also interfere with RNA expression.","The over-expression of a protein with DNA helicase properties could facilitate the generation of more dsRNA which could in turn enhance the RNAi effect.","Furthermore, CDC45 mutants show an increased rate of plasmid segregation.","If plasmid loss is inhibited by over-expression of sna41 then more dsRNA may be generated leading to more effective RNAi in this system.","In this light it is not unreasonable to expect that other proteins normally involved in DNA and/or RNA metabolism and function could also have a role in RNAi modulation and/or enhancement.","The L7a protein is part of the 60s ribosomal sub-unit.","Without wishing to be bound by any particular mechanism of action, RNAi augmentation by over-expression of this protein is reasonable as it is hypothesised that the short dsRNA species may undergo strand displacement with target mRNA at the ribosome.","The L7a ribosomal protein may act in RNAi by i) mediating docking of dsRNA or its unwound form into the A site of the ribosome, assisting in association of the antisense strand with the target mRNA, and/or shuttling of dsRNA to the ribosomal complex.","Nucleotide sequences representative of the aes factors referred to above are provided below and identified as Seq ID Nos 1 to 4: DNA sequence for aes1 factor (Seq ID No 1) TTTAACTTAGTTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAA GCCGCAATTGTAACAGTAGATTTTTTGCATCATTATTACTCTCCGAAACA TGACTGAACACTCATTTAAGCAAATAGACGTGTTTTCTAATAAAGGTTTT CGAGGTAATCCTGTTGCAGTTTTTTTTGATGCAGATAATTTATCACAAAA GGAAATGCAGCAGATTGCCAAGTGGACAAATTTATCTGAGACAACATTTG TTCAAAAGCCGACAATCGATAAAGCAGATTACAGACTTCGTATATTTACC CCAGAATGTGAATTAAGCTTTGCTGGTCACCCAACAATTGGATCGTGCTT TGCTGTTGTTGAAAGTGGATATTGTACTCCAAAAAACTGTAAAATTATTC AGGAATGTTTAGCCGGTTTAGTTGAATTAACTATCGATGGGGAAAAGGAT GAAGACACTTGGATTTCTTTCAAACTTCCGTATTACAAAATTTTACAGAC TTCTGAAACTGCAATTTCAGAAGTAGAAAATGCATTGGGTATTCCTCTGA ATTATAGTTCTCAAGTTTCTCCTCCTGTGTTAATAGATGATGGACCAAAG TGGCTTGTAATTCAACTTCCAAACGCTACAGATGTGCTCAACCTCGTTCC GAAATTTCAGTCCCTTTCCCAAGTTTGTAAAAACAATGATTGGATAGGCG TCACCCGTCTTTGGTGAATTAGAAAAGACTCGTTTGAAAGCCCGAAGCTT TGCGCCTTTAATACATGTCAATGAGGATCCGGCTTGCGGTAGTGGTGCAG GAGCTGTCGGTGTGTATATTGGAAGCTCTCAAAAAACTCCAACTTCTCTA TCATTTACGATTTCTCAAGGTACAAAATTAAGTAGACAAGCAATTTCCAA AGTCAGCGTAGACGTTTCCTCCAATAAATCAATTGCTGTTTTTGTCGGTG GACAGGCAAAAACTTGTATTTCTGGAAAATCGTTTATTTAATGTTTTTAT TACAAATATTCACTTGCGAGTTTATTTTCCAATACTGAAGACTTTCAATC AATAGCAAATATGCTACTCAAGGAAGTTCACTCATTCAAAAGCAATTGGT TTACTATATCGTTTTTTCTAACTAGTTACTAGTCATTGAACAATCTACCG AATGATAAAATGAAATTTTGGTTTTTCCCCGGGTAAAAGGAATGTCTCCC TTGCCAGTACTGCTAGGGTTTTTCTTTCGAACTATAAGA DNA sequence for aes2 factor (Seq ID No 2) AGTCCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAGCCTGATA TGATCGAGCTTGTCGAAATGGAAATGCGTGAGCTACTCTCCGAATACGGA TTTGATGGTGACAATACTCCAATTGTTAGCGGCAGTGCTTTATGTGCCTT AGAGGGTCGTGAGCCTGAGATTGGTCTCAATAGTATTACTAAATTGATGG AAGCTGTTGATAGTTATATTACTCTTCCTGAAAGAAAAACGGATGTCCCT TTCTTGATGGCCATCGAGGACGTTTTTTCAATTTCAGGTCGCGGAACTGT AGTCACTGGCCGTGTCGAGCGCGGTACTTTAAAGAAGGGTGCTGAAATCG AAATCGTCGGTTATGGTAGCCATTTAAAGACTACCGTTACTGGAATTGAA ATGTTCAAAAAGCAGCTTGATGCCGCCGTTGCCGGTGACAATTGTGGCCT TTTACTTCGTTCTATCAAGCGAGAGCAATTAAAACGTGGAATGATTGTCG CTCAACCAGGAACCGTTGCTCCTCATCAGAAATTCAAGGCATCATTCTAT ATTTTGACAAAAGAGGAAGGAGGTCGTCGTACCCGGTTTCGTTGACAAGT ATCGTCCCCAACTGTACAGTCCGTACTTCCGACGTTACTGTCGAACTTAC CCACCCTGATCCTAACGACTCAACAAAATGGTTATGCCTGGAGACAATGT CGAGATGATCTGTACGCTTATTCACCCCATTGTCATCGAAAAAGGACAAC GCTTCACAGTTCGTGAGGGTGGAAGCACTGTAGGCACAGCTTTGGTTACT GAACTTTTGGATTAGTGCATTTATGAACTTATTGGCTTTAAAAATTTTGC ATGCTGAATACCAATATTATGTCCCTTCTCAGAATTCTATAACTACAGTG TCATTATTGTAATAAGACTTTTGCATCCATTGACAATGGTATTTGATACT TTTATAGTTTCTACTATTGTTAGCCAAAGTTATAAAACAAATAATAAAAT AACGTTGAATCAAAAAAAAAAAAAAAAAAAGCGGCCGCGGATCCCCGGGT AAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCTTTCAAACTA TGGGA DNA sequence for aes3 factor (Seq ID No 3) ATTTCAGACGCAATTCACATGGCTTTTGACTGTATTGCTATTCTTGTCGG TTTAGTTGCTACGACGCTTGCCAAGATGCCTCTAAATTATGCTTACCCCT TTGGATTTGCAAAAATTGAGGCTCTTTCGGGTTTCACTAATGGTATTTTT TTAGTTTTGATTTCATTTTCTATCGTCGGCGAGGCATTATATAGGTTATT TCATCCGCCCCAAATGAATACCGACCAATTGTTGTTGGTTAGTTTTTTGG GCCTTGTTGTGAATTTGGTAGGTATCCTAGCGTTCAATCATGGGCATAAT CATGATCATGGGTCTCATCACCATCATTCCCATAGTAATCATAGTATGTG TCTGCCTAACACTACAAATGATATAAATATTTTTGAAGAGTTTGAAGAAG AAAAAGATAATGTTGAAGCCCAGAAAATGGGCTATACGAATGACGATCAC GTATCCCAACATGAACATACCCATGAGAATAGTCAGGAACATCACCATGA GCATAACCACAATCATGATCACATCCATAAATACAATGAAAAATGCGACC ATGAAAGCATAAGTCTCCAGAATTTAGACAATGATCATCACTGTCATCAT CACCATGAAAATCATAATATGCATGGCATATTTCTGCATATTATCGCAGA TACTATGGGCTCTGTTGGAGTTATTGTCTCTACTATATTAATACAGTGGT TTTCATGGACCGGTTTTGATCCTTCGGCATCTCTAATAATTGCTGCATTA ATATTTGTTTCTGTACTTCCATTAATTAAAGATTCGGCGAAGAATTTGCT CTCTGTGACTGATCCAGAATCGGAATATTTATTGAAGCAGTGTTTGTCGA ACATCAGTTTAAGTCACTCCGTTGTCAGTTTATCCAACCCTAAGTTCTGG ACAAACGAAAGAGGTGAAGTGTATGGAATACTCCATATTCAGGTGAGCAT AGACGGTGATTTAAACGTGGTTCGTAATGAAGTATTTAGGAAGCTCTCAA TCGCTGTACCAAATTTAAAACACATTTGTATACAATCTGAACGGCCAAAC AATTGCTGGTGTGGAAAATAGTTCTTACATCAGTTGATATCCATACTTAT TTACGTGTAATTTTAATTAGATGAATTAATATTTTCTTTATTAGC DNA sequence for aes4 factor (Seq ID No 4) TTTACTTTAGTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAG CTTTTTTTTTAAGAGATATAACATATGTCAACGCGTCATTGATTAACTAC ATAACACGCCAATTATAAACTTCTCCCAAAAGAACTTAAGAATTTCCATT TTCAATCCAGATGAATTTATTTAAGAGACGAACAGTAGCGGCAGCAGCCT TAGCACGCTTAGCGAGCAAAGCTTGGGTCTTACCACCCATGATACCCACC ACCCCACTTACGACGGGCTTTCGTCGTACTTAGCAGAGAAGTTAGCATCA ACGGCGGAGACAATAGAAGCGAGTTCGTTCTTGTCTTCCTCACGGACCTC AGTGACAGCTAAAACAGCAGCAGTCTTTTGGTGAATGACAGTACCAAGGC GGGCCTTGTTCTTGACAATGGCATAAGGAACACCCATCTTCTTGCACAAA GCAGGCAAGAAAACGACGAGTTCAATGGGGTCGACATCGCTGGCAATGAG AACCAACTTAGCCTTCTTGGCCTCAATGAGAGCTACAACATGGTTCAAAC CATATTTAACATTGTAAGGCTTCTTAGAGACGTCTTGAGCAGACTTGCCG TTGGCAACAGCCTCGGCTTCAGCAACCAAACGTTGCTTCTTTTCAGCAGC AGTCTCAGGACGGTACTTGTTAAGCAACTTGAAGACCTGAGTAGCAGTGT TTTTGTCCAAAGTCTTCTGGAACTGAGCAATGGCAGGAGGAACCTTCAAA CGCAAGTTCAAAATCTTGCGACGGCGTTGAAGGCGGATATACTCAGGCCA CTTAACAAAACGGCTCAAGTCACGCTTAGGTTGGATGTCTTGTCCCCCGG GTAAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCGTTCGAAT AAGGCC Example 9 Effect of Novel RNAi Factors on Antisense RNA and dsRNA-Mediated Gene Regulation With recent studies on PTGS suggesting that antisense RNA, co-suppression, and dsRNA-mediated interference may share similar mechanisms, we wanted to determine whether over-expression of an aes factor would also enhance dsRNA-mediated regulation.","Having demonstrated that dsRNA could mediate gene suppression in this fission yeast model, the effect of an antisense enhancing sequence on dsRNA-mediated regulation was tested.","To this end, the aes2 gene was co-expressed with the lacZ panhandle construct in a yeast strain containing the lacZ target gene.","Under these conditions, this transformant displayed an additional 30% suppression of β-galactosidase activity when compared to the transformant expressing only the panhandle lacZ RNA (see FIG.","10).","This result shows that the aes2 gene, encoding the two domains of the mitochondrial elongation factor EF Tu, stimulated not only antisense RNA but also dsRNA-mediated gene inhibition.","Further it was shown that these two forms of regulation are related by over-expressing the ATP-dependent RNA helicase ded1 in the presence of lacZ antisense RNA and showing that this helicase enhanced gene suppression by a further 50% compared to the control strain (example 6).","ded1 was tested on both active and inactive antisense plasmids and demonstrated that ded1 augmentation of gene silencing was dependent on an active antisense RNA (see FIG.","11).","This could be due to the absence of RNA duplex formation with the inactive antisense RNA and the consequent lack of a substrate for the RNA helicase.","The methods of the present invention have utility in demonstrating a range of RNAi efficacies, in identifying new factors which enhance or reduce gene silencing, in inhibiting gene expression or increasing sensitivity to antisense inhibition of gene expression, in the treatment or prevention of disorders which require inhibition or down-regulation of gene expression.","Although the present invention has been described with reference to specific examples and preferred embodiments it will be clear to those skilled in the art that variations and modifications which do not depart from the concept and the spirit of the invention described herein are also contemplated as being within the scope of the present invention.","REFERENCES 1.Raponi, M., Atkins, D., Dawes, I.","& Arndt, G. (2000) Antisense and Nucleic Acid Drug Development 10, 29-34.2.Arndt, G., Atkins, D., Patrikakis, M. & Izant, J.","(1995) Molecular and General Genetics 248, 293-300.3.Arndt, G., Patrikakis, M. & Atkins, D. (2000) Nucleic Acids Research 28, e15.4.Clarke, M., Patrikakis, M. & Atkins, D. (2000) Biochemical and Biophysical Research Communications 268, 8-13.5.Moreno, S. & Nurse, P. (1994) Nature 367, 236-242.6.Moreno, S., Klar, A.","& Nurse, P. (1991) Methods in Enzymology 194, 795-823.7.Maundrell, K. (1990) Journal of Biological Chemistry 265, 10857-10864.8.Prentice, H. (1992) Nucleic Acids Research 20, 621.9.Hoffman, C. & Winston, F. (1987) Gene 57, 267-272.10.Rose, M., Winston, F. & Hieter, P. (1990) Methods in yeast genetics: A laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor).","11.Maundrell, K. (1993) Gene 123, 127-1390.12.Basi, G., Schmid, E. & Maundrell, K. (1993) Gene 123, 131-136.13.Carr, A., MacNeill, S., Hayles, J.","& Nurse, P. (1989) Molecular and General Genetics 218, 41-49.14.Sambrook, J., Fritsch, E. & Maniatis, T. (1989) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).","15.Patrikakis, M., Izant, J.","& Atkins, D. (1996) Current Genetics 30, 151-158.16.Brun, C., Dubey, D. & Huberman, J.","(1995) Gene 164, 173-177.17.Heyer, W.-D., Sipiczki, M. & Kohli, J.","(1986) Molecular and Cellular Biology 6, 80-89."]],"string":"[\n [\n \"METHODS FOR MEDIATING GENE SUPPRESSION\",\n \"The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.\",\n \"The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.\",\n \"It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs.\"\n ],\n [\n \"1.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"2.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"3.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"4.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"5.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"6.A method according to any one of claims 1 to 5, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.\",\n \"7.A method according to any one of claims 1 to 6, wherein the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.\",\n \"8.A method according to any one of claims 1 to 7, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"9.A method according to any one of claims 1 to 8, wherein the factor is selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"10.A method according to any one of claims 1 to 9, wherein the RNAi factor is an res sequence.\",\n \"11.A method according to claim 10, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"12.A method according to claim 11, wherein the res sequence is obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.13.A method according to claim 11, wherein the res sequence is represented by Seq ID Nos 1 to 4.14.A method according to any one of claims 1 to 13, wherein the cell is a eukaryotic cell.\",\n \"15.A method according to 14, wherein the eukaryotic cell is a mammalian.\",\n \"16.A method according to claim 1 or claim 2, wherein the cell is a Schizosaccharomyces pombe cell.\",\n \"17.A method according to any one of claims 1 to 16, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.\",\n \"18.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"19.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"20.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"21.A cell according to claim 20, wherein the cell is a eukaryotic cell.\",\n \"22.A cell according to claim 20 or claim 21, wherein the cell is a Schizosaccharomyces pombe cell.\",\n \"23.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.\",\n \"24.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"25.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"26.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"27.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"28.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"29.A method according to any one of claims 23 to 28, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.\",\n \"30.A method according to any one of claims 23 to 29, wherein the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.\",\n \"31.A method according to any one of claims 23 to 30, wherein the cell is a eukaryotic cell.\",\n \"32.A method according to 31, wherein the eukaryotic cell is a mammalian.\",\n \"33.A method according to claim 23 or claim 24, wherein the cell is a Schizosaccharomyces pombe cell.\",\n \"34.A method according to any one of claims 23 to 33, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.\",\n \"35.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"36.A cell according to claim 35, wherein the cell is a eukaryotic cell.\",\n \"37.A cell according to claim 35 or claim 36, wherein the cell is a Schizosaccharomyces pombe cell.\",\n \"38.Method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\",\n \"39.A factor identified by the method of claim 38.40.A factor according to claim 39, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"41.A factor according to claim 39 or claim 40, wherein the factor is selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"42.A factor according to any one of claims 39 to 41, wherein the factor is an res sequence.\",\n \"43.A factor according to claim 42, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"44.An RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.45.An RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.46.A Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.\",\n \"Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.\",\n \"It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.\",\n \"One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.\",\n \"The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.\",\n \"Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.\",\n \"The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.\",\n \"These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.\",\n \"The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.\",\n \"The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.\",\n \"Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.\",\n \"The second strategy for finding key players in RNAi has involved the use of cell free assays.\",\n \"These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.\",\n \"However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.\",\n \"Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.\",\n \"This would enable the identification of factors which can enhance or reduce the gene silencing effect.\",\n \"It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative.\"\n ],\n [\n \" SUMMARY OF THE INVENTION Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.\",\n \"Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.\",\n \"This is indicative of an RNAi-like mechanism of gene suppression.\",\n \"It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.\",\n \"This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.\",\n \"The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.\",\n \"The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.\",\n \"According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.\",\n \"Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.\",\n \"It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.\",\n \"Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"Even more preferred is an RNAi factor which is an res sequence.\",\n \"Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.\",\n \"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.\",\n \"According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.\",\n \"As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.\",\n \"According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.\",\n \"Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.\",\n \"Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.\",\n \"Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.\",\n \"However it will be understood that the entire native sequence of the target nucleic acid may be employed.\",\n \"Conveniently, the target nucleic acid may be coupled to a selectable marker.\",\n \"The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.\",\n \"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.\",\n \"According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.\",\n \"According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\",\n \"According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.\",\n \"The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"Even more preferred is a factor having an res sequence.\",\n \"Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\",\n \"The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.\",\n \"The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.\",\n \"This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).\",\n \"The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).\",\n \"It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.\",\n \"The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.\",\n \"It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.\",\n \"The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.\",\n \"The effective concentration range can be determined using routine methodology known in the art.\",\n \"Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.\",\n \"Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.\",\n \"The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics.\"\n ],\n [\n \"TECHNICAL FIELD The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.\",\n \"The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.\",\n \"It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs.\",\n \"BACKGROUND OF THE INVENTION The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.\",\n \"Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.\",\n \"It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.\",\n \"One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.\",\n \"The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.\",\n \"Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.\",\n \"The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.\",\n \"These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.\",\n \"The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.\",\n \"The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.\",\n \"Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.\",\n \"The second strategy for finding key players in RNAi has involved the use of cell free assays.\",\n \"These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.\",\n \"However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.\",\n \"Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.\",\n \"This would enable the identification of factors which can enhance or reduce the gene silencing effect.\",\n \"It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative.\",\n \"SUMMARY OF THE INVENTION Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.\",\n \"Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.\",\n \"This is indicative of an RNAi-like mechanism of gene suppression.\",\n \"It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.\",\n \"This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.\",\n \"The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.\",\n \"The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.\",\n \"According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.\",\n \"Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.\",\n \"It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.\",\n \"Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"Even more preferred is an RNAi factor which is an res sequence.\",\n \"Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.\",\n \"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.\",\n \"According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.\",\n \"According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.\",\n \"As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.\",\n \"According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.\",\n \"According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.\",\n \"According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.\",\n \"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.\",\n \"Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.\",\n \"Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.\",\n \"Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.\",\n \"However it will be understood that the entire native sequence of the target nucleic acid may be employed.\",\n \"Conveniently, the target nucleic acid may be coupled to a selectable marker.\",\n \"The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.\",\n \"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.\",\n \"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.\",\n \"According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.\",\n \"The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.\",\n \"According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\",\n \"According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.\",\n \"The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.\",\n \"More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.\",\n \"Even more preferred is a factor having an res sequence.\",\n \"Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.\",\n \"According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.\",\n \"The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.\",\n \"The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.\",\n \"This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).\",\n \"The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).\",\n \"It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.\",\n \"The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.\",\n \"It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.\",\n \"The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.\",\n \"The effective concentration range can be determined using routine methodology known in the art.\",\n \"Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.\",\n \"Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.\",\n \"The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics.\",\n \"BRIEF DESCRIPTION OF THE FIGURES FIG.\",\n \"1.Target genes and antisense fragments.\",\n \"a, The strain KC4-6 expresses low levels of lacZ RNA.\",\n \"The lacZ expression cassette is integrated at the ura4 locus on chromosome III and is composed of the SV40 early promoter, the E. coli lacZ gene and the SV40 3′ processing signal.\",\n \"The 5′ and 3′ flanking DNA sequences of the ura4 locus are as indicated.\",\n \"b, The strain RB3-2, which expresses high levels of lacZ RNA, contains an expression cassette containing the S. pombe adh1 promoter, the lacZ coding region, and the S. pombe ura4 3′ processing signal.\",\n \"c, The full-length 3.5 kb antisense lacZ fragment is shown.\",\n \"The crippled sense fragment was generated by end-filling the ClaI cut lacZ vector and religating on itself.\",\n \"d, The AML1 strain contains a c-myc-lacZ fusion cassette integrated at the ura4 locus.\",\n \"Exons 2 and 3 of the human c-myc gene were employed as a target.\",\n \"The relative position of the c-myc antisense fragment is shown.\",\n \"Transcription initiation sites are indicated by the bent arrows.\",\n \"The straight arrows represent the normal direction of transcription for a particular DNA fragment.\",\n \"FIG.\",\n \"2.dsRNA-mediated gene silencing in fission yeast.\",\n \"a, A fission yeast strain containing the integrated lacZ gene under control of the strong adh1 promoter was transformed with lacZ antisense vectors.\",\n \"The antisense gene was under control of the weak nmt41 promoter (low antisense), the strong nmt1 promoter in single copy (15) (medium antisense), or the nmt1 promoter in multi-copy (high antisense).\",\n \"For the strain expressing maximum antisense RNA two episomal nmt1-driven antisense vectors containing different selectable markers were co-transformed into the target strain.\",\n \"b, A fission yeast strain containing the integrated lacZ gene under control of the weak SV40 promoter (low target) or strong adh1 promoter (high target) was transformed with the antisense lacZ vector or antisense lacZ and sense lacZ vectors.\",\n \"Strains were co-transformed with vectors containing appropriate selectable markers to complement auxotrophy.\",\n \"c, The lacZ inverted repeat vector was expressed in the high-expressing lacZ target strain.\",\n \"The strain expressing the antisense construct only is also indicated.\",\n \"d, A fission yeast strain containing an integrated c-myc-lacZ fusion gene was transformed with the sense construct, the antisense construct, or both.\",\n \"Antisense and sense constructs were also expressed in the high-expressing lacZ strain.\",\n \"All strains were transformed with appropriate control plasmids to complement auxotrophy.\",\n \"FIG.\",\n \"3.Effect of co-expressing single copies of sense and antisense genes.\",\n \"Strains containing target lacZ alone (RB3-2), the target and a single copy of the antisense gene (K40-7), and the target and both the antisense and frameshifted lacZ genes (M62-1) were assayed for β-galactosidase activity.\",\n \"pREP2 and pREP4 are parental plasmids containing the LEU2 and ura4 selectable markers respectively.\",\n \"Strains were transformed with the appropriate control plasmids to complement auxotrophy.\",\n \"For each sample three independent colonies were assayed in triplicate.\",\n \"FIG.\",\n \"4.RNA expression profiles in fission yeast strains a, The target strain RB3-2 was transformed with the lacZ inverted repeat vector (pM53-1) or co-transformed with antisense (pGT2) and sense (pM54-3) vectors and grown to mid-log in the absence of thiamine.\",\n \"Total RNA from each strain was separated on a 1% denaturing agarose gel, transferred to a nylon membrane and probed with the radioactively labeled 2.2 kb nmt1 fragment.\",\n \"The episomal lacZ signal was normalized with the endogenous nmt1 transcript and quantitated by phosphorimage analysis.\",\n \"The relative level of episomal lacZ expression is shown.\",\n \"b, The strain M38-1 which contains a single copy stably integrated lacZ inverted repeat gene was grown to mid-log in the absence of thiamine, its RNA isolated and analyzed by Northern hybridization.\",\n \"The relative steady-state level of the “panhandle” lacZ RNA is indicated in comparison to episomally expressed antisense lacZ RNA.\",\n \"FIG.\",\n \"5.In vivo dsRNA assay.\",\n \"a, The constructs employed for assaying the ability of the lacZ inverted repeat to form an intramolecular RNA duplex are shown.\",\n \"Each vector contains a functional lacZ fragment which gives the transformed strain a blue color phenotype when grown in the presence of X-GAL.\",\n \"The predicted secondary structure of the pM81-2 generated transcript is indicated.\",\n \"b, The strain 2037 was transformed with the vectors pM85-1, pM91-1 and pM81-2 in the absence of thiamine Single colonies were streaked on minimal media plates and overlayed with 0.5% agarose medium containing 500 μg/ml X-gal and 0.01% SDS.\",\n \"FIG.\",\n \"6.The effect of ded1 on dsRNA-mediated gene regulation.\",\n \"a, The ded1 gene was co-expressed in a fission yeast strain containing the integrated lacZ gene.\",\n \"β-galactosidase activity of the strain transformed with and without the antisense lacZ vector is shown.\",\n \"b, The target strain was transformed with a vector expressing a short lacZ antisense gene or with both the short lacZ antisense vector and the ded1-expressing vector.\",\n \"Strains were transformed with appropriate control plasmids to complement auxotrophy.\",\n \"FIG.\",\n \"7.Over-expression of thi1 in an antisense lacZ expressing strain.\",\n \"β-galactosidase activity of the target strain RB3-2 transformed with and without the antisense lacZ vector is shown.\",\n \"Over-expression of the thi1 gene in the antisense expressing strain is also indicated.\",\n \"Strains were transformed with appropriate control plasmids to complement auxotrophy.\",\n \"FIG.\",\n \"8.Over-expression screening strategy for RNAi modulating factors.\",\n \"A target strain containing the integrated lacZ gene under control of the adh1 promoter and the episomal vector containing the nmt1-driven lacZ antisense gene is transformed with an S. pombe cDNA library.\",\n \"Library fragments are driven by the nmt1 promoter.\",\n \"Transformants are individually screened for a change in the lacZ-encoded blue colony colour phenotype.\",\n \"These transformants are then quantitatively assayed for β-galactosidase activity in the presence and absence of thiamine.\",\n \"The antisense vector is then segregated out of transformants showing a cDNA dependent modification of lacZ suppression.\",\n \"A tertiary β-galactosidase assay is performed to determine if the effect is dependent on the presence of dsRNA.\",\n \"cDNA vectors are recovered from strains of interest, sequenced, and subjected to BLASTN analysis.\",\n \"FIG.\",\n \"9.Over-expression screen of a S. pombe cDNA library.\",\n \"a, Transformants were grown on minimal media plates and over-layed with X-GAL-containing medium.\",\n \"Those which showed a reduced blue colour-phenotype (black arrow) were analysed further.\",\n \"Transformants demonstrating an enhanced blue-colour phenotype were also identified (white arrow).\",\n \"b, Transformants which showed a visual reduction in the blue phenotype were assayed for β-galactosidase activity in liquid culture in the absence of thiamine.\",\n \"Thiamine was added to the medium to demonstrate that the enhanced lacZ suppression was dependent on RNA expression.\",\n \"Transformants were again assayed for β-galactosidase activity following antisense vector segregation.\",\n \"c, Over-expression of unique res genes in dsRNA-expressing strains.\",\n \"FIG.\",\n \"10.lacZ panhandle-mediated gene silencing.\",\n \"(A) The lacZ panhandle construct contains the full-length crippled lacZ gene which is followed by the inverted 5′ 2.5 kb lacZ fragment.\",\n \"Intramolecular hybridization generates an RNA with 2.5 kb RNA duplex and a 1 kb loop.\",\n \"(B) The relative steady-state level of the episomally expressed panhandle lacZ RNA (7.0 kb) is shown in comparison to episomally expressed antisense lacZ RNA (4.5 kb).\",\n \"The lacZ signals were normalized to the endogenous nmt1 transcript (1.3 kb) and quantitated by phosphorimage analysis.\",\n \"(C) The target strain was transformed with the episomally expressed lacZ panhandle and analyzed for b-galactosidase activity.\",\n \"The appropriate plasmids were co-introduced to complement auxotrophy.\",\n \"At least three independent colonies were assayed in triplicate for each strain.\",\n \"Transformants were assayed in the presence of thiamine to abrogate expression of the panhandle RNA (hatched).\",\n \"(D) The target lacZ strain was transformed with the panhandle vector only or both the panhandle and aes2 vector.\",\n \"FIG.\",\n \"11.Co-expression of ded1 and lacZ antisense genes.\",\n \"b-galactosidase activity of the target strain co-transformed with the ded1 gene and the antisense lacZ vectors is shown.\",\n \"Ded1 was expressed from the ura4-based plasmid pREP4 while the antisense genes were expressed from the LEU2-based plasmid pREP2.Strains expressing antisense RNAs or ded1 RNA alone are also indicated.\",\n \"Strains were transformed with appropriate control plasmids to complement auxotrophy.\",\n \"Three independent colonies were assayed in triplicate for each strain.\",\n \"FIG.\",\n \"12.DNA sequence for aes1 factor FIG.\",\n \"13.DNA sequence for aes2 factor FIG.\",\n \"14.DNA sequence for aes3 factor FIG.\",\n \"15.DNA sequence for aes4 factor DESCRIPTION OF THE PREFERRED EMBODIMENTS Using the lacZ fission yeast model to investigate features of antisense-RNA technology in vivo (1-4), it has been shown that the degree of target gene suppression is dependent on the level of antisense RNA (FIG.\",\n \"2A)(1).\",\n \"During the course of developing this model it was surprisingly found that by increasing the steady-state levels of the lacZ target mRNA 20-fold in the presence of the same pool of antisense RNA that antisense-mediated suppression was not only maintained, but enhanced (FIG.\",\n \"2B).\",\n \"To verify that this effect was independent of the target mRNA a lacZ gene containing a frameshift mutation (leading to an inability to translate the β-galactosidase enzyme) was co-expressed in antisense-expressing strains.\",\n \"Again, additional stimulation of lacZ inhibition was observed (FIG.\",\n \"2B).\",\n \"These observations are consistent with the potential formation of additional dsRNA and consequent stimulation of gene silencing.\",\n \"They also indicate that the dsRNA-mediated gene silencing is dose-dependent in this system.\",\n \"To further show that dsRNA was the key component in target gene inhibition in fission yeast dsRNA was generated by expressing a lacZ inverted repeat with 2.5 kb of internal complementarity in the presence of lacZ target mRNA.\",\n \"Expression of this construct also reduced β-galactosidase activity in a lacZ expressing strain to a similar level to the full-length antisense RNA (FIG.\",\n \"2C).\",\n \"Overall, these data suggested that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, resulted in efficient interference of target gene expression in S. pombe.\",\n \"To test the ability of dsRNA to specifically interfere with another target sequence in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (3).\",\n \"A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene was previously found to suppress β-galactosidase activity within the c-myc-lacZ fusion target strain by 47% (3).\",\n \"β-galactosidase assays demonstrated that co-expression of the antisense and sense c-myc constructs in the target strain enhanced c-myc suppression compared with the antisense c-myc vector alone (FIG.\",\n \"2D).\",\n \"Transformation of the strain expressing only the lacZ target with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is sequence-specific (FIG.\",\n \"2D).\",\n \"All of the above observations, as well as the reduction in target mRNA levels in response to additional dsRNA (data not shown), are consistent with features associated with RNAi.\",\n \"This is the first example of dsRNA-mediated gene regulation in yeast and, unlike RNAi in all other organisms except zebrafish, appears to be dependent on the concentration of intracellular dsRNA.\",\n \"The variation in RNAi efficacy between organisms may be due to the absence or under-expression of some components of the RNAi pathway or the presence of RNAi inhibitors.\",\n \"We have now further developed this genetic model for identification of factors involved in RNAi.\",\n \"Without wishing to be bound by any particular mechanism, it is probable that a multi-protein complex exists which mediates the post-transcriptional degradation of target mRNA in RNAi.\",\n \"Several genes which are involved in the RNAi phenomenon have been identified through genetic screens in Neurospora and nematodes.\",\n \"These genes include an RNA-dependent RNA polymerase (qde-1, ego-1) a RecQ DNA helicase (qde-3), an RNase D homologue (mut-7), and a putative translation initiation factor (rde-1, qde-2).\",\n \"The genes mut-2, rde-2, rde-4, and rde-7 have also been identified as being involved in either the initiation or maintenance of RNAi.\",\n \"Additionally, Drosophila cell free assays have shown that RNAi is mediated by nuclease degradation of the targeted mRNA while a 21-25 nt RNA species appears to be integral to specific post-transcriptional genetic interference.\",\n \"RNAi has also been shown to be dependent on ATP which may be required for strand dissociation of dsRNA.\",\n \"However, a missing component of the proposed multi-protein complex in the RNAi pathway is the implied RNA helicase.\",\n \"The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.\",\n \"In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.\",\n \"Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.\",\n \"The first gene that was tested in mediating RNAi activity in the present model was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.\",\n \"As we have previously shown that antisense RNA-mediated gene suppression is dose dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.\",\n \"The thi1 open reading frame was PCR amplified and subcloned into pREP4 as a BamHI fragment.\",\n \"This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.\",\n \"As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.\",\n \"7).\",\n \"This result indicated that host-encoded factors are present which affect the robustness of dsRNA-mediated gene suppression in fission yeast and also validated the overexpression strategy.\",\n \"The second gene investigated has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.\",\n \"According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.\",\n \"It was therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.\",\n \"Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.\",\n \"6A).\",\n \"When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.\",\n \"6A).\",\n \"The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (16).\",\n \"Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.\",\n \"6B).\",\n \"It was thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.\",\n \"By over-expressing a cDNA library in a dsRNA fission yeast model we have identified four novel, genes with potential roles in the RNAi pathway.\",\n \"All of the known genes identified are essential and have natural associations with DNA, RNA or nucleic acid binding proteins, consistent with the expectation of RNAi cellular factors.\",\n \"In addition, two of the three known cDNAs encoded proteins involved in the process of translation.\",\n \"These findings along with our previous identification of an ATP-dependent RNA helicase (Raponi & Arndt, submitted) and reports of the involvement of a translation initiation factor in RNAi and the co-purification of ribonuclease activity with ribosomal fractions, suggests that this may be one site at which RNAi functions.\",\n \"Alternatively, the identification of such varied proteins as DNA helicases and translational components as part of the RNAi machinery implies that particular cellular proteins may be recruited into more than one multiprotein complex.\",\n \"Under the latter conditions, over-expression of these specific proteins may result in the generation of an RNAi complex that recognises dsRNA and mediates target gene suppression.\",\n \"Certain of these proteins may be rate-limiting or rate-determining in RNAi and only through supplementation of these factors are their roles in RNAi uncovered.\",\n \"It is proposed, without wishing to be bound by any particular mechanism, that in addition to the previously identified core proteins in the RNAi multiprotein complex(es), additional factors such as EF Tu, L7a, sna41 and res1 may be enlisted for a role in dsRNA-mediated gene suppression.\",\n \"The over-expression strategy described overcomes some limitations associated with mutagenesis by identifying essential genes with a role in RNAi.\",\n \"In addition, this strategy complements these other systems by allowing the isolation of cellular factors that modify the efficacy of RNAi in vivo.\",\n \"The roles of these modulators of RNAi may be varied and include recognition and amplification of the dsRNA, delivery of the small 21-25 nt dsRNAs to the target mRNA, association between the antisense and target mRNA strands, and RNAi complex formation.\",\n \"Alternatively, these modulators may control the rate of RNAi or the formation of different complexes within cell types or for different forms of post-transcriptional gene silencing.\",\n \"The fact that over-expression of specific RNAi modulators enhanced dsRNA-mediated gene regulation in fission yeast indicates that a similar approach could be used to identify RNAi modulators in other organisms.\",\n \"In addition, the co-expression of these factors with different forms of post-transcriptional gene silencing including co-suppression, quelling, and antisense RNA could be one way of enhancing the efficacy of these methods.\",\n \"This may be especially important for application of RNAi to mammalian cells and tissues or to genes which have been somewhat recalcitrant to this form of regulation.\",\n \"The present invention demonstrates for the first time the intrinsic involvement of an ATP-dependent RNA helicase as a key component in RNAi.\",\n \"Further, it can be rate limiting, as the over-expression leads to increased RNAi activity in this system.\",\n \"This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.\",\n \"These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.\",\n \"The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.\",\n \"The present invention also provides for the first time a novel and quantitative genetic system based on S.pombe, for rapidly identifying essential cellular factors involved in RNAi.\",\n \"The use of this model has enabled verification of RNA helicase activity as a critical contributor to efficient RNAi activity and isolated novel RNAi factors including EF Tu, L7a, sna41, and an unidentified gene res1.The present invention also demonstrates that gene silencing may be enhanced by concomitant expression of such RNAi factors.\",\n \"The invention will now be described more particularly with reference to non-limiting examples of certain preferred embodiments of the invention.\",\n \"Example 1 Materials and Methods Used to Exemplify the Invention S. pombe Media and Manipulations.\",\n \"All yeast strains were maintained on standard YES or EMM media (6).\",\n \"Repression of nmt1 transcription was achieved by the addition of thiamine to EMM media at a final concentration of 4 μM (7).\",\n \"Yeast cells were transformed with plasmid DNA by electroporation (8) and stable integrants were identified as previously described (6).\",\n \"A glass bead procedure (9) was used to isolate genomic DNA which was used for Southern analysis and PCR diagnosis.\",\n \"Total RNA was extracted as previously described (10).\",\n \"S. pombe Strain and Plasmid Construction.\",\n \"Construction of the low expressing lacZ strain, KC4-6 (h−, ura4::SV40-lacZ, leu1-32), has been previously described (2).\",\n \"The strain RB3-2 (h−, ura4::adh1-lacZ, leu1-32) which expresses higher levels β-galactosidase has also been previously described (1).\",\n \"The target strain which contains the c-myc:lacZ fusion has previously been described (3).\",\n \"The construction of the long lacZ antisense containing episomal plasmid, pGT2, and corresponding control plasmids have been described (2).\",\n \"Plasmid pREP4-As was generated by subcloning the lacZ BamHI fragment contained in pGT2 into the plasmid pREP4 (11).\",\n \"pREP4 is identical to pREP1 except that the S. cerevisiae LEU2 gene has been replaced with the S. pombe ura4 selectable marker.\",\n \"To decrease the steady-state level of episomally expressed antisense lacZ, plasmids pREP42-As and pREP82-As were constructed by subcloning the lacZ BamHI fragment from pGT2 into pREP42 and pREP82, respectively.\",\n \"These plasmids are derivatives of pREP4 with mutations in the TATA box of the nmt1 promoter (12).\",\n \"The crippled lacZ vector, pGT62, was generated by end-filling the ClaI site of pGT2 and re-ligating (2).\",\n \"This frameshifted fragment was then subcloned into the BamHI site of pREP4 to generate the plasmid pM54-3.The lacZ panhandle integration vector, pM30-8, was generated by first introducing a NotI site into the XmaI site of pRIP1/s (11) using the self-complementary linker 5′ CCG GGC GGC CGC 3′ to generate pL121-14.The 2.5 kb sequence of the 5′ end of the frameshifted lacZ gene (2) was then PCR-amplified to give it NotI ends using the forward primer 5′ ATGCGGCCGCAATTCCCGGGGATCGAAAGA 3′ and reverse primer 5′ ATGCGGCCGCAATGGGGTCGCTTCACTTA 3′.\",\n \"This product was cloned using a TA cloning kit (TOPO: Invitrogen, San Diego, Calif., USA) and then subcloned into the NotI site of pL121-14 in the antisense orientation.\",\n \"The integrating vector was introduced into target strains in single copy by using the sup3-5/ade6-704 complementation system (13).\",\n \"The full-length frameshifted lacZ fragment was then introduced into the BamHI site of this vector in the sense orientation upstream of the 2.5 kb antisense fragment to generate pM30-8.The episomal version of this vector (pM53-1) was made by removing the Pst1 sup3-5 fragment and introducing the autonomous replicating sequence as an EcoRI fragment (11).\",\n \"For testing the ability of the panhandle construct to form dsRNA in vivo, the frameshifted lacZ fragment was replaced with the functional lacZ fragment in the vectors pM54-3 and pM53-1, to generate the vectors pM85-1 and pM81-2, respectively.\",\n \"The vector pM91-1, which is unable to form a lacZ panhandle transcript, was generated by removing the 2.5 kb NotI lacZ fragment from pM81-2 and reintroducing it in the sense orientation.\",\n \"Generation of the c-myc antisense construct, pCM-17, has been described elsewhere (3).\",\n \"The 792 by BglII c-myc fragment from pCM-17 was subcloned into the BamHI site of pREP4 in the sense orientation to generate pN12-1.The ded1, and thi1, open reading frames were amplified from fission yeast genomic DNA (strain 1913).\",\n \"ded1 was amplified to give it BamHI ends using the forward primer 5′ ATGGGATCCCAACCAAACACTTCAACTCAG 3′ and the reverse primer 5′ ATGGGATCCTCAGAAGCCTGTGCATAACAC 3′.\",\n \"thi1 was amplified to give it BglII ends using the forward primer 5′ ATGAGATCTGTGGTTGGTATTCTAGAGAGA 3′ and the reverse primer 5′ ATGAGATCTAACAAAGACCTGCAAAAAACC 3′.\",\n \"PCR products were purified (Qiagen PCR purification kit), digested with either BamHI or BglII, gel-purified (Qiagen), and subcloned into the BamHI site of pREP4 in the sense orientation.\",\n \"Southern and Northern Analysis.\",\n \"Nucleic acid electrophoresis and membrane transfer was performed as described (14).\",\n \"Southern and Northern blots were hybridized using ExpressHyb solution according to the manufacturer's instructions (Clontech Laboratories).\",\n \"DNA probes were 32P-labelled using the Megaprime labelling kit (Amersham).\",\n \"Probes included a 960 bp BamHI/ClaI lacZ fragment from pI2-1 (1), a 570 by HindIII/EcoRI ura4-3′ fragment from pGT113 (15), and a 2.2 kb PstI/SacI nmt1 fragment from pRIP1/s.\",\n \"Radioactive signals were detected by autoradiography and quantitated by phosphorimage analysis (ImageQuant; Molecular Dynamics).\",\n \"Plasmid Distribution Analysis.\",\n \"Plasmid co-transformants of strain RB3-2 were grown under selective conditions to a cell density of 1-2×107 cells/ml.\",\n \"A serial dilution of each culture was performed and cells plated for single colonies in triplicate onto each of YES, EMM, EMM+leucine, and EMM+uracil agar media.\",\n \"The number of colonies grown on YES was taken as the total number of viable cells, while colonies growing on EMM represented the cells in the sampled population that contained both the ura4-containing (pREP4-based) and the LEU2-containing (pREP1-based) plasmids.\",\n \"Cells containing either the ura4-containing plasmid or the LEU2-containing plasmid were identified from the EMM+leucine and EMM+uracil plates respectively.\",\n \"The ratio of the number of colonies grown on selective media to the total number of viable colonies was used as the quantitative measure of the proportion of cells in the population, grown under selection, which contained plasmids.\",\n \"β-galactosidase Assays.\",\n \"The expression of the lacZ gene-encoded product, β-galactosidase, was quantitated using a cell permeabilization protocol as previously described (Raponi et al., 2000).\",\n \"A semi-quantitative overlay assay was also employed for rapid screening of yeast transformants (3).\",\n \"Isolation of S. pombe cDNA Clones That Alter Antisense RNA Efficacy.\",\n \"The S. pombe cDNA library was originally constructed in pREP3Xho by Bruce Edgar and Chris Norbury (5).\",\n \"The vector pREP3Xho is derived from pREP3 which contains the LEU2 marker and inserts are under control of the nmt1 promoter (11).\",\n \"A total of 5 μg of library DNA was transformed into the strain RB3-2 containing the episomal antisense lacZ vector pREP4-lacZAS and grown in EMM liquid media to the mid-logarithmic phase.\",\n \"Transformants were then plated on EMM solid media and grown at 30° C. for 3 days.\",\n \"Colonies were over-layed with medium containing 0.5 M sodium phosphate, 0.5% agarose, 2% dimethylformamide, 0.01% SDS, and 500 μg/ml X-GAL (Progen, Australia).\",\n \"Plates were then incubated at 37° C. for 3 hrs, colonies of interest recovered and assayed for β-galactosidase activity.\",\n \"Plasmid Segregation.\",\n \"Strains were plated on EMM containing limiting uracil and 1 mg/ml 5-fluoroorotic acid (6).\",\n \"Strains were then replica plated on both selective and non-selective media.\",\n \"Those colonies that did not grow on selective media were identified as having lost the ura4-containing antisense plasmid.\",\n \"To determine the mitotic stability of pREP-based plasmids we used the plasmid segregation method previously described (16).\",\n \"This method indicated that up to 30% of the cells grown in selective media did not contain the resident plasmid.\",\n \"Example 2 Effect of Steady-State Level of Target mRNA and Antisense RNA on Antisense Efficacy To determine the role of target mRNA steady-state levels in antisense-mediated gene suppression, we investigated the ability of a long lacZ antisense RNA (2) to regulate lacZ target genes under control of both weak and strong constitutive promoters.\",\n \"The low-level expressing strain, KC4-6, contained the lacZ gene driven by the SV40 early promoter integrated at the ura4 locus in chromosome III (FIG.\",\n \"1A; (2)).\",\n \"The high-level lacZ expressing strain, RB3-2, was constructed by placing the lacZ gene under control of the strong fission yeast adh1 promoter and the 3′ processing signal from the ura4 gene and integrating this expression cassette at the ura4 locus (FIG.\",\n \"1B; (1)).\",\n \"Characterization of both strains indicated that RB3-2 expressed 20-fold more lacZ mRNA than KC4-6 while an approximate 40-fold increase in β-galactosidase was also detected (data not shown).\",\n \"Both strains were transformed with the lacZ antisense gene-containing plasmid (pGT2) and its corresponding sense control plasmid (pGT62).\",\n \"β-galactosidase assays indicated that the antisense RNA suppressed β-galactosidase activity by 45% in KC4-6 while the same RNA reduced β-galactosidase activity by 52% in RB3-2 (FIG.\",\n \"2B).\",\n \"No reduction in β-galactosidase activity was seen in either strain when transformed with the control plasmid pGT62 which expresses a crippled version of lacZ.\",\n \"These results indicated that despite a 20-fold increase in the steady state level of the lacZ target mRNA in RB3-2 the efficacy of antisense-mediated down regulation was not only maintained, but enhanced.\",\n \"The increase in gene suppression demonstrated with strain RB3-2 was also seen when antisense molecules targeted to subregions of the lacZ target gene were used, but was not as pronounced.\",\n \"These data suggested an increase in target mRNA can result in enhanced target gene suppression.\",\n \"It has previously, been demonstrated that a stably integrated antisense lacZ gene acts in a dose dependent fashion with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number (1).\",\n \"Here the role of the steady-state level of episomally expressed antisense RNA in both the low lacZ expressing target strain, KC4-6, and the high lacZ expressing target strain, RB3-2, was investigated.\",\n \"The expression of lacZ antisense RNA was increased by co-transforming KC4-6 and RB3-2 with plasmids pGT2 and pREP4-As, each of which contains the lacZ antisense gene under control of the nmt1 promoter, but different selectable markers.\",\n \"To decrease the steady-state level of antisense RNA, the plasmids pREP42-As and pREP82-AS were employed.\",\n \"The nmt1 promoter in these vectors contain deletions in the TATA box sequence which affect the level of transcription, but have no impact on the site of transcription initiation or thiamine repressibility (12).\",\n \"Each of the different antisense gene-containing plasmids was co-transformed with a control plasmid to complement auxotrophy where appropriate.\",\n \"This resulted in a set of co-transformants of both RB3-2 and KC4-6 each containing the same lacZ antisense gene, but with different promoter capacities.\",\n \"Each co-transformant was analyzed for antisense RNA steady-state levels and β-galactosidase activity.\",\n \"Table 1 indicates that with both strains the degree of target suppression is enhanced with the increase of antisense lacZ RNA expression.\",\n \"TABLE 1 Effects of varying the level of antisense RNA in strains expressing both low and high level target.\",\n \"Relative β-gal.\",\n \"activity antisense Strain Plasmids Descriptions (units) % suppressiona RNA levelb KC4-6 pREP2, pREP4 Vector 1.07 0 0 pREP2, pREP82-AS Low As 1.10 0 0.04 pREP2, pREP42-AS Medium As 1.07 0.1 0.14 pREP2, pREP4-AS Full As-ura4 0.75 30 1.0 pGT2, pREP4 Full As-LEU2 0.70 34.6 2.2 pGT2, pREP4-AS Double-As 0.54 49.2 5.6 RB3-2 pREP2, pREP4 Vector 48.5 0 0 pREP2, pREP82-AS Low As 44.0 9.4 0.05 pREP2, pREP42-AS Medium As 43.9 9.6 0.18 pREP2, pREP4-AS Full As-ura4 27.2 43.9 1.0 pGT2, pREP4 Full As-LEU2 24.9 48.7 1.9 pGT2, pREP4-AS Double-As 16.6 65.8 5.4 aThe % suppression of β-galactosidase activity was determined by expressing each β-galactosidase activity as a percentage of the activity found in the pREP2, pREP4 co-transformant.\",\n \"bWithin each strain the steady-state level of long lacZ antisense RNA was normalized to nmt1 mRNA and then expressed relative to the level observed in the pREP2, pREP4-As co-transformant.\",\n \"These data agree with the previous report that antisense RNA-mediated gene inhibition is dose dependent in S. pombe (1).\",\n \"However, for each co-transformant the degree of β-galactosidase suppression was greater by 10-15% in the high lacZ expressing strain, RB3-2, than the low lacZ expressing strain, KC4-6, again demonstrating that low levels of target mRNA can limit antisense efficacy.\",\n \"Example 3 Effect of Increasing Sense RNA on Antisense RNA-Mediated Target Gene Suppression To determine whether the increase in gene suppression was due to formation of an antisense RNA:target mRNA hybrid or an antisense RNA:sense RNA hybrid, a version of lacZ which is unable to be translated into functional β-galactosidase was co-expressed in strains expressing the lacZ antisense gene.\",\n \"If antisense RNA was required to hybridize to target mRNA for inhibition of the gene expression pathway, then over-expression of the sense RNA would compete with the target for the available antisense molecules and a decrease in lacZ gene suppression would result.\",\n \"Initially, both antisense and sense lacZ vectors were integrated in single copy into separate target strains and then crossed with each other.\",\n \"In strains containing the antisense gene alone, β-galactosidase activity was reduced by approximately 40% (FIG.\",\n \"3; (1)) while there was no reduction in the strain expressing sense lacZ alone (data not shown).\",\n \"However, in the strain expressing both complementary transcripts (M62-1), β-galactosidase activity was reduced by 50% (FIG.\",\n \"3).\",\n \"These results suggested that increasing the intracellular concentration of sense RNA in the presence of antisense RNA, and therefore potentially generating more dsRNA, stimulates target gene suppression.\",\n \"To further increase the potential formation of intracellular dsRNA an episomal sense lacZ plasmid (pM54-3; FIG.\",\n \"2B) was co-transformed with the episomal antisense lacZ plasmid, pGT2, into the target strain R133-2.Both of these sequences were expressed by the strong conditional nmt1 promoter and RNA analysis showed that each transcript was being expressed at high levels when grown in the absence of thiamine (FIG.\",\n \"4A).\",\n \"When both RNAs were co-expressed in RB3-2, the target lacZ was suppressed by 65% compared to 50% in the strain expressing the antisense plasmid alone (FIG.\",\n \"2B).\",\n \"Northern analysis demonstrated that the total relative level of episomally expressed nmt1-driven transgenes was approximately 60% higher than when either vector was expressed alone (FIG.\",\n \"4A).\",\n \"Although expression of these complementary RNA transcripts is at a high level compared with single copy integrants only an additional 15% suppression was observed.\",\n \"One explanation for the absence of higher levels of target gene suppression is plasmid segregation.\",\n \"S. pombe undergoes asymmetric segregation, with the result that mitosis produces a daughter cell which lacks the segregating plasmid (17).\",\n \"Therefore, when the 65% suppression level seen in the total population is corrected for only those cells containing the ura4-based and LEU2-based plasmids the level of suppression approaches approximately 100% (data not shown).\",\n \"Overall these data suggest that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, is required for efficient interference of target gene expression in S. pombe.\",\n \"However, unlike the phenomenon of RNAi seen in plants and nematodes, the dsRNA-mediated gene suppression demonstrated in fission yeast seems to be dependent on the concentration of intracellular dsRNA or a threshold level of dsRNA is required to invoke potent gene silencing.\",\n \"Example 4 Effect of a lacZ Inverted Repeat on lacZ Gene Expression To further investigate the ability of dsRNA to inhibit gene expression in fission yeast we generated a vector containing the full-length 3.5 kb frameshifted lacZ sequence with a 2.5 kb inverted repeat.\",\n \"This construct generates a transcript of approximately 7 kb in length with 2.5 kb of self-complementarity which, predictably, will form a strong intramolecular RNA duplex.\",\n \"This gene was initially integrated into a fission yeast strain in single copy and then crossed with the strain RB3-2.The resulting strain which contained the single copy inverted repeat gene and target lacZ showed no reduction in β-galactosidase activity when transcription of the inverted repeat was activated.\",\n \"Southern analysis confirmed that the cassette was intact (data not shown) while RNA analysis indicated that the 7 kb transcript was being generated but approximately 10-fold less than episomally expressed antisense lacZ (FIG.\",\n \"4A&B).\",\n \"To increase the steady-state level of the inverted repeat RNA we generated the episomal plasmid pM53-1 containing this cassette.\",\n \"RNA analysis demonstrated that this vector is expressed at a similar level to episomally expressed antisense lacZ (FIG.\",\n \"4A).\",\n \"When expressed in RB3-2 pM53-1 inhibited β-galactosidase activity by 40% (FIG.\",\n \"2C), while addition of thiamine to the culture medium returned β-galactosidase activity to control level.\",\n \"To confirm that gene inhibition was due to this construct forming an RNA duplex, an in vivo assay for dsRNA was developed.\",\n \"To this end, a series of vectors were generated which contained functional lacZ sequences including lacZ alone (pM85-1), a lacZ inverted repeat (pM81-2), and the lacZ repeat with both sequences in the sense orientation (pM91-1) (FIG.\",\n \"5A).\",\n \"These vectors were then introduced into a strain lacking the integrated target sequence (NCYC2037; h+, ura4-D18) and resulting transformants were overlayed with X-GAL-containing agarose.\",\n \"Both strains containing control vectors generated high levels of β-galactosidase while the strain expressing the lacZ inverted repeat did not (FIG.\",\n \"5B).\",\n \"In each case Northern analysis demonstrated that strains were expressing the transgene.\",\n \"These results indicate that the inverted repeat RNA could not be efficiently translated to produce β-galactosidase, most probably due to the formation of a strong intramolecular hairpin structure.\",\n \"Taken together, these results show that a construct which has the ability to form dsRNA by intramolecular hybridization will interfere with target gene expression when expressed at high levels, again indicating that dsRNA mediates target gene suppression in a dose-dependent manner in fission yeast.\",\n \"Example 5 Gene-Specificity of dsRNA-Meditated Gene Silencing in Fission Yeast To test the ability of dsRNA to specifically interfere with other target sequences in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (FIG.\",\n \"1D; (3)).\",\n \"A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene (named CM-17) was previously found to suppress β-galactosidase activity within a c-myc-lacZ fusion target strain (AML1) by 47% (3).\",\n \"CM-17 was subsequently subcloned into pREP4 in the sense orientation to generate pN12-1.The antisense c-myc vector (pCM-17) and the sense c-myc vector were transformed into AML1 both independently and together.\",\n \"β-galactosidase assays demonstrated that co-expression of the antisense and sense constructs enhanced c-myc suppression by an additional 13% compared with the antisense c-myc vector alone (FIG.\",\n \"2D).\",\n \"Transformation of RB3-2 with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is gene-specific (FIG.\",\n \"2D).\",\n \"These results demonstrate that dsRNA can specifically interfere with multiple targets in fission yeast including one of human origin.\",\n \"Example 6 Over-Expression of a Translation Factor can Enhance RNAi in Fission Yeast The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.\",\n \"In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.\",\n \"Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.\",\n \"The first gene that we have tested in mediating RNAi activity in the present model has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.\",\n \"According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.\",\n \"We therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.\",\n \"Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.\",\n \"6A).\",\n \"When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.\",\n \"6A).\",\n \"The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (2).\",\n \"Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.\",\n \"6B).\",\n \"We have thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.\",\n \"Although an ATP-dependent RNA helicase has recently been suggested to be a key component in RNAi, this is the first demonstration of its intrinsic involvement.\",\n \"Further, it can clearly be rate limiting, as the over-expression leads to increased RNAi activity in this system.\",\n \"This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.\",\n \"These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.\",\n \"The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.\",\n \"Example 7 Over-Expression of a Transcription Factor can Enhance RNAi in Fission Yeast The second gene investigated was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.\",\n \"As we have previously shown that antisense RNA-mediated gene suppression is dose-dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.\",\n \"The thi1 open reading frame was PCR-amplified and subcloned into pREP4 as a BamHI fragment.\",\n \"This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.\",\n \"As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.\",\n \"7).\",\n \"This result indicated that host-encoded factors are present which modulate the efficacy of dsRNA-mediated gene suppression in fission yeast and also validated the over-expression strategy.\",\n \"Example 8 Screening for Novel RNAi Modulating Factors Over-expression of a fission yeast cDNA library (5) in an antisense lacZ expressing strain, revealed a series of transformants in which β-galactosidase activity was significantly reduced from that demonstrated in antisense RNA-expressing strains alone.\",\n \"The screening strategy is shown in FIG.\",\n \"8.Transformants were grown on selective plates and overlayed with X-GAL-containing media to generate a blue phenotype which could be analysed visually in a semi-quantitative manner (FIG.\",\n \"9A)(3).\",\n \"From 12,000 transformants screened, 48 were initially identified as having a reduced blue phenotype compared with background transformants.\",\n \"Of these, 25 demonstrated a reproducible enhancement in antisense RNA-mediated lacZ suppression when quantified by a liquid β-galactosidase assay (FIG.\",\n \"9B).\",\n \"To demonstrate that the observed effects were due to transcription of the cDNA insert, transformants were grown in the presence of thiamine.\",\n \"This repression of the nmt1 promoter resulted in a return to control levels of β-galactosidase activity in all transformants (FIG.\",\n \"9B).\",\n \"This indicated that the enhanced suppression was due to expression of the cDNA rather than other genetic events such as lacZ reporter mutations.\",\n \"To determine if the effect was dependent on the presence of dsRNA, the antisense plasmid was segregated out of the transformants and the strains were again assayed for β-galactosidase activity.\",\n \"Nine of the 25 transformants demonstrated a return to the level of β-galactosidase seen in the parental strain indicating that the effect was dsRNA-dependent (FIG.\",\n \"9B).\",\n \"The cDNAs being expressed in these transformants were named RNAi enhancing sequences (res).\",\n \"All transformants displayed normal growth phenotypes indicating that over-expression of the resident cDNAs did not affect general biology of the cell.\",\n \"In the absence of antisense RNA the remaining 16 transformants retained a reduced β-galactosidase activity indicating that the enhanced gene silencing was not dependent on the presence of dsRNA.\",\n \"This suggests that the cDNA-encoded proteins in these strains could have been affecting lacZ at the level of transcription or the stability or function of the protein.\",\n \"The library plasmids were recovered from the aes-containing strains (also referred to as res-containing strains) and their cDNA inserts sequenced.\",\n \"BLASTN and BLASTP analysis identified clones W18, W20, and W30 (named aes2) as homologues of domains 2 and 3 of the mitochondrial elongation factor Tu (EF Tu).\",\n \"EF Tu is an essential protein which plays a role in transporting tRNA to the A site in the ribosome for peptide elongation.\",\n \"The cDNA in transformants W21, W23, and W32 (named aes3) was homologous to a putative protein that was identified in a screen for fission yeast ORFs.\",\n \"Interestingly, the cDNA insert was also homologous to the antisense strand of the 3′ UTR of the fission yeast gene sna41 which has previously been shown to be involved in DNA replication.\",\n \"It is thus possible that aes3 may operate through more than one mechanism in enhancing antisense RNA activity.\",\n \"The cDNA in transformant W47 (named aes4) was homologous to the antisense strand of the ribosomal protein L7a, a component of the 60S ribosomal subunit.\",\n \"aes4 also contained a small ORF of unknown biological function.\",\n \"The inserts in transformants W27 and W28 (named aes1) shared homology with a putative protein from C. albicans that was identified in a screen for genes essential for cell growth.\",\n \"A tertiary quantitative β-galactosidase assay was performed to obtain accurate levels of gene silencing augmentation in the antisense lacZ strains co-expressing these unique factors (FIG.\",\n \"9C).\",\n \"This analysis showed that these co-factors enhanced antisense suppression by up to 50%.\",\n \"These results demonstrated that over-expression of a cDNA library was an effective way of identifying novel co-factors that magnify the suppressive effect mediated by antisense RNA.\",\n \"aes1 shared 43% identity with amino acids 4 to 202 of a C. albicans hypothetical protein (AJ390519).\",\n \"aes2 shared 99% identity with nucleotides 10452 to 9484 of the translation elongation factor EF Tu (AL049769).\",\n \"aes3 shared 94% identity with nucleotides 776 to 1145 of D89239 S. pombe ORF (D89239) and 93% identity with nucleotides 3246 to 2876 of the antisense strand of the DNA replication factor sna41 (AB001379).\",\n \"aes3 also contained a 220 nt stretch of a GA repeat sequence at its 3′ end.\",\n \"aes4 shared 99% identity with nucleotides 9678 to 8897 of the antisense strand of ribosomal protein L7a (AJ001133) and 99% identity with nucleoStides 1365 to 584 of the antisense strand of ribosomal protein L4 (AB005750).\",\n \"The reference numerals in brackets refer to accession numbers in the GeneBank database (the GeneBank database can be accessed from the following web site: http://www.ncbi.nlm.nih.gov/).\",\n \"EF Tu is analogous to the eukaryotic EF1α and acts by transporting tRNA to the A site in the ribosome for peptide elongation.\",\n \"EF1α is an essential protein which has also been implicated in a large array of cellular activities including actin binding, microtubule severing, cellular transformation, cell senescence, protein ubiquitination, and protein folding.\",\n \"Detailed analysis of the EF Tu-expressing strain showed that it enhanced antisense RNA-mediated lacZ silencing by an additional 15% (FIG.\",\n \"9C).\",\n \"It has been proposed that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and dissociated by an ATP-dependent RNA helicase.\",\n \"The small antisense fragments are then free to attack homologous mRNA by RNA nuclease-mediated degradation.\",\n \"It has recently been suggested that an associative mechanism could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.\",\n \"This may involve the transport of fragmented dsRNA to the ribosome where, in a competitive reaction with tRNA complexes, the complementary antisense strand binds to the mRNA.\",\n \"This would permit the association of the antisense strand of the dsRNA with the target mRNA when the latter is in a structurally exposed state and both inhibit translation and target the mRNA for ribonuclease degradation.\",\n \"Without wishing to be bound by any particular mechanism of action, EF Tu could act by binding to RNAi-dependent short dsRNA species and bring them to the site of action.\",\n \"The protein encoded by sna41 has previously been shown to be involved in DNA replication.\",\n \"sna41 has low homology with CDC45 and might have DNA helicase properties which could facilitate the expression of complementary sequences.\",\n \"It is conceivable that the antisense plasmid and target DNA sequences may ectopically pair by intermolecular complementarity.\",\n \"Such pairing may inhibit RNA expression and consequently reduce the level of intracellular dsRNA.\",\n \"Similarly the intramolecular pairing of inverted repeat DNA sequences may also interfere with RNA expression.\",\n \"The over-expression of a protein with DNA helicase properties could facilitate the generation of more dsRNA which could in turn enhance the RNAi effect.\",\n \"Furthermore, CDC45 mutants show an increased rate of plasmid segregation.\",\n \"If plasmid loss is inhibited by over-expression of sna41 then more dsRNA may be generated leading to more effective RNAi in this system.\",\n \"In this light it is not unreasonable to expect that other proteins normally involved in DNA and/or RNA metabolism and function could also have a role in RNAi modulation and/or enhancement.\",\n \"The L7a protein is part of the 60s ribosomal sub-unit.\",\n \"Without wishing to be bound by any particular mechanism of action, RNAi augmentation by over-expression of this protein is reasonable as it is hypothesised that the short dsRNA species may undergo strand displacement with target mRNA at the ribosome.\",\n \"The L7a ribosomal protein may act in RNAi by i) mediating docking of dsRNA or its unwound form into the A site of the ribosome, assisting in association of the antisense strand with the target mRNA, and/or shuttling of dsRNA to the ribosomal complex.\",\n \"Nucleotide sequences representative of the aes factors referred to above are provided below and identified as Seq ID Nos 1 to 4: DNA sequence for aes1 factor (Seq ID No 1) TTTAACTTAGTTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAA GCCGCAATTGTAACAGTAGATTTTTTGCATCATTATTACTCTCCGAAACA TGACTGAACACTCATTTAAGCAAATAGACGTGTTTTCTAATAAAGGTTTT CGAGGTAATCCTGTTGCAGTTTTTTTTGATGCAGATAATTTATCACAAAA GGAAATGCAGCAGATTGCCAAGTGGACAAATTTATCTGAGACAACATTTG TTCAAAAGCCGACAATCGATAAAGCAGATTACAGACTTCGTATATTTACC CCAGAATGTGAATTAAGCTTTGCTGGTCACCCAACAATTGGATCGTGCTT TGCTGTTGTTGAAAGTGGATATTGTACTCCAAAAAACTGTAAAATTATTC AGGAATGTTTAGCCGGTTTAGTTGAATTAACTATCGATGGGGAAAAGGAT GAAGACACTTGGATTTCTTTCAAACTTCCGTATTACAAAATTTTACAGAC TTCTGAAACTGCAATTTCAGAAGTAGAAAATGCATTGGGTATTCCTCTGA ATTATAGTTCTCAAGTTTCTCCTCCTGTGTTAATAGATGATGGACCAAAG TGGCTTGTAATTCAACTTCCAAACGCTACAGATGTGCTCAACCTCGTTCC GAAATTTCAGTCCCTTTCCCAAGTTTGTAAAAACAATGATTGGATAGGCG TCACCCGTCTTTGGTGAATTAGAAAAGACTCGTTTGAAAGCCCGAAGCTT TGCGCCTTTAATACATGTCAATGAGGATCCGGCTTGCGGTAGTGGTGCAG GAGCTGTCGGTGTGTATATTGGAAGCTCTCAAAAAACTCCAACTTCTCTA TCATTTACGATTTCTCAAGGTACAAAATTAAGTAGACAAGCAATTTCCAA AGTCAGCGTAGACGTTTCCTCCAATAAATCAATTGCTGTTTTTGTCGGTG GACAGGCAAAAACTTGTATTTCTGGAAAATCGTTTATTTAATGTTTTTAT TACAAATATTCACTTGCGAGTTTATTTTCCAATACTGAAGACTTTCAATC AATAGCAAATATGCTACTCAAGGAAGTTCACTCATTCAAAAGCAATTGGT TTACTATATCGTTTTTTCTAACTAGTTACTAGTCATTGAACAATCTACCG AATGATAAAATGAAATTTTGGTTTTTCCCCGGGTAAAAGGAATGTCTCCC TTGCCAGTACTGCTAGGGTTTTTCTTTCGAACTATAAGA DNA sequence for aes2 factor (Seq ID No 2) AGTCCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAGCCTGATA TGATCGAGCTTGTCGAAATGGAAATGCGTGAGCTACTCTCCGAATACGGA TTTGATGGTGACAATACTCCAATTGTTAGCGGCAGTGCTTTATGTGCCTT AGAGGGTCGTGAGCCTGAGATTGGTCTCAATAGTATTACTAAATTGATGG AAGCTGTTGATAGTTATATTACTCTTCCTGAAAGAAAAACGGATGTCCCT TTCTTGATGGCCATCGAGGACGTTTTTTCAATTTCAGGTCGCGGAACTGT AGTCACTGGCCGTGTCGAGCGCGGTACTTTAAAGAAGGGTGCTGAAATCG AAATCGTCGGTTATGGTAGCCATTTAAAGACTACCGTTACTGGAATTGAA ATGTTCAAAAAGCAGCTTGATGCCGCCGTTGCCGGTGACAATTGTGGCCT TTTACTTCGTTCTATCAAGCGAGAGCAATTAAAACGTGGAATGATTGTCG CTCAACCAGGAACCGTTGCTCCTCATCAGAAATTCAAGGCATCATTCTAT ATTTTGACAAAAGAGGAAGGAGGTCGTCGTACCCGGTTTCGTTGACAAGT ATCGTCCCCAACTGTACAGTCCGTACTTCCGACGTTACTGTCGAACTTAC CCACCCTGATCCTAACGACTCAACAAAATGGTTATGCCTGGAGACAATGT CGAGATGATCTGTACGCTTATTCACCCCATTGTCATCGAAAAAGGACAAC GCTTCACAGTTCGTGAGGGTGGAAGCACTGTAGGCACAGCTTTGGTTACT GAACTTTTGGATTAGTGCATTTATGAACTTATTGGCTTTAAAAATTTTGC ATGCTGAATACCAATATTATGTCCCTTCTCAGAATTCTATAACTACAGTG TCATTATTGTAATAAGACTTTTGCATCCATTGACAATGGTATTTGATACT TTTATAGTTTCTACTATTGTTAGCCAAAGTTATAAAACAAATAATAAAAT AACGTTGAATCAAAAAAAAAAAAAAAAAAAGCGGCCGCGGATCCCCGGGT AAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCTTTCAAACTA TGGGA DNA sequence for aes3 factor (Seq ID No 3) ATTTCAGACGCAATTCACATGGCTTTTGACTGTATTGCTATTCTTGTCGG TTTAGTTGCTACGACGCTTGCCAAGATGCCTCTAAATTATGCTTACCCCT TTGGATTTGCAAAAATTGAGGCTCTTTCGGGTTTCACTAATGGTATTTTT TTAGTTTTGATTTCATTTTCTATCGTCGGCGAGGCATTATATAGGTTATT TCATCCGCCCCAAATGAATACCGACCAATTGTTGTTGGTTAGTTTTTTGG GCCTTGTTGTGAATTTGGTAGGTATCCTAGCGTTCAATCATGGGCATAAT CATGATCATGGGTCTCATCACCATCATTCCCATAGTAATCATAGTATGTG TCTGCCTAACACTACAAATGATATAAATATTTTTGAAGAGTTTGAAGAAG AAAAAGATAATGTTGAAGCCCAGAAAATGGGCTATACGAATGACGATCAC GTATCCCAACATGAACATACCCATGAGAATAGTCAGGAACATCACCATGA GCATAACCACAATCATGATCACATCCATAAATACAATGAAAAATGCGACC ATGAAAGCATAAGTCTCCAGAATTTAGACAATGATCATCACTGTCATCAT CACCATGAAAATCATAATATGCATGGCATATTTCTGCATATTATCGCAGA TACTATGGGCTCTGTTGGAGTTATTGTCTCTACTATATTAATACAGTGGT TTTCATGGACCGGTTTTGATCCTTCGGCATCTCTAATAATTGCTGCATTA ATATTTGTTTCTGTACTTCCATTAATTAAAGATTCGGCGAAGAATTTGCT CTCTGTGACTGATCCAGAATCGGAATATTTATTGAAGCAGTGTTTGTCGA ACATCAGTTTAAGTCACTCCGTTGTCAGTTTATCCAACCCTAAGTTCTGG ACAAACGAAAGAGGTGAAGTGTATGGAATACTCCATATTCAGGTGAGCAT AGACGGTGATTTAAACGTGGTTCGTAATGAAGTATTTAGGAAGCTCTCAA TCGCTGTACCAAATTTAAAACACATTTGTATACAATCTGAACGGCCAAAC AATTGCTGGTGTGGAAAATAGTTCTTACATCAGTTGATATCCATACTTAT TTACGTGTAATTTTAATTAGATGAATTAATATTTTCTTTATTAGC DNA sequence for aes4 factor (Seq ID No 4) TTTACTTTAGTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAG CTTTTTTTTTAAGAGATATAACATATGTCAACGCGTCATTGATTAACTAC ATAACACGCCAATTATAAACTTCTCCCAAAAGAACTTAAGAATTTCCATT TTCAATCCAGATGAATTTATTTAAGAGACGAACAGTAGCGGCAGCAGCCT TAGCACGCTTAGCGAGCAAAGCTTGGGTCTTACCACCCATGATACCCACC ACCCCACTTACGACGGGCTTTCGTCGTACTTAGCAGAGAAGTTAGCATCA ACGGCGGAGACAATAGAAGCGAGTTCGTTCTTGTCTTCCTCACGGACCTC AGTGACAGCTAAAACAGCAGCAGTCTTTTGGTGAATGACAGTACCAAGGC GGGCCTTGTTCTTGACAATGGCATAAGGAACACCCATCTTCTTGCACAAA GCAGGCAAGAAAACGACGAGTTCAATGGGGTCGACATCGCTGGCAATGAG AACCAACTTAGCCTTCTTGGCCTCAATGAGAGCTACAACATGGTTCAAAC CATATTTAACATTGTAAGGCTTCTTAGAGACGTCTTGAGCAGACTTGCCG TTGGCAACAGCCTCGGCTTCAGCAACCAAACGTTGCTTCTTTTCAGCAGC AGTCTCAGGACGGTACTTGTTAAGCAACTTGAAGACCTGAGTAGCAGTGT TTTTGTCCAAAGTCTTCTGGAACTGAGCAATGGCAGGAGGAACCTTCAAA CGCAAGTTCAAAATCTTGCGACGGCGTTGAAGGCGGATATACTCAGGCCA CTTAACAAAACGGCTCAAGTCACGCTTAGGTTGGATGTCTTGTCCCCCGG GTAAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCGTTCGAAT AAGGCC Example 9 Effect of Novel RNAi Factors on Antisense RNA and dsRNA-Mediated Gene Regulation With recent studies on PTGS suggesting that antisense RNA, co-suppression, and dsRNA-mediated interference may share similar mechanisms, we wanted to determine whether over-expression of an aes factor would also enhance dsRNA-mediated regulation.\",\n \"Having demonstrated that dsRNA could mediate gene suppression in this fission yeast model, the effect of an antisense enhancing sequence on dsRNA-mediated regulation was tested.\",\n \"To this end, the aes2 gene was co-expressed with the lacZ panhandle construct in a yeast strain containing the lacZ target gene.\",\n \"Under these conditions, this transformant displayed an additional 30% suppression of β-galactosidase activity when compared to the transformant expressing only the panhandle lacZ RNA (see FIG.\",\n \"10).\",\n \"This result shows that the aes2 gene, encoding the two domains of the mitochondrial elongation factor EF Tu, stimulated not only antisense RNA but also dsRNA-mediated gene inhibition.\",\n \"Further it was shown that these two forms of regulation are related by over-expressing the ATP-dependent RNA helicase ded1 in the presence of lacZ antisense RNA and showing that this helicase enhanced gene suppression by a further 50% compared to the control strain (example 6).\",\n \"ded1 was tested on both active and inactive antisense plasmids and demonstrated that ded1 augmentation of gene silencing was dependent on an active antisense RNA (see FIG.\",\n \"11).\",\n \"This could be due to the absence of RNA duplex formation with the inactive antisense RNA and the consequent lack of a substrate for the RNA helicase.\",\n \"The methods of the present invention have utility in demonstrating a range of RNAi efficacies, in identifying new factors which enhance or reduce gene silencing, in inhibiting gene expression or increasing sensitivity to antisense inhibition of gene expression, in the treatment or prevention of disorders which require inhibition or down-regulation of gene expression.\",\n \"Although the present invention has been described with reference to specific examples and preferred embodiments it will be clear to those skilled in the art that variations and modifications which do not depart from the concept and the spirit of the invention described herein are also contemplated as being within the scope of the present invention.\",\n \"REFERENCES 1.Raponi, M., Atkins, D., Dawes, I.\",\n \"& Arndt, G. (2000) Antisense and Nucleic Acid Drug Development 10, 29-34.2.Arndt, G., Atkins, D., Patrikakis, M. & Izant, J.\",\n \"(1995) Molecular and General Genetics 248, 293-300.3.Arndt, G., Patrikakis, M. & Atkins, D. (2000) Nucleic Acids Research 28, e15.4.Clarke, M., Patrikakis, M. & Atkins, D. (2000) Biochemical and Biophysical Research Communications 268, 8-13.5.Moreno, S. & Nurse, P. (1994) Nature 367, 236-242.6.Moreno, S., Klar, A.\",\n \"& Nurse, P. (1991) Methods in Enzymology 194, 795-823.7.Maundrell, K. (1990) Journal of Biological Chemistry 265, 10857-10864.8.Prentice, H. (1992) Nucleic Acids Research 20, 621.9.Hoffman, C. & Winston, F. (1987) Gene 57, 267-272.10.Rose, M., Winston, F. & Hieter, P. (1990) Methods in yeast genetics: A laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor).\",\n \"11.Maundrell, K. (1993) Gene 123, 127-1390.12.Basi, G., Schmid, E. & Maundrell, K. (1993) Gene 123, 131-136.13.Carr, A., MacNeill, S., Hayles, J.\",\n \"& Nurse, P. (1989) Molecular and General Genetics 218, 41-49.14.Sambrook, J., Fritsch, E. & Maniatis, T. (1989) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).\",\n \"15.Patrikakis, M., Izant, J.\",\n \"& Atkins, D. (1996) Current Genetics 30, 151-158.16.Brun, C., Dubey, D. & Huberman, J.\",\n \"(1995) Gene 164, 173-177.17.Heyer, W.-D., Sipiczki, M. & Kohli, J.\",\n \"(1986) Molecular and Cellular Biology 6, 80-89.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297167"}}},{"rowIdx":95,"cells":{"text":{"kind":"list like","value":[["Ligands of Integrin Receptors","The invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds."],["1.The use of compounds of the formula I B-G-L I as ligand of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3, together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4 RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —C1-NRG12RG13—NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11 B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.","2.The use as claimed in claim 1, wherein the structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally contained or the carbon atoms can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.3.The use as claimed in one of claims 1 or 2, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18, where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—Cl1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms-O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both: radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA1, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11 Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.","4.The use as claimed in one of claims 1 to 3, wherein the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE9RE10)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 1-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NHC1-C6-alkoxyalkyl, CO—NHC1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an, optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.","5.The use as claimed in one of claims 1 to 4, wherein the spacer structural-element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6—IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14 (ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical or independently of one another are in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle, which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms together with RW2 or RW2* a saturated or unsaturated C3-C7-heterocycle, which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.","6.The use of the structural element of the formula IGL -G-L, IGL for the preparation of compounds which bind to integrin receptors, where G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 und RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted, C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, with the proviso that in the case of this fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle substituents are excluded which contain a structural element —V—CO—R8, where V is an optionally substituted C1-C2-alkylene radical R8 is a hydroxyl group, a C1-C8-alkoxy, aryl-C0-C6-alkoxy, C1-C8-alkylcarbonyloxy-C1-C4-alkoxy or aryl-C1-C8-alkylcarbonyloxy-C1-C4-alkoxy group or an L- or D-amino acid, which is bonded by an amide bond and in which the carboxylic acid component of said amino acid is present as a free acid or esterified with C1-C6-alkyl, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—N12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and which can contain up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11.7.The use of the compounds as claimed in one of claims 1 to 5 as ligands of the αVβ3 integrin receptor.","8.The use of the compounds as claimed in one of claims 1 to 5 for the production of drugs for treating diseases in which the interaction between integrins and their natural ligands is excessive or decreased.","9.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 8 for the treatment of diseases in which the interaction between αVβ3 ingegrin and its natural ligands is excessive or decreased.","10.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 9 for the treatment of atherosclerosis, rheumatoid arthritis, restenosis after vascular injury or stent implantation, angioplasty, acute kidney failure, angiogenesis-associated microangiopathies, diabetic angiopathies, blood platelet-mediated vascular occlusion, arterial thrombosis, congestive heart failure, myocardial infarct, stroke, cancer, osteoporosis, high blood pressure, psoriasis or viral, parasitic or bacterial conditions, inflammation, wound healing, hyperparathyroidism, Paget's disease, malignant hypercalcemia or metastatic osteolytic lesions.","11.A compound of the formula I′ A-E′-G′-L I′ where A, E′, G′ and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G′ is a structural element of the formula IG where the structural element A-E′ is bonded to the structural element G′ via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG′12RG′13, C1-C4-alkylene-CO—NRG′12RG′13, C1-C4-alkylene-O—CO—NRG′12RG′13, C1-C4-alkylene-NRG′12RG′13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG′12RG′13, —SO2—NRG′12RG′13, —CO—NRG′12RG′13, —NRG′12RG′13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG′12, RG′13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-heterocycloalkyl, C1-C4-alkylene-heterocycloalkenyl or hetarylalkyl radical, E′ is a structural element, composed of two to four substructural elements, selected from the group E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO-o, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE8 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—C—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW12)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded, A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon, which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.","12.A compound as claimed in claim 11, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where both radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* is a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6 are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms Q, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.","13.A compound as claimed in claim 11 or 12, wherein the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 (NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon, which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group-O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded.","14.A compound as claimed in one of claims 11 to 13 for use as a drug.","15.The use of the compounds as claimed in one of claims 11 to 13 for the production of drugs for treating diseases.","16.A pharmaceutical preparation comprising, in addition to the customary pharmaceutical excipients, at least one compound as claimed in one of claims 11 to 13.17.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet adhesion, activation or aggregation, anticoagulants which prevent thrombin activity or formation, antagonists of blood platelet-activating compounds or selectin antagonists.","18.The use of the pharmaceutical preparation as claimed in claim 17 for the production of a drug for treating blood platelet-mediated vascular occlusion or thrombosis.","19.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet activation or aggregation, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vascular wall transmigration, fibrinolysis-modulating compounds, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists.","20.The use of the pharmaceutical preparation as claimed in claim 19 for the production of a drug for treating myocardial infarct or stroke.","21.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors.","22.The use of the pharmaceutical preparation as claimed in claim 21 for the production of a drug for treating congestive heart failure.","23.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, inhibitors of blood platelet adhesion, activation or aggregation, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants.","24.The use of the pharmaceutical preparation as claimed in claim 23 for the production of a drug for treating restenosis after vascular injury or stent implantation.","25.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, glycosylation inhibitors and AGE formation inhibitors or AGE breakers and antagonists of their receptors.","26.The use of the pharmaceutical preparation as claimed in claim 25 for the production of a drug for treating diabetic angiopathies.","27.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers.","28.The use of the pharmaceutical preparation as claimed in claim 27 for the production of a drug for treating atherosclerosis.","29.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group cytostatic or antineoplastic compounds, compounds which inhibit proliferation and heparin or low-molecular weight heparins or further GAGs.","30.The use of the pharmaceutical preparation as claimed in claim 29 for the production of a drug for the treatment of cancer.","31.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group compounds for antiresorptive therapy, compounds for hormone exchange therapy, recombinant human growth hormone, bisphosphonates, compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, interleukin-6 antagonists and Src tyrosine kinase inhibitors.","32.The use of the pharmaceutical preparation as claimed in claim 31 for the production of a drug for the treatment of osteoporosis.","33.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors.","34.The use of the pharmaceutical preparation as claimed in claim 33 for the production of a drug for treating rheumatoid arthritis.","35.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group collagenase, PDGF antagonists and MMPs.","36.The use of the pharmaceutical preparation as claimed in claim 35 for the production of a drug for improving wound healing."],["The present invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds.","Integrins are cell surface glycoprotein receptors which mediate interactions between similar and different cells as well as between cells and extracellular matrix proteins.","They are involved in physiological processes, such as embryogenesis, hemostasis, wound healing, immune response and formation/maintenance of the tissue architecture.","Disturbances in the gene expression of cell adhesion molecules and functional disorders of the receptors can contribute to the pathogenesis of many disorders, such as tumors, thromboembolic events, cardiovascular disorders, lung diseases, disorders of the CNS, the kidney, the gastrointestinal tract or inflammation.","Integrins are heterodimers of an α- and a β-transmembrane subunit in each case, which are noncovalently bonded.","Up to now, 16 different α- and 8 different β-subunits and 22 different combinations have been identified.","Integrin αvβ3, also called the vitronectin receptor, mediates adhesion to a multiplicity of ligands—plasma proteins, extracellular matrix proteins, cell surface proteins, of which the majority contain the amino acid sequence RGD (Cell, 1986, 44, 517-518; Science 1987, 238, 491-497), such as vitronectin, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, osteopontin, laminin, collagen, thrombin, tenascin, MMP-2, bone sialoprotein II, various viral, fungal, parasitic and bacterial proteins, natural integrin antagonists such as disintegrins, neurotoxins—mambin—and blood fluke proteins—decorsin, ornatin—and also some non-RGD ligands, such as Cyr-61 and PECAM-1 (L. Piali, J.","Cell Biol.","1995, 130, 451-460; Buckley, J.","Cell Science 1996, 109, 437-445, J. Biol.","Chem.","1998, 273, 3090-3096).","A number of integrin receptors show cross-reactivity with ligands which contain the RGD motif.","Thus integrin αIIbβ3, also called the platelet fibrinogen receptor, recognizes fibronectin, vitronectin, thrombospondin, von Willebrand factor and fibrinogen.","Integrin αvβ3 is expressed, inter alia, on endothelial cells, blood platelets, monocytes/macrophages, smooth muscle cells, some B cells, fibroblasts, osteoclasts and various tumor cells, such as melanoma, glioblastoma, lung, breast, prostate and bladder carcinomas, osteosarcomas or neuroblastomas.","Increased expression is observed under various pathological conditions, such as in the prothrombotic state, in vascular injury, tumor growth or metastasis or reperfusion and on activated cells, in particular on endothelial cells, smooth muscle cells or macrophages.","An involvement of integrin αvβ3 has been demonstrated, inter alia, in the following syndromes: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury, and angioplasty (neointima formation, smooth muscle cell migration and proliferation) (J. Vasc.","Surg.","1994, 19, 125-134; Circulation 1994, 90, 2203-2206), acute kidney failure (Kidney Int.","1994, 46, 1050-1058; Proc.","Natl.","Acad.","Sci.","1993, 90, 5700-5704; Kidney Int.","1995, 48, 1375-1385), angiogenesis-associated microangiopathies such as diabetic retinopathy or rheumatoid arthritis (Ann.","Rev.","Physiol 1987, 49, 453-464; Int.","Opthalmol.","1987, 11, 41-50; Cell 1994, 79, 1157-1164; J. Biol.","Chem.","1992, 267, 1093.1-10934), arterial thrombosis, stroke (phase II studies with ReoPro, Centocor Inc., 8th annual European Stroke Meeting), carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis) (Cell 1991, 64, 327-336; Nature 1989, 339, 58-61; Science 1995, 270, 1500-1502), osteoporosis (bone resorption after proliferation, chemotaxis and adhesion of osteoclasts to bone matrix) (FASEB J.","1993, 7, 1475-1482; Exp.","Cell Res.","1991, 195, 368-375, Cell 1991, 64, 327-336), high blood pressure (Am.","J. Physiol.","1998, 275, H1449-H1454), psoriasis (Am.","J. Pathol.","1995, 147, 1661-1667), hyperparathyroidism, Paget's disease (J. Clin.","Endocrinol.","Metab.","1996, 81, 1810-1820), malignant hypercalcemia (Cancer Res.","1998, 58, 1930-1935), metastatic osteolytic lesions (Am.","J. Pathol.","1997, 150, 1383-1393), pathogenic protein (e.g.","HIV-1 tat)-induced processes (e.g.","angiogenesis, Kaposi's sarcoma) (Blood 1999, 94, 663-672) inflammation (J.","Allergy Clin.","Immunol.","1998, 102, 376-381), cardiac insufficiency, CHF, and also in antiviral, antiparasitic, antifungal or antibacterial therapy and prophylaxis (adhesion and internalization) (J. Infect.","Dis.","1999, 180, 156-166; J. Virology 1995, 69, 2664-2666; Cell 1993, 73, 309-319).","On account of its key role, pharmaceutical preparations which contain low-molecular weight integrin αvβ3 ligands are of high therapeutic or diagnostic benefit, inter alia, in the indications mentioned.","Advantageous αvβ3 integrin receptor ligands bind to the integrin αvβ3 receptor with an increased affinity.","In contrast to integrin αvβ3, particularly advantageous αvβ3 integrin receptor ligands additionally have an increased selectivity and are less active with respect to the integrin αIIbβ3 by at least a factor of 10, preferably at least a factor of 100.For a multiplicity of compounds, such as anti-αvβ3 monoclonal antibodies, peptides which contain the RGD binding sequence, natural, RGD-containing proteins (e.g.","disintegrins) and low-molecular weight compounds, an integrin αvβ3 antagonistic action has been shown and a positive in vivo effect demonstrated (FEBS Letts 1991, 291, 50-54; J. Biol.","Chem.","1990, 265, 12267-12271; J. Biol.","Chem.","1994, 269, 20233-20238; J.","Cell Biol 1993, 51, 206-218; J. Biol.","Chem.","1987, 262, 17703-17711; Bioorg.","Med.","Chem.","1998, 6, 1185-1208).","Antagonists of the αVβ3 integrin receptor based on a bicyclic structural element are described in WO 9906049, WO 9905107, WO 9814192, WO 9724124, WO 9724122 and WO 9626190.EP 540 334 and WO 9308174 describe bicyclic antagonists of the αIIbβ3 integrin receptor.","WO 9407488 A1 describes compounds having a bicyclic molecular structure and which accelerate the release of growth hormone.","Further, vasopressin antagonists having a bicyclic molecular structure are described in the specifications EP 620216, WO 9534540, WO 9408582, WO 9802432, WO 9420473, JP 09221476 A1, JP 11060488 A1, WO 9404525, JP 04321669 A1, WO 9722591, as well as in Matsuhisa et al., Chem.","Pharm.","Bull.","1999, 47, 3, 329-339.It is an object of the present invention to make available novel integrin receptor ligands having advantageous properties.","We have found that this object is achieved by the use of compounds of the formula I B-G-L I as ligands of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.","In the structural element L, T is understood as meaning a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH.","A radical hydrolyzable to COOH is understood as meaning a radical which changes into a group COOH after hydrolysis.","A group which may be mentioned by way of example as a radical T hydrolyzable to COOH is in which R1 has the following meanings: a) OM, where M can be a metal cation, such as an alkali metal cation, such as lithium, sodium, potassium, the equivalent of an alkaline earth metal cation, such as calcium, magnesium and barium, or an environmentally tolerable organic ammonium ion such as primary, secondary, tertiary or quaternary C1-C4-alkylammonium or ammonium ion, such as ONa, OK or OLi, b) a branched or unbranched, optionally halogen-substituted C1-C8-alkoxy radical, such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, in particular methoxy, ethoxy, 1-methylethoxy, pentoxy, hexoxy, heptoxy, octoxy, difluoromethoxy, trifluoromethoxy, chlorodifluoromethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2,2,2-trifluoroethoxy, 2-chloro-1,1,2-trifluoroethoxy or pentafluoroethoxy c) a branched or unbranched, optionally halogen-substituted C1-C4-alkylthio radical such as methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio or 1,1-dimethylethylthio radical d) an optionally substituted —O-alkylenearyl radical, such as —O-benzyl e) R1 is further a radical —(O)m—N(R18)(R19), in which m is 0 or 1 and R18 and R19, which can be identical or different, have the following meanings: hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl or the corresponding substituted radicals, preferably methyl, ethyl, propyl, butyl or i-butyl, C2-C6-alkenyl radical, such as vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl or the corresponding substituted radicals, C2-C6-alkynyl radical, such as ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl or the corresponding substituted radicals, C3-C8-cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl und cycloheptyl, cyclooctyl or the corresponding substituted radicals, or a phenyl radical, optionally mono- or polysubstituted, for example mono- to trisubstituted, by halogen, nitro, cyano, C1-C4-alkyl, C1-C4-halogenoalkyl, C1-C4-alkoxy, C1-C4-halogenoalkoxy or C1-C4-alkylthio such as 2-fluorophenyl, 3-chlorophenyl, 4-bromophenyl, 2-methylphenyl, 3-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-methoxyphenyl, 4-trifluoroethoxyphenyl, 2-methylthiophenyl, 2,4-dichlorophenyl, 2-methoxy-3-methylphenyl, 2,4-dimethoxyphenyl, 2-nitro-5-cyanophenyl, 2,6-difluorophenyl, or R18 and R19 together form an optionally substituted, e.g.","C1-C4-alkyl-substituted, C4-C7-alkylene chain closed to give a cycle, which can contain a heteroatom selected from the group consisting of oxygen, sulfur and nitrogen, such as —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)2—O—(CH2)2—, —CH2—S—(CH2)3—, —(CH2)2—O—(CH2)3—, —NH—(CH2)3—, —CH2—NH—(CH2)2—, —CH2—CH═CH—CH2—, —CH═CH—(CH2)3—, —CO— (CH2)2—CO— or —CO— (CH2)3—CO—.","A radical bioisosteric to COOH is understood as meaning radicals which can replace the function of a group COOH in active compounds by equivalent bond donor/acceptor capabilities or by equivalent charge distribution.","Radicals which may be mentioned by way of example as radicals bioisosteric to —COOH are those such as described in “The Practice of Medicinal Chemistry, Editor: C. G. Wermuth, Academic Press 1996, pages 125 and 216, in particular the radicals —P═O(OH)2, —SO3H, tetrazole or acylsulfonamides.","Preferred radicals T are —COOH, —CO—O—C1-C8-alkyl or —CO—O-benzyl.","The radical —U— in the structural element L is a spacer selected from the group —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1-.","In the case of the radical ═CRL1—, the structural element L is linked to the structural element G via a double bond.","XL is a radical CRL3RL4, NRL5, oxygen or sulfur.","Preferred radicals —U— are the radicals —CRL1═CRL2—, ethynylene or —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a=1).","Particularly preferred radicals —U— are the radicals —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a 1).","Under RL11, RL2, RL3 or RL4 in the structural element L, a halogen radical is understood as meaning, for example, F, Cl, Br or I, preferably F. Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C6-alkyl radical is understood as meaning, for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, preferably branched or unbranched C1-C4-alkyl radicals such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, particularly preferably methyl.","Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkenyl radical is understood as meaning, for example, vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl.","Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkynyl radical is understood as meaning, for example, ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably ethynyl, 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl.","Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C3-C7-cycloalkyl radical is understood as meaning, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.","Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C4-alkoxy radical is understood as meaning, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy or 1,1-dimethylethoxy.","The radicals —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 are secondary or tertiary amides and are composed of the amide bond and the corresponding C1-C6-alkyl radicals such as described above for RL1, RL2, RL3 or RL4.The radicals RL1, RL2, RL3 or RL4 can furthermore be a radical C1-C2-alkylene-T, such as methylene-T or ethylene-T, C2-alkenylene-T, such as ethenylene-T or C2-alkynylene-T, such as ethynylene-T, an aryl radical, such as phenyl, 1-naphthyl or 2-naphthyl or an arylalkyl radical, such as benzyl or ethylenephenyl (homobenzyl), where the radicals can optionally be substituted.","Furthermore, two radicals RL1 and RL2 or RL3 and RL4 or optionally RL1 and RL3 can in each case independently of one another together be an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. All radicals for RL1, RL2, RL3 or RL4 can be optionally substituted.","For the radicals RL1, RL2, RL3 oder RL4 and all further substituted radicals of the description below, suitable substituents, if the substituents are not specified in greater detail, are independently of one another up to 5 substituents, for example selected from the following group: —NO2, —NH2, —OH, —CN, —COOH, —O—CH2—COOH, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl, CF3, C2F5 or CH2F, —CO—O—C1-C4-alkyl, C3-C6-cycloalkyl, C1-C4-alkoxy, C1-C4-thioalkyl, —NH—CO—O—C1-C4-alkyl, —O—CH2—COO—C1-C4-alkyl, —NH—CO—C1-C4-alkyl, —CO—NH—C1-C4-alkyl, —NH—SO2—C1-C4-alkyl, —SO2—NH—C1-C4-alkyl, —N(C1-C4-alkyl)2, —NH—C1-C4-alkyl, or —SO2—C1-C4-alkyl radical, such as —SO2—CF3, an optionally substituted —NH—CO-aryl, —CO—NH-aryl, —NH—CO—O-aryl, —NH—CO—O-alkylenearyl, —NH—SO2-aryl, —SO2—NH-aryl, —CO—NH-benzyl, —NH—SO2-benzyl or —SO2—NH-benzyl radical, an optionally substituted radical —SO2—NR2R3 or —CO—NR2R3 where the radicals R2 and R3 independently of one another can have the meaning RL5 as below or both radicals R2 and R3 together can be a 3- to 6-membered, optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three further different or identical heteroatoms O, N, S, and optionally two radicals substituted on this heterocycle can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted cycle can be fused to this cycle.","If not specified in greater detail, in all terminally bonded, substituted-hetaryl radicals of the description, two substituents can form a fused 5- to 7-membered, unsaturated or aromatic carbocycle.","Preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical or the radical —NRL6RL7.Particularly preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, fluorine or a branched or unbranched, optionally substituted C1-C4-alkyl radical, preferably methyl.","The radicals RL5, RL6, RL7 in structural element L are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, for example as described above for RL1, C3-C7-cycloalkyl radical, for example as described above for RL1, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the C1-C6-alkyl radicals described above for RL1, or an optionally substituted CO—O-alkylenearyl, SO2-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the aryl or arylalkyl radicals described above for RL1.Preferred radicals for RL6 in structural element L are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—O—C1-C4-alkyl, CO—C1-C4-alkyl or SO2—C1-C4-alkyl radical or an optionally substituted CO—O-benzyl, SO2-aryl, SO2-alkylenearyl or CO-aryl radical.","Preferred radicals for RL7 in structural element L are hydrogen or a branched or unbranched, optionally substituted C1-C4-alkyl radical.","Preferred structural elements L are composed of the preferred radicals of the structural element.","Particularly preferred structural elements L are composed of the particularly preferred radicals of the structural element.","G is a structural element of the formula IG where the structural element B is bonded via the ring nitrogen and the structural element L is bonded via WG to the structural element G, optionally via a double bond.","YG in structural element G is CO, CS, C═NRG2 or CRG3RG4, preferably CO, C═NRG2 or CRG3RG4, particularly preferably CO or CRG3RG4.RG2 in structural element G is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical, for example as described above for RL1 in each case, an optionally substituted —O—C3-C7-cycloalkyl radical, which is composed of an ether group and, for example, of the C3-C7-cycloalkyl radical described above for RL1, an optionally substituted aryl or arylalkyl radical, for example as described above for RL1 in each case or an optionally substituted —O-aryl or —O-alkylenearyl radical, which is composed of a group —O— and, for example, of the aryl or arylalkyl radicals described above for RL1.Branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radicals for RG3 or RG4 in structural element G independently of one another are understood as meaning, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG3 and RG4 can together form a cyclic acetal, such as —O—CH2—CH2—O— or —O—CH2—O—.","Furthermore, both radicals RG3 and RG4 can together form an optionally substituted C3-C7-cycloalkyl radical.","Preferred radicals for RG3 or RG4 are independently of one another hydrogen, C1-C4-alkyl or C1-C4-alkoxy.","Branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radicals and optionally substituted aryl or arylalkyl radicals for RG5 and RG6 in structural element G independently of one another are, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG5 and RG6 can together form an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. Preferred radicals for RG5 and RG6 are independently of one another hydrogen or optionally substituted aryl radicals, preferably phenyl or arylalkyl radicals, preferably benzyl, and in each case both radicals RG5 and RG6 together can contain an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S. In particularly preferred radicals for RG5 and RG6, both radicals RG5 and RG6 together form an optionally substituted, fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle, for example selected from one of the following doubly bonded structural formulae: in particular selected from one of the following, doubly bonded structural formulae: Suitable substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are in particular substituents such as generally described above.","Particularly preferred substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are independently of one another up to four substituents selected from the following group: hydroxyl, —CN, F or Cl or a branched or unbranched, optionally substituted C1-C4-alkoxy or C1-C4-alkyl radical, such as methoxy, methyl, CF3, C2F5 or CH2F.","WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, where the dashed lines intersect the atomic bonds within the structural element G and the carbon atom substituted by RG7 and RG8 is bonded to YG.","In a preferred embodiment, WG is a structural element selected from the group of structural elements of the formulae IWG2 and IWG3, in particular the structural element of the formula IWG2.RG1 in structural element WG is hydrogen, halogen, such as Cl, F, Br or I, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl radical, preferably C1-C4-alkyl or C1-C4-alkoxy radical such as in each case described above for RL1.Particularly preferred radicals for RG1 are hydrogen, methoxy or hydroxyl.","RG7, RG8, RG9 and RG10 in structural element G are independently of one another hydrogen, a hydroxyl group, CN, halogen, such as F, Cl, Br, I, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, C2-C6-alkenyl radical, such as optionally substituted vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl or 1-ethyl-2-methyl-2-propenyl, C2-C6-alkynyl radical, such as optionally substituted ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl or 1-ethyl-1-methyl-2-propynyl, an optionally substituted C3-C7-cycloalkyl radical, such as optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, C3-C7-heterocycloalkyl radical, such as optionally substituted aziridinyl, diaziridinyl, oxiranyl, oxaziridinyl, oxetanyl, thiiranyl, thietanyl, pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,4-dioxanyl, hexahydroazepinyl, oxepanyl, 1,2-oxathiolanyl or oxazolidinyl, C3-C7-heterocycloalkenyl radical, such as optionally substituted azirinyl, diazirinyl, thiirenyl, thietyl, pyrrolinyls, oxazolinyls, azepinyl, oxepinyl, α-pyranyl, β-pyranyl, γ-pyranyl, dihydropyranyls, 2,5-dihydropyrrolinyl or 4,5-dihydrooxazolyl, a branched or unbranched, optionally substituted C1-C4-alkylene-C3-C7-cycloalkyl radical, which is composed, for example, of branched or unbranched C1-C4-alkylene radicals such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-cycloalkyl radicals, a branched or unbranched optionally substituted C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, which is composed of optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-heterocycloalkyl or C3-C7-heterocycloalkenyl radicals, the radicals being preferred which in the cyclic moiety contain one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds, a branched or unbranched, optionally substituted radical C1-C4-alkylene-O—RG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13, C1-C4-alkylene-SRG11 or C1-C4-alkylene-SO—RG11, which is composed of branched or unbranched, optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene, the corresponding groups —O—, —CO—, —S—, —N and the terminal radicals RG11, RG12 and RG13 described below, an optionally substituted aryl radical, preferably optionally substituted phenyl, 1-naphthyl or 2-naphthyl, arylalkyl radical, preferably optionally substituted benzyl or ethylenephenyl (homobenzyl), hetaryl radical, preferably optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, thiadiazolyl, oxadiazolyl or triazinyl or their fused derivatives such as indazolyl, indolyl, benzothiophenyl, benzofuranyl, indolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolinyl or isoquinolinyl, hetarylalkyl radical, preferably optionally substituted —CH2-2-pyridyl, —CH2-3-pyridyl, —CH2-4-pyridyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-thiazolyl, —CH2-4-thiazolyl, CH2-5-thiazolyl, —CH2—CH2-2-pyridyl, —CH2—CH2-3-pyridyl, —CH2—CH2-4-pyridyl, —CH2—CH2-2-thienyl, —CH2—CH2-3-thienyl, —CH2—CH2-2-thiazolyl, —CH2—CH2-4-thiazolyl or —CH2—CH2-5-thiazolyl or a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13, CO—RG11.Further, two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 can in each case independently of one another together form an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group consisting of O, N, S and up to two double bonds.","Preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical, as described above in each case.","Particularly preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, F or a branched or unbranched, optionally substituted C1-C4-alkyl radical, as described above.","A branched or unbranched, optionally substituted C1-C8-alkyl radical for RG11, RG12 and RG13 is understood as meaning independently of one another, for example, the C1-C6-alkyl radicals mentioned above for RG1, plus the radicals heptyl and octyl.","Preferred substituents of the branched or unbranched, optionally substituted C1-C8-alkyl radicals for RG11, RG12 and RG13 independently of one another are the radicals halogen, hydroxyl, C1-C4-alkoxy, —CN, —COOH and —CO—O—C1-C4-alkyl.","A branched or unbranched, optionally substituted C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkylene-C3-C7-cycloalkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RG11, RG12 and RG13 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Preferred, branched or unbranched, optionally substituted —C1-C5-alkylene-C1-C4-alkoxy radicals for RG11, RG12 and RG13 are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.","Preferred, branched or unbranched, optionally substituted mono- and bis-alkylaminoalkylene or acylaminoalkylene radicals for RG11, RG12 and RG13 are independently of one another branched or unbranched, optionally substituted radicals —C1-C4-alkylene-NH(C1-C4-alkyl), —C1-C4-alkylene-N(C1-C4-alkyl)2 or —C1-C4-alkylene-NH—CO—C1-C4-alkyl.","Preferred optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1.Particularly preferred, optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1, one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds being contained in the cyclic moiety.","Further, RG12 and RG13 can independently of one another be a radical —SO2—RG11, —CO—O—RG11, —CO—NRG11RG11* or —CO—RG11, RG11* being a radical RG11 which is independent of RG11.Preferred structural elements G are composed of at least one preferred radical of the structural element G, while the remaining radicals are widely variable.","Particularly preferred structural elements G are composed of the preferred radicals of the structural element G. Very particularly preferred structural elements G are composed of the particularly preferred radicals of the structural element G. Structural element B is understood as meaning a structural element comprising at least one atom which under physiological conditions can form hydrogen bridges as a hydrogen acceptor, at least one hydrogen acceptor atom having a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton.","The arrangement of the structural skeleton of structural element B is widely variable.","Suitable atoms which under physiological conditions can form hydrogen bridges as hydrogen acceptors are, for example, atoms having Lewis base properties, such as the heteroatoms nitrogen, oxygen or sulfur.","Physiological conditions is understood as meaning a pH which prevails at the site in a body at which the ligands interact with the receptors.","In the present case, the physiological conditions have a pH of, for example, 5 to 9.In a preferred embodiment, structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—R11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.In a particularly preferred embodiment, the structural element A is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, g independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1), —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.","In a further very particularly preferred embodiment, the structural element A is a structural element of the formula IA1, IA4, IA7, IA8 or IA9.For RA1 or RA2 independently of one another a branched or unbranched, optionally substituted C1-C6-alkyl radical is understood as meaning, for example, the corresponding radicals described above for RG1, preferably methyl or trifluoromethyl.","For RA1 or RA2 in the structural elements IA1, IA2, IA3 or IA17, the branched or unbranched, optionally substituted radical CO—C1-C6-alkyl is composed, for example, of the group CO and the branched or unbranched, optionally substituted C1-C6-alkyl radicals described above for RA1 or RA2.Optionally substituted hetaryl, hetarylalkyl, aryl, arylalkyl or C3-C7-cycloalkyl radicals for RA1 or RA2 independently of one another are understood as meaning, for example, the corresponding radicals described above for RG7.For RA1 or RA2, the optionally substituted radicals CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 are composed, for example, of the groups CO—O, O, S, N, CO—N or SO2—N and the radicals RA14, RA15 or RA16 described in greater detail below.","Further, both radicals RA1 and RA2 can together form a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group consisting of O, N and S. RA13 and RA13* are independently of one another hydrogen, CN, halogen, such as fluorine, chlorine, bromine or iodine, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as described above for RG1, preferably methyl or trifluoromethyl or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2NRA15RA16 or CO—NRA15RA16 as in each case described above for RA1.Preferred radicals for RA13 and RA13* are the radicals hydrogen, F, Cl, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or arylalkyl or a radical CO—O—RA14, O—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16.A branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, alkylenecycloalkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl or C2-C6-alkynyl radical for RA14 in structural element A is understood as meaning, for example, the corresponding radicals described above for RG7.Optionally substituted aryl, arylalkyl, hetaryl or alkylhetaryl radicals for RA14 in structural element A are understood as meaning, for example, the corresponding radicals described above for RG7.Preferred radicals for RA14 are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical and optionally substituted benzyl.","A branched or unbranched, optionally substituted C1-C6-alkyl or arylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or hetarylalkyl radical for RA15 or RA16 independently of one another is understood as meaning, for example, the corresponding radicals described above for RA14.The branched or unbranched, optionally substituted CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, COO-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or SO2-alkylenearyl radicals or the optionally substituted CO-aryl, SO2-aryl, CO—NH-aryl, CO—NH-hetaryl or CO-hetaryl radicals for RA15 or RA16 are composed, for example, of the corresponding groups —CO—, —SO2—, —CO—O—, —CO—NH— and the corresponding branched or unbranched, optionally substituted C1-C6-alkyl, hetarylalkyl or arylalkyl radicals or the corresponding optionally substituted aryl or hetaryl radicals described above.","A radical —(CH2)n—(XA)j—RA12 for RA3 or RA4 independently of one another is understood as meaning a radical which is composed of the corresponding radicals —(CH2)n—, (XA)j and RA12.Here, n can be: 0, 1, 2 or 3 and j can be: 0 or 1.XA is a doubly bonded radical selected from the group —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— and —N(RX1)—SO2—.","RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, as described above for RG7.a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, or a 3- to 6-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, triazinyl.","Further, RA12 and RX1 or RX1* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radical RA12 together with the radical RX1 or RX1* forms a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl, where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.","A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C12-alkynyl, preferably C2-C6-alkynyl or C2-C6-alkenyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl or hetaryl radical for RX1 and RX1* independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.Preferred, branched or unbranched, optionally substituted C1-C6-alkoxyalkyl for RX1 and RX1* are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.","Preferred, branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl are preferably composed of the C1-C6-alkyl, arylalkyl, aryl or hetaryl radicals and the radicals —CO—, —O—, —SO2— described above.","Preferred radicals for RX1 and RX1* are independently of one another hydrogen, methyl, cyclopropyl, allyl and propargyl.","RA3 and RA4 can further together form a 3- to 8-membered saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, RA5 is a branched or unbranched, optionally substituted C1-C6-alkyl, arylalkyl, C1-C4-alkyl-C3-C7-cycloalkyl or C3-C7-cycloalkyl radical or an optionally substituted aryl, hetaryl, heterocycloalkyl or heterocycloalkenyl radical, such as described above for RG7.RA6 and RA6* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, —CO—O—C1-C4-alkyl or —CO—C1-C4-alkyl radical such as composed of the group —CO—O— or —CO— and the C1-C4-alkyl radicals described above, arylalkyl radical, as described above for RG7, —CO—O-alkylenearyl or —CO-alkylenearyl radical such as composed of the group —CO—O— or —CO— and the arylalkyl radicals described above, —CO—O-allyl or —CO-allyl radical, or C3-C7-cycloalkyl radical, such as described above for RG7.Further, both radicals RA6 and RA6* in structural element IA7 can together form an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl radical, for example as described above for RA6, C1-C4-alkoxy, arylalkyl or C3-C7-cycloalkyl radical, for example as described above for RL14, a branched or unbranched, optionally substituted —O—CO—C1-C4-alkyl radical, which is composed of the group —O—CO— and, for example, of the C1-C4-alkyl radicals mentioned above or an optionally substituted —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical which is composed of the groups —O— or —O—CO— and, for example, of the corresponding radicals described above for RG7.Further, both radicals RA6 and RA7 can together form an optionally substituted unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. For RAS in structural element A, the branched or unbranched, optionally substituted C1-C4-alkyl radical or an optionally substituted aryl or arylalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA15, where the radicals CO—C1-C4-alkyl, SO2—C1-C4-alkyl, CO—O—C1-C4-alkyl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl or CO—O-alkylenearyl are composed analogously to the other composed radicals of the group consisting of CO, SO2 and COO and, for example, of the corresponding C1-C4-alkyl, aryl or arylalkyl radicals described above for RA15 and these radicals can be optionally substituted.","In each case, for RA9 or RA10 independently of one another, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA14, preferably methyl or trifluoromethyl.","In each case, for RA9 or RA10 independently of one another, a radical CO—O—RA14, O—RA14, S—RA14, SO2—NRA15RA16, NRA15RA16 or CO—NRA15RA16 is understood as meaning, for example, the corresponding radicals described above for RA13.Further, both radicals RA9 and RA10 together in structural element IA14 can form a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.","Substituents in this case are in particular understood as meaning halogen, CN, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl or trifluoromethyl or the radicals O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or —((RA8)HN)C═N—RA7.A branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16 for RA11 is understood, for example, as meaning the corresponding radicals described above for RA9.Further, in structural element IA16, both radicals RA9 and RA17 together can form a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.","A branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 for RA18 and RA19 independently of one another is understood as meaning, for example, the radicals described above for RG12, preferably hydrogen or a branched or unbranched, optionally substituted C1-C8-alkyl radical.","Z1, Z2, Z3, Z4 are independently of one another nitrogen, C—H, C-halogen, such as C—F, C—Cl, C—Br or C—I or a branched or unbranched, optionally substituted C—C1-C4-alkyl radical which is composed of a carbon radical and, for example, a C1-C4-alkyl radical described above for RA6 or a branched or unbranched optionally substituted C—C1-C4-alkoxy radical which is composed of a carbon radical and, for example, a C1-C4-alkoxy radical described above for RA7.Z5 is oxygen, sulfur or a radical NRA8.Preferred structural elements A are composed of at least one preferred radical of the radicals belonging to the structural element A, while the remaining radicals are widely variable.","Particularly preferred structural elements A are composed of the preferred radicals of the structural element A.","In a preferred embodiment, the spacer structural element E is understood as meaning a structural element that consists of a branched or unbranched aliphatic C2-C30-hydrocarbon radical which is optionally substituted and contains heteroatoms and/or of a 4- to 20-membered aliphatic or aromatic mono- or polycyclic hydrocarbon radical which is optionally substituted and contains heteroatoms.","In a further preferred embodiment, the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently, of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.","The coefficient c is preferably 0 or 1, the coefficient d is preferably 1 or 2, and the coefficients f, g, h independently of one another are preferably 0 or 1.An optionally substituted 4- to 11-membered mono- or polycyclic aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, S, where the ring carbons or ring nitrogens can optionally be substituted, for QE and XE independently of one another is preferably understood as meaning optionally substituted arylene, such as optionally substituted phenylene or naphthylene, or optionally substituted hetarylene such as the radicals and their substituted or fused derivatives, or radicals of the formulae IE1 to IE11, where the incorporation of the radicals can take place in both orientations.","Aliphatic hydrocarbons are understood as meaning, for example, saturated and unsaturated hydrocarbons.","Z6 and Z7 are independently of one another CH or nitrogen.","Z8 is oxygen, sulfur or NH.","Z9 is oxygen, sulfur or NRE20.r1, r2, r3 and t are independently of one another 0, 1, 2 or 3.s and u are independently of one another 0, 1 or 2.Particularly preferably, XE and QE independently of one another are optionally substituted phenylene, a radical and their substituted or fused derivatives, or radicals of the formulae IE1, IE2, IE3, IE4 and IE7, where the incorporation of the radicals can take place in both orientations.","RE18 and RE19 are independently of one another hydrogen, —NO2, —NH2, —CN, —COOH, a hydroxyl group, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, as in each case described above.","RE20 is independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C3-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, preferably hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl radical.","YE and ZE are independently of one another CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, preferably CO, SO2 and oxygen.","RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C8-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical, such as correspondingly described above for RG7 or a radical CO—RE16, COORE16 or SO2—RE16, preferably hydrogen, methyl, allyl, propargyl and cyclopropyl.","A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE13, RE14 or RE15 independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.A branched or unbranched, optionally substituted C1-C4-alkoxy radical for RE13 or RE14 independently of one another is understood as meaning, for example, the C1-C4-alkoxy radicals described above for RA14.Preferred alkylenecycloalkyl radicals for RE13, RE14 or RE15 independently of one another are, for example, the C1-C4-alkylene-C3-C7-cycloalkyl radicals described above for RG7.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical for RE16 is understood as meaning, for example, the corresponding radicals described above for RG11.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Further, two radicals RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 can in each case independently of one another together form a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle which can contain up to three heteroatoms from the group O, N and S. The radical —(CH2)x—(WE)z—RE17 is composed of a C0-C4-alkylene radical, optionally a bonding element WE selected from the group —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, preferably selected from the group —CO—N(RW2)—, —N(RW2)—CO—, —O—, —SO2—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, and the radical RE17, where RW2 and RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, preferably independently of one another are hydrogen, methyl, cyclopropyl, allyl, propargyl, and RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3 to 8-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl or triazinyl.","Further, RE17 and RW2 or RW2* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radicals RE17 and RW2 or RW2* together form a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.","Preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or the radical —(CH2)x—(WE)z—RE17.Particularly preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, F, a branched or unbranched, optionally substituted C1-C4-alkyl radical, in particular methyl.","A branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical or an optionally substituted aryl, hetaryl or C3-C7-cycloalkyl for RE11 and RE11* in structural element E independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.The branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or the optionally substituted radicals CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl for RE11 and RE11* independently of one another are composed, for example, of the corresponding groups CO, COO, CONH or SO2 and the corresponding radicals mentioned above.","Preferred radicals for RE11 or RE11* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical, or an optionally substituted hetaryl or C3-C7-cycloalkyl radical.","Particularly preferred radicals for RE11 or RE11* are hydrogen, methyl, cyclopropyl, allyl or propargyl.","In a particularly preferred embodiment of structural element E1, structural element E1 is a radical —CH2—CH2—CO—, —CH2—CH2—CH2—CO— or a C1-C5-alkylene radical.","In a particularly preferred embodiment of structural element E, the spacer structural element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where the structural elements E2 and E1 have the meanings described above.","Preferred structural elements E are composed of at least one preferred radical of the radicals belonging to structural element E, while the remaining radicals are widely variable.","Particularly preferred structural elements E are composed of the preferred radicals of structural element E. Preferred structural elements B are composed either of the preferred structural element A, while E is widely variable, or of the preferred structural element E, while A is widely variable.","In a further preferred embodiment, the structural element E used is the structural element E′ described below for the novel compounds of the formula I′.","In a further preferred embodiment, the structural element G used is the structural element G′ described below for the novel compounds of the formula I′.","The compounds of the formula I, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.","The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.","The use of an enantiomerically pure compound as the active compound is preferred.","The compounds of the formula I can also be present in other tautomeric forms.","The compounds of the formula I can also be present in the form of physiologically tolerable salts.","The compounds of the formula I can also be present as prodrugs in a form in which the compounds of the formula I are liberated under physiological conditions.","By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.","Derivatized structural elements B or A are also suitable which liberate the structural element B or A respectively under physiological conditions.","In preferred compounds of the formula I, in each case one of the three structural elements B, G or L has the preferred range, while the remaining structural elements are widely variable.","In particularly preferred compounds of the formula I, in each case two of the three structural elements B, G and L have the preferred range, while the remaining structural elements are widely variable.","In very particularly preferred compounds of the formula I, in each case all three structural elements B, G and L have the preferred range, while the remaining structural element is widely variable.","Preferred compounds of the formula I contain, for example, the preferred structural element G, while the structural elements B and L are widely variable.","In particularly preferred compounds of the formula I, for example, B is replaced by the structural element A-E- and the compounds contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.","Further particularly preferred compounds of the formula I contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.","The invention further relates to the use of the structural element of the formula IGL -G-L IGL for the preparation of compounds which bind to integrin receptors.","The compounds of the formula I bind to integrin receptors.","The compounds of the formula I are therefore preferably suitable as integrin receptor ligands and for the production of drugs for treating diseases in which an integrin receptor is involved, in particular for treating diseases in which the interaction between integrins and their natural ligands is dysregulated, i.e.","excessive or decreased.","Integrin receptor ligands are understood as meaning agonists and antagonists.","An excessive or decreased interaction is understood as meaning both an excessive or decreased expression of the natural ligand and/or of the integrin receptor and thus an excessive or decreased amount of natural ligand and/or integrin receptor or an increased or decreased affinity of the natural ligand for the integrin receptor.","The interaction between integrins and their natural ligands is dysregulated compared with the normal state, i.e.","excessive or decreased, if this dysregulation does not correspond to the physiological state.","An increased or decreased interaction can lead to pathophysiological situations.","The level of dysregulation which leads to a pathophysiological situation is dependent on the individual organism and on the site and nature of the disorder.","Preferred integrin receptors for which the compounds of the formula I according to the invention can be used are the α5β1, α4β1, αVβ5 and αVβ3 integrin receptors.","The compounds of the formula I particularly preferably bind to the αVβ3 integrin receptor and can thus be particularly preferably used as ligands of the αVβ3 integrin receptor and for the treatment of illnesses in which the interaction between αVβ3 integrin receptor and its natural ligand is excessive or reduced.","The compounds of the formula I are preferably used for the treatment of the following illnesses: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury or stent implantation, and angioplasty (neointima formation, smooth muscle cell migration and proliferation), acute kidney failure, angiogenesis-associated microangiopathies such as diabetic angiopathies or retinopathy or rheumatoid arthritis, blood platelet-mediated vascular occlusion, arterial thrombosis, stroke, reperfusion damage after myocardial infarct or stroke, carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis), osteoporosis (bone resorption after chemotaxis and adhesion of osteoclasts to the bone matrix), high blood pressure, psoriasis, hyperparathyroidism, Paget's disease, malignant hypercalcemia, metastatic osteolytic lesions, inflammation, wound healing, cardiac insufficiency, congestive heart failure CHF, as well as in antiviral, antimycotic, antiparasitic or antibacterial therapy and prophylaxis (adhesion and internalization).","Furthermore, the invention relates in particular to the use of the compounds of the formula I as ligands of the αVβ3 integrin receptor.","The invention furthermore relates to the novel compounds of the formula I′ A-E′-G′-L I′ where the structural elements A and L have the meanings described above.","Preferred structural elements A and L are described.","Structural element E′ is composed of two to four substructural elements selected from the group consisting of E1 and E2, the linkage sequence of the substructural elements being arbitrary and E1 and E2 having the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(X)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case in which YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical of the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.","In a further preferred embodiment of structural element E′, the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.","Further preferred embodiments of structural element E′ correspond to the preferred embodiments of structural element E with the proviso described above.","Structural element G′ is identical to structural element G, as described above, except for the radicals RG12 and RG13.In structural element G′, the radicals RG12 and RG13 of structural element G are replaced by the radicals RG′12 and RG′13.The radicals RG12 and RG′13 in structural element G′ have the following meanings: independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG14, where RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical.","Preferred radicals of RG′12 and RG13 are the corresponding radicals described above for RG12 and RG13.Further preferred embodiments of the structural element G′ correspond to the preferred embodiments of structural element G. The compounds of the formula I′, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.","The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.","The use of an enantiomerically pure compound as active compound is preferred.","The compounds of the formula I′ can also be present in other tautomeric forms.","The compounds of the formula I′ can also be present in the form of physiologically tolerable salts.","The compounds of the formula I′ can also be present as prodrugs in a form in which the compounds of the formula I′ are liberated under physiological conditions.","By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.","Derivatized structural elements A which liberate the structural element A under physiological conditions are also suitable.","Analogously to the compounds of the formula I, the following applies for the compounds of the formula I′ as mentioned above: In preferred compounds of the formula I′, one of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.","In particularly preferred compounds of the formula I′, two of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.","In further particularly preferred compounds of the formula I′, three of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural element is widely variable.","In very particularly preferred compounds of the formula I′, all four structural elements A, E′, G′ or L have the preferred range.","Preferred compounds of the formula I, have, for example, the preferred structural element G′, while the structural elements A, E′ and L are widely variable.","Further particularly preferred compounds of the formula I′ have, for example, the preferred structural element G′ and the preferred structural element A, while the structural elements E′ and L are widely variable.","Very particularly preferred compounds of the formula I′ are listed below, the number before the text block being the number of an individualized compound of the formula I′, and in the text block A-E′-G′-L the abbreviations separated by a bonding dash in each case being an individual structural element A, E′, G′ or L and the meaning of the abbreviations of the structural elements being explained after the table.","No.","A-E′-G′-L 1 bhs-edia3-phen-es 2 2py-inda2-phen-es 3 bhs-35thirna2-meph-es 4 bim-dibema2-dmeph-es 5 2py-bam2-4-clph-es 6 2py-dibema2-dmeph-es 7 bhs-a24thima2-dmeph-es 8 bhs-aaf-phen-es 9 bhs-a24thima2-4-clph-es 10 bim-me42thiaz2-phen-es 11 2py-edia2-phen-es 12 bim-a24thima2-hdb-es 13 2py-apma2-4-clph-es 14 gua-chex2-phen-es 15 bhs-dibema2-4-clph-es 16 bhs-bam2-phen-es 17 bim-a23thima2-4-clph-es 18 bim-dibema2-phen-es 19 bim-bam2-4-clph-es 20 2py-pipa2-4-clph-es 21 2py-me25thima2-phen-es 22 gua-a24thima2-ioph-es 23 imhs-apma2-phen-es 24 2py-apma2-ioph-es 25 gua-edia3-phen-es 26 bhs-a23thima2-4-clph-es 27 2py-a24thima2-2pyph-es 28 2py-bam2-thoph-es 29 gua-apma2-phen-es 30 2py-a24thima2-reph-es 31 gua-hexa-phen-es 32 dimethpym-apma2-phen-es 33 2py-dibema2-meph-es 34 bhs-apma2-phen-es 35 bim-edia3-phen-es 36 gua-apma2-meph-es 37 bim-apma2-reph-es 38 2py-a24thima2-dmeph-es 39 gua-aaf-phen-es 40 gua-apma2-yrph-es 41 gua-pipeme2-phen-es 42 2py-35thima2-phen-es 43 gua-a24thima2-reph-es 44 bim-bam2-phen-es 45 gua-a24thima2-phen-ms 46 gua-apma2-reph-es 47 mam2py-a24thima2-phen-es 48 2py-a24thima2-phen-gs 49 2py-apma2-phen-nes 50 2py-a24thima2-4-clph-es 51 bhs-penta-phen-es 52 gua-35thima2-dmeph-es 53 bim-dibema2-thoph-es 54 bim-a24thima2-reph-es 55 2py-a23thima2-4-clph-es 56 gua-a23thima2-phen-es 57 dhim-a24thima2-phen-es 58 gua-penta-phen-es 59 bhs-a24thima2-reph-es 60 2py-a23thima2-meph-es 61 gua-prodia2-phen-es 62 bhs-apma2-reph-es 63 2py-apma2-meph-es 64 gua-bam2-4-clph-es 65 2py-me35thima2-phen-es 66 gua-apma2-2pyph-es 67 2py-35thima2-thoph-es 68 clim-a24thima2-phen-es 69 2py-buta-phen-es 70 am2py-apma2-phen-es 71 gua-a24thima2-dmeph-es 72 bhs-apma2-hdb-es 73 bhs-dibema2-thoph-es 74 bim-dibema2-meph-es 75 bhs-bam2-meph-es 76 bhs-apma2-yrph-es 77 bhs-apma2-dmeph-es 78 gua-me25thima2-phen-es 79 2py-a24thima2-meph-es 80 gua-inda2-phen-es 81 bhs-mepipe2-phen-es 82 bhs-a24thima2-phen-as 83 bim-pipa2-thoph-es 84 bhs-bam2-4-clph-es 85 bhs-a24thima2-phen-es 86 bhs-35thima2-phen-es 87 bim-penta-phen-es 88 bhs-apma2-dbph-es 89 gua-42thiaz2-phen-es 90 bhs-a24thima2-24pym-es 91 2py-dibema2-phen-es 92 bim-a24thima2-dm-es 93 bhs-pipa2-4-clph-es 94 2py-apma2-phen-ps 95 2py-apma2-dmeph-es 96 2py-mepipe2-phen-es 97 bhs-35thima2-4-clph-es 98 gua-apma2-phen-mals 99 gua-35thima2-4-clph-es 100 mam2py-apma2-phen-es 101 gua-buta-phen-es 102 2py-a23thima2-thoph-es 103 2py-pyma2-phen-es 104 gua-apma2-phen-gs 105 bim-apma2-phen-as 106 bhs-apma2-4-clph-es 107 bhs-a24thima2-4-pyph-es 108 thpym-apma2-phen-es 109 gua-pipa2-dmeph-es 110 amim-a24thima2-phen-es 111 bim-aepi2-phen-es 112 bim-a23thima2-thoph-es 113 bhs-a23thima2-thoph-es 114 gua-pdagk-phen-es 115 2py-hexa-phen-es 116 bhs-a24thima2-meph-es 117 gua-bam2-meph-es 118 2py-35thima2-meph-es 119 2py-pipa2-meph-es 120 bhs-a23thima2-phen-es 121 bim-pipeme2-phen-es 122 bhs-buta-phen-es 123 bhs-pipa2-dmeph-es 124 bhs-pipa2-meph-es 125 2py-35thima2-dmeph-es 126 bim-bam2-thoph-es 127 gua-35thima2-thoph-es 128 bim-edia2-phen-es 129 bim-apma2-phen-nes 130 gua-bam2-thoph-es 131 gua-bam2-phen-es 132 pippy-a24thima2-phen-es 133 gua-35thima2-phen-es 134 bim-a23thima2-phen-es 135 gua-dibema2-dmeph-es 136 bhs-apma2-phen-gs 137 bhs-apma2-thoph-es 138 2py-apma2-phen-es 139 im-a24thima2-phen-es 140 gua-aepi2-phen-es 141 2py-mea2-phen-es 142 gua-a24thima2-phen-es 143 2py-a24thima2-thaph-es 144 gua-apma2-4-clph-es 145 bhs-apma2-phen-f2es 146 bhs-inda2-phen-es 147 bim-a24thima2-dbph-es 148 bim-apma2-phen-ms 149 gua-a23thima2-thoph-es 150 pippy-apma2-phen-es 151 bhs-apma2-meph-es 152 2py-apma2-phen-as 153 gua-mea2-phen-es 154 bhs-me35thima2-phen-es 155 bhs-pdagk-phen-es 156 bim-42thiaz2-phen-es 157 2py-pipeme2-phen-es 158 bim-me25thima2-phen-es 159 gua-dibema2-phen-es 160 2py-apma2-oxph-es 161 bhs-a24thima2-3clph-es 162 bim-pyma2-phen-es 163 bhs-edia2-phen-es 164 imhs-a24thima2-phen-es 165 gua-dibema2-thoph-es 166 bim-a24thima2-4-clph-es 167 bim-apma2-phen-ps 168 gua-apma2-dm-es 169 2py-a24thima2-phen-mals 170 2py-dibema2-4-clph-es 171 bhs-dibema2-meph-es 172 bim-aof-phen-es 173 bhs-pipeme2-phen-es 174 gua-35thima2-meph-es 175 bim-aaf-phen-es 176 bim-dibema2-4-clph-es 177 bim-a24thima2-2pyph-es 178 bim-a24thima2-yrph-es 179 bim-35thima2-4-clph-es 180 bhs-aepi2-phen-es 181 bim-pipa2-phen-es 182 gua-a23thima2-dmeph-es 183 2py-a24thima2-yrph-es 184 gua-a24thima2-dmeoph-es 185 bhs-42thiaz2-phen-es 186 bim-mepipe2-phen-es 187 2py-chex2-phen-es 188 bhs-prodia2-phen-es 189 gua-bam2-dmeph-es 190 bhs-me25thima2-phen-es 191 thpym-a2.4thima2-phen-es 192 bim-pipa2-dmeph-es 193 bhs-dibema2-phen-es 194 gua-a24thima2-thoph-es 195 2py-pipa2-thoph-es 196 clim-apma2-phen-es 197 bhs-chex2-phen-es 198 gua-a24thima2-phen-nes 199 gua-a24thima2-meph-es 200 bhs-a24thima2-dmeoph-es 201 2py-apma2-24pym-es 202 2py-a24thima2-phen-pms 203 gua-a24thima2-4-piph-es 204 bim-apma2-3clph-es 205 gua-apma2-hdb-es 206 2py-bam2-meph-es 207 bhs-a24thima2-phen-nes 208 gua-pyma2-phen-es 209 bim-a24thima2-thoph-es 210 gua-mepipe2-phen-es 211 bim-apma2-4-clph-es 212 2py-42thiaz2-phen-es 213 bim-apma2-24pym-es 214 bhs-a23thima2-dmeph-es 215 gua-apma2-dbph-es 216 gua-me35thima2-phen-es 217 bim-35thima2-phen-es 218 gua-apma2-thaph-es 219 bim-35thima2-thoph-es 220 gua-apma2-phen-f2es 221 2py-apma2-3clph-es 222 gua-apma2-thoph-es 223 bim-pipa2-meph-es 224 2py-aof-phen-es 225 bhs-35thima2-dmeph-es 226 bhs-hexa-phen-es 227 bim-pipa2-4-clph-es 228 bhs-apma2-phen-pms 229 bim-a23thima2-dmeph-es 230 2py-me42thiaz2-phen-es 231 bim-a24thima2-phen-gs 232 bim-apma2-dmeph-es 233 gua-a23thima2-4-clph-es 234 bim-a24thima2-phen-mals 235 2py-apma2-phen-ms 236 bhs-a24thima2-ioph-es 237 bim-a24thima2-phen-es 238 2py-pdagk-phen-es 239 gua-a24thima2-phen-ps 240 2py-pipa2-phen-es 241 2py-aepi2-phen-es 242 2py-a24thima2-thoph-es 243 bhs-bam2-dmeph-es 244 bim-hexa-phen-es 245 bim-a24thima2-meph-es 246 bhs-me42thiaz2-phen-es 247 am2py-a24thima2-phen-es 248 bim-apma2-meph-es 249 bim-me35thima2-phen-es 250 gua-pipa2-phen-es 251 bhs-a24thima2-oxph-es 252 2py-pipa2-dmeph-es 253 2py-apma2-4-pyph-es 254 bhs-35thima2-thoph-es 255 gua-me42thiaz2-phen-es 256 bim-a24thima2-thaph-es 257 gua-a24thima2-phen-as 258 2py-a24thima2-phen-f2es 259 2py-a24thima2-dbph-es 260 bim-35thima2-meph-es 261 bim-apma2-phen-es 262 bim-a24thima2-phen-pms 263 bim-chex2-phen-es 264 bim-a24thima2-dmeph-es 265 bim-mea2-phen-es 266 2py-apma2-thoph-es 267 dimethpym-a24thima2-phen-es 268 2py-dibema2-thoph-es 269 2py-apma2-dmeoph-es 270 gua-dibema2-4-clph-es 271 bhs-pipa2-phen-es 272 gua-edia2-phen-es 273 gua-apma2-dmeph-es 274 2py-edia3-phen-es 275 gua-a23thima2-meph-es 276 bim-pdagk-phen-es 277 gua-apma2-phen-pms 278 bhs-mea2-phen-es 279 bim-35thima2-dmeph-es 280 bhs-aof-phen-es 281 2py-prodia2-phen-es 282 bim-inda2-phen-es 283 bhs-bam2-thoph-es 284 bim-apma2-4-pyph-es 285 2py-aaf-phen-es 286 2py-bam2-phen-es 287 bhs-apma2-dm-es 288 2py-penta-phen-es 289 gua-aof-phen-es 290 im-apma2-phen-es 291 gua-pipa2-4-clph-es 292 bim-apma2-ioph-es 293 bim-bam2-meph-es 294 gua-pipa2-meph-es 295 bhs-apma2-thaph-es 296 bhs-apma2-2pyph-es 297 bim-apma2-dmeoph-es 298 amim-apma2-phen-es 299 dhim-apma2-phen-es 300 bhs-a24thima2-phen-ps 301 2py-a23thima2-dmeph-es 302 gua-pipa2-thoph-es 303 bim-a23thima2-meph-es 304 2py-bam2-dmeph-es 305 bhs-a24thima2-thoph-es 306 bhs-apma2-phen-mals 307 bhs-a23thima2-meph-es 308 bim-buta-phen-es 309 2py-apma2-reph-es 310 gua-dibema2-meph-es 311 2py-a24thima2-hdb-es 312 gua-a24thima2-4-clph-es 313 bhs-pipa2-thoph-es 314 gua-a24thima2-3clph-es 315 gua-a24thima2-oxph-es 316 bim-bam2-dmeph-es 317 bim-apma2-thoph-es 318 bim-apma2-oxph-es 319 2py-a24thima2-phen-es 320 bhs-a24thima2-phen-ms 321 bim-prodia2-phen-es 322 2py-a24thima2-dm-es 323 bhs-pyma2-phen-es 324 bim-a24thima2-phen-f2es 325 2py-a23thima2-phen-es 326 gua-a24thima2-24pym-es 327 2py-35thima2-4-clph-es 328 bhs-dibema2-dmeph-es In the above list, the following abbreviations are used for the structural units A, E′, G′ and L. A= Abbreviation 2py dhim bim imhs dimethpym mam2py am2py thpym bhs gua amim clim im pippy E′= Abbreviation edia2 pyma2 pipa2 aepi2 me35thima2 dibema2 edia3 buta aaf 42thiaz2 chex2 bam2 apma2 pdagk mepipe2 prodia2 inda2 35thima2 me25thima2 penta aof hexa mea2 pipeme2 me42thiaz2 a23thima2 a24thima2 The bond to the structural unit L should be understood as meaning a double bond in the compound where L=as.","G′= Abbreviation 24pym dmeph dm thoph thaph 2pyph 4clph phen dbph hdb dmeoph meph 3clph oxph ioph yrph 4pyph reph L= Abbreviation es gs pms f2es mals ps ms nes as The compounds of the general formula I and thus also the compounds of the formula I′ as well as the starting substances used for their preparation can be prepared by methods of organic chemistry known to the person skilled in the art, such as are described in standard works such as Houben-Weyl, “Methoden der Organischen Chemie” [Methods of Organic Chemistry], Thieme-verlag, Stuttgart, or March “Advanced Organic Chemistry”, 4th Edition, Wiley & Sons.","Further preparation methods are also described in R. Larock, “Comprehensive Organic Transformations”, Weinheim 1989, in particular the preparation of alkenes, alkynes, halides, amines, ethers, alcohols, phenols, aldehydes, ketones, nitrites, carboxylic acids, esters, amides and acid chlorides.","The synthesis of compounds of the formula I can be carried out either according to the “classical” method in solution or on a polymeric support, in each case reaction conditions as are known and suitable for the respective reactions being used.","Use can also be made in this case of variants which are known per se but not mentioned here.","The general synthesis of compounds of the formula I is described in schemes 1-7.If not stated otherwise, all starting materials and reagents are commercially available, or can be prepared from commercially obtainable precursors by customary methods.","Fused 2,3,4,5-tetrahydro-1H-azepinediones of type II are known and can be prepared by known methods, e.g.","starting from anthranilic acid esters or the corresponding heterocyclic analogs via Dieckmann condensation and subsequent decarboxylation, as is described in the following publications: J.","Am.","Chem.","Soc.","80, 1958, 2172-2178; J. Chem.","Soc.","1959, 3111; J. Chem.","Soc.","1934, 1326; Arch.","Pharm.","324, 1991, 579-581.The preparation of 3,4-dihydro-1H-azepine-2,5-dione is described in Heterocycles 8, 1977, 345-350.The conversion into compounds of the type III is generally carried out by methods known to the person skilled in the art, such as are described, for example, in Larock, “Comprehensive Organic Transformations”, Weinheim 1989, pp.","167ff, although methods which are not mentioned here can also be used.","Preferably, compounds of the general formula III can be prepared by reaction of the ketones II with a phosphonic ester of the general formula (EtO)2P(═O)(XL)a(CRL1RL2)b—COOSG1 in the presence of a base.","The reaction preferably takes place in a polar aprotic solvent, such as tetrahydrofuran, dioxane; dimethylformamide (DMF), dimethylacetamide or acetamide; dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; in a temperature range depending on the nature of the solvent used from −40° C. up to the boiling point of the corresponding solvent.","The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.","sodium carbonate or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such lithium diisopropylamide, or lithium, sodium or potassium bis(trimethylsilyl)amide.","The reaction to give IV is carried out by hydrogenation of the double bond under standard conditions.","Use can also be made here of variants which are known per se but not mentioned.","Preferably, the hydrogenation is carried out in the presence of a noble metal catalyst, such as Pd on activated carbon, Pt, PtO2, Rh on Al2O3 in an inert solvent at a temperature of 0-150° C. and a pressure of 1-200 bar; the addition of an acid such as acetic acid or hydrochloric acid can be advantageous.","Hydrogenation in the presence of 5-10% Pd on activated carbon is particularly preferred.","Solvents which can be used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; sulfoxides such as dimethyl sulfoxide, sulfolane; pyridine, N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, water or mixtures of the solvents mentioned.","Compounds of type V are prepared by reaction with compounds of the general formula A-E-X1 (VI), the radical X1 being a customary leaving group, for example halogen such as chlorine, bromine, iodine or aryl- or alkylsulfonyl optionally substituted by halogen, alkyl or haloalkyl, such as toluenesulfonyl, trifluoromethanesulfonyl and methylsulfonyl or another equivalent leaving group.","The reaction preferably takes place in an inert solvent (such as previously described) with addition of a suitable base, i.e.","of a base which brings about deprotonation of the intermediate IV, in a temperature range from −40° C. up to the boiling point of the corresponding solvent.","The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.","sodium or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such as lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide or potassium bis(trimethylsilyl)amide.","Removal of the protective group SG1 according to standard conditions (see below) leads to the compounds of the general formula I.","If SG1 is C1-C4-alkyl or benzyl, the compounds of the general formula V correspond directly to the compounds of type I. Alternatively to this synthesis strategy, compounds of type I can also be prepared via VII as an intermediate, here too reaction conditions being used such as are known to the person skilled in the art and described in standard works.","Compound VII is prepared by reaction of compounds of type IV having radio of the general formula DE-E-X2 (VIII) under reaction conditions such as have already been described for the preparation of V from IV and VI.","X2 here is a suitable leaving group, as has already been described for X1, and DE is CN, N3 or a protected amino or acid function of the general formula NSG3 or COOSG2.The synthesis of the fragments DE-E or A-E is carried out—depending on the actual structure of E—by removal of the protective groups and coupling of the remaining fragments according to standard methods, e.g.","amide couplings.","The introduction of A is then carried out analogously to the reactions described in schemes 3-7.Compounds of the formula I in which WG in structural element G is a structural element of the formula IWG1 can be prepared according to scheme 2.The starting point of the synthesis is compounds of type IX, which are either known or are accessible by methods known to the person skilled in the art, such as are described, for example, in J.","Am.","Chem.","Soc.","71, 1949, 1985.Alkylation with a compound of the general formula XIII (X3, X4=a customary leaving group) under customary reaction conditions leads to X.","The further reactions to give I then proceed analogously to scheme 1 via compounds of type XI.","In the case in which WG in structural element G is a structural element of the formula IWG3, compounds of type III can be converted into compounds of type XII and then into I analogously to the preparation of V (scheme 2).","The coupling of the individual fragments and the removal of the protective groups can be carried out according to known processes (see Larock, “Comprehensive Organic Transformations; protective groups: Greene, T., “Protective Groups in Organic Synthesis”, New York 1991), in the case of amide bonds also analogously to the methods of peptide synthesis, such as are described in standard works, e.g.","in Bodanszky “The Practice of Peptide Synthesis”, 2nd Edition, Springer-Verlag 1994, and Bodanszky “Principles of Peptide Synthesis”, Springer-Verlag 1984.A general survey of the customary methods for peptide synthesis and a listing of suitable reagents can furthermore be found in NOVABIOCHEM 1999 “Catalog and Peptide Synthesis Handbook”.","The amide couplings mentioned can be carried out with the aid of customary coupling reagents using suitably protected amino and carboxylic acid derivatives.","Another method consists in the use of preactivated carboxylic acid derivatives, preferably of carboxylic acid halides, symmetrical or mixed anhydrides or so-called active esters, which are customarily used for the acylation of amines.","These activated carboxylic acid derivatives can also be prepared in situ.","As a rule, the couplings can be carried out in inert solvents in the presence of an acid-binding agent, preferably of an organic base such as triethylamine, pyridine, diisopropylethylamine, N-methylmorpholine, quinoline; the addition of an alkali metal or alkaline earth metal hydroxide, carbonate or hydrogencarbonate or of another salt of a weak acid of the alkali metals or alkaline earth metals, preferably of potassium, sodium, calcium or cesium, can also be favorable.","Depending on the conditions used, the reaction time is between minutes and 14 days, the reaction temperature between −40° C. and 140° C., preferably between −20° C. and 100° C. Suitable inert solvents are, for example, hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate; water; or mixtures of the solvents mentioned.","Protective groups SG which can be used are all protective groups which are known from peptide synthesis and customary to the person skilled in the art, such as are also described in the abovementioned standard works.","The protective groups in the compounds of the formulae V, VII, XI and XII are likewise removed according to conditions such as are known to the person skilled in the art and are described by Greene and Wuts in “Protective Groups in organic Synthesis”, 2nd Edition, Wiley & Sons, 1991.Protective groups such as SG3 are so-called N-terminal amino protective groups; Boc, Fmoc, benzyloxycarbonyl (Z), acetyl, Mtr are preferred here.","SG1 and SG2 are acid protective groups; C1-C4-alkyl is preferred here, such as methyl, ethyl, tert-butyl, or alternatively benzyl or trityl, or alternatively polymer-bonded protective groups in the form of the commercially available polystyrene resins such as 2-chlorotrityl chloride resin or Wang resin (Bachem, Novabiochem).","Acid-labile protective groups (e.g.","Boc, tert-butyl, Mtr, trityl) can be removed—depending on the protective group used—using organic acids such as trifluoroacetic acid (TFA), trichloroacetic acid, perchloric acid, trifluoroethanol; but also inorganic acids such as hydrochloric acid or sulfuric acid, sulfonic acids such as benzene- or p-toluenesulfonic acid, the acids generally being employed in an excess.","HCl or TFA is preferably used.","In the case of trityl, the addition of thiols such as thioanisole or thiophenol can be advantageous.","The presence of an additional inert solvent is possible, but not always necessary.","Suitable inert solvents are preferably organic solvents, for example carboxylic acids such as acetic acid; ethers such as THF or dioxane; amides such as DMF or dimethylacetamide; halogenated hydrocarbons such as dichloromethane; alcohols such as methanol, isopropanol; or water.","Suitable solvents are also mixtures of those mentioned.","The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Base-labile protective groups such as Fmoc are cleaved by treatment with organic amines such as dimethylamine, diethylamine, morpholine or piperidine as 5-50% solutions in CH2Cl2 or DMF.","The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Acid protective groups such as methyl or ethyl are preferably cleaved by basic hydrolysis in an inert solvent.","The bases used are preferably alkali metal or alkaline earth metal hydroxides, preferably NaOH, KOH or LiOH; the solvents used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyltert-butyl ether, diisopropyl-ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; nitro compounds such as nitromethane or nitrobenzene; water or mixtures of the solvents mentioned.","The addition of a phase-transfer catalyst—depending on the solvent or solvent mixture used—may be advantageous.","The reaction temperature for these reactions is generally between −10° C. and 100° C. Hydrogenolytically removable protective groups such as benzyloxycarbonyl (Z) or benzyl can be removed, for example, by hydrogenolysis in the presence of a catalyst (e.g.","of a noble metal catalyst on activated carbon as support).","Suitable solvents are those mentioned above, in particular alcohols such as methanol, ethanol; amides such as DMF or dimethylacetamide; esters such as ethyl acetate.","As a rule, the hydrogenolysis is carried out at a pressure of 1-200 bar and temperatures between 0° and 100° C.; the addition of an acid such as acetic acid or hydrochloric acid may be advantageous.","The catalyst used is preferably 5-10% Pd on activated carbon.","The synthesis of structural units of type E is generally carried out by methods known to the person skilled in the art; the structural units used are either commercially available or accessible by methods known from the literature.","The synthesis of some of these structural units is described by way of example in the experimental section.","In the case in which the fragments QE or XE contained in the compounds of type VI and VIII are a hetaryl radical, the structural units used are either commercially available or accessible by methods known to the person-skilled in the art.","A multiplicity of preparation methods are described in detail in Houben-Weyl's “Methoden der organischen Chemie” (Vol.","E6: furans, thiophenes, pyrroles, indoles, benzothiophenes, -furans, -pyrroles; Vol.","E7: quinolines, pyridines, Vol.","E8: isoxazoles, oxazoles, thiazoles, pyrazoles, imidazoles and their benzo-fused representatives, as well as oxadiazoles, thiadiazoles and triazoles; Vol.","E9: pyridazines, pyrimidines, triazines, azepines and their benzo-fused representatives as well as purines).","The linkage of these fragments to E, depending on the structure of E, can also take place via the amino or acid function according to methods which are known to the person skilled in the art.","Structures of the general formula A-E-DE are synthesized according to methods known to the person skilled in the art, which are described, for example, in WO 97/08145.Examples of these are the conversion of compounds of the general formula: HNRE11EA1-DE (XIV) NC-EA2-DE (XV) into compounds of the general formula: A-HNRE11-EA1-DE (XVI) A-E-DE (XVII) The groups EA1 and EA in the formulae XIV-XVIII represent structural fragments which, after an appropriate modification (e.g.","reaction with suitable reagents or coupling with appropriate structural units) as a whole form the structural fragment A-E.","These structural units can then be reacted either directly—in the case of the corresponding free amines or carboxylic acids—or after removal of the protective groups—to give compounds of the general formula I (scheme 1 and 2).","In principle, A, however, can also be introduced into compounds of the type IV, as described in scheme 1, where the reaction conditions mentioned can be used just as variants which are not described here.","In schemes 3-7, a number of the methods for the introduction of A are described by way of example, in each case reaction conditions being used which are known and suitable for the respective reactions.","Use can also be made in this case of variants which are known per se but not mentioned here.","Ureas and thioureas (AE-1 to AE-3) can be prepared by customary methods of organic chemistry, e.g.","by reaction of an isocyanate or of an isothiocyanate with an amine, if appropriate in an inert solvent with heating (Houben-Weyl Volume VIII, 157ff.)","(scheme 3): Scheme 4 shows, by way of example, the preparation of compounds of the type AE-4, as is described, for example, by Blakemoore et al.","in Eur.","J. Med.","Chem.","1987 (22) 2, 91-100, or Misra et al.","in Bioorg.","Ned.","Chem.","Lett.","1994 4 (18), 2165-2170.Unsubstituted or cyclic guanidine derivatives of the general formulae AE-5 and AE-6 can be prepared by means of commercially available or simply accessible reagents, such as are described, for example, in Synlett 1990, 745, J. Org.","Chem.","1992, 57, 2497, Bioorg.","Ned.","Chem.","1996, 6, 1185-1208; Bioorg.","Med.","Chem.","1998, 1185, or Synth.","Comm.","1998, 28, 741-746 (scheme 5).","The preparation of compounds of the general formula AE-7 can be carried out analogously to U.S. Pat.","No.","3,202,660, compounds of the formulae AE-9, AE-10, AE-11 and AE-12 analogously to WO 97/08145.Compounds of the formula AE-8 can be prepared, as shown in scheme 6, e.g.","according to the method described by Perkins et al., Tetrahedron Lett.","1999, 40, 1103-1106.Scheme 5 gives a survey of the synthesis of the compounds mentioned, the circle in AE-8 representing a fused cycle, such as aryl or hetaryl.","Compounds of the general formula AE-13 can be prepared analogously to Froeyen et al., Phosphorus Sulfur silicon Relat.","Elem.","1991, 63, 283-293; AE-14 analogously to Yoneda et al., Heterocycles 1998, 15 N′-1, Spec.","Issue, 341-344 (scheme 6).","The preparation of corresponding compounds can also be carried out analogously to WO 97/36859.Compounds of the general formula AE-15 can be prepared according to Synthesis 1981, 963-965 or Synth.","Comm.","1997, 27 (15), 2701-2707; AE-16 analogously to J. Org.","Chem.","1991, 56 (6), 2260-2262 (scheme 7), the circle being a fused cycle, such as aryl, hetaryl or cycloalkyl.","The invention further relates to pharmaceutical preparations, comprising at least one compound of the formula I′ in addition to the customary pharmaceutical excipients.","The compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally) in the customary manner.","Administration can also be carried out through the nasopharynx using vapors or sprays.","Further, the compounds according to the invention can be introduced by direct contact with the affected tissue.","The dose depends on the age, condition and weight of the patient and on the manner of administration.","As a rule, the daily dose of active compound is between approximately 0.5 and 50 mg/kg of body weight in the case of oral administration and between approximately 0.1 and 10 mg/kg of body weight in the case of parenteral administration.","The novel compounds can be administered in solid or liquid form in the customary pharmaceutical administration forms, e.g.","as tablets, film-coated tablets, capsules, powders, granules, coated tablets, suppositories, solutions, ointments, creams or sprays.","These are prepared in a customary manner.","The active compounds can in this case be processed using the customary pharmaceutical excipients such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, release-delaying agents, antioxidants and/or propellants (cf.","H. Sucker et al.",": Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991).","The administration forms thus obtained normally contain the active compound in an amount from 0.1 to 90% by weight.","The invention further relates to the use of compounds of the formula I′ for the production of drugs for treating illnesses.","The compounds of the formula I′ can be used for treating human and animal illnesses.","The compounds of the formula I′ which represent the novel compounds of the formula I bind, as mentioned above, to integrin receptors.","They are therefore suitable, as mentioned above, preferably as integrin receptor ligands and for the production of drugs for treating illnesses in which an integrin receptor is involved, in particular for the treatment of illnesses in which the interaction between integrins and their natural ligands is dysregulated, i.e.","excessive or reduced, as described above.","Advantageously, the compounds of the formula I, preferably the compounds of the formula I′, can be administered in combination with at least one further compound in order to achieve an improved curative action in a number of indications.","These further compounds can have the same or a different mechanism of action as/from the compounds of the formula I.","In addition to the compounds of the formula I, preferably in addition to the compounds of the formula I′ and the customary pharmaceutical excipients, the pharmaceutical preparations can therefore contain at least one further compound, depending on the indication, in each case selected from one of the 10 groups below.","Group 1: inhibitors of blood platelelet adhesion, activation or aggregation, such as acetylsalicylic acid, lysine acetylsalicylate, piracetam, dipyridamol, abciximab, thromboxane antagonists, fibrinogen antagonists, such as tirofiban, or inhibitors of ADP-induced aggregation such as ticlopidine or clopidogrel, anticoagulants which prevent thrombin activity or formation, such as inhibitors of IIa, Xa, XIa, IXa or VIIa, antagonists of blood platelet-activating compounds and selectin antagonists for the treatment of blood platelet-mediated vascular occlusion or thrombosis, or Group 2: inhibitors of blood platelet activation or aggregation, such as GPIIb/IIIa antagonists, thrombin or factor Xa inhibitors or ADP receptor antagonists, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vessel wall transmigration, fibrinolysis-modulating compounds, such as streptokinase, tPA, plasminogen-activating stimulants, TAFI inhibitors, XIa inhibitors or PAI-1 antagonists, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists for the treatment of myocardial infarct or stroke, or Group 3: endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors for the treatment of congestive heart failure, or Group 4: thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, such as heparin or low-molecular weight heparins, inhibitors of blood platelet adhesion, activation or aggregation, such as GPIIb-IIIa antagonists or antagonists of the blood platelet adhesion and activation mediated by vWF or GPIb, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants for the treatment of restenosis after vascular injury or stent implantation, or Group 5: antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists and glycosylation inhibitors or AGE formation inhibitors or AGE breakers and antagonists of their receptors, such as RAGE, for the treatment of diabetic angiopathies, or Group 6: lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers for the treatment of atherosclerosis, or Group 7: cytostatic or antineoplastic compounds, compounds which inhibit proliferation, such as kinase inhibitors and heparin or low-molecular weight heparins or further GAGS for the treatment of cancer, preferably for the inhibition of tumor growth or metastasis, or Group 8: compounds for antiresorptive therapy, compounds for hormone exchange therapy, such as estrogen or progesterone antagonists, recombinant human growth hormone, bisphosphonates, such as alendronates compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, such as growth factor antagonists, interleukin-6 antagonists and Src tyrosine kinase inhibitors for the treatment of osteoporosis, or Group 9: TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors for the treatment of rheumatoid arthritis, or Group 10: collagenase, PDGF antagonists and MMPs for improved wound healing.","A pharmaceutical preparation comprising at least one compound of the formula I, preferably comprising at least one compound of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, is understood as meaning a combined administration of at least one of the compounds of the formula I, preferably of one of the compounds of the formula I′, with at least one further compound in each case selected from one of the groups described above and, if appropriate, pharmaceutical excipients.","Combined administration can be carried out by means of a substance mixture comprising at least one compound of the formula I, preferably of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, but also spatially and/or chronologically separate.","In the case of the spatially and/or chronologically separate administration, the administration of the components of the pharmaceutical preparation, the compounds of the formula I, preferably of the formula I′ and the compounds selected from one of the abovementioned groups, takes place spatially and/or chronologically separately.","For the treatment of restenosis after vascular injury or stenting, the administrations of the compounds of the formula I, preferably of the formula I′, can be carried out locally at the affected sites, on their own or in combination with at least one compound selected from group 4.It may also be advantageous to coat the stents with these compounds.","For the treatment of osteoporosis, it may be advantageous to carry out the administration of the compounds of the formula I, preferably of the formula I′, in combination with an antiresorptive or hormone exchange therapy.","The invention accordingly relates to the use of the abovementioned pharmaceutical preparations for the production of drugs for treating illnesses.","In a preferred embodiment, the invention relates to the use of the abovementioned combined pharmaceutical preparations for the production of drugs for treating blood platelet-mediated vascular occlusion or thrombosis when using compounds of group 1, myocardial infarct or stroke when using compounds of group 2, congestive heart failure when using compounds of group 3, restenosis after vascular injury or stent implantation when using compounds of group 4, diabetic angiopathies when using compounds of group 5, atherosclerosis when using compounds of group 6, cancer when using compounds of group 7, osteoporosis when using compounds of group 8, rheumatoid arthritis when using compounds of group 9, wound healing when using compounds of group 10.The following examples illustrate the invention, the selection of these examples being non-limiting.","I. SYNTHESIS EXAMPLES I.A Precursors Example 1 t-Butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) 22.3 g (80 mmol) of t-butyl diethylphosphonate (95%) were added dropwise at 0° C. to a suspension of 3.27 g of NaH (60%; deoiled) in ml of DMF.","The mixture was stirred until a clear solution was formed and then 12.4 g (70.9 mmol) of 3,4-dihydro-1H-1-benzazepine-2,5-dione (preparation according to Arch.","Pharm.","1991, 324, 579) in 90 ml of DMF were added dropwise at 0° C. The reaction mixture then remained standing at RT for about 3 days.","For workup, the mixture was poured into 700 ml of cold 5% NaCl solution, and the resulting yellow precipitate was filtered off with suction and washed with H2O.","The moist residue was taken up in CH2Cl2, washed with 5% NaHCO3 solution and dried over Na2SO4.The residue which remained after evaporation was treated with 150 ml of cyclohexane in the presence of heat, and after cooling, filtering off with suction and washing with n-hexane 17.5 g (90.5%) of white crystals remained; m.p.",": 136-138° C. Example 2 t-Butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetate (2) A suspension of 3 g of 10% Pd/C in 50 ml of ethanol was prehydrogenated, then a solution of compound 1 (14.7 g; 53.8 mmol) in 125 ml of ethanol and 75 ml of dioxane was added, and the mixture was hydrogenated under standard conditions until the absorption of hydrogen was complete.","After filtering off the catalyst with suction and washing it with ethanol, the filtrate was concentrated in vacuo, the oily residue was dissolved in diethyl ether and the crystallization commencing was completed by addition of n-hexane.","After filtering off the precipitate with suction and washing it with n-hexane, 14.2 g (96%) of white crystals remained; m.p.",": 101-103° C. Example 3 [5-(2-t-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) a.)","A solution of compound 2 (16.8 g; 61.1 mmol) in 60 ml of DMF was added dropwise at 10-20° C. to a suspension of 2.6 g of NaH (60%, deoiled) in 35 ml of DMF and the mixture was stirred until the appearance of an almost clear yellowish solution.","t-Methyl bromoacetate (10 g; 63.4 mmol) was then added dropwise and the mixture was stirred overnight.","For workup, the reaction mixture was poured into 400 ml of 5% cold NaCl solution and extracted 3× with 100 ml each of a diethyl ether/n-hexane mixture.","The combined extracts were then washed with H2O, 10% NaHCO3 solution and NaCl solution, dried over Na2SO4, filtered and evaporated.","The residual yellowish oil was reacted further without further purification; FAB-MS: 348 [M−H+].","b.)","Crude product 3a was dissolved in 100 ml of dioxane and 65 ml of 1N NaOH were added dropwise at RT with stirring.","After about 45′, the reaction mixture was adjusted to pH 7 using 1N KHSO4 solution, the dioxane was largely distilled off in vacuo, and the residue was diluted with H2O, adjusted to pH 9 using 1N NaOH and extracted 3× with diethyl ether.","The aqueous phase was then rendered acidic using 1N KHSO4 solution, the acid precipitating was extracted with a mixture of diethyl ether/n-hexane 4:1, and the organic phase was washed with H2O, 1N NaOH solution and NaCl solution and dried over Na2SO4.Filtration and evaporation afforded an oily residue, which could be crystallized by treatment with diethyl ether/n-hexane 1:4 (water-saturated).","Filtering with suction, washing with n-hexane and drying afforded 17.8 g (87.5%) of white crystals: m.p.",": 117-119° C. Example 4 N-[4-(Aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) a.)","20 g of tert-butyl-4-aminobenzyl carbamate (89.97 mmol)—dissolved in 100 ml of CH3CN—were added dropwise at 0° C. to a solution of 24.5 g of thiocarbonyldiimidazole and 1.56 g of imidazole in 600 ml of CH3CN and the mixture was stirred at RT overnight.","19.5 g of 1,2-phenylenediamine were then added and the mixture was again stirred at RT for 2 h. For workup, the reaction mixture was evaporated in vacuo, the residue was taken up in CH2Cl2, and the solution was washed 7× with 10% citric acid solution and 2× with satd.","NaCl solution, dried over Na2SO4, filtered and concentrated.","The crude product thus obtained (31.78 g; brown foam) was reacted directly without further purification; ESI-MS [M+H+]=373.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.5 and 9.05 (each s, 1H), 7.45 (d, 2H), 7.35 (m, 1H), 7.20 (d, 1H), 7.15, 6.95, 6.75, 6.60 (each m, 1H), 4.85 (s, 2H), 4.10 (d, 2H), 1.35 (s, 9H).","b.)","Crude product 4a was dissolved in 750 ml of ethanol together with 36.7 g of HgO (yellow) and 0.4 g of sulfur and heated to reflux for 2 h. The reaction mixture was then filtered twice through Celite and evaporated to dryness; 20.7 g, ESI-MS [M+H+]=339.15.c.)","7 g of the crude product 4b were introduced into 70 ml of CH2Cl2, 35 ml of HCl in diethyl ether (satd.","at 0° C.) were added and the mixture was stirred at RT for 2 h. The resulting precipitate was filtered off with suction, washed with CH2Cl2 and dried.","6.7 g of brown amorphous solid; ESI-MS [M+H+]=239.15 1H-NMR (360 MHz, DMSO) δ ppm: 11.6 (s broad, 1H), 8.4 (s broad, 3H), 8.25 (s broad, 1H), 7.65 and 7.55 (each d, 2H), 7.45 and 7.3 (each m, 2H), 4.19 (m, 2H).","Example 5 N-[5-(Aminomethyl)-1,3-thiazol-2-yl]guanidine (dihydrochloride) (5) a.)","31 g (130 mmol) of 2-chloro-3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal (preparation according to THL 39 (1998), 8085-8088) and 15.4 g of amidinothiourea were heated at 110° C. for 75′ in 200 ml of n-butanol, then the mixture was evaporated and the residue was treated with CH2Cl2 and conc.","NH3.Evaporation of the organic phase, purification of the residue by chromatography on silica gel (CH2Cl2/CH3OH 0-5%) and crystallization from acetone afforded 12.3 g of N-{5-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]-1,3-thiazol-2-yl}guanidine.","b.)","1 g of 5a in 20 ml of CH3OH was treated with 0.81 ml of hydrazine hydrate and stirred at RT for 2 h. The mixture was then cooled to 0° C., filtered, and the filtrate was concentrated and stirred with dilute HCl.","This process was repeated a number of times, and the crude product obtained in this way was then stirred with ethanol; 0.92 g of white solid, ESI-MS [M+H+]=172.05.Example 6 N-[4-(Aminomethyl)phenyl]-N′-benzylurea (6) a.)","4-Aminobenzylamine (10.0 g, 81.85 mmol) in 150 ml CH2Cl2 was treated with triethylamine (6.8 g, 67.12 mmol) and then treated at 0° C. with di-t-butyl dicarbonate (18.6 g, 85.0 mmol).","The mixture was stirred at 0° C. for 1 h and then at RT for 2 h. For workup, 150 ml of a 1%-aqueous citric acid solution were added, the phases were separated and the aqueous phase was reextracted 2 times with CH2Cl2 (150 ml).","Fresh washing with H2O, drying of the combined organic phases using Na2SO4 and evaporation afforded a solid, which was washed with stirring with a little diisopropyl ether, filtered off with suction and dried.","13.0 g; ESI-MS [M+H+-tBu]=167.05.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (2H, d), 6.61 (2H, d), 4.78 (1H, s br.","), 4.17 (2H, d), 3.67 (2H, s br.","), 1.46 (9H, s).","b.)","Benzyl isocyanate (2.40 g, 18.0 mmol) was added with ice-cooling to a solution of the protected amine 6a (4.0 g, 17.99 mmol) and triethylamine (1.82 g, 18.0 mmol) in 220 ml of toluene/DMF 10:1.The reaction mixture was stirred at RT overnight.","It was possible to filter off some of the urea formed directly as a precipitate and dry it.","The filtrate was washed 2× with H2O, with dilute tartaric acid to pH 3 and again 2 times with H2O to pH 5, and the organic phase was then dried and evaporated.","Altogether, 6.0 g were thus obtained; ESI-MS [M+H+-tBu]=300.15.c.)","The urea 6b thus obtained was introduced in 90 ml of CH2Cl2, and TFA (2.24 g, 196.25 mmol)—dissolved in 90 ml of CH2Cl2—was added dropwise at 0° C. After 3 h, 1 ml of TFA was added again, then the mixture was stirred at RT overnight.","After fresh addition of 1 ml of TFA, the mixture was stirred for a further 5 h, then poured onto ice water and extracted with ethyl acetate (2×50 ml).","The water phase was rendered basic with 2N NaOH solution and extracted with CH2Cl2 (2×50 ml).","The insoluble portion between the phases was filtered off and dried.","4 g; ESI-MS [2M+H+]=511.35 1H-NMR (200 MHz, DMSO) δ (ppm): 8.52 (1H, s), 7.39-7.07 (9H, m), 6.62 (1H, t), 4.27 (2H, d), 3.61 (2H, s).","Example 7 [4-(1H-Benzimidazol-2-yl)phenyl]methaneamine (hydrochloride) (7) a.)","Di(tert-butyl) 4-cyanobenzylimidodicarbonate (10 g, 30.08 mmol; preparation according to Synth.","Comm.","28, 23, 1998, 4419ff) in 200 ml of pyridine was treated with 45 ml of triethylamine and saturated at 0° C. with H2S for 1.5 h. The reaction mixture was allowed to stand at RT overnight and then evaporated.","The residue thus obtained was then stirred with diethyl ether, filtered off with suction and dried (8.5 g).","b.)","6 g of the thioamide 7a (16.37 mmol) in 40 ml of dry CH2Cl2 were alkylated at RT overnight using 23.2 g of CH3I and the mixture was then evaporated.","The residue thus obtained was taken up in 40 ml of CH3OH, 1.95 g of 1,2-phenylenediamine were added and it was again stirred overnight.","Evaporating the reaction mixture and stirring the solid with n-pentane afforded 6.9 g of the desired benzimidazole.","M.p.",": >170° C. (decomposition); ESI-MS: [M+H+]=424.25 c.) 1 g of the bis-Boc compound 7b was dissolved in 5 ml of CH2Cl2, 5 ml of TFA were added at 0° C. and the mixture was stirred at room temperature for 1 h. Evaporating the reaction mixture, treating with HCl in diethyl ether and stirring the isolated solid with diethyl ether afforded 0.6 g of the amine as the hydrochloride; ESI-MS: [M+H+]=224.05.Example 8 N1-(1H-Benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) Preparation was carried out analogously to the preparation of compound 4 starting from 7 g of N-Boc-1,5-diaminopentane hydrochloride (29.3 mmol).","After reaction analogously to 4a, 10.3 g of N-Boc 5-{[(2-aminoanilino)carbothioyl]amino}pentane-1-amine were obtained; ESI-MS [M+H+]=353.25.Cyclodesulfurization and subsequent removal of the Boc group with TFA afforded an oily crude product, which was taken up in CH3OH and converted into the corresponding hydrochloride using 250 ml of ethereal HCl (saturated at 0° C.).","Stirring the solid obtained with a mixture of CH3OH/methyl t-butyl ether afforded 1.8 g of a reddish amorphous solid.","1H-NMR (360 MHz, DMSO) δ ppm: 9.30 (t, 1H), 8.15 (s broad, 3H), 7.40 and 7.25 (each m, 2H), 3.35 (m, 2H superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).","Example 9 N1-(1H-Benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) Preparation was carried out analogously to the preparation of compound 4 starting from 9.87 g of N-Boc-1,4-diaminobutane (52.3 mmol).","After reaction analogously to 4a, 17.08 g of N-Boc 4-{[(2-aminoanilino)carbothioyl]amino}butane-1-amine were obtained; ESI-MS [M+H+]=338.99.Subsequent cyclodesulfurization and Boc removal using TFA afforded a brown solid, which was stirred a number of times with n-pentane and then recrystallized from a mixture of CH3OH/methyl t-butyl ether; 14.35 g, ESI-MS [M+H+]=205.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.20 (t, 1H), 7.80 (s broad, 3H), 7.35 and 7.20 (each m, 2H), 3.40 (m, 2H partially superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H).","Example 10 trans-N-[(4-Aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) Preparation was carried out analogously to compound 4 starting from 5.4 g of tert-butyl-4-(aminomethyl)cyclohexylamine carbamate (WO 9603374; Bioorg.","Med.","Chem.","Lett.","1997, 7 (1), 67).","After removal of the Boc group, 3.3 g of white dihydrochloride were obtained; FAB-MS [M+H+]: 245.Example 11 trans-N-{[4-(Aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) Preparation was carried out analogously to compound 4 starting from 10 g of benzyl {4-[(tert-butoxycarbonyl)amino]cyclohexyl}-methylcarbamate (EP 669317) by removal of the Boc group using 4N HCl in dioxane, synthesis of the benzimidazole and subsequent hydrogenolysis.","3.6 g of white dihydrochloride were isolated; FAB-MS [M+H+]: 245.Example 12 [6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) a.)","Preparation was carried out analogously to 7 starting from tert-butyl (6-cyanopyridin-3-yl)methylcarbamate (6.0 g, 25.72 mmol); crystallization of the crude product from ethanol afforded 5.15 g; ESI-MS [M+H+]=325.b.)","0.55 g of the Boc-protected amine 12a in 10 ml of CH2Cl2 was treated with 5 ml of TFA and stirred at RT for 2 h. Evaporation of the reaction mixture afforded 0.95 g of a white solid; ESI-MS [M+H+]: 225.25.Example 13 N-[4-(Aminomethyl)phenyl]-2-pyridineamine (13) tert-Butyl 4-aminobenzylcarbamate (2 g; 9 mmol) was heated to reflux for 32 h with 8.74 g of 2-fluoropyridine.","The reaction mixture was evaporated in vacuo and the residue obtained was stirred with n-pentane (1.9 g).","The Boc group was cleaved using TFA, and the crude product obtained was precipitated from diethyl ether as the hydrochloride and then converted into the free-base (0.8 g) using NH3; ESI-MS [M+H+]: 200.25.N-[4-(Aminomethyl)benzyl]-2-pyridineamine (14) a.)","20 g 2-Aminopyridine were dissolved in 100 ml CH3OH, adjusted to pH 6 with isopropanolic HCl and 36 g p-cyanobenzaldehyde were added.","9.35 g Sodium cyanoborohydride were added portionwise over one hour and stirred overnight.","For workup, the suspension was evaporated, the residue taken up in 100 ml water and with KOH adjusted to pH>10.The watery phase was saturated with NaCl and extracted 3× with diethylether.","The ether phase was washed after filtration of a precipitate 3× with a FeSO4 solution, dried and evaporated.","Purification of the residue by chromatography on silica gel (heptane/ethyl acetate 1:1) afforded 28.15 g 4-[2-pyridinyl-amino)methyl]benzonitrile.","b.)","10 g 4-[2-Pyridinyl-amino)methyl]benzonitrile were dissolved in 280 ml ammonia-alkali CH3OH, 10 g Raney-nickel added and it was hydrogenated for 24 h. It was filtered, evaporated and the residue purified by chromatography on silica gel (ethyl acetate/ethyl alcohol 1:3).","5.18 g, ESI-MS: [M+H+]=214.","[5-(1H-Benzimidazole-2-yl(thiene-2-yl]methaneamine (15) Preparation was carried out starting from 5-(aminomethyl)thiophene-2-carbonitrile (preparation according to WO 95/23609), which was reacted to the respective Boc-derivative according to standard methods.","1.1 eq.","Sodium methanolate solution was added to t-butyl-5-cyanothiene-2-ylcarbamate (25 g; 104.9 mmol) in 330 ml CH3OH and stirred overnight at RT, then for 2 h at 40-50°.","18.95 g Phenylenediaminebihydrochloride were then added and again stirred at RT.","For workup, water was added, the resulting precipitate filtered off and carefully dried.","19.6 yellow solid; ESI-MS: [M+H+]=330.The following cleavage of the Boc-group with TFA afforded a raw product, which was dissolved in water, 2× extracted with diethyl ether, the watery phase adjusted to pH 10-11 and then extracted 2× with ethyl acetate.","The watery phase was saturated with NaCl and again extracted with ethyl acetate.","The combined organic phases were dried and evaporated (6.3 g); ESI-MS [M+H+]=230.1.tert-Butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) Preparation was carried out analogously to the preparation of [4-(1H-benzimidazole-2-yl)phenyl]methaneamine (hydrochloride) (7) starting from tert-butyl-2-(4-cyanophenyl)ethylcarbamate.","The raw product obtained after reaction with H2S, alkylation with CH3I and reaction with 1,2-phenylenediamine was purified by chromatography on silica gel (CH2Cl2/CH3OH 4-50%) (4.8 g); ESI-MS [M+H+]=338.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with TFA (under standard conditions); the isolated TFA-salt was then directly utilized in the respective couplings.","N-(Piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoroacetate) (17) a) A solution of tert-butyloxycarbonyl-4-(aminomethyl)-1-piperidine (5.39 g; 25 mmol) in 25 ml CH3CN was added dropwise to 6.75 g thiocarbonyldiimidazole and 0.5 g imidazole in 100 ml CH3CN at 0° C. and then stirred for 3 h at RT.","1,2-Phenylenediamine (5.5 g; 50.86 mmol) was then added and heated for about 1 h to 60° C. The solid obtained upon cooling was filtered off with suction and dried.","6.79 g; ESI-MS [M+H+-tBu]=309.15 b) tert-Butyoxycarbonyl-4-({[(2-aminoanilino)carbothioyl]amino}methyl)1-piperidine (5 g; 13.72 mmol), 5.94 g HgO (yellow) and 0.6 g sulfur in 150 ml ethyl alcohol were heated under reflux for 1 h. The mixture was filtered 2× over Celite, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 5-25%).","2.65 g; ESI-MS [M+H+]=331.25 1H-NMR (360 MHz, DMSO) δ ppm: 7.15 and 6.9 (each m, 2H), 3.95 (d, 2H) 3.2 (m, 2H, 2.7 (br m; 2H), 1.8 (m, 1H), 1.7 (m, 2H), 1.35 (s, 9H), 1.05 (m, 2H).","c) tert-Butyloxycarbonyl-4-[(1H-benzimidazole-2-ylamino)methyl]-1-piperidine (2.65 g; 8.02 mmol) was treated with 10 ml TFA according to standard conditions.","Evaporation and mixing the raw product with n-pentane afforded 2.39; ESI-MS [M+H+]=231.15.1H-NMR (360 MHz, DMSO δ ppm: 13.25 (s, 1H), 9.35 (m, 1H), 8.8 and 8.5 (each br s, 1H), 7.4 and 7.20 (each m, 2H), 3.3 (m, 4H), 2.85 (m, 2H), 1.9 (m, 3H), 1.35 (m, 2H).","N-{[5-(Aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoroacetate) (18) a) A solution of tert-butyl-(4-cyanothiene-2-yl)methylcarbamate (7 g; 29.4 mmol) in 120 ml ethyl alcohol was saturated with NH3 and then hydrogenated under standard conditions in the presence of Ra—Ni (9 g watery suspension; decanted with ethyl alcohol).","Filtration of the reaction mixture, evaporation and chromatography of the obtained residue on silica gel (CH2Cl2/CH3OH plus watery NH3) afforded 4.4 g of the amine as a yellow oil.","b) 1.2.of the amine 18a (4.3 mmol), 06.g ethyldiisopropylamine and 15 g 2-fluoropyridine were heated for 20 h to reflux.","The residue obtained after evaporation of the mixture was taken up in CH2Cl2, washed with 0.1n HCl and saturated NaCl solution, dried and again evaporated.","1 g; ESI-MS [M+H+]=320.15 c) 0.9 of the Boc-protected amine 18b were dissolved in 10 ml CH2Cl2, 5 ml TFA was added at 0° C. and it was stirred at room temperature for 1 h. Evaporation of the reaction mixture afforded 1.65 g of a brownish oil which was reacted directly without further purification (ESI-MS [M+H+]=220.05).","3-Amino-N-(1H-imidazole-2-yl)propaneamide (19) a) Z-β-Alanine (10 g; 44.8 mmol) was dissolved in 200 ml DMF and 15.86 g (3.5 eq) N-methylmorpholine and 5.9 g (0.5 eq) 2-aminoimidazolesulfate were added.","7.87 g (1.3.eq) HOBt and 11.16 g (1.3 eq) N′-(dimethylaminopropyl)-N-ethylcarbodiimide were added at −10° C. and stirred for 1 h whilst to RT and then for 18 h. 150 ml diethyl ether were added whereupon a wide residue precipitated which was filtered with suction.","The residue was washed with cold diethyl ether, suspended in ethyl acetate and 1n HCl was added up to an acid reaction.","The watery solution was extracted 1× with ethyl acetate, the watery phase was then adjusted to a basic pH with 10% NaOH at 4° C. The resulting precipitate was filtered with suction and washed with water.","5.4 g; ESI-MS [M+H+]=289.05.b) 5.3 g of the Z-compound 19a were suspended in 250 ml ethyl alcohol and 530 mg 10% Pd on activated carbon was added.","It was hydrogenated with H2 for 18 h at RT, then diluted with CH3OH and the suspension was boiled up whereon the precipitate of the product disintegrated.","Filtering and evaporation of the solution afforded 1.5 g; ESI-MS [M+H+]=155.05.N-[4-(Aminomethyl)phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) tert-Butyl-4-aminobenzylcarbamate (2 g; 9 mmol), 2.2 g ethyldiisopropylamine and 4.4 g (2-(3,5-dimethylpyrazole)-4,5-dihydroimidazole×HBr in 15 ml DMF were stirred at 110° C. After the reaction had completed, the mixture was evaporated, the residue taken up in ethyl acetate, each 2× washed with saturated NaHCO3 and NaCl solution, dried and again evaporated.","The basic watery phase was also evaporated, mixed with acetone, the precipitate filtered with suction and the mother liquor evaporated.","The combined residues were purified by means of MPLC (silica gel: Fa.","Bischoff Prontoprep 60-2540-C18E, 32 μm; solvent: CH3CN/H2O+0.1% acetic acid).","0.22 g; ESI-MS [M+H+]=291.15.The product thus obtained was treated with 4n HCl in dioxane for 2 h at RT.","The resulting precipitate was filtered with suction, washed with pentane and dried.","110 mg; ESI-MS [M+H+]=191.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.85 (s, 1H), 8.45 (s broad, 3H), 8.40 (s, 1H), 7.60 and 7.30 (each d, 2H), 4.05 (m, 2H), 3.70 (s, 4H).","N-{[5-Aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) Amine 18a (6.5 g; 23.31 mmol) was reacted to the respective aminobenzimidazole by reaction with thiocarbonyldiimidazole, imidazole and then phenylenediamine analogously to the preparation of 17.1.6 g: ESI-MS [M+H+]=359.15.The subsequent cleavage of the Boc-group by means of 4n HCl in dioxane afforded 1.3 g of slightly yellow solids: ESI-MS [M+H+]=191.15.N1-Pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) Preparation analogously to 18b by reaction of N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 20.3 g 2-fluoropyridine, 5.64 g clear oil; ESI-MS [M+H+]=280.15.Cleavage of the Boc-group with 4n HCl in dioxane afforded 3.46 g white solids; ESI-MS [M+H+]=180.20.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (s broad, 1H), 8.05 (s broad, 3H), 7.85 (m, 2H), 7.20 (m, 2H), 6.80 (m, 1H), 3.45 (m, superimposed by H2O), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).","N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol), 5.4 g ethyldiisopropylamine and 5.11 g 2-(methylsulfanyl)-4,5-dihydro-1H-imidazole×HI in 30 ml DMF were stirred overnight at RT.","The reaction mixture was evaporated, taken up in CH2Cl2, washed with water and saturated NaCl solution, dried and again evaporated.","5.05 g clear oil, ESI-MS [M+H+]: 271.18.Cleavage of the Boc-group with 4n HCl in dioxane and purification of the raw product by means of MPLC afforded 2.57 g; ESI-MS [M+H+]: 171.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 160.3, 43.3 (2 signals superimposed).","42.85, 39.70, 28.90, 27.2, 23.60.N-[4-(Aminomethyl)phenyl]-1H-imidazole-2-amine (24) a) 5.24 g BrCN—dissolved in 50 ml CH3OH-were added dropwise to a mixture of tert-butyl-4-aminobenzylcarbamate (10 g; 44.99 mmol) and 11.07 g sodium acetate in 100 ml CH3OH at 0° C., and it was stirred for 3 hours at 0° C. and overnight at RT.","The mixture was evaporated, the obtained residue taken up in water and 2× extracted with methyl-tert-butylether.","Drying and evaporation of the organic phases afforded 12.99 of a yellow-orange oil.","b) 28.6 g triethylamine were added to 7 g tert-butyl-4-[(iminomethylene)amino]benzyl carbamate in 50 ml pyridine, H2S was introduced for 1 hour at 0° C. and the mixture was allowed to stand for 48 hours.","Evaporation afforded 9.79 g of a rosa foam; ESI-MS [M+H+]: 282.05.1H-NMR (360 MHz.","D6DMSO) δ ppm: 9.70 (s, 1H), 7.35 (m, 4H), 7.20 (m, 2H), 4.15 (d, 2H), 1.45 (s, 9H).","c) 5 g of the thioamide in 50 ml CH3OH where methylated with 5.05 g CH3I and the obtained raw product was directly reacted with 1.73 g aminoacetaldehyde-diethylacetal in 7.5 ml CH3CN for 3 hours at RT.","Evaporation of the reaction mixture afforded 6.36 g of a reddish oil (ESI-MS [M+H+]: 381.25) which was dissolved in 50 ml 6n HCl and stirred for 3 hours at 0° C. A pH of 12 was then adjusted with a 25% NaOH solution and it was again stirred for 48 hours at RT.","The mixture was extracted 4× with ethyl acetate, the combined organic phases dried and evaporated.","The oil thus obtained was stirred 2× with methyl-tert-butylether, the obtained solids filtered with suction and dried.","0.6 g red solid; ESI-MS [M+H+]: 189.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 145.5, 141.60, 130.75, 128.4, 119.8, 115.6, 44.85.N1-(1,4,5,6-Tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (25) Preparation was carried out analogously to 23 starting from N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 5.4 g 2-(methylsulfanyl)-1,4,5,6-tetrahydropyrimidine×HI.","After workup, 1.3 g of a yellowish oil was obtained; [M+H+]: 282.2.Cleavage of the Boc-group with 4n HCl in dioxane and purification by means of MPLC afforded 0.46 g; ESI-MS [M+H+]: 185.15.N-[4-(Aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26) Benzyl (4-aminocyclohexyl)methylcarbamate (TFA-salt) (5 g; 13.28 mmol)-preparation by TFA cleavage starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317)-was heated to reflux analogously to 18 with 1.71 g ethyldiisopropylamine in 50 ml 2-fluoropyridine.","Usual workup and crystallization of the raw products from methyl-tert-butylether/methanol afforded 4.15 g; ESI-MS [M+H+]: 340.29.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.75 (d broad, 1H), 7.85 (m, 2H), 7.35 (m, 5H), 7.05 (d, 1H), 6.85 (m, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.75 (each m, 2H), 1.45-0.90 (m, 6H).","Cleavage of the Z group under standard conditions (H2; Pd-activated carbon), precipitation of the resulting amine as hydrochloride and drying of the obtained precipitates afforded 1.5 g; ESI-MS [M+H+]: 206.15.tert-Butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-ylacetate (27) 75 ml of a 1.0 m BH3-THF solution were added to a solution of tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10 g; 36.32 mmol) in 100 ml THF and it was stirred at RT.","For workup, water was carefully added, it was 2× extracted with diethyl ether and then the organic phases were washed 2× with water.","Evaporation and drying afforded 9.3 g; ESI-MS [M+H+]: 262.04.","[5-(2-tert-Butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) a) 3 g K2CO3, 0.05 g KI and 5 g methylbromoacetate were added to a solution of tert-butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl acetate (27) in 50 ml DMF and it was stirred for 12 hours at 90° C. Thereafter, 4 g methylbromoacetate were added and it was stirred for further 5 hours at 120° C. For workup, the mixture was concentrated, diluted with CH2Cl2, washed with saturated NaCl solution, dried and again evaporated.","Chromatography on silica gel (CH2Cl2/CH3OH 1-5%) afforded 4.6 g of a bright yellow oil; ESI-MS [M+H+]: 334.12.b) 4.19 of the methylester in 20 ml dioxane/15 ml H2O were saponified with 19 KOH at RT.","For workup, the mixture was concentrated, adjusted to a pH of 2 with 2n HCl and extracted with CH2Cl2.The combined organic phases were dried, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 2-7%).","2.4 g oil, ESI-MS [M+H+]: 320.15.1H-NMR (360 MHz, CDCl3) δ ppm: 7.20-7.10 (m, 1H), 6.90 and 6.80 (each m, 1H), 4.0 (s, 2H), 3.55 (m, 1H), 3.10 (m, 2H), 2.85 and 2.70 (each m, 1H), 2.90-2.50 (m, 4H), 1.35 (s, 9H).","N-[4-(Aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) Preparation analogously to the preparation of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (23) starting from benzyl-(4-aminocyclohexyl)methylcarbamate (TFA salt) (3 g; 7.97 mmol).","a) Reaction with BrCN and subsequent purification (1.52 g; ESI-MS [M+H+]: 288.15).","1H-NMR (360 MHz, d6-DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.75 (d, 1H), 5.05 (d, 2H), 2.85 (m, 3H), 1.85 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.20 and 0.95 (each m, 2H).","b) Conversion to the corresponding thiourea and subsequent methylation (1.489; ESI-MS [M+H+]: 336.15.c) Reaction with aminoacetaldehyde-diethylacetal and subsequent cyclization afforded 0.79 g; ESI-MS [M+H+]: 329.15.1H-NMR (360 MHz, DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.45 (s, 2H), 5.35 (d, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.15 and 0.95 (each m, 2H).","d) For cleavage of the Z-group it was dissolved in 30 ml HBr/glacial acidic acid and stirred for 3 h at RT.","For workup, the mixture was evaporated and several times co-evaporated with acetone.","0.89 g; ESI-MS [M+H+]: 195.15.tert-Butyloxycarbonyl-4-[(2-pyridinylamino)methyl]-1-piperidine (30) tert-Butyloxycarbonyl-4-(aminomethyl)-1-piperidine (3 g; 14 mmol) and 10 ml 2-fluoropyridine were heated for 4 h to reflux.","Evaporation and mixing the raw product in n-pentane afforded 3 g of a wide solid, mp: 126-130° C.; ESI-MS [M+H+]=292.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with HCl in dioxane (under standard conditions); the isolated HCl-salt was then directly utilized.","Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-oxo-2,3,4,6-tetrahydro H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) a) A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) in 20 ml DMF was added dropwise to a suspension of 1.28 g NaH (60%; deoiled) in 10 ml DMF at 5° C. and it was stirred for 1 h. 3.49 g Bromoacetonitrile-dissolved in 20 ml DMF-were then added dropwise and it was stirred for 4 h at RT.","For workup, water was added carefully, it was diluted with CH2Cl2, washed several times with H2O and saturated NaCl solution, dried and evaporated.","Purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 5%) afforded 7.61 g; ESI-MS [M+H+-tBu]: 259.05.b) 5 g tert-Butyl-[1-(cyanomethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate in 70 ml pyridine were saturated for 1 h with H2S and then allowed to stand overnight at RT.","Evaporation of the mixture and mixing of the obtained residue with pentane afforded 5.5 g of a rosa solid which was directly reacted.","c) A mixture of 2 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.62 g ethylpyruvate and 0.86 g KHCO3 in 30 ml dioxane was stirred for 2.5 h at RT.","Dilution with CH2Cl2, washing with saturated NaCl solution, drying and evaporation afforded 2.65 g of a yellow oil; ESI-MS [M+H+]: 445.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.50 (s, 1H), 7.50 and 7.35 (each m, 1H), 7.30-7.20 (m, 2H), 7.35 (d, 1H), 5.20 (broad, 1H), 4.30 (q, 2H), 4.20 (m, 1H), 3.65-3.50 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 3H), 1.70-1.50 (m, 2H), 1.30 (s, 9H; superimposed by t, 3H).","2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) 2.6 g Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) was provided in a mixture of 31 ml dioxane and 4 ml H2O, 1.5 eq.","KOH was added and it was heated to reflux.","After 5 h, again 1 eq.","KOH was added and it was further stirred for 12 h at RT.","For workup, it was concentrated, the residue was taken up in water, a pH of 4-5 was adjusted with 2n HCl and it was extracted several times with CH2Cl2.The combined org.","phases were washed with saturated NaCl solution, dried and evaporated.","Mixing with n-pentane afforded 2.1 g of a white solid; ESI-MS [M+H+]: 417.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 7.5 (m, 1H), 7.35 (m, 1H), 7.30-7.20 (m, 3H), 5.25 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 6H), 1.70 (m, 1H), 1.30 (s, 9H).","2-Ammonio-6-(ammoniomethyl)pyridinium trichloride (33) a) 2-Amino-6-methylpyridine (0.14 mol, 15.0 g) and phthalanhydride (0.14 mol, 20.55 g) were heated to 190° C. at the water separator.","Distributing between H2O and CH2Cl2, evaporation of the org.","phase and recrystallisation of the residue (diethyl ether) afforded 28.25 g of a slightly yellowish solid; ESI-MS [M+H+]=239.15.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.95 (2H, m), 7.78 (3H, m), 7.23 (2H, m), 2.64 (3H, s).","b.)","N-bromosuccimide (25.18 mmol, 4.48 g) was added portionwise to a boiling suspension of 2-(6-methyl-2-pyridinyl)-1H-isoindole-1,3(2H)-dione (33a, 20.99 mmol, 5.0 g), AIBN (2.10 mmol, 0.35 g) and dibenzoylperoxide (2.10 mmol, 0.51 g) in CCl4.The reaction mixture was boiled for 20 h, filtered and the filtrate evaporated.","Chromatography (CH2Cl2) afforded 3.12 g of the target product and 1.20 g of the dibromo compound; ESI-MS 318.95, 316.95.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98 (2H, m), 7.95 (1H, t), 7.81 (2H, m), 7.58 (1H, d), 7.35 (1H, d), 4.60 (2H, s).","c) Potassium phthalimide (9.46 mmol, 1.75 g) was added to a solution of 2-[6-(bromomethyl)-2-pyridinyl]-1H-isoindole-1,3(2H)-dione (33b, 6.31 mmol, 2.0 g) in DMF (30 ml), the reaction mixture was heated for 15 h to 60° C., stirred for 24 h at RT, water (60 ml) was added and it was stirred for 2 h at 0° C. The residue was filtrated and washed with a mixture of H2O-DMF and then with diethyl ether; 2.12 g; ESI-MS [M+H+]=384.05.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98-7.91 (4H, m), 7.88 (1H, t), 7.85-7.75 (4H, m), 7.38-7.32 (2H, m), 5.10 (2H, s).","d) 2-{6-[(1,3-Dioxo-1,3-dihydro-2H-isoindole-2-yl)methyl]-2-pyridinyl}-1H-isoindole-1,3(2H)-dione (33c, 5.22 mmol, 2.09) was heated to reflux for 3 h with hydrazinium hydroxide (13.04 mmol, 0.65 g) in methanol (50 ml).","For workup, water was added, it was evaporated and the watery phase acidified with conc.","HCl.","Anew evaporation and recrystallising (ethyl alcohol) afforded 1.20 g of a white solid; ESI-MS [M+H+]=124.05.1H-NMR (270 MHz, D2O) δ (ppm): 7.94 (1H, t), 7.08 (1H, d), 6.99 (1H, d), 4.33 (2H, s).","trans-N-[4-(Aminomethyl)cyclohexyl]-N′-benzylurea (34) Preparation was carried out analogously to compound 6 starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317) by cleavage of the boc-group with 4n HCl in dioxane.","Build-up of the benzyl urea by reaction with benzyl isocyanate and triethylamine in DMF and subsequent hydrogenolysis afforded 0.55 g of the target product; ESI-MS [M+H+]=262.20.7-(4-Aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoroacetate) (35) a.)","A solution of 5-tert-butoxycarbonylaminovaleric acid (50.0 mmol, 10.86 g), O,N-dimethylhydroxylamine hydrochloride (50 mmol, 4.88 g), N-Methylmorpholine (0.30 mol, 30.35 g), HOBT (53.90 mmol, 8.42 g) and EDCI*HCl (55.0 mmol, 10.54 g) in CH3CN (200 ml) were stirred for 2 days at RT.","After evaporation the residue was taken up in ethyl acetate, and then washed with water, a 10% KHSO4-solution, a saturated watery NaHCO3 solution and a saturated watery NaCl-solution, subsequently.","Drying and evaporation of the organic phase afforded 6.96 g of a yellowish oil; ESI-MS: [2M+Na+]=543.3, [M+Na+]=283.1, 205.1, 161.1.1H-NMR (270 MHz, CDCl3) δ (ppm): 4.63 (1H, s.","br.","), 3.68 (3H, s), 3.21-3.05 (3+2H, m), 2.44 (2H, t), 1.76-1.48 (2+2H, m), 1.43 (9H, s).","b.)","Methyl magnesia bromide (60.0 mmol, 17.30 ml of a 3M solution in Et2O) at 0° C. was added dropwise to a solution of tert-butyl 5-[methoxy(methyl)amino]-5-oxopentylcarbamate (35a, 30.0 mmol, 6.9 g) in THF (120 ml) and stirred for 5 h at 0° C. The reaction mixture was then carefully acidified with a 10% KHSO4-solution, extracted with ethyl acetate and the organic phase then washed with saturated watery NaHCO3— and saturated watery NaCl-solution, dried and evaporated: 5.5 g yellowish oil; ESI-MS: [M-BOC+H+]=116.15.c.)","A mixture of tert-butyl 5-oxohexylcarbamate (35b, 9.29 mmol, 2.0 g), 2-aminonicotinaldehyde (Heterocycl.","1993, 36, 2518; 11.20 mmol, 1.379) and KOH (0.37 ml of a 20% watery solution) was heated to reflux for 8 h. Evaporation and column chromatography afforded 1.60 g of the target product; ESI-MS: [M+H+]=302.15.d.)","A suspension of tert-butyl 4-[1,8]naphthyridine-2-ylbutylcarbamate (35c, 5.31 mmol, 1.60 g) and Pd/C (10%, 1.59) in ethyl alcohol (40 ml) were stirred overnight under H2 atmosphere, then filtered over celite and washed with ethyl alcohol.","Column chromatography afforded 290 mg; ESI-MS: [M+H+]=306.25.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (1H, d), 6.29 (1H, d), 4.97 (1H, s.br.","), 4.81 (1H, s.br.","), 3.37 (2H, m sym.","), 3.12 (2H, q br.","), 2.65 (2H, t), 2.53 (2H, t), 1.89 (2H, quint.","), 1.67 (2H, quint.","), 1.51 (2H, quint.","), 1.43 (9H, s).","e.) TFA (18.30 mmol, 2.099) was added to a solution of tert-butyl 4-(5,6,7,8-tetrahydro[1,8]naphthyridine-2-yl)butylcarbamate (35d, 0.92 mmol, 0.289) in CH2Cl2 (8 ml), the solution was stirred for 20 h and evaporated: 380 mg; ESI-MS: 206.1, 130.7.1H-NMR (400 MHz, CDCl3) δ (ppm): 7.07 (1H, d), 6.31 (1H, d), 5.58 (1H, s.br.","), 3.39 (2H, m sym.","), 2.96 (2H, s br.","), 2.76 (2H, t), 2.68 (2H, t), 2.56 (2H, t), 1.88 (2H, quint.","), 1.69 (2H, quint.","), 1.51 (2H, quint.).","tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36) Tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10.9 mmol, 3.0 g)—dissolved in THF—was added at 0° C. to a solution of diisopropylamine (11.0 mmol, 1.119) and butyl lithium (11.0 mmol, 6.91 ml of a 15% solution in hexane) in THF (100 ml) and the solution further stirred for 1 h. About 100 ml ethylene oxide were then added and the mixture was stirred overnight at RT.","The solution was distributed between saturated NH4Cl and ethyl acetate, the organic phase was washed with water and evaporated; 2.7 g; ESI-MS: [2M+Na+]=661.3, [M+K+]=358.1, 321.1, [M+H+]=320.1, 264.0.tert-Butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37) tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36, 6.26 mmol, 2.00 g) dissolved in CH2Cl2 was added dropwise within 10 minutes to a solution of oxalyl chloride (7.93 mmol, 1.0 g) and DMSO (16.59 mmol, 1.26 g) in little CH2Cl2.After 30 min.","triethyl amine (38.22 mmol, 3.87 g) was added, stirred for 5 min., left to reach RT and stirred overnight at RT.","For workup, water was added, the mixture extracted with CH2Cl2 and the organic phase washed with saturated NaCl—, 1%-H2SO4— and with 5%-NaHCO3-solution.","Evaporation afforded 1.8 g of the target product; ESI-MS: 693.2, [M+K+]=358.1, 319.1, [M+H+]=318.1, 262.0.Methyl-2-amino-5-chlorobenzoic acid (38) Thionyl chloride (0.47 mmol, 55.46 g) was added dropwise at 0° C. to a solution of 2-chloro-5-aminobenzoic acid (0.23 mmol, 40.09) in methanol (400 ml) and the mixture heated to 50-60° C. After the reaction had finished, water was added and it was extracted with ethyl acetate.","Then the organic phase was washed with 1n NaOH and diluted HCl solution (pH 1-2), subsequently, and evaporated; 23.0 g; ESI-MS: [M+H+]=186.05.Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39) Ethyl succinic acid-chloride (0.14 mol, 22.44 g) in toluen (15 ml) was added dropwise at 0° C. to a solution of methyl 2-amino-5-chloro-benzoic acid (38, 123.9 mmol, 23.0 g) and pyridine (0.26 mol, 20.58 g) in toluen (40 ml).","The solution was stirred overnight at RT, water was added and it was extracted with ethyl acetate.","The organic phase was washed with 1N HCl-solution, with water, with a saturated NaHCO3-solution and with a saturated NaCl-solution.","Evaporation, recrystallisation (methanol) afforded 34.1 g of the target product; ESI-MS: [2M+Na+]=649.0, [M+K+]=352.0, [M+H+]=314.05; 1H-NMR (270.MHz, CDCl3) δ (ppm): 11.06 (1H, s br.","), 8.68 (1H, d), 7.99 (1H, m), 7.47 (1H, dd), 4.16 (2H, q), 3.92 (3H, s), 2.74 (4H, m), 1.24 (3H, t).","Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40) Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39, 0.16 mol, 50.20 g) in DMSO (250 ml) was added dropwise at 15° C. to a suspension of deoiled NaH (0.27 mol) in THF (50 ml) and DMSO (80 ml) and the mixture was stirred for 2 h at RT.","At 0° C. glacial acetic acid (24 ml) was added and stirred for 20 min.","Water (25 ml) was added, the resulting precipitate filtered off, washed with water, taken up in CH2Cl2, extracted with water and the organic phase evaporated.","The residue was then stirred with diethyl ether (50 ml), filtered and dried; 32 g of a 6:4-mixture methyl/ethyl ester, which was not separated; ESI-MS (Me-ester): [M+K+]=307.9, [M+Na+]=290.0, [M+H+]=268.0; ESI-MS (Et-ester): [M+K+]=321.9, [M+Na+]=304.0, [M+H+]=282.0.7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41) Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40, 0.11 mol, 32.0 g) was heated to 150° C. in DMSO (500 ml) and water (0.23 mol, 4.09 g) and stirred for 2 h. Water was added at 100° C., the mixture cooled to 0° C. and the resulting precipitate filtered off.","Drying afforded 19.09; ESI-MS: [M+Na+]=251.1, [M+H+]=211.9, 209.95, 130.1.tert-Butyl (2E,Z)-(7-Chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene) ethane acid (42) t-Butyl diethylphosphono acetic acid (0.10 mol, 25.849) was added dropwise at 0° C. to a solution of deoiled NaH (0.10 mol) in DMF (40 ml) and stirred until a clear solution develops.","7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41, 90.63 mmol, 19.09) in DMF (185 ml) was added dropwise at 0° C. and stirred at RT.","Water was added to the reaction mixture, it was stirred for 1 h and the resulting yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.","Recrystallisation afforded 23.5 g of the target product; ESI-MS: [2M+H+]=615.2, [M+Na+]=330.0, 293.0, 254.1, 252.1.1H-NMR (400 MHz, CDCl3) δ (ppm): 9.59 (1H, s br.","), 7.45 (1H, m), 7.25 (1H, m), 7.06 (1H, m), 5.99 (1H, t), 3.43 (2H, s), 2.84 (2H, d), 1.32 (9H, s).","t-Butyl (7-chloro-2-oxo-2,3,4,4-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43) tert-Butyl (2E,Z)-(7-chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)ethane acid (42, 75.0 mmol, 23.089) in ethyl alcohol/dioxane (250 ml/100 ml) was hydrogenated for 4 days with Pt/carbon (5%, 4.1 g) under standard conditions.","Water was added to the reaction mixture, stirred for 1 h, the yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.","Recrystallisation afforded 23.5 g of a solid (mixture of target product and the corresponding dechlorinated compound; the mixture was reacted directly); ESI-MS: [2M+H+]=618.94, [M+K+]=350.66, 309.75, 254.11.Methyl [5-(2-tert-butoxy-2-oxo ethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44) tert-Butyl (7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43, 60.00 mmol, 18.59 g) in DMF (10 ml) was added dropwise at 15° C. to deoiled NaH (69.00 mmol) in DMF (5 ml) and the mixture was stirred overnight at RT.","Ice water was added to the reaction mixture, extracted (2×) with ethyl acetate and the organic phase washed with a 10% CH3COOH-solution, with water and then with 1n NaOH.","Evaporation afforded 20.4 g of a raw product, which was reacted without further purification.","[5-(2-tert-Butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) KOH (80.45 mmol, 4.51 g) in water (150 ml) was added to methyl [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44, 50.28 mmol, 19.29) dissolved in dioxane (250 ml) and stirred for 1 h at RT.","The reaction mixture was evaporated, water (100 ml) was added and it was extracted with ethyl acetate (2×).","After the evaporation, the residue was taken up in diethyl ether and precipitated by addition of n-pentane.","Recrystallisation (diisopropyl ether, 2×) afforded 4.8 g (comprises about 15% of the corresponding dechloro compound); ESI-MS: [M+Na+]=390.0, 314.0, 312.0.","[4-(Aminomethyl)phenyl]guanidine (Bihydrochloride) (46) p-Aminobenzylamine (6.7 g; 54.84 mmol) was suspended in 20 ml 6n HCl and 5.3 g cyanamide—dissolved in 5 ml H20—added slowly under reflux.","After the reaction had completed, 50% NaOH-solution was added at 0° C. to the solution, the resulting precipitate filtered with suction, boiled up in 50 ml ethyl alcohol and filtered off.","Evaporation of the mother liquor and mixing of the obtained residue with diethyl ether afforded 1.4 g of yellow solids; Fp.",": 255° C. 7,8-Dimethoxy-3,4-dihydro-1H-1-benzazepine-2,5-dione (47) Analogously to the preparation of the corresponding building blocks 39, 40 and 41 first ethyl-2-amino-4,5-dimethoxybenzoate (20 g; 88.8 mmol) was reacted with ethyl succinic acid-chloride to the respective amide.","After mixing with n-pentane 30.4 g of a solid was obtained; ESI-MS [M+H+]: 354.15.1H-NMR (400 MHz, DMSO) δ (ppm): 10.65 (s, 1H), 8.10 and 7.40 (each s, 1H), 4.30 and 4.10 (each q, 2H), 3.85 and 3.80 (each s, 3H), 2.70 (m, 4H), 1.30 and 1.20 (each t, 3H).","Subsequent cyclization with 25 g of the obtained amide under utilization of NaH analogously to 40 and usual workup afforded 19.5 g of a white solid; ESI-MS [M+H+]: 308.05.1H-NMR (400 MHz, DMSO) δ (ppm): 13.3 (s, 1H), 10.10 (s, 1H), 7.25 and 6.75 (each s, 1H), 4.30 (q, 2H), 3.80 (s, 6H), 2.95 (s, 2H), 1.35 (t, 3H).","Decarboxylation analogously to 41 starting from 17 g afforded 10.5 g of the target product as a solid; ESI-MS [M+H+]: 236.15.1H-NMR (400 MHz, DMSO) δ (ppm): 9.90 (s, 1H), 7.35 and 6.80 (each s, 1H), 3.80 and 3.75 (each s, 3H), 2.85 and 2.65 (each m, 2H).","tert-Butyl (7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate (48) Preparation analogously to building blocks 42 and 43 by Wittig-Homer-reaction and subsequent hydrogenation; after mixing with n-pentane 8.16 g of solids were obtained; ESI-MS [M+H+-tBu]: 280.15.","[5-(2-tert-Butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (49) Analogously to 44 and 45, starting from 4 g tert-butyl-(7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 2.6 g of the target product were isolated as a bright foam; [M+H+-tBu]: 338.15.1H-NMR (400 MHz, DMSO) δ (ppm): 6.85 and 6.75 (each s, 1H), 4.35 (s broad, 2H), 3.80 and 3.75 (each s, 3H), 3.60 (s, 2H), 2.70 (m, 2H), 2.25 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H), 1.35 (s, 9H).","[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) a.)","56.1 g (406 mmol) powdered K2CO3 were added at room temperature to a solution of 37 g (135.3 mmol) t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl)acetic acid (1) and 3.7 g tetrabutylammoniumbromide in 370 ml DMF, 22.7 g (148.9 mmol) methylbromoacetic acid was added dropwise, then it was stirred for 3 h at 40° C. and overnight at room temperature.","The reaction mixture was poured into 1000 ml of a water-ice-mixture, and 2× extracted with each 200 ml methyl-tert.butyl ether.","The combined extracts were washed with H2O, 5% NaHCO3— and NaCl-solution, dried over Na2SO4, filtered off and evaporated.","The remaining yellow oil (about 52 g, purity about 90%) was reacted without further purification; ESI-MS [M+H+]: 346.b.)","9.2 g (26.6 mmol) of the raw product 50a were dissolved in 66.6 ml 4n HCl in dioxane and stirred for 24 h at 50° C., then the dioxane was extensively distilled off, 5% NaHCO3-solution and diethyl ether added to the residue, the watery phase again washed with diethyl ether and acidified with 1n KHSO4-solution.","The precipitating acid was extracted with diethyl ether, the ether phase washed with a NaCl-solution, dried over Na2SO4, filtered off with suction and evaporated.","It remained 3.2 g of a slightly yellow oil; ESI-MS [M+H+]: 290.trans-[4-(1H-Benzimidazole-2-yl)cyclohexyl]methaneamine (hydrochloride) (51) 39.3 g (0.25 mol) trans-4-(Aminomethyl)cyclohexanecarbonic acid and 27.0 g (0.25 mol) 1,2-phenylene diamine were heated to reflux for 18 h in a mixture of 167 ml conc.","hydrochloric acid and 333 ml H2O analogously to J. Heterocycl.","Chem.","26, 541 (1989).","The green reaction solution was concentrated until the occurrence of a yellow crystalline pulp and stirred with 400 ml isopropanol, filtered off, washed with 90% isopropanol and finally with diethyl ether.","After 2 times recrystallisation from a isopropanol-water mixture (70/30), 30 g of a white monohydrochloride remained; ESI-MS [M+H+]: 230.Methyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5benzodiazepine-1-yl)acetic acid (52) 1.99 (79.8 mmol) NaH (60% dispersion in mineral oil) were added to an ice cooled solution of 12.2 g (75.3 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-One (preparation: J.","Am.","Chem.","Soc.","1949, 71, 1985) in 350 ml DMF, stirred for 30 min.","at 0-5° C. and for 10 min.","at room temperature.","Then 11.5 g (75.3 mmol) methyl bromo acetic acid were added dropwise at 0° C. and it was then stirred for 30 min.","at the same temperature.","The reaction solution was poured onto 600 ml ice water and 3× extracted with each 150 ml ethyl acetate.","The organic phase was washed with a NaCl-solution, dried over MgSO4 and ethyl acetate was distilled off.","The residue was purified by column chromatography (eluent: CH2Cl2/CH3OH 9/1).","9.6 g of a yellowish oil were isolated; ESI-MS [M+H+]: 235.tert-butyl (2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) Preparation was carried out analogously to building block 52 by reaction of 11.99 (73 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-one with 14.3 g (73 mmol) tert-butyl bromo acetic acid.","17 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 277.","[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) a.)","14.2 g (102 mmol) powdered K2CO3 were added at 0° C. to a solution of 9.6 g (41 mmol) compound 52 and 8.09 (41 mmol) tert-butyl bromo acetic acid in 90 ml DMF, and stirred for 1 h at 0° C. and then for 14 h at room temperature.","The reaction mixture was poured onto 300 ml ice water and 3× extracted with each 100 ml methyl-tert.butyl ether.","The combined organic phases were washed several times with a NaCl-solution, dried over MgSO4 and evaporated to dryness.","The residue was purified by column chromatography (eluent: ethyl acetate/cyclohexane 7/3).","7.0 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 349.b.)","The alkaline saponification of the methyl ester was carried out analogously to 3b.","3.8 g white crystals were obtained; Fp.",": 140-142° C.; ESI-MS [M+H+]: 335.","[5-(2-tert-Butoxy-2 oxoethyl)-4-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) Analogously to building block 54a tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) was reacted with methyl bromoacetic acid and then alkaline saponified analogously to 3b.","After purification by column chromatography 2.9 g white crystals were obtained; Fp.",": 82-84° C.; ESI-MS [M+H+]: 335.","[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) 14.5 g (42 mmol) building block 3a were dissolved in 105 ml 4n HCl in dioxane and stirred for 2 days at 50° C. After evaporation of the solvent, the residue was dissolved in 5% NaHCO3-solution, 2× extracted with methyl-tert.butyl ether, then the watery phase was acidified with a 1n KHSO4-solution and the precipitating acid extracted with methyl-tert.butyl ether.","After drying over MgSO4, evaporation of the solvent and purification by column chromatography (eluent: CH2Cl2/CH3OH/glacial acetic acid 451511) 1.6 g of a viscous, slightly yellowish oil remained; ESI-MS [M+H+]: 292.","[(5R)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (57) 14.2 g (42.6 mmol) [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) were suspended in 170 ml diethylether and dissolved by addition of 7.3 g (42.64 mmol) (1S)-(−)-1-napthyl)ethylamine.","The yellow solution was inoculated with (1S)-1-napthyl)ethaneaminium[(5R)-5-(2-tert.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetate—prepared by preparative HPLC-separation of compound 3 by means of a chiral column (Chiralpak AD 500×; 50 mm; 20 μm) and subsequent salification-, the deposited precipitate filtered off with suction after 3.5 h and 3× recrystallized from a ethyl acetate/isopropanol mixture.","The purity of enantiomers was checked by means of a chiral HPLC.","3.5 g. of the salt were suspended in 30 ml of a diethylether/hexane-mixture 10/3 and after addition of 50 ml of a 5% watery amidosulfonic acid solution stirred until occurrence of a clear phase.","After separation of the watery phase, the organic phase was washed 3× with 5 ml amidosulfonic acid- and then with a NaCl-solution, dried over Na2SO4 and evaporated.","2.25 g amorphous residue; ESI-MS [M+H+]: 505.","[(5S)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (58) From the combined mother liquors of building block 57, 8 g of the acid were isolated as a yellowish amorphous residue as described with a amidosulfonic acid solution, said acid was reacted with (1R)-(+)-1-napthyl)ethyl amine into the diastereomeric salt and recrystallized until the achievement of enantiomeric purity.","Analogously to example 57, 2.5 g of the dextrorotatory acid were isolated as an amorphous solid, ESI-MS [M+H+]: 505.A sample of the acid was reacted with 4-bromobenzylamine into the good crystallizing amide and the absolute configuration figured out by means of a x-ray structure analysis.","tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate (59) A solution of 5.3 g (29.2 mmol) 6,7-dihydro-4H-thieno[3,2-b]azepine-5,8-dione (Arch.","Pharm.","1991 324, 579) and 12 g (32.2 mmol) (tert-butoxycarbonylmethylene)-triphenyl phosphorane in 15 ml toluen was heated to reflux for 10 h, then toluen was distilled off and the black residue purified by chromatography (eluent: ethyl acetate/cyclohexane 7/3).","The consistent fraction was again digested with 40 ml boiling cyclohexane, cooled and filtered with suction.","3 g yellowish crystals; ESI-MS [M+H+]: 280.tert-Butyl-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl)acetate (60) 39 (11 mol) tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate were hydrogenated analogously to building block 2 in the presence of 10% Pd/C.","Since educt was still present after 6 h according to HPLC, the catalyst was filtered with suction and after addition of new catalyst it was again hydrogenated for 6 h. After workup and chromatographic purification (eluent: ethyl acetate/cyclohexane 7/3), 1.4 g yellowish crystals were isolated, which comprised according to HPLC still about 2.5% educt; ESI-MS [M+H+]: 282.","[8-(2-tert-Butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) Preparation was carried out analogously to building block 50.Ester stage: 1.5 g yellowish crystals; ESI-MS [M+H+]: 354.Target product: 1.2 g yellowish crystals; ESI-MS [M+H+]: 340.I.B Compounds of the Formula I or I′ Example I t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetate 1.1 g of N-methylmorpholine were added dropwise at 0° C. to 1.5 g (4.65 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) and 1.22 g (4.8 mmol) of N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) in 20 ml of DMF, and 1.55 g (4.7 mmol) of TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetrafluoroborate) were then introduced in portions over the course of 35′.","The yellow reaction solution was stirred at 0° C. for 1 h and then largely evaporated in vacuo.","The residue was then digested a number of times with H2O, taken up in a mixture of 120 ml of ethyl acetate and 40 ml of diethyl ether, washed with 10% K2CO3 and NaCl solution, dried over Na2SO4 and concentrated, the crude product thoroughly crystallizing.","Purification by chromatography on silica gel (CH2Cl2/CH3OH/NH3 42:8:0.1) and crystallization from ethyl acetate/n-hexane afforded 1.45 g (65%) of white crystals.","M.p.",": 190-193° C. (dec.); FAB-MS [M+H+]: 487.Example II [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid 1.2 g of the t-butyl ester from Example I were suspended in 70 ml of CH2Cl2, treated with 45 ml of 4N HCl in dioxane and stirred overnight at RT.","The solution was evaporated, and the residue was digested a number of times with CH2Cl2 and then dried.","In this way, 1.07 g of a slightly yellowish amorphous powder were isolated; FAB-MS [M−H+]: 432.Example III [1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine hydrochloride (4) and subsequent removal of the t-butyl group analogously to Example II.","A slightly yellowish amorphous residue was obtained, FAB-MS [M−H+]: 554.Example IV t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetate Analogously to the preparation of compound 3a, 0.9 g (3.3 mmol) of t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) was alkylated with methyl bromoacetate (FAB-MS [M−H+]: 346) and then hydrolyzed analogously to 3b (0.44 g; FAB-MS [M−H+]: 332).","Coupling of 0.57 g (1.72 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-1-yl]acetic acid with N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) analogously to Example I afforded the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 485.Example V [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetic acid The t-butyl ester was removed analogously to Example II and afforded 0.42 g of the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 429.Example VI (1-{2-[(4-{[(Benzylamino)carbonyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-N′-benzylurea (6) and subsequent removal of the t-butyl group analogously to Example II.","Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 27 mg as an amorphous solid; ESI-MS [M+H+]: 515.2; [M+K+]: 553.2.Example VII [1-(2-{[4-(1H-Benzimidazol-2-yl)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [4-(1H-benzimidazol-2-yl)phenyl]methaneamine (7) and subsequent cleavage of the t-butyl group analogously to Example II.","Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 9 mg as an amorphous solid; ESI-MS [M+H+]: 485.2.Example VIII [1-(2-{[4-(1H-Benzimidazol-2-ylamino)butyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) and subsequent cleavage of the t-butyl group analogously to Example II.","After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.5 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 464.Example IX [1-(2-{[5-(1H-Benzimidazol-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) and subsequent cleavage of the t-butyl group analogously to Example II.","After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.48 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 478.Example X {1-[2-({4-[(1H-Benzimidazol-2-ylamino)methyl]cyclohexyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with trans-N-[(4-aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) and subsequent cleavage of the t-butyl group analogously to Example II.","After chromatographic purification on silica gel, 0.7 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XI {1-[2-({[4-(1H-Benzimidazol-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with trans-N-{[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) and subsequent cleavage of the t-butyl group analogously to Example II.","After chromatographic purification on silica gel, 0.5 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XII [2-Oxo-1-(2-oxo-2-{[4-(pyridin-2-ylamino)benzyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-2-pyridineamine (13) and subsequent cleavage of the t-butyl group analogously to Example II (14 mg); ESI-MS [M+H+]: 459.15.Example XIII {1-[2-({[6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid (bishydrochloride) Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [6-(1H-benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) and subsequent cleavage of the t-butyl group analogously to Example II.","(13 mg); ESI-MS [M+H+]: 484.15.Compounds of the general formula I analogously to example II were prepared: Example 14 {2-Oxo-1-[2-oxo-2-({4-[(pyridine-2-ylamino)methyl]benzyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of N-[4-(aminomethyl)benzyl]-2-pyridine amine (14) as educt, 207 mg as an amorphous solid were obtained according to MPLC 207 mg; ESI-MS [M+H+]: 473.28.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.5 (m, 1H), 7.95 (d, 1H), 7.45 (m, 1H), 7.35-72.0 (m, 9H), 6.60 (m, 2H), 4.45 (m, 3H), 4.25 (m, 3H), 3.55 (m, 1H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m 1H).","Example 15 {1-[2-({[5-(1H-Benzimidazole-2-yl)thiene-2-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of [5-(1H-benzimidazole-2-yl)thiene-2-yl]methaneamine (15) as educt, 210 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 489.27.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 7.70 (m, 1H), 7.65 (m, 2H), 7.35-7.15 (m, 6H), 7.05 (m, 1H), 4.55 (m, 3H), 4.20 (m, 1H), 3.55 (m, 1H), 2.75 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H).","Example 16 {1-[2-({2-[4-(1H-Benzimidazole-2-yl)phenyl]ethyl}amino)-2-oxoethyl]-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of tert-butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) as educt, 190 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 497.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.15 (d, 2H), 7.65 (d, 2H), 7.45 (m, 2H), 7.30-7.15 (m, 6H), 4.45 and 4.15 (each m, 1H), 3.6-3.25 (m, superimposed by H2O), 2.80 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.70 (m, 1H).","Example 17 [1-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of (3 N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) as educt, 134 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 490.29.Example 18 [2-Oxo-1-(2-oxo-2-{[2-(pyridine-2-ylamino)ethyl]amino}ethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N1-pyridine-2-ylethane-1,2-diamine, the subsequent MPLC afforded 278 mg as amorphous white solids; ESI-MS [M+H+]: 397.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.95 (m, 1H), 7.70 (m, 2H), 7.35-7.20 (m, 4H) 6.80 and 6.70 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.0 (m, superimposed by H2O), 2.70 (m, 2H), 2.30 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).","Example 19 (2-Oxo-1-{2-oxo-2-[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]ethyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18), the subsequent MPLC afforded 135 mg as amorphous white solids; ESI-MS [M+H+]: 479.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.65 (m, 1H), 8.0 (m, 1H), 7.45 (m, 1H), 7.35-7.15 (m, 6H), 6.95 (s, 1H), 6.60 and 6.55 (each m, 1H), 4.60-4.30 (m, 5H), 4.15 (m 1H), 3.50 (m, 1H), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.70 (m, 1H).","Example 20 [1-(2-{[3-(1H-imidazole-2-ylamino)-3-oxopropyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of 3-amino-N-(1H-imidazole-2-yl)propane amide (19) and following purification of the raw product by MPLC 50 mg were obtained as amorphous white solids; ESI-MS [M+H+]: 414.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.30-7.20 (m, 7H), 4.45 and 4.15 (each m, 1H), 3.75-3.25 (m, superimposed by H2O), 2.80-2.65 (m, 4H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).","Example 21 [1-(2-{[4-(4,5-Dihydro-1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N-[4-(aminomethyl)-phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) as educt, 12 mg were obtained as amorphous white solids after MPLC; ESI-MS [M+H+]: 450.3.Example 22 (1-{2-[({4-[(1H-Benzimidazole-2-ylamino)methyl]thiene-2-yl}methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-6-yl)acetic acid Utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) as educt; purification by MPLC afforded 90 mg; ESI-MS [M+H+]: 518.29.Example 23 [2-Oxo-1-(2-oxo-2-{[5-(pyridine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) as educt; after MPLC, 210 mg white solids were obtained; ESI-MS [M+H+]: 439.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (m, 1H), 7.95 (m, 1H), 7.75 (m broad, 1H), 7.65 (m, 1H), 7.30-7.15 (m, 4H), 6.75 and 6.60 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.20 (m, superimposed by H2O), 2.70 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60, 1.45 and 1.30 (each m, 2H).","Example 24 N-[5-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)pentyl]-4,5-dihydro-1H-imidazole-2-amine (acetate) Utilization of N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) as educt; 22 mg were obtained after MPLC; ESI-MS [M+H+]: 430.15.Example 25 [1-(2-{[4-(1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (24) as educt; purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 448.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.95 (s, 1H), 8.45 (m, 1H), 7.35-7.10 (m, 8H), 6.75 (s, 2H), 4.50 (m, 1H), 4.20 (m, 3H), 3.5-3.1 (m, superimposed by H2O).","2.70 (m, 1H), 2.35 (m 1H), 2.20 (m, 2H), 1.65 (m, 1H).","Example 26 [2-Oxo-1-(2-oxo-2-{[1-(1,4,6,6-tetrahydropyrimidine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-(1,4,5,6-tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (26) as educt; 40 mg were obtained after MPLC; ESI-MS [M+H+]: 444.9.Example 27 {2-Oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Utilization of N-[4-(aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26), purification by means of an elution over a Chromabond-C18-cartridge afforded 85 mg; ESI-MS [M+H+]: 465.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.55 (s broad, 1H), 8.05 (m, 1H), 7.90 (d, 1H), 7.85 (m, 1H), 7.35 (m, 2H), 7.25 (m, 2H), 6.95 (d, 1H), 6.80 (m, 1H), 4.40 and 4.20 (each m 1H), 3.55 (m, superimposed by H2O), 2.95 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.15, 1.95 and 1.75 (each m, 2H), 1.60 and 1.40 (each m, 1H), 1.25 (m, 2H), 1.05 (m, 2H).","Example 28 Ethyl-{2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 30 ml 4n HCl in dioxane were added to 100 mg {2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid in 10 ml EtOH and it was stirred for 48 h at RT.","Evaporation and elution over a Chromabond-C8-cartridge afforded 26 mg; ESI-MS [M+H+]: 493.25.Example 29 (1-{4-[4-(1H-Benzimidazole-2-ylamino)phenyl]butyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid a.)","A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (0.8 g; 2.91 mmol) in 20 ml DMF was added dropwise at 0° C. to a suspension of 0.169 NaH (60%; deoiled) in 10 ml DMF and stirred for 1 h. A spatula tip full of KI was then added likewise at 0° C., 1 g 1-(4-bromo butyl)-4-nitrobenzene (preparation according to J. Med.","Chem.","38, 13 (1995), 2418-2420)—dissolved in 10 ml DMF—was slowly added dropwise and stirred for 2 h at RT.","For workup, water was carefully added to the reaction mixture, it was diluted with CH2Cl2 and several times extracted with a saturated NaCl-solution.","After drying and evaporation, the obtained raw product was purified by chromatography on silica gel (CH2Cl2/CH3OH 2-10%); 0.429 yellow oil, ESI-MS [M+H+-Boc]: 397.15.b.)","Hydrogenation of the nitro group in 75 ml CH3OH with 100 mg 10% Pd on activated carbon under standard conditions afforded 0.36 mg of a bright oil; ESI-MS [M+H+]: 423.25.c.)","The build-up of the corresponding aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and then phenylene diamine.","Chromatography on silica gel (CH2Cl2/CH3OH 2-4%) afforded 220 mg of a bright foam; ESI-MS [M+H+]: 539.25.d.)","200 mg tert-Butyl-(1-(4-[4-(1H-benzimidazole-2-ylamino)phenyl]butyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate was treated at RT with 10 ml TFA.","Evaporation and lyophilizing the obtained oil afforded 213 mg of a solid; ESI-MS [M+H+]: 483.25.Example 30 2-(4-{[{[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}(methyl)amino]methyl}anilino)-1H-benzimidazole (trifluoro acetate) a.)","Standard coupling of [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (2.2 g; 6.6 mmol) with 1.32 g N-methyl(4-nitrophenyl)methane amine (J.","Am.","Chem.","Soc.","65 (1943), 1984-1990) in 40 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.","Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 2-4%) afforded 2.269 of a slightly yellow oil; ESI-MS [M+H+]: 482.03.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 8.25/8.20 (d, 2H), 7.60/7.50 (d, 2H), 7.35-7.20 (m, 4H), 4.85-4.5 (m, 4H), 3.05/2.85 (s, 3H), 2.70 (m, 1H), 2.30 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H), 1.30 (s, 9H).","b.)","5 ml Hydrazine hydrate were added at 60° C. to 1.26 g nitro compound a and 15 mg FeCl3×6H2O in 20 ml CH3OH and for 1 h stirred at 60° C. The mixture was filtered over Celite and the filtrate evaporated.","1.17 g; ESI-MS [M+H+]: 452.17.c.)","The build-up of the respective aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and phenylene diamine, subsequently.","Chromatography on silica gel (CH2Cl2/CH3OH 4-7.5%) afforded 0.9 g of a slightly beige foam; ESI-MS [M+H+]: 568.25.d.)","Cleavage of the t-butylester with 50 ml TFA and subsequent purification by chromatography on silica gel (CH2Cl2/CH3OH 8-10%) afforded 0.88 g; ESI-MS [M+H+]: 512.25.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.05 (broad, 1H), 7.60-7.20 (m, 1.2H), 4.85 (m, 1H), 4.75-4.45 (m, 3H), 3.65 (m, 1H), 3.20 (s, 3H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).","Example 31 tert-Butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Coupling of [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) analogously to I and purification by chromatography on silica gel (CH2Cl2/CH3OH 2-3%) afforded 200 mg of a beige foam; ESI-MS [M+H+]: 540.25.Example 32 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) TFA-cleavage of tert-butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate afforded 240 mg; ESI-MS [M+H+]: 484.36.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.10 (s, 1H), 8.30 (t, 1H), 7.5-7.25 (m, 8H), 7.15 (m, 2H), 6.85 (m, 2H), 4.40 (m, 2H), 3.75-3.55 (m, superimposed by H2O), 3.5 (m, 1H), 3.0 (m, 1H), 2.70 (m, 2H), 1.70 (m, 2H), 1.45 (m, 1H).","Example 33 tert-Butyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).","410 mg of a white foam; ESI-MS [M+H+]: 546.35.Example 34 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) Analogously to example 32; 420 mg of white solids; ESI-MS [M+H+]: 490.47.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.0 (d, 1H), 7.65 (m, 1H), 7.40 and 7.30 (each m, 2H), 7.15 and 6.85 (each m, 2H), 3.80 (m, 2H), 3.50 (m, 1H), 3.0 (m, 4H), 2.75 (m, 2H), 2.0 (m, 2H), 1.75-1.50 (m, 5H), 1.50-1.20 (m, 5H), 0.95 (m, 2H).","Example 35 tert-Butyl-[1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8).","300 mg of a foam; ESI-MS [M+H+]: 520.39.Example 36 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butyl ester from example 35 with 15 ml 4nHCl in dioxane afforded 300 mg solid; ESI-MS [M+H+]: 464.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (m, 1H), 7.70 (m, 1H), 7.35, 7.25, 7.10 and 6.85 (each m, 2H), 4.75 (m 2H), 3.30, 3.15, 2.95, 2.70 (each m, 2H), 1.65 (m, 5H), 1.45 and 1.30 (each m, 2H).","Example 37 tert-Butyl-{1-[2-({[4-(1H-imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example I under utilization of N-[4-(aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) as educt; chromatography on silica gel (CH2Cl2/CH3OH 4-15%) afforded 530 mg of a solid foam; ESI-MS [M+H+]: 510.35.Example 38 {1-[2-({[4-(1H-Imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Cleavage of the t-butyl ester from example 37 mit TFA and subsequent purification by means of chromatography on silica gel (CH2Cl2/CH3OH 10-15%+2% glacial acetic acid) afforded 570 mg; ESI-MS [M+H+]: 454.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.0 (broad, 1H), 7.30 and 7.25 (each m, 2H), 6.80 (broad, 1H), 6.70 (s, 2H), 4.40 and 4.20 (each broad, 1H), 4.75-4.25 (superimposed by H2O), 3.0 (m, 2H), 2.65 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.95 (m, 1H), 1.75 (m, 2H), 1.60 and 1.35 (each m, 1H), 1.20 (m, 4H), 0.95 (m, 2H).","Example 39 tert-Butyl-(2-oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate Standard coupling of 2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) (0.5 g; 1.2 mmol) with N-(piperidine-4-ylmethyl)pyridine-2-amine (hydrochloride; accessibly from 29) in 15 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.","Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 3-20%) afforded 0.44 g of a slightly yellow oil; ESI-MS [M+H+]: 590.35.Example 40 (2-Oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 31 with TFA afforded 385 mg; ESI-MS [M+H+]: 534.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (s, 1H), 7.90 (m, 2H), 7.55 (m, 1H), 7.35 (m, 1H), 7.25 (m, 2H), 7.05 and 6.80 (each m, 1H), 5.40-5.20 (m, 2H), 4.20 and 4.0 (each m, 1H), 3.25 (m, 2H), 2.95 (m, 1H), 2.80-2.60 (m, 3H), 2.30 (m, 1H), 2.25-2.05 (m, 2H), 1.80 and 1.65 (each m, 2H), 1.20-1.10 (m, 3H).","Example 41 tert-Butyl-[1-({4-[({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino) carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Analogously to example 29 under utilization of trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11) as educt; chromatography of the raw product on silica gel (CH2Cl2/CH3OH 5-8%) afforded 350 mg of a bright yellow oil; ESI-MS [M+H+]: 643.45.Example 42 [1-({4-[({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example 31 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 300 mg; ESI-MS [M+H+]: 587.35.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 8.20 (m, 1H), 8.20 (s, 1H), 7.35-7.20 (m, 5H), 7.15 (m, 2H), 5.40 and 5.25 (each d, 1H), 3.15 (m, superimposed by H2O), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 2.05 and 1.80 (each m, 2H), 1.65, 1.30 and 1.15 (each m, 2H).","Example 43 tert-Butyl-(1-{[4 ({4-[(1H-benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 430 mg were obtained analogously to example 29 under utilization of N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) and subsequent purification; ESI-MS [M+H+]: 629.45.Example 44 (1-{[4-({4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 43 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 340 mg; ESI-MS [M+H+]: 573.35.1H-NMR (360 MHz, d-DMSO) δ ppm: 9.20 (m, 1H), 7.95 (s, 1H), 7.45 (m, 1H), 7.40 (m, 2H), 7.30-7.15 (m, 5H), 5.35-5.10 (m, 2H), 4.45 (m, 1H), 4.0-3.25 (m, superimposed by H2O), 2.95 (m, 1H), 2.8-2.60 (m, 2H), 2.30 and 2.20 (each m, 1H), 2.0-1.75 (m, 3H), 1.70-1.50 (m, 2H), 1.20 (m, 3H).","Example 45 tert-Butyl-{2-oxo-1-[(4-{[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 330 mg were obtained analogously to example 39 under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18) and purification; ESI-MS [M+H+]: 618.25.Example 46 {2-Oxo-1-[(4-{([{4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Cleavage of the t-butylesters of example 45 and purification by means of MPLC afforded 150 mg; ESI-MS [M+H+]: 562.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 8.20 (m, 1H), 8.0 (d, 1H), 7.50 (d, 1H), 7.30-7.20 (m, 4H), 7.10 and 6.95 (each s, 1H), 6.85 (m, 1H), 6.50 (m, 2H), 5.40 and 5.20 (each d, 1H), 4.55 and 4.35 (each m, 2H), 2.80-2.55 (m, 2H), 2.30 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).","Example 47 tert-Butyl-[1-({4-[4-(1H-benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate a.)","A mixture of 1.5 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.36 g 2-bromo-4-nitroacetophenone and 0.65 g KHCO3 in 30 ml dioxane were stirred for 12 h at RT.","Workup analogously to building block 30c and mixing of the raw product with n-pentane afforded 2.1 g of a brown solid; ESI-MS [M+H+]: 494.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (s, 1H), 8.30 and 8.20 (each m, 2H), 7.60 and 7.35 (each m, 1H), 7.25 (m, 2H), 5.50 and 5.30 (each d, 1H), 2.70 (m, 2H), 2.30 (m, 1H), 2.20 (m, 3H), 1.65 (m, 1H), 1.25 (s, 9H).","b.)","Reduction of the nitro group analogously to example 30b (1.6 g; ESI-MS [M+H+]: 464.15) and build-up of the aminobenzimidazole as already described afforded after chromatography on silica gel (CH2Cl2/CH3OH 24%) 0.58 g of a slightly yellow foam; ESI-MS [M+H+]: 614.35.Example 48 [1-({4-[4-(1H-Benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example XXXXVII and purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 524.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.95 and 9.60 (each broad, 1H), 7.85-7.75 (m, 4H), 7.65 (m, 1H), 7.40-7.25 (m, 5H), 7.0 (m, 2H), 5.40 and 5.30 (each d, 1H), 3.50 (m, 1H), 2.75 (m, 1H), 2.45 (m, 2H), 2.20 (m, 2H), 1.70 (m, 1H).","Example 49 tert-Butyl (1-{2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Ethyldiisopropylamine (0.42 mmol, 54.50 mg) and HATU (0.50 mmol, 190.11 mg) were added at 0° C. to a solution of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.42 mmol, 140.59 mg) in CH2Cl2 (10 ml), the mixture was stirred for 1 h at 0° C. and [4-(aminomethyl)phenyl]guanidine (bihydrochloride) (46) (0.42 mmol, 100 mg), ethyldiisopropylamine (0.63 mmol, 81.76 mg) added.","The mixture was stirred for 1 h at 0° C. and overnight at RT.","After evaporation, the residue was taken up in CH2Cl2/water, washed with watery NaHCO3—, 5%-citric acid- and finally with watery saturated NaCl-solution.","Evaporation and chromatography on silica gel (CH2Cl2/CH3OH 0-100%) afforded 6.0 mg target product; ESI-MS [M+H+]: 426.1, 425.1.Example 50 {4-[({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]anilino}(imino)methaneamine (trifluoro acetate) tert-Butyl-({2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (example 49, 0.01 mmol, 6 mg) was dissolved in 5 ml CH2Cl2, TFA (1.25 mmol, 142.53 mg) added at 0° C. and stirred overnight at room temperature.","After evaporation, the residue was taken up in CHCl3/water and the watery phase washed with CHCl3; evaporation of the watery phase affords 4.8 mg target product; ESI-MS [M+H+]=425.1.Example 51 tert-Butyl [1-(2-{[(6-amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49, under utilization of 2-ammonio-6-(ammonio methyl)pyridinium trichloride (33) (0.30 mmol, 69.76 mg) and following purification and subsequent purification (chromatography; CH2Cl2/MeOH 0-100%) afforded 120 mg of the target product; ESI-MS: [M+H+]=439.28, 383.36.Example 52 [1-(2-{([(6-Amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (hydrochloride) Cleavage of the t-butylester, chromatography of the raw product (CHCl3/MeOH 0-5%) and ester interchange with HCl in diethyl ether afforded 15.0 mg; the target product as hydrochloride; ESI-MS [M+H+]=383.1.Example 53 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 2 Drops of a solution of HCl in diethylether were added to N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (0.63 mmol, 196.10 mg) in methanol (40 mL) and stirred for 5 min.","at RT.","tert-Butyl.","[2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) was added, NaBH3CN (3 mmol, 188.5 mg) portionwise added after 10 min.","and the mixture stirred for 17 h at RT.","After evaporation, the residue was taken up in ethyl acetate and washed with NaHCO3— (pH 8-9), saturated NaCl— (1×) and a 10% FeSO4-solution (2×).","Chromatography (CHCl3/MeOH 0-5%) afforded 84 mg of the target product; ESI-MS [M+H+]=540.24, 270.75.Example 54 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester and chromatography (CHCl3/MeOH 0-100%) afforded 4 mg of the target product; ESI-MS [M+H+]=484.1.Example 55 tert-Butyl [1-(2-{[(4-{[(benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49 under utilization of N-[4-(aminomethyl)cyclohexyl]-N′-benzyl urea (34) (0.50 mmol, 130 mg) afforded 320 mg; ESI-MS [M+H+]=577.11.Example 56 [1-(2-{[(4-{[(Benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogous cleavage of the t-butylester afforded a raw product which after evaporation was distributed between ethyl acetate/water.","The watery phase was then adjusted to pH 10 with 0.1n NaOH and extracted with ethyl acetate.","It was adjusted then with 1nN HCl to pH4, extracted with CH2Cl2, washed and evaporated; 80 mg; ESI-MS [M+H+]=521.3.Example 57 tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 53 under utilization of N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (0.63 mmol, 183.47 mg) and tert-butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) afforded 50.0 mg of the target product; ESI-MS [M+H+]=520.41, 260.79.Example 58 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (example 57, 0.04 mmol, 20.00 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.","Evaporation of the reaction mixture, chromatography (CHCl3/MeOH 0-100%) afforded 10 mg of the target product; ESI-MS [M+H+]=464.25.Example 59 tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 53 under utilization of ethyldiisopropylamine and EDCI*HCl starting from trans-N-[[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (0.63 mmol, 0.209) afforded 84 mg; ESI-MS [M+H+]=546.32, 273.79.Example 60 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (example 59, 0.01 mmol, 8 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.","The reaction mixture was evaporated, the watery phase washed with a 3:1 mixture CHCl3/EtOH.","Evaporation of the watery phase afforded 6.0 mg of the target product; ESI-MS [M+H+]=490.25.Example 61 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (1.70 mmol, 467.08 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).","TFA (1.73 mmol, 197.72 mg) was added at 0° C. to the obtained raw product and it was stirred overnight at room temperature.","After evaporation, the residue was distributed between ethyl acetate and water, the watery phase adjusted to pH 10 with 2n NaOH and extracted with ethyl acetate; purification by means of MPLC afforded 90 mg of the target product as TFA-salt; ESI-MS [M+K+]=570.2, 534.2, 532.2, 266.5, 101.1.Example 62 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (1.7 mmol, 433.10 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600.00 mg).","Treatment of the raw product with TFA analogously to example 61 afforded 48 mg as a TFA-salt; ESI-MS: 514.2, 512.2, 101.2.Example 63 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)butyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)butane-1,4-diamine (trifluoro acetate) (9) (1.7 mmol, 541.1 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).","Treatment of the raw product with TFA analogously to example 61 afforded 15 mg as a TFA-salt; ESI-MS: 536.2, 500.1, 498.2, 105.1, 101.2.Example 64 [7-Chloro-1-(2-{[5-(4,5-dihydro-1H-imidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(4,5-dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) (1.70 mmol, 351.43 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).","Treatment of the raw product with TFA analogously to example 61 afforded 85 mg as a TFA-salt; ESI-MS: 464.15.Example 65 tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate Ethyldiisopropylamine (2.08 mmol, 268.41 mg) and EDCI*HCl (0.78 mmol, 150.44 mg) were added at 0° C. to a solution of [5-(2-t.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.46 mmol, 0.15 g) in CH3CN (45 ml).","After 1 h at 0° C., 7-(4-aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoro acetate) (35) (0.46 mmol, 0.2 g) was added, stirred for 1 h at 0° C. and overnight at RT.","Since by chromatography a purification could not be achieved, the obtained was reacted to the corresponding Boc-derivative according to standard methods.","Chromatography (heptane/CH2Cl2 0-100%, CH2Cl2/MeOH 0-100%) afforded 15 mg of the target product (about 95% purity); ESI-MS [M+Na+]: 643.5, 622.5, [M+H+]=621.5.Example 66 7-[4-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-1,2,3,4-tetrahydro[1,8]naphthyridine (trifluoro acetate) tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate (example 65; 0.02 mmol, 15 mg) was dissolved in 2 ml CH2Cl2, TFA (0.53 mmol, 60.7 mg) added at 0° C. and stirred overnight at RT.","Usual workup afforded 9.3 mg of the target product as TFA-salt; ESI-MS: 466.2, [M+H+]=465.2, 233.3.Example 67 N-{4-[({[5-(Carboxymethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]cyclohexyl})-1H-benzimidazole (acetate) Coupling analogously to example 49 starting from trans-N-[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (2.99 mmol, 948.78 mg) and [5-(2-tert-butoxy-2-oxoethyl) 7 chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (2.72 mmol, 1.00 g) afforded 760 mg raw product.","Treatment of the raw product with TFA analogously to example 61 and purification of the raw product by means of MPLC afforded 400 mg; ESI-MS: [M+H+]=540.3, 538.25, 269.6, 101.1.Example 68 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 15 ml HCl in diethyl ether (saturated at 0° C.) were added to 300 mg {1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-1-benzazepine-5-yl}acetic acid in 30 ml ethyl alcohol and allowed to stand for 3 days at room temperature.","The mixture was evaporated, the remaining residue purified by chromatography on silica gel (CH2Cl2/CH3OH 5%+1% glacial acetic acid) and lyophilized.","230 mg; ESI-MS: [M+H+]=518.35.Example 69 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (1 g; 2.54 mmol) with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); after chromatography on silica gel (CH2Cl2/CH3OH 5-7%), 1.03 g were isolated as a white glass; ESI-MS: [M+H+]=620.35.Cleavage of the ester under utilization of TFA and subsequent purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 10%+2% glacial acetic acid) afforded 0.8 g of the target product; ESI-MS: [M+H+]=564.25.Example 70 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (0.5 g; 1.27 mmol) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); after chromatography on silica gel (CH2Cl2/CH3OH 3-5%) 0.67 g were isolated as a foam; ESI-MS: [M+H+]=614.35.Cleavage of the ester under utilization of TFA and lyophilization of the obtained product afforded 0.61 g; ESI-MS: [M+H+]=558.35.Example 80 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl}acetic acid 0.4 g (1.4 mmol) [1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.37 g (1.4 mmol) [4-(1H-benzimidazole-2-yl)cyclohexyl]methane amine (hydrochloride) (51) were reacted analogously to example I, the reaction product then purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.","NH3 45/5/0.2) and then the methyl ester cleaved with 0.8 ml 1n NaOH in 8 ml dioxane at room temperature.","After neutralization with 1n HCl, evaporation of the solvent, extraction with ethyl acetate and drying with Na2SO4, the raw product was purified by chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1).","After lyophilization, 0.22 g remained as a white amorphous residue; ESI-MS: [M+H+]=487.Example 81 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.36 g (1.4 mmol) N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) were reacted.","Purification of the raw product, alkaline saponification of the methyl ester and purification of the end stage was conducted analogously to example 80.0.3 g of a white amorphous residue; ESI-MS: [M+H+]=476.Example 82 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1H-benzazepine-1-yl]acetic acid Analogously to example I 0.49 (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) was reacted with 0.63 g (1.4 mmol) N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17).","After purification, alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.3 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=488.Example 83 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo 2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) were reacted with 0.43 g trans-N-J[4-(aminomethyl)cyclohexyl]1H-benzimidazole-2-amine (dihydrochloride) (11).","After purification of the ester (eluent: CH2Cl2/CH3OH/25% NH3 45/5/0.2), alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.18 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=502.Example 84 [5-(2-{[5-H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Analogously to example I 0.65 g (2 mmol) [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) were reacted with N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.","NH3 45/5/0.2); ESI-MS: [M+H+]=535.Then the tert-butyl ester was cleaved with 4n HCl in dioxane, and after chromatographic purification 40 mg of a white amorphous powder were isolated; ESI-MS: [M+H+]=479.Example 85 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); ester stage ESI-MS: [M+H+]=561.Purification after ester cleavage was conducted by means of preparative thick-layer chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 12/3/1); 0.35 g of a white amorphous powder; ESI-MS: [M+H+]=505.Example 86 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); ester stage ESI-MS: [M+H+]=555; target product: 0.4 g of a white amorphous powder; ESI-MS: [M+H+]=499.Example 87 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid Analogously to example I 1.2 g (3.6 mmol) [5-(2-tert-butoxy-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) was reacted with 0.86 g (3.6 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.","NH3 45/5/0.1); ESI-MS: [M+H+]=: 555.Subsequent cleavage of the tert-butylester with 4n HCl in dioxane afforded 0.8 g of a white amorphous powder; ESI-MS [M+H+]=499.Example 88 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid Preparation and purification analogously to example 87; ester stage ESI-MS: [M+H+]=561; 0.9 g of the target product were obtained as a white amorphous powder; ESI-MS [M+H+]=505.Example 89 (1-{2-[4-(1H-Benzimidazole-2-yl)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (acetate) Analogously to example I 0.5 g (1.5 mmol) [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) and 0.36 g (1.5 mmol) 2-(4-piperidinyl)-1H-benzimidazole (J. Heterocycl.","Chem.","1989, 26, 541) were reacted, purified by means of preparative thick-layer chromatography (eluent CH2Cl2/CH3OH/conc.","NH3 45/5/0.2); ESI-MS: [M+H+]=517.Subsequent cleavage of the tert-butyl ester with 4n HCl in a mixture of dioxane/glacial acetic acid and chromatographic purification afforded 0.259 of a white amorphous powder; ESI-MS [M+H+]=: 461.Example 90 (1-{2-[[3-(1H-Benzimidazole-2-yl)propyl](methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (hydrochloride) Preparation analogously to example I by reaction with N-[3-(1H-benzimidazole-2-yl)propyl]-N-methylamine (WO 9849148); after chromatographic purification 0.69 were obtained as a white amorphous powder; ESI-MS [M+H+]=: 449.Example 91 (1-{2-[4-(1H-Benzimidazole-2-ylamino)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Preparation analogously to example I by reaction with N-piperidine-4-yl-1H-benzimidazole-2-amine (J. Med.","Chem.","1985, 28, 1925); after chromatographic purification 0.45 g were obtained as a white amorphous powder; ESI-MS [M+H+]=: 476.Example 92 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) and 0.33 g (1.4 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted and purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.","NH3 45/5/0.2); ESI-MS: [M+H+]=512.Subsequent cleavage of the methyl ester with 1n NaOH in dioxane and chromatographic purification of the residue (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) afforded 0.15 g, isolated as a white amorphous powder; ESI-MS [M+H+]=498.Example 93 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=503; chromatographic purification after cleavage of the ester afforded 0.3 g of the target product as a white amorphous powder; ESI-MS [M+H+]=489.Example 94 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=492.Chromatographic purification after cleavage of the ester afforded 20 mg as a white amorphous powder ESI-MS [M+H+]=478.Example 95 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=518.Chromatographic purification after cleavage of the ester afforded 0.15 g as a white amorphous powder; ESI-MS [M+H+]=504.Example 96 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.6 g (2.4 mmol) N,N-Bis(2-oxo-3-oxazolidinyl)phosphoryl chloride were added to a solution of 0.8 g (1.6 mmol) of the acid of example XI, 0.15 g (3.2 mmol) ethyl alcohol, 0.2 g (1.6 mmol) 4-dimethylaminopyridine and 0.4 g (4 mmol) triethylamine in 60 ml CH2Cl2 and stirred overnight.","After the reaction had completed 2 g glacial acetic acid and 3.7 g ethyl alcohol were added to the reaction solution and stirred for another 20 h at room temperature.","For workup, 50 ml H2O were added to the mixture, the organic phase washed with 10% K2CO3-solution and H2O, dried over MgSO4 and evaporated.","Chromatographic purification of the residue (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 15/5/1) afforded 0.35 g of a white amorphous powder; ESI-MS [M+H+]=532.Analogously to example 96 were prepared: Example 97 Cyclohexyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.26 g; ESI-MS [M+H+]=586.Example 98 Neopentyl{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.21 g; ESI-MS [M+H+]=574.Example 99 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation was carried out analogously to example I by reaction of [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4).","After chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 45/5/0.3) the residue was dissolved in 70 ml CH2Cl2 and 5 ml CH3OH, washed with 5% K2CO3-solution and H2O, dried over Na2SO4 and evaporated to dryness.","The amorphous residue was mixed with 25 ml CH3OH in heat.","0.51 g of white crystals; Fp.",": 231° C. (decomposition); ESI-MS [M+H+]=554.Example 100 Cyclohexyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.64 g (1.28 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml cyclohexanol, 1.0 g HCl-gas introduced with stirring and the resulting yellow solution allowed to stand for 4 days at room temperature.","Since an acid was still detectable by chromatography, it was heated for 7 h to 40-50° C. For workup, 100 ml diethylether were added, washed with K2CO3-solution and H2O, dried over Na2SO4 and the solvent—finally for removal of cyclohexanol under oil pump vacuum and at a bath temperature of 50° C.—distilled off.","The residue was purified chromatographically (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45151410.3).","0.659 of a slightly beige coloured powder; ESI-MS [M+H+]=580.Example 101 Ethyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-6-yl]acetate 0.619 (1.23 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml ethyl alcohol, 0.4 g HCl-gas introduced and allowed to stand for 4 days at room temperature.","The ethyl alcohol was distilled off in vacuum, the residue taken up in ethyl acetate, washed with 5% NaHCO3- and NaCl-solution, dried over Na2SO4 and evaporated.","After thick-layer chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% acetic acid 43/7/0.5) the eluate was diluted with some CH2Cl2 and for removal of the acetic acid washed with 50% NaHCO3-solution.","After drying over Na2SO4 it was evaporated and the residue converted to an amorphous powder filterable with suction by means of diethylether/n-hexane; 0.55 g; ESI-MS [M+H+]=526.Example 102 1-{[(Cyclohexyloxy)carbonyl]oxy}ethyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.3 g K2CO3 were added at 3° C. to a solution of 0.6 g (1.2 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (add of example III) in 10 ml DMF with stirring, some crystals 18-crown-6, 0.4 g cyclohexyl-1-iodo ethylcarbonate (preparation from cyclohexyl-1-chloroethylcarbonate and NaI analogously to J. Antibiot.","1987, 40 (1), 81-90) dissolved in 5 ml CH3CN were added dropwise and stirred for 20 min.","After addition of 100 ml cold NaCl-solution it was extracted several times with ethyl acetate, the combined organic phases washed with NaCl-solution, dried over Na2SO4 and evaporated.","The residue was purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/5/0.3), after evaporation of the solvent taken up in 50 ml CH2Cl2, washed with 5% NaHCO3-solution, dried over Na2SO4 and again evaporated.","90 mg of a white amorphous powder; ESI-MS [M+H+]=668.Example 103 [(5R)-1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 1.0 g (3.0 mmol) of the left-rotating acid (building block 57) and 0.72 g (3.0 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted analogously to example I and the reaction product purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/4/0.3); ester stage ESI-MS [M+H+]=554.Cleavage of the tert-butyl ester with 4n HCl in dioxane afforded 0.82 g of a white amorphous powder; ESI-MS [M+H+]=498; [α]D20=−107.7° (K+-salt, c=1 in H2O).","Example 104 [(5S)-1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 1.5 g of a amorphous powder; ESI-MS [M+H+]=554.Target product: 0.79 g of a white amorphous powder; ESI-MS [M+H+]=498.Example 105 {(5R)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example I from the left-rotating acid building block 57 and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).","Ester stage: 0.9 g of a white amorphous powder; ESI-MS [M+H+]=560.Target product: 0.67 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=−104° (K+-salt, c=1 in H2O).","Example 106 {(SS)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 0.72 g of a amorphous powder; ESI-MS [M+H+]=560.Target product: 0.56 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=+101.60 (K+-salt, c=1 in H2O).","Example 107 [4-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl]acetic acid Analogously to example I 0.6 g (1.8 mmol) [8-(2-tert-butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) were reacted with 0.42 g (1.8 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) and the reaction product purified by thick-layer chromatography (eluent: CH2Cl2/methanol/conc.","NH3 451510.2); ESI-MS [M+H+]=560.Cleavage of the t-butyl group with 4n HCl in dioxane afforded 0.34 g of a slightly yellowish powder; ESI-MS [M+H+]=504.Example 108 {4-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl}acetic acid Analogously to example 107.Ester stage: 100 mg of a white amorphous powder, ESI-MS [M+H+]=566.Target product: 98 mg of a white amorphous powder; ESI-MS [M+H+]=510.II.","BIOLOGICAL EXAMPLES Example 1 Integrin αvβ3 Assay For the identification and assessment of integrin αvβ3 ligands, a test system was used which was based on competition between the natural integrin αvβ3 ligand vitronectin and the test substance for binding to solid phase-bound integrin αvβ3.Procedure Microtiter plates coated with 250 ng/ml of integrin αvβ3 in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturation with 1% powdered milk/assay buffer; 0.3 ml/well; 0.5 h/RT 3× washing with 0.05% Tween 20/assay buffer test substance in 0.1% powdered milk/assay buffer, 50 μl/well+0 μg/ml or 2 μg/ml of human vitronectin (Boehringer Ingelheim T007) in 0.1% powdered milk/assay buffer, 50 μl/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 1 μg/ml of anti human vitronectin antibody coupled to peroxidase (Kordia SAVN-APHRP) in 0.1% powdered milk/assay buffer; 0.1 ml/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 0.1 ml/well of peroxidase substrate stop reaction with 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Integrin αvβ3: Human placenta is solubilized with Nonidet and integrin αvβ3 affinity-purified on a GRGDSPK matrix (elution with EDTA).","Impurities due to integrin αIIbβ3 and human serum albumin, and the detergent and EDTA are removed by anion-exchange chromatography.","Assay buffer: 50 mM Tris pH 7.5; 100 mM NaCl; 1 mM CaCl2; 1 mM MgCl2; 10 μM MnCl2 Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M sodium acetate pH 4.9), then add 14.7 μl of 3% H2O2.Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the ligand at which 50% of the ligand is displaced).","The compound from Example VII showed the best result here.","Example 2 Integrin αIIbβ3 Assay The assay is based on competition between the natural integrin αIIbβ3 ligand fibrinogen and the test substance for binding to integrin αIIbβ3.Procedure Coat microtiter plates with 10 μg/ml of fibrinogen (Calbiochem 341578) in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturate with 1% BSA/PBS; 0.3 ml/well; 30 min/RT 3× washing with 0.05% Tween 20/PBS test substance in 0.1% BSA/PBS; 50 μl/well+200 μg/ml of integrin αIIbβ3 (Kordia) in 0.1% BSA/PBS; 50 μl/well; 2 to 4 h/RT 3× washing as above biotinylated anti-integrin αIIbβ3 antibody (Dianova CBL 130 B); 1:1000 in 0.1% BSA/PBS; 0.1 ml/well; 2 to 4 h/RT.","3× washing as above streptavidin-peroxidase complex (B.M.","1089153) 1:10000 in 0.1% BSA/PBS; 0.1 ml/well; 30 min/RT 3× washing as above 0.1 ml/well of peroxidase substrate stop reaction using 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M Na acetate pH 4.9), then add 14.7 μl of 3% H2O2 Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the antagonist at which 50% of the ligand is displaced).","By comparison of the IC50 values in the integrin αIIbβ3 and integrin αvb3 assay, the selectivity of the substances can be determined.","Example 3 CAM Assay The CAM (chorioallantoic membrane) assay serves as a generally recognized model for the assessment of the in vivo activity of integrin αvβ3 antagonists.","It is based on the inhibition of angiogenesis and neovascularization of tumor tissue (Am.","J. Pathol.","1975, 79, 597-618; Cancer Res.","1980, 40, 2300-2309; Nature 1987, 329, 630).","The procedure is carried out analogously to the prior art.","The growth of the chicken embryo blood vessels and of the transplanted tumor tissue can be readily monitored and assessed.","Example 4 Rabbit Eye Assay In this in vivo model, the inhibition of angiogenesis and neovascularization in the presence of integrin αvβ3 antagonists can be monitored and assessed analogously to Example 3.The model is generally recognized and is based on the growth of rabbit blood vessels starting from the edge in the corner of the eye (Proc.","Natl.","Acad.","Sci.","USA.","1994, 91, 4082-4085; Science 1976, 193, 70-72).","The procedure is carried out analogously to the prior art."]],"string":"[\n [\n \"Ligands of Integrin Receptors\",\n \"The invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds.\"\n ],\n [\n \"1.The use of compounds of the formula I B-G-L I as ligand of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3, together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4 RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —C1-NRG12RG13—NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11 B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.\",\n \"2.The use as claimed in claim 1, wherein the structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally contained or the carbon atoms can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.3.The use as claimed in one of claims 1 or 2, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18, where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—Cl1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms-O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both: radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA1, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11 Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.\",\n \"4.The use as claimed in one of claims 1 to 3, wherein the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE9RE10)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 1-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NHC1-C6-alkoxyalkyl, CO—NHC1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an, optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.\",\n \"5.The use as claimed in one of claims 1 to 4, wherein the spacer structural-element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6—IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14 (ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical or independently of one another are in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle, which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms together with RW2 or RW2* a saturated or unsaturated C3-C7-heterocycle, which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.\",\n \"6.The use of the structural element of the formula IGL -G-L, IGL for the preparation of compounds which bind to integrin receptors, where G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 und RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted, C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, with the proviso that in the case of this fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle substituents are excluded which contain a structural element —V—CO—R8, where V is an optionally substituted C1-C2-alkylene radical R8 is a hydroxyl group, a C1-C8-alkoxy, aryl-C0-C6-alkoxy, C1-C8-alkylcarbonyloxy-C1-C4-alkoxy or aryl-C1-C8-alkylcarbonyloxy-C1-C4-alkoxy group or an L- or D-amino acid, which is bonded by an amide bond and in which the carboxylic acid component of said amino acid is present as a free acid or esterified with C1-C6-alkyl, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—N12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and which can contain up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11.7.The use of the compounds as claimed in one of claims 1 to 5 as ligands of the αVβ3 integrin receptor.\",\n \"8.The use of the compounds as claimed in one of claims 1 to 5 for the production of drugs for treating diseases in which the interaction between integrins and their natural ligands is excessive or decreased.\",\n \"9.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 8 for the treatment of diseases in which the interaction between αVβ3 ingegrin and its natural ligands is excessive or decreased.\",\n \"10.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 9 for the treatment of atherosclerosis, rheumatoid arthritis, restenosis after vascular injury or stent implantation, angioplasty, acute kidney failure, angiogenesis-associated microangiopathies, diabetic angiopathies, blood platelet-mediated vascular occlusion, arterial thrombosis, congestive heart failure, myocardial infarct, stroke, cancer, osteoporosis, high blood pressure, psoriasis or viral, parasitic or bacterial conditions, inflammation, wound healing, hyperparathyroidism, Paget's disease, malignant hypercalcemia or metastatic osteolytic lesions.\",\n \"11.A compound of the formula I′ A-E′-G′-L I′ where A, E′, G′ and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G′ is a structural element of the formula IG where the structural element A-E′ is bonded to the structural element G′ via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG′12RG′13, C1-C4-alkylene-CO—NRG′12RG′13, C1-C4-alkylene-O—CO—NRG′12RG′13, C1-C4-alkylene-NRG′12RG′13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG′12RG′13, —SO2—NRG′12RG′13, —CO—NRG′12RG′13, —NRG′12RG′13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG′12, RG′13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-heterocycloalkyl, C1-C4-alkylene-heterocycloalkenyl or hetarylalkyl radical, E′ is a structural element, composed of two to four substructural elements, selected from the group E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO-o, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE8 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—C—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW12)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded, A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon, which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.\",\n \"12.A compound as claimed in claim 11, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where both radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* is a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6 are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms Q, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.\",\n \"13.A compound as claimed in claim 11 or 12, wherein the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 (NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon, which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group-O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded.\",\n \"14.A compound as claimed in one of claims 11 to 13 for use as a drug.\",\n \"15.The use of the compounds as claimed in one of claims 11 to 13 for the production of drugs for treating diseases.\",\n \"16.A pharmaceutical preparation comprising, in addition to the customary pharmaceutical excipients, at least one compound as claimed in one of claims 11 to 13.17.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet adhesion, activation or aggregation, anticoagulants which prevent thrombin activity or formation, antagonists of blood platelet-activating compounds or selectin antagonists.\",\n \"18.The use of the pharmaceutical preparation as claimed in claim 17 for the production of a drug for treating blood platelet-mediated vascular occlusion or thrombosis.\",\n \"19.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet activation or aggregation, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vascular wall transmigration, fibrinolysis-modulating compounds, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists.\",\n \"20.The use of the pharmaceutical preparation as claimed in claim 19 for the production of a drug for treating myocardial infarct or stroke.\",\n \"21.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors.\",\n \"22.The use of the pharmaceutical preparation as claimed in claim 21 for the production of a drug for treating congestive heart failure.\",\n \"23.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, inhibitors of blood platelet adhesion, activation or aggregation, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants.\",\n \"24.The use of the pharmaceutical preparation as claimed in claim 23 for the production of a drug for treating restenosis after vascular injury or stent implantation.\",\n \"25.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, glycosylation inhibitors and AGE formation inhibitors or AGE breakers and antagonists of their receptors.\",\n \"26.The use of the pharmaceutical preparation as claimed in claim 25 for the production of a drug for treating diabetic angiopathies.\",\n \"27.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers.\",\n \"28.The use of the pharmaceutical preparation as claimed in claim 27 for the production of a drug for treating atherosclerosis.\",\n \"29.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group cytostatic or antineoplastic compounds, compounds which inhibit proliferation and heparin or low-molecular weight heparins or further GAGs.\",\n \"30.The use of the pharmaceutical preparation as claimed in claim 29 for the production of a drug for the treatment of cancer.\",\n \"31.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group compounds for antiresorptive therapy, compounds for hormone exchange therapy, recombinant human growth hormone, bisphosphonates, compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, interleukin-6 antagonists and Src tyrosine kinase inhibitors.\",\n \"32.The use of the pharmaceutical preparation as claimed in claim 31 for the production of a drug for the treatment of osteoporosis.\",\n \"33.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors.\",\n \"34.The use of the pharmaceutical preparation as claimed in claim 33 for the production of a drug for treating rheumatoid arthritis.\",\n \"35.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group collagenase, PDGF antagonists and MMPs.\",\n \"36.The use of the pharmaceutical preparation as claimed in claim 35 for the production of a drug for improving wound healing.\"\n ],\n [\n \"The present invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds.\",\n \"Integrins are cell surface glycoprotein receptors which mediate interactions between similar and different cells as well as between cells and extracellular matrix proteins.\",\n \"They are involved in physiological processes, such as embryogenesis, hemostasis, wound healing, immune response and formation/maintenance of the tissue architecture.\",\n \"Disturbances in the gene expression of cell adhesion molecules and functional disorders of the receptors can contribute to the pathogenesis of many disorders, such as tumors, thromboembolic events, cardiovascular disorders, lung diseases, disorders of the CNS, the kidney, the gastrointestinal tract or inflammation.\",\n \"Integrins are heterodimers of an α- and a β-transmembrane subunit in each case, which are noncovalently bonded.\",\n \"Up to now, 16 different α- and 8 different β-subunits and 22 different combinations have been identified.\",\n \"Integrin αvβ3, also called the vitronectin receptor, mediates adhesion to a multiplicity of ligands—plasma proteins, extracellular matrix proteins, cell surface proteins, of which the majority contain the amino acid sequence RGD (Cell, 1986, 44, 517-518; Science 1987, 238, 491-497), such as vitronectin, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, osteopontin, laminin, collagen, thrombin, tenascin, MMP-2, bone sialoprotein II, various viral, fungal, parasitic and bacterial proteins, natural integrin antagonists such as disintegrins, neurotoxins—mambin—and blood fluke proteins—decorsin, ornatin—and also some non-RGD ligands, such as Cyr-61 and PECAM-1 (L. Piali, J.\",\n \"Cell Biol.\",\n \"1995, 130, 451-460; Buckley, J.\",\n \"Cell Science 1996, 109, 437-445, J. Biol.\",\n \"Chem.\",\n \"1998, 273, 3090-3096).\",\n \"A number of integrin receptors show cross-reactivity with ligands which contain the RGD motif.\",\n \"Thus integrin αIIbβ3, also called the platelet fibrinogen receptor, recognizes fibronectin, vitronectin, thrombospondin, von Willebrand factor and fibrinogen.\",\n \"Integrin αvβ3 is expressed, inter alia, on endothelial cells, blood platelets, monocytes/macrophages, smooth muscle cells, some B cells, fibroblasts, osteoclasts and various tumor cells, such as melanoma, glioblastoma, lung, breast, prostate and bladder carcinomas, osteosarcomas or neuroblastomas.\",\n \"Increased expression is observed under various pathological conditions, such as in the prothrombotic state, in vascular injury, tumor growth or metastasis or reperfusion and on activated cells, in particular on endothelial cells, smooth muscle cells or macrophages.\",\n \"An involvement of integrin αvβ3 has been demonstrated, inter alia, in the following syndromes: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury, and angioplasty (neointima formation, smooth muscle cell migration and proliferation) (J. Vasc.\",\n \"Surg.\",\n \"1994, 19, 125-134; Circulation 1994, 90, 2203-2206), acute kidney failure (Kidney Int.\",\n \"1994, 46, 1050-1058; Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"1993, 90, 5700-5704; Kidney Int.\",\n \"1995, 48, 1375-1385), angiogenesis-associated microangiopathies such as diabetic retinopathy or rheumatoid arthritis (Ann.\",\n \"Rev.\",\n \"Physiol 1987, 49, 453-464; Int.\",\n \"Opthalmol.\",\n \"1987, 11, 41-50; Cell 1994, 79, 1157-1164; J. Biol.\",\n \"Chem.\",\n \"1992, 267, 1093.1-10934), arterial thrombosis, stroke (phase II studies with ReoPro, Centocor Inc., 8th annual European Stroke Meeting), carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis) (Cell 1991, 64, 327-336; Nature 1989, 339, 58-61; Science 1995, 270, 1500-1502), osteoporosis (bone resorption after proliferation, chemotaxis and adhesion of osteoclasts to bone matrix) (FASEB J.\",\n \"1993, 7, 1475-1482; Exp.\",\n \"Cell Res.\",\n \"1991, 195, 368-375, Cell 1991, 64, 327-336), high blood pressure (Am.\",\n \"J. Physiol.\",\n \"1998, 275, H1449-H1454), psoriasis (Am.\",\n \"J. Pathol.\",\n \"1995, 147, 1661-1667), hyperparathyroidism, Paget's disease (J. Clin.\",\n \"Endocrinol.\",\n \"Metab.\",\n \"1996, 81, 1810-1820), malignant hypercalcemia (Cancer Res.\",\n \"1998, 58, 1930-1935), metastatic osteolytic lesions (Am.\",\n \"J. Pathol.\",\n \"1997, 150, 1383-1393), pathogenic protein (e.g.\",\n \"HIV-1 tat)-induced processes (e.g.\",\n \"angiogenesis, Kaposi's sarcoma) (Blood 1999, 94, 663-672) inflammation (J.\",\n \"Allergy Clin.\",\n \"Immunol.\",\n \"1998, 102, 376-381), cardiac insufficiency, CHF, and also in antiviral, antiparasitic, antifungal or antibacterial therapy and prophylaxis (adhesion and internalization) (J. Infect.\",\n \"Dis.\",\n \"1999, 180, 156-166; J. Virology 1995, 69, 2664-2666; Cell 1993, 73, 309-319).\",\n \"On account of its key role, pharmaceutical preparations which contain low-molecular weight integrin αvβ3 ligands are of high therapeutic or diagnostic benefit, inter alia, in the indications mentioned.\",\n \"Advantageous αvβ3 integrin receptor ligands bind to the integrin αvβ3 receptor with an increased affinity.\",\n \"In contrast to integrin αvβ3, particularly advantageous αvβ3 integrin receptor ligands additionally have an increased selectivity and are less active with respect to the integrin αIIbβ3 by at least a factor of 10, preferably at least a factor of 100.For a multiplicity of compounds, such as anti-αvβ3 monoclonal antibodies, peptides which contain the RGD binding sequence, natural, RGD-containing proteins (e.g.\",\n \"disintegrins) and low-molecular weight compounds, an integrin αvβ3 antagonistic action has been shown and a positive in vivo effect demonstrated (FEBS Letts 1991, 291, 50-54; J. Biol.\",\n \"Chem.\",\n \"1990, 265, 12267-12271; J. Biol.\",\n \"Chem.\",\n \"1994, 269, 20233-20238; J.\",\n \"Cell Biol 1993, 51, 206-218; J. Biol.\",\n \"Chem.\",\n \"1987, 262, 17703-17711; Bioorg.\",\n \"Med.\",\n \"Chem.\",\n \"1998, 6, 1185-1208).\",\n \"Antagonists of the αVβ3 integrin receptor based on a bicyclic structural element are described in WO 9906049, WO 9905107, WO 9814192, WO 9724124, WO 9724122 and WO 9626190.EP 540 334 and WO 9308174 describe bicyclic antagonists of the αIIbβ3 integrin receptor.\",\n \"WO 9407488 A1 describes compounds having a bicyclic molecular structure and which accelerate the release of growth hormone.\",\n \"Further, vasopressin antagonists having a bicyclic molecular structure are described in the specifications EP 620216, WO 9534540, WO 9408582, WO 9802432, WO 9420473, JP 09221476 A1, JP 11060488 A1, WO 9404525, JP 04321669 A1, WO 9722591, as well as in Matsuhisa et al., Chem.\",\n \"Pharm.\",\n \"Bull.\",\n \"1999, 47, 3, 329-339.It is an object of the present invention to make available novel integrin receptor ligands having advantageous properties.\",\n \"We have found that this object is achieved by the use of compounds of the formula I B-G-L I as ligands of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.\",\n \"In the structural element L, T is understood as meaning a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH.\",\n \"A radical hydrolyzable to COOH is understood as meaning a radical which changes into a group COOH after hydrolysis.\",\n \"A group which may be mentioned by way of example as a radical T hydrolyzable to COOH is in which R1 has the following meanings: a) OM, where M can be a metal cation, such as an alkali metal cation, such as lithium, sodium, potassium, the equivalent of an alkaline earth metal cation, such as calcium, magnesium and barium, or an environmentally tolerable organic ammonium ion such as primary, secondary, tertiary or quaternary C1-C4-alkylammonium or ammonium ion, such as ONa, OK or OLi, b) a branched or unbranched, optionally halogen-substituted C1-C8-alkoxy radical, such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, in particular methoxy, ethoxy, 1-methylethoxy, pentoxy, hexoxy, heptoxy, octoxy, difluoromethoxy, trifluoromethoxy, chlorodifluoromethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2,2,2-trifluoroethoxy, 2-chloro-1,1,2-trifluoroethoxy or pentafluoroethoxy c) a branched or unbranched, optionally halogen-substituted C1-C4-alkylthio radical such as methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio or 1,1-dimethylethylthio radical d) an optionally substituted —O-alkylenearyl radical, such as —O-benzyl e) R1 is further a radical —(O)m—N(R18)(R19), in which m is 0 or 1 and R18 and R19, which can be identical or different, have the following meanings: hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl or the corresponding substituted radicals, preferably methyl, ethyl, propyl, butyl or i-butyl, C2-C6-alkenyl radical, such as vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl or the corresponding substituted radicals, C2-C6-alkynyl radical, such as ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl or the corresponding substituted radicals, C3-C8-cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl und cycloheptyl, cyclooctyl or the corresponding substituted radicals, or a phenyl radical, optionally mono- or polysubstituted, for example mono- to trisubstituted, by halogen, nitro, cyano, C1-C4-alkyl, C1-C4-halogenoalkyl, C1-C4-alkoxy, C1-C4-halogenoalkoxy or C1-C4-alkylthio such as 2-fluorophenyl, 3-chlorophenyl, 4-bromophenyl, 2-methylphenyl, 3-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-methoxyphenyl, 4-trifluoroethoxyphenyl, 2-methylthiophenyl, 2,4-dichlorophenyl, 2-methoxy-3-methylphenyl, 2,4-dimethoxyphenyl, 2-nitro-5-cyanophenyl, 2,6-difluorophenyl, or R18 and R19 together form an optionally substituted, e.g.\",\n \"C1-C4-alkyl-substituted, C4-C7-alkylene chain closed to give a cycle, which can contain a heteroatom selected from the group consisting of oxygen, sulfur and nitrogen, such as —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)2—O—(CH2)2—, —CH2—S—(CH2)3—, —(CH2)2—O—(CH2)3—, —NH—(CH2)3—, —CH2—NH—(CH2)2—, —CH2—CH═CH—CH2—, —CH═CH—(CH2)3—, —CO— (CH2)2—CO— or —CO— (CH2)3—CO—.\",\n \"A radical bioisosteric to COOH is understood as meaning radicals which can replace the function of a group COOH in active compounds by equivalent bond donor/acceptor capabilities or by equivalent charge distribution.\",\n \"Radicals which may be mentioned by way of example as radicals bioisosteric to —COOH are those such as described in “The Practice of Medicinal Chemistry, Editor: C. G. Wermuth, Academic Press 1996, pages 125 and 216, in particular the radicals —P═O(OH)2, —SO3H, tetrazole or acylsulfonamides.\",\n \"Preferred radicals T are —COOH, —CO—O—C1-C8-alkyl or —CO—O-benzyl.\",\n \"The radical —U— in the structural element L is a spacer selected from the group —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1-.\",\n \"In the case of the radical ═CRL1—, the structural element L is linked to the structural element G via a double bond.\",\n \"XL is a radical CRL3RL4, NRL5, oxygen or sulfur.\",\n \"Preferred radicals —U— are the radicals —CRL1═CRL2—, ethynylene or —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a=1).\",\n \"Particularly preferred radicals —U— are the radicals —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a 1).\",\n \"Under RL11, RL2, RL3 or RL4 in the structural element L, a halogen radical is understood as meaning, for example, F, Cl, Br or I, preferably F. Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C6-alkyl radical is understood as meaning, for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, preferably branched or unbranched C1-C4-alkyl radicals such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, particularly preferably methyl.\",\n \"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkenyl radical is understood as meaning, for example, vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl.\",\n \"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkynyl radical is understood as meaning, for example, ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably ethynyl, 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl.\",\n \"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C3-C7-cycloalkyl radical is understood as meaning, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.\",\n \"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C4-alkoxy radical is understood as meaning, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy or 1,1-dimethylethoxy.\",\n \"The radicals —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 are secondary or tertiary amides and are composed of the amide bond and the corresponding C1-C6-alkyl radicals such as described above for RL1, RL2, RL3 or RL4.The radicals RL1, RL2, RL3 or RL4 can furthermore be a radical C1-C2-alkylene-T, such as methylene-T or ethylene-T, C2-alkenylene-T, such as ethenylene-T or C2-alkynylene-T, such as ethynylene-T, an aryl radical, such as phenyl, 1-naphthyl or 2-naphthyl or an arylalkyl radical, such as benzyl or ethylenephenyl (homobenzyl), where the radicals can optionally be substituted.\",\n \"Furthermore, two radicals RL1 and RL2 or RL3 and RL4 or optionally RL1 and RL3 can in each case independently of one another together be an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. All radicals for RL1, RL2, RL3 or RL4 can be optionally substituted.\",\n \"For the radicals RL1, RL2, RL3 oder RL4 and all further substituted radicals of the description below, suitable substituents, if the substituents are not specified in greater detail, are independently of one another up to 5 substituents, for example selected from the following group: —NO2, —NH2, —OH, —CN, —COOH, —O—CH2—COOH, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl, CF3, C2F5 or CH2F, —CO—O—C1-C4-alkyl, C3-C6-cycloalkyl, C1-C4-alkoxy, C1-C4-thioalkyl, —NH—CO—O—C1-C4-alkyl, —O—CH2—COO—C1-C4-alkyl, —NH—CO—C1-C4-alkyl, —CO—NH—C1-C4-alkyl, —NH—SO2—C1-C4-alkyl, —SO2—NH—C1-C4-alkyl, —N(C1-C4-alkyl)2, —NH—C1-C4-alkyl, or —SO2—C1-C4-alkyl radical, such as —SO2—CF3, an optionally substituted —NH—CO-aryl, —CO—NH-aryl, —NH—CO—O-aryl, —NH—CO—O-alkylenearyl, —NH—SO2-aryl, —SO2—NH-aryl, —CO—NH-benzyl, —NH—SO2-benzyl or —SO2—NH-benzyl radical, an optionally substituted radical —SO2—NR2R3 or —CO—NR2R3 where the radicals R2 and R3 independently of one another can have the meaning RL5 as below or both radicals R2 and R3 together can be a 3- to 6-membered, optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three further different or identical heteroatoms O, N, S, and optionally two radicals substituted on this heterocycle can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted cycle can be fused to this cycle.\",\n \"If not specified in greater detail, in all terminally bonded, substituted-hetaryl radicals of the description, two substituents can form a fused 5- to 7-membered, unsaturated or aromatic carbocycle.\",\n \"Preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical or the radical —NRL6RL7.Particularly preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, fluorine or a branched or unbranched, optionally substituted C1-C4-alkyl radical, preferably methyl.\",\n \"The radicals RL5, RL6, RL7 in structural element L are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, for example as described above for RL1, C3-C7-cycloalkyl radical, for example as described above for RL1, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the C1-C6-alkyl radicals described above for RL1, or an optionally substituted CO—O-alkylenearyl, SO2-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the aryl or arylalkyl radicals described above for RL1.Preferred radicals for RL6 in structural element L are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—O—C1-C4-alkyl, CO—C1-C4-alkyl or SO2—C1-C4-alkyl radical or an optionally substituted CO—O-benzyl, SO2-aryl, SO2-alkylenearyl or CO-aryl radical.\",\n \"Preferred radicals for RL7 in structural element L are hydrogen or a branched or unbranched, optionally substituted C1-C4-alkyl radical.\",\n \"Preferred structural elements L are composed of the preferred radicals of the structural element.\",\n \"Particularly preferred structural elements L are composed of the particularly preferred radicals of the structural element.\",\n \"G is a structural element of the formula IG where the structural element B is bonded via the ring nitrogen and the structural element L is bonded via WG to the structural element G, optionally via a double bond.\",\n \"YG in structural element G is CO, CS, C═NRG2 or CRG3RG4, preferably CO, C═NRG2 or CRG3RG4, particularly preferably CO or CRG3RG4.RG2 in structural element G is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical, for example as described above for RL1 in each case, an optionally substituted —O—C3-C7-cycloalkyl radical, which is composed of an ether group and, for example, of the C3-C7-cycloalkyl radical described above for RL1, an optionally substituted aryl or arylalkyl radical, for example as described above for RL1 in each case or an optionally substituted —O-aryl or —O-alkylenearyl radical, which is composed of a group —O— and, for example, of the aryl or arylalkyl radicals described above for RL1.Branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radicals for RG3 or RG4 in structural element G independently of one another are understood as meaning, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG3 and RG4 can together form a cyclic acetal, such as —O—CH2—CH2—O— or —O—CH2—O—.\",\n \"Furthermore, both radicals RG3 and RG4 can together form an optionally substituted C3-C7-cycloalkyl radical.\",\n \"Preferred radicals for RG3 or RG4 are independently of one another hydrogen, C1-C4-alkyl or C1-C4-alkoxy.\",\n \"Branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radicals and optionally substituted aryl or arylalkyl radicals for RG5 and RG6 in structural element G independently of one another are, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG5 and RG6 can together form an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. Preferred radicals for RG5 and RG6 are independently of one another hydrogen or optionally substituted aryl radicals, preferably phenyl or arylalkyl radicals, preferably benzyl, and in each case both radicals RG5 and RG6 together can contain an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S. In particularly preferred radicals for RG5 and RG6, both radicals RG5 and RG6 together form an optionally substituted, fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle, for example selected from one of the following doubly bonded structural formulae: in particular selected from one of the following, doubly bonded structural formulae: Suitable substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are in particular substituents such as generally described above.\",\n \"Particularly preferred substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are independently of one another up to four substituents selected from the following group: hydroxyl, —CN, F or Cl or a branched or unbranched, optionally substituted C1-C4-alkoxy or C1-C4-alkyl radical, such as methoxy, methyl, CF3, C2F5 or CH2F.\",\n \"WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, where the dashed lines intersect the atomic bonds within the structural element G and the carbon atom substituted by RG7 and RG8 is bonded to YG.\",\n \"In a preferred embodiment, WG is a structural element selected from the group of structural elements of the formulae IWG2 and IWG3, in particular the structural element of the formula IWG2.RG1 in structural element WG is hydrogen, halogen, such as Cl, F, Br or I, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl radical, preferably C1-C4-alkyl or C1-C4-alkoxy radical such as in each case described above for RL1.Particularly preferred radicals for RG1 are hydrogen, methoxy or hydroxyl.\",\n \"RG7, RG8, RG9 and RG10 in structural element G are independently of one another hydrogen, a hydroxyl group, CN, halogen, such as F, Cl, Br, I, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, C2-C6-alkenyl radical, such as optionally substituted vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl or 1-ethyl-2-methyl-2-propenyl, C2-C6-alkynyl radical, such as optionally substituted ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl or 1-ethyl-1-methyl-2-propynyl, an optionally substituted C3-C7-cycloalkyl radical, such as optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, C3-C7-heterocycloalkyl radical, such as optionally substituted aziridinyl, diaziridinyl, oxiranyl, oxaziridinyl, oxetanyl, thiiranyl, thietanyl, pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,4-dioxanyl, hexahydroazepinyl, oxepanyl, 1,2-oxathiolanyl or oxazolidinyl, C3-C7-heterocycloalkenyl radical, such as optionally substituted azirinyl, diazirinyl, thiirenyl, thietyl, pyrrolinyls, oxazolinyls, azepinyl, oxepinyl, α-pyranyl, β-pyranyl, γ-pyranyl, dihydropyranyls, 2,5-dihydropyrrolinyl or 4,5-dihydrooxazolyl, a branched or unbranched, optionally substituted C1-C4-alkylene-C3-C7-cycloalkyl radical, which is composed, for example, of branched or unbranched C1-C4-alkylene radicals such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-cycloalkyl radicals, a branched or unbranched optionally substituted C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, which is composed of optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-heterocycloalkyl or C3-C7-heterocycloalkenyl radicals, the radicals being preferred which in the cyclic moiety contain one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds, a branched or unbranched, optionally substituted radical C1-C4-alkylene-O—RG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13, C1-C4-alkylene-SRG11 or C1-C4-alkylene-SO—RG11, which is composed of branched or unbranched, optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene, the corresponding groups —O—, —CO—, —S—, —N and the terminal radicals RG11, RG12 and RG13 described below, an optionally substituted aryl radical, preferably optionally substituted phenyl, 1-naphthyl or 2-naphthyl, arylalkyl radical, preferably optionally substituted benzyl or ethylenephenyl (homobenzyl), hetaryl radical, preferably optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, thiadiazolyl, oxadiazolyl or triazinyl or their fused derivatives such as indazolyl, indolyl, benzothiophenyl, benzofuranyl, indolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolinyl or isoquinolinyl, hetarylalkyl radical, preferably optionally substituted —CH2-2-pyridyl, —CH2-3-pyridyl, —CH2-4-pyridyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-thiazolyl, —CH2-4-thiazolyl, CH2-5-thiazolyl, —CH2—CH2-2-pyridyl, —CH2—CH2-3-pyridyl, —CH2—CH2-4-pyridyl, —CH2—CH2-2-thienyl, —CH2—CH2-3-thienyl, —CH2—CH2-2-thiazolyl, —CH2—CH2-4-thiazolyl or —CH2—CH2-5-thiazolyl or a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13, CO—RG11.Further, two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 can in each case independently of one another together form an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group consisting of O, N, S and up to two double bonds.\",\n \"Preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical, as described above in each case.\",\n \"Particularly preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, F or a branched or unbranched, optionally substituted C1-C4-alkyl radical, as described above.\",\n \"A branched or unbranched, optionally substituted C1-C8-alkyl radical for RG11, RG12 and RG13 is understood as meaning independently of one another, for example, the C1-C6-alkyl radicals mentioned above for RG1, plus the radicals heptyl and octyl.\",\n \"Preferred substituents of the branched or unbranched, optionally substituted C1-C8-alkyl radicals for RG11, RG12 and RG13 independently of one another are the radicals halogen, hydroxyl, C1-C4-alkoxy, —CN, —COOH and —CO—O—C1-C4-alkyl.\",\n \"A branched or unbranched, optionally substituted C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkylene-C3-C7-cycloalkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RG11, RG12 and RG13 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Preferred, branched or unbranched, optionally substituted —C1-C5-alkylene-C1-C4-alkoxy radicals for RG11, RG12 and RG13 are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.\",\n \"Preferred, branched or unbranched, optionally substituted mono- and bis-alkylaminoalkylene or acylaminoalkylene radicals for RG11, RG12 and RG13 are independently of one another branched or unbranched, optionally substituted radicals —C1-C4-alkylene-NH(C1-C4-alkyl), —C1-C4-alkylene-N(C1-C4-alkyl)2 or —C1-C4-alkylene-NH—CO—C1-C4-alkyl.\",\n \"Preferred optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1.Particularly preferred, optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1, one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds being contained in the cyclic moiety.\",\n \"Further, RG12 and RG13 can independently of one another be a radical —SO2—RG11, —CO—O—RG11, —CO—NRG11RG11* or —CO—RG11, RG11* being a radical RG11 which is independent of RG11.Preferred structural elements G are composed of at least one preferred radical of the structural element G, while the remaining radicals are widely variable.\",\n \"Particularly preferred structural elements G are composed of the preferred radicals of the structural element G. Very particularly preferred structural elements G are composed of the particularly preferred radicals of the structural element G. Structural element B is understood as meaning a structural element comprising at least one atom which under physiological conditions can form hydrogen bridges as a hydrogen acceptor, at least one hydrogen acceptor atom having a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton.\",\n \"The arrangement of the structural skeleton of structural element B is widely variable.\",\n \"Suitable atoms which under physiological conditions can form hydrogen bridges as hydrogen acceptors are, for example, atoms having Lewis base properties, such as the heteroatoms nitrogen, oxygen or sulfur.\",\n \"Physiological conditions is understood as meaning a pH which prevails at the site in a body at which the ligands interact with the receptors.\",\n \"In the present case, the physiological conditions have a pH of, for example, 5 to 9.In a preferred embodiment, structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—R11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.In a particularly preferred embodiment, the structural element A is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, g independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1), —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.\",\n \"In a further very particularly preferred embodiment, the structural element A is a structural element of the formula IA1, IA4, IA7, IA8 or IA9.For RA1 or RA2 independently of one another a branched or unbranched, optionally substituted C1-C6-alkyl radical is understood as meaning, for example, the corresponding radicals described above for RG1, preferably methyl or trifluoromethyl.\",\n \"For RA1 or RA2 in the structural elements IA1, IA2, IA3 or IA17, the branched or unbranched, optionally substituted radical CO—C1-C6-alkyl is composed, for example, of the group CO and the branched or unbranched, optionally substituted C1-C6-alkyl radicals described above for RA1 or RA2.Optionally substituted hetaryl, hetarylalkyl, aryl, arylalkyl or C3-C7-cycloalkyl radicals for RA1 or RA2 independently of one another are understood as meaning, for example, the corresponding radicals described above for RG7.For RA1 or RA2, the optionally substituted radicals CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 are composed, for example, of the groups CO—O, O, S, N, CO—N or SO2—N and the radicals RA14, RA15 or RA16 described in greater detail below.\",\n \"Further, both radicals RA1 and RA2 can together form a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group consisting of O, N and S. RA13 and RA13* are independently of one another hydrogen, CN, halogen, such as fluorine, chlorine, bromine or iodine, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as described above for RG1, preferably methyl or trifluoromethyl or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2NRA15RA16 or CO—NRA15RA16 as in each case described above for RA1.Preferred radicals for RA13 and RA13* are the radicals hydrogen, F, Cl, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or arylalkyl or a radical CO—O—RA14, O—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16.A branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, alkylenecycloalkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl or C2-C6-alkynyl radical for RA14 in structural element A is understood as meaning, for example, the corresponding radicals described above for RG7.Optionally substituted aryl, arylalkyl, hetaryl or alkylhetaryl radicals for RA14 in structural element A are understood as meaning, for example, the corresponding radicals described above for RG7.Preferred radicals for RA14 are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical and optionally substituted benzyl.\",\n \"A branched or unbranched, optionally substituted C1-C6-alkyl or arylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or hetarylalkyl radical for RA15 or RA16 independently of one another is understood as meaning, for example, the corresponding radicals described above for RA14.The branched or unbranched, optionally substituted CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, COO-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or SO2-alkylenearyl radicals or the optionally substituted CO-aryl, SO2-aryl, CO—NH-aryl, CO—NH-hetaryl or CO-hetaryl radicals for RA15 or RA16 are composed, for example, of the corresponding groups —CO—, —SO2—, —CO—O—, —CO—NH— and the corresponding branched or unbranched, optionally substituted C1-C6-alkyl, hetarylalkyl or arylalkyl radicals or the corresponding optionally substituted aryl or hetaryl radicals described above.\",\n \"A radical —(CH2)n—(XA)j—RA12 for RA3 or RA4 independently of one another is understood as meaning a radical which is composed of the corresponding radicals —(CH2)n—, (XA)j and RA12.Here, n can be: 0, 1, 2 or 3 and j can be: 0 or 1.XA is a doubly bonded radical selected from the group —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— and —N(RX1)—SO2—.\",\n \"RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, as described above for RG7.a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, or a 3- to 6-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, triazinyl.\",\n \"Further, RA12 and RX1 or RX1* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radical RA12 together with the radical RX1 or RX1* forms a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl, where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.\",\n \"A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C12-alkynyl, preferably C2-C6-alkynyl or C2-C6-alkenyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl or hetaryl radical for RX1 and RX1* independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.Preferred, branched or unbranched, optionally substituted C1-C6-alkoxyalkyl for RX1 and RX1* are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.\",\n \"Preferred, branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl are preferably composed of the C1-C6-alkyl, arylalkyl, aryl or hetaryl radicals and the radicals —CO—, —O—, —SO2— described above.\",\n \"Preferred radicals for RX1 and RX1* are independently of one another hydrogen, methyl, cyclopropyl, allyl and propargyl.\",\n \"RA3 and RA4 can further together form a 3- to 8-membered saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, RA5 is a branched or unbranched, optionally substituted C1-C6-alkyl, arylalkyl, C1-C4-alkyl-C3-C7-cycloalkyl or C3-C7-cycloalkyl radical or an optionally substituted aryl, hetaryl, heterocycloalkyl or heterocycloalkenyl radical, such as described above for RG7.RA6 and RA6* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, —CO—O—C1-C4-alkyl or —CO—C1-C4-alkyl radical such as composed of the group —CO—O— or —CO— and the C1-C4-alkyl radicals described above, arylalkyl radical, as described above for RG7, —CO—O-alkylenearyl or —CO-alkylenearyl radical such as composed of the group —CO—O— or —CO— and the arylalkyl radicals described above, —CO—O-allyl or —CO-allyl radical, or C3-C7-cycloalkyl radical, such as described above for RG7.Further, both radicals RA6 and RA6* in structural element IA7 can together form an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl radical, for example as described above for RA6, C1-C4-alkoxy, arylalkyl or C3-C7-cycloalkyl radical, for example as described above for RL14, a branched or unbranched, optionally substituted —O—CO—C1-C4-alkyl radical, which is composed of the group —O—CO— and, for example, of the C1-C4-alkyl radicals mentioned above or an optionally substituted —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical which is composed of the groups —O— or —O—CO— and, for example, of the corresponding radicals described above for RG7.Further, both radicals RA6 and RA7 can together form an optionally substituted unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. For RAS in structural element A, the branched or unbranched, optionally substituted C1-C4-alkyl radical or an optionally substituted aryl or arylalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA15, where the radicals CO—C1-C4-alkyl, SO2—C1-C4-alkyl, CO—O—C1-C4-alkyl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl or CO—O-alkylenearyl are composed analogously to the other composed radicals of the group consisting of CO, SO2 and COO and, for example, of the corresponding C1-C4-alkyl, aryl or arylalkyl radicals described above for RA15 and these radicals can be optionally substituted.\",\n \"In each case, for RA9 or RA10 independently of one another, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA14, preferably methyl or trifluoromethyl.\",\n \"In each case, for RA9 or RA10 independently of one another, a radical CO—O—RA14, O—RA14, S—RA14, SO2—NRA15RA16, NRA15RA16 or CO—NRA15RA16 is understood as meaning, for example, the corresponding radicals described above for RA13.Further, both radicals RA9 and RA10 together in structural element IA14 can form a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.\",\n \"Substituents in this case are in particular understood as meaning halogen, CN, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl or trifluoromethyl or the radicals O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or —((RA8)HN)C═N—RA7.A branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16 for RA11 is understood, for example, as meaning the corresponding radicals described above for RA9.Further, in structural element IA16, both radicals RA9 and RA17 together can form a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.\",\n \"A branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 for RA18 and RA19 independently of one another is understood as meaning, for example, the radicals described above for RG12, preferably hydrogen or a branched or unbranched, optionally substituted C1-C8-alkyl radical.\",\n \"Z1, Z2, Z3, Z4 are independently of one another nitrogen, C—H, C-halogen, such as C—F, C—Cl, C—Br or C—I or a branched or unbranched, optionally substituted C—C1-C4-alkyl radical which is composed of a carbon radical and, for example, a C1-C4-alkyl radical described above for RA6 or a branched or unbranched optionally substituted C—C1-C4-alkoxy radical which is composed of a carbon radical and, for example, a C1-C4-alkoxy radical described above for RA7.Z5 is oxygen, sulfur or a radical NRA8.Preferred structural elements A are composed of at least one preferred radical of the radicals belonging to the structural element A, while the remaining radicals are widely variable.\",\n \"Particularly preferred structural elements A are composed of the preferred radicals of the structural element A.\",\n \"In a preferred embodiment, the spacer structural element E is understood as meaning a structural element that consists of a branched or unbranched aliphatic C2-C30-hydrocarbon radical which is optionally substituted and contains heteroatoms and/or of a 4- to 20-membered aliphatic or aromatic mono- or polycyclic hydrocarbon radical which is optionally substituted and contains heteroatoms.\",\n \"In a further preferred embodiment, the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently, of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.\",\n \"The coefficient c is preferably 0 or 1, the coefficient d is preferably 1 or 2, and the coefficients f, g, h independently of one another are preferably 0 or 1.An optionally substituted 4- to 11-membered mono- or polycyclic aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, S, where the ring carbons or ring nitrogens can optionally be substituted, for QE and XE independently of one another is preferably understood as meaning optionally substituted arylene, such as optionally substituted phenylene or naphthylene, or optionally substituted hetarylene such as the radicals and their substituted or fused derivatives, or radicals of the formulae IE1 to IE11, where the incorporation of the radicals can take place in both orientations.\",\n \"Aliphatic hydrocarbons are understood as meaning, for example, saturated and unsaturated hydrocarbons.\",\n \"Z6 and Z7 are independently of one another CH or nitrogen.\",\n \"Z8 is oxygen, sulfur or NH.\",\n \"Z9 is oxygen, sulfur or NRE20.r1, r2, r3 and t are independently of one another 0, 1, 2 or 3.s and u are independently of one another 0, 1 or 2.Particularly preferably, XE and QE independently of one another are optionally substituted phenylene, a radical and their substituted or fused derivatives, or radicals of the formulae IE1, IE2, IE3, IE4 and IE7, where the incorporation of the radicals can take place in both orientations.\",\n \"RE18 and RE19 are independently of one another hydrogen, —NO2, —NH2, —CN, —COOH, a hydroxyl group, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, as in each case described above.\",\n \"RE20 is independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C3-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, preferably hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl radical.\",\n \"YE and ZE are independently of one another CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, preferably CO, SO2 and oxygen.\",\n \"RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C8-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical, such as correspondingly described above for RG7 or a radical CO—RE16, COORE16 or SO2—RE16, preferably hydrogen, methyl, allyl, propargyl and cyclopropyl.\",\n \"A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE13, RE14 or RE15 independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.A branched or unbranched, optionally substituted C1-C4-alkoxy radical for RE13 or RE14 independently of one another is understood as meaning, for example, the C1-C4-alkoxy radicals described above for RA14.Preferred alkylenecycloalkyl radicals for RE13, RE14 or RE15 independently of one another are, for example, the C1-C4-alkylene-C3-C7-cycloalkyl radicals described above for RG7.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical for RE16 is understood as meaning, for example, the corresponding radicals described above for RG11.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Further, two radicals RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 can in each case independently of one another together form a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle which can contain up to three heteroatoms from the group O, N and S. The radical —(CH2)x—(WE)z—RE17 is composed of a C0-C4-alkylene radical, optionally a bonding element WE selected from the group —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, preferably selected from the group —CO—N(RW2)—, —N(RW2)—CO—, —O—, —SO2—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, and the radical RE17, where RW2 and RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, preferably independently of one another are hydrogen, methyl, cyclopropyl, allyl, propargyl, and RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3 to 8-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl or triazinyl.\",\n \"Further, RE17 and RW2 or RW2* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radicals RE17 and RW2 or RW2* together form a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.\",\n \"Preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or the radical —(CH2)x—(WE)z—RE17.Particularly preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, F, a branched or unbranched, optionally substituted C1-C4-alkyl radical, in particular methyl.\",\n \"A branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical or an optionally substituted aryl, hetaryl or C3-C7-cycloalkyl for RE11 and RE11* in structural element E independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.The branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or the optionally substituted radicals CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl for RE11 and RE11* independently of one another are composed, for example, of the corresponding groups CO, COO, CONH or SO2 and the corresponding radicals mentioned above.\",\n \"Preferred radicals for RE11 or RE11* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical, or an optionally substituted hetaryl or C3-C7-cycloalkyl radical.\",\n \"Particularly preferred radicals for RE11 or RE11* are hydrogen, methyl, cyclopropyl, allyl or propargyl.\",\n \"In a particularly preferred embodiment of structural element E1, structural element E1 is a radical —CH2—CH2—CO—, —CH2—CH2—CH2—CO— or a C1-C5-alkylene radical.\",\n \"In a particularly preferred embodiment of structural element E, the spacer structural element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where the structural elements E2 and E1 have the meanings described above.\",\n \"Preferred structural elements E are composed of at least one preferred radical of the radicals belonging to structural element E, while the remaining radicals are widely variable.\",\n \"Particularly preferred structural elements E are composed of the preferred radicals of structural element E. Preferred structural elements B are composed either of the preferred structural element A, while E is widely variable, or of the preferred structural element E, while A is widely variable.\",\n \"In a further preferred embodiment, the structural element E used is the structural element E′ described below for the novel compounds of the formula I′.\",\n \"In a further preferred embodiment, the structural element G used is the structural element G′ described below for the novel compounds of the formula I′.\",\n \"The compounds of the formula I, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.\",\n \"The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.\",\n \"The use of an enantiomerically pure compound as the active compound is preferred.\",\n \"The compounds of the formula I can also be present in other tautomeric forms.\",\n \"The compounds of the formula I can also be present in the form of physiologically tolerable salts.\",\n \"The compounds of the formula I can also be present as prodrugs in a form in which the compounds of the formula I are liberated under physiological conditions.\",\n \"By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.\",\n \"Derivatized structural elements B or A are also suitable which liberate the structural element B or A respectively under physiological conditions.\",\n \"In preferred compounds of the formula I, in each case one of the three structural elements B, G or L has the preferred range, while the remaining structural elements are widely variable.\",\n \"In particularly preferred compounds of the formula I, in each case two of the three structural elements B, G and L have the preferred range, while the remaining structural elements are widely variable.\",\n \"In very particularly preferred compounds of the formula I, in each case all three structural elements B, G and L have the preferred range, while the remaining structural element is widely variable.\",\n \"Preferred compounds of the formula I contain, for example, the preferred structural element G, while the structural elements B and L are widely variable.\",\n \"In particularly preferred compounds of the formula I, for example, B is replaced by the structural element A-E- and the compounds contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.\",\n \"Further particularly preferred compounds of the formula I contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.\",\n \"The invention further relates to the use of the structural element of the formula IGL -G-L IGL for the preparation of compounds which bind to integrin receptors.\",\n \"The compounds of the formula I bind to integrin receptors.\",\n \"The compounds of the formula I are therefore preferably suitable as integrin receptor ligands and for the production of drugs for treating diseases in which an integrin receptor is involved, in particular for treating diseases in which the interaction between integrins and their natural ligands is dysregulated, i.e.\",\n \"excessive or decreased.\",\n \"Integrin receptor ligands are understood as meaning agonists and antagonists.\",\n \"An excessive or decreased interaction is understood as meaning both an excessive or decreased expression of the natural ligand and/or of the integrin receptor and thus an excessive or decreased amount of natural ligand and/or integrin receptor or an increased or decreased affinity of the natural ligand for the integrin receptor.\",\n \"The interaction between integrins and their natural ligands is dysregulated compared with the normal state, i.e.\",\n \"excessive or decreased, if this dysregulation does not correspond to the physiological state.\",\n \"An increased or decreased interaction can lead to pathophysiological situations.\",\n \"The level of dysregulation which leads to a pathophysiological situation is dependent on the individual organism and on the site and nature of the disorder.\",\n \"Preferred integrin receptors for which the compounds of the formula I according to the invention can be used are the α5β1, α4β1, αVβ5 and αVβ3 integrin receptors.\",\n \"The compounds of the formula I particularly preferably bind to the αVβ3 integrin receptor and can thus be particularly preferably used as ligands of the αVβ3 integrin receptor and for the treatment of illnesses in which the interaction between αVβ3 integrin receptor and its natural ligand is excessive or reduced.\",\n \"The compounds of the formula I are preferably used for the treatment of the following illnesses: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury or stent implantation, and angioplasty (neointima formation, smooth muscle cell migration and proliferation), acute kidney failure, angiogenesis-associated microangiopathies such as diabetic angiopathies or retinopathy or rheumatoid arthritis, blood platelet-mediated vascular occlusion, arterial thrombosis, stroke, reperfusion damage after myocardial infarct or stroke, carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis), osteoporosis (bone resorption after chemotaxis and adhesion of osteoclasts to the bone matrix), high blood pressure, psoriasis, hyperparathyroidism, Paget's disease, malignant hypercalcemia, metastatic osteolytic lesions, inflammation, wound healing, cardiac insufficiency, congestive heart failure CHF, as well as in antiviral, antimycotic, antiparasitic or antibacterial therapy and prophylaxis (adhesion and internalization).\",\n \"Furthermore, the invention relates in particular to the use of the compounds of the formula I as ligands of the αVβ3 integrin receptor.\",\n \"The invention furthermore relates to the novel compounds of the formula I′ A-E′-G′-L I′ where the structural elements A and L have the meanings described above.\",\n \"Preferred structural elements A and L are described.\",\n \"Structural element E′ is composed of two to four substructural elements selected from the group consisting of E1 and E2, the linkage sequence of the substructural elements being arbitrary and E1 and E2 having the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(X)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case in which YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical of the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.\",\n \"In a further preferred embodiment of structural element E′, the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.\",\n \"Further preferred embodiments of structural element E′ correspond to the preferred embodiments of structural element E with the proviso described above.\",\n \"Structural element G′ is identical to structural element G, as described above, except for the radicals RG12 and RG13.In structural element G′, the radicals RG12 and RG13 of structural element G are replaced by the radicals RG′12 and RG′13.The radicals RG12 and RG′13 in structural element G′ have the following meanings: independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG14, where RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical.\",\n \"Preferred radicals of RG′12 and RG13 are the corresponding radicals described above for RG12 and RG13.Further preferred embodiments of the structural element G′ correspond to the preferred embodiments of structural element G. The compounds of the formula I′, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.\",\n \"The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.\",\n \"The use of an enantiomerically pure compound as active compound is preferred.\",\n \"The compounds of the formula I′ can also be present in other tautomeric forms.\",\n \"The compounds of the formula I′ can also be present in the form of physiologically tolerable salts.\",\n \"The compounds of the formula I′ can also be present as prodrugs in a form in which the compounds of the formula I′ are liberated under physiological conditions.\",\n \"By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.\",\n \"Derivatized structural elements A which liberate the structural element A under physiological conditions are also suitable.\",\n \"Analogously to the compounds of the formula I, the following applies for the compounds of the formula I′ as mentioned above: In preferred compounds of the formula I′, one of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.\",\n \"In particularly preferred compounds of the formula I′, two of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.\",\n \"In further particularly preferred compounds of the formula I′, three of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural element is widely variable.\",\n \"In very particularly preferred compounds of the formula I′, all four structural elements A, E′, G′ or L have the preferred range.\",\n \"Preferred compounds of the formula I, have, for example, the preferred structural element G′, while the structural elements A, E′ and L are widely variable.\",\n \"Further particularly preferred compounds of the formula I′ have, for example, the preferred structural element G′ and the preferred structural element A, while the structural elements E′ and L are widely variable.\",\n \"Very particularly preferred compounds of the formula I′ are listed below, the number before the text block being the number of an individualized compound of the formula I′, and in the text block A-E′-G′-L the abbreviations separated by a bonding dash in each case being an individual structural element A, E′, G′ or L and the meaning of the abbreviations of the structural elements being explained after the table.\",\n \"No.\",\n \"A-E′-G′-L 1 bhs-edia3-phen-es 2 2py-inda2-phen-es 3 bhs-35thirna2-meph-es 4 bim-dibema2-dmeph-es 5 2py-bam2-4-clph-es 6 2py-dibema2-dmeph-es 7 bhs-a24thima2-dmeph-es 8 bhs-aaf-phen-es 9 bhs-a24thima2-4-clph-es 10 bim-me42thiaz2-phen-es 11 2py-edia2-phen-es 12 bim-a24thima2-hdb-es 13 2py-apma2-4-clph-es 14 gua-chex2-phen-es 15 bhs-dibema2-4-clph-es 16 bhs-bam2-phen-es 17 bim-a23thima2-4-clph-es 18 bim-dibema2-phen-es 19 bim-bam2-4-clph-es 20 2py-pipa2-4-clph-es 21 2py-me25thima2-phen-es 22 gua-a24thima2-ioph-es 23 imhs-apma2-phen-es 24 2py-apma2-ioph-es 25 gua-edia3-phen-es 26 bhs-a23thima2-4-clph-es 27 2py-a24thima2-2pyph-es 28 2py-bam2-thoph-es 29 gua-apma2-phen-es 30 2py-a24thima2-reph-es 31 gua-hexa-phen-es 32 dimethpym-apma2-phen-es 33 2py-dibema2-meph-es 34 bhs-apma2-phen-es 35 bim-edia3-phen-es 36 gua-apma2-meph-es 37 bim-apma2-reph-es 38 2py-a24thima2-dmeph-es 39 gua-aaf-phen-es 40 gua-apma2-yrph-es 41 gua-pipeme2-phen-es 42 2py-35thima2-phen-es 43 gua-a24thima2-reph-es 44 bim-bam2-phen-es 45 gua-a24thima2-phen-ms 46 gua-apma2-reph-es 47 mam2py-a24thima2-phen-es 48 2py-a24thima2-phen-gs 49 2py-apma2-phen-nes 50 2py-a24thima2-4-clph-es 51 bhs-penta-phen-es 52 gua-35thima2-dmeph-es 53 bim-dibema2-thoph-es 54 bim-a24thima2-reph-es 55 2py-a23thima2-4-clph-es 56 gua-a23thima2-phen-es 57 dhim-a24thima2-phen-es 58 gua-penta-phen-es 59 bhs-a24thima2-reph-es 60 2py-a23thima2-meph-es 61 gua-prodia2-phen-es 62 bhs-apma2-reph-es 63 2py-apma2-meph-es 64 gua-bam2-4-clph-es 65 2py-me35thima2-phen-es 66 gua-apma2-2pyph-es 67 2py-35thima2-thoph-es 68 clim-a24thima2-phen-es 69 2py-buta-phen-es 70 am2py-apma2-phen-es 71 gua-a24thima2-dmeph-es 72 bhs-apma2-hdb-es 73 bhs-dibema2-thoph-es 74 bim-dibema2-meph-es 75 bhs-bam2-meph-es 76 bhs-apma2-yrph-es 77 bhs-apma2-dmeph-es 78 gua-me25thima2-phen-es 79 2py-a24thima2-meph-es 80 gua-inda2-phen-es 81 bhs-mepipe2-phen-es 82 bhs-a24thima2-phen-as 83 bim-pipa2-thoph-es 84 bhs-bam2-4-clph-es 85 bhs-a24thima2-phen-es 86 bhs-35thima2-phen-es 87 bim-penta-phen-es 88 bhs-apma2-dbph-es 89 gua-42thiaz2-phen-es 90 bhs-a24thima2-24pym-es 91 2py-dibema2-phen-es 92 bim-a24thima2-dm-es 93 bhs-pipa2-4-clph-es 94 2py-apma2-phen-ps 95 2py-apma2-dmeph-es 96 2py-mepipe2-phen-es 97 bhs-35thima2-4-clph-es 98 gua-apma2-phen-mals 99 gua-35thima2-4-clph-es 100 mam2py-apma2-phen-es 101 gua-buta-phen-es 102 2py-a23thima2-thoph-es 103 2py-pyma2-phen-es 104 gua-apma2-phen-gs 105 bim-apma2-phen-as 106 bhs-apma2-4-clph-es 107 bhs-a24thima2-4-pyph-es 108 thpym-apma2-phen-es 109 gua-pipa2-dmeph-es 110 amim-a24thima2-phen-es 111 bim-aepi2-phen-es 112 bim-a23thima2-thoph-es 113 bhs-a23thima2-thoph-es 114 gua-pdagk-phen-es 115 2py-hexa-phen-es 116 bhs-a24thima2-meph-es 117 gua-bam2-meph-es 118 2py-35thima2-meph-es 119 2py-pipa2-meph-es 120 bhs-a23thima2-phen-es 121 bim-pipeme2-phen-es 122 bhs-buta-phen-es 123 bhs-pipa2-dmeph-es 124 bhs-pipa2-meph-es 125 2py-35thima2-dmeph-es 126 bim-bam2-thoph-es 127 gua-35thima2-thoph-es 128 bim-edia2-phen-es 129 bim-apma2-phen-nes 130 gua-bam2-thoph-es 131 gua-bam2-phen-es 132 pippy-a24thima2-phen-es 133 gua-35thima2-phen-es 134 bim-a23thima2-phen-es 135 gua-dibema2-dmeph-es 136 bhs-apma2-phen-gs 137 bhs-apma2-thoph-es 138 2py-apma2-phen-es 139 im-a24thima2-phen-es 140 gua-aepi2-phen-es 141 2py-mea2-phen-es 142 gua-a24thima2-phen-es 143 2py-a24thima2-thaph-es 144 gua-apma2-4-clph-es 145 bhs-apma2-phen-f2es 146 bhs-inda2-phen-es 147 bim-a24thima2-dbph-es 148 bim-apma2-phen-ms 149 gua-a23thima2-thoph-es 150 pippy-apma2-phen-es 151 bhs-apma2-meph-es 152 2py-apma2-phen-as 153 gua-mea2-phen-es 154 bhs-me35thima2-phen-es 155 bhs-pdagk-phen-es 156 bim-42thiaz2-phen-es 157 2py-pipeme2-phen-es 158 bim-me25thima2-phen-es 159 gua-dibema2-phen-es 160 2py-apma2-oxph-es 161 bhs-a24thima2-3clph-es 162 bim-pyma2-phen-es 163 bhs-edia2-phen-es 164 imhs-a24thima2-phen-es 165 gua-dibema2-thoph-es 166 bim-a24thima2-4-clph-es 167 bim-apma2-phen-ps 168 gua-apma2-dm-es 169 2py-a24thima2-phen-mals 170 2py-dibema2-4-clph-es 171 bhs-dibema2-meph-es 172 bim-aof-phen-es 173 bhs-pipeme2-phen-es 174 gua-35thima2-meph-es 175 bim-aaf-phen-es 176 bim-dibema2-4-clph-es 177 bim-a24thima2-2pyph-es 178 bim-a24thima2-yrph-es 179 bim-35thima2-4-clph-es 180 bhs-aepi2-phen-es 181 bim-pipa2-phen-es 182 gua-a23thima2-dmeph-es 183 2py-a24thima2-yrph-es 184 gua-a24thima2-dmeoph-es 185 bhs-42thiaz2-phen-es 186 bim-mepipe2-phen-es 187 2py-chex2-phen-es 188 bhs-prodia2-phen-es 189 gua-bam2-dmeph-es 190 bhs-me25thima2-phen-es 191 thpym-a2.4thima2-phen-es 192 bim-pipa2-dmeph-es 193 bhs-dibema2-phen-es 194 gua-a24thima2-thoph-es 195 2py-pipa2-thoph-es 196 clim-apma2-phen-es 197 bhs-chex2-phen-es 198 gua-a24thima2-phen-nes 199 gua-a24thima2-meph-es 200 bhs-a24thima2-dmeoph-es 201 2py-apma2-24pym-es 202 2py-a24thima2-phen-pms 203 gua-a24thima2-4-piph-es 204 bim-apma2-3clph-es 205 gua-apma2-hdb-es 206 2py-bam2-meph-es 207 bhs-a24thima2-phen-nes 208 gua-pyma2-phen-es 209 bim-a24thima2-thoph-es 210 gua-mepipe2-phen-es 211 bim-apma2-4-clph-es 212 2py-42thiaz2-phen-es 213 bim-apma2-24pym-es 214 bhs-a23thima2-dmeph-es 215 gua-apma2-dbph-es 216 gua-me35thima2-phen-es 217 bim-35thima2-phen-es 218 gua-apma2-thaph-es 219 bim-35thima2-thoph-es 220 gua-apma2-phen-f2es 221 2py-apma2-3clph-es 222 gua-apma2-thoph-es 223 bim-pipa2-meph-es 224 2py-aof-phen-es 225 bhs-35thima2-dmeph-es 226 bhs-hexa-phen-es 227 bim-pipa2-4-clph-es 228 bhs-apma2-phen-pms 229 bim-a23thima2-dmeph-es 230 2py-me42thiaz2-phen-es 231 bim-a24thima2-phen-gs 232 bim-apma2-dmeph-es 233 gua-a23thima2-4-clph-es 234 bim-a24thima2-phen-mals 235 2py-apma2-phen-ms 236 bhs-a24thima2-ioph-es 237 bim-a24thima2-phen-es 238 2py-pdagk-phen-es 239 gua-a24thima2-phen-ps 240 2py-pipa2-phen-es 241 2py-aepi2-phen-es 242 2py-a24thima2-thoph-es 243 bhs-bam2-dmeph-es 244 bim-hexa-phen-es 245 bim-a24thima2-meph-es 246 bhs-me42thiaz2-phen-es 247 am2py-a24thima2-phen-es 248 bim-apma2-meph-es 249 bim-me35thima2-phen-es 250 gua-pipa2-phen-es 251 bhs-a24thima2-oxph-es 252 2py-pipa2-dmeph-es 253 2py-apma2-4-pyph-es 254 bhs-35thima2-thoph-es 255 gua-me42thiaz2-phen-es 256 bim-a24thima2-thaph-es 257 gua-a24thima2-phen-as 258 2py-a24thima2-phen-f2es 259 2py-a24thima2-dbph-es 260 bim-35thima2-meph-es 261 bim-apma2-phen-es 262 bim-a24thima2-phen-pms 263 bim-chex2-phen-es 264 bim-a24thima2-dmeph-es 265 bim-mea2-phen-es 266 2py-apma2-thoph-es 267 dimethpym-a24thima2-phen-es 268 2py-dibema2-thoph-es 269 2py-apma2-dmeoph-es 270 gua-dibema2-4-clph-es 271 bhs-pipa2-phen-es 272 gua-edia2-phen-es 273 gua-apma2-dmeph-es 274 2py-edia3-phen-es 275 gua-a23thima2-meph-es 276 bim-pdagk-phen-es 277 gua-apma2-phen-pms 278 bhs-mea2-phen-es 279 bim-35thima2-dmeph-es 280 bhs-aof-phen-es 281 2py-prodia2-phen-es 282 bim-inda2-phen-es 283 bhs-bam2-thoph-es 284 bim-apma2-4-pyph-es 285 2py-aaf-phen-es 286 2py-bam2-phen-es 287 bhs-apma2-dm-es 288 2py-penta-phen-es 289 gua-aof-phen-es 290 im-apma2-phen-es 291 gua-pipa2-4-clph-es 292 bim-apma2-ioph-es 293 bim-bam2-meph-es 294 gua-pipa2-meph-es 295 bhs-apma2-thaph-es 296 bhs-apma2-2pyph-es 297 bim-apma2-dmeoph-es 298 amim-apma2-phen-es 299 dhim-apma2-phen-es 300 bhs-a24thima2-phen-ps 301 2py-a23thima2-dmeph-es 302 gua-pipa2-thoph-es 303 bim-a23thima2-meph-es 304 2py-bam2-dmeph-es 305 bhs-a24thima2-thoph-es 306 bhs-apma2-phen-mals 307 bhs-a23thima2-meph-es 308 bim-buta-phen-es 309 2py-apma2-reph-es 310 gua-dibema2-meph-es 311 2py-a24thima2-hdb-es 312 gua-a24thima2-4-clph-es 313 bhs-pipa2-thoph-es 314 gua-a24thima2-3clph-es 315 gua-a24thima2-oxph-es 316 bim-bam2-dmeph-es 317 bim-apma2-thoph-es 318 bim-apma2-oxph-es 319 2py-a24thima2-phen-es 320 bhs-a24thima2-phen-ms 321 bim-prodia2-phen-es 322 2py-a24thima2-dm-es 323 bhs-pyma2-phen-es 324 bim-a24thima2-phen-f2es 325 2py-a23thima2-phen-es 326 gua-a24thima2-24pym-es 327 2py-35thima2-4-clph-es 328 bhs-dibema2-dmeph-es In the above list, the following abbreviations are used for the structural units A, E′, G′ and L. A= Abbreviation 2py dhim bim imhs dimethpym mam2py am2py thpym bhs gua amim clim im pippy E′= Abbreviation edia2 pyma2 pipa2 aepi2 me35thima2 dibema2 edia3 buta aaf 42thiaz2 chex2 bam2 apma2 pdagk mepipe2 prodia2 inda2 35thima2 me25thima2 penta aof hexa mea2 pipeme2 me42thiaz2 a23thima2 a24thima2 The bond to the structural unit L should be understood as meaning a double bond in the compound where L=as.\",\n \"G′= Abbreviation 24pym dmeph dm thoph thaph 2pyph 4clph phen dbph hdb dmeoph meph 3clph oxph ioph yrph 4pyph reph L= Abbreviation es gs pms f2es mals ps ms nes as The compounds of the general formula I and thus also the compounds of the formula I′ as well as the starting substances used for their preparation can be prepared by methods of organic chemistry known to the person skilled in the art, such as are described in standard works such as Houben-Weyl, “Methoden der Organischen Chemie” [Methods of Organic Chemistry], Thieme-verlag, Stuttgart, or March “Advanced Organic Chemistry”, 4th Edition, Wiley & Sons.\",\n \"Further preparation methods are also described in R. Larock, “Comprehensive Organic Transformations”, Weinheim 1989, in particular the preparation of alkenes, alkynes, halides, amines, ethers, alcohols, phenols, aldehydes, ketones, nitrites, carboxylic acids, esters, amides and acid chlorides.\",\n \"The synthesis of compounds of the formula I can be carried out either according to the “classical” method in solution or on a polymeric support, in each case reaction conditions as are known and suitable for the respective reactions being used.\",\n \"Use can also be made in this case of variants which are known per se but not mentioned here.\",\n \"The general synthesis of compounds of the formula I is described in schemes 1-7.If not stated otherwise, all starting materials and reagents are commercially available, or can be prepared from commercially obtainable precursors by customary methods.\",\n \"Fused 2,3,4,5-tetrahydro-1H-azepinediones of type II are known and can be prepared by known methods, e.g.\",\n \"starting from anthranilic acid esters or the corresponding heterocyclic analogs via Dieckmann condensation and subsequent decarboxylation, as is described in the following publications: J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"80, 1958, 2172-2178; J. Chem.\",\n \"Soc.\",\n \"1959, 3111; J. Chem.\",\n \"Soc.\",\n \"1934, 1326; Arch.\",\n \"Pharm.\",\n \"324, 1991, 579-581.The preparation of 3,4-dihydro-1H-azepine-2,5-dione is described in Heterocycles 8, 1977, 345-350.The conversion into compounds of the type III is generally carried out by methods known to the person skilled in the art, such as are described, for example, in Larock, “Comprehensive Organic Transformations”, Weinheim 1989, pp.\",\n \"167ff, although methods which are not mentioned here can also be used.\",\n \"Preferably, compounds of the general formula III can be prepared by reaction of the ketones II with a phosphonic ester of the general formula (EtO)2P(═O)(XL)a(CRL1RL2)b—COOSG1 in the presence of a base.\",\n \"The reaction preferably takes place in a polar aprotic solvent, such as tetrahydrofuran, dioxane; dimethylformamide (DMF), dimethylacetamide or acetamide; dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; in a temperature range depending on the nature of the solvent used from −40° C. up to the boiling point of the corresponding solvent.\",\n \"The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.\",\n \"sodium carbonate or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such lithium diisopropylamide, or lithium, sodium or potassium bis(trimethylsilyl)amide.\",\n \"The reaction to give IV is carried out by hydrogenation of the double bond under standard conditions.\",\n \"Use can also be made here of variants which are known per se but not mentioned.\",\n \"Preferably, the hydrogenation is carried out in the presence of a noble metal catalyst, such as Pd on activated carbon, Pt, PtO2, Rh on Al2O3 in an inert solvent at a temperature of 0-150° C. and a pressure of 1-200 bar; the addition of an acid such as acetic acid or hydrochloric acid can be advantageous.\",\n \"Hydrogenation in the presence of 5-10% Pd on activated carbon is particularly preferred.\",\n \"Solvents which can be used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; sulfoxides such as dimethyl sulfoxide, sulfolane; pyridine, N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, water or mixtures of the solvents mentioned.\",\n \"Compounds of type V are prepared by reaction with compounds of the general formula A-E-X1 (VI), the radical X1 being a customary leaving group, for example halogen such as chlorine, bromine, iodine or aryl- or alkylsulfonyl optionally substituted by halogen, alkyl or haloalkyl, such as toluenesulfonyl, trifluoromethanesulfonyl and methylsulfonyl or another equivalent leaving group.\",\n \"The reaction preferably takes place in an inert solvent (such as previously described) with addition of a suitable base, i.e.\",\n \"of a base which brings about deprotonation of the intermediate IV, in a temperature range from −40° C. up to the boiling point of the corresponding solvent.\",\n \"The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.\",\n \"sodium or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such as lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide or potassium bis(trimethylsilyl)amide.\",\n \"Removal of the protective group SG1 according to standard conditions (see below) leads to the compounds of the general formula I.\",\n \"If SG1 is C1-C4-alkyl or benzyl, the compounds of the general formula V correspond directly to the compounds of type I. Alternatively to this synthesis strategy, compounds of type I can also be prepared via VII as an intermediate, here too reaction conditions being used such as are known to the person skilled in the art and described in standard works.\",\n \"Compound VII is prepared by reaction of compounds of type IV having radio of the general formula DE-E-X2 (VIII) under reaction conditions such as have already been described for the preparation of V from IV and VI.\",\n \"X2 here is a suitable leaving group, as has already been described for X1, and DE is CN, N3 or a protected amino or acid function of the general formula NSG3 or COOSG2.The synthesis of the fragments DE-E or A-E is carried out—depending on the actual structure of E—by removal of the protective groups and coupling of the remaining fragments according to standard methods, e.g.\",\n \"amide couplings.\",\n \"The introduction of A is then carried out analogously to the reactions described in schemes 3-7.Compounds of the formula I in which WG in structural element G is a structural element of the formula IWG1 can be prepared according to scheme 2.The starting point of the synthesis is compounds of type IX, which are either known or are accessible by methods known to the person skilled in the art, such as are described, for example, in J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"71, 1949, 1985.Alkylation with a compound of the general formula XIII (X3, X4=a customary leaving group) under customary reaction conditions leads to X.\",\n \"The further reactions to give I then proceed analogously to scheme 1 via compounds of type XI.\",\n \"In the case in which WG in structural element G is a structural element of the formula IWG3, compounds of type III can be converted into compounds of type XII and then into I analogously to the preparation of V (scheme 2).\",\n \"The coupling of the individual fragments and the removal of the protective groups can be carried out according to known processes (see Larock, “Comprehensive Organic Transformations; protective groups: Greene, T., “Protective Groups in Organic Synthesis”, New York 1991), in the case of amide bonds also analogously to the methods of peptide synthesis, such as are described in standard works, e.g.\",\n \"in Bodanszky “The Practice of Peptide Synthesis”, 2nd Edition, Springer-Verlag 1994, and Bodanszky “Principles of Peptide Synthesis”, Springer-Verlag 1984.A general survey of the customary methods for peptide synthesis and a listing of suitable reagents can furthermore be found in NOVABIOCHEM 1999 “Catalog and Peptide Synthesis Handbook”.\",\n \"The amide couplings mentioned can be carried out with the aid of customary coupling reagents using suitably protected amino and carboxylic acid derivatives.\",\n \"Another method consists in the use of preactivated carboxylic acid derivatives, preferably of carboxylic acid halides, symmetrical or mixed anhydrides or so-called active esters, which are customarily used for the acylation of amines.\",\n \"These activated carboxylic acid derivatives can also be prepared in situ.\",\n \"As a rule, the couplings can be carried out in inert solvents in the presence of an acid-binding agent, preferably of an organic base such as triethylamine, pyridine, diisopropylethylamine, N-methylmorpholine, quinoline; the addition of an alkali metal or alkaline earth metal hydroxide, carbonate or hydrogencarbonate or of another salt of a weak acid of the alkali metals or alkaline earth metals, preferably of potassium, sodium, calcium or cesium, can also be favorable.\",\n \"Depending on the conditions used, the reaction time is between minutes and 14 days, the reaction temperature between −40° C. and 140° C., preferably between −20° C. and 100° C. Suitable inert solvents are, for example, hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate; water; or mixtures of the solvents mentioned.\",\n \"Protective groups SG which can be used are all protective groups which are known from peptide synthesis and customary to the person skilled in the art, such as are also described in the abovementioned standard works.\",\n \"The protective groups in the compounds of the formulae V, VII, XI and XII are likewise removed according to conditions such as are known to the person skilled in the art and are described by Greene and Wuts in “Protective Groups in organic Synthesis”, 2nd Edition, Wiley & Sons, 1991.Protective groups such as SG3 are so-called N-terminal amino protective groups; Boc, Fmoc, benzyloxycarbonyl (Z), acetyl, Mtr are preferred here.\",\n \"SG1 and SG2 are acid protective groups; C1-C4-alkyl is preferred here, such as methyl, ethyl, tert-butyl, or alternatively benzyl or trityl, or alternatively polymer-bonded protective groups in the form of the commercially available polystyrene resins such as 2-chlorotrityl chloride resin or Wang resin (Bachem, Novabiochem).\",\n \"Acid-labile protective groups (e.g.\",\n \"Boc, tert-butyl, Mtr, trityl) can be removed—depending on the protective group used—using organic acids such as trifluoroacetic acid (TFA), trichloroacetic acid, perchloric acid, trifluoroethanol; but also inorganic acids such as hydrochloric acid or sulfuric acid, sulfonic acids such as benzene- or p-toluenesulfonic acid, the acids generally being employed in an excess.\",\n \"HCl or TFA is preferably used.\",\n \"In the case of trityl, the addition of thiols such as thioanisole or thiophenol can be advantageous.\",\n \"The presence of an additional inert solvent is possible, but not always necessary.\",\n \"Suitable inert solvents are preferably organic solvents, for example carboxylic acids such as acetic acid; ethers such as THF or dioxane; amides such as DMF or dimethylacetamide; halogenated hydrocarbons such as dichloromethane; alcohols such as methanol, isopropanol; or water.\",\n \"Suitable solvents are also mixtures of those mentioned.\",\n \"The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Base-labile protective groups such as Fmoc are cleaved by treatment with organic amines such as dimethylamine, diethylamine, morpholine or piperidine as 5-50% solutions in CH2Cl2 or DMF.\",\n \"The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Acid protective groups such as methyl or ethyl are preferably cleaved by basic hydrolysis in an inert solvent.\",\n \"The bases used are preferably alkali metal or alkaline earth metal hydroxides, preferably NaOH, KOH or LiOH; the solvents used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyltert-butyl ether, diisopropyl-ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; nitro compounds such as nitromethane or nitrobenzene; water or mixtures of the solvents mentioned.\",\n \"The addition of a phase-transfer catalyst—depending on the solvent or solvent mixture used—may be advantageous.\",\n \"The reaction temperature for these reactions is generally between −10° C. and 100° C. Hydrogenolytically removable protective groups such as benzyloxycarbonyl (Z) or benzyl can be removed, for example, by hydrogenolysis in the presence of a catalyst (e.g.\",\n \"of a noble metal catalyst on activated carbon as support).\",\n \"Suitable solvents are those mentioned above, in particular alcohols such as methanol, ethanol; amides such as DMF or dimethylacetamide; esters such as ethyl acetate.\",\n \"As a rule, the hydrogenolysis is carried out at a pressure of 1-200 bar and temperatures between 0° and 100° C.; the addition of an acid such as acetic acid or hydrochloric acid may be advantageous.\",\n \"The catalyst used is preferably 5-10% Pd on activated carbon.\",\n \"The synthesis of structural units of type E is generally carried out by methods known to the person skilled in the art; the structural units used are either commercially available or accessible by methods known from the literature.\",\n \"The synthesis of some of these structural units is described by way of example in the experimental section.\",\n \"In the case in which the fragments QE or XE contained in the compounds of type VI and VIII are a hetaryl radical, the structural units used are either commercially available or accessible by methods known to the person-skilled in the art.\",\n \"A multiplicity of preparation methods are described in detail in Houben-Weyl's “Methoden der organischen Chemie” (Vol.\",\n \"E6: furans, thiophenes, pyrroles, indoles, benzothiophenes, -furans, -pyrroles; Vol.\",\n \"E7: quinolines, pyridines, Vol.\",\n \"E8: isoxazoles, oxazoles, thiazoles, pyrazoles, imidazoles and their benzo-fused representatives, as well as oxadiazoles, thiadiazoles and triazoles; Vol.\",\n \"E9: pyridazines, pyrimidines, triazines, azepines and their benzo-fused representatives as well as purines).\",\n \"The linkage of these fragments to E, depending on the structure of E, can also take place via the amino or acid function according to methods which are known to the person skilled in the art.\",\n \"Structures of the general formula A-E-DE are synthesized according to methods known to the person skilled in the art, which are described, for example, in WO 97/08145.Examples of these are the conversion of compounds of the general formula: HNRE11EA1-DE (XIV) NC-EA2-DE (XV) into compounds of the general formula: A-HNRE11-EA1-DE (XVI) A-E-DE (XVII) The groups EA1 and EA in the formulae XIV-XVIII represent structural fragments which, after an appropriate modification (e.g.\",\n \"reaction with suitable reagents or coupling with appropriate structural units) as a whole form the structural fragment A-E.\",\n \"These structural units can then be reacted either directly—in the case of the corresponding free amines or carboxylic acids—or after removal of the protective groups—to give compounds of the general formula I (scheme 1 and 2).\",\n \"In principle, A, however, can also be introduced into compounds of the type IV, as described in scheme 1, where the reaction conditions mentioned can be used just as variants which are not described here.\",\n \"In schemes 3-7, a number of the methods for the introduction of A are described by way of example, in each case reaction conditions being used which are known and suitable for the respective reactions.\",\n \"Use can also be made in this case of variants which are known per se but not mentioned here.\",\n \"Ureas and thioureas (AE-1 to AE-3) can be prepared by customary methods of organic chemistry, e.g.\",\n \"by reaction of an isocyanate or of an isothiocyanate with an amine, if appropriate in an inert solvent with heating (Houben-Weyl Volume VIII, 157ff.)\",\n \"(scheme 3): Scheme 4 shows, by way of example, the preparation of compounds of the type AE-4, as is described, for example, by Blakemoore et al.\",\n \"in Eur.\",\n \"J. Med.\",\n \"Chem.\",\n \"1987 (22) 2, 91-100, or Misra et al.\",\n \"in Bioorg.\",\n \"Ned.\",\n \"Chem.\",\n \"Lett.\",\n \"1994 4 (18), 2165-2170.Unsubstituted or cyclic guanidine derivatives of the general formulae AE-5 and AE-6 can be prepared by means of commercially available or simply accessible reagents, such as are described, for example, in Synlett 1990, 745, J. Org.\",\n \"Chem.\",\n \"1992, 57, 2497, Bioorg.\",\n \"Ned.\",\n \"Chem.\",\n \"1996, 6, 1185-1208; Bioorg.\",\n \"Med.\",\n \"Chem.\",\n \"1998, 1185, or Synth.\",\n \"Comm.\",\n \"1998, 28, 741-746 (scheme 5).\",\n \"The preparation of compounds of the general formula AE-7 can be carried out analogously to U.S. Pat.\",\n \"No.\",\n \"3,202,660, compounds of the formulae AE-9, AE-10, AE-11 and AE-12 analogously to WO 97/08145.Compounds of the formula AE-8 can be prepared, as shown in scheme 6, e.g.\",\n \"according to the method described by Perkins et al., Tetrahedron Lett.\",\n \"1999, 40, 1103-1106.Scheme 5 gives a survey of the synthesis of the compounds mentioned, the circle in AE-8 representing a fused cycle, such as aryl or hetaryl.\",\n \"Compounds of the general formula AE-13 can be prepared analogously to Froeyen et al., Phosphorus Sulfur silicon Relat.\",\n \"Elem.\",\n \"1991, 63, 283-293; AE-14 analogously to Yoneda et al., Heterocycles 1998, 15 N′-1, Spec.\",\n \"Issue, 341-344 (scheme 6).\",\n \"The preparation of corresponding compounds can also be carried out analogously to WO 97/36859.Compounds of the general formula AE-15 can be prepared according to Synthesis 1981, 963-965 or Synth.\",\n \"Comm.\",\n \"1997, 27 (15), 2701-2707; AE-16 analogously to J. Org.\",\n \"Chem.\",\n \"1991, 56 (6), 2260-2262 (scheme 7), the circle being a fused cycle, such as aryl, hetaryl or cycloalkyl.\",\n \"The invention further relates to pharmaceutical preparations, comprising at least one compound of the formula I′ in addition to the customary pharmaceutical excipients.\",\n \"The compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally) in the customary manner.\",\n \"Administration can also be carried out through the nasopharynx using vapors or sprays.\",\n \"Further, the compounds according to the invention can be introduced by direct contact with the affected tissue.\",\n \"The dose depends on the age, condition and weight of the patient and on the manner of administration.\",\n \"As a rule, the daily dose of active compound is between approximately 0.5 and 50 mg/kg of body weight in the case of oral administration and between approximately 0.1 and 10 mg/kg of body weight in the case of parenteral administration.\",\n \"The novel compounds can be administered in solid or liquid form in the customary pharmaceutical administration forms, e.g.\",\n \"as tablets, film-coated tablets, capsules, powders, granules, coated tablets, suppositories, solutions, ointments, creams or sprays.\",\n \"These are prepared in a customary manner.\",\n \"The active compounds can in this case be processed using the customary pharmaceutical excipients such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, release-delaying agents, antioxidants and/or propellants (cf.\",\n \"H. Sucker et al.\",\n \": Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991).\",\n \"The administration forms thus obtained normally contain the active compound in an amount from 0.1 to 90% by weight.\",\n \"The invention further relates to the use of compounds of the formula I′ for the production of drugs for treating illnesses.\",\n \"The compounds of the formula I′ can be used for treating human and animal illnesses.\",\n \"The compounds of the formula I′ which represent the novel compounds of the formula I bind, as mentioned above, to integrin receptors.\",\n \"They are therefore suitable, as mentioned above, preferably as integrin receptor ligands and for the production of drugs for treating illnesses in which an integrin receptor is involved, in particular for the treatment of illnesses in which the interaction between integrins and their natural ligands is dysregulated, i.e.\",\n \"excessive or reduced, as described above.\",\n \"Advantageously, the compounds of the formula I, preferably the compounds of the formula I′, can be administered in combination with at least one further compound in order to achieve an improved curative action in a number of indications.\",\n \"These further compounds can have the same or a different mechanism of action as/from the compounds of the formula I.\",\n \"In addition to the compounds of the formula I, preferably in addition to the compounds of the formula I′ and the customary pharmaceutical excipients, the pharmaceutical preparations can therefore contain at least one further compound, depending on the indication, in each case selected from one of the 10 groups below.\",\n \"Group 1: inhibitors of blood platelelet adhesion, activation or aggregation, such as acetylsalicylic acid, lysine acetylsalicylate, piracetam, dipyridamol, abciximab, thromboxane antagonists, fibrinogen antagonists, such as tirofiban, or inhibitors of ADP-induced aggregation such as ticlopidine or clopidogrel, anticoagulants which prevent thrombin activity or formation, such as inhibitors of IIa, Xa, XIa, IXa or VIIa, antagonists of blood platelet-activating compounds and selectin antagonists for the treatment of blood platelet-mediated vascular occlusion or thrombosis, or Group 2: inhibitors of blood platelet activation or aggregation, such as GPIIb/IIIa antagonists, thrombin or factor Xa inhibitors or ADP receptor antagonists, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vessel wall transmigration, fibrinolysis-modulating compounds, such as streptokinase, tPA, plasminogen-activating stimulants, TAFI inhibitors, XIa inhibitors or PAI-1 antagonists, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists for the treatment of myocardial infarct or stroke, or Group 3: endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors for the treatment of congestive heart failure, or Group 4: thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, such as heparin or low-molecular weight heparins, inhibitors of blood platelet adhesion, activation or aggregation, such as GPIIb-IIIa antagonists or antagonists of the blood platelet adhesion and activation mediated by vWF or GPIb, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants for the treatment of restenosis after vascular injury or stent implantation, or Group 5: antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists and glycosylation inhibitors or AGE formation inhibitors or AGE breakers and antagonists of their receptors, such as RAGE, for the treatment of diabetic angiopathies, or Group 6: lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers for the treatment of atherosclerosis, or Group 7: cytostatic or antineoplastic compounds, compounds which inhibit proliferation, such as kinase inhibitors and heparin or low-molecular weight heparins or further GAGS for the treatment of cancer, preferably for the inhibition of tumor growth or metastasis, or Group 8: compounds for antiresorptive therapy, compounds for hormone exchange therapy, such as estrogen or progesterone antagonists, recombinant human growth hormone, bisphosphonates, such as alendronates compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, such as growth factor antagonists, interleukin-6 antagonists and Src tyrosine kinase inhibitors for the treatment of osteoporosis, or Group 9: TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors for the treatment of rheumatoid arthritis, or Group 10: collagenase, PDGF antagonists and MMPs for improved wound healing.\",\n \"A pharmaceutical preparation comprising at least one compound of the formula I, preferably comprising at least one compound of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, is understood as meaning a combined administration of at least one of the compounds of the formula I, preferably of one of the compounds of the formula I′, with at least one further compound in each case selected from one of the groups described above and, if appropriate, pharmaceutical excipients.\",\n \"Combined administration can be carried out by means of a substance mixture comprising at least one compound of the formula I, preferably of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, but also spatially and/or chronologically separate.\",\n \"In the case of the spatially and/or chronologically separate administration, the administration of the components of the pharmaceutical preparation, the compounds of the formula I, preferably of the formula I′ and the compounds selected from one of the abovementioned groups, takes place spatially and/or chronologically separately.\",\n \"For the treatment of restenosis after vascular injury or stenting, the administrations of the compounds of the formula I, preferably of the formula I′, can be carried out locally at the affected sites, on their own or in combination with at least one compound selected from group 4.It may also be advantageous to coat the stents with these compounds.\",\n \"For the treatment of osteoporosis, it may be advantageous to carry out the administration of the compounds of the formula I, preferably of the formula I′, in combination with an antiresorptive or hormone exchange therapy.\",\n \"The invention accordingly relates to the use of the abovementioned pharmaceutical preparations for the production of drugs for treating illnesses.\",\n \"In a preferred embodiment, the invention relates to the use of the abovementioned combined pharmaceutical preparations for the production of drugs for treating blood platelet-mediated vascular occlusion or thrombosis when using compounds of group 1, myocardial infarct or stroke when using compounds of group 2, congestive heart failure when using compounds of group 3, restenosis after vascular injury or stent implantation when using compounds of group 4, diabetic angiopathies when using compounds of group 5, atherosclerosis when using compounds of group 6, cancer when using compounds of group 7, osteoporosis when using compounds of group 8, rheumatoid arthritis when using compounds of group 9, wound healing when using compounds of group 10.The following examples illustrate the invention, the selection of these examples being non-limiting.\",\n \"I. SYNTHESIS EXAMPLES I.A Precursors Example 1 t-Butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) 22.3 g (80 mmol) of t-butyl diethylphosphonate (95%) were added dropwise at 0° C. to a suspension of 3.27 g of NaH (60%; deoiled) in ml of DMF.\",\n \"The mixture was stirred until a clear solution was formed and then 12.4 g (70.9 mmol) of 3,4-dihydro-1H-1-benzazepine-2,5-dione (preparation according to Arch.\",\n \"Pharm.\",\n \"1991, 324, 579) in 90 ml of DMF were added dropwise at 0° C. The reaction mixture then remained standing at RT for about 3 days.\",\n \"For workup, the mixture was poured into 700 ml of cold 5% NaCl solution, and the resulting yellow precipitate was filtered off with suction and washed with H2O.\",\n \"The moist residue was taken up in CH2Cl2, washed with 5% NaHCO3 solution and dried over Na2SO4.The residue which remained after evaporation was treated with 150 ml of cyclohexane in the presence of heat, and after cooling, filtering off with suction and washing with n-hexane 17.5 g (90.5%) of white crystals remained; m.p.\",\n \": 136-138° C. Example 2 t-Butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetate (2) A suspension of 3 g of 10% Pd/C in 50 ml of ethanol was prehydrogenated, then a solution of compound 1 (14.7 g; 53.8 mmol) in 125 ml of ethanol and 75 ml of dioxane was added, and the mixture was hydrogenated under standard conditions until the absorption of hydrogen was complete.\",\n \"After filtering off the catalyst with suction and washing it with ethanol, the filtrate was concentrated in vacuo, the oily residue was dissolved in diethyl ether and the crystallization commencing was completed by addition of n-hexane.\",\n \"After filtering off the precipitate with suction and washing it with n-hexane, 14.2 g (96%) of white crystals remained; m.p.\",\n \": 101-103° C. Example 3 [5-(2-t-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) a.)\",\n \"A solution of compound 2 (16.8 g; 61.1 mmol) in 60 ml of DMF was added dropwise at 10-20° C. to a suspension of 2.6 g of NaH (60%, deoiled) in 35 ml of DMF and the mixture was stirred until the appearance of an almost clear yellowish solution.\",\n \"t-Methyl bromoacetate (10 g; 63.4 mmol) was then added dropwise and the mixture was stirred overnight.\",\n \"For workup, the reaction mixture was poured into 400 ml of 5% cold NaCl solution and extracted 3× with 100 ml each of a diethyl ether/n-hexane mixture.\",\n \"The combined extracts were then washed with H2O, 10% NaHCO3 solution and NaCl solution, dried over Na2SO4, filtered and evaporated.\",\n \"The residual yellowish oil was reacted further without further purification; FAB-MS: 348 [M−H+].\",\n \"b.)\",\n \"Crude product 3a was dissolved in 100 ml of dioxane and 65 ml of 1N NaOH were added dropwise at RT with stirring.\",\n \"After about 45′, the reaction mixture was adjusted to pH 7 using 1N KHSO4 solution, the dioxane was largely distilled off in vacuo, and the residue was diluted with H2O, adjusted to pH 9 using 1N NaOH and extracted 3× with diethyl ether.\",\n \"The aqueous phase was then rendered acidic using 1N KHSO4 solution, the acid precipitating was extracted with a mixture of diethyl ether/n-hexane 4:1, and the organic phase was washed with H2O, 1N NaOH solution and NaCl solution and dried over Na2SO4.Filtration and evaporation afforded an oily residue, which could be crystallized by treatment with diethyl ether/n-hexane 1:4 (water-saturated).\",\n \"Filtering with suction, washing with n-hexane and drying afforded 17.8 g (87.5%) of white crystals: m.p.\",\n \": 117-119° C. Example 4 N-[4-(Aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) a.)\",\n \"20 g of tert-butyl-4-aminobenzyl carbamate (89.97 mmol)—dissolved in 100 ml of CH3CN—were added dropwise at 0° C. to a solution of 24.5 g of thiocarbonyldiimidazole and 1.56 g of imidazole in 600 ml of CH3CN and the mixture was stirred at RT overnight.\",\n \"19.5 g of 1,2-phenylenediamine were then added and the mixture was again stirred at RT for 2 h. For workup, the reaction mixture was evaporated in vacuo, the residue was taken up in CH2Cl2, and the solution was washed 7× with 10% citric acid solution and 2× with satd.\",\n \"NaCl solution, dried over Na2SO4, filtered and concentrated.\",\n \"The crude product thus obtained (31.78 g; brown foam) was reacted directly without further purification; ESI-MS [M+H+]=373.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.5 and 9.05 (each s, 1H), 7.45 (d, 2H), 7.35 (m, 1H), 7.20 (d, 1H), 7.15, 6.95, 6.75, 6.60 (each m, 1H), 4.85 (s, 2H), 4.10 (d, 2H), 1.35 (s, 9H).\",\n \"b.)\",\n \"Crude product 4a was dissolved in 750 ml of ethanol together with 36.7 g of HgO (yellow) and 0.4 g of sulfur and heated to reflux for 2 h. The reaction mixture was then filtered twice through Celite and evaporated to dryness; 20.7 g, ESI-MS [M+H+]=339.15.c.)\",\n \"7 g of the crude product 4b were introduced into 70 ml of CH2Cl2, 35 ml of HCl in diethyl ether (satd.\",\n \"at 0° C.) were added and the mixture was stirred at RT for 2 h. The resulting precipitate was filtered off with suction, washed with CH2Cl2 and dried.\",\n \"6.7 g of brown amorphous solid; ESI-MS [M+H+]=239.15 1H-NMR (360 MHz, DMSO) δ ppm: 11.6 (s broad, 1H), 8.4 (s broad, 3H), 8.25 (s broad, 1H), 7.65 and 7.55 (each d, 2H), 7.45 and 7.3 (each m, 2H), 4.19 (m, 2H).\",\n \"Example 5 N-[5-(Aminomethyl)-1,3-thiazol-2-yl]guanidine (dihydrochloride) (5) a.)\",\n \"31 g (130 mmol) of 2-chloro-3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal (preparation according to THL 39 (1998), 8085-8088) and 15.4 g of amidinothiourea were heated at 110° C. for 75′ in 200 ml of n-butanol, then the mixture was evaporated and the residue was treated with CH2Cl2 and conc.\",\n \"NH3.Evaporation of the organic phase, purification of the residue by chromatography on silica gel (CH2Cl2/CH3OH 0-5%) and crystallization from acetone afforded 12.3 g of N-{5-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]-1,3-thiazol-2-yl}guanidine.\",\n \"b.)\",\n \"1 g of 5a in 20 ml of CH3OH was treated with 0.81 ml of hydrazine hydrate and stirred at RT for 2 h. The mixture was then cooled to 0° C., filtered, and the filtrate was concentrated and stirred with dilute HCl.\",\n \"This process was repeated a number of times, and the crude product obtained in this way was then stirred with ethanol; 0.92 g of white solid, ESI-MS [M+H+]=172.05.Example 6 N-[4-(Aminomethyl)phenyl]-N′-benzylurea (6) a.)\",\n \"4-Aminobenzylamine (10.0 g, 81.85 mmol) in 150 ml CH2Cl2 was treated with triethylamine (6.8 g, 67.12 mmol) and then treated at 0° C. with di-t-butyl dicarbonate (18.6 g, 85.0 mmol).\",\n \"The mixture was stirred at 0° C. for 1 h and then at RT for 2 h. For workup, 150 ml of a 1%-aqueous citric acid solution were added, the phases were separated and the aqueous phase was reextracted 2 times with CH2Cl2 (150 ml).\",\n \"Fresh washing with H2O, drying of the combined organic phases using Na2SO4 and evaporation afforded a solid, which was washed with stirring with a little diisopropyl ether, filtered off with suction and dried.\",\n \"13.0 g; ESI-MS [M+H+-tBu]=167.05.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (2H, d), 6.61 (2H, d), 4.78 (1H, s br.\",\n \"), 4.17 (2H, d), 3.67 (2H, s br.\",\n \"), 1.46 (9H, s).\",\n \"b.)\",\n \"Benzyl isocyanate (2.40 g, 18.0 mmol) was added with ice-cooling to a solution of the protected amine 6a (4.0 g, 17.99 mmol) and triethylamine (1.82 g, 18.0 mmol) in 220 ml of toluene/DMF 10:1.The reaction mixture was stirred at RT overnight.\",\n \"It was possible to filter off some of the urea formed directly as a precipitate and dry it.\",\n \"The filtrate was washed 2× with H2O, with dilute tartaric acid to pH 3 and again 2 times with H2O to pH 5, and the organic phase was then dried and evaporated.\",\n \"Altogether, 6.0 g were thus obtained; ESI-MS [M+H+-tBu]=300.15.c.)\",\n \"The urea 6b thus obtained was introduced in 90 ml of CH2Cl2, and TFA (2.24 g, 196.25 mmol)—dissolved in 90 ml of CH2Cl2—was added dropwise at 0° C. After 3 h, 1 ml of TFA was added again, then the mixture was stirred at RT overnight.\",\n \"After fresh addition of 1 ml of TFA, the mixture was stirred for a further 5 h, then poured onto ice water and extracted with ethyl acetate (2×50 ml).\",\n \"The water phase was rendered basic with 2N NaOH solution and extracted with CH2Cl2 (2×50 ml).\",\n \"The insoluble portion between the phases was filtered off and dried.\",\n \"4 g; ESI-MS [2M+H+]=511.35 1H-NMR (200 MHz, DMSO) δ (ppm): 8.52 (1H, s), 7.39-7.07 (9H, m), 6.62 (1H, t), 4.27 (2H, d), 3.61 (2H, s).\",\n \"Example 7 [4-(1H-Benzimidazol-2-yl)phenyl]methaneamine (hydrochloride) (7) a.)\",\n \"Di(tert-butyl) 4-cyanobenzylimidodicarbonate (10 g, 30.08 mmol; preparation according to Synth.\",\n \"Comm.\",\n \"28, 23, 1998, 4419ff) in 200 ml of pyridine was treated with 45 ml of triethylamine and saturated at 0° C. with H2S for 1.5 h. The reaction mixture was allowed to stand at RT overnight and then evaporated.\",\n \"The residue thus obtained was then stirred with diethyl ether, filtered off with suction and dried (8.5 g).\",\n \"b.)\",\n \"6 g of the thioamide 7a (16.37 mmol) in 40 ml of dry CH2Cl2 were alkylated at RT overnight using 23.2 g of CH3I and the mixture was then evaporated.\",\n \"The residue thus obtained was taken up in 40 ml of CH3OH, 1.95 g of 1,2-phenylenediamine were added and it was again stirred overnight.\",\n \"Evaporating the reaction mixture and stirring the solid with n-pentane afforded 6.9 g of the desired benzimidazole.\",\n \"M.p.\",\n \": >170° C. (decomposition); ESI-MS: [M+H+]=424.25 c.) 1 g of the bis-Boc compound 7b was dissolved in 5 ml of CH2Cl2, 5 ml of TFA were added at 0° C. and the mixture was stirred at room temperature for 1 h. Evaporating the reaction mixture, treating with HCl in diethyl ether and stirring the isolated solid with diethyl ether afforded 0.6 g of the amine as the hydrochloride; ESI-MS: [M+H+]=224.05.Example 8 N1-(1H-Benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) Preparation was carried out analogously to the preparation of compound 4 starting from 7 g of N-Boc-1,5-diaminopentane hydrochloride (29.3 mmol).\",\n \"After reaction analogously to 4a, 10.3 g of N-Boc 5-{[(2-aminoanilino)carbothioyl]amino}pentane-1-amine were obtained; ESI-MS [M+H+]=353.25.Cyclodesulfurization and subsequent removal of the Boc group with TFA afforded an oily crude product, which was taken up in CH3OH and converted into the corresponding hydrochloride using 250 ml of ethereal HCl (saturated at 0° C.).\",\n \"Stirring the solid obtained with a mixture of CH3OH/methyl t-butyl ether afforded 1.8 g of a reddish amorphous solid.\",\n \"1H-NMR (360 MHz, DMSO) δ ppm: 9.30 (t, 1H), 8.15 (s broad, 3H), 7.40 and 7.25 (each m, 2H), 3.35 (m, 2H superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).\",\n \"Example 9 N1-(1H-Benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) Preparation was carried out analogously to the preparation of compound 4 starting from 9.87 g of N-Boc-1,4-diaminobutane (52.3 mmol).\",\n \"After reaction analogously to 4a, 17.08 g of N-Boc 4-{[(2-aminoanilino)carbothioyl]amino}butane-1-amine were obtained; ESI-MS [M+H+]=338.99.Subsequent cyclodesulfurization and Boc removal using TFA afforded a brown solid, which was stirred a number of times with n-pentane and then recrystallized from a mixture of CH3OH/methyl t-butyl ether; 14.35 g, ESI-MS [M+H+]=205.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.20 (t, 1H), 7.80 (s broad, 3H), 7.35 and 7.20 (each m, 2H), 3.40 (m, 2H partially superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H).\",\n \"Example 10 trans-N-[(4-Aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) Preparation was carried out analogously to compound 4 starting from 5.4 g of tert-butyl-4-(aminomethyl)cyclohexylamine carbamate (WO 9603374; Bioorg.\",\n \"Med.\",\n \"Chem.\",\n \"Lett.\",\n \"1997, 7 (1), 67).\",\n \"After removal of the Boc group, 3.3 g of white dihydrochloride were obtained; FAB-MS [M+H+]: 245.Example 11 trans-N-{[4-(Aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) Preparation was carried out analogously to compound 4 starting from 10 g of benzyl {4-[(tert-butoxycarbonyl)amino]cyclohexyl}-methylcarbamate (EP 669317) by removal of the Boc group using 4N HCl in dioxane, synthesis of the benzimidazole and subsequent hydrogenolysis.\",\n \"3.6 g of white dihydrochloride were isolated; FAB-MS [M+H+]: 245.Example 12 [6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) a.)\",\n \"Preparation was carried out analogously to 7 starting from tert-butyl (6-cyanopyridin-3-yl)methylcarbamate (6.0 g, 25.72 mmol); crystallization of the crude product from ethanol afforded 5.15 g; ESI-MS [M+H+]=325.b.)\",\n \"0.55 g of the Boc-protected amine 12a in 10 ml of CH2Cl2 was treated with 5 ml of TFA and stirred at RT for 2 h. Evaporation of the reaction mixture afforded 0.95 g of a white solid; ESI-MS [M+H+]: 225.25.Example 13 N-[4-(Aminomethyl)phenyl]-2-pyridineamine (13) tert-Butyl 4-aminobenzylcarbamate (2 g; 9 mmol) was heated to reflux for 32 h with 8.74 g of 2-fluoropyridine.\",\n \"The reaction mixture was evaporated in vacuo and the residue obtained was stirred with n-pentane (1.9 g).\",\n \"The Boc group was cleaved using TFA, and the crude product obtained was precipitated from diethyl ether as the hydrochloride and then converted into the free-base (0.8 g) using NH3; ESI-MS [M+H+]: 200.25.N-[4-(Aminomethyl)benzyl]-2-pyridineamine (14) a.)\",\n \"20 g 2-Aminopyridine were dissolved in 100 ml CH3OH, adjusted to pH 6 with isopropanolic HCl and 36 g p-cyanobenzaldehyde were added.\",\n \"9.35 g Sodium cyanoborohydride were added portionwise over one hour and stirred overnight.\",\n \"For workup, the suspension was evaporated, the residue taken up in 100 ml water and with KOH adjusted to pH>10.The watery phase was saturated with NaCl and extracted 3× with diethylether.\",\n \"The ether phase was washed after filtration of a precipitate 3× with a FeSO4 solution, dried and evaporated.\",\n \"Purification of the residue by chromatography on silica gel (heptane/ethyl acetate 1:1) afforded 28.15 g 4-[2-pyridinyl-amino)methyl]benzonitrile.\",\n \"b.)\",\n \"10 g 4-[2-Pyridinyl-amino)methyl]benzonitrile were dissolved in 280 ml ammonia-alkali CH3OH, 10 g Raney-nickel added and it was hydrogenated for 24 h. It was filtered, evaporated and the residue purified by chromatography on silica gel (ethyl acetate/ethyl alcohol 1:3).\",\n \"5.18 g, ESI-MS: [M+H+]=214.\",\n \"[5-(1H-Benzimidazole-2-yl(thiene-2-yl]methaneamine (15) Preparation was carried out starting from 5-(aminomethyl)thiophene-2-carbonitrile (preparation according to WO 95/23609), which was reacted to the respective Boc-derivative according to standard methods.\",\n \"1.1 eq.\",\n \"Sodium methanolate solution was added to t-butyl-5-cyanothiene-2-ylcarbamate (25 g; 104.9 mmol) in 330 ml CH3OH and stirred overnight at RT, then for 2 h at 40-50°.\",\n \"18.95 g Phenylenediaminebihydrochloride were then added and again stirred at RT.\",\n \"For workup, water was added, the resulting precipitate filtered off and carefully dried.\",\n \"19.6 yellow solid; ESI-MS: [M+H+]=330.The following cleavage of the Boc-group with TFA afforded a raw product, which was dissolved in water, 2× extracted with diethyl ether, the watery phase adjusted to pH 10-11 and then extracted 2× with ethyl acetate.\",\n \"The watery phase was saturated with NaCl and again extracted with ethyl acetate.\",\n \"The combined organic phases were dried and evaporated (6.3 g); ESI-MS [M+H+]=230.1.tert-Butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) Preparation was carried out analogously to the preparation of [4-(1H-benzimidazole-2-yl)phenyl]methaneamine (hydrochloride) (7) starting from tert-butyl-2-(4-cyanophenyl)ethylcarbamate.\",\n \"The raw product obtained after reaction with H2S, alkylation with CH3I and reaction with 1,2-phenylenediamine was purified by chromatography on silica gel (CH2Cl2/CH3OH 4-50%) (4.8 g); ESI-MS [M+H+]=338.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with TFA (under standard conditions); the isolated TFA-salt was then directly utilized in the respective couplings.\",\n \"N-(Piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoroacetate) (17) a) A solution of tert-butyloxycarbonyl-4-(aminomethyl)-1-piperidine (5.39 g; 25 mmol) in 25 ml CH3CN was added dropwise to 6.75 g thiocarbonyldiimidazole and 0.5 g imidazole in 100 ml CH3CN at 0° C. and then stirred for 3 h at RT.\",\n \"1,2-Phenylenediamine (5.5 g; 50.86 mmol) was then added and heated for about 1 h to 60° C. The solid obtained upon cooling was filtered off with suction and dried.\",\n \"6.79 g; ESI-MS [M+H+-tBu]=309.15 b) tert-Butyoxycarbonyl-4-({[(2-aminoanilino)carbothioyl]amino}methyl)1-piperidine (5 g; 13.72 mmol), 5.94 g HgO (yellow) and 0.6 g sulfur in 150 ml ethyl alcohol were heated under reflux for 1 h. The mixture was filtered 2× over Celite, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 5-25%).\",\n \"2.65 g; ESI-MS [M+H+]=331.25 1H-NMR (360 MHz, DMSO) δ ppm: 7.15 and 6.9 (each m, 2H), 3.95 (d, 2H) 3.2 (m, 2H, 2.7 (br m; 2H), 1.8 (m, 1H), 1.7 (m, 2H), 1.35 (s, 9H), 1.05 (m, 2H).\",\n \"c) tert-Butyloxycarbonyl-4-[(1H-benzimidazole-2-ylamino)methyl]-1-piperidine (2.65 g; 8.02 mmol) was treated with 10 ml TFA according to standard conditions.\",\n \"Evaporation and mixing the raw product with n-pentane afforded 2.39; ESI-MS [M+H+]=231.15.1H-NMR (360 MHz, DMSO δ ppm: 13.25 (s, 1H), 9.35 (m, 1H), 8.8 and 8.5 (each br s, 1H), 7.4 and 7.20 (each m, 2H), 3.3 (m, 4H), 2.85 (m, 2H), 1.9 (m, 3H), 1.35 (m, 2H).\",\n \"N-{[5-(Aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoroacetate) (18) a) A solution of tert-butyl-(4-cyanothiene-2-yl)methylcarbamate (7 g; 29.4 mmol) in 120 ml ethyl alcohol was saturated with NH3 and then hydrogenated under standard conditions in the presence of Ra—Ni (9 g watery suspension; decanted with ethyl alcohol).\",\n \"Filtration of the reaction mixture, evaporation and chromatography of the obtained residue on silica gel (CH2Cl2/CH3OH plus watery NH3) afforded 4.4 g of the amine as a yellow oil.\",\n \"b) 1.2.of the amine 18a (4.3 mmol), 06.g ethyldiisopropylamine and 15 g 2-fluoropyridine were heated for 20 h to reflux.\",\n \"The residue obtained after evaporation of the mixture was taken up in CH2Cl2, washed with 0.1n HCl and saturated NaCl solution, dried and again evaporated.\",\n \"1 g; ESI-MS [M+H+]=320.15 c) 0.9 of the Boc-protected amine 18b were dissolved in 10 ml CH2Cl2, 5 ml TFA was added at 0° C. and it was stirred at room temperature for 1 h. Evaporation of the reaction mixture afforded 1.65 g of a brownish oil which was reacted directly without further purification (ESI-MS [M+H+]=220.05).\",\n \"3-Amino-N-(1H-imidazole-2-yl)propaneamide (19) a) Z-β-Alanine (10 g; 44.8 mmol) was dissolved in 200 ml DMF and 15.86 g (3.5 eq) N-methylmorpholine and 5.9 g (0.5 eq) 2-aminoimidazolesulfate were added.\",\n \"7.87 g (1.3.eq) HOBt and 11.16 g (1.3 eq) N′-(dimethylaminopropyl)-N-ethylcarbodiimide were added at −10° C. and stirred for 1 h whilst to RT and then for 18 h. 150 ml diethyl ether were added whereupon a wide residue precipitated which was filtered with suction.\",\n \"The residue was washed with cold diethyl ether, suspended in ethyl acetate and 1n HCl was added up to an acid reaction.\",\n \"The watery solution was extracted 1× with ethyl acetate, the watery phase was then adjusted to a basic pH with 10% NaOH at 4° C. The resulting precipitate was filtered with suction and washed with water.\",\n \"5.4 g; ESI-MS [M+H+]=289.05.b) 5.3 g of the Z-compound 19a were suspended in 250 ml ethyl alcohol and 530 mg 10% Pd on activated carbon was added.\",\n \"It was hydrogenated with H2 for 18 h at RT, then diluted with CH3OH and the suspension was boiled up whereon the precipitate of the product disintegrated.\",\n \"Filtering and evaporation of the solution afforded 1.5 g; ESI-MS [M+H+]=155.05.N-[4-(Aminomethyl)phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) tert-Butyl-4-aminobenzylcarbamate (2 g; 9 mmol), 2.2 g ethyldiisopropylamine and 4.4 g (2-(3,5-dimethylpyrazole)-4,5-dihydroimidazole×HBr in 15 ml DMF were stirred at 110° C. After the reaction had completed, the mixture was evaporated, the residue taken up in ethyl acetate, each 2× washed with saturated NaHCO3 and NaCl solution, dried and again evaporated.\",\n \"The basic watery phase was also evaporated, mixed with acetone, the precipitate filtered with suction and the mother liquor evaporated.\",\n \"The combined residues were purified by means of MPLC (silica gel: Fa.\",\n \"Bischoff Prontoprep 60-2540-C18E, 32 μm; solvent: CH3CN/H2O+0.1% acetic acid).\",\n \"0.22 g; ESI-MS [M+H+]=291.15.The product thus obtained was treated with 4n HCl in dioxane for 2 h at RT.\",\n \"The resulting precipitate was filtered with suction, washed with pentane and dried.\",\n \"110 mg; ESI-MS [M+H+]=191.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.85 (s, 1H), 8.45 (s broad, 3H), 8.40 (s, 1H), 7.60 and 7.30 (each d, 2H), 4.05 (m, 2H), 3.70 (s, 4H).\",\n \"N-{[5-Aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) Amine 18a (6.5 g; 23.31 mmol) was reacted to the respective aminobenzimidazole by reaction with thiocarbonyldiimidazole, imidazole and then phenylenediamine analogously to the preparation of 17.1.6 g: ESI-MS [M+H+]=359.15.The subsequent cleavage of the Boc-group by means of 4n HCl in dioxane afforded 1.3 g of slightly yellow solids: ESI-MS [M+H+]=191.15.N1-Pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) Preparation analogously to 18b by reaction of N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 20.3 g 2-fluoropyridine, 5.64 g clear oil; ESI-MS [M+H+]=280.15.Cleavage of the Boc-group with 4n HCl in dioxane afforded 3.46 g white solids; ESI-MS [M+H+]=180.20.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (s broad, 1H), 8.05 (s broad, 3H), 7.85 (m, 2H), 7.20 (m, 2H), 6.80 (m, 1H), 3.45 (m, superimposed by H2O), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).\",\n \"N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol), 5.4 g ethyldiisopropylamine and 5.11 g 2-(methylsulfanyl)-4,5-dihydro-1H-imidazole×HI in 30 ml DMF were stirred overnight at RT.\",\n \"The reaction mixture was evaporated, taken up in CH2Cl2, washed with water and saturated NaCl solution, dried and again evaporated.\",\n \"5.05 g clear oil, ESI-MS [M+H+]: 271.18.Cleavage of the Boc-group with 4n HCl in dioxane and purification of the raw product by means of MPLC afforded 2.57 g; ESI-MS [M+H+]: 171.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 160.3, 43.3 (2 signals superimposed).\",\n \"42.85, 39.70, 28.90, 27.2, 23.60.N-[4-(Aminomethyl)phenyl]-1H-imidazole-2-amine (24) a) 5.24 g BrCN—dissolved in 50 ml CH3OH-were added dropwise to a mixture of tert-butyl-4-aminobenzylcarbamate (10 g; 44.99 mmol) and 11.07 g sodium acetate in 100 ml CH3OH at 0° C., and it was stirred for 3 hours at 0° C. and overnight at RT.\",\n \"The mixture was evaporated, the obtained residue taken up in water and 2× extracted with methyl-tert-butylether.\",\n \"Drying and evaporation of the organic phases afforded 12.99 of a yellow-orange oil.\",\n \"b) 28.6 g triethylamine were added to 7 g tert-butyl-4-[(iminomethylene)amino]benzyl carbamate in 50 ml pyridine, H2S was introduced for 1 hour at 0° C. and the mixture was allowed to stand for 48 hours.\",\n \"Evaporation afforded 9.79 g of a rosa foam; ESI-MS [M+H+]: 282.05.1H-NMR (360 MHz.\",\n \"D6DMSO) δ ppm: 9.70 (s, 1H), 7.35 (m, 4H), 7.20 (m, 2H), 4.15 (d, 2H), 1.45 (s, 9H).\",\n \"c) 5 g of the thioamide in 50 ml CH3OH where methylated with 5.05 g CH3I and the obtained raw product was directly reacted with 1.73 g aminoacetaldehyde-diethylacetal in 7.5 ml CH3CN for 3 hours at RT.\",\n \"Evaporation of the reaction mixture afforded 6.36 g of a reddish oil (ESI-MS [M+H+]: 381.25) which was dissolved in 50 ml 6n HCl and stirred for 3 hours at 0° C. A pH of 12 was then adjusted with a 25% NaOH solution and it was again stirred for 48 hours at RT.\",\n \"The mixture was extracted 4× with ethyl acetate, the combined organic phases dried and evaporated.\",\n \"The oil thus obtained was stirred 2× with methyl-tert-butylether, the obtained solids filtered with suction and dried.\",\n \"0.6 g red solid; ESI-MS [M+H+]: 189.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 145.5, 141.60, 130.75, 128.4, 119.8, 115.6, 44.85.N1-(1,4,5,6-Tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (25) Preparation was carried out analogously to 23 starting from N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 5.4 g 2-(methylsulfanyl)-1,4,5,6-tetrahydropyrimidine×HI.\",\n \"After workup, 1.3 g of a yellowish oil was obtained; [M+H+]: 282.2.Cleavage of the Boc-group with 4n HCl in dioxane and purification by means of MPLC afforded 0.46 g; ESI-MS [M+H+]: 185.15.N-[4-(Aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26) Benzyl (4-aminocyclohexyl)methylcarbamate (TFA-salt) (5 g; 13.28 mmol)-preparation by TFA cleavage starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317)-was heated to reflux analogously to 18 with 1.71 g ethyldiisopropylamine in 50 ml 2-fluoropyridine.\",\n \"Usual workup and crystallization of the raw products from methyl-tert-butylether/methanol afforded 4.15 g; ESI-MS [M+H+]: 340.29.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.75 (d broad, 1H), 7.85 (m, 2H), 7.35 (m, 5H), 7.05 (d, 1H), 6.85 (m, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.75 (each m, 2H), 1.45-0.90 (m, 6H).\",\n \"Cleavage of the Z group under standard conditions (H2; Pd-activated carbon), precipitation of the resulting amine as hydrochloride and drying of the obtained precipitates afforded 1.5 g; ESI-MS [M+H+]: 206.15.tert-Butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-ylacetate (27) 75 ml of a 1.0 m BH3-THF solution were added to a solution of tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10 g; 36.32 mmol) in 100 ml THF and it was stirred at RT.\",\n \"For workup, water was carefully added, it was 2× extracted with diethyl ether and then the organic phases were washed 2× with water.\",\n \"Evaporation and drying afforded 9.3 g; ESI-MS [M+H+]: 262.04.\",\n \"[5-(2-tert-Butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) a) 3 g K2CO3, 0.05 g KI and 5 g methylbromoacetate were added to a solution of tert-butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl acetate (27) in 50 ml DMF and it was stirred for 12 hours at 90° C. Thereafter, 4 g methylbromoacetate were added and it was stirred for further 5 hours at 120° C. For workup, the mixture was concentrated, diluted with CH2Cl2, washed with saturated NaCl solution, dried and again evaporated.\",\n \"Chromatography on silica gel (CH2Cl2/CH3OH 1-5%) afforded 4.6 g of a bright yellow oil; ESI-MS [M+H+]: 334.12.b) 4.19 of the methylester in 20 ml dioxane/15 ml H2O were saponified with 19 KOH at RT.\",\n \"For workup, the mixture was concentrated, adjusted to a pH of 2 with 2n HCl and extracted with CH2Cl2.The combined organic phases were dried, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 2-7%).\",\n \"2.4 g oil, ESI-MS [M+H+]: 320.15.1H-NMR (360 MHz, CDCl3) δ ppm: 7.20-7.10 (m, 1H), 6.90 and 6.80 (each m, 1H), 4.0 (s, 2H), 3.55 (m, 1H), 3.10 (m, 2H), 2.85 and 2.70 (each m, 1H), 2.90-2.50 (m, 4H), 1.35 (s, 9H).\",\n \"N-[4-(Aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) Preparation analogously to the preparation of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (23) starting from benzyl-(4-aminocyclohexyl)methylcarbamate (TFA salt) (3 g; 7.97 mmol).\",\n \"a) Reaction with BrCN and subsequent purification (1.52 g; ESI-MS [M+H+]: 288.15).\",\n \"1H-NMR (360 MHz, d6-DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.75 (d, 1H), 5.05 (d, 2H), 2.85 (m, 3H), 1.85 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.20 and 0.95 (each m, 2H).\",\n \"b) Conversion to the corresponding thiourea and subsequent methylation (1.489; ESI-MS [M+H+]: 336.15.c) Reaction with aminoacetaldehyde-diethylacetal and subsequent cyclization afforded 0.79 g; ESI-MS [M+H+]: 329.15.1H-NMR (360 MHz, DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.45 (s, 2H), 5.35 (d, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.15 and 0.95 (each m, 2H).\",\n \"d) For cleavage of the Z-group it was dissolved in 30 ml HBr/glacial acidic acid and stirred for 3 h at RT.\",\n \"For workup, the mixture was evaporated and several times co-evaporated with acetone.\",\n \"0.89 g; ESI-MS [M+H+]: 195.15.tert-Butyloxycarbonyl-4-[(2-pyridinylamino)methyl]-1-piperidine (30) tert-Butyloxycarbonyl-4-(aminomethyl)-1-piperidine (3 g; 14 mmol) and 10 ml 2-fluoropyridine were heated for 4 h to reflux.\",\n \"Evaporation and mixing the raw product in n-pentane afforded 3 g of a wide solid, mp: 126-130° C.; ESI-MS [M+H+]=292.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with HCl in dioxane (under standard conditions); the isolated HCl-salt was then directly utilized.\",\n \"Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-oxo-2,3,4,6-tetrahydro H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) a) A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) in 20 ml DMF was added dropwise to a suspension of 1.28 g NaH (60%; deoiled) in 10 ml DMF at 5° C. and it was stirred for 1 h. 3.49 g Bromoacetonitrile-dissolved in 20 ml DMF-were then added dropwise and it was stirred for 4 h at RT.\",\n \"For workup, water was added carefully, it was diluted with CH2Cl2, washed several times with H2O and saturated NaCl solution, dried and evaporated.\",\n \"Purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 5%) afforded 7.61 g; ESI-MS [M+H+-tBu]: 259.05.b) 5 g tert-Butyl-[1-(cyanomethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate in 70 ml pyridine were saturated for 1 h with H2S and then allowed to stand overnight at RT.\",\n \"Evaporation of the mixture and mixing of the obtained residue with pentane afforded 5.5 g of a rosa solid which was directly reacted.\",\n \"c) A mixture of 2 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.62 g ethylpyruvate and 0.86 g KHCO3 in 30 ml dioxane was stirred for 2.5 h at RT.\",\n \"Dilution with CH2Cl2, washing with saturated NaCl solution, drying and evaporation afforded 2.65 g of a yellow oil; ESI-MS [M+H+]: 445.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.50 (s, 1H), 7.50 and 7.35 (each m, 1H), 7.30-7.20 (m, 2H), 7.35 (d, 1H), 5.20 (broad, 1H), 4.30 (q, 2H), 4.20 (m, 1H), 3.65-3.50 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 3H), 1.70-1.50 (m, 2H), 1.30 (s, 9H; superimposed by t, 3H).\",\n \"2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) 2.6 g Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) was provided in a mixture of 31 ml dioxane and 4 ml H2O, 1.5 eq.\",\n \"KOH was added and it was heated to reflux.\",\n \"After 5 h, again 1 eq.\",\n \"KOH was added and it was further stirred for 12 h at RT.\",\n \"For workup, it was concentrated, the residue was taken up in water, a pH of 4-5 was adjusted with 2n HCl and it was extracted several times with CH2Cl2.The combined org.\",\n \"phases were washed with saturated NaCl solution, dried and evaporated.\",\n \"Mixing with n-pentane afforded 2.1 g of a white solid; ESI-MS [M+H+]: 417.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 7.5 (m, 1H), 7.35 (m, 1H), 7.30-7.20 (m, 3H), 5.25 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 6H), 1.70 (m, 1H), 1.30 (s, 9H).\",\n \"2-Ammonio-6-(ammoniomethyl)pyridinium trichloride (33) a) 2-Amino-6-methylpyridine (0.14 mol, 15.0 g) and phthalanhydride (0.14 mol, 20.55 g) were heated to 190° C. at the water separator.\",\n \"Distributing between H2O and CH2Cl2, evaporation of the org.\",\n \"phase and recrystallisation of the residue (diethyl ether) afforded 28.25 g of a slightly yellowish solid; ESI-MS [M+H+]=239.15.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.95 (2H, m), 7.78 (3H, m), 7.23 (2H, m), 2.64 (3H, s).\",\n \"b.)\",\n \"N-bromosuccimide (25.18 mmol, 4.48 g) was added portionwise to a boiling suspension of 2-(6-methyl-2-pyridinyl)-1H-isoindole-1,3(2H)-dione (33a, 20.99 mmol, 5.0 g), AIBN (2.10 mmol, 0.35 g) and dibenzoylperoxide (2.10 mmol, 0.51 g) in CCl4.The reaction mixture was boiled for 20 h, filtered and the filtrate evaporated.\",\n \"Chromatography (CH2Cl2) afforded 3.12 g of the target product and 1.20 g of the dibromo compound; ESI-MS 318.95, 316.95.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98 (2H, m), 7.95 (1H, t), 7.81 (2H, m), 7.58 (1H, d), 7.35 (1H, d), 4.60 (2H, s).\",\n \"c) Potassium phthalimide (9.46 mmol, 1.75 g) was added to a solution of 2-[6-(bromomethyl)-2-pyridinyl]-1H-isoindole-1,3(2H)-dione (33b, 6.31 mmol, 2.0 g) in DMF (30 ml), the reaction mixture was heated for 15 h to 60° C., stirred for 24 h at RT, water (60 ml) was added and it was stirred for 2 h at 0° C. The residue was filtrated and washed with a mixture of H2O-DMF and then with diethyl ether; 2.12 g; ESI-MS [M+H+]=384.05.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98-7.91 (4H, m), 7.88 (1H, t), 7.85-7.75 (4H, m), 7.38-7.32 (2H, m), 5.10 (2H, s).\",\n \"d) 2-{6-[(1,3-Dioxo-1,3-dihydro-2H-isoindole-2-yl)methyl]-2-pyridinyl}-1H-isoindole-1,3(2H)-dione (33c, 5.22 mmol, 2.09) was heated to reflux for 3 h with hydrazinium hydroxide (13.04 mmol, 0.65 g) in methanol (50 ml).\",\n \"For workup, water was added, it was evaporated and the watery phase acidified with conc.\",\n \"HCl.\",\n \"Anew evaporation and recrystallising (ethyl alcohol) afforded 1.20 g of a white solid; ESI-MS [M+H+]=124.05.1H-NMR (270 MHz, D2O) δ (ppm): 7.94 (1H, t), 7.08 (1H, d), 6.99 (1H, d), 4.33 (2H, s).\",\n \"trans-N-[4-(Aminomethyl)cyclohexyl]-N′-benzylurea (34) Preparation was carried out analogously to compound 6 starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317) by cleavage of the boc-group with 4n HCl in dioxane.\",\n \"Build-up of the benzyl urea by reaction with benzyl isocyanate and triethylamine in DMF and subsequent hydrogenolysis afforded 0.55 g of the target product; ESI-MS [M+H+]=262.20.7-(4-Aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoroacetate) (35) a.)\",\n \"A solution of 5-tert-butoxycarbonylaminovaleric acid (50.0 mmol, 10.86 g), O,N-dimethylhydroxylamine hydrochloride (50 mmol, 4.88 g), N-Methylmorpholine (0.30 mol, 30.35 g), HOBT (53.90 mmol, 8.42 g) and EDCI*HCl (55.0 mmol, 10.54 g) in CH3CN (200 ml) were stirred for 2 days at RT.\",\n \"After evaporation the residue was taken up in ethyl acetate, and then washed with water, a 10% KHSO4-solution, a saturated watery NaHCO3 solution and a saturated watery NaCl-solution, subsequently.\",\n \"Drying and evaporation of the organic phase afforded 6.96 g of a yellowish oil; ESI-MS: [2M+Na+]=543.3, [M+Na+]=283.1, 205.1, 161.1.1H-NMR (270 MHz, CDCl3) δ (ppm): 4.63 (1H, s.\",\n \"br.\",\n \"), 3.68 (3H, s), 3.21-3.05 (3+2H, m), 2.44 (2H, t), 1.76-1.48 (2+2H, m), 1.43 (9H, s).\",\n \"b.)\",\n \"Methyl magnesia bromide (60.0 mmol, 17.30 ml of a 3M solution in Et2O) at 0° C. was added dropwise to a solution of tert-butyl 5-[methoxy(methyl)amino]-5-oxopentylcarbamate (35a, 30.0 mmol, 6.9 g) in THF (120 ml) and stirred for 5 h at 0° C. The reaction mixture was then carefully acidified with a 10% KHSO4-solution, extracted with ethyl acetate and the organic phase then washed with saturated watery NaHCO3— and saturated watery NaCl-solution, dried and evaporated: 5.5 g yellowish oil; ESI-MS: [M-BOC+H+]=116.15.c.)\",\n \"A mixture of tert-butyl 5-oxohexylcarbamate (35b, 9.29 mmol, 2.0 g), 2-aminonicotinaldehyde (Heterocycl.\",\n \"1993, 36, 2518; 11.20 mmol, 1.379) and KOH (0.37 ml of a 20% watery solution) was heated to reflux for 8 h. Evaporation and column chromatography afforded 1.60 g of the target product; ESI-MS: [M+H+]=302.15.d.)\",\n \"A suspension of tert-butyl 4-[1,8]naphthyridine-2-ylbutylcarbamate (35c, 5.31 mmol, 1.60 g) and Pd/C (10%, 1.59) in ethyl alcohol (40 ml) were stirred overnight under H2 atmosphere, then filtered over celite and washed with ethyl alcohol.\",\n \"Column chromatography afforded 290 mg; ESI-MS: [M+H+]=306.25.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (1H, d), 6.29 (1H, d), 4.97 (1H, s.br.\",\n \"), 4.81 (1H, s.br.\",\n \"), 3.37 (2H, m sym.\",\n \"), 3.12 (2H, q br.\",\n \"), 2.65 (2H, t), 2.53 (2H, t), 1.89 (2H, quint.\",\n \"), 1.67 (2H, quint.\",\n \"), 1.51 (2H, quint.\",\n \"), 1.43 (9H, s).\",\n \"e.) TFA (18.30 mmol, 2.099) was added to a solution of tert-butyl 4-(5,6,7,8-tetrahydro[1,8]naphthyridine-2-yl)butylcarbamate (35d, 0.92 mmol, 0.289) in CH2Cl2 (8 ml), the solution was stirred for 20 h and evaporated: 380 mg; ESI-MS: 206.1, 130.7.1H-NMR (400 MHz, CDCl3) δ (ppm): 7.07 (1H, d), 6.31 (1H, d), 5.58 (1H, s.br.\",\n \"), 3.39 (2H, m sym.\",\n \"), 2.96 (2H, s br.\",\n \"), 2.76 (2H, t), 2.68 (2H, t), 2.56 (2H, t), 1.88 (2H, quint.\",\n \"), 1.69 (2H, quint.\",\n \"), 1.51 (2H, quint.).\",\n \"tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36) Tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10.9 mmol, 3.0 g)—dissolved in THF—was added at 0° C. to a solution of diisopropylamine (11.0 mmol, 1.119) and butyl lithium (11.0 mmol, 6.91 ml of a 15% solution in hexane) in THF (100 ml) and the solution further stirred for 1 h. About 100 ml ethylene oxide were then added and the mixture was stirred overnight at RT.\",\n \"The solution was distributed between saturated NH4Cl and ethyl acetate, the organic phase was washed with water and evaporated; 2.7 g; ESI-MS: [2M+Na+]=661.3, [M+K+]=358.1, 321.1, [M+H+]=320.1, 264.0.tert-Butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37) tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36, 6.26 mmol, 2.00 g) dissolved in CH2Cl2 was added dropwise within 10 minutes to a solution of oxalyl chloride (7.93 mmol, 1.0 g) and DMSO (16.59 mmol, 1.26 g) in little CH2Cl2.After 30 min.\",\n \"triethyl amine (38.22 mmol, 3.87 g) was added, stirred for 5 min., left to reach RT and stirred overnight at RT.\",\n \"For workup, water was added, the mixture extracted with CH2Cl2 and the organic phase washed with saturated NaCl—, 1%-H2SO4— and with 5%-NaHCO3-solution.\",\n \"Evaporation afforded 1.8 g of the target product; ESI-MS: 693.2, [M+K+]=358.1, 319.1, [M+H+]=318.1, 262.0.Methyl-2-amino-5-chlorobenzoic acid (38) Thionyl chloride (0.47 mmol, 55.46 g) was added dropwise at 0° C. to a solution of 2-chloro-5-aminobenzoic acid (0.23 mmol, 40.09) in methanol (400 ml) and the mixture heated to 50-60° C. After the reaction had finished, water was added and it was extracted with ethyl acetate.\",\n \"Then the organic phase was washed with 1n NaOH and diluted HCl solution (pH 1-2), subsequently, and evaporated; 23.0 g; ESI-MS: [M+H+]=186.05.Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39) Ethyl succinic acid-chloride (0.14 mol, 22.44 g) in toluen (15 ml) was added dropwise at 0° C. to a solution of methyl 2-amino-5-chloro-benzoic acid (38, 123.9 mmol, 23.0 g) and pyridine (0.26 mol, 20.58 g) in toluen (40 ml).\",\n \"The solution was stirred overnight at RT, water was added and it was extracted with ethyl acetate.\",\n \"The organic phase was washed with 1N HCl-solution, with water, with a saturated NaHCO3-solution and with a saturated NaCl-solution.\",\n \"Evaporation, recrystallisation (methanol) afforded 34.1 g of the target product; ESI-MS: [2M+Na+]=649.0, [M+K+]=352.0, [M+H+]=314.05; 1H-NMR (270.MHz, CDCl3) δ (ppm): 11.06 (1H, s br.\",\n \"), 8.68 (1H, d), 7.99 (1H, m), 7.47 (1H, dd), 4.16 (2H, q), 3.92 (3H, s), 2.74 (4H, m), 1.24 (3H, t).\",\n \"Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40) Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39, 0.16 mol, 50.20 g) in DMSO (250 ml) was added dropwise at 15° C. to a suspension of deoiled NaH (0.27 mol) in THF (50 ml) and DMSO (80 ml) and the mixture was stirred for 2 h at RT.\",\n \"At 0° C. glacial acetic acid (24 ml) was added and stirred for 20 min.\",\n \"Water (25 ml) was added, the resulting precipitate filtered off, washed with water, taken up in CH2Cl2, extracted with water and the organic phase evaporated.\",\n \"The residue was then stirred with diethyl ether (50 ml), filtered and dried; 32 g of a 6:4-mixture methyl/ethyl ester, which was not separated; ESI-MS (Me-ester): [M+K+]=307.9, [M+Na+]=290.0, [M+H+]=268.0; ESI-MS (Et-ester): [M+K+]=321.9, [M+Na+]=304.0, [M+H+]=282.0.7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41) Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40, 0.11 mol, 32.0 g) was heated to 150° C. in DMSO (500 ml) and water (0.23 mol, 4.09 g) and stirred for 2 h. Water was added at 100° C., the mixture cooled to 0° C. and the resulting precipitate filtered off.\",\n \"Drying afforded 19.09; ESI-MS: [M+Na+]=251.1, [M+H+]=211.9, 209.95, 130.1.tert-Butyl (2E,Z)-(7-Chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene) ethane acid (42) t-Butyl diethylphosphono acetic acid (0.10 mol, 25.849) was added dropwise at 0° C. to a solution of deoiled NaH (0.10 mol) in DMF (40 ml) and stirred until a clear solution develops.\",\n \"7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41, 90.63 mmol, 19.09) in DMF (185 ml) was added dropwise at 0° C. and stirred at RT.\",\n \"Water was added to the reaction mixture, it was stirred for 1 h and the resulting yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.\",\n \"Recrystallisation afforded 23.5 g of the target product; ESI-MS: [2M+H+]=615.2, [M+Na+]=330.0, 293.0, 254.1, 252.1.1H-NMR (400 MHz, CDCl3) δ (ppm): 9.59 (1H, s br.\",\n \"), 7.45 (1H, m), 7.25 (1H, m), 7.06 (1H, m), 5.99 (1H, t), 3.43 (2H, s), 2.84 (2H, d), 1.32 (9H, s).\",\n \"t-Butyl (7-chloro-2-oxo-2,3,4,4-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43) tert-Butyl (2E,Z)-(7-chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)ethane acid (42, 75.0 mmol, 23.089) in ethyl alcohol/dioxane (250 ml/100 ml) was hydrogenated for 4 days with Pt/carbon (5%, 4.1 g) under standard conditions.\",\n \"Water was added to the reaction mixture, stirred for 1 h, the yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.\",\n \"Recrystallisation afforded 23.5 g of a solid (mixture of target product and the corresponding dechlorinated compound; the mixture was reacted directly); ESI-MS: [2M+H+]=618.94, [M+K+]=350.66, 309.75, 254.11.Methyl [5-(2-tert-butoxy-2-oxo ethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44) tert-Butyl (7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43, 60.00 mmol, 18.59 g) in DMF (10 ml) was added dropwise at 15° C. to deoiled NaH (69.00 mmol) in DMF (5 ml) and the mixture was stirred overnight at RT.\",\n \"Ice water was added to the reaction mixture, extracted (2×) with ethyl acetate and the organic phase washed with a 10% CH3COOH-solution, with water and then with 1n NaOH.\",\n \"Evaporation afforded 20.4 g of a raw product, which was reacted without further purification.\",\n \"[5-(2-tert-Butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) KOH (80.45 mmol, 4.51 g) in water (150 ml) was added to methyl [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44, 50.28 mmol, 19.29) dissolved in dioxane (250 ml) and stirred for 1 h at RT.\",\n \"The reaction mixture was evaporated, water (100 ml) was added and it was extracted with ethyl acetate (2×).\",\n \"After the evaporation, the residue was taken up in diethyl ether and precipitated by addition of n-pentane.\",\n \"Recrystallisation (diisopropyl ether, 2×) afforded 4.8 g (comprises about 15% of the corresponding dechloro compound); ESI-MS: [M+Na+]=390.0, 314.0, 312.0.\",\n \"[4-(Aminomethyl)phenyl]guanidine (Bihydrochloride) (46) p-Aminobenzylamine (6.7 g; 54.84 mmol) was suspended in 20 ml 6n HCl and 5.3 g cyanamide—dissolved in 5 ml H20—added slowly under reflux.\",\n \"After the reaction had completed, 50% NaOH-solution was added at 0° C. to the solution, the resulting precipitate filtered with suction, boiled up in 50 ml ethyl alcohol and filtered off.\",\n \"Evaporation of the mother liquor and mixing of the obtained residue with diethyl ether afforded 1.4 g of yellow solids; Fp.\",\n \": 255° C. 7,8-Dimethoxy-3,4-dihydro-1H-1-benzazepine-2,5-dione (47) Analogously to the preparation of the corresponding building blocks 39, 40 and 41 first ethyl-2-amino-4,5-dimethoxybenzoate (20 g; 88.8 mmol) was reacted with ethyl succinic acid-chloride to the respective amide.\",\n \"After mixing with n-pentane 30.4 g of a solid was obtained; ESI-MS [M+H+]: 354.15.1H-NMR (400 MHz, DMSO) δ (ppm): 10.65 (s, 1H), 8.10 and 7.40 (each s, 1H), 4.30 and 4.10 (each q, 2H), 3.85 and 3.80 (each s, 3H), 2.70 (m, 4H), 1.30 and 1.20 (each t, 3H).\",\n \"Subsequent cyclization with 25 g of the obtained amide under utilization of NaH analogously to 40 and usual workup afforded 19.5 g of a white solid; ESI-MS [M+H+]: 308.05.1H-NMR (400 MHz, DMSO) δ (ppm): 13.3 (s, 1H), 10.10 (s, 1H), 7.25 and 6.75 (each s, 1H), 4.30 (q, 2H), 3.80 (s, 6H), 2.95 (s, 2H), 1.35 (t, 3H).\",\n \"Decarboxylation analogously to 41 starting from 17 g afforded 10.5 g of the target product as a solid; ESI-MS [M+H+]: 236.15.1H-NMR (400 MHz, DMSO) δ (ppm): 9.90 (s, 1H), 7.35 and 6.80 (each s, 1H), 3.80 and 3.75 (each s, 3H), 2.85 and 2.65 (each m, 2H).\",\n \"tert-Butyl (7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate (48) Preparation analogously to building blocks 42 and 43 by Wittig-Homer-reaction and subsequent hydrogenation; after mixing with n-pentane 8.16 g of solids were obtained; ESI-MS [M+H+-tBu]: 280.15.\",\n \"[5-(2-tert-Butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (49) Analogously to 44 and 45, starting from 4 g tert-butyl-(7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 2.6 g of the target product were isolated as a bright foam; [M+H+-tBu]: 338.15.1H-NMR (400 MHz, DMSO) δ (ppm): 6.85 and 6.75 (each s, 1H), 4.35 (s broad, 2H), 3.80 and 3.75 (each s, 3H), 3.60 (s, 2H), 2.70 (m, 2H), 2.25 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H), 1.35 (s, 9H).\",\n \"[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) a.)\",\n \"56.1 g (406 mmol) powdered K2CO3 were added at room temperature to a solution of 37 g (135.3 mmol) t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl)acetic acid (1) and 3.7 g tetrabutylammoniumbromide in 370 ml DMF, 22.7 g (148.9 mmol) methylbromoacetic acid was added dropwise, then it was stirred for 3 h at 40° C. and overnight at room temperature.\",\n \"The reaction mixture was poured into 1000 ml of a water-ice-mixture, and 2× extracted with each 200 ml methyl-tert.butyl ether.\",\n \"The combined extracts were washed with H2O, 5% NaHCO3— and NaCl-solution, dried over Na2SO4, filtered off and evaporated.\",\n \"The remaining yellow oil (about 52 g, purity about 90%) was reacted without further purification; ESI-MS [M+H+]: 346.b.)\",\n \"9.2 g (26.6 mmol) of the raw product 50a were dissolved in 66.6 ml 4n HCl in dioxane and stirred for 24 h at 50° C., then the dioxane was extensively distilled off, 5% NaHCO3-solution and diethyl ether added to the residue, the watery phase again washed with diethyl ether and acidified with 1n KHSO4-solution.\",\n \"The precipitating acid was extracted with diethyl ether, the ether phase washed with a NaCl-solution, dried over Na2SO4, filtered off with suction and evaporated.\",\n \"It remained 3.2 g of a slightly yellow oil; ESI-MS [M+H+]: 290.trans-[4-(1H-Benzimidazole-2-yl)cyclohexyl]methaneamine (hydrochloride) (51) 39.3 g (0.25 mol) trans-4-(Aminomethyl)cyclohexanecarbonic acid and 27.0 g (0.25 mol) 1,2-phenylene diamine were heated to reflux for 18 h in a mixture of 167 ml conc.\",\n \"hydrochloric acid and 333 ml H2O analogously to J. Heterocycl.\",\n \"Chem.\",\n \"26, 541 (1989).\",\n \"The green reaction solution was concentrated until the occurrence of a yellow crystalline pulp and stirred with 400 ml isopropanol, filtered off, washed with 90% isopropanol and finally with diethyl ether.\",\n \"After 2 times recrystallisation from a isopropanol-water mixture (70/30), 30 g of a white monohydrochloride remained; ESI-MS [M+H+]: 230.Methyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5benzodiazepine-1-yl)acetic acid (52) 1.99 (79.8 mmol) NaH (60% dispersion in mineral oil) were added to an ice cooled solution of 12.2 g (75.3 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-One (preparation: J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"1949, 71, 1985) in 350 ml DMF, stirred for 30 min.\",\n \"at 0-5° C. and for 10 min.\",\n \"at room temperature.\",\n \"Then 11.5 g (75.3 mmol) methyl bromo acetic acid were added dropwise at 0° C. and it was then stirred for 30 min.\",\n \"at the same temperature.\",\n \"The reaction solution was poured onto 600 ml ice water and 3× extracted with each 150 ml ethyl acetate.\",\n \"The organic phase was washed with a NaCl-solution, dried over MgSO4 and ethyl acetate was distilled off.\",\n \"The residue was purified by column chromatography (eluent: CH2Cl2/CH3OH 9/1).\",\n \"9.6 g of a yellowish oil were isolated; ESI-MS [M+H+]: 235.tert-butyl (2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) Preparation was carried out analogously to building block 52 by reaction of 11.99 (73 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-one with 14.3 g (73 mmol) tert-butyl bromo acetic acid.\",\n \"17 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 277.\",\n \"[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) a.)\",\n \"14.2 g (102 mmol) powdered K2CO3 were added at 0° C. to a solution of 9.6 g (41 mmol) compound 52 and 8.09 (41 mmol) tert-butyl bromo acetic acid in 90 ml DMF, and stirred for 1 h at 0° C. and then for 14 h at room temperature.\",\n \"The reaction mixture was poured onto 300 ml ice water and 3× extracted with each 100 ml methyl-tert.butyl ether.\",\n \"The combined organic phases were washed several times with a NaCl-solution, dried over MgSO4 and evaporated to dryness.\",\n \"The residue was purified by column chromatography (eluent: ethyl acetate/cyclohexane 7/3).\",\n \"7.0 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 349.b.)\",\n \"The alkaline saponification of the methyl ester was carried out analogously to 3b.\",\n \"3.8 g white crystals were obtained; Fp.\",\n \": 140-142° C.; ESI-MS [M+H+]: 335.\",\n \"[5-(2-tert-Butoxy-2 oxoethyl)-4-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) Analogously to building block 54a tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) was reacted with methyl bromoacetic acid and then alkaline saponified analogously to 3b.\",\n \"After purification by column chromatography 2.9 g white crystals were obtained; Fp.\",\n \": 82-84° C.; ESI-MS [M+H+]: 335.\",\n \"[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) 14.5 g (42 mmol) building block 3a were dissolved in 105 ml 4n HCl in dioxane and stirred for 2 days at 50° C. After evaporation of the solvent, the residue was dissolved in 5% NaHCO3-solution, 2× extracted with methyl-tert.butyl ether, then the watery phase was acidified with a 1n KHSO4-solution and the precipitating acid extracted with methyl-tert.butyl ether.\",\n \"After drying over MgSO4, evaporation of the solvent and purification by column chromatography (eluent: CH2Cl2/CH3OH/glacial acetic acid 451511) 1.6 g of a viscous, slightly yellowish oil remained; ESI-MS [M+H+]: 292.\",\n \"[(5R)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (57) 14.2 g (42.6 mmol) [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) were suspended in 170 ml diethylether and dissolved by addition of 7.3 g (42.64 mmol) (1S)-(−)-1-napthyl)ethylamine.\",\n \"The yellow solution was inoculated with (1S)-1-napthyl)ethaneaminium[(5R)-5-(2-tert.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetate—prepared by preparative HPLC-separation of compound 3 by means of a chiral column (Chiralpak AD 500×; 50 mm; 20 μm) and subsequent salification-, the deposited precipitate filtered off with suction after 3.5 h and 3× recrystallized from a ethyl acetate/isopropanol mixture.\",\n \"The purity of enantiomers was checked by means of a chiral HPLC.\",\n \"3.5 g. of the salt were suspended in 30 ml of a diethylether/hexane-mixture 10/3 and after addition of 50 ml of a 5% watery amidosulfonic acid solution stirred until occurrence of a clear phase.\",\n \"After separation of the watery phase, the organic phase was washed 3× with 5 ml amidosulfonic acid- and then with a NaCl-solution, dried over Na2SO4 and evaporated.\",\n \"2.25 g amorphous residue; ESI-MS [M+H+]: 505.\",\n \"[(5S)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (58) From the combined mother liquors of building block 57, 8 g of the acid were isolated as a yellowish amorphous residue as described with a amidosulfonic acid solution, said acid was reacted with (1R)-(+)-1-napthyl)ethyl amine into the diastereomeric salt and recrystallized until the achievement of enantiomeric purity.\",\n \"Analogously to example 57, 2.5 g of the dextrorotatory acid were isolated as an amorphous solid, ESI-MS [M+H+]: 505.A sample of the acid was reacted with 4-bromobenzylamine into the good crystallizing amide and the absolute configuration figured out by means of a x-ray structure analysis.\",\n \"tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate (59) A solution of 5.3 g (29.2 mmol) 6,7-dihydro-4H-thieno[3,2-b]azepine-5,8-dione (Arch.\",\n \"Pharm.\",\n \"1991 324, 579) and 12 g (32.2 mmol) (tert-butoxycarbonylmethylene)-triphenyl phosphorane in 15 ml toluen was heated to reflux for 10 h, then toluen was distilled off and the black residue purified by chromatography (eluent: ethyl acetate/cyclohexane 7/3).\",\n \"The consistent fraction was again digested with 40 ml boiling cyclohexane, cooled and filtered with suction.\",\n \"3 g yellowish crystals; ESI-MS [M+H+]: 280.tert-Butyl-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl)acetate (60) 39 (11 mol) tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate were hydrogenated analogously to building block 2 in the presence of 10% Pd/C.\",\n \"Since educt was still present after 6 h according to HPLC, the catalyst was filtered with suction and after addition of new catalyst it was again hydrogenated for 6 h. After workup and chromatographic purification (eluent: ethyl acetate/cyclohexane 7/3), 1.4 g yellowish crystals were isolated, which comprised according to HPLC still about 2.5% educt; ESI-MS [M+H+]: 282.\",\n \"[8-(2-tert-Butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) Preparation was carried out analogously to building block 50.Ester stage: 1.5 g yellowish crystals; ESI-MS [M+H+]: 354.Target product: 1.2 g yellowish crystals; ESI-MS [M+H+]: 340.I.B Compounds of the Formula I or I′ Example I t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetate 1.1 g of N-methylmorpholine were added dropwise at 0° C. to 1.5 g (4.65 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) and 1.22 g (4.8 mmol) of N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) in 20 ml of DMF, and 1.55 g (4.7 mmol) of TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetrafluoroborate) were then introduced in portions over the course of 35′.\",\n \"The yellow reaction solution was stirred at 0° C. for 1 h and then largely evaporated in vacuo.\",\n \"The residue was then digested a number of times with H2O, taken up in a mixture of 120 ml of ethyl acetate and 40 ml of diethyl ether, washed with 10% K2CO3 and NaCl solution, dried over Na2SO4 and concentrated, the crude product thoroughly crystallizing.\",\n \"Purification by chromatography on silica gel (CH2Cl2/CH3OH/NH3 42:8:0.1) and crystallization from ethyl acetate/n-hexane afforded 1.45 g (65%) of white crystals.\",\n \"M.p.\",\n \": 190-193° C. (dec.); FAB-MS [M+H+]: 487.Example II [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid 1.2 g of the t-butyl ester from Example I were suspended in 70 ml of CH2Cl2, treated with 45 ml of 4N HCl in dioxane and stirred overnight at RT.\",\n \"The solution was evaporated, and the residue was digested a number of times with CH2Cl2 and then dried.\",\n \"In this way, 1.07 g of a slightly yellowish amorphous powder were isolated; FAB-MS [M−H+]: 432.Example III [1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine hydrochloride (4) and subsequent removal of the t-butyl group analogously to Example II.\",\n \"A slightly yellowish amorphous residue was obtained, FAB-MS [M−H+]: 554.Example IV t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetate Analogously to the preparation of compound 3a, 0.9 g (3.3 mmol) of t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) was alkylated with methyl bromoacetate (FAB-MS [M−H+]: 346) and then hydrolyzed analogously to 3b (0.44 g; FAB-MS [M−H+]: 332).\",\n \"Coupling of 0.57 g (1.72 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-1-yl]acetic acid with N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) analogously to Example I afforded the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 485.Example V [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetic acid The t-butyl ester was removed analogously to Example II and afforded 0.42 g of the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 429.Example VI (1-{2-[(4-{[(Benzylamino)carbonyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-N′-benzylurea (6) and subsequent removal of the t-butyl group analogously to Example II.\",\n \"Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 27 mg as an amorphous solid; ESI-MS [M+H+]: 515.2; [M+K+]: 553.2.Example VII [1-(2-{[4-(1H-Benzimidazol-2-yl)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [4-(1H-benzimidazol-2-yl)phenyl]methaneamine (7) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 9 mg as an amorphous solid; ESI-MS [M+H+]: 485.2.Example VIII [1-(2-{[4-(1H-Benzimidazol-2-ylamino)butyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.5 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 464.Example IX [1-(2-{[5-(1H-Benzimidazol-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.48 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 478.Example X {1-[2-({4-[(1H-Benzimidazol-2-ylamino)methyl]cyclohexyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with trans-N-[(4-aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"After chromatographic purification on silica gel, 0.7 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XI {1-[2-({[4-(1H-Benzimidazol-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with trans-N-{[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"After chromatographic purification on silica gel, 0.5 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XII [2-Oxo-1-(2-oxo-2-{[4-(pyridin-2-ylamino)benzyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-2-pyridineamine (13) and subsequent cleavage of the t-butyl group analogously to Example II (14 mg); ESI-MS [M+H+]: 459.15.Example XIII {1-[2-({[6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid (bishydrochloride) Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [6-(1H-benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) and subsequent cleavage of the t-butyl group analogously to Example II.\",\n \"(13 mg); ESI-MS [M+H+]: 484.15.Compounds of the general formula I analogously to example II were prepared: Example 14 {2-Oxo-1-[2-oxo-2-({4-[(pyridine-2-ylamino)methyl]benzyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of N-[4-(aminomethyl)benzyl]-2-pyridine amine (14) as educt, 207 mg as an amorphous solid were obtained according to MPLC 207 mg; ESI-MS [M+H+]: 473.28.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.5 (m, 1H), 7.95 (d, 1H), 7.45 (m, 1H), 7.35-72.0 (m, 9H), 6.60 (m, 2H), 4.45 (m, 3H), 4.25 (m, 3H), 3.55 (m, 1H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m 1H).\",\n \"Example 15 {1-[2-({[5-(1H-Benzimidazole-2-yl)thiene-2-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of [5-(1H-benzimidazole-2-yl)thiene-2-yl]methaneamine (15) as educt, 210 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 489.27.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 7.70 (m, 1H), 7.65 (m, 2H), 7.35-7.15 (m, 6H), 7.05 (m, 1H), 4.55 (m, 3H), 4.20 (m, 1H), 3.55 (m, 1H), 2.75 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H).\",\n \"Example 16 {1-[2-({2-[4-(1H-Benzimidazole-2-yl)phenyl]ethyl}amino)-2-oxoethyl]-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of tert-butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) as educt, 190 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 497.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.15 (d, 2H), 7.65 (d, 2H), 7.45 (m, 2H), 7.30-7.15 (m, 6H), 4.45 and 4.15 (each m, 1H), 3.6-3.25 (m, superimposed by H2O), 2.80 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.70 (m, 1H).\",\n \"Example 17 [1-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of (3 N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) as educt, 134 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 490.29.Example 18 [2-Oxo-1-(2-oxo-2-{[2-(pyridine-2-ylamino)ethyl]amino}ethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N1-pyridine-2-ylethane-1,2-diamine, the subsequent MPLC afforded 278 mg as amorphous white solids; ESI-MS [M+H+]: 397.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.95 (m, 1H), 7.70 (m, 2H), 7.35-7.20 (m, 4H) 6.80 and 6.70 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.0 (m, superimposed by H2O), 2.70 (m, 2H), 2.30 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).\",\n \"Example 19 (2-Oxo-1-{2-oxo-2-[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]ethyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18), the subsequent MPLC afforded 135 mg as amorphous white solids; ESI-MS [M+H+]: 479.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.65 (m, 1H), 8.0 (m, 1H), 7.45 (m, 1H), 7.35-7.15 (m, 6H), 6.95 (s, 1H), 6.60 and 6.55 (each m, 1H), 4.60-4.30 (m, 5H), 4.15 (m 1H), 3.50 (m, 1H), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.70 (m, 1H).\",\n \"Example 20 [1-(2-{[3-(1H-imidazole-2-ylamino)-3-oxopropyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of 3-amino-N-(1H-imidazole-2-yl)propane amide (19) and following purification of the raw product by MPLC 50 mg were obtained as amorphous white solids; ESI-MS [M+H+]: 414.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.30-7.20 (m, 7H), 4.45 and 4.15 (each m, 1H), 3.75-3.25 (m, superimposed by H2O), 2.80-2.65 (m, 4H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).\",\n \"Example 21 [1-(2-{[4-(4,5-Dihydro-1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N-[4-(aminomethyl)-phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) as educt, 12 mg were obtained as amorphous white solids after MPLC; ESI-MS [M+H+]: 450.3.Example 22 (1-{2-[({4-[(1H-Benzimidazole-2-ylamino)methyl]thiene-2-yl}methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-6-yl)acetic acid Utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) as educt; purification by MPLC afforded 90 mg; ESI-MS [M+H+]: 518.29.Example 23 [2-Oxo-1-(2-oxo-2-{[5-(pyridine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) as educt; after MPLC, 210 mg white solids were obtained; ESI-MS [M+H+]: 439.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (m, 1H), 7.95 (m, 1H), 7.75 (m broad, 1H), 7.65 (m, 1H), 7.30-7.15 (m, 4H), 6.75 and 6.60 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.20 (m, superimposed by H2O), 2.70 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60, 1.45 and 1.30 (each m, 2H).\",\n \"Example 24 N-[5-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)pentyl]-4,5-dihydro-1H-imidazole-2-amine (acetate) Utilization of N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) as educt; 22 mg were obtained after MPLC; ESI-MS [M+H+]: 430.15.Example 25 [1-(2-{[4-(1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (24) as educt; purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 448.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.95 (s, 1H), 8.45 (m, 1H), 7.35-7.10 (m, 8H), 6.75 (s, 2H), 4.50 (m, 1H), 4.20 (m, 3H), 3.5-3.1 (m, superimposed by H2O).\",\n \"2.70 (m, 1H), 2.35 (m 1H), 2.20 (m, 2H), 1.65 (m, 1H).\",\n \"Example 26 [2-Oxo-1-(2-oxo-2-{[1-(1,4,6,6-tetrahydropyrimidine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-(1,4,5,6-tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (26) as educt; 40 mg were obtained after MPLC; ESI-MS [M+H+]: 444.9.Example 27 {2-Oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Utilization of N-[4-(aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26), purification by means of an elution over a Chromabond-C18-cartridge afforded 85 mg; ESI-MS [M+H+]: 465.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.55 (s broad, 1H), 8.05 (m, 1H), 7.90 (d, 1H), 7.85 (m, 1H), 7.35 (m, 2H), 7.25 (m, 2H), 6.95 (d, 1H), 6.80 (m, 1H), 4.40 and 4.20 (each m 1H), 3.55 (m, superimposed by H2O), 2.95 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.15, 1.95 and 1.75 (each m, 2H), 1.60 and 1.40 (each m, 1H), 1.25 (m, 2H), 1.05 (m, 2H).\",\n \"Example 28 Ethyl-{2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 30 ml 4n HCl in dioxane were added to 100 mg {2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid in 10 ml EtOH and it was stirred for 48 h at RT.\",\n \"Evaporation and elution over a Chromabond-C8-cartridge afforded 26 mg; ESI-MS [M+H+]: 493.25.Example 29 (1-{4-[4-(1H-Benzimidazole-2-ylamino)phenyl]butyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid a.)\",\n \"A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (0.8 g; 2.91 mmol) in 20 ml DMF was added dropwise at 0° C. to a suspension of 0.169 NaH (60%; deoiled) in 10 ml DMF and stirred for 1 h. A spatula tip full of KI was then added likewise at 0° C., 1 g 1-(4-bromo butyl)-4-nitrobenzene (preparation according to J. Med.\",\n \"Chem.\",\n \"38, 13 (1995), 2418-2420)—dissolved in 10 ml DMF—was slowly added dropwise and stirred for 2 h at RT.\",\n \"For workup, water was carefully added to the reaction mixture, it was diluted with CH2Cl2 and several times extracted with a saturated NaCl-solution.\",\n \"After drying and evaporation, the obtained raw product was purified by chromatography on silica gel (CH2Cl2/CH3OH 2-10%); 0.429 yellow oil, ESI-MS [M+H+-Boc]: 397.15.b.)\",\n \"Hydrogenation of the nitro group in 75 ml CH3OH with 100 mg 10% Pd on activated carbon under standard conditions afforded 0.36 mg of a bright oil; ESI-MS [M+H+]: 423.25.c.)\",\n \"The build-up of the corresponding aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and then phenylene diamine.\",\n \"Chromatography on silica gel (CH2Cl2/CH3OH 2-4%) afforded 220 mg of a bright foam; ESI-MS [M+H+]: 539.25.d.)\",\n \"200 mg tert-Butyl-(1-(4-[4-(1H-benzimidazole-2-ylamino)phenyl]butyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate was treated at RT with 10 ml TFA.\",\n \"Evaporation and lyophilizing the obtained oil afforded 213 mg of a solid; ESI-MS [M+H+]: 483.25.Example 30 2-(4-{[{[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}(methyl)amino]methyl}anilino)-1H-benzimidazole (trifluoro acetate) a.)\",\n \"Standard coupling of [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (2.2 g; 6.6 mmol) with 1.32 g N-methyl(4-nitrophenyl)methane amine (J.\",\n \"Am.\",\n \"Chem.\",\n \"Soc.\",\n \"65 (1943), 1984-1990) in 40 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.\",\n \"Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 2-4%) afforded 2.269 of a slightly yellow oil; ESI-MS [M+H+]: 482.03.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 8.25/8.20 (d, 2H), 7.60/7.50 (d, 2H), 7.35-7.20 (m, 4H), 4.85-4.5 (m, 4H), 3.05/2.85 (s, 3H), 2.70 (m, 1H), 2.30 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H), 1.30 (s, 9H).\",\n \"b.)\",\n \"5 ml Hydrazine hydrate were added at 60° C. to 1.26 g nitro compound a and 15 mg FeCl3×6H2O in 20 ml CH3OH and for 1 h stirred at 60° C. The mixture was filtered over Celite and the filtrate evaporated.\",\n \"1.17 g; ESI-MS [M+H+]: 452.17.c.)\",\n \"The build-up of the respective aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and phenylene diamine, subsequently.\",\n \"Chromatography on silica gel (CH2Cl2/CH3OH 4-7.5%) afforded 0.9 g of a slightly beige foam; ESI-MS [M+H+]: 568.25.d.)\",\n \"Cleavage of the t-butylester with 50 ml TFA and subsequent purification by chromatography on silica gel (CH2Cl2/CH3OH 8-10%) afforded 0.88 g; ESI-MS [M+H+]: 512.25.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.05 (broad, 1H), 7.60-7.20 (m, 1.2H), 4.85 (m, 1H), 4.75-4.45 (m, 3H), 3.65 (m, 1H), 3.20 (s, 3H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).\",\n \"Example 31 tert-Butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Coupling of [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) analogously to I and purification by chromatography on silica gel (CH2Cl2/CH3OH 2-3%) afforded 200 mg of a beige foam; ESI-MS [M+H+]: 540.25.Example 32 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) TFA-cleavage of tert-butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate afforded 240 mg; ESI-MS [M+H+]: 484.36.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.10 (s, 1H), 8.30 (t, 1H), 7.5-7.25 (m, 8H), 7.15 (m, 2H), 6.85 (m, 2H), 4.40 (m, 2H), 3.75-3.55 (m, superimposed by H2O), 3.5 (m, 1H), 3.0 (m, 1H), 2.70 (m, 2H), 1.70 (m, 2H), 1.45 (m, 1H).\",\n \"Example 33 tert-Butyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).\",\n \"410 mg of a white foam; ESI-MS [M+H+]: 546.35.Example 34 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) Analogously to example 32; 420 mg of white solids; ESI-MS [M+H+]: 490.47.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.0 (d, 1H), 7.65 (m, 1H), 7.40 and 7.30 (each m, 2H), 7.15 and 6.85 (each m, 2H), 3.80 (m, 2H), 3.50 (m, 1H), 3.0 (m, 4H), 2.75 (m, 2H), 2.0 (m, 2H), 1.75-1.50 (m, 5H), 1.50-1.20 (m, 5H), 0.95 (m, 2H).\",\n \"Example 35 tert-Butyl-[1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8).\",\n \"300 mg of a foam; ESI-MS [M+H+]: 520.39.Example 36 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butyl ester from example 35 with 15 ml 4nHCl in dioxane afforded 300 mg solid; ESI-MS [M+H+]: 464.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (m, 1H), 7.70 (m, 1H), 7.35, 7.25, 7.10 and 6.85 (each m, 2H), 4.75 (m 2H), 3.30, 3.15, 2.95, 2.70 (each m, 2H), 1.65 (m, 5H), 1.45 and 1.30 (each m, 2H).\",\n \"Example 37 tert-Butyl-{1-[2-({[4-(1H-imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example I under utilization of N-[4-(aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) as educt; chromatography on silica gel (CH2Cl2/CH3OH 4-15%) afforded 530 mg of a solid foam; ESI-MS [M+H+]: 510.35.Example 38 {1-[2-({[4-(1H-Imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Cleavage of the t-butyl ester from example 37 mit TFA and subsequent purification by means of chromatography on silica gel (CH2Cl2/CH3OH 10-15%+2% glacial acetic acid) afforded 570 mg; ESI-MS [M+H+]: 454.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.0 (broad, 1H), 7.30 and 7.25 (each m, 2H), 6.80 (broad, 1H), 6.70 (s, 2H), 4.40 and 4.20 (each broad, 1H), 4.75-4.25 (superimposed by H2O), 3.0 (m, 2H), 2.65 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.95 (m, 1H), 1.75 (m, 2H), 1.60 and 1.35 (each m, 1H), 1.20 (m, 4H), 0.95 (m, 2H).\",\n \"Example 39 tert-Butyl-(2-oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate Standard coupling of 2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) (0.5 g; 1.2 mmol) with N-(piperidine-4-ylmethyl)pyridine-2-amine (hydrochloride; accessibly from 29) in 15 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.\",\n \"Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 3-20%) afforded 0.44 g of a slightly yellow oil; ESI-MS [M+H+]: 590.35.Example 40 (2-Oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 31 with TFA afforded 385 mg; ESI-MS [M+H+]: 534.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (s, 1H), 7.90 (m, 2H), 7.55 (m, 1H), 7.35 (m, 1H), 7.25 (m, 2H), 7.05 and 6.80 (each m, 1H), 5.40-5.20 (m, 2H), 4.20 and 4.0 (each m, 1H), 3.25 (m, 2H), 2.95 (m, 1H), 2.80-2.60 (m, 3H), 2.30 (m, 1H), 2.25-2.05 (m, 2H), 1.80 and 1.65 (each m, 2H), 1.20-1.10 (m, 3H).\",\n \"Example 41 tert-Butyl-[1-({4-[({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino) carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Analogously to example 29 under utilization of trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11) as educt; chromatography of the raw product on silica gel (CH2Cl2/CH3OH 5-8%) afforded 350 mg of a bright yellow oil; ESI-MS [M+H+]: 643.45.Example 42 [1-({4-[({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example 31 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 300 mg; ESI-MS [M+H+]: 587.35.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 8.20 (m, 1H), 8.20 (s, 1H), 7.35-7.20 (m, 5H), 7.15 (m, 2H), 5.40 and 5.25 (each d, 1H), 3.15 (m, superimposed by H2O), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 2.05 and 1.80 (each m, 2H), 1.65, 1.30 and 1.15 (each m, 2H).\",\n \"Example 43 tert-Butyl-(1-{[4 ({4-[(1H-benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 430 mg were obtained analogously to example 29 under utilization of N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) and subsequent purification; ESI-MS [M+H+]: 629.45.Example 44 (1-{[4-({4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 43 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 340 mg; ESI-MS [M+H+]: 573.35.1H-NMR (360 MHz, d-DMSO) δ ppm: 9.20 (m, 1H), 7.95 (s, 1H), 7.45 (m, 1H), 7.40 (m, 2H), 7.30-7.15 (m, 5H), 5.35-5.10 (m, 2H), 4.45 (m, 1H), 4.0-3.25 (m, superimposed by H2O), 2.95 (m, 1H), 2.8-2.60 (m, 2H), 2.30 and 2.20 (each m, 1H), 2.0-1.75 (m, 3H), 1.70-1.50 (m, 2H), 1.20 (m, 3H).\",\n \"Example 45 tert-Butyl-{2-oxo-1-[(4-{[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 330 mg were obtained analogously to example 39 under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18) and purification; ESI-MS [M+H+]: 618.25.Example 46 {2-Oxo-1-[(4-{([{4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Cleavage of the t-butylesters of example 45 and purification by means of MPLC afforded 150 mg; ESI-MS [M+H+]: 562.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 8.20 (m, 1H), 8.0 (d, 1H), 7.50 (d, 1H), 7.30-7.20 (m, 4H), 7.10 and 6.95 (each s, 1H), 6.85 (m, 1H), 6.50 (m, 2H), 5.40 and 5.20 (each d, 1H), 4.55 and 4.35 (each m, 2H), 2.80-2.55 (m, 2H), 2.30 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).\",\n \"Example 47 tert-Butyl-[1-({4-[4-(1H-benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate a.)\",\n \"A mixture of 1.5 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.36 g 2-bromo-4-nitroacetophenone and 0.65 g KHCO3 in 30 ml dioxane were stirred for 12 h at RT.\",\n \"Workup analogously to building block 30c and mixing of the raw product with n-pentane afforded 2.1 g of a brown solid; ESI-MS [M+H+]: 494.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (s, 1H), 8.30 and 8.20 (each m, 2H), 7.60 and 7.35 (each m, 1H), 7.25 (m, 2H), 5.50 and 5.30 (each d, 1H), 2.70 (m, 2H), 2.30 (m, 1H), 2.20 (m, 3H), 1.65 (m, 1H), 1.25 (s, 9H).\",\n \"b.)\",\n \"Reduction of the nitro group analogously to example 30b (1.6 g; ESI-MS [M+H+]: 464.15) and build-up of the aminobenzimidazole as already described afforded after chromatography on silica gel (CH2Cl2/CH3OH 24%) 0.58 g of a slightly yellow foam; ESI-MS [M+H+]: 614.35.Example 48 [1-({4-[4-(1H-Benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example XXXXVII and purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 524.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.95 and 9.60 (each broad, 1H), 7.85-7.75 (m, 4H), 7.65 (m, 1H), 7.40-7.25 (m, 5H), 7.0 (m, 2H), 5.40 and 5.30 (each d, 1H), 3.50 (m, 1H), 2.75 (m, 1H), 2.45 (m, 2H), 2.20 (m, 2H), 1.70 (m, 1H).\",\n \"Example 49 tert-Butyl (1-{2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Ethyldiisopropylamine (0.42 mmol, 54.50 mg) and HATU (0.50 mmol, 190.11 mg) were added at 0° C. to a solution of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.42 mmol, 140.59 mg) in CH2Cl2 (10 ml), the mixture was stirred for 1 h at 0° C. and [4-(aminomethyl)phenyl]guanidine (bihydrochloride) (46) (0.42 mmol, 100 mg), ethyldiisopropylamine (0.63 mmol, 81.76 mg) added.\",\n \"The mixture was stirred for 1 h at 0° C. and overnight at RT.\",\n \"After evaporation, the residue was taken up in CH2Cl2/water, washed with watery NaHCO3—, 5%-citric acid- and finally with watery saturated NaCl-solution.\",\n \"Evaporation and chromatography on silica gel (CH2Cl2/CH3OH 0-100%) afforded 6.0 mg target product; ESI-MS [M+H+]: 426.1, 425.1.Example 50 {4-[({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]anilino}(imino)methaneamine (trifluoro acetate) tert-Butyl-({2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (example 49, 0.01 mmol, 6 mg) was dissolved in 5 ml CH2Cl2, TFA (1.25 mmol, 142.53 mg) added at 0° C. and stirred overnight at room temperature.\",\n \"After evaporation, the residue was taken up in CHCl3/water and the watery phase washed with CHCl3; evaporation of the watery phase affords 4.8 mg target product; ESI-MS [M+H+]=425.1.Example 51 tert-Butyl [1-(2-{[(6-amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49, under utilization of 2-ammonio-6-(ammonio methyl)pyridinium trichloride (33) (0.30 mmol, 69.76 mg) and following purification and subsequent purification (chromatography; CH2Cl2/MeOH 0-100%) afforded 120 mg of the target product; ESI-MS: [M+H+]=439.28, 383.36.Example 52 [1-(2-{([(6-Amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (hydrochloride) Cleavage of the t-butylester, chromatography of the raw product (CHCl3/MeOH 0-5%) and ester interchange with HCl in diethyl ether afforded 15.0 mg; the target product as hydrochloride; ESI-MS [M+H+]=383.1.Example 53 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 2 Drops of a solution of HCl in diethylether were added to N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (0.63 mmol, 196.10 mg) in methanol (40 mL) and stirred for 5 min.\",\n \"at RT.\",\n \"tert-Butyl.\",\n \"[2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) was added, NaBH3CN (3 mmol, 188.5 mg) portionwise added after 10 min.\",\n \"and the mixture stirred for 17 h at RT.\",\n \"After evaporation, the residue was taken up in ethyl acetate and washed with NaHCO3— (pH 8-9), saturated NaCl— (1×) and a 10% FeSO4-solution (2×).\",\n \"Chromatography (CHCl3/MeOH 0-5%) afforded 84 mg of the target product; ESI-MS [M+H+]=540.24, 270.75.Example 54 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester and chromatography (CHCl3/MeOH 0-100%) afforded 4 mg of the target product; ESI-MS [M+H+]=484.1.Example 55 tert-Butyl [1-(2-{[(4-{[(benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49 under utilization of N-[4-(aminomethyl)cyclohexyl]-N′-benzyl urea (34) (0.50 mmol, 130 mg) afforded 320 mg; ESI-MS [M+H+]=577.11.Example 56 [1-(2-{[(4-{[(Benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogous cleavage of the t-butylester afforded a raw product which after evaporation was distributed between ethyl acetate/water.\",\n \"The watery phase was then adjusted to pH 10 with 0.1n NaOH and extracted with ethyl acetate.\",\n \"It was adjusted then with 1nN HCl to pH4, extracted with CH2Cl2, washed and evaporated; 80 mg; ESI-MS [M+H+]=521.3.Example 57 tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 53 under utilization of N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (0.63 mmol, 183.47 mg) and tert-butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) afforded 50.0 mg of the target product; ESI-MS [M+H+]=520.41, 260.79.Example 58 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (example 57, 0.04 mmol, 20.00 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.\",\n \"Evaporation of the reaction mixture, chromatography (CHCl3/MeOH 0-100%) afforded 10 mg of the target product; ESI-MS [M+H+]=464.25.Example 59 tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 53 under utilization of ethyldiisopropylamine and EDCI*HCl starting from trans-N-[[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (0.63 mmol, 0.209) afforded 84 mg; ESI-MS [M+H+]=546.32, 273.79.Example 60 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (example 59, 0.01 mmol, 8 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.\",\n \"The reaction mixture was evaporated, the watery phase washed with a 3:1 mixture CHCl3/EtOH.\",\n \"Evaporation of the watery phase afforded 6.0 mg of the target product; ESI-MS [M+H+]=490.25.Example 61 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (1.70 mmol, 467.08 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).\",\n \"TFA (1.73 mmol, 197.72 mg) was added at 0° C. to the obtained raw product and it was stirred overnight at room temperature.\",\n \"After evaporation, the residue was distributed between ethyl acetate and water, the watery phase adjusted to pH 10 with 2n NaOH and extracted with ethyl acetate; purification by means of MPLC afforded 90 mg of the target product as TFA-salt; ESI-MS [M+K+]=570.2, 534.2, 532.2, 266.5, 101.1.Example 62 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (1.7 mmol, 433.10 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600.00 mg).\",\n \"Treatment of the raw product with TFA analogously to example 61 afforded 48 mg as a TFA-salt; ESI-MS: 514.2, 512.2, 101.2.Example 63 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)butyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)butane-1,4-diamine (trifluoro acetate) (9) (1.7 mmol, 541.1 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).\",\n \"Treatment of the raw product with TFA analogously to example 61 afforded 15 mg as a TFA-salt; ESI-MS: 536.2, 500.1, 498.2, 105.1, 101.2.Example 64 [7-Chloro-1-(2-{[5-(4,5-dihydro-1H-imidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(4,5-dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) (1.70 mmol, 351.43 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).\",\n \"Treatment of the raw product with TFA analogously to example 61 afforded 85 mg as a TFA-salt; ESI-MS: 464.15.Example 65 tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate Ethyldiisopropylamine (2.08 mmol, 268.41 mg) and EDCI*HCl (0.78 mmol, 150.44 mg) were added at 0° C. to a solution of [5-(2-t.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.46 mmol, 0.15 g) in CH3CN (45 ml).\",\n \"After 1 h at 0° C., 7-(4-aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoro acetate) (35) (0.46 mmol, 0.2 g) was added, stirred for 1 h at 0° C. and overnight at RT.\",\n \"Since by chromatography a purification could not be achieved, the obtained was reacted to the corresponding Boc-derivative according to standard methods.\",\n \"Chromatography (heptane/CH2Cl2 0-100%, CH2Cl2/MeOH 0-100%) afforded 15 mg of the target product (about 95% purity); ESI-MS [M+Na+]: 643.5, 622.5, [M+H+]=621.5.Example 66 7-[4-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-1,2,3,4-tetrahydro[1,8]naphthyridine (trifluoro acetate) tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate (example 65; 0.02 mmol, 15 mg) was dissolved in 2 ml CH2Cl2, TFA (0.53 mmol, 60.7 mg) added at 0° C. and stirred overnight at RT.\",\n \"Usual workup afforded 9.3 mg of the target product as TFA-salt; ESI-MS: 466.2, [M+H+]=465.2, 233.3.Example 67 N-{4-[({[5-(Carboxymethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]cyclohexyl})-1H-benzimidazole (acetate) Coupling analogously to example 49 starting from trans-N-[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (2.99 mmol, 948.78 mg) and [5-(2-tert-butoxy-2-oxoethyl) 7 chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (2.72 mmol, 1.00 g) afforded 760 mg raw product.\",\n \"Treatment of the raw product with TFA analogously to example 61 and purification of the raw product by means of MPLC afforded 400 mg; ESI-MS: [M+H+]=540.3, 538.25, 269.6, 101.1.Example 68 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 15 ml HCl in diethyl ether (saturated at 0° C.) were added to 300 mg {1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-1-benzazepine-5-yl}acetic acid in 30 ml ethyl alcohol and allowed to stand for 3 days at room temperature.\",\n \"The mixture was evaporated, the remaining residue purified by chromatography on silica gel (CH2Cl2/CH3OH 5%+1% glacial acetic acid) and lyophilized.\",\n \"230 mg; ESI-MS: [M+H+]=518.35.Example 69 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (1 g; 2.54 mmol) with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); after chromatography on silica gel (CH2Cl2/CH3OH 5-7%), 1.03 g were isolated as a white glass; ESI-MS: [M+H+]=620.35.Cleavage of the ester under utilization of TFA and subsequent purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 10%+2% glacial acetic acid) afforded 0.8 g of the target product; ESI-MS: [M+H+]=564.25.Example 70 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (0.5 g; 1.27 mmol) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); after chromatography on silica gel (CH2Cl2/CH3OH 3-5%) 0.67 g were isolated as a foam; ESI-MS: [M+H+]=614.35.Cleavage of the ester under utilization of TFA and lyophilization of the obtained product afforded 0.61 g; ESI-MS: [M+H+]=558.35.Example 80 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl}acetic acid 0.4 g (1.4 mmol) [1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.37 g (1.4 mmol) [4-(1H-benzimidazole-2-yl)cyclohexyl]methane amine (hydrochloride) (51) were reacted analogously to example I, the reaction product then purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.\",\n \"NH3 45/5/0.2) and then the methyl ester cleaved with 0.8 ml 1n NaOH in 8 ml dioxane at room temperature.\",\n \"After neutralization with 1n HCl, evaporation of the solvent, extraction with ethyl acetate and drying with Na2SO4, the raw product was purified by chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1).\",\n \"After lyophilization, 0.22 g remained as a white amorphous residue; ESI-MS: [M+H+]=487.Example 81 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.36 g (1.4 mmol) N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) were reacted.\",\n \"Purification of the raw product, alkaline saponification of the methyl ester and purification of the end stage was conducted analogously to example 80.0.3 g of a white amorphous residue; ESI-MS: [M+H+]=476.Example 82 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1H-benzazepine-1-yl]acetic acid Analogously to example I 0.49 (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) was reacted with 0.63 g (1.4 mmol) N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17).\",\n \"After purification, alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.3 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=488.Example 83 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo 2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) were reacted with 0.43 g trans-N-J[4-(aminomethyl)cyclohexyl]1H-benzimidazole-2-amine (dihydrochloride) (11).\",\n \"After purification of the ester (eluent: CH2Cl2/CH3OH/25% NH3 45/5/0.2), alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.18 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=502.Example 84 [5-(2-{[5-H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Analogously to example I 0.65 g (2 mmol) [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) were reacted with N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.\",\n \"NH3 45/5/0.2); ESI-MS: [M+H+]=535.Then the tert-butyl ester was cleaved with 4n HCl in dioxane, and after chromatographic purification 40 mg of a white amorphous powder were isolated; ESI-MS: [M+H+]=479.Example 85 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); ester stage ESI-MS: [M+H+]=561.Purification after ester cleavage was conducted by means of preparative thick-layer chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 12/3/1); 0.35 g of a white amorphous powder; ESI-MS: [M+H+]=505.Example 86 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); ester stage ESI-MS: [M+H+]=555; target product: 0.4 g of a white amorphous powder; ESI-MS: [M+H+]=499.Example 87 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid Analogously to example I 1.2 g (3.6 mmol) [5-(2-tert-butoxy-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) was reacted with 0.86 g (3.6 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.\",\n \"NH3 45/5/0.1); ESI-MS: [M+H+]=: 555.Subsequent cleavage of the tert-butylester with 4n HCl in dioxane afforded 0.8 g of a white amorphous powder; ESI-MS [M+H+]=499.Example 88 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid Preparation and purification analogously to example 87; ester stage ESI-MS: [M+H+]=561; 0.9 g of the target product were obtained as a white amorphous powder; ESI-MS [M+H+]=505.Example 89 (1-{2-[4-(1H-Benzimidazole-2-yl)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (acetate) Analogously to example I 0.5 g (1.5 mmol) [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) and 0.36 g (1.5 mmol) 2-(4-piperidinyl)-1H-benzimidazole (J. Heterocycl.\",\n \"Chem.\",\n \"1989, 26, 541) were reacted, purified by means of preparative thick-layer chromatography (eluent CH2Cl2/CH3OH/conc.\",\n \"NH3 45/5/0.2); ESI-MS: [M+H+]=517.Subsequent cleavage of the tert-butyl ester with 4n HCl in a mixture of dioxane/glacial acetic acid and chromatographic purification afforded 0.259 of a white amorphous powder; ESI-MS [M+H+]=: 461.Example 90 (1-{2-[[3-(1H-Benzimidazole-2-yl)propyl](methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (hydrochloride) Preparation analogously to example I by reaction with N-[3-(1H-benzimidazole-2-yl)propyl]-N-methylamine (WO 9849148); after chromatographic purification 0.69 were obtained as a white amorphous powder; ESI-MS [M+H+]=: 449.Example 91 (1-{2-[4-(1H-Benzimidazole-2-ylamino)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Preparation analogously to example I by reaction with N-piperidine-4-yl-1H-benzimidazole-2-amine (J. Med.\",\n \"Chem.\",\n \"1985, 28, 1925); after chromatographic purification 0.45 g were obtained as a white amorphous powder; ESI-MS [M+H+]=: 476.Example 92 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) and 0.33 g (1.4 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted and purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.\",\n \"NH3 45/5/0.2); ESI-MS: [M+H+]=512.Subsequent cleavage of the methyl ester with 1n NaOH in dioxane and chromatographic purification of the residue (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) afforded 0.15 g, isolated as a white amorphous powder; ESI-MS [M+H+]=498.Example 93 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=503; chromatographic purification after cleavage of the ester afforded 0.3 g of the target product as a white amorphous powder; ESI-MS [M+H+]=489.Example 94 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=492.Chromatographic purification after cleavage of the ester afforded 20 mg as a white amorphous powder ESI-MS [M+H+]=478.Example 95 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=518.Chromatographic purification after cleavage of the ester afforded 0.15 g as a white amorphous powder; ESI-MS [M+H+]=504.Example 96 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.6 g (2.4 mmol) N,N-Bis(2-oxo-3-oxazolidinyl)phosphoryl chloride were added to a solution of 0.8 g (1.6 mmol) of the acid of example XI, 0.15 g (3.2 mmol) ethyl alcohol, 0.2 g (1.6 mmol) 4-dimethylaminopyridine and 0.4 g (4 mmol) triethylamine in 60 ml CH2Cl2 and stirred overnight.\",\n \"After the reaction had completed 2 g glacial acetic acid and 3.7 g ethyl alcohol were added to the reaction solution and stirred for another 20 h at room temperature.\",\n \"For workup, 50 ml H2O were added to the mixture, the organic phase washed with 10% K2CO3-solution and H2O, dried over MgSO4 and evaporated.\",\n \"Chromatographic purification of the residue (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 15/5/1) afforded 0.35 g of a white amorphous powder; ESI-MS [M+H+]=532.Analogously to example 96 were prepared: Example 97 Cyclohexyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.26 g; ESI-MS [M+H+]=586.Example 98 Neopentyl{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.21 g; ESI-MS [M+H+]=574.Example 99 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation was carried out analogously to example I by reaction of [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4).\",\n \"After chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 45/5/0.3) the residue was dissolved in 70 ml CH2Cl2 and 5 ml CH3OH, washed with 5% K2CO3-solution and H2O, dried over Na2SO4 and evaporated to dryness.\",\n \"The amorphous residue was mixed with 25 ml CH3OH in heat.\",\n \"0.51 g of white crystals; Fp.\",\n \": 231° C. (decomposition); ESI-MS [M+H+]=554.Example 100 Cyclohexyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.64 g (1.28 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml cyclohexanol, 1.0 g HCl-gas introduced with stirring and the resulting yellow solution allowed to stand for 4 days at room temperature.\",\n \"Since an acid was still detectable by chromatography, it was heated for 7 h to 40-50° C. For workup, 100 ml diethylether were added, washed with K2CO3-solution and H2O, dried over Na2SO4 and the solvent—finally for removal of cyclohexanol under oil pump vacuum and at a bath temperature of 50° C.—distilled off.\",\n \"The residue was purified chromatographically (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45151410.3).\",\n \"0.659 of a slightly beige coloured powder; ESI-MS [M+H+]=580.Example 101 Ethyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-6-yl]acetate 0.619 (1.23 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml ethyl alcohol, 0.4 g HCl-gas introduced and allowed to stand for 4 days at room temperature.\",\n \"The ethyl alcohol was distilled off in vacuum, the residue taken up in ethyl acetate, washed with 5% NaHCO3- and NaCl-solution, dried over Na2SO4 and evaporated.\",\n \"After thick-layer chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% acetic acid 43/7/0.5) the eluate was diluted with some CH2Cl2 and for removal of the acetic acid washed with 50% NaHCO3-solution.\",\n \"After drying over Na2SO4 it was evaporated and the residue converted to an amorphous powder filterable with suction by means of diethylether/n-hexane; 0.55 g; ESI-MS [M+H+]=526.Example 102 1-{[(Cyclohexyloxy)carbonyl]oxy}ethyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.3 g K2CO3 were added at 3° C. to a solution of 0.6 g (1.2 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (add of example III) in 10 ml DMF with stirring, some crystals 18-crown-6, 0.4 g cyclohexyl-1-iodo ethylcarbonate (preparation from cyclohexyl-1-chloroethylcarbonate and NaI analogously to J. Antibiot.\",\n \"1987, 40 (1), 81-90) dissolved in 5 ml CH3CN were added dropwise and stirred for 20 min.\",\n \"After addition of 100 ml cold NaCl-solution it was extracted several times with ethyl acetate, the combined organic phases washed with NaCl-solution, dried over Na2SO4 and evaporated.\",\n \"The residue was purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/5/0.3), after evaporation of the solvent taken up in 50 ml CH2Cl2, washed with 5% NaHCO3-solution, dried over Na2SO4 and again evaporated.\",\n \"90 mg of a white amorphous powder; ESI-MS [M+H+]=668.Example 103 [(5R)-1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 1.0 g (3.0 mmol) of the left-rotating acid (building block 57) and 0.72 g (3.0 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted analogously to example I and the reaction product purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/4/0.3); ester stage ESI-MS [M+H+]=554.Cleavage of the tert-butyl ester with 4n HCl in dioxane afforded 0.82 g of a white amorphous powder; ESI-MS [M+H+]=498; [α]D20=−107.7° (K+-salt, c=1 in H2O).\",\n \"Example 104 [(5S)-1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 1.5 g of a amorphous powder; ESI-MS [M+H+]=554.Target product: 0.79 g of a white amorphous powder; ESI-MS [M+H+]=498.Example 105 {(5R)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example I from the left-rotating acid building block 57 and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).\",\n \"Ester stage: 0.9 g of a white amorphous powder; ESI-MS [M+H+]=560.Target product: 0.67 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=−104° (K+-salt, c=1 in H2O).\",\n \"Example 106 {(SS)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 0.72 g of a amorphous powder; ESI-MS [M+H+]=560.Target product: 0.56 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=+101.60 (K+-salt, c=1 in H2O).\",\n \"Example 107 [4-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl]acetic acid Analogously to example I 0.6 g (1.8 mmol) [8-(2-tert-butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) were reacted with 0.42 g (1.8 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) and the reaction product purified by thick-layer chromatography (eluent: CH2Cl2/methanol/conc.\",\n \"NH3 451510.2); ESI-MS [M+H+]=560.Cleavage of the t-butyl group with 4n HCl in dioxane afforded 0.34 g of a slightly yellowish powder; ESI-MS [M+H+]=504.Example 108 {4-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl}acetic acid Analogously to example 107.Ester stage: 100 mg of a white amorphous powder, ESI-MS [M+H+]=566.Target product: 98 mg of a white amorphous powder; ESI-MS [M+H+]=510.II.\",\n \"BIOLOGICAL EXAMPLES Example 1 Integrin αvβ3 Assay For the identification and assessment of integrin αvβ3 ligands, a test system was used which was based on competition between the natural integrin αvβ3 ligand vitronectin and the test substance for binding to solid phase-bound integrin αvβ3.Procedure Microtiter plates coated with 250 ng/ml of integrin αvβ3 in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturation with 1% powdered milk/assay buffer; 0.3 ml/well; 0.5 h/RT 3× washing with 0.05% Tween 20/assay buffer test substance in 0.1% powdered milk/assay buffer, 50 μl/well+0 μg/ml or 2 μg/ml of human vitronectin (Boehringer Ingelheim T007) in 0.1% powdered milk/assay buffer, 50 μl/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 1 μg/ml of anti human vitronectin antibody coupled to peroxidase (Kordia SAVN-APHRP) in 0.1% powdered milk/assay buffer; 0.1 ml/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 0.1 ml/well of peroxidase substrate stop reaction with 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Integrin αvβ3: Human placenta is solubilized with Nonidet and integrin αvβ3 affinity-purified on a GRGDSPK matrix (elution with EDTA).\",\n \"Impurities due to integrin αIIbβ3 and human serum albumin, and the detergent and EDTA are removed by anion-exchange chromatography.\",\n \"Assay buffer: 50 mM Tris pH 7.5; 100 mM NaCl; 1 mM CaCl2; 1 mM MgCl2; 10 μM MnCl2 Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M sodium acetate pH 4.9), then add 14.7 μl of 3% H2O2.Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the ligand at which 50% of the ligand is displaced).\",\n \"The compound from Example VII showed the best result here.\",\n \"Example 2 Integrin αIIbβ3 Assay The assay is based on competition between the natural integrin αIIbβ3 ligand fibrinogen and the test substance for binding to integrin αIIbβ3.Procedure Coat microtiter plates with 10 μg/ml of fibrinogen (Calbiochem 341578) in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturate with 1% BSA/PBS; 0.3 ml/well; 30 min/RT 3× washing with 0.05% Tween 20/PBS test substance in 0.1% BSA/PBS; 50 μl/well+200 μg/ml of integrin αIIbβ3 (Kordia) in 0.1% BSA/PBS; 50 μl/well; 2 to 4 h/RT 3× washing as above biotinylated anti-integrin αIIbβ3 antibody (Dianova CBL 130 B); 1:1000 in 0.1% BSA/PBS; 0.1 ml/well; 2 to 4 h/RT.\",\n \"3× washing as above streptavidin-peroxidase complex (B.M.\",\n \"1089153) 1:10000 in 0.1% BSA/PBS; 0.1 ml/well; 30 min/RT 3× washing as above 0.1 ml/well of peroxidase substrate stop reaction using 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M Na acetate pH 4.9), then add 14.7 μl of 3% H2O2 Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the antagonist at which 50% of the ligand is displaced).\",\n \"By comparison of the IC50 values in the integrin αIIbβ3 and integrin αvb3 assay, the selectivity of the substances can be determined.\",\n \"Example 3 CAM Assay The CAM (chorioallantoic membrane) assay serves as a generally recognized model for the assessment of the in vivo activity of integrin αvβ3 antagonists.\",\n \"It is based on the inhibition of angiogenesis and neovascularization of tumor tissue (Am.\",\n \"J. Pathol.\",\n \"1975, 79, 597-618; Cancer Res.\",\n \"1980, 40, 2300-2309; Nature 1987, 329, 630).\",\n \"The procedure is carried out analogously to the prior art.\",\n \"The growth of the chicken embryo blood vessels and of the transplanted tumor tissue can be readily monitored and assessed.\",\n \"Example 4 Rabbit Eye Assay In this in vivo model, the inhibition of angiogenesis and neovascularization in the presence of integrin αvβ3 antagonists can be monitored and assessed analogously to Example 3.The model is generally recognized and is based on the growth of rabbit blood vessels starting from the edge in the corner of the eye (Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"USA.\",\n \"1994, 91, 4082-4085; Science 1976, 193, 70-72).\",\n \"The procedure is carried out analogously to the prior art.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297202"}}},{"rowIdx":96,"cells":{"text":{"kind":"list like","value":[["Method, apparatus and system for building a compact language model for large vocabulary continous speech recognition (lvcsr) system","According to one aspect of the invention, a method is provided in which a set of probabilistic attributes in an N-gram language model is classified into a plurality of classes.","Each resultant class is clustered into a plurality of segments to build a code-book for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process.","A probabilistic attribute in each class is then represented by the centroid of the corresponding segment to which the respective probabilistic attribute belongs."],["1.A method comprising: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.","2.The method of claim 1 wherein a probabilistic attribute is represented by a first number of data bits before clustering.","3.The method of claim 2 wherein the respective probabilistic attribute is represented by an index value having a second number of data bits after clustering, the second number being smaller than the first number, the respective index value being used to reference a corresponding codeword in the respective codebook.","4.The method of claim 1 wherein a probabilistic attribute is a probability value associated with an n-gram node in the N-gram language model.","5.The method of claim 1 wherein a probabilistic attribute is a back-off weight associated with an n-gram node in the N-gram language model.","6.The method of claim 1 wherein the N-gram language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.","7.The method of claim 1 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.","8.The method of claim 7 wherein computing the total deviation includes: computing a deviation of each segment based upon the corresponding centroid of the respective segment; and summing the deviations of all segments to generate the total deviation.","9.The method of claim 7 wherein performing the set of operations includes: determining, for each segment in the plurality of segments, whether the new edge value equals the previous edge value; and indicating that the one or more optimization criteria are met if the new edge value equals the previous edge value.","10.The method of claim 7 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more optimization criteria are met if the predetermined number of iterations is reached.","11.The method of claim 7 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more optimization criteria are met if the changing rate in the total deviation is less than the predetermined threshold.","12.A method comprising: clustering a set of probabilistic values associated with each data class of a plurality of data classes of an N-gram language model into a plurality of clusters using a modified K-means clustering process which dynamically adjusts the size and centroid of each respective cluster during each iteration in the modified K-means clustering process until one or more predetermined conditions are satisfied, the clustering including: initializing the plurality of clusters such that all clusters have equivalent areas; setting the centroid of each resultant cluster such that the area of each respective cluster is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more predetermined conditions are satisfied, including: computing a total deviation of the clusters; replacing the edge of each cluster by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.","13.The method of claim 12 wherein performing the set of operations includes: determining, for each cluster in the plurality of clusters, whether the new edge value equals the previous edge value; and indicating that the one or more predetermined conditions are satisfied if the new edge value equals the previous edge value.","14.The method of claim 12 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more predetermined conditions are satisfied if the predetermined number of iterations is reached.","15.The method of claim 12 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more predetermined conditions are satisfied if the changing rate in the total deviation is less than the predetermined threshold.","16.An apparatus comprising: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in a language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria.","17.The apparatus of claim 16 wherein the language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding tp a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.","18.The apparatus of claim 16 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.","19.The apparatus of claim 18 wherein logic to compute the total deviation includes: logic to compute a deviation of each cluster based upon the corresponding centroid of the respective cluster; and logic to sum the deviations of all clusters to generate the total deviation.","20.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value.","21.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a predetermined number of iterations is reached.","22.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.","23.A system comprising: a feature extraction unit to convert an input signal representing an input speech into a set of feature vectors each representing a corresponding frame of the input signal; an acoustic model comprising a set of phonetic statistical models each representing a distinct phonetic unit; a language model including: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in the language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria; and a decoder coupled to the feature extraction unit, the acoustic model, and the language model, the decoder to recognize the input speech based, at least in part, upon the feature vectors, the acoustic model, and the language model.","24.The system of claim 23 wherein the language model is an tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.","25.The system of claim 23 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.","26.The system of claim 25 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value; logic to determine whether a predetermined number of iterations is reached; and logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.","27.A machine-readable medium comprising instructions which, when executed by a machine, cause the machine to perform operations including: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.","28.The machine-readable medium of claim 27 wherein the N-gram language model is a tri-gram language model and wherein thy plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigrarn back-off weights, and a fifth class corresponding to a set of trigram probability values.","29.The machine-readable medium of claim 27 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.","30.The machine-readable medium of claim 29 wherein performing the set of operations includes: determining, for each cluster, whether the new edge value equals the previous edge value; determining whether a predetermined number of iterations is reached; and determining whether a changing rate in the total deviation is less than a predetermined threshold."],[" BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.","In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.","In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).","In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.","In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.","The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.","Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.","N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).","In a typical LVCSR system, the size of the language model is usually very large.","For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.","Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.","One solution is to use a memory-map file format to access language model files in LVCSR system.","Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.","In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.","In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed."],[" BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.","1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.","2 is a diagram illustrating an HMM-based phone model; FIG.","3 is a diagram illustrating an N-gram language model; FIG.","4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.","5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.","6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.","7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.","8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.","9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.","detailed-description description=\"Detailed Description\" end=\"lead\"?"],["FIELD OF THE INVENTION The present invention relates to the field of speech recognition.","More specifically, the present invention relates to a method, apparatus, and system for building a compact language model for a large vocabulary continuous speech recognition (LVCSR) system.","BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.","In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.","In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).","In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.","In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.","The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.","Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.","N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).","In a typical LVCSR system, the size of the language model is usually very large.","For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.","Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.","One solution is to use a memory-map file format to access language model files in LVCSR system.","Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.","In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.","In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed.","BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.","1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.","2 is a diagram illustrating an HMM-based phone model; FIG.","3 is a diagram illustrating an N-gram language model; FIG.","4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.","5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.","6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.","7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.","8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.","9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.","DETAILED DESCRIPTION In the following detailed description numerous specific details are set forth in order to provide a thorough understanding of the present invention.","However, it will be appreciated by one skilled in the art that the present invention may be understood and practiced without these specific details.","In the discussion below, the teachings of the present invention are utilized to implement a method, apparatus, system, and machine-readable medium for building a compact language model for a speech recognition system.","In one embodiment, a set of N-gram probabilistic attributes that represent conditional probabilities of a set of words in an N-gram language model is classified into a plurality of classes based upon a relative order of the respective N-gram probabilistic attributes.","Each resultant class is then clustered into a plurality of segments or clusters to build a corresponding codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and the centroid of each segment during each iteration in the modified K-means clustering process.","In one embodiment, the modified K-means clustering process is performed until one or more optimization criteria are met.","In one embodiment, after the codebook for the respective class has been built, each N-gram probabilistic attribute in the respective class can be represented by the centroid of the corresponding segment to which the respective N-gram probabilistic attribute belongs.","For example, after a corresponding codebook has been built for a class of uni-gram probability values, a probability value associated with a given uni-gram node can be represented by a corresponding codeword in the respective codebook which can be referenced by a corresponding index value.","In one embodiment, an N-gram probabilistic attribute is represented by a first number of data bits prior to the construction of the corresponding codebook.","After the construction of the corresponding codebook, the respective N-gram probabilistic attribute can be represented by a corresponding index value having a second number of data bits which is smaller than the first number of data bits.","In one embodiment, an N-gram probabilistic attribute corresponds to an N-gram probability value or an N-gram back-off weight.","In one embodiment, the N-gram language model can be a tri-gram language model and the plurality of class can include a first class corresponding to a set of uni-gram probability values, a second class corresponding to a set of uni-gram back-off weights, a third class corresponding to a set of bi-gram probability values, a fourth class corresponding to a set of bi-gram back-off weights, and a fifth class corresponding to a set of tri-gram probability values.","In one embodiment, the clustering of each class of probabilistic attributes is performed as follows.","First, the plurality of segments are initialized such that all segments have equivalent areas.","The centroid for each segment is set such that the area of the respective segment is approximately equally split based on the corresponding centroid.","A set of clustering operations is then performed iteratively until one or more optimization criteria are met to create a corresponding codebook for the respective class of probabilistic attributes.","During each iteration, a total deviation of the plurality of segments is computed.","The edge of each segment is then replaced by the arithmetic average of the nearest two centroids.","A new centroid is then set for each resultant segment such that each resultant segment is approximately equally split by the corresponding centroid.","A new total deviation of the resultant segments is then computed.","In one embodiment, the one or more optimization criteria are met (e.g., the total deviation becomes steady) if one of the following conditions is satisfied: a) For each segment, the new edge value equals the previous edge value.","In other words, if the arithmetic average of the nearest two centroids equals the previous edge value then the total deviation is considered steady; b) A predetermined number of iterations is reached; or c) A change of total deviation is less than a predetermined threshold.","The teachings of the present invention are applicable to any method, apparatus, and system for speech recognition that employs N-gram language models.","However, the present invention is not limited to N-gram language models in speech recognition systems and can be applied to the other types of language modeling in speech recognition.","In addition, the present invention can also be applied to data modeling in other fields or disciplines including, but not limited to, image processing, signal processing, geometric modeling, computer-aided-design (CAD), computer-aided-manufacturing (CAM), etc.","FIG.","1 illustrates a block diagram of one embodiment of a speech recognition system 100 according to the teachings of the present invention.","The system 100, as shown in FIG.","1, includes an analog to digital converter (A/D) 110, a feature extractor or spectral analysis unit 120, a decoder 130, an acoustic model 140, and a language model 150.An input signal 105 representing the input speech is first digitized using the A/D 110.In one embodiment, the digital signal is then sliced up into frames typically of 10, 15, or 20 ms. Each frame of the signal is then converted into a corresponding feature vector which is used to characterize the spectral properties of the input signal.","In one embodiment, the feature vector is multi-dimensional and has a plurality of feature components.","The feature vectors generated by the feature extractor unit 120 are then inputted into the decoder 130 which determines the sentence or sequence of words that has the highest probability given the acoustic events characterized by the feature vectors, based upon the acoustic model 140 and the language model 150.The language model 150, in one embodiment, is an N-gram language model which includes a plurality of codebooks.","Each codebook corresponds to a class of probabilistic attributes (e.g., uni-gram probability values, uni-gram back-off weights, bi-gram probability values, etc.)","that are used to represent various conditional probabilities of a set of words.","For example, one of the codebook may correspond to the uni-gram probability class, another codebook may correspond to the uni-gram back-off weight class, and so on.","The language model 150 and the construction of the corresponding codebooks are described in greater detail below.","FIG.","2 illustrates a diagram of one embodiment of an HMM-based phone model structure used in the acoustic model 140 to model a phonetic unit (e.g., a phoneme or a subword unit, etc.).","Each individual phonetic unit is represented or modeled by a corresponding HM.","As shown in FIG.","2, an “MM has a set of sequence of states (1-5) that are connected by a set of transition probabilities (a12, a23, a34, a45), and a set of observation probabilities or likelihoods (b2 (o1),b2(o2),b3(o3),b4(o4),b4(o5),b4(o6)).","Each transition probability aij represents the probability of transitioning from a state i to a state j.","Each observation probability or distribution bi(oj) represents the probability of an observation vector oj being generated from a state i.","Thus, the transition probabilities model the durational variability in speech and the output probabilities model the spectral variability.","Accordingly, the set of states, the set of transition probabilities and the set of output probabilities are the parameters that are used to define an HMM.","The HMM shown in FIG.","2 has a left-right topology.","In many modern speech recognition systems, each state output distribution or observation probability function is modeled by a multivariate mixture Gaussian as follows: b j ( o t ) = ∑ k = 1 M c jk N ( o t , m jk , V jk ) where cjk is the weight of mixture component k in state j and N(oi, mjk, Vjk) denotes a multivariate Gaussian of mean mjk and covanance Vjk for the kth mixture component in state j.","FIG.","3 shows a structure diagram of one embodiment of an N-gram language model.","As described above, the purpose of a language model is to provide a mechanism for estimating the probability of some word, wk, in an utterance given the preceding words, W1k-1=w1 .",".",".","wk1.An N-gram language model assumes that wk depends only on the preceding N-1 words, that is P ( w k ❘ W 1 k - 1 ) = P ( w k ❘ W k - n + 1 k - 1 ) .","Because of the very large amount of models and associated data that need to be stored and processed, N is usually limited to two (for a bi-gram or 2-gram language model) or three (for a tri-gram or 3-gram language model).","A back-off mechanism can be used to compute a conditional probability of a given word when some N-gram context (e.g., a tri-gram or a bi-gram) does not exist or cannot be found for the given word.","For example, for given words “a” “b” “c”, there may be no bi-gram probability for “b→a” or tri-gram probability for “c→b→a” in certain contexts.","In this case, a back-off mechanism is used to compute the bi-gram or tri-gram probability for the given word “a”.","For example, for a tri-gram of words “c→b→a” (meaning word “a” preceded by word “b” preceded by word “c”), the corresponding tri-gram and bi-gram probabilities of this word sequence can be computed as follows using a back-off mechanism: P ( a / b , c ) = { P ( a / b , c ) , if P ( a / b , c ) exists , P ( a / b ) × w ( b , c ) , if w ( b , c ) exists , P ( a / b ) otherwise } P ( a / b ) = { P ( a / b ) , if P ( a / b ) exists , P ( a ) × w ( b ) otherwise } where: P(a/b,c) represents the tri-gram probability of the tri-gram c→b→a; P(a/b) represents the bi-gram probability of the bi-gram b→a; P(a) represents the uni-gram probability of word a; w(b,c) represents the back-off weight for the bi-gram c→b; and w(b) represents the back-off weight for the word b.","In various LVCSR systems, language model look-head techniques can be used to speed up the search in the corresponding N-gram language model.","The look-head techniques are used to incorporate the language model probabilities as early as possible in the pruning process of the beam search.","In general, look-head probabilities are pre-computed and stored in certain file formats or data structures.","Referring to FIG.","3, an N-gram language model in a typical LVSR system can be logically represented as a set of nodes 300 which includes a set of uni-gram nodes 310, a set of bi-gram nodes 320, and so on.","The uni-gram nodes are used to represent single words and their associated probability data (e.g., probability value and back-off weight).","The bi-gram nodes are used to represent word pairs and their associated probability data, etc.","In one embodiment, the second words in the bi-gram nodes that follow the same first word in a uni-gram node can be grouped together and point them to the corresponding first word in the uni-gram node.","Thus, only the second words and the corresponding bi-gram probability data need to be stored in the bi-gram nodes.","Tri-gram nodes can be structured similarly.","For example, all the third words in the tri-gram nodes that follow the same bi-gram node can be grouped together and point to the corresponding bi-gram node.","Thus, only the third words and their associated probability data need to be stored in the tri-gram nodes.","As shown in FIG.","3, each uni-gram node can be linked to its corresponding bi-gram nodes via a pointer.","Similarly, each bi-gram node can be linked to its corresponding tri-gram nodes via a pointer, and so on.","In other words, each uni-gram node points to the head of a set of bi-gram nodes whose words follow the word stored in the respective uni-gram node, each bi-gram node points to the head of a set of tri-gram nodes, and so on.","Each n-gram node (e.g., uni-gram node, bi-gram node, tri-gram node, etc.)","typically has the following components: the word identity of the word that the respective node represents (which can be the word itself or a word index corresponding to the word), an n-gram probability value, a back-off weight, and a pointer to the head of the corresponding (n+1)-gram nodes.","For example, a uni-gram node typically contains an index value of a particular word that is represented by the respective uni-gram node, a uni-gram probability value, a uni-gram back-off weight, and a pointer to a corresponding set of bi-gram nodes that are linked with the respective uni-gram node.","In one embodiment, as described above, each n-gram node only needs to store the last word of the corresponding n-gram.","For example, a bi-gram node only needs to store the second word of the corresponding bi-gram that the respective bi-gram node represents, a tri-gram node only needs to store the third word of the corresponding tri-gram that the respective tri-gram node represents, and so on.","In general, for computational efficiency and to avoid numerical underflow, probability values and back-off weights associated with the n-gram nodes can be represented in logarithmic space.","A n-gram node may be represented by various data structures depending upon the particular implementations and applications of the language model.","For example, an n-gram node may be represented by a corresponding data structure as shown below: typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID is an index of the word W that this node represents; P and W are the corresponding probability value and back-off weight, respectively; PTR is a pointer that points to the (n+1)-gram area linked by this node.","If there is no (n+1)-gram linked with this node, PTR can be set to a pre-defined value.","In this example, the word index WID is an unsigned 16-bit value (e.g., unsigned short data type); the probability value P and the back-off weight are 32-bit float values (e.g., 32-bit float data type); and the PTR is a 32-bit integer value.","The whole size of the data structure in this example is therefore 112 bits or 14 bytes.","An example of a data structure that can be used to represent a look-head node is shown below: typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; In this example, the NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.","It can be understood from the above description that a typical language model for a LVCSR system can contain a very large number of nodes each of which may contain corresponding probability value and/or backup weight that are represented by very large number of data bits (e.g., probability value and back-off weight are 32-bit float values in the above examples).","Thus, a language model for a typical LVCSR requires a very large amount of memory space.","As discussed above and described in more details below, the present invention provides a method and mechanism for reducing the size of the language model and the access time while maintaining a high level of recognition accuracy.","According to the teachings of the present invention, a method is introduced and described in detail herein to reduce the size of a language model in a LVCSR system.","In one embodiment, the probability values and back-off weights (also referred to as probabilistic attributes herein) associated with the respective n-gram nodes in the respective language model are quantized or clustered by a modified K-means clustering algorithm to compress the size of the respective language model.","In one embodiment, the probability values or back-off weights are divided or grouped into separate classes or groups based on the relative n-th order of the corresponding n-gram nodes and a modified K-means clustering algorithm is applied to each class to cluster the probability values or back-off weights in each class to build a corresponding codebook for the respective class.","For example, uni-gram probability values associated with uni-gram nodes are grouped into one class, uni-gram back-off weights are grouped into another class, bi-gram probability values are grouped into yet another class, and so on.","Thus, for a tri-gram language model (which includes uni-gram nodes, bi-gram nodes, and tri-gram nodes), there can be five separate classes (also referred to as data classes herein) that can be quantized or clustered to reduce the size of the language model.","These classes include a uni-gram probability class, a uni-gram back-off weight class, a bi-gram probability class, a bi-gram back-off weight class, and a tri-gram probability class.","For each class, the field of probability values (also referred to as the range of probability values) is quantized or clustered into a plurality of clusters or segments using a modified K-means clustering algorithm described in more detail below.","A centroid is defined for each cluster or segment.","Thus, a separate codebook can be constructed for each class of probability values or each class of back-off weights.","After quantization or clustering, a given probability value or back-off weight can be represented by the centroid (also called codeword) of the corresponding segment or cluster to which the respective probability value or back-off weight belongs.","By quantizing or clustering the field of probability values in each class, the probability values or back-off weights (which originally were represented by large-size data types such as 32-bit float type) can now be represented by smaller-size index values such as 8-bit char type that points to the corresponding codeword (centroid) in the corresponding codebook.","In one embodiment, the field of probability value for each class is divided into 256 segments or clusters using the modified K-means clustering process described in greater details below.","As such, the corresponding codebook will have 256 codewords.","Each codeword can be referenced by an 8-bit index value.","Thus, assuming that the original probability values or back-off weights use 32-bit float type, these probability values or back-off weights can now be replaced by 8-bit index values which are used to reference the corresponding codewords in the codebook.","It should be appreciated and understood by one skilled in the art that the teachings of the present invention are not limited to any particular number of segments or clusters.","Similarly, the teachings of the present invention are not limited to any particular data types that are used to represent the probability values and back-off weights before and after the quantization.","The number of segments or clusters for each class (hence the size of the corresponding codebook) and the data types that are used to represent the probability values, the back-off weights, or the indices to the codebook can vary depending upon the particular applications and implementations of the present invention.","FIG.","4 illustrates a flow diagram of one embodiment of a method 400 according to the teachings of the present invention.","At block 410, the probabilistic attributes (e.g., the probability values and back-off weights) in a N-gram language model are classified or grouped into a plurality of separate classes.","For example, in a tri-gram language model that includes uni-gram nodes, bi-gram nodes, and tri-gram nodes, the probabilistic attributes can be classified into five separate classes as follows: uni-gram probability, uni-gram back-off weight, bi-gram probability, bi-gram back-off weight, and tri-gram probability.","At block 420, the field of value for each class is clustered or quantized using a modified K-means clustering process to build a corresponding codebook for the respective class.","The modified K-means clustering process is described in more details below.","At block 430, each probabilistic attribute (e.g., a probability value or a back-off weight) can be represented by the corresponding codeword in the respective codebook.","In one embodiment, as described herein, after quantization or clustering, the probability value or back-off weight for any given node in the language model can then be replaced by a corresponding index value that is used to reference the corresponding codeword.","For example, assuming that the original probability value or back-off weight for a given node was 32-bit data type (e.g., 32-bit float value) and that there are 256 codewords in the corresponding codebook, then after quantization or clustering, the respective probability value or back-off weight for the given node can be represented by an 8-bit index value which is used to reference or point to the corresponding codeword in the codebook.","FIG.","5 shows a block diagram of one embodiment of a method according to teachings of the present invention.","As shown in FIG.","5, the probabilistic attributes (e.g., probability values and back-off weights) 510 in an N-gram language model are classified or grouped into separate classes based on the n-gram order of the respective nodes.","In this example, the classes include: uni-gram probability 520A, uni-gram back-off weight 520B, bi-gram probability 520C, bi-gram back-off weight 520D, tri-gram probability 520E, etc.","Each class is then clustered or quantized using a modified K-means clustering process to create a corresponding codebook (e.g., 530A-E) for each respective class.","As described herein, the size of the codebook for each class may or may not be the same, depending on the particular applications and implementations of the present invention.","Each probabilistic attribute (e.g., a uni-gram probability value associated with a uni-gram node) can then be represented by a corresponding codeword in the respective codebook (e.g., the codebook for the uni-gram probability class).","FIG.","6 illustrates a flow diagram of one embodiment of a modified K-means clustering process 600 to create a corresponding codebook for a probabilistic class (e.g., uni-gram probability class or bi-gram probability class, etc.","), according to the teachings of the present invention.","The process starts at block 601 and proceeds to block 605 to initialize the codebook as follows: At block 605: initialization of the process is performed by clustering the field of values for the respective class into a plurality of segments (e.g., n segments in this example) and initialize the segments such that all the segments have equivalent areas.","The segments are also called clusters herein.","The centroid of each segment is then set such that the respective segment is about equally split (i.e., the segment can be split into two equivalent sub-areas by the corresponding centroid).","As shown in FIGS.","7 and 8, the segment initialization is performed according to the following conditions: E 1 = min ( x ) , E 2 … E n , E n + 1 = max ( x ) , C 1 , C 2 … C n - 1 , C n ∫ E 1 E 2 P ( x ) ⅆ x = ∫ E 2 E 3 P ( x ) ⅆ x = … = ∫ E n - 1 E n P ( x ) ⅆ x = ∫ E n E n + 1 P ( x ) ⅆ x ∫ E i C i P ( x ) ⅆ x = ∫ C i E i + 1 P ( x ) ⅆ x i = 1 , 2 … n Where: x is a possible probability value in the respective class; P(x) is the probability that x occurs; the i segment is defined as [Ei, Ei+1; and Ci is defined as the centroid of the i segment.","At this point, there are n segments or clusters which have equivalent areas and each i segment has a corresponding centroid Ci which can split the respective segment into two equivalent sub-areas.","Set the number of iterations R=0 At block 610: compute the total deviation Dev as sum of the deviation of each segment Devi as follows: Dev=ΣDevi.","for the i segment, Devi is defined as ∫ E i E i + 1 P ( x ) * abs ( x - C i ) ⅆ x , (illustrated in FIG.","8) At block 615: replace the edge Ei of each segment by the arithmetic average of the nearest two centroids as shown below: Ei=(Ci-1+Ci)/2 (illustrated in FIG.","9) set the new centroid Ci for each resultant segment compute the new total deviation Dev increment the number of iterations R=R+1 At block 625: if the total deviation is not steady, then the process 600 loops back to block 615.Otherwise, the corresponding codebook is obtained at block 635 and the process 600 proceeds to ends at block 691.In one embodiment, the total deviation is considered steady if one of the following conditions (also called optimization criteria) is met: a.","For each i, Ei=Ei (the new edge value is the same as the previous edge value); b.","The number of iterations performed reaches a predetermined number (R=Rmax); or c. The change of total deviation is less than a pre-determined threshold value Δth, |Dev−Dev|/Dev<Δth Significant reduction in memory requirement and access time for a language model can be attained according to the teachings of the present invention.","For example, assuming that before compression or quantization, the following data structures were used for n-gram nodes and look-head nodes, respectively: n-gram node data structure (before compression): typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (32-bit float value) and W (32-bit float value) are the corresponding probability value and back-off weight, respectively, PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.","If there is no (n+1)-gram linked with this node, PTK is a pre-defined value.","Thus the whole size of an-gram data structure in this example is 112 bits or 14 bytes.","look-head node data structure (before compression): typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; where: NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.","Thus the whole size of a look-head data structure is 64 bits.","After compression, an n-gram node and a look-head node can be represented by the following data structures, respectively.","It is assumed in this example that 8-bit codebooks are used (e.g., each codebook has 256 codewords): n-gram node data structure (after compression): typedef struct _asNGram{ unsigned short WID; unsigned char P; int PTR; unsigned char W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (8-bit unsigned char type) is the index value used to reference the corresponding quantized probability value (codeword) in the corresponding codebook; W (8-bit unsigned char type) is the index value used to reference the corresponding quantized back-off weight (codeword) in the corresponding codebook; PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.","If there is no (n+1)-gram linked with this node, PTR is a pre-defined value.","Thus the whole size of an n-gram data structure after compression in this example is 64 bits or 8 bytes.","look-head node data structure (after compression): typedef struct _asTreeBi-gramt{ int NODENO; unsigned P; } asTreeBi-gram; where: NODENO is now an 8-bit index value that is used to reference the corresponding quantized probability value in the corresponding codebook.","Thus the size of the look-head data structure after compression is 40 bits.","From the above example, it can be seen that the size of the language model can be significantly reduced according to the teachings of the present invention.","In this example, the size of each n-gram node is reduced from 112 bits to 64 bits and the size of each look-head node is reduced from 64 bits to 40 bits.","For a language model in a typical LVCSR system which usually has a very large number of nodes, the reduction in memory requirement can be very substantial.","The additional memory requirement for the codebooks is minimal or insignificant compared to the reduction in the memory requirement for the whole language model.","For example, the memory requirement for an 8-bit codebook which has 256 codewords and each codeword is a 32-bit float value is only: 256(codewords)*32(bits/codeword)=8192 bits Tables 1 and 2 below show some experimental results based upon various experiments conducted according to the teachings of the present invention.","These experiments were conducted on a 51K Mandarin LVCSR system and using a testing machine such as a double-PIII866 with 512M memory and 256K L2 Cache.","Table 1 illustrates the improvements with respect to memory requirement and decoding speed using 8-bit codebooks (e.g., each codebook has 256 codewords).","It can be seen from table 1 that the memory requirement and decoding speed have been reduced after compression while the increase in the word error rate (WER) of the system after compression is minimal or insignificant.","The XRT notation in table 1 is used to mean times of real time.","TABLE 1 System using BaseLine modified K-Means System (before clustering (after compression) compression) WER 10.0% 10.1% Tri-gram file size 130M 75M Treebi-gram file size 109M 68M MapMode XRT 1.18XRT 1.08XRT Loading directly mode 1.11XRT 1.02XRT XRT As shown in table 1, the experiments and comparisons were done to show the improvements with respect to two different embodiments: one embodiment using memory-map file format for the language model files and the other embodiment loading the language model files directly into memory.","After quantization the size of the language model file is reduced by 40% from 239M(130M+109M) to 143M (75M+68M).","In addition, the decoding speed has been improved by 8% from 1.18×RT to 1.08×RT using memory-map file format while WER only increases from 10.0% to 10.1%.","Furthermore, if language model files are loaded directly to memory, the decoding speed can be improved by another 6%.","Table 2 shows the experimental results using smaller-size codebooks.","In this example, 6-bit codebooks and 4bit codebooks were used.","TABLE 2 System using modified K-means clustering (after compression) 6-bit 4-bit codebooks codebooks WER 10.1% 12.8% MapMode XRT 1.07XRT 1.03XRT Loading directly 1.01XRT 0.98XRT mode XRT The invention has been described in conjunction with the preferred embodiment.","It is evident that numerous alternatives, modifications, variations and uses will be apparent to those skilled in the art in light of the foregoing description."]],"string":"[\n [\n \"Method, apparatus and system for building a compact language model for large vocabulary continous speech recognition (lvcsr) system\",\n \"According to one aspect of the invention, a method is provided in which a set of probabilistic attributes in an N-gram language model is classified into a plurality of classes.\",\n \"Each resultant class is clustered into a plurality of segments to build a code-book for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process.\",\n \"A probabilistic attribute in each class is then represented by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.\"\n ],\n [\n \"1.A method comprising: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.\",\n \"2.The method of claim 1 wherein a probabilistic attribute is represented by a first number of data bits before clustering.\",\n \"3.The method of claim 2 wherein the respective probabilistic attribute is represented by an index value having a second number of data bits after clustering, the second number being smaller than the first number, the respective index value being used to reference a corresponding codeword in the respective codebook.\",\n \"4.The method of claim 1 wherein a probabilistic attribute is a probability value associated with an n-gram node in the N-gram language model.\",\n \"5.The method of claim 1 wherein a probabilistic attribute is a back-off weight associated with an n-gram node in the N-gram language model.\",\n \"6.The method of claim 1 wherein the N-gram language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.\",\n \"7.The method of claim 1 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.\",\n \"8.The method of claim 7 wherein computing the total deviation includes: computing a deviation of each segment based upon the corresponding centroid of the respective segment; and summing the deviations of all segments to generate the total deviation.\",\n \"9.The method of claim 7 wherein performing the set of operations includes: determining, for each segment in the plurality of segments, whether the new edge value equals the previous edge value; and indicating that the one or more optimization criteria are met if the new edge value equals the previous edge value.\",\n \"10.The method of claim 7 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more optimization criteria are met if the predetermined number of iterations is reached.\",\n \"11.The method of claim 7 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more optimization criteria are met if the changing rate in the total deviation is less than the predetermined threshold.\",\n \"12.A method comprising: clustering a set of probabilistic values associated with each data class of a plurality of data classes of an N-gram language model into a plurality of clusters using a modified K-means clustering process which dynamically adjusts the size and centroid of each respective cluster during each iteration in the modified K-means clustering process until one or more predetermined conditions are satisfied, the clustering including: initializing the plurality of clusters such that all clusters have equivalent areas; setting the centroid of each resultant cluster such that the area of each respective cluster is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more predetermined conditions are satisfied, including: computing a total deviation of the clusters; replacing the edge of each cluster by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.\",\n \"13.The method of claim 12 wherein performing the set of operations includes: determining, for each cluster in the plurality of clusters, whether the new edge value equals the previous edge value; and indicating that the one or more predetermined conditions are satisfied if the new edge value equals the previous edge value.\",\n \"14.The method of claim 12 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more predetermined conditions are satisfied if the predetermined number of iterations is reached.\",\n \"15.The method of claim 12 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more predetermined conditions are satisfied if the changing rate in the total deviation is less than the predetermined threshold.\",\n \"16.An apparatus comprising: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in a language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria.\",\n \"17.The apparatus of claim 16 wherein the language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding tp a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.\",\n \"18.The apparatus of claim 16 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.\",\n \"19.The apparatus of claim 18 wherein logic to compute the total deviation includes: logic to compute a deviation of each cluster based upon the corresponding centroid of the respective cluster; and logic to sum the deviations of all clusters to generate the total deviation.\",\n \"20.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value.\",\n \"21.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a predetermined number of iterations is reached.\",\n \"22.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.\",\n \"23.A system comprising: a feature extraction unit to convert an input signal representing an input speech into a set of feature vectors each representing a corresponding frame of the input signal; an acoustic model comprising a set of phonetic statistical models each representing a distinct phonetic unit; a language model including: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in the language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria; and a decoder coupled to the feature extraction unit, the acoustic model, and the language model, the decoder to recognize the input speech based, at least in part, upon the feature vectors, the acoustic model, and the language model.\",\n \"24.The system of claim 23 wherein the language model is an tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.\",\n \"25.The system of claim 23 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.\",\n \"26.The system of claim 25 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value; logic to determine whether a predetermined number of iterations is reached; and logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.\",\n \"27.A machine-readable medium comprising instructions which, when executed by a machine, cause the machine to perform operations including: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.\",\n \"28.The machine-readable medium of claim 27 wherein the N-gram language model is a tri-gram language model and wherein thy plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigrarn back-off weights, and a fifth class corresponding to a set of trigram probability values.\",\n \"29.The machine-readable medium of claim 27 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.\",\n \"30.The machine-readable medium of claim 29 wherein performing the set of operations includes: determining, for each cluster, whether the new edge value equals the previous edge value; determining whether a predetermined number of iterations is reached; and determining whether a changing rate in the total deviation is less than a predetermined threshold.\"\n ],\n [\n \" BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.\",\n \"In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.\",\n \"In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).\",\n \"In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.\",\n \"In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.\",\n \"The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.\",\n \"Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.\",\n \"N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).\",\n \"In a typical LVCSR system, the size of the language model is usually very large.\",\n \"For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.\",\n \"Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.\",\n \"One solution is to use a memory-map file format to access language model files in LVCSR system.\",\n \"Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.\",\n \"In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.\",\n \"In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed.\"\n ],\n [\n \" BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.\",\n \"1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.\",\n \"2 is a diagram illustrating an HMM-based phone model; FIG.\",\n \"3 is a diagram illustrating an N-gram language model; FIG.\",\n \"4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.\",\n \"5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.\",\n \"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.\",\n \"7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.\",\n \"8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.\",\n \"9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.\",\n \"detailed-description description=\\\"Detailed Description\\\" end=\\\"lead\\\"?\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention relates to the field of speech recognition.\",\n \"More specifically, the present invention relates to a method, apparatus, and system for building a compact language model for a large vocabulary continuous speech recognition (LVCSR) system.\",\n \"BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.\",\n \"In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.\",\n \"In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).\",\n \"In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.\",\n \"In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.\",\n \"The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.\",\n \"Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.\",\n \"N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).\",\n \"In a typical LVCSR system, the size of the language model is usually very large.\",\n \"For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.\",\n \"Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.\",\n \"One solution is to use a memory-map file format to access language model files in LVCSR system.\",\n \"Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.\",\n \"In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.\",\n \"In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.\",\n \"1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.\",\n \"2 is a diagram illustrating an HMM-based phone model; FIG.\",\n \"3 is a diagram illustrating an N-gram language model; FIG.\",\n \"4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.\",\n \"5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.\",\n \"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.\",\n \"7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.\",\n \"8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.\",\n \"9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.\",\n \"DETAILED DESCRIPTION In the following detailed description numerous specific details are set forth in order to provide a thorough understanding of the present invention.\",\n \"However, it will be appreciated by one skilled in the art that the present invention may be understood and practiced without these specific details.\",\n \"In the discussion below, the teachings of the present invention are utilized to implement a method, apparatus, system, and machine-readable medium for building a compact language model for a speech recognition system.\",\n \"In one embodiment, a set of N-gram probabilistic attributes that represent conditional probabilities of a set of words in an N-gram language model is classified into a plurality of classes based upon a relative order of the respective N-gram probabilistic attributes.\",\n \"Each resultant class is then clustered into a plurality of segments or clusters to build a corresponding codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and the centroid of each segment during each iteration in the modified K-means clustering process.\",\n \"In one embodiment, the modified K-means clustering process is performed until one or more optimization criteria are met.\",\n \"In one embodiment, after the codebook for the respective class has been built, each N-gram probabilistic attribute in the respective class can be represented by the centroid of the corresponding segment to which the respective N-gram probabilistic attribute belongs.\",\n \"For example, after a corresponding codebook has been built for a class of uni-gram probability values, a probability value associated with a given uni-gram node can be represented by a corresponding codeword in the respective codebook which can be referenced by a corresponding index value.\",\n \"In one embodiment, an N-gram probabilistic attribute is represented by a first number of data bits prior to the construction of the corresponding codebook.\",\n \"After the construction of the corresponding codebook, the respective N-gram probabilistic attribute can be represented by a corresponding index value having a second number of data bits which is smaller than the first number of data bits.\",\n \"In one embodiment, an N-gram probabilistic attribute corresponds to an N-gram probability value or an N-gram back-off weight.\",\n \"In one embodiment, the N-gram language model can be a tri-gram language model and the plurality of class can include a first class corresponding to a set of uni-gram probability values, a second class corresponding to a set of uni-gram back-off weights, a third class corresponding to a set of bi-gram probability values, a fourth class corresponding to a set of bi-gram back-off weights, and a fifth class corresponding to a set of tri-gram probability values.\",\n \"In one embodiment, the clustering of each class of probabilistic attributes is performed as follows.\",\n \"First, the plurality of segments are initialized such that all segments have equivalent areas.\",\n \"The centroid for each segment is set such that the area of the respective segment is approximately equally split based on the corresponding centroid.\",\n \"A set of clustering operations is then performed iteratively until one or more optimization criteria are met to create a corresponding codebook for the respective class of probabilistic attributes.\",\n \"During each iteration, a total deviation of the plurality of segments is computed.\",\n \"The edge of each segment is then replaced by the arithmetic average of the nearest two centroids.\",\n \"A new centroid is then set for each resultant segment such that each resultant segment is approximately equally split by the corresponding centroid.\",\n \"A new total deviation of the resultant segments is then computed.\",\n \"In one embodiment, the one or more optimization criteria are met (e.g., the total deviation becomes steady) if one of the following conditions is satisfied: a) For each segment, the new edge value equals the previous edge value.\",\n \"In other words, if the arithmetic average of the nearest two centroids equals the previous edge value then the total deviation is considered steady; b) A predetermined number of iterations is reached; or c) A change of total deviation is less than a predetermined threshold.\",\n \"The teachings of the present invention are applicable to any method, apparatus, and system for speech recognition that employs N-gram language models.\",\n \"However, the present invention is not limited to N-gram language models in speech recognition systems and can be applied to the other types of language modeling in speech recognition.\",\n \"In addition, the present invention can also be applied to data modeling in other fields or disciplines including, but not limited to, image processing, signal processing, geometric modeling, computer-aided-design (CAD), computer-aided-manufacturing (CAM), etc.\",\n \"FIG.\",\n \"1 illustrates a block diagram of one embodiment of a speech recognition system 100 according to the teachings of the present invention.\",\n \"The system 100, as shown in FIG.\",\n \"1, includes an analog to digital converter (A/D) 110, a feature extractor or spectral analysis unit 120, a decoder 130, an acoustic model 140, and a language model 150.An input signal 105 representing the input speech is first digitized using the A/D 110.In one embodiment, the digital signal is then sliced up into frames typically of 10, 15, or 20 ms. Each frame of the signal is then converted into a corresponding feature vector which is used to characterize the spectral properties of the input signal.\",\n \"In one embodiment, the feature vector is multi-dimensional and has a plurality of feature components.\",\n \"The feature vectors generated by the feature extractor unit 120 are then inputted into the decoder 130 which determines the sentence or sequence of words that has the highest probability given the acoustic events characterized by the feature vectors, based upon the acoustic model 140 and the language model 150.The language model 150, in one embodiment, is an N-gram language model which includes a plurality of codebooks.\",\n \"Each codebook corresponds to a class of probabilistic attributes (e.g., uni-gram probability values, uni-gram back-off weights, bi-gram probability values, etc.)\",\n \"that are used to represent various conditional probabilities of a set of words.\",\n \"For example, one of the codebook may correspond to the uni-gram probability class, another codebook may correspond to the uni-gram back-off weight class, and so on.\",\n \"The language model 150 and the construction of the corresponding codebooks are described in greater detail below.\",\n \"FIG.\",\n \"2 illustrates a diagram of one embodiment of an HMM-based phone model structure used in the acoustic model 140 to model a phonetic unit (e.g., a phoneme or a subword unit, etc.).\",\n \"Each individual phonetic unit is represented or modeled by a corresponding HM.\",\n \"As shown in FIG.\",\n \"2, an “MM has a set of sequence of states (1-5) that are connected by a set of transition probabilities (a12, a23, a34, a45), and a set of observation probabilities or likelihoods (b2 (o1),b2(o2),b3(o3),b4(o4),b4(o5),b4(o6)).\",\n \"Each transition probability aij represents the probability of transitioning from a state i to a state j.\",\n \"Each observation probability or distribution bi(oj) represents the probability of an observation vector oj being generated from a state i.\",\n \"Thus, the transition probabilities model the durational variability in speech and the output probabilities model the spectral variability.\",\n \"Accordingly, the set of states, the set of transition probabilities and the set of output probabilities are the parameters that are used to define an HMM.\",\n \"The HMM shown in FIG.\",\n \"2 has a left-right topology.\",\n \"In many modern speech recognition systems, each state output distribution or observation probability function is modeled by a multivariate mixture Gaussian as follows: b j ( o t ) = ∑ k = 1 M c jk N ( o t , m jk , V jk ) where cjk is the weight of mixture component k in state j and N(oi, mjk, Vjk) denotes a multivariate Gaussian of mean mjk and covanance Vjk for the kth mixture component in state j.\",\n \"FIG.\",\n \"3 shows a structure diagram of one embodiment of an N-gram language model.\",\n \"As described above, the purpose of a language model is to provide a mechanism for estimating the probability of some word, wk, in an utterance given the preceding words, W1k-1=w1 .\",\n \".\",\n \".\",\n \"wk1.An N-gram language model assumes that wk depends only on the preceding N-1 words, that is P ( w k ❘ W 1 k - 1 ) = P ( w k ❘ W k - n + 1 k - 1 ) .\",\n \"Because of the very large amount of models and associated data that need to be stored and processed, N is usually limited to two (for a bi-gram or 2-gram language model) or three (for a tri-gram or 3-gram language model).\",\n \"A back-off mechanism can be used to compute a conditional probability of a given word when some N-gram context (e.g., a tri-gram or a bi-gram) does not exist or cannot be found for the given word.\",\n \"For example, for given words “a” “b” “c”, there may be no bi-gram probability for “b→a” or tri-gram probability for “c→b→a” in certain contexts.\",\n \"In this case, a back-off mechanism is used to compute the bi-gram or tri-gram probability for the given word “a”.\",\n \"For example, for a tri-gram of words “c→b→a” (meaning word “a” preceded by word “b” preceded by word “c”), the corresponding tri-gram and bi-gram probabilities of this word sequence can be computed as follows using a back-off mechanism: P ( a / b , c ) = { P ( a / b , c ) , if P ( a / b , c ) exists , P ( a / b ) × w ( b , c ) , if w ( b , c ) exists , P ( a / b ) otherwise } P ( a / b ) = { P ( a / b ) , if P ( a / b ) exists , P ( a ) × w ( b ) otherwise } where: P(a/b,c) represents the tri-gram probability of the tri-gram c→b→a; P(a/b) represents the bi-gram probability of the bi-gram b→a; P(a) represents the uni-gram probability of word a; w(b,c) represents the back-off weight for the bi-gram c→b; and w(b) represents the back-off weight for the word b.\",\n \"In various LVCSR systems, language model look-head techniques can be used to speed up the search in the corresponding N-gram language model.\",\n \"The look-head techniques are used to incorporate the language model probabilities as early as possible in the pruning process of the beam search.\",\n \"In general, look-head probabilities are pre-computed and stored in certain file formats or data structures.\",\n \"Referring to FIG.\",\n \"3, an N-gram language model in a typical LVSR system can be logically represented as a set of nodes 300 which includes a set of uni-gram nodes 310, a set of bi-gram nodes 320, and so on.\",\n \"The uni-gram nodes are used to represent single words and their associated probability data (e.g., probability value and back-off weight).\",\n \"The bi-gram nodes are used to represent word pairs and their associated probability data, etc.\",\n \"In one embodiment, the second words in the bi-gram nodes that follow the same first word in a uni-gram node can be grouped together and point them to the corresponding first word in the uni-gram node.\",\n \"Thus, only the second words and the corresponding bi-gram probability data need to be stored in the bi-gram nodes.\",\n \"Tri-gram nodes can be structured similarly.\",\n \"For example, all the third words in the tri-gram nodes that follow the same bi-gram node can be grouped together and point to the corresponding bi-gram node.\",\n \"Thus, only the third words and their associated probability data need to be stored in the tri-gram nodes.\",\n \"As shown in FIG.\",\n \"3, each uni-gram node can be linked to its corresponding bi-gram nodes via a pointer.\",\n \"Similarly, each bi-gram node can be linked to its corresponding tri-gram nodes via a pointer, and so on.\",\n \"In other words, each uni-gram node points to the head of a set of bi-gram nodes whose words follow the word stored in the respective uni-gram node, each bi-gram node points to the head of a set of tri-gram nodes, and so on.\",\n \"Each n-gram node (e.g., uni-gram node, bi-gram node, tri-gram node, etc.)\",\n \"typically has the following components: the word identity of the word that the respective node represents (which can be the word itself or a word index corresponding to the word), an n-gram probability value, a back-off weight, and a pointer to the head of the corresponding (n+1)-gram nodes.\",\n \"For example, a uni-gram node typically contains an index value of a particular word that is represented by the respective uni-gram node, a uni-gram probability value, a uni-gram back-off weight, and a pointer to a corresponding set of bi-gram nodes that are linked with the respective uni-gram node.\",\n \"In one embodiment, as described above, each n-gram node only needs to store the last word of the corresponding n-gram.\",\n \"For example, a bi-gram node only needs to store the second word of the corresponding bi-gram that the respective bi-gram node represents, a tri-gram node only needs to store the third word of the corresponding tri-gram that the respective tri-gram node represents, and so on.\",\n \"In general, for computational efficiency and to avoid numerical underflow, probability values and back-off weights associated with the n-gram nodes can be represented in logarithmic space.\",\n \"A n-gram node may be represented by various data structures depending upon the particular implementations and applications of the language model.\",\n \"For example, an n-gram node may be represented by a corresponding data structure as shown below: typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID is an index of the word W that this node represents; P and W are the corresponding probability value and back-off weight, respectively; PTR is a pointer that points to the (n+1)-gram area linked by this node.\",\n \"If there is no (n+1)-gram linked with this node, PTR can be set to a pre-defined value.\",\n \"In this example, the word index WID is an unsigned 16-bit value (e.g., unsigned short data type); the probability value P and the back-off weight are 32-bit float values (e.g., 32-bit float data type); and the PTR is a 32-bit integer value.\",\n \"The whole size of the data structure in this example is therefore 112 bits or 14 bytes.\",\n \"An example of a data structure that can be used to represent a look-head node is shown below: typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; In this example, the NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.\",\n \"It can be understood from the above description that a typical language model for a LVCSR system can contain a very large number of nodes each of which may contain corresponding probability value and/or backup weight that are represented by very large number of data bits (e.g., probability value and back-off weight are 32-bit float values in the above examples).\",\n \"Thus, a language model for a typical LVCSR requires a very large amount of memory space.\",\n \"As discussed above and described in more details below, the present invention provides a method and mechanism for reducing the size of the language model and the access time while maintaining a high level of recognition accuracy.\",\n \"According to the teachings of the present invention, a method is introduced and described in detail herein to reduce the size of a language model in a LVCSR system.\",\n \"In one embodiment, the probability values and back-off weights (also referred to as probabilistic attributes herein) associated with the respective n-gram nodes in the respective language model are quantized or clustered by a modified K-means clustering algorithm to compress the size of the respective language model.\",\n \"In one embodiment, the probability values or back-off weights are divided or grouped into separate classes or groups based on the relative n-th order of the corresponding n-gram nodes and a modified K-means clustering algorithm is applied to each class to cluster the probability values or back-off weights in each class to build a corresponding codebook for the respective class.\",\n \"For example, uni-gram probability values associated with uni-gram nodes are grouped into one class, uni-gram back-off weights are grouped into another class, bi-gram probability values are grouped into yet another class, and so on.\",\n \"Thus, for a tri-gram language model (which includes uni-gram nodes, bi-gram nodes, and tri-gram nodes), there can be five separate classes (also referred to as data classes herein) that can be quantized or clustered to reduce the size of the language model.\",\n \"These classes include a uni-gram probability class, a uni-gram back-off weight class, a bi-gram probability class, a bi-gram back-off weight class, and a tri-gram probability class.\",\n \"For each class, the field of probability values (also referred to as the range of probability values) is quantized or clustered into a plurality of clusters or segments using a modified K-means clustering algorithm described in more detail below.\",\n \"A centroid is defined for each cluster or segment.\",\n \"Thus, a separate codebook can be constructed for each class of probability values or each class of back-off weights.\",\n \"After quantization or clustering, a given probability value or back-off weight can be represented by the centroid (also called codeword) of the corresponding segment or cluster to which the respective probability value or back-off weight belongs.\",\n \"By quantizing or clustering the field of probability values in each class, the probability values or back-off weights (which originally were represented by large-size data types such as 32-bit float type) can now be represented by smaller-size index values such as 8-bit char type that points to the corresponding codeword (centroid) in the corresponding codebook.\",\n \"In one embodiment, the field of probability value for each class is divided into 256 segments or clusters using the modified K-means clustering process described in greater details below.\",\n \"As such, the corresponding codebook will have 256 codewords.\",\n \"Each codeword can be referenced by an 8-bit index value.\",\n \"Thus, assuming that the original probability values or back-off weights use 32-bit float type, these probability values or back-off weights can now be replaced by 8-bit index values which are used to reference the corresponding codewords in the codebook.\",\n \"It should be appreciated and understood by one skilled in the art that the teachings of the present invention are not limited to any particular number of segments or clusters.\",\n \"Similarly, the teachings of the present invention are not limited to any particular data types that are used to represent the probability values and back-off weights before and after the quantization.\",\n \"The number of segments or clusters for each class (hence the size of the corresponding codebook) and the data types that are used to represent the probability values, the back-off weights, or the indices to the codebook can vary depending upon the particular applications and implementations of the present invention.\",\n \"FIG.\",\n \"4 illustrates a flow diagram of one embodiment of a method 400 according to the teachings of the present invention.\",\n \"At block 410, the probabilistic attributes (e.g., the probability values and back-off weights) in a N-gram language model are classified or grouped into a plurality of separate classes.\",\n \"For example, in a tri-gram language model that includes uni-gram nodes, bi-gram nodes, and tri-gram nodes, the probabilistic attributes can be classified into five separate classes as follows: uni-gram probability, uni-gram back-off weight, bi-gram probability, bi-gram back-off weight, and tri-gram probability.\",\n \"At block 420, the field of value for each class is clustered or quantized using a modified K-means clustering process to build a corresponding codebook for the respective class.\",\n \"The modified K-means clustering process is described in more details below.\",\n \"At block 430, each probabilistic attribute (e.g., a probability value or a back-off weight) can be represented by the corresponding codeword in the respective codebook.\",\n \"In one embodiment, as described herein, after quantization or clustering, the probability value or back-off weight for any given node in the language model can then be replaced by a corresponding index value that is used to reference the corresponding codeword.\",\n \"For example, assuming that the original probability value or back-off weight for a given node was 32-bit data type (e.g., 32-bit float value) and that there are 256 codewords in the corresponding codebook, then after quantization or clustering, the respective probability value or back-off weight for the given node can be represented by an 8-bit index value which is used to reference or point to the corresponding codeword in the codebook.\",\n \"FIG.\",\n \"5 shows a block diagram of one embodiment of a method according to teachings of the present invention.\",\n \"As shown in FIG.\",\n \"5, the probabilistic attributes (e.g., probability values and back-off weights) 510 in an N-gram language model are classified or grouped into separate classes based on the n-gram order of the respective nodes.\",\n \"In this example, the classes include: uni-gram probability 520A, uni-gram back-off weight 520B, bi-gram probability 520C, bi-gram back-off weight 520D, tri-gram probability 520E, etc.\",\n \"Each class is then clustered or quantized using a modified K-means clustering process to create a corresponding codebook (e.g., 530A-E) for each respective class.\",\n \"As described herein, the size of the codebook for each class may or may not be the same, depending on the particular applications and implementations of the present invention.\",\n \"Each probabilistic attribute (e.g., a uni-gram probability value associated with a uni-gram node) can then be represented by a corresponding codeword in the respective codebook (e.g., the codebook for the uni-gram probability class).\",\n \"FIG.\",\n \"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process 600 to create a corresponding codebook for a probabilistic class (e.g., uni-gram probability class or bi-gram probability class, etc.\",\n \"), according to the teachings of the present invention.\",\n \"The process starts at block 601 and proceeds to block 605 to initialize the codebook as follows: At block 605: initialization of the process is performed by clustering the field of values for the respective class into a plurality of segments (e.g., n segments in this example) and initialize the segments such that all the segments have equivalent areas.\",\n \"The segments are also called clusters herein.\",\n \"The centroid of each segment is then set such that the respective segment is about equally split (i.e., the segment can be split into two equivalent sub-areas by the corresponding centroid).\",\n \"As shown in FIGS.\",\n \"7 and 8, the segment initialization is performed according to the following conditions: E 1 = min ( x ) , E 2 … E n , E n + 1 = max ( x ) , C 1 , C 2 … C n - 1 , C n ∫ E 1 E 2 P ( x ) ⅆ x = ∫ E 2 E 3 P ( x ) ⅆ x = … = ∫ E n - 1 E n P ( x ) ⅆ x = ∫ E n E n + 1 P ( x ) ⅆ x ∫ E i C i P ( x ) ⅆ x = ∫ C i E i + 1 P ( x ) ⅆ x i = 1 , 2 … n Where: x is a possible probability value in the respective class; P(x) is the probability that x occurs; the i segment is defined as [Ei, Ei+1; and Ci is defined as the centroid of the i segment.\",\n \"At this point, there are n segments or clusters which have equivalent areas and each i segment has a corresponding centroid Ci which can split the respective segment into two equivalent sub-areas.\",\n \"Set the number of iterations R=0 At block 610: compute the total deviation Dev as sum of the deviation of each segment Devi as follows: Dev=ΣDevi.\",\n \"for the i segment, Devi is defined as ∫ E i E i + 1 P ( x ) * abs ( x - C i ) ⅆ x , (illustrated in FIG.\",\n \"8) At block 615: replace the edge Ei of each segment by the arithmetic average of the nearest two centroids as shown below: Ei=(Ci-1+Ci)/2 (illustrated in FIG.\",\n \"9) set the new centroid Ci for each resultant segment compute the new total deviation Dev increment the number of iterations R=R+1 At block 625: if the total deviation is not steady, then the process 600 loops back to block 615.Otherwise, the corresponding codebook is obtained at block 635 and the process 600 proceeds to ends at block 691.In one embodiment, the total deviation is considered steady if one of the following conditions (also called optimization criteria) is met: a.\",\n \"For each i, Ei=Ei (the new edge value is the same as the previous edge value); b.\",\n \"The number of iterations performed reaches a predetermined number (R=Rmax); or c. The change of total deviation is less than a pre-determined threshold value Δth, |Dev−Dev|/Dev<Δth Significant reduction in memory requirement and access time for a language model can be attained according to the teachings of the present invention.\",\n \"For example, assuming that before compression or quantization, the following data structures were used for n-gram nodes and look-head nodes, respectively: n-gram node data structure (before compression): typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (32-bit float value) and W (32-bit float value) are the corresponding probability value and back-off weight, respectively, PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.\",\n \"If there is no (n+1)-gram linked with this node, PTK is a pre-defined value.\",\n \"Thus the whole size of an-gram data structure in this example is 112 bits or 14 bytes.\",\n \"look-head node data structure (before compression): typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; where: NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.\",\n \"Thus the whole size of a look-head data structure is 64 bits.\",\n \"After compression, an n-gram node and a look-head node can be represented by the following data structures, respectively.\",\n \"It is assumed in this example that 8-bit codebooks are used (e.g., each codebook has 256 codewords): n-gram node data structure (after compression): typedef struct _asNGram{ unsigned short WID; unsigned char P; int PTR; unsigned char W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (8-bit unsigned char type) is the index value used to reference the corresponding quantized probability value (codeword) in the corresponding codebook; W (8-bit unsigned char type) is the index value used to reference the corresponding quantized back-off weight (codeword) in the corresponding codebook; PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.\",\n \"If there is no (n+1)-gram linked with this node, PTR is a pre-defined value.\",\n \"Thus the whole size of an n-gram data structure after compression in this example is 64 bits or 8 bytes.\",\n \"look-head node data structure (after compression): typedef struct _asTreeBi-gramt{ int NODENO; unsigned P; } asTreeBi-gram; where: NODENO is now an 8-bit index value that is used to reference the corresponding quantized probability value in the corresponding codebook.\",\n \"Thus the size of the look-head data structure after compression is 40 bits.\",\n \"From the above example, it can be seen that the size of the language model can be significantly reduced according to the teachings of the present invention.\",\n \"In this example, the size of each n-gram node is reduced from 112 bits to 64 bits and the size of each look-head node is reduced from 64 bits to 40 bits.\",\n \"For a language model in a typical LVCSR system which usually has a very large number of nodes, the reduction in memory requirement can be very substantial.\",\n \"The additional memory requirement for the codebooks is minimal or insignificant compared to the reduction in the memory requirement for the whole language model.\",\n \"For example, the memory requirement for an 8-bit codebook which has 256 codewords and each codeword is a 32-bit float value is only: 256(codewords)*32(bits/codeword)=8192 bits Tables 1 and 2 below show some experimental results based upon various experiments conducted according to the teachings of the present invention.\",\n \"These experiments were conducted on a 51K Mandarin LVCSR system and using a testing machine such as a double-PIII866 with 512M memory and 256K L2 Cache.\",\n \"Table 1 illustrates the improvements with respect to memory requirement and decoding speed using 8-bit codebooks (e.g., each codebook has 256 codewords).\",\n \"It can be seen from table 1 that the memory requirement and decoding speed have been reduced after compression while the increase in the word error rate (WER) of the system after compression is minimal or insignificant.\",\n \"The XRT notation in table 1 is used to mean times of real time.\",\n \"TABLE 1 System using BaseLine modified K-Means System (before clustering (after compression) compression) WER 10.0% 10.1% Tri-gram file size 130M 75M Treebi-gram file size 109M 68M MapMode XRT 1.18XRT 1.08XRT Loading directly mode 1.11XRT 1.02XRT XRT As shown in table 1, the experiments and comparisons were done to show the improvements with respect to two different embodiments: one embodiment using memory-map file format for the language model files and the other embodiment loading the language model files directly into memory.\",\n \"After quantization the size of the language model file is reduced by 40% from 239M(130M+109M) to 143M (75M+68M).\",\n \"In addition, the decoding speed has been improved by 8% from 1.18×RT to 1.08×RT using memory-map file format while WER only increases from 10.0% to 10.1%.\",\n \"Furthermore, if language model files are loaded directly to memory, the decoding speed can be improved by another 6%.\",\n \"Table 2 shows the experimental results using smaller-size codebooks.\",\n \"In this example, 6-bit codebooks and 4bit codebooks were used.\",\n \"TABLE 2 System using modified K-means clustering (after compression) 6-bit 4-bit codebooks codebooks WER 10.1% 12.8% MapMode XRT 1.07XRT 1.03XRT Loading directly 1.01XRT 0.98XRT mode XRT The invention has been described in conjunction with the preferred embodiment.\",\n \"It is evident that numerous alternatives, modifications, variations and uses will be apparent to those skilled in the art in light of the foregoing description.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297354"}}},{"rowIdx":97,"cells":{"text":{"kind":"list like","value":[["Refrigeration unit","The refrigerating unit can be operated by means of a thermal solar system as energy source.","Therein it is provided that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.","The refrigerating unit has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to form a closed fuel circuit with one another."],["1-12.Cancelled.","12.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit, comprising: a closed fuel circuit having a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for rectification of a mixture of fuel and water vapor.","13.The refrigerating unit according to claim 12, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.","14.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","15.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","16.The refrigerating unit according to claim 13, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.","17.The refrigerating unit according to claim 13, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.","18.The refrigerating unit according to claim 17, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.","19.The refrigerating unit according to claim 13, further comprising a rising tube having an inner structure promoting the formation of bubbles.","20.The refrigerating unit according to claim 12, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).","21.The refrigerating unit according to claim 13, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.","22.The refrigerating unit according to claim 13, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 23.The refrigeration unit of claim 12, where the closed fuel circuit comprises an expeller condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit.","24.The refrigerating unit according to claim 23, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.","25.The refrigerating unit according to claim 23, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","26.The refrigerating unit according to claim 23, wherein the the expeller has at leapt one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","27.The refrigerating unit according to claim 12, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.","28.The refrigerating unit according to claim 23, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.","29.The refrigerating unit according to claim 28, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.","30.The refrigerating unit according to claim 23, further comprising a rising tube having an inner structure promoting the formation of bubbles.","31.The refrigerating unit according to claim 23, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).","32.The refrigerating unit according to claim 23, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.","33.The refrigerating unit according to claim 23, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 34.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit and can be operated by means of a thermal solar system as energy source, with an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit, wherein the refrigerating unit has, in the fuel circuit, a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for the rectification of a mixture of fuel and water vapor.","35.A method for refrigerating a unit, the method comprising: forming a fuel circuit having a triple head exchanger; and preheating a solution which is high in fuel, precooling a solution which is low in fuel, and rectifying a mixture of fuel and water vapor."],[" BACKGROUND The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1 .","Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.","For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.","In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.","Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.","These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range."],[" SUMMARY OF THE INVENTION It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.","For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.","A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.","This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.","A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.","Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.","from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.","A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.","The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.","The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.","It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.","Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.","Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.","Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.","A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.","Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).","According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.","of the still not vaporized fuel.","Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.","If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.","In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.","This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.","Instead of ribbing, deflecting plates can also be provided.","Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.","This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.","The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.","Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.","The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.","In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.","Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.","An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.","Advantageously, the rising tube has an inner structure promoting the formation of bubbles.","An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.","The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.","Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H 2 O).","NH3 as fuel and a mixture of NH3 and H 2 O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.","Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).","According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.","By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.","In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).","Additional advantageous developments of the invention follow from the description."],["RELATED APPLICATIONS The present application is the U.S. National Phase of PCT Application PCT/EP01/06412, filed Jun.","6, 2001, which claims priority to German Patent Application No.","10028543.0, filed Jun.","8, 2000.BACKGROUND The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1.Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.","For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.","In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.","Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.","These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range.","SUMMARY OF THE INVENTION It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.","For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.","A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.","This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.","A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.","Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.","from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.","A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.","The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.","The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.","It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.","Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.","Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.","Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.","A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.","Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).","According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.","The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.","of the still not vaporized fuel.","Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.","If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.","In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.","This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.","Instead of ribbing, deflecting plates can also be provided.","Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.","This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.","The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.","Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.","The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.","In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.","Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.","An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.","Advantageously, the rising tube has an inner structure promoting the formation of bubbles.","An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.","The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.","Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).","NH3 as fuel and a mixture of NH3 and H2O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.","Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).","According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.","By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.","In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).","Additional advantageous developments of the invention follow from the description.","BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained below in several embodiment examples with the aid of the accompanying drawings.","Shown are: FIG.","1 shows a block diagram of a refrigerating unit according to the invention; FIG.","2a shows a schematic representation of a first part of the refrigerating unit according to FIG.","1; FIG.","2b shows a schematic representation of a second part of the refrigerating unit according to FIG.","1; FIG.","3 shows an alternative form of embodiment of an expeller for the refrigerating unit according to FIG.","2; FIG.","4 shows a schematic representation of a conveyance line having a capillary sleeve on an enlarged scale.","DETAILED DESCRIPTION FIG.","1 shows a block diagram of a refrigerating unit 10.Furthermore, FIGS.","2a and 2b, which complement one another as partial figures, show the refrigerating unit 10 according to FIG.","1 in schematic representation.","The refrigerating unit 10 has an expeller 12 which is actively connected to a thermal solar system 11 by means of heat transfer medium lines represented as arrows 13 and 14 (FIG.","2a: 43, 43a, 43b, 44).","A fuel vapor supply line represented as an arrow 16 (FIG.","2a: 51) and a supply line, represented as an arrow 17 (FIG.","2a: 47) and serving to convey a solution which is low in fuel, leads from the expeller 12 to a triple heat exchanger 15, from which a supply line, represented as an arrow 18 (FIG.","2a: 48) and serving to convey a solution which is high in fuel, leads to the expeller 12.The triple heat exchanger 15 is actively connected to a condenser 19 by means of a supply line, represented as an arrow 20 (FIGS.","2a/2b: 55), for fuel vapor and, if needed or desired, with low percentages of water (H2O).","The condenser 19 is actively connected to a refrigerating medium circuit 21 by means of cold transfer medium lines, represented as arrows 22, 23 (FIG.","2b: 56, 57).","A supply line, represented as an arrow 25 (FIG.","2b: 58) for liquid fuel leads from the condenser 19 to an evaporator 24 which is actively connected to a refrigerating medium circuit 26 by means of cold transfer medium lines, represented as arrows 27, 28 (FIG.","2b: 60).","A supply line, represented as an arrow 30 (FIG.","2b: 59) for a gaseous mixture of vaporized fuel and an auxiliary gas leads from the evaporator 24 to a gas heat exchanger 29 which, in operation, is actively connected to the evaporator 24 by means of a supply line, represented as an arrow 31 (FIG.","2b: 76), for the precooled auxiliary gas.","By means of a supply line, for the heated gas mixture consisting of fuel and auxiliary gas and represented as an arrow 33 (FIG.","2b: 77), the gas heat exchanger 29 is actively connected to an absorber 32, from which a supply line, for warm auxiliary gas and represented as an arrow 34 (FIG.","2b: 61), leads to the gas heat exchanger 29.The absorber 32 is actively connected to a refrigerating medium circuit 35 by means of cold transfer medium lines represented as arrows 36, 37 (FIG.","2b: 63, 64).","A supply line, for a solution which is high in fuel and represented as an arrow 38 (FIGS.","2a/2b: 68), leads from the absorber 32 to a triple gas heat exchanger 15 and from the latter a supply line, for a solution which is low in fuel and represented as an arrow 39 (FIGS.","2a/2b: 69), leads to the absorber 32.The refrigerating unit 10 is divided, by means of a dotted line 40 according to arrow 42, into an upper area in which the total operational pressure is equal to the fuel pressure and, according to arrow 41, into a lower area in which the total operational pressure is equal to the sum of the fuel pressure and the auxiliary gas pressure.","Preferably the fuel is ammonia (NH3), the auxiliary gas is helium (He), and each of the solution which is high in fuel and the solution which is low in fuel is a mixture of ammonia and water (NH3/H2O).","NH3 vapor with low percentages of H2O is thus conveyed from the expeller 12 to the triple heat exchanger 15 (arrow 16), from which NH3 vapor flows into the condenser 19 (arrow 20) and is cooled to liquid NH3 where the liquid NH3 subsequently flows into the evaporator 24 (arrow 25) in which NH3 diffuses into a precooled He atmosphere forming a “heavy” cold He-NH3 gas mixture.","This cold gas mixture flows into the gas heat exchanger 29 (arrow 30) in which the same is heated due to a heat transfer from a warm He stream (arrow 34) flowing in the opposite direction through the gas heat exchanger 29 and subsequently flows from the absorber 32 to the absorber 32 (arrow 33).","The solution which is high in NH3/H2O flows on the contrary from the absorber 32 to the triple heat exchanger 15 (arrow 38) and from there to the expeller 12 (arrow 18).","The solution which is low in NH3/H2O flows on the contrary to the triple heat exchanger 15 (arrow 17) and from there to the absorber 32 (arrow 39).","Furthermore, the withdrawal of cooling capacity of the refrigerating unit 10 is done by means of the refrigerating medium circuit 26, which is actively connected to the evaporator 24.FIG.","2a shows in addition the expeller 12 preferably formed as a gas bubble pump, said expeller being actively connected to the thermal solar system 11 and to the triple heat exchanger 15.The thermal solar system 11 has a solar cell unit 72 in the form of a solar collector cell unit which is actively connected, by means of the heat transfer medium lines 43, 43a, 43b, and 44, to a plurality of rising tubes 45 (in FIG.","2a two rising tubes 45 are represented) of the expeller 12.The heat transfer medium lines 43, 43a, and 43b are connected to one another in operation by means of a change-over valve 78 in such a manner that a heat transfer medium is conveyed according to arrow 13 from the solar cell unit through the heat transfer medium line 43 to the change-over valve 78, which conducts the heat transfer medium through the heat transfer medium line 43a and/or 43b according to arrow 13a or 13b to the expeller 12.The rising tubes 45 have on one side a ribbing 50 (or deflecting plate not represented) in order to insure a rapid, spatially defined, and reliable heat transfer from the heat carrier of the solar cell unit 11 to the solution which is high in fuel, said solution being located in the rising tubes 45.Between the rising tubes 45 disposed in parallel at a distance from one another, a parallel recycling line 49 is disposed to convey a solution which is low in fuel.","The expeller 12 is thus formed as a compact bundle of tubes (rising tubes 45 for solution which is high in fuel, recycling line 49 for solution which is low in fuel).","The solution which is low in fuel is conducted, by means of a supply line 47, from the expeller 12 to the triple heat exchanger 15 and conducted from there to the absorber 32 by means of the supply line 69 (see also FIG.","2b).","The fuel vapor is conducted, by means of a conducting line 51, from the expeller 12 to the triple heat exchanger 15 in which water is condensed out from the mixture of fuel and water vapor and conducted, by means of the line 52, according to the arrow 53 to the supply line 48 which serves to convey the solution which is high in fuel into the expeller 12.Pure, or nearly pure, fuel is conducted, by means of the supply line 55, from the triple heat exchanger 15 to the condenser 19 (see also FIG.","2b).","The supply line 48 for conveying the solution which is high in fuel is actively connected to a fuel reservoir 54.A dotted line 70 denotes the level of liquid in the fuel reservoir 54 while a dotted line 71 represents the level of liquid in the inlet of the absorber 32 (see also FIG.","2b).","The expeller 12 is formed as a compact bundle of tubes with a plurality of cold transfer medium lines 65 and a plurality of auxiliary gas conveyance lines 62.Likewise, the gas heat exchanger 29, the evaporator 24, and the condenser 19 are each formed as a compact bundle of tubes.","The absorber 32 can however also be formed in such a manner that the cold transfer medium lines 65 are provided with intermediate spaces for the auxiliary gas conveyance lines 62 and preferably with deflecting plates.","To expel the fuel from the solution which is high in fuel as well as to convey the same, the expeller 12 needs a certain minimum supply of energy from the solar cell unit 72.A supply of energy into the expeller 12 below this limit would lead, when conveying the heat transfer medium into the expeller 12 through the heat transfer medium line 43a below the liquid level denoted as the dotted line 70, to undesirable reduction of the solution which is high in fuel without the same being conveyed.","With longer operation of the refrigerating unit in a state of this type, the solution which is high in fuel, said solution being located in the expeller 12, would be reduced still further in fuel so that higher and higher operating temperatures would be required in order to expel additional fuel from the solution which is high in fuel.","This has as a consequence that the refrigerating unit could no longer be put in operation without additional effort.","Providing a second heat transfer medium line 43b, which is actively connected to the expeller 12 above the liquid level denoted as the dotted line 70, advantageously makes it possible that, on starting the refrigerating unit, that energy which is not sufficient to expel the fuel from the solution which is high in fuel can still be used to preheat the expeller 12.Thereby it is avoided that a “high-value” energy has to be used to heat the expeller 12 (material of the tubes) so that this energy can be used exclusively for the conveyance of the solution which is high in fuel, said solution being located in the expeller 12, and to expel the fuel from the same.","Furthermore, the solution which is high in fuel can be preheated in the triple heat exchanger 15, the fuel reservoir 54, and in the supply line 55 (vapor line) which leads to the condenser 19 by means of “low-value” solar energy.","FIG.","3 shows an expeller 12 according to an alternative development.","This expeller 12 corresponds essentially to that of FIG.","2 and is different in that according to FIG.","3 the fuel vapor supply line 51 leads coaxially through the recycling line 49.In order to guarantee reliable separation in the expeller 12 of the fuel vapor (supply line 51) and the solution which is low in fuel (recycling line 49), an encircling, conically tapering baffle plate 73 is provided, disposed on the outlet side with respect to the rising tubes 45.The baffle plate 73 serves to conduct the solution which is high in fuel according to the arrows 75 into the recycling line 49 while the fuel vapor is conducted through a gap formed between the baffle plate 73 and the supply line 51 according to the arrow 74 into the supply line 51.The expeller 12 according to FIG.","3 is thus formed as a compact bundle of tubes with a plurality of rising tubes 45, a central recycling line 49, and a supply line 51 guided coaxially through the recycling line 49.The additional active connections of the expeller 12, or of the corresponding supply lines, to the corresponding functional units of the refrigerating unit 10 correspond to the above-described form of embodiment according to FIGS.","1 and 2.The individual functional units of the refrigerating unit 10 are actively connected to one another and disposed spatially so as to form three gravity-based gas or liquid circuits.","In so doing, only the external circuits for cooling (cooling circuits 21, 26, and 35) and for the thermal solar system 11 are maintained by means of conventional pumps.","Within the refrigerating unit, each of the solution circuit, refrigerating medium circuit, and auxiliary gas circuit is closed.","The solution circuit begins in the expeller 12 formed as a gas bubble pump, where, by means of the solar system 11, thermal energy is supplied to convert a solution which is high in fuel into a mixture of vaporous fuel and water and into a solution which is low in fuel.","The solution which is low in fuel is conveyed from the expeller 12 to the absorber 32 in which the solution which is low in fuel backs up in the supply line 69 up to a certain level of liquid.","The solution which is low in fuel is thus raised from the level of liquid in the fuel reservoir 54 (dotted line 70) to the level of the inlet of the absorber 32 (dotted line 71).","In the process, the conveyance height and the mass flow of the solution which is low in fuel depend on the geometric configuration of the expeller 12, the level of liquid in the fuel reservoir 54 (dotted line 70), and thus the immersion depth of the rising tubes 45, and the recycling line 49 of the expeller 12 as well as on the number of conveyance tubes (rising tubes 45, recycling line 49) of the expeller 12.The solution which is low in fuel conveyed to the absorber 32 absorbs the fuel supplied by the evaporator 24 to form a solution which is high in fuel, said solution being conducted for preheating through the triple heat exchanger 15 and flowing back into the expeller 12.The solution which is high in fuel flowing back into the expeller 12 is thus available once again to expel the fuel in the same.","In the refrigerating medium circuit, the mixture of hot fuel and water vapor, said mixture having been vaporized in the expeller 12, flows into the triple heat exchanger 15 and is precooled and rectified there by the cold solution which is high in fuel coming from the absorber 32 and flowing in the opposite direction.","The pure fuel vapor, near to the condensation temperature, rises from the triple heat exchanger 15 into condenser 19 in which, through additional withdrawal of heat by means of the refrigerating medium circuit 21, the pure fuel is cooled to under its condensation temperature so that the fuel vapor condenses.","The liquid fuel subsequently flows from the condenser 19 into the evaporator 24 in which the liquid fuel diffuses into an atmosphere of auxiliary gas and draws heat from the refrigerating medium of the refrigerating medium circuit 26.Subsequently the relatively heavy mixture of auxiliary gas and fuel sinks into the absorber 32.In the auxiliary gas circuit, the atmosphere of auxiliary gas in the evaporator 24 is constantly pressurized with pure auxiliary gas in order to avoid the atmosphere of auxiliary gas reaching its saturation limit through the diffusing fuel and thus sufficient diffusion of liquid fuel into the atmosphere of auxiliary gas not taking place.","The relatively heavy mixture of auxiliary gas and fuel sinks through the lines 59 from the evaporator 24 into the gas heat exchanger 29 and flows from it through the supply line 77 into the absorber 32.In the absorber 32 the fuel contained in the mixture is absorbed by the solution which is low in fuel, where the then lighter, pure auxiliary gas rises through the lines 62 from the absorber 32, flowing in the opposite direction, and with precooling by means of the refrigerating medium circuit 35 in the gas heat exchanger 29.From there, the pure auxiliary gas flows through the supply lines 61 and 76 into the upper area of the evaporator 24 in which it is enriched with diffusing fuel and subsequently sinks back into the absorber 32 once again as a mixture of fuel and auxiliary gas.","Advantageously, the triple heat exchanger 15 simultaneously fulfills three different functions, namely preheating of the solution which is high in fuel coming from the expeller 12, precooling of the low solution flowing to the absorber 32, and rectification of the NH3-H2O vapor coming from the expeller 12.By conducting the hot vapor mixture along the solution which is high in fuel cooled in the absorber 32, the water vapor contained condenses and falls back as condensate into the solution which is high in fuel.","The heat of condensation or rectification being released is thus also used to preheat the rich solution.","The triple heat exchanger 15 thus provides for a nearly complete recovery of the heat of rectification which advantageously no longer has to be supplied to the expeller 12 from outside.","FIG.","4 shows a detail of the absorber 32 on an enlarged scale.","According to this preferred form of embodiment, the given auxiliary gas conveyance line 62 of the absorber 32 is provided with a capillary sleeve 79 which is actively connected to the line 62, on the outlet side with respect to the auxiliary gas conveyance line (arrow 82).","The capillary sleeve 79 forms, together with the line 62, a capillary gap 81, extending circumferentially, through which the solution which is low in fuel (liquid) can flow according to the arrows 83 and 84.The solution which is low in fuel is thus conducted by means of the supply line 69 into the absorber 32 and, due to the capillary action present, conveyed according to the arrows 83 and 84 into the given auxiliary gas conveyance line 62 of the absorber 32 in which the same slides downwards along its walls (arrow 84).","The auxiliary gas is conducted according to the arrows 82 through the auxiliary gas conveyance line 62 and through a central longitudinal hole 80 of the capillary sleeve 79 and flows according to the arrows 34 to the supply line 61 which actively connects the absorber 32 to the gas heat exchanger 29.A separating plate 85 connected to a jacket tube 86 of the absorber 32 makes it possible that the solution which is low in fuel, said solution being located in the absorber 32, can be backed up to a certain level of liquid (FIG.","2b: dotted line 71).","A given auxiliary gas conveyance line 62 equipped with a capillary sleeve 79 is advantageous since, due to the capillary action present at the time in the corresponding capillary gap 81, a uniform distribution of the solution which is low in fuel to the individual auxiliary gas conveyance line 62 in the absorber 32 is guaranteed independently of the level, present at the time, of the solution which is low in fuel in the absorber 32 or on the amount of the solution which is low in fuel flowing through the supply line 69 into the absorber 32 according to the arrows 39.In particular in the case of an oblique position (not represented) of the absorber 32, it is avoided by means of the capillary sleeves 79, due to capillary action setting in, that several auxiliary gas conveyance lines 62 are completely filled with solution which is low in fuel and the remaining auxiliary gas conveyance lines 62 are not pressurized with solution which is low in fuel so that the absorber 32 would not be functional, at least not completely.","According to a preferred form of embodiment not represented, each of the auxiliary gas conveyance line 62 can be provided with a plurality of projections acting as space holders at its free end projecting into the interior of the auxiliary gas conveyance line 62, by means of which projections a coaxial, with respect to the corresponding auxiliary gas conveyance lines 62, positioning of the respective capillary sleeve 79 is possible in a relatively simple manner.","Projections of this type at the free end of the respective capillary sleeve 79 are preferably distributed uniformly over its circumference and spaced sufficiently far from one another that a free flow of the solution which is low in fuel through the capillary gap according to arrows 83 and 84 is guaranteed.","Corresponding to another additional form of embodiment not represented, the lines 59 of the evaporator 24 are provided at their upper free end with a corresponding capillary sleeve 79 according to FIG.","4."]],"string":"[\n [\n \"Refrigeration unit\",\n \"The refrigerating unit can be operated by means of a thermal solar system as energy source.\",\n \"Therein it is provided that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.\",\n \"The refrigerating unit has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to form a closed fuel circuit with one another.\"\n ],\n [\n \"1-12.Cancelled.\",\n \"12.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit, comprising: a closed fuel circuit having a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for rectification of a mixture of fuel and water vapor.\",\n \"13.The refrigerating unit according to claim 12, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.\",\n \"14.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"15.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"16.The refrigerating unit according to claim 13, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.\",\n \"17.The refrigerating unit according to claim 13, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.\",\n \"18.The refrigerating unit according to claim 17, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.\",\n \"19.The refrigerating unit according to claim 13, further comprising a rising tube having an inner structure promoting the formation of bubbles.\",\n \"20.The refrigerating unit according to claim 12, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).\",\n \"21.The refrigerating unit according to claim 13, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.\",\n \"22.The refrigerating unit according to claim 13, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 23.The refrigeration unit of claim 12, where the closed fuel circuit comprises an expeller condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit.\",\n \"24.The refrigerating unit according to claim 23, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.\",\n \"25.The refrigerating unit according to claim 23, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"26.The refrigerating unit according to claim 23, wherein the the expeller has at leapt one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"27.The refrigerating unit according to claim 12, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.\",\n \"28.The refrigerating unit according to claim 23, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.\",\n \"29.The refrigerating unit according to claim 28, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.\",\n \"30.The refrigerating unit according to claim 23, further comprising a rising tube having an inner structure promoting the formation of bubbles.\",\n \"31.The refrigerating unit according to claim 23, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).\",\n \"32.The refrigerating unit according to claim 23, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.\",\n \"33.The refrigerating unit according to claim 23, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 34.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit and can be operated by means of a thermal solar system as energy source, with an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit, wherein the refrigerating unit has, in the fuel circuit, a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for the rectification of a mixture of fuel and water vapor.\",\n \"35.A method for refrigerating a unit, the method comprising: forming a fuel circuit having a triple head exchanger; and preheating a solution which is high in fuel, precooling a solution which is low in fuel, and rectifying a mixture of fuel and water vapor.\"\n ],\n [\n \" BACKGROUND The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1 .\",\n \"Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.\",\n \"For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.\",\n \"In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.\",\n \"Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.\",\n \"These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range.\"\n ],\n [\n \" SUMMARY OF THE INVENTION It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.\",\n \"For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.\",\n \"A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.\",\n \"This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.\",\n \"A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.\",\n \"Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.\",\n \"from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.\",\n \"A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.\",\n \"The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.\",\n \"The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.\",\n \"It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.\",\n \"Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.\",\n \"Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.\",\n \"Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.\",\n \"A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.\",\n \"Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).\",\n \"According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.\",\n \"of the still not vaporized fuel.\",\n \"Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.\",\n \"If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.\",\n \"In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.\",\n \"This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.\",\n \"Instead of ribbing, deflecting plates can also be provided.\",\n \"Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.\",\n \"This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.\",\n \"The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.\",\n \"Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.\",\n \"The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.\",\n \"In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.\",\n \"Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.\",\n \"An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.\",\n \"Advantageously, the rising tube has an inner structure promoting the formation of bubbles.\",\n \"An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.\",\n \"The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.\",\n \"Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H 2 O).\",\n \"NH3 as fuel and a mixture of NH3 and H 2 O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.\",\n \"Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).\",\n \"According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.\",\n \"By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.\",\n \"In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).\",\n \"Additional advantageous developments of the invention follow from the description.\"\n ],\n [\n \"RELATED APPLICATIONS The present application is the U.S. National Phase of PCT Application PCT/EP01/06412, filed Jun.\",\n \"6, 2001, which claims priority to German Patent Application No.\",\n \"10028543.0, filed Jun.\",\n \"8, 2000.BACKGROUND The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1.Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.\",\n \"For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.\",\n \"In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.\",\n \"Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.\",\n \"These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range.\",\n \"SUMMARY OF THE INVENTION It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.\",\n \"For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.\",\n \"A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.\",\n \"This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.\",\n \"A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.\",\n \"Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.\",\n \"from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.\",\n \"A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.\",\n \"The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.\",\n \"The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.\",\n \"It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.\",\n \"Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.\",\n \"Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.\",\n \"Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.\",\n \"A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.\",\n \"Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).\",\n \"According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.\",\n \"The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.\",\n \"of the still not vaporized fuel.\",\n \"Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.\",\n \"If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.\",\n \"In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.\",\n \"This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.\",\n \"Instead of ribbing, deflecting plates can also be provided.\",\n \"Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.\",\n \"This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.\",\n \"The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.\",\n \"Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.\",\n \"The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.\",\n \"In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.\",\n \"Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.\",\n \"An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.\",\n \"Advantageously, the rising tube has an inner structure promoting the formation of bubbles.\",\n \"An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.\",\n \"The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.\",\n \"Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).\",\n \"NH3 as fuel and a mixture of NH3 and H2O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.\",\n \"Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).\",\n \"According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.\",\n \"By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.\",\n \"In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).\",\n \"Additional advantageous developments of the invention follow from the description.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained below in several embodiment examples with the aid of the accompanying drawings.\",\n \"Shown are: FIG.\",\n \"1 shows a block diagram of a refrigerating unit according to the invention; FIG.\",\n \"2a shows a schematic representation of a first part of the refrigerating unit according to FIG.\",\n \"1; FIG.\",\n \"2b shows a schematic representation of a second part of the refrigerating unit according to FIG.\",\n \"1; FIG.\",\n \"3 shows an alternative form of embodiment of an expeller for the refrigerating unit according to FIG.\",\n \"2; FIG.\",\n \"4 shows a schematic representation of a conveyance line having a capillary sleeve on an enlarged scale.\",\n \"DETAILED DESCRIPTION FIG.\",\n \"1 shows a block diagram of a refrigerating unit 10.Furthermore, FIGS.\",\n \"2a and 2b, which complement one another as partial figures, show the refrigerating unit 10 according to FIG.\",\n \"1 in schematic representation.\",\n \"The refrigerating unit 10 has an expeller 12 which is actively connected to a thermal solar system 11 by means of heat transfer medium lines represented as arrows 13 and 14 (FIG.\",\n \"2a: 43, 43a, 43b, 44).\",\n \"A fuel vapor supply line represented as an arrow 16 (FIG.\",\n \"2a: 51) and a supply line, represented as an arrow 17 (FIG.\",\n \"2a: 47) and serving to convey a solution which is low in fuel, leads from the expeller 12 to a triple heat exchanger 15, from which a supply line, represented as an arrow 18 (FIG.\",\n \"2a: 48) and serving to convey a solution which is high in fuel, leads to the expeller 12.The triple heat exchanger 15 is actively connected to a condenser 19 by means of a supply line, represented as an arrow 20 (FIGS.\",\n \"2a/2b: 55), for fuel vapor and, if needed or desired, with low percentages of water (H2O).\",\n \"The condenser 19 is actively connected to a refrigerating medium circuit 21 by means of cold transfer medium lines, represented as arrows 22, 23 (FIG.\",\n \"2b: 56, 57).\",\n \"A supply line, represented as an arrow 25 (FIG.\",\n \"2b: 58) for liquid fuel leads from the condenser 19 to an evaporator 24 which is actively connected to a refrigerating medium circuit 26 by means of cold transfer medium lines, represented as arrows 27, 28 (FIG.\",\n \"2b: 60).\",\n \"A supply line, represented as an arrow 30 (FIG.\",\n \"2b: 59) for a gaseous mixture of vaporized fuel and an auxiliary gas leads from the evaporator 24 to a gas heat exchanger 29 which, in operation, is actively connected to the evaporator 24 by means of a supply line, represented as an arrow 31 (FIG.\",\n \"2b: 76), for the precooled auxiliary gas.\",\n \"By means of a supply line, for the heated gas mixture consisting of fuel and auxiliary gas and represented as an arrow 33 (FIG.\",\n \"2b: 77), the gas heat exchanger 29 is actively connected to an absorber 32, from which a supply line, for warm auxiliary gas and represented as an arrow 34 (FIG.\",\n \"2b: 61), leads to the gas heat exchanger 29.The absorber 32 is actively connected to a refrigerating medium circuit 35 by means of cold transfer medium lines represented as arrows 36, 37 (FIG.\",\n \"2b: 63, 64).\",\n \"A supply line, for a solution which is high in fuel and represented as an arrow 38 (FIGS.\",\n \"2a/2b: 68), leads from the absorber 32 to a triple gas heat exchanger 15 and from the latter a supply line, for a solution which is low in fuel and represented as an arrow 39 (FIGS.\",\n \"2a/2b: 69), leads to the absorber 32.The refrigerating unit 10 is divided, by means of a dotted line 40 according to arrow 42, into an upper area in which the total operational pressure is equal to the fuel pressure and, according to arrow 41, into a lower area in which the total operational pressure is equal to the sum of the fuel pressure and the auxiliary gas pressure.\",\n \"Preferably the fuel is ammonia (NH3), the auxiliary gas is helium (He), and each of the solution which is high in fuel and the solution which is low in fuel is a mixture of ammonia and water (NH3/H2O).\",\n \"NH3 vapor with low percentages of H2O is thus conveyed from the expeller 12 to the triple heat exchanger 15 (arrow 16), from which NH3 vapor flows into the condenser 19 (arrow 20) and is cooled to liquid NH3 where the liquid NH3 subsequently flows into the evaporator 24 (arrow 25) in which NH3 diffuses into a precooled He atmosphere forming a “heavy” cold He-NH3 gas mixture.\",\n \"This cold gas mixture flows into the gas heat exchanger 29 (arrow 30) in which the same is heated due to a heat transfer from a warm He stream (arrow 34) flowing in the opposite direction through the gas heat exchanger 29 and subsequently flows from the absorber 32 to the absorber 32 (arrow 33).\",\n \"The solution which is high in NH3/H2O flows on the contrary from the absorber 32 to the triple heat exchanger 15 (arrow 38) and from there to the expeller 12 (arrow 18).\",\n \"The solution which is low in NH3/H2O flows on the contrary to the triple heat exchanger 15 (arrow 17) and from there to the absorber 32 (arrow 39).\",\n \"Furthermore, the withdrawal of cooling capacity of the refrigerating unit 10 is done by means of the refrigerating medium circuit 26, which is actively connected to the evaporator 24.FIG.\",\n \"2a shows in addition the expeller 12 preferably formed as a gas bubble pump, said expeller being actively connected to the thermal solar system 11 and to the triple heat exchanger 15.The thermal solar system 11 has a solar cell unit 72 in the form of a solar collector cell unit which is actively connected, by means of the heat transfer medium lines 43, 43a, 43b, and 44, to a plurality of rising tubes 45 (in FIG.\",\n \"2a two rising tubes 45 are represented) of the expeller 12.The heat transfer medium lines 43, 43a, and 43b are connected to one another in operation by means of a change-over valve 78 in such a manner that a heat transfer medium is conveyed according to arrow 13 from the solar cell unit through the heat transfer medium line 43 to the change-over valve 78, which conducts the heat transfer medium through the heat transfer medium line 43a and/or 43b according to arrow 13a or 13b to the expeller 12.The rising tubes 45 have on one side a ribbing 50 (or deflecting plate not represented) in order to insure a rapid, spatially defined, and reliable heat transfer from the heat carrier of the solar cell unit 11 to the solution which is high in fuel, said solution being located in the rising tubes 45.Between the rising tubes 45 disposed in parallel at a distance from one another, a parallel recycling line 49 is disposed to convey a solution which is low in fuel.\",\n \"The expeller 12 is thus formed as a compact bundle of tubes (rising tubes 45 for solution which is high in fuel, recycling line 49 for solution which is low in fuel).\",\n \"The solution which is low in fuel is conducted, by means of a supply line 47, from the expeller 12 to the triple heat exchanger 15 and conducted from there to the absorber 32 by means of the supply line 69 (see also FIG.\",\n \"2b).\",\n \"The fuel vapor is conducted, by means of a conducting line 51, from the expeller 12 to the triple heat exchanger 15 in which water is condensed out from the mixture of fuel and water vapor and conducted, by means of the line 52, according to the arrow 53 to the supply line 48 which serves to convey the solution which is high in fuel into the expeller 12.Pure, or nearly pure, fuel is conducted, by means of the supply line 55, from the triple heat exchanger 15 to the condenser 19 (see also FIG.\",\n \"2b).\",\n \"The supply line 48 for conveying the solution which is high in fuel is actively connected to a fuel reservoir 54.A dotted line 70 denotes the level of liquid in the fuel reservoir 54 while a dotted line 71 represents the level of liquid in the inlet of the absorber 32 (see also FIG.\",\n \"2b).\",\n \"The expeller 12 is formed as a compact bundle of tubes with a plurality of cold transfer medium lines 65 and a plurality of auxiliary gas conveyance lines 62.Likewise, the gas heat exchanger 29, the evaporator 24, and the condenser 19 are each formed as a compact bundle of tubes.\",\n \"The absorber 32 can however also be formed in such a manner that the cold transfer medium lines 65 are provided with intermediate spaces for the auxiliary gas conveyance lines 62 and preferably with deflecting plates.\",\n \"To expel the fuel from the solution which is high in fuel as well as to convey the same, the expeller 12 needs a certain minimum supply of energy from the solar cell unit 72.A supply of energy into the expeller 12 below this limit would lead, when conveying the heat transfer medium into the expeller 12 through the heat transfer medium line 43a below the liquid level denoted as the dotted line 70, to undesirable reduction of the solution which is high in fuel without the same being conveyed.\",\n \"With longer operation of the refrigerating unit in a state of this type, the solution which is high in fuel, said solution being located in the expeller 12, would be reduced still further in fuel so that higher and higher operating temperatures would be required in order to expel additional fuel from the solution which is high in fuel.\",\n \"This has as a consequence that the refrigerating unit could no longer be put in operation without additional effort.\",\n \"Providing a second heat transfer medium line 43b, which is actively connected to the expeller 12 above the liquid level denoted as the dotted line 70, advantageously makes it possible that, on starting the refrigerating unit, that energy which is not sufficient to expel the fuel from the solution which is high in fuel can still be used to preheat the expeller 12.Thereby it is avoided that a “high-value” energy has to be used to heat the expeller 12 (material of the tubes) so that this energy can be used exclusively for the conveyance of the solution which is high in fuel, said solution being located in the expeller 12, and to expel the fuel from the same.\",\n \"Furthermore, the solution which is high in fuel can be preheated in the triple heat exchanger 15, the fuel reservoir 54, and in the supply line 55 (vapor line) which leads to the condenser 19 by means of “low-value” solar energy.\",\n \"FIG.\",\n \"3 shows an expeller 12 according to an alternative development.\",\n \"This expeller 12 corresponds essentially to that of FIG.\",\n \"2 and is different in that according to FIG.\",\n \"3 the fuel vapor supply line 51 leads coaxially through the recycling line 49.In order to guarantee reliable separation in the expeller 12 of the fuel vapor (supply line 51) and the solution which is low in fuel (recycling line 49), an encircling, conically tapering baffle plate 73 is provided, disposed on the outlet side with respect to the rising tubes 45.The baffle plate 73 serves to conduct the solution which is high in fuel according to the arrows 75 into the recycling line 49 while the fuel vapor is conducted through a gap formed between the baffle plate 73 and the supply line 51 according to the arrow 74 into the supply line 51.The expeller 12 according to FIG.\",\n \"3 is thus formed as a compact bundle of tubes with a plurality of rising tubes 45, a central recycling line 49, and a supply line 51 guided coaxially through the recycling line 49.The additional active connections of the expeller 12, or of the corresponding supply lines, to the corresponding functional units of the refrigerating unit 10 correspond to the above-described form of embodiment according to FIGS.\",\n \"1 and 2.The individual functional units of the refrigerating unit 10 are actively connected to one another and disposed spatially so as to form three gravity-based gas or liquid circuits.\",\n \"In so doing, only the external circuits for cooling (cooling circuits 21, 26, and 35) and for the thermal solar system 11 are maintained by means of conventional pumps.\",\n \"Within the refrigerating unit, each of the solution circuit, refrigerating medium circuit, and auxiliary gas circuit is closed.\",\n \"The solution circuit begins in the expeller 12 formed as a gas bubble pump, where, by means of the solar system 11, thermal energy is supplied to convert a solution which is high in fuel into a mixture of vaporous fuel and water and into a solution which is low in fuel.\",\n \"The solution which is low in fuel is conveyed from the expeller 12 to the absorber 32 in which the solution which is low in fuel backs up in the supply line 69 up to a certain level of liquid.\",\n \"The solution which is low in fuel is thus raised from the level of liquid in the fuel reservoir 54 (dotted line 70) to the level of the inlet of the absorber 32 (dotted line 71).\",\n \"In the process, the conveyance height and the mass flow of the solution which is low in fuel depend on the geometric configuration of the expeller 12, the level of liquid in the fuel reservoir 54 (dotted line 70), and thus the immersion depth of the rising tubes 45, and the recycling line 49 of the expeller 12 as well as on the number of conveyance tubes (rising tubes 45, recycling line 49) of the expeller 12.The solution which is low in fuel conveyed to the absorber 32 absorbs the fuel supplied by the evaporator 24 to form a solution which is high in fuel, said solution being conducted for preheating through the triple heat exchanger 15 and flowing back into the expeller 12.The solution which is high in fuel flowing back into the expeller 12 is thus available once again to expel the fuel in the same.\",\n \"In the refrigerating medium circuit, the mixture of hot fuel and water vapor, said mixture having been vaporized in the expeller 12, flows into the triple heat exchanger 15 and is precooled and rectified there by the cold solution which is high in fuel coming from the absorber 32 and flowing in the opposite direction.\",\n \"The pure fuel vapor, near to the condensation temperature, rises from the triple heat exchanger 15 into condenser 19 in which, through additional withdrawal of heat by means of the refrigerating medium circuit 21, the pure fuel is cooled to under its condensation temperature so that the fuel vapor condenses.\",\n \"The liquid fuel subsequently flows from the condenser 19 into the evaporator 24 in which the liquid fuel diffuses into an atmosphere of auxiliary gas and draws heat from the refrigerating medium of the refrigerating medium circuit 26.Subsequently the relatively heavy mixture of auxiliary gas and fuel sinks into the absorber 32.In the auxiliary gas circuit, the atmosphere of auxiliary gas in the evaporator 24 is constantly pressurized with pure auxiliary gas in order to avoid the atmosphere of auxiliary gas reaching its saturation limit through the diffusing fuel and thus sufficient diffusion of liquid fuel into the atmosphere of auxiliary gas not taking place.\",\n \"The relatively heavy mixture of auxiliary gas and fuel sinks through the lines 59 from the evaporator 24 into the gas heat exchanger 29 and flows from it through the supply line 77 into the absorber 32.In the absorber 32 the fuel contained in the mixture is absorbed by the solution which is low in fuel, where the then lighter, pure auxiliary gas rises through the lines 62 from the absorber 32, flowing in the opposite direction, and with precooling by means of the refrigerating medium circuit 35 in the gas heat exchanger 29.From there, the pure auxiliary gas flows through the supply lines 61 and 76 into the upper area of the evaporator 24 in which it is enriched with diffusing fuel and subsequently sinks back into the absorber 32 once again as a mixture of fuel and auxiliary gas.\",\n \"Advantageously, the triple heat exchanger 15 simultaneously fulfills three different functions, namely preheating of the solution which is high in fuel coming from the expeller 12, precooling of the low solution flowing to the absorber 32, and rectification of the NH3-H2O vapor coming from the expeller 12.By conducting the hot vapor mixture along the solution which is high in fuel cooled in the absorber 32, the water vapor contained condenses and falls back as condensate into the solution which is high in fuel.\",\n \"The heat of condensation or rectification being released is thus also used to preheat the rich solution.\",\n \"The triple heat exchanger 15 thus provides for a nearly complete recovery of the heat of rectification which advantageously no longer has to be supplied to the expeller 12 from outside.\",\n \"FIG.\",\n \"4 shows a detail of the absorber 32 on an enlarged scale.\",\n \"According to this preferred form of embodiment, the given auxiliary gas conveyance line 62 of the absorber 32 is provided with a capillary sleeve 79 which is actively connected to the line 62, on the outlet side with respect to the auxiliary gas conveyance line (arrow 82).\",\n \"The capillary sleeve 79 forms, together with the line 62, a capillary gap 81, extending circumferentially, through which the solution which is low in fuel (liquid) can flow according to the arrows 83 and 84.The solution which is low in fuel is thus conducted by means of the supply line 69 into the absorber 32 and, due to the capillary action present, conveyed according to the arrows 83 and 84 into the given auxiliary gas conveyance line 62 of the absorber 32 in which the same slides downwards along its walls (arrow 84).\",\n \"The auxiliary gas is conducted according to the arrows 82 through the auxiliary gas conveyance line 62 and through a central longitudinal hole 80 of the capillary sleeve 79 and flows according to the arrows 34 to the supply line 61 which actively connects the absorber 32 to the gas heat exchanger 29.A separating plate 85 connected to a jacket tube 86 of the absorber 32 makes it possible that the solution which is low in fuel, said solution being located in the absorber 32, can be backed up to a certain level of liquid (FIG.\",\n \"2b: dotted line 71).\",\n \"A given auxiliary gas conveyance line 62 equipped with a capillary sleeve 79 is advantageous since, due to the capillary action present at the time in the corresponding capillary gap 81, a uniform distribution of the solution which is low in fuel to the individual auxiliary gas conveyance line 62 in the absorber 32 is guaranteed independently of the level, present at the time, of the solution which is low in fuel in the absorber 32 or on the amount of the solution which is low in fuel flowing through the supply line 69 into the absorber 32 according to the arrows 39.In particular in the case of an oblique position (not represented) of the absorber 32, it is avoided by means of the capillary sleeves 79, due to capillary action setting in, that several auxiliary gas conveyance lines 62 are completely filled with solution which is low in fuel and the remaining auxiliary gas conveyance lines 62 are not pressurized with solution which is low in fuel so that the absorber 32 would not be functional, at least not completely.\",\n \"According to a preferred form of embodiment not represented, each of the auxiliary gas conveyance line 62 can be provided with a plurality of projections acting as space holders at its free end projecting into the interior of the auxiliary gas conveyance line 62, by means of which projections a coaxial, with respect to the corresponding auxiliary gas conveyance lines 62, positioning of the respective capillary sleeve 79 is possible in a relatively simple manner.\",\n \"Projections of this type at the free end of the respective capillary sleeve 79 are preferably distributed uniformly over its circumference and spaced sufficiently far from one another that a free flow of the solution which is low in fuel through the capillary gap according to arrows 83 and 84 is guaranteed.\",\n \"Corresponding to another additional form of embodiment not represented, the lines 59 of the evaporator 24 are provided at their upper free end with a corresponding capillary sleeve 79 according to FIG.\",\n \"4.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297462"}}},{"rowIdx":98,"cells":{"text":{"kind":"list like","value":[["Acetyl glucosaminyl inositol deacetylase, a mycothiol biosynthetic enzyme, and methods of use","The present invention provides a family of bacterial acetyl glucosaminyl inositol deacetylases (MshB) with deacetylase activity against acyl glucosaminyl inositol and which play a key role in mycothiol biosynthesis.","The invention deacetylases are characterized by a conserved 100 amino acid N-terminal region and three highly conserved histidine-containing regions and by having deacetylase activity as well as amide hydrolase activity.","The invention further provides methods for using the invention deacetylases in drug screening assays to determine compounds that inhibit activity.","The invention provides for treatment of actino-mycete infections in mammals using antibiotics that inhibit production or activity of MshB and thereby reduce the production of mycothiol and the virulence of the infecting bacteria."],["1.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, b) three domains of conservation, wherein two of the domains contain conserved histidine residues, and c) deacetylase activity against acetyl glucosaminyl inositol.","2.The purified deacetylase of claim 1, wherein the deacetylase hydrolyzes a C2-amide bond of the glucosaminyl inositol moiety.","3.The purified deacetylase of claim 1, wherein the acetyl glucosaminyl inositol is a precursor of mycothiol.","4.The purified deacetylase of claim 1, wherein the acetyl glucoasaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).","5.The purified deacetylase of claim 1, wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6, conservative variations thereof, and any combination of two or more thereof.","6.The purified deacetylase of claim 1, wherein the deacetylase is derived from an actinomycetes.","7.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis.","8.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. tuberculosis.","9.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. leprae.","10.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. bovis.","11.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum.","M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.","12.The purified deacetylase of claim 6, wherein the bacterium is selected from the group consisting of Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.","13.The purified deacetylase of claim 1, wherein the deacetylase has amidase activity against acyl glucosaminyl inositols.","14.The purified deacetylase of claim 1, wherein the deacetylase has an amino acid sequence as set forth in SEQ ID NO:1 15.The purified deacetylase of claim 1, wherein the deacetylase is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO:7.16.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, b) one or more domains of conservation containing conserved metal chelating residues, and c) deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.","17.The purified deacetylase of claim 16, wherein the metal ion is selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+.","18.The purified deacetylase of claim 17 wherein the metal ion is Zn2+.","19.The purified deacetylase of claim 16, wherein the metal chelating residues are selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.","20.An antibody, or functional fragment thereof, that binds specifically to an deacetylase of claim 1.21.An isolated polynucleotide that encodes a deacetylase of claim 1.22.A vector containing a polynucleotide that encodes an deacetylase of claim 1.23.A cell transformed with a vector of claim 22.24.A method for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase, said method comprising: a) contacting a candidate compound with a deacetylase of claim 1 in the presence of an acyl glucosaminyl inositol under suitable conditions and b) determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity or amidase activity, wherein the substantial absence of the deacetylase activity or the amidase activity is indicative of a candidate compound that inhibits activity of the deacetylase.","25.The method of claim 24, wherein the deacetylase is an acetyl glucosaminyl inositol deacetylase.","26.The method of claim 24, wherein a breakdown product is a free amine.","27.The method of claim 26, wherein the free amine is GlcN-Ins.","28.The method of claim 24, wherein a breakdown product is acetate 29.The method of claim 24, wherein a breakdown product is a derivative of cysteine.","30.The method of claim 24, wherein the acyl glucosaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside.","31.The method of claim 24, wherein the acyl glucosaminyl inositol is an S-conjugate of mycothiol.","32.The method of claim 31, wherein the S-conjugate of mycothiol is the monobromobimane derivative of mycothiol (MSmB).","33.The method of claim 24, wherein the three domains in the deacetylase that contain conserved histidine residues have amino acid sequences selected from SEQ ID NOs: 3, 5, 6 and any combination of two or more thereof.","34.The method of claim 24, wherein the deacetylase is produced in an actinomycete.","35.The method of claim 34, wherein the actinomycete is M. smegmatis.","36.The method of claim 34, wherein the actinomycete is M. tuberculosis.","37.The method of claim 34, wherein the actinomycete is M. leprae.","38.The method of claim 34, wherein the actinomycete is M. bovis.","39.The method of claim 34, wherein the actinomycete is M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.","40.The method of claim 24, wherein candidate compound is a polypeptide, polynucleotide or small molecule.","41.A high throughput screening method for identifying inhibitors of the deacetylase of claim 1, said method comprising: a) contacting each of a plurality of candidate compounds with a deacetylase of claim 1 in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and b) determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine, wherein the substantial absence of binding of the marker to a reaction mixture is indicative of a candidate compound that inhibits activity of the deacetylase.","42.The method of claim 41, wherein the plurality of reaction mixtures are formed in the wells of a microtiter plate.","43.The method of claim 41, wherein the detectable marker is colorometric or fluorometric.","44.The method of claim 43 wherein the fluorometric marker is fluorescamine or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.","45.An inhibitor of the deacetylase of claim 1 wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.","46.The inhibitor of claim 45 wherein the inhibitor additionally chelates one or more metal ions metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.","47.A derivative of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.","48.The derivative of claim 47, wherein the reactive residue is a nitroso residue.","49.The derivative of claim 47, wherein the reactive residue is a nitroalkyl residue comprising 1 to 3 carbon atoms.","50.The derivative of claim 47, wherein the reactive residue is a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms.","51.The derivative of claim 47, wherein the reactive residue is a peroxyalkyl residue comprising 2 to 4 carbon atoms.","52.A live mutant actinomycete, whose genome comprises a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein said disruption results in said mutant actinomycetes exhibiting transient survival in mammalian white blood cells for an immune response-raising period of time.","53.The live mutant actinomycete of claim 52, wherein the period of time is from 1 to 30 days.","54.The live mutant actinomycete of claim 52, wherein the survival of the mutant actinomycetes in mammalian white blood cells does not exceed 30 days.","55.The live mutant actinomycete of claim 52, wherein the mutant actinomycete is derived from a pathogen selected from the group consisting of M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, and M. scrofulaceum.","56.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes.","is derived from M. smegmatis.","57.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. tuberculosis.","58.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. leprae.","59.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. bovis and said mammal is bovine.","60.A method for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells, said method comprising: introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity, wherein the intracellular presence of the inhibitor decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.","61.The method of claim 60, wherein the inhibitor inhibits intracellular production of the deacetylase.","62.The method of claim 60, wherein the inhibitor inhibits intracellular deacetylase activity of the deacetylase.","63.The method of claim 60, wherein the introducing comprises culturing the bacterium in the presence of the inhibitor.","64.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.","65.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide that hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.66.The method of claim 60, wherein the bacterium is an actinomycete and the inhibitor inhibits intracellular production of mycothiol.","67.The method of claim 60, wherein the bacterium is selected from the group consisting of the pathogenic bacteria M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M abscessus, and M. scrofulaceum.","68.The method of claim 60, wherein the bacterium is an actinomycete.","69.A method for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, said method comprising administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.","70.The method of claim 69, wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates a metal ion selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+, or otherwise binds the enzyme active site in the deacetylase.","71.The method of claim 70, wherein the moiety is ClCH2CONH—.","72.The method of claim 70 wherein the moiety is HONHCONH—.","73.The method of claim 70, wherein the moiety is HONHCOCH2—.","74.The method of claim 70, wherein the moiety is HOPO(CH3)NH—.","75.The method of claim 70, wherein the moiety is HOPO(CH3)nCH2—; wherein n=1-5.76.The method of claim 70 wherein the moiety is HSCH2(CH2)nNH—; wherein n=2-5.77.The method of claim 70 wherein the moiety is HS(CH2)nCONH—; wherein n=1-3.78.The method of claim 69 wherein the acetyl glucosaminyl inositol-producing bacterium is a mycothiol-producing bacterium.","79.A process for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), said method comprising: contacting an acyl glucosaminyl inositol with an deacetylase of claim 1 under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.","80.A method for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample, said method comprising: a) contacting a sample containing GlcNAc-Ins with a deacetylase of claim 1 under suitable conditions and b) determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.","81.The method of claim 80, wherein the amount of GlcN-Ins is determined by HPLC after labeling thereof with a fluorometric or calorimetric reagent.","82.The method of claim 81, wherein the fluorometric reagent is fluorescamine.","83.The method of claim 81, wherein the fluorometric reagent is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate."],[" BACKGROUND INFORMATION Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.","However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.","Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.","Actinomycetes, including Streptomyces and Mycobacteria , do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).","The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.","However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.","First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.","(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)","pp 517-551, John Wiley & Sons, et al.).","An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.","(1997) FEBS Lett.","409:221-222).","This enzyme has been sequenced (A. Norin, et al.","(1997) Eur.","J. Biochem.","248:282-289).","The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.","(1999) J. Amer.","Chem.","Soc.","120:11538-11539; M. P. Patel et al.","(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).","The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.","Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.","Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis , is a well-known problem faced by medical practitioners in treatment of bacterial diseases.","Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals."],[" SUMMARY OF THE INVENTION The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.","In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.","Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.","In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.","Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.","In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.","The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.","Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.","In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.","In still another embodiment, the present invention provides live mutant actinomycetes.","The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.","In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.","The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.","By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.","For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.","In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn 2+ , or otherwise binds the enzyme active site of the deacetylase.","In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.","In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins."],["FIELD OF THE INVENTION The present invention generally relates to a family of enzymatic compounds produced by bacteria and methods of their use in drug discovery and disease control, and more specifically to acetyl glucosaminyl inositol deacetylases and methods of their use.","BACKGROUND INFORMATION Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.","However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.","Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.","Actinomycetes, including Streptomyces and Mycobacteria, do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).","The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.","However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.","First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.","(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)","pp 517-551, John Wiley & Sons, et al.).","An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.","(1997) FEBS Lett.","409:221-222).","This enzyme has been sequenced (A. Norin, et al.","(1997) Eur.","J. Biochem.","248:282-289).","The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.","(1999) J. Amer.","Chem.","Soc.","120:11538-11539; M. P. Patel et al.","(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).","The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.","Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.","Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases.","Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals.","SUMMARY OF THE INVENTION The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.","In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.","Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.","In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.","Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.","In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.","The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.","Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.","In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.","In still another embodiment, the present invention provides live mutant actinomycetes.","The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.","In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.","The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.","By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.","For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.","In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.","In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.","In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.","BRIEF DESCRIPTION OF THE DRAWINGS FIGS.","1A and B show the nucleotide sequence of M. tuberculosis deacetylase gene mshB (Rv1170) (SEQ ID NO:7).","FIG.","1C shows the deduced amino acid sequence of M. tuberculosis GlcNAc-Ins deacetylase (SEQ ID NO:1) encoded by mshB.","FIG.","2 is a schematic drawing showing a map of plasmid pYA1170b employed to express Rv1170 in E. coli.","FIG.","3 is a chart showing the chemical structures of the substrates for assaying deacetylase and amidase activities of the invention acetyl glucosaminyl inositol deacetylases (MshB), such as MshB Rv1170 and homologs.","FIG.","4 is a graph showing growth of a mutant M. smegmatis (mutant 49) in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins.","Mycothiol (MSH) and MSH precursor contents in cells are expressed in micromoles per gram of residual dry weight (RDW).","FIG.","5 is a schematic representation of the proposed MSH biosynthesis pathway.","FIG.","6 is a chart showing alignment of the amino acid sequences of four homologs of MshB of M. tuberculosis H37Rv (Rv1170) GenBank accession number B70875 and M. leprae GenBank accession number CAC30445.Sequences for M. avium (The Institute for Genomic Research (TIGR)), and M. smegmatis (Sanger Center) are from unfinished genomes.","FIG.","7 is a graph showing MshB deacetylase activity with 0.1 mM GlcNAc-Ins in the presence of 1,10-phenanthroline.","FIG.","8A is a graph showing MshB deacetylase reaction specific activity with 0.8 mM GlcNAc-Ins as substrate in the presence of increasing concentrations of thiol inhibitors.","FIG.","8B is a graph showing MshB amidase reaction specific activity with 0.55 mM MSmB as substrate in the presence of increasing concentrations of inhibitors.","FIG.","9 is a map of the plasmid pYA1170C used to express the recombinant His-tagged acetyl glucosaminyl inositol deacetylase encoded by M. tuberculosis Rv1170.FIG.","10 is a graph showing percent survival of strains of M. smegmatis versus hydrogen peroxide concentration after treatment as described in Example 11 herein.","Open triangles=mutant TN3, which produces no MSH; open circles=chemical mutant 164, which produces a low residual MSH content; and darkened squares=high MSH content mc2 155.FIG.","11 is a graph showing percent survival over time of strains of M. smegmatis in murine macrophages as described in Example 11 herein.","Open circles=chemical mutant TN3 (0% MSH content), Experiment 1; open triangles=mutant TN3 (0% MSH content), Experiment 2; and darkened squares=mc2155 (100% MSH content).","DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there are provided a family of purified acetyl glucosaminyl inositol deacetylase polypeptides with enzymatic deacetylase activity for acetyl glucosaminyl inositol.","The invention acetyl glucosaminyl inositol deacetylases are characterized by having an N-terminal region with an amino acid sequence with 40% or greater sequence identity to an amino acid sequence as set forth in SEQ ID NO:2 and conservative variation thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.","SEQ ID NO:2 corresponds to amino acid residues 1-100 of amino acids of SEQ ID NO:1 (FIG.","1C).","The members of the family of invention deacetylases hydrolyze a C2-amide bond of acetyl glucosaminyl inositol moiety, such as N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).","Preferably the acetyl glucosaminyl inositol is a precursor of mycothiol (e.g., in a mycothiol producing bacterium).","In preferred embodiments the three histidine-containing conserved domains in the invention deacetylases are selected from VHAHPDDE (SEQ ID NO:3), which corresponds to amino acids 10 through 17 of SEQ ID NO:1; VTCTLGEXGEV (SEQ ID NO:4), which corresponds to amino acids 38 through 49 of amino acid SEQ ID NO:1; YDPXGGYGHPDH (SEQ ID NO:5), which corresponds to amino acids 136 through 147 of SEQ ID NO:1; and AXXAHATQ (SEQ ID NO:6), which corresponds to amino acids 240 through 247 of amino acid SEQ ID NO:1 (FIG.","1C) wherein X is any amino acid; and conservative variations thereof, or any combination of any two or more thereof (See FIG.","6).","Members of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides are metalloproteases.","Accordingly, the invention further provides purified acetyl glucosaminyl inositol deacetylases that are characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.","The invention deacetylases have been shown to contain an active site that contains a metal ion selected from the group consisting of Mn2+, Ni2, Cd2+, Co2+, and Zn2+, with the preferred metal ion being Zn2+.","The metal chelating residues in the invention deacetylases are preferably selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.","In preferred embodiments, the invention deacetylases have additional activity as amidases, catalyzing the hydrolysis of acyl glucosaminyl inositols.","As used herein, the term “acyl glucosaminyl inositol” means substrate compounds for the invention family of deacetylases having the chemical formula R—CONH-GlcN-Ins wherein R═CH3(CH2)n— where n=0-6.Included in the group of substrate compounds is the natural substrate, acetyl-GlcN-Ins, where n=0.Members of this group of substrate compounds are derived from an alkanoic acid.","In addition, compounds having the chemical structure R—CONH-GlcN-Ins wherein R=aryl-(CH2)n, where n=0-6 are encompassed by the term.","Members of this group of substrate compounds are derived from an aryl alkanoic acid such as phenyl-(CH2)nCOOH.","The acyl glucosaminyl inositols of the above chemical formula can also be derived from the reaction of GlcN-Ins with any commercial acid chloride in the form RCOCl, wherein R is: o-tolyl-, 4-ethylphenyl-, 4-propylphenyl-, 4-biphenyl-, 3,4-dimethoxyphenyl-, 3,4,5-trimethoxyphenyl-, 2-furyl-, and the like.","The acyl glucosaminyl inositols of the above chemical formula can also be derived from an amino acid or an N-acetyl amino acid.","Preferred amino acids include N-acetylcysteinyl-S—R′ or cysteinyl-S—R′, wherein R′ is an organic group attached to the cysteine sulfur, such as may be derived from commercial thiol labeling reagents like 2-bromoacetophenone, monobromobinane, N-ethylmaleimide, cerulenin, 7-diethylamino-3-(4′-maleimidyl phenyl)-4-methyl coumarin, 3(N-maleimidopropionyl)biocytin, and the like.","The studies described herein further elaborate the pathway involved in MSH biosynthesis.","The present studies demonstrate that GlcNAc-Ins is a major intracellular MSH component in M. smegmatis and is converted to GlcN-Ins by GlcNAc-Ins deacetylase.","This conversion defines the second step in MSH biosynthesis.","The discovery described herein that Rv1170 is the gene encoding the deacetylase in the M. tuberculosis genome represents the first gene of the MSH biosynthesis pathway to be identified.","Assuming that a single enzyme produces GlcNAc-Ins, it is proposed that the genes for the MSH biosynthesis pathway be designated mshA, mshB, mshC, and mshD, with the corresponding enzymes labeled as shown in FIG.","5.Identification of mshA, mshC, and mshD would be greatly simplified if they were clustered with mshB in a single operon, but inspection of the gene assignments and open reading frames surrounding mshB (Rv1170) in the M. tuberculosis genome indicates that this is not the case.","All mycobacteria thus far examined have high MSH content (G. L. Newton et al.","(1996) J. Bacteriol.","178:1990-1995) and, based on the studies described herein, are expected to have an ortholog of the MSH biosynthesis enzyme MshB (Rv1170).","The available unfinished mycobacterial genome databases yield homologs close to Rv1170 for Mycobacterium leprae (Sanger Centre), Mycobacterium bovis (Sanger Centre), M tuberculosis CDC1551 (The Institute for Genomic Research [TIGR]), M. smegmatis (TIGR), and Mycobacterium avium (TIGR).","This sequence homology (FIG.","6) indicates that MSH biosynthesis in these organisms utilizes a GlcNAc-Ins deacetylase (MshB) in the same manner as that described here for M. smegmatis.","Other actinomycetes that produce MSH are also expected to have a GlcNAc-Ins deacetylase (MshB) gene homologous to Rv1170.Sequence BLAST searches to determine molecules with amino acid homology to the Rv1170 sequence of SEQ ID NO:1 (FIG.","1C) also show M. tuberculosis homolog Rv1082 (mycothiol S-conjugate deacetylase) and the Rv1082 orthologs from M. leprae, M. bovis (Sanger Centre), M. tuberculosis CDC1551 (TIGR), M. smegmatis (TIGR), and M. avium (TIGR) to be close homologs.","Sequences with partial homology to Rv1170 were also found in the yeast, rat, and human genome databases.","These genes code for PIG-L, the second enzyme in glycosylphosphatidylinositol (GPI) anchor biosynthesis (Nakamura et al., (1997) J. Biol.","Chem.","272:15834-15840), and catalyze the deacetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI).","The studies described in the Examples herein indicate that the invention deacetylase is rather specific for GlcNAc-Ins and is not a broad spectrum deacetylase.","For example, removal of the inositol residue from the substrate GlcNAc-Ins resulted in a more than 300-fold decrease in reactivity (i.e., GlcNAc-Ins versus GlcNAc) (Table 1).","Also, the studies described herein (Table 1) show that neither MSH nor MSmB is deacetylated by the invention deacetylases.","Therefore, the AcCys moiety of MSH is inert to the deacetylase.","This finding shows that the invention deacetylase has no significant activity for the degradation of MSH.","The invention deacetylase appears to be the control point for MSH biosynthesis.","In cells tested, a very large endogenous pool of its substrate GlcNAc-Ins (12 to 15 μmol per g of residual dry weight (RDW)) was found (Table 2), but quite low levels were found of its product GlcN-Ins (˜0.2 μmol per g of RDW) (Table 2) and of the final intermediate Cys-GlcN-Ins (<0.006 μmol per g of RDW) leading to MSH.","This finding shows that substantial quantities of MSH can be produced upon demand from the endogenous pool of GlcNAc-Ins under control of the invention family of acetyl glucosaminyl inositol deacetylases.","If a particular member of the invention family of polypeptide deacetylases hydrolyzes its substrate (GlcNAc-Ins), cleavage of the substrate molecule will form breakdown products wherein one product is a free amine (e.g., 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins)) and the other product is acetate.","If a particular member of the invention family of polypeptide deacetylases has activity as an amide hydrolase (amidase), cleavage of the substrate acyl glucosaminyl inositol will form breakdown products wherein one product is a carboxylic acid, and the other product is a free amine (e.g., GlcN-Ins).","If the substrate amide is a mycothiol-derived amide, one of the breakdown products will be GlcN-Ins and the other breakdown product will be a sulfur-containing carboxylic acid, such as an AcCys S-conjugate.","AcCys S-conjugates are termed mercapturic acids, the final excreted product in the mercapturic acid pathway of glutathione-dependent detoxification in mammals.","Mycothiol (1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside) (MSH) is present in a variety of actinomycetes and plays an essential role in a pathway of detoxification in such bacteria.","Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes.","In the mycothiol-dependent detoxification process in actinomycetes, an alkylating agent is converted to a S-conjugate of mycothiol, the latter is cleaved to release a mercapturic acid, and the mercapturic acid is excreted from the cell (Newton et al.","(2000) Biochemistry 39:10739-10746).","It has been discovered that invention acetyl glucosaminyl inositol deacetylases participate in the pathway of mycothiol biosynthesis in which substrate GlcNAc-Ins is an intermediate.","In the mycothiol pathway as shown schematically in FIG.","5, GlcNAc-Ins is converted to GlcN-Ins by the invention deacetylase.","In fact, the studies disclosed herein have shown that the invention deacetylases are the critical step in the production of mycothiol in actinomycetes.","For example, it has been shown by the studies described herein (Example 10) that MshB is inhibited by mycothiol in a manner consistent with a negative feedback loop dependent upon the level of mycothiol for control of mycothiol biosynthesis in the cell.","Thus, a falling level of mycothiol in the cell available for detoxification of intracellular toxins increases activity of the deacetylase so that substrate GlcNAc-Ins will be cleaved by the expressed deacetylase, in turn, providing the GlcN-Ins moiety necessary for mycothiol production.","A member of the invention family of polypeptide deacetylases shown to be responsible for cleavage of GlcNAc-Ins has been cloned from M. tuberculosis gene Rv1170 and has an amino acid sequence as set for in SEQ ID NO:1 (shown in FIG.","1C; see also GenBank Accession No.","B70875) and a nucleotide sequence as set forth in SEQ ID NO:7 (shown in FIG.","1; see also nucleic acid residues 4222-5133 of GenBank Accession No.","AL010186).","The N-terminal region 100 residues of this newly discovered GlcNAc-Ins deacetylase has the amino acid sequence as shown in SEQ ID NO:2.Members of invention family of acetyl glucosaminyl inositol deacetylases are formed in vivo by bacteria as part of a mycothiol biosynthesis pathway, most usually in bacteria characterized by intracellular production of mycothiol.","Additional bacteria from which the invention deacetylase polypeptides can be derived include actinomycetes, such as M. smegmatis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC) (Holzman, in Tuberculosis ed.","by Rom and Gary (Little, Brown, and Company, 1996) Chapter 56), M. ulcerans, M. abscessus, or M. scrofulaceum, and the like.","Homologous non-mycobacterial deacetylase proteins can also be derived from the antibiotic producers Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei, and the polyketide erythromycin antibiotic producer Saccharopolyspora erythraea.","Inhibitors of the invention acetyl glucosaminyl inositol deacetylases are particularly well suited as antibiotics since mycothiol production will cease in the absence of the product GlcN-Ins produced by activity of the invention deacetylases.","Accordingly, in another embodiment according to the present invention, there are provided methods for identifying an inhibitor of an invention acetyl glucosaminyl inositol deacetylase, MshB.","The invention methods comprise contacting a candidate compound with a member of the invention family of deacetylases in the presence of a substrate for the enzyme under suitable conditions and determining the presence or absence of breakdown products of the substrate indicative of deacetylase (or optionally S-conjugate amidase) activity.","For example, if the test compound is a putative inhibitor of amide hydrolase activity of the invention polypeptide deacetylase, the absence of acetate or other carboxylic acid and/or free amine, such as GlcN-Ins, as breakdown products indicates the candidate compound is an inhibitor of the activity of the invention polypeptide as a deacetylase.","The preferred substrate for testing for deacetylase activity of the invention polypeptides is the acetyl glucosaminyl inositol (GlcNAc-Ins).","On the other hand, if the test compound is a putative inhibitor of MshB, the appearance of free amine, such as GlcN-Ins, and/or acetate or carboxylate indicates that the candidate compound is not an inhibitor of the activity of the invention deacetylase as a deacetylase or as an amidase of an acyl glucosaminyl inositol.","A preferred substrate for use in screening for inhibitors of deacetylase activity is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).","Substantial absence of the breakdown products of enzymatic activity is indicative of a candidate compound that inhibits enzymatic activity of the deacetylase.","In a preferred embodiment, the deacetylase used in the invention method is an acetyl glucosaminyl inositol deacetylase and the method is designed to assay for production of free amine.","Such assays can be conducted en masse using a high throughput screening method for identifying inhibitors of the invention deacetylase.","For example, a plurality of candidate compounds can be contacted with an invention deacetylase in the presence of a substrate for the enzyme (e.g.","an acyl glucosaminyl inositol) under suitable conditions (e.g., as shown in the Examples herein) for activity of the deacetylase.","Binding of a marker selected to bind to a breakdown product can then be assayed using any technique known in the art for high throughput screening.","For example, a colorometric or fluorometric marker that binds to free amine can be used to screen a plurality of reaction mixtures formed in the wells of a microtitre plate (Wong, et al.","(2001) Anal.","Biochem.","290:338-346).","A preferred fluorometric marker for use in the invention high throughput screening methods is fluorescamine, which binds to free amine.","In another embodiment of the invention, methods are provided for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample by contacting a sample containing GlcNAc-Ins with an invention deacetylase under suitable conditions and determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.","The amount of GlcN-Ins in the sample can be determined by HPLC after labeling thereof with a fluorometric or colorimetric reagent, such as 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (sold as AccQFluor® reagent by Waters), and the like.","In an alternative embodiment of the invention, methods are provided for decreasing the virulence in mammalian cells of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium, such as an actinomycete.","In the invention method for decreasing the virulence of pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria, an inhibitor of acetyl glucosaminyl inositol deacetylase (for example, one identified by the above-described screening method) is introduced into the bacterium.","Intracellular uptake by the treated bacterium of the inhibitor results in decreased activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.","For example, for treatment of isolated mammalian cells, the introducing can comprise culturing the bacterium in the presence of the inhibitor.","Alternatively, for treatment of mammalian cells contained in a living organism, the inhibitor may be administered systemically to the living organism.","Pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria whose virulence can be reduced according to the invention methods include such actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis particularly in bovine subjects), M. intracellulare, M. africanum, and M. marinarum.","M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.","The inhibitors used in the invention methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium may either inhibit intracellular production of the deacetylase or inhibit intracellular deacetylase activity of the deacetylase.","In one embodiment according to the present invention, there are provided inhibitors of the members of the invention family of deacetylase polypeptides.","The invention inhibitors are characterized by being derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+ or otherwise binds the enzyme active site.","The invention inhibitors may also chelate one or more metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.","The following are examples of moieties that can substitute for the amino group in GlcN-Ins in the invention inhibitors: ClCH2CONH— HONHCONH— HONHCOCH2— HOPO(CH3)NH— HOPO(CH3)nCH2—; wherein n=1-5 HSCH2(CH2)nNH—; wherein n=2-5 HS(CH2)nCONH—; wherein n=1-3.In another embodiment according to the present invention, there are provided derivatives of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress (e.g., including nitrosative stress) so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.","Examples of reactive residues that can be attached to the amino group of GlcN-Ins to cause the compound to be selectively toxic to mycothiol-producing actinomycetes by intracellular concentration include, but are not limited to a nitroso residue, a nitroalkyl residue comprising 1 to 3 carbon atoms, a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms, or a peroxyalkyl residue comprising 2 to 4 carbon atoms.","In another embodiment according to the present invention, the invention inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or 80% or more sequence identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.","In another embodiment the anti-sense oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.For example, in one embodiment, the candidate compound inhibits intracellular production or activity of the acetyl glucosaminyl inositol deacetylase.","A presently preferred drug candidate for screening in live bacteria for activity that inhibits intracellular production or activity of acetyl glucosaminyl inositol deacetylase is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence with 40% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:2, or a conservative variation thereof, for example, 50%, 55%, 60% or 65% sequence identity.","Suitable conditions for conducting invention drug screening methods are well known in the art and are described, for example, in the Examples hereinbelow.","Alternatively, an anti-sense oligonucleotide can be designed to hybridize under in vivo conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2, or contains an amino acid segment as set forth in SEQ ID NOs:3, 4, 5, and 6, or a conservative variation thereof.","The anti-sense oligonucleotide can comprise from about 10 to about 60 nucleic acid residues, for example from 10 to about 50, or from 10 to about 40, 30 or 20 nucleic acid residues.","“Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing.","Hybridization reactions can be sensitive and selective so that a particular sequence of interest will join with a complementary strand even in samples in which it is present at low concentrations.","Suitable intracellular conditions for hybridization of an anti-sense oligonucleotide to messenger RNA will be determined by the particular bacterium used in the invention method.","In general, the pH, temperature and salt concentration must be comparable to intracellular conditions in the test bacterium.","In yet another embodiment according to the present invention, there are provided live mutant actinomycetes, whose genomes comprise a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene and thereby prevents mycothiol synthesis.","Disruption of genes for mycothiol biosynthesis in mycobacteria reduces their survival in mammalian macrophages (FIG.","11).","Disruption of the endogenous acetyl glucosaminyl inositol deacetylase gene will prevent function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids should be substantially unaffected.","As a result, invention live mutant actinomycetes exhibit the phenotype of transient survival in mammalian white blood cells, such as murine or human white blood cells, for an immune response raising period of time.","Such genetically engineered live mutant actinomycetes will survive in mammalian white blood cells for a period of time from 1 to 30 days, for example from 4 to 25 days or from 5 to 20 days, but in no event for more than 30 days.","Due to lack of intracellular acetyl glucosaminyl inositol deacetylase the invention live mutant bacterium will fail to produce mycothiol.","Hence, the mutant live bacterium is unable to survive the oxidative stress inherent in the intracellular environment of mammalian white blood cells long enough to establish infection in the cells or to establish infection in an immunocompetent mammal containing such white blood cells.","Thus, invention live mutant actinomycetes possess a combination of features desired for a vaccine effective in mammals against infection by such pathogenic actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.","An individual (e.g., an animal, such as a mouse, a farm animal or a human) to which the live mutant is administered according to a protocol appropriate for inducing a protective immune response and who has not previously been infected by the counterpart wild type will mount an immune response to the vaccine, for example an immune response sufficient to protect the individual against future infection by the corresponding wild-type live pathogen.","Alternatively, the invention live mutant actinomycetes are useful as a research tool to investigate the properties desirable in a live mutant vaccine.","In still another embodiment according to the present invention, there are provided processes for preparation of GlcN-Ins by contacting an N-acyl glucosaminyl inositol under suitable conditions with an acetyl glucosaminyl inositol deacetylase so as to hydrolyze the amide bond therein to obtain stereochemically pure α(1-1) GlcN-Ins (1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside).","For example, the N-acyl glucosaminyl inositol can be a mycothiol S-conjugate, such as the bimane derivative of mycothiol.","The deacetylase cleaves the N-acyl glucosaminyl inositol, freeing GlcN-Ins as one of the cleavage breakdown products.","GlcN-Ins has utility in conducting research regarding deacetylase activity and mycothiol biochemistry in bacteria, development of products and procedures for overcoming the antibiotic resistance of pathogenic bacteria, such as actinomycetes, and as a precursor for formation of acyl glucosaminyl inositol derivatives and inhibitors of deacetylases thereof as disclosed herein.","A “conservative variation” in an amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.","A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).","One or more amino acids can be deleted, for example, from an deacetylase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.","For example, carboxyl-terminal amino acids that are not required for deacetylase or deacetylase activity of the invention polypeptides can be removed.","Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.","Such vectors are used to incorporate DNA sequences of the invention.","In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted eukaryotic genetic sequence are used in connection with the host.","The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells.","In addition to expression vectors known in the art such as bacterial, yeast and mammalian expression systems, baculovirus vectors may also be used.","One advantage to expression of foreign genes in this invertebrate virus expression vector is that it is capable of expression of high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts.","Baculovirus vectors and the appropriate insect host cells used in conjunction with the vectors will be known to those skilled in the art.","The term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the invention acetyl glucosaminyl inositol deacetylase genetic sequences.","Such expression vectors contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host.","The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.","Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol.","Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.","The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).","The vector may include a phenotypically selectable marker to identify host cells which contain the expression vector.","Examples of markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin (β-lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase).","Examples of such markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and xanthine guanine phosphoribosyltransferse (XGPRT, gpt).","The isolation and purification of host cell expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody.","Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art.","Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by electroporation or the CaCl2 method using procedures well known in the art.","Alternatively, MgCl2 or RbCl could be used.","Where the host used is a eukaryote, various methods of DNA transfer can be used.","These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or the use of virus vectors.","Eukaryotic cells can also be cotransformed with DNA sequences encoding the polypeptides of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.","Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.","(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).","Examples of mammalian host cells include COS, BHK, 293, and CHO cells.","Eukaryotic host cells may also include yeast.","For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells.","Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987).","The invention provides antibodies that are specifically reactive with invention deacetylase polypeptides or fragments thereof.","Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.","Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).","Monoclonal antibodies specific for acetyl glucosaminyl inositol deacetylase polypeptide can be selected, for example, by screening for hybridoma culture supernatants that react with acetyl glucosaminyl inositol deacetylase polypeptides, but do not react with other bacterial deacetylases.","Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.","Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).","The term “antibody” as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2 and Fv, which are capable of binding the epitopic determinant.","These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.","Methods of making these fragments are known in the art.","(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).","As used in this invention, the term “epitope” means any antigenic determinant on an antigen to which the paratope of an antibody binds.","Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.","Antibodies that bind to acetyl glucosaminyl inositol deacetylase polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.","The polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis and can be conjugated to a carrier protein, if desired.","Such commonly used carriers, which are chemically coupled to the peptide, include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.","The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).","If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.","Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated herein by reference).","It is also possible to use the anti-idiotype technology to produce monoclonal antibodies that mimic an epitope.","For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the “image” of the epitope bound by the first monoclonal antibody.","In yet other preferred embodiments, the recombinant acetyl glucosaminyl inositol deacetylase polypeptide is a fusion protein further-comprising a second polypeptide portion having an amino acid sequence from a protein unrelated to the acetyl glucosaminyl inositol deacetylase.","Such fusion proteins can be functional in a two-hybrid assay.","Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an acetyl glucosaminyl inositol deacetylase polypeptide, or a fragment thereof, having an amino acid sequence at least 60% homologous to one of SEQ ID NOs:2, 3, 4 or 5.In a more preferred embodiment, the nucleic acid encodes a protein having an amino acid sequence 40% or more homologous to SEQ ID NO:2, more preferably at least 50% homologous to SEQ ID NO:2, and most preferably at least 65% homologous to SEQ ID NO:2.In another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 24 consecutive nucleotides encoding SEQ ID NO:3.In a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:4.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:5.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:6; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:6.Furthermore, in certain embodiments, the acetyl glucosaminyl inositol deacetylase nucleic acid will comprise a transcriptional regulatory sequence, e.g.","at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the acetyl glucosaminyl inositol deacetylase-gene sequence so as to render the recombinant acetyl glucosaminyl inositol deacetylase gene sequence suitable for use as an expression vector.","The present invention also features transgenic non-human organisms, e.g.","live mutant actinomycetes, which either express a heterologous acetyl glucosaminyl inositol deacetylase gene, or in which expression of their own acetyl glucosaminyl inositol deacetylase gene is disrupted.","In addition to the other utilities of such organisms disclosed herein, such a transgenic organism has utility for overproduction of acetyl glucosaminyl inositol needed for screening (particularly high throughput screening) for compounds that inhibit acetyl glucosaminyl inositol deacetylase activity in mycothiol-producing bacteria.","The present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or anti-sense sequence encoding one of the amino acid sequences encompassed by SEQ ID NOs:2, 3, 4 5, or 6, or naturally occurring mutants thereof.","Yet another aspect of the invention pertains to a peptidomimetic that binds to or interferes with an acetyl glucosaminyl inositol deacetylase polypeptide and inhibits its binding to or cleavage of substrate acyl glucosaminyl inositols.","For example, a preferred peptidomimetic is an analog of a peptide having the sequence of one of the SEQ ID NOs.","1, 2, 3, 4, 5, or 6.Non-hydrolyzable peptide analogs of such residues can be generated using, for example, benzodiazepine, azepine, substituted gama-lactam rings, keto-methylene pseudopeptides, beta-turn dipeptide cores, or beta-aminoalcohols.","Other features and advantages of the invention will be apparent from the detailed description herein, and from the claims.","The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.","Such techniques are explained fully in the literature.","See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.","by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al.","U.S. Pat.","No.","4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.","1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.","1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.","154 and 155 (Wu et al.","eds.","), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).","As used herein, the term “actinomycetes” and “an actinomycete” encompasses any bacterium of the order Actinomycetales.","As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).","The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or anti-sense) and double-stranded polynucleotides.","As used herein, the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an open reading frame encoding an invention acetyl glucosaminyl inositol deacetylase, including both exon and (optionally) intron sequences.","The term “intron” refers to a DNA sequence present in a given acetyl glucosaminyl inositol deacetylase gene which is not translated into protein and is generally found between exons.","“Homology” refers to sequence similarity between two peptides or between two nucleic acid molecules.","Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.","When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position.","Percent homology between sequences is a function of the number of matching or homologous positions shared by the sequences.","The sequence data of a test clone is aligned to the sequences in the database or databases using algorithms designed to measure homology between two or more sequences.","Sequence alignment methods include, for example, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), and FASTA (Person & Lipman, 1988).","For example, optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.","The term “transfection” or “transforming” and grammatical equivalents thereof, refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.","“Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of one of the invention family of acetyl glucosaminyl inositol deacetylases.","“Cells” or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context.","These terms include the immediate subject cells that express the acetyl glucosaminyl inositol deacetylases of the present invention, and, of course, the progeny thereof.","It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment.","However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell.","In the present case, such a characteristic might be the ability to produce a recombinant acetyl glucosaminyl inositol deacetylase polypeptide.","As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.","The term “expression vector” includes plasmids, cosmids or phages capable of synthesizing the subject acetyl glucosaminyl inositol deacetylase polypeptide encoded by the respective recombinant gene carried by the vector.","Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.","In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.","Moreover, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.","“Transcriptional regulatory sequence” is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, as well as polyadenylation sites, which induce or control transcription of protein coding sequences with which they are operably linked.","In preferred embodiments, transcription of a recombinant acetyl glucosaminyl inositol deacetylase gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in a cell-type in which expression is intended.","It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences that control transcription of the naturally occurring form of the regulatory protein.","As used herein, a “transgenic organism” is any organism, preferably a bacteria in which one or more of the cells of the organism contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.","The nucleic acid is introduced into the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or a vector.","The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.","This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.","In the typical transgenic organisms described herein, the transgene causes cells to express a recombinant form of the subject acetyl glucosaminyl inositol deacetylase polypeptides.","As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., an acetyl glucosaminyl inositol deacetylase polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic organism or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic organism or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the organism's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).","A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.","The term “evolutionarily related to”, with respect to nucleic acid sequences encoding acetyl glucosaminyl inositol deacetylase polypeptides, refers to nucleic acid sequences that have arisen naturally in an organism, including naturally occurring mutants.","The term also refers to nucleic acid sequences which, while derived from a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide.","One aspect of the present invention pertains to an isolated nucleic acid comprising the nucleotide sequence encoding an acetyl glucosaminyl inositol deacetylase polypeptide, fragments thereof encoding polypeptides having acetyl glucosaminyl inositol deacetylase activity, and/or equivalents of such nucleic acids.","The term-nucleic acid as used herein is intended to include such fragments and equivalents.","The term equivalent is understood to include nucleotide sequences encoding functionally equivalent acetyl glucosaminyl inositol deacetylase polypeptides or functionally equivalent peptides having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide such as described herein.","Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will also include sequences that differ from the nucleotide sequence encoding native acetyl glucosaminyl inositol deacetylases due to the degeneracy of the genetic code.","Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27° C. below the melting temperature of the DNA duplex formed in about 1 M salt) to the nucleotide sequence of an acetyl glucosaminyl inositol deacetylase gene, such as that as set forth in SEQ ID NO:7, particularly those segments encoding the polypeptides shown in one of SEQ ID NOs.","2, 3, 4, 5 or 6, and conservative variations thereof.","In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to such nucleotide sequences.","The term “isolated” or “purified” as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.","For example, an isolated nucleic acid encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the acetyl glucosaminyl inositol deacetylase gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.","The term isolated or purified as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.","Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state.","In yet another embodiment, the nucleic acid of the invention encodes a peptide having an amino acid sequence as shown SEQ ID NO:1.Preferred nucleic acids encode a peptide having acetyl glucosaminyl inositol deacetylase polypeptide activity and being 40% or more homologous, more preferably 50% homologous and most preferably 65% homologous with an amino acid sequence as set forth in SEQ ID NO:2 (encoded by a nucleic acid sequence as set forth in SEQ ID NO:7).","Nucleic acids that encode peptides having an activity of an invention acetyl glucosaminyl inositol deacetylase polypeptide are also within the scope of the invention.","Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes an acetyl glucosaminyl inositol deacetylase polypeptide having all or a portion of an amino acid sequence shown in one of SEQ ID NOs.","2, 3, 4, 5 or 6.Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Isolated nucleic acids that differ from the nucleotide sequences disclosed herein due to degeneracy in the genetic code are also within the scope of the invention.","For example, more than one triplet designates a number of amino acids.","Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of the protein.","However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject acetyl glucosaminyl inositol deacetylase polypeptides will exist among prokaryotic cells.","One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding a particular member of the acetyl glucosaminyl inositol deacetylase polypeptide family may exist among individuals of a given species due to natural allelic variation.","Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.","Fragments of the nucleic acid encoding a biologically active portion of the subject acetyl glucosaminyl inositol deacetylase polypeptides are also within the scope of the invention.","As used herein, a fragment of the nucleic acid encoding an active portion of an acetyl glucosaminyl inositol deacetylase polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, for example, the deacetylase polypeptides represented in nucleic acid SEQ ID NO:1, and which encodes a peptide which retains at least a portion of the biological activity of the full-length protein (i.e., a peptide capable of acetyl glucosaminyl inositol deacetylase activity) as defined herein, or alternatively, which is functional as an antagonist of the deacetylase activity of the full-length protein.","Nucleic acid fragments within the scope of the invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species, e.g.","for use in screening protocols to detect homologs of the subject acetyl glucosaminyl inositol deacetylase polypeptides.","Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant peptides.","As indicated by the examples set out below, a nucleic acid encoding a peptide having an activity of an invention deacetylase polypeptide may be obtained from mRNA or genomic DNA present in any of a number of antibiotic-producing or pathogenic bacteria, particularly actinomycetes, in accordance with protocols described herein, as well as those generally known to those skilled in the art.","A cDNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide, for example, can be obtained by isolating total mRNA from a bacterial cell.","Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.","A gene encoding an acetyl glucosaminyl inositol deacetylase polypeptide can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.","Another aspect of the invention relates to the use of an “anti-sense” isolated nucleic acid.","As used herein, an “anti-sense” inhibition of endogenous production of an acetyl glucosaminyl inositol deacetylase molecule is carried out by administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g.","bind) under intracellular conditions, with the cellular mRNA and/or genomic DNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide so as to inhibit expression of that protein or a constituent thereof, e.g.","by inhibiting transcription and/or translation.","The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.","In general, “anti-sense” therapy refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.","An anti-sense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the transformed cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an acetyl glucosaminyl inositol deacetylase polypeptide.","Alternatively, the anti-sense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one of the subject acetyl glucosaminyl inositol deacetylase proteins.","Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g.","exonucleases and/or endonucleases, and is therefore stable in vivo.","Exemplary nucleic acid molecules for use as anti-sense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.","Nos.","5,176,996; 5,264,564; and 5,256,775).","Additionally, general approaches to constructing oligomers useful in anti-sense techniques have been reviewed, for example, by van der Krol et al.","(1988) Biotechniques 6:958-976; and Stein et al.","(1988) Cancer Res 48:2659-2668.In addition, the oligomers of the invention may be used as reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind.","Such diagnostic tests are described in further detail below.","This invention also provides expression vectors comprising a nucleotide sequence encoding a member of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides and operably linked to at least one regulatory sequence.","Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence.","Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.","Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements.","Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).","For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the acetyl glucosaminyl inositol deacetylase polypeptides of this invention.","Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.","It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.","Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.","As will be apparent, the subject gene constructs can be used to cause expression of the subject acetyl glucosaminyl inositol deacetylase polypeptides in cells propagated in culture, e.g.","to produce proteins or peptides, including fusion proteins or peptides, for purification.","This invention also pertains to a host cell transfected with a recombinant acetyl glucosaminyl inositol deacetylase gene in order to express a polypeptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.","The host cell may be any prokaryotic or eukaryotic cell.","For example, an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells.","Other suitable host cells are known to those skilled in the art.","Another aspect of the present invention concerns recombinant acetyl glucosaminyl inositol deacetylase polypeptides that have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide, or which are naturally occurring mutants thereof.","The term “recombinant protein” refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the acetyl glucosaminyl inositol deacetylase polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.","Moreover, the phrase “derived from”, with respect to a recombinant gene encoding the recombinant acetyl glucosaminyl inositol deacetylase polypeptide, is meant to include within the meaning of “recombinant protein” those proteins having an amino acid sequence of a native acetyl glucosaminyl inositol deacetylase polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions of a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide of a organism.","The present invention further pertains to methods of producing the subject acetyl glucosaminyl inositol deacetylase polypeptides.","For example, a host cell transfected with expression vector encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptide can be cultured under appropriate conditions to allow expression of the peptide to occur.","The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide.","Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.","A cell culture includes host cells, media and other byproducts.","Suitable media for cell culture are well known in the art.","The peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the subject acetyl glucosaminyl inositol deacetylase polypeptides.","Thus, a nucleotide sequence derived from the cloning of an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention, encoding all or a selected portion of the protein, can be used to produce a recombinant form of the protein via microbial cellular processes.","The recombinant acetyl glucosaminyl inositol deacetylase polypeptide can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in bacterial cells.","Expression vehicles for production of a recombinant acetyl glucosaminyl inositol deacetylase polypeptide include plasmids and other vectors.","For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.","A number of vectors exist for the expression of recombinant proteins in yeast.","For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al.","(1983) in Experimental Manipulation of Gene Expression, ed.","M. Inouye Academic Press, p. 83, incorporated by reference herein).","These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.","In addition, drug resistance markers such as ampicillin can be used.","The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.","For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.","by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.In some instances, it may be desirable to express the recombinant acetyl glucosaminyl inositol deacetylase polypeptide by the use of a baculovirus expression system.","Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).","This invention further contemplates a method of generating sets of combinatorial mutants of the present acetyl glucosaminyl inositol deacetylase polypeptides, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g.","homologs) that are functional in cleaving acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide molecules.","In a representative embodiment of this method, the amino acid sequences for a population of acetyl glucosaminyl inositol deacetylase polypeptide homologs are aligned, preferably to promote the highest homology possible.","Such a population of variants can include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation.","Amino acids that appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.","The presence or absence of amino acids from an aligned sequence of a particular variant is relative to a chosen consensus length of a reference sequence, which can be real or artificial.","In order to maintain the highest homology in alignment of sequences, deletions in the sequence of a variant relative to the reference sequence can be represented by an amino acid space (*), while insertional mutations in the variant relative to the reference sequence can be disregarded and left out of the sequence of the variant when aligned.","Further expansion of the combinatorial library can be made by, for example, by including amino acids that would represent conservative mutations at one or more of the degenerate positions.","Inclusion of such conservative mutations can give rise to a library of potential acetyl glucosaminyl inositol deacetylase sequences.","Alternatively, amino acid replacement at degenerate positions can be based on steric criteria, e.g.","isosteric replacement, without regard for polarity or charge of amino acid sidechains.","Similarly, completely random mutagenesis of one or more of the variant positions can be carried out.","In a preferred embodiment, the combinatorial acetyl glucosaminyl inositol deacetylase library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential acetyl glucosaminyl inositol deacetylase polypeptide sequences.","For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential acetyl glucosaminyl inositol deacetylase nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g.","for phage display) containing the set of acetyl glucosaminyl inositol deacetylase polypeptide sequences therein.","There are many ways by which the library of potential acetyl glucosaminyl inositol deacetylase homologs can be generated from a degenerate oligonucleotide sequence.","Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression.","The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential acetyl glucosaminyl inositol deacetylase sequences.","The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.","(1981) Recombinant DNA, Proc 3rd Cleveland Sympos.","Macromolecules, ed.","A G Walton, Amsterdam: Elsevier pp.","273-289; Itakura et al.","(1984) Annu.","Rev.","Biochem.","53:323; Itakura et al.","(1984) Science 198:1056; Ike et al.","(1983) Nucleic Acid Res.","11:477.A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and, for that matter, for screening cDNA libraries for gene products having a certain property.","Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of acetyl glucosaminyl inositol deacetylase homologs.","The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene product was detected.","Each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.","The invention also provides for reduction of the subject acetyl glucosaminyl inositol deacetylase polypeptides to generate mimetics, e.g.","peptide or non-peptide agents, which are able to mimic binding of the authentic acetyl glucosaminyl inositol deacetylase polypeptide to a substrate molecule.","Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of an acetyl glucosaminyl inositol deacetylase polypeptide which participate in protein-protein interactions involved in, for example, binding of the subject acetyl glucosaminyl inositol deacetylase polypeptide to a substrate.","To illustrate, the critical residues of a subject acetyl glucosaminyl inositol deacetylase polypeptide which are involved in molecular recognition of substrate can be determined and used to generate acetyl glucosaminyl inositol deacetylase-derived peptidomimetics which cleave acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide substrates and, like the authentic acetyl glucosaminyl inositol deacetylase polypeptide, cleave the substrate molecule, for example by amide hydrolase activity.","By employing, for example, scanning mutagenesis to map the amino acid residues of a particular acetyl glucosaminyl inositol deacetylase polypeptide involved in binding to a substrate, peptidomimetic compounds (e.g.","diazepine or isoquinoline derivatives) can be generated which mimic those residues cleaving substrate.","For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al; in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al.","in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al.","in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.","(1986) J Med Chem 29:295; and Ewenson et al.","in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), beta-turn dipeptide cores (Nagai et al.","(1985) Tetrahedron Lett 26:647; and Sato et al.","(1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols (Gordon et al.","(1985) Biochem Biophys Res Commun 126:419; and Dann et al.","(1986) Biochem Biophys Res Commun 134:71).","The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.","EXAMPLE 1 Preparation of Reagents and Assay Methods Used.","Mycothiol (MSH) was isolated from M. smegmatis and derivatized with monobromobimane to form the MSH-bimane derivative (MSmB).","Mycothiol can be purified from Streptomyces clavuligerus, M. smegmatis, or from cells of any other mycothiol containing bacterium.","Early stationary phase cells (25 gm wet weight) were extracted with 250 ml of 50% acetonitirile containing 25 mM methanesulfonic acid and 1 mM DTT at 60° C. for 15 min.","The cell debris was removed by centrifugation for 30 min (4° C.) at 20,000×g.","The excess acetonitrile was removed using a rotary evaporator.","The aqueous extract was neutralized with Tris base to pH 8.0 and clarified by centrifugation for 30 min (4° C.) at 20,000×g The extract was loaded on a 25×70 mm propyl thiol Sepharose 6B resin activated with 2-mercaptopyridine with 1 mmole total thiol binding capacity.","The column was washed with 10 column volumes of 20 mM Tris-HCl pH 8.0 to remove unbound extract components.","The bound thiols were eluted with 200 ml of 20 mM Tris-HCl pH 8.0 containing 3 mM DTT.","The eluate was extracted 5 times with an equal volume of ethyl acetate to remove excess DTT.","The ethyl acetate washed aqueous extract was reduced to dryness by lyophilization.","The extract was suspended in 5 ml of aqueous 0.1% trifluoroacetic acid (TFA).","The mycothiol was purified by preparative HPLC using a 10×250 mm Vydac C-18 (Cat.","# 218TP152022) reverse phase column (or equivalent).","Mycothiol eluted at 26 min using an isocratic elution in aqueous 0.1% TFA at 5 ml per min.","The volatile solvent was removed by lyophilization to yield pure mycothiol.","Mycothiol is stable for years when stored as a mildly acidic aqueous solution of 30-100 mM at −70° C. The bimane derivative of mycothiol is readily made using monobromobimane (Molecular Probes) at 2 mM excess over the thiol concentration.","The derivatization is carried out in aqueous 20 mM Tris-HCl pH 8.0 for 15 min in the dark at room temperature.","The derivatization mixture was acidified to pH<3 with TFA and extracted 2 times with equal volumes of dichloromethane to remove excess monobromobimane.","The derivatization mixture was purified on the preparative reverse phase column described above using a gradient elution.","The A-buffer was 0.1% aqueous TFA and the B buffer was methanol.","The linear gradient was 15-30% methanol over 30 min with a flow rate of 5 ml per min.","The MSmB was detected at 385 nm and eluted as a symmetrical peak at 33 min.","The volatile solvent was removed by lyophilization to yield>98% pure MSmB.","This derivative was stored as a 10-30 mM solution in water at −70° C. The purified M. smegmatis mycothiol S-conjugate amidase was used to quantitatively hydrolyze MSMB to stereochemically pure GlcN-Ins, and the latter was purified from the other hydrolysis product, AcCySmB, using a Sep-Pak® C18 (Waters) cartridge.","A 10 mM stock solution of GlcNAc-Ins was prepared by addition of a tenfold excess of acetic anhydride (Fisher) to 10 mM GlcN-Ins in 10 mM NaHCO3 over 20 min while adjusting the pH to 8.5 with NaOH.","The reaction was monitored for GlcN-Ins loss as described below to insure that the reaction was complete and that no residual GlcN-Ins was present.","This stock solution of GlcNAc-Ins was assayed as described below and used without further purification.","Preparation of Mycothiol S-conjugates.","Mycothiol S-conjugates were prepared by reaction of excess electrophile with mycothiol followed by removal of excess electrophile.","Stock solutions (100 mM) of iodoacetamide (Sigma) or bromoacetophenone (Sigma) were prepared in acetonitrile.","Reaction of these electrophiles at 2 mM in 25 mM HEPES chloride pH 7.5 was initiated by addition of mycothiol to 1 mM from a 32 mM stock solution in H2O (pH˜3) and was allowed to proceed 15 min at 22° C. Excess reagent was removed by extracting 3 times with an equal volume of H2O-saturated dichloromethane.","Prior to extraction a 1 μl aliquot was withdrawn for reaction with mBBr and analysis of residual mycothiol by HPLC (see above) to verify complete reaction.","This showed that>99% of the mycothiol had reacted.","The MS-acetophenone was purified to homogeneity by reverse phase preparative HPLC as described above for MSmB, except that MS-acetophenone was detected at 260 nm.","Assay of MSH and MSH precursors.","Mycothiol and its precursors cysteine, and Cys-GlcN-Ins were assayed as the bimane derivative by high performance liquid chromatography (HPLC) on a Ultrasphere® ODS IP (Beckman) reversed phase column.","The bimane derivatives were detected by fluorescence using a Fluoromonitor III® monitor (Thermo Separation products) with excitation filters 340-380 nm and emission filters 418-700 nm.","The A buffer was 0.1% TFA and the B buffer was 7.5% methanol in acetonitrile.","The separation was achieved at 22° C. and a flow rate of 1 ml/min with linear gradients as follows: time 0 min 0% B, 10 min 0% B, 30 min 20% B, 33 min 100% B, 36 min 100% B, 38 min 0% B, 50 min reinject.","CySmB-GlcN-Ins, CySmB and mycothiol-mB (MSmB) eluted at 28, 31 and 33 min, respectively.","GlcN-Ins was assayed by precolumn derivatization using AccQFluor® (Waters) reagent followed by HPLC analysis.","A sample, 7.5 μl of 50% acetonitrile sample containing 5 mM NEM or 5 mM NEM and 1 mM 1,10 phenanthroline was mixed with 39.4 μl of buffer stock.","The buffer stock was composed of 30% acetonitrile in aqueous 160 mM HEPES pH 7.5.This was mixed with 15.6 μl of AccQ-Fluor® reagent (Waters) and incubated for 1 min at 22° C. followed by 10 min at 60° C. The sample was diluted with 188 μl of water and the sample was analyzed by HPLC.","The sample was centrifuged for 3 min 14,000×g in a microcentrifuge.","The HPLC separation conditions were the same as described above for bimane derivatives, except the B buffer was 0.1% TFA in methanol.","The product was detected by fluorescence using 254 nm excitation and 370-700 nm emission filters.","AccQ-GlcN and AccQ-GlcN-Ins eluted at 12 and 24 min respectively.","Mycothiol S-conjugate amidase assay.","The enzymatic activity was routinely assayed by quantitation of the bimane derivative of N-acetylcysteine (AcCySmB) produced from the bimane derivative of mycothiol (MSmB), prepared from purified mycothiol (see above).","To conduct an amidase assay a sample (2-10 μL) of extract was mixed with 40 μL of 30 μM MSmB in 3 mM 2-mercaptoethanol, 25 mM HEPES chloride pH 7.5 and reacted 10-30 min at 30° C. before quenching the reaction with 50 μL of 40 mM methanesulfonic acid on ice.","The mixture was centrifuged for 3 min at 14,000×g in a microcentrifuge at room temperature and the supernatant analyzed by HPLC without dilution.","Separation of MSmB and AcCySmB was achieved by HPLC on a 0.46×250 mM Ultrasphere®ODS column (Beckman) using a linear gradients: time 0 min 10% B, 15 min 18% B, 30 min 27% B, 32 min 100% B, 34 min 10% B, 45 min reinject.","Buffer A (aqueous 0.25% acetic acid titrated to pH 3.6 with NaOH) and buffer B (methanol) were pumped at 1.2 ml/min at ambient temperature (22° C.).","The bimane derivative of mycothiol (MSmB) eluted at 23.5 min and AcCySmB eluted at 27 min.","EXAMPLE 2 Cloning and Expression of the Rv1170 gene from M. tuberculosis and demonstration of its GlcNAc-Ins deacetylase activity.","Since the invention deacetylases and the mycothiol S-conjugate amidase both cleave an amide bond involving glucosamine, further studies were conducted to determine whether these proteins might be structurally related.","A search was conducted of the Sanger Centre database for homologs of the 288 amino acid M. tuberculosis deacetylase, Rv1082.The closest homolog found was Rv1170, with a length of 304 residues and 36% identity to Rv1082 with homology throughout the sequence.","To isolate the Rv1170 gene from M. tuberculosis H37Rv genomic DNA was prepared using known methods.","The open reading frame Rv1170 was amplified from this DNA with the following primers: 5′-TAGCCATGGTGTCTGAGACGCCGCG-3′ (SEQ ID NO:8) and 5′-GGATCCCGGGGTGAAGCCCAGAC-3′ (SEQ ID NO:9) These primers contain NcoI and BamHI restriction sites, respectively.","PCR was performed with Taq polymerase (Gibco BRL), using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.","The 30 cycles of PCR included denaturation at 94° C. for 40 s, annealing at 55° C. for 1 min, and amplification at 72° C. The PCR products were separated on a 1% agarose gel.","The appropriate PCR product was ligated into vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into Escherichia coli DH5α by standard chemical transformation procedures.","Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml), and plasmid DNA was digested with the restriction endonucleases NcoI and BamHI (Fermentas).","Restriction enzyme-digested plasmids were isolated with a QIAquick® gel extraction kit (Qiagen Ltd.).","A corresponding digestion was applied to the plasmid pET-22b, and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170.In order to express Rv1170 in E. coli without the pelB leader sequence, the gene from pYA1170 was excised using NcoI and Bpu1102I (Fermentas) and ligated to an aliquot of pET16b cut with NcoI and Bpu1102I to generate the plasmid pYA1170b (FIG.","2).","This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg ampicillin/ml.","Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).","After overnight incubation at 37° C. with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37° C. with shaking.","Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.4 mM when the A600 reached 0.6, and incubation was continued overnight at 25° C. before harvesting by centrifugation at 5000×g for 15 min at room temperature.","The pellets were lysed by sonication.","Proteins were separated by centrifugation (15,000×g, 4° C., 30 min) into soluble and insoluble fractions.","Total proteins were separated by sodium dodecyl sulfate 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride membranes (Bio-Rad).","The N-terminal amino acid sequence was verified using Edman degradation after separation of samples by SDS-PAGE and electroblotting to a polyvinylidene difluoride membrane.","E. coli BL21 (DE3) was transformed with the blank cloning vector (pET16b) or with vector containing Rv1170 (pYA1170b).","SDS-PAGE was used to separate desalted 20% to 50% saturated ammonium sulfate extracts of these isopropyl-β-D-thiogalactopyranoside induced E. coli.","Bio-Rad Broad Range molecular mass standards and purified M. smegmatis mycothiol S-conjugate deacetylase were also assayed for controls.","The results of the SDS-PAGE showed that the 20% to 50% saturated ammonium sulfate fraction from E. coli carrying the Rv 1170 gene contained elevated levels of a protein of the expected size (36 kDa) as compared to those from E. coli prepared with a blank cloning vector.","Another, smaller protein (˜26 kDa) was also present at elevated levels and is believed to be a degradation product of the deacetylase.","The identity of the 36-kDa protein was confirmed by determining the N-terminal amino acid sequence and finding it identical to that predicted for the protein encoded by open reading frame Rv1170.The amidase and deacetylase activity of the 20 to 50% saturated ammonium sulfate extracts of E. coli transformed with pYA1170b or with the blank cloning vector pET16b after desalting and concentrating was assayed as described above.","The results of these assays are shown in Table 1 below.","TABLE 1 Substrate specificity of crude deacetylase from E. coli transformed with pYA1170b Activity (nmol min−1 mg of protein−1)(n = 3) Substrate Rv1170 Blank vector MsmBa 1.2 ± 0.1 <0.002 MSmBb <10−4 NDf GlcNAc-Insc 27 ± 2 <0.002 GlcNAcc 0.087 ± 0.005 <0.005 MSHd 0.002 ± 0.001 ND MSHe <10−4 ND aAnalysis for AcCySmB produced by amidase cleavage.","bAnalysis for production of the deacetylase product CySmB-GlcN-Ins.","cAnalysis for corresponding amine resulting from deacetylase reaction.","dAnalysis for AcCys produced by amidase cleavage.","eAnalysis for production of the deacetylase product Cys-GlcN-Ins.","fNot determined.","As shown by the results summarized in Table 1, extract from cells expressing Rv1170 exhibited substantial deacetylase activity with GlcNAc-Ins as the substrate, over 300-fold greater than the deacetylase activity determined with GlcNAc and 23-fold greater than the amidase activity measured with MSMB (FIG.","2).","Thus, Rv1170 was putatively identified as the GlcNAc-Ins deacetylase gene in the MSH biosynthesis pathway of M. tuberculosis.","M. smegmatis mycothiol S-conjugate amidase lacks deacetylase activity.","Mycothiol S-conjugate amidase from M. smegmatis, a homolog of the protein encoded by M. tuberculosis Rv1082, cleaves the amide bond linking Cys to GlcN in MSH derivatives having an alkylated sulfur residue (G. L. Newton et al., Biochemistry 39:10739-10746, 2000).","Tests were conducted to determine whether the mycothiol S-conjugate amidase also cleaves the corresponding bond in GlcNAc-Ins, functioning as a deacetylase (FIG.","3).","GlcNAc-Ins was prepared from GlcN-Ins and the ability of the amidase purified from M. smegmatis to hydrolyze GlcNAc-Ins (i.e., to function as a deacetylase) was compared with its ability to function as an amidase by cleaving the amide bond in the monobromobimane conjugate of MSH (MSmB).","The assays utilized are described above.","For a test with 100 μM substrate, the activity measured with deacetylase substrate MSmB was 4.5±1.0 μmol min−1 mg of protein−1, whereas that measured with the invention deacetylase substrate GlcNAc-Ins was <1 nmol min−1 mg of protein−1 (n=4).","The comparative assays showed that the mycothiol S-conjugate deacetylase does not exhibit measurable deacetylase activity and cannot serve this function in the MSH biosynthesis pathway of M. smegmatis.","EXAMPLE 3 Extracts of M. smegmatis and mutant 49 have deacetylase activity.","If the deacetylase is involved in MSH biosynthesis and if mutant 49 is blocked at an earlier step in the MSH-production pathway, then strains mc2155 and 49 should both produce deacetylase activity.","To test this hypothesis, centrifuged extracts of exponentially growing cells were assayed in duplicate for GlcN-Ins production with and without the addition of 100 μM GlcNAc-Ins.","The background rate for production of GlcN-Ins measured without added GlcNAc-Ins was high for strain mc2155 (6.1±0.1 pmol min−1 mg of protein−1) presumably reflecting the substantial endogenous level of GlcNAc-Ins present in the undialyzed extract.","Addition of 100 μM GlcNAc-Ins increased the rate to 19.7±0.4 pmol min−1 mg of protein−1, giving a net rate increase of 13.6±0.5 pmol min−1 mg of protein−1.For mutant 49, which does not contain GlcNAc-Ins, the background rate was <0.06 pmol min−1 mg of protein−1 and the rate with 100 μM GlcNAc-Ins was 16.4±1.4 pmol min−1 mg of protein−1.Thus, the deacetylase activity of strain 49 is essentially the same as that of the parent strain.","EXAMPLE 4 Assay for GlcNAc-Ins.","To confirm that GlcNAc-Ins is present in mycobacteria, an assay for this component was needed.","The availability of cloned Rv1170 allowed the development of such an assay.","Cell extracts were prepared in warm 50% acetonitrile containing NEM, as employed previously for an assay of GlcN-Ins (Anderberg et al.","(1998) J. Biol.","Chem.","273:30391-30397).","One portion of the extract was assayed directly for GlcN-Ins content while a second portion was assayed after treatment with a preparation of expressed Rv1170 under conditions sufficient to convert all GlcNAc-Ins to GlcN-Ins.","The difference in these two assay results yields the GlcNAc-Ins content for the sample.","In most cases, GlcN-Ins levels were at least an order of magnitude lower than those of GlcNAc-Ins, so this method gives reliable results.","Recovery experiments for GlcNAc-Ins were conducted using a M. smegmatis MSH-deficient mutant (“mutant 49”), derived from mc2155 by chemical mutagenesis.","Mutant 49 was cultured in Middlebrook 7H9 medium at 37° C. as previously described (Newton, et al.","(1999) Biochem.","Biophys.","Res.","Comm.","255:239-244).","In the uptake and metabolism assay, 50% acetonitrile-NEM extracts were analyzed after the addition of 0.7 and 7.0 μM GlcNAc-Ins.","Extracts of mutant 49 contained no detectable GlcN-Ins or MSH, so that any GlcN-Ins found in the extracts after treatment with the deacetylase preparation was derived solely from the deacetylation of GlcNAc-Ins.","The relationship between A600 and RDW was determined for aliquots of mc2155 cells producing ˜30 mg of RDW and found to be 0.42±0.02 mg of RDW per ml of cells with A600 at 1.00.No variation of >15% in this value was seen between log- and stationary-phase cells.","The method was tested with extracts prepared from log-phase cells of mutant 49 by spiking the extract with GlcNAc-Ins at levels equivalent to 0.13 and 0.013 μmol per g of RDW.","The recoveries from triplicate determinations were 97%±5% and 132%±3%, respectively.","At the lowest level of added GlcNAc-Ins, a significant correction had to be applied for background peaks present in unspiked controls that overlapped the GlcN-Ins peak.","The greater than 100% recovery found for the samples spiked at lowest level indicates that this correction produces a systematic error and thus defines the lower limit of useful sensitivity for this assay.","EXAMPLE 5 M. smegmatis mc2155 produces GlcNAc-Ins but MSH-deficient mutant 49 does not.","M. smegmatis mc2155 cells were examined in exponential and stationary phases for their content of MSH and its precursors using the methods described above.","The results of these studies are summarized in Table 2 below: TABLE 2 Levels of MSH and precursors in M. smegmatis mc2155 Content (μmol [g of RDW]−1) ( n = 3) Growth phase A600 MSH GlcNAc-Ins GlcN-Ins Log 0.54 7.8 ± 0.6 14.5 ± 1.2 0.2 ± 0.1a Stationary 3.3 6.6 ± 0.6 12.6 ± 0.6 <0.03 aBest estimate after correction for overlapping peak in HPLC analysis.","These results show that M. smegmatis accumulates GlcNAc-Ins to a substantial level, almost twice the level of MSH content.","The GlcN-Ins content was very much lower in log phase and declined further in stationary phase, as had been observed previously (Anderberg et al; (1998) Supra.","EXAMPLE 6 MSH-deficient mutant 49 can import GlcNAc-Ins and utilize it to sythesize MSH.","The foregoing results imply that mutant 49 should be able to synthesize MSH if supplied with GlcNAc-Ins.","The concentration dependence of the uptake of GlcNAc-Ins was examined with cultures of M. smegmatis mutant 49.For this study mutant 49 was cultured at 37° C. for 23 hr in 5 ml of 7H9 Middlebrook medium as described below, supplemented with 0, 4, 8, 21 μM GlcNAc-Ins.","The cells were collected by centrifugation and extracted for amine and thiol analysis as described above.","The absence of GlcNAc-Ins in mutant 49 shows that it is defective in an initial step of MSH biosynthesis.","When mutant 49 was grown in media supplemented with GlcNAc-Ins, the cells did show the presence of MSH and mycothiol precursors in increasing concentrations with the media content of GlcNAc-Ins.","These results established that GlcNAc-Ins is indeed imported intact, deacetylated and utilized for the production of MSH in a concentration dependent manner (Table 3).","TABLE 3 Levels of MSH and precursors in MSH-deficient strain 49 (M. smegmatis) Content (μmol [g of RDW]−1) [GlcNAc-Ins]a (μM) MSH GlcNAc-Ins GlcN-Ins 0 <0.04 <0.01 <0.02 4 0.47 0.39 <0.02 8 0.91 0.80 0.026 21 2.1 1.4 0.047 aIn Middlebrook 7H9 medium.","; [GlcNAc-Ins] = 0).","In another experiment mutant 49 was cultured in 7H9 Middlebrook medium with 0.4% glucose and 0.5% TWEEN 80 detergent to log phase (A600=0.6) and 10-ml aliquots transferred to sterile 25 ml erlenmeyer flasks.","Duplicate flasks contained cells only (control), 20 μM inositol, 20 μM glucosamine, 20 μM N-acetylglucosamine, or 17 μM GlcNAc-Ins and were cultured at 37° C., shaking at 225 rpm.","Samples (2.3×108 cells) were taken at 2, 6, 19 and 46 hr of culture.","the cells were pelleted by centrifugation for 2 min at 14,000×g and were extracted in 50% acetonitrile containing 20 mM HEPES (pH 8.0) and either 2 mM monobromobimane for thiol analysis or 5 mM NEM for amine analysis and thiol control samples.","Cells from duplicate cultures were analyzed for GlcN-Ins, GlcNAc-Ins, Cys-GlcN-Ins, cysteine, and MSH.","No significant MSH was found in any sample except for the cultures supplemented with GlcNAc-Ins (FIG.","4).","Strain 49 cultured in 17 μM GlcNAc-Ins showed a cysteine level that remained unchanged at 0.36±0.08 μmol per gm of residual dry weight over the experiment.","Cells contained 2.9 μmole per gm of residual dry weight of GlcNAc-Ins at the first measurement (2 hr), which corresponds to a level of GlcN-Ins approaching millimolar.","This occurred prior to the appearance of significant GlcN-Ins in the cell, showing that strain 49 is able to import and concentrate GlcNAc-Ins intact prior to its deacetylation.","The level of GlcNAc-Ins appeared to fall at 6 hr before rising to an even higher level in stationary phase.","The MSH content was measurable as early as 2 hr, but was about 10-fold less than the GlcNAc-Ins content at that time.","The MSH content increased thereafter, and the level at 48 hr was about 60% of that measured for the parent strain (Table 2).","The level of GlcN-Ins ranged from 0.025 (2 hr) to 0.1 (48 hr) μmole per gm of residual dry weight, values in the range of those measured for mc2155 (Table 2).","These results show that exogenous GlcNAc-Ins is efficiently imported by strain 49 and substantially restores its defective biosynthesis of MSH.","The intracellular level of GlcN-Ins found in mutant 49 at 48 hours indicates that GlcNAc-Ins has been concentrated more than 100 fold from the supplemented medium.","This implies that toxic analogs of GlcNAc-Ins, such as those with oxidizing or nitrosating moieties, will be concentrated inside pathogenic mycobacteria or other actinomycetes and function as antimicrobial agents.","EXAMPLE 7 Cloning, expression and purification of M. tuberculosis His-tagged MshB.","To facilitate the purification of the M. tuberculosis deacetylase, Rv1170 was cloned to contain a C-terminal HisTag permitting its easy isolation by affinity chromatography on a Ni2+ chelate resin.","Genomic DNA of M. tuberculosis H37Rv was prepared as described previously (Av-Gay et al., Infect.","Immun.","67:5676-5682, 1999).","The open reading frame Rv1170 was amplified from this DNA with the primers 5′-TTCATATGGTGTCTGAGACGCCG-3′; (SEQ ID NO:10 and 5′-ATAAGCTTGTAGCCGGACGCGGTG-3′ (SEQ ID NO:11) containing NdeI and HindIII restriction sites, respectively.","PCR was performed with Taq polymerase obtained from Gibco BRL, using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.","The 30 cycles of PCR included denaturation at 94° C. for 40 seconds, annealing at 55° C. for 1 min and amplification at 72° C. The PCR products were separated on a 1% agarose gel.","The appropriate PCR product was ligated into the vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into E. coli DH5a by standard chemical transformation procedure.","Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml) and plasmid DNA was digested with restriction endonucleases NdeI and HindIII (Fermentas).","Restriction enzyme-digested plasmids were isolated with a QIAquick gel extraction kit (Qiagen Ltd.).","A corresponding digestion was applied to the plasmid pET-22b and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170c (shown in FIG.","9).","This provides a recombinant protein without a pelB leader sequence, but with a c-terminal His tag.","This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg/ml ampicillin.","Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).","After over-night incubation at 37° C. with shaking, 5 1-liter cultures were inoculated to 1% with the starter culture and incubated at 37° C. with shaking.","Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM when the A600 reached 0.6 and incubation was continued overnight at 22° C. before harvesting by centrifugation at 5000×g for 20 minutes at 4° C. The pellets were lysed by sonication in 3 mM 2-mercaptoethanol with 50 mM HEPES pH 7.4 (lysis buffer) containing 35 μM each protease inhibitors (N-α-p-tosyl-L-lysinechloromethyl ketone and N-α-p-tosyl-L-phenylalaninechloromethyl ketone).","Proteins were separated by centrifugation (1-5,000×g, 4° C., 30 min) into-soluble and insoluble-fractions.","The soluble proteins were applied to a 2.5×11 cm Fast Flow® chelating resin (Pharmacia) in the Ni2+ form in lysis buffer containing 10 mM imidazole.","The column was washed with 10 volumes of the loading buffer and then step eluted with lysis buffer containing 150 mM imidazole followed by lysis buffer containing 500 mM imidazole.","Fractions were collected and the purity of the eluted protein was assayed by SDS PAGE gel electrophoresis.","The early 150 mM imidazole fractions were contaminated with E. coli proteins, but the later fractions of the 150 mM and the early 500 mM imidazole fractions were judged>95% pure and pooled.","The yield of pure recombinant MshB (Rv1170) was estimated to be ˜125 mg of protein.","The protein was concentrated and the buffer exchanged repeatedly with 50 mM HEPES containing 50 mM NaCl (assay buffer) in a Centricon® 10 spin ultra filter, adjusted to 10% glycerol and frozen at −70° C. The enzyme as purified was assayed for deacetylase activity with 0.1 mM GlcNAc-Ins as substrate and was found to be sensitive to chelating agents such as 1,10-phenanthroline.","The deacetylase reaction was substantially inhibited by the presence of 0.1 mM 1,10-phenanthroline in the reaction mixture (FIG.","7).","1 mM 1,10-phenanthroline completely inhibited the deacetylase reaction, indicating a dependence of the reaction on divalent metal ions.","The apoenzyme was generated by treatment of the concentrated enzyme with 5 mM 1,10-phenanthroline in assay buffer for 30 min on ice.","The chelator was removed by repeated dilution with assay buffer and concentration until the 1,10-phenanthroline concentration was estimated to be below 1 μM.","All solutions were treated with Chelex-100® chelator to remove extraneous divalent cations.","The apoenzyme was stored at 10 mg/ml in 50 mM NaCl+50 mM HEPES pH 7.5 with 10% glycerol at −70° C. The zinc form (Zn2+) of the deacetylase was prepared by treating the apoenzyme with 0.1 mM ZnCl2 in the assay buffer for 30 min on ice.","The excess Zn2+ was removed by repeated dilution and concentration with assay buffer until the Zn2+ was estimated to be below 1 μM.","The Zn2+ form of the enzyme was stored as described above for the apoenzyme and is herein referred to as “His-tagged MshB”.","EXAMPLE 8 Metal ion dependence of the deacetylase.","The metal ion dependence of the deacetylase activity of MshB was assayed using GlcNAc-Ins in the presence of 0.1 mM divalent metal ion.","Previous studies with ZnCl2 have shown that excess Zn2+ is not inhibitory at 0.1 mM and only slightly inhibitory at 1 mM (see above).","The metal salts tested, ZnCl2, CuSO4, MgCl2, MnCl2, CaCl2, COCl2, CdCl2 and NiCl2, were of reagent grade or higher purity and solutions of the metals were prepared as 10 mM stock solutions in water treated with Chelex-100 chelator.","Enzymatic activity was assayed in Chelex-100® treated 50 mM NaCl with 50 mM HEPES pH 7.4 (assay buffer) containing 0.1 mM of the desired metal salt and 0.1 mM GlcNAc-Ins as substrate.","Buffer and 10 μg of purified apoenzyme were preincubated the divalent metal ion in an 80 μl total reaction volume for 10 min at 37° C. and the assay initiated by the addition of the substrate.","Duplicate reactions were sampled at 3, 10, and 30 min by removing a 20 μl sample of the reaction mixture and adding it to 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.","The samples were incubated for 10 min at 60° C., placed on ice, and subsequently reacted with AccQ Fluor® (Waters) reagent as described above assayed for AccQ-GlcN-Ins by HPLC as described above.","The MshB apoenzyme was able to catalyze both the deacetylase (GlcNAc-Ins as substrate) and the amidase (MSmB as substrate) reactions at substantially reduced rates, as shown in Table 4 below.","TABLE 4 M. tuberculosis MshB (Rv1170) metal ion dependencea Amidase Specific Deacetylase Activityb Amidase Specific Deacetylase Metalloenzyme nmole/ Activity Activityc Activity Form min/mg relative rate nmole/min/mg Relative rate Apoenzyme 3.4 ± 0.5 1.0 35 ± 6 1.0 Untreated 15 ± 0.2 4.4 70 ± 19 2.0 Zn2+ 17 ± 0.7 5.0 81 ± 16 2.3 Ni2+ 13 ± 0.7 3.8 62 ± 23 1.8 Mn2+ 22 ± 0.7 6.5 69 ± 5 2.0 Ca2+ 3.3 ± 0.1 0.97 34 ± 5 0.97 Mg2+ 3.5 ± 0.2 1.0 41 ± 4 1.2 Cu2+ 3.2 ± 0.1 0.94 32 ± 10 0.9 Cd2+ 21 ± 1.5 6.2 34 ± 2 0.97 Co2+ 29 ± 0.3 8.5 236 ± 23 6.7 Zn2+ + Mg2+ ND — 75 ± 14 2.1 Ni2+ + Mg2+ ND — 64 ± 15 1.8 aRecombinant Rv1170 purified to homogeneity over Ni2+ Sepharose.","b0.1 mM MSmB as substrate with 0.1 mM metal ion.","c0.1 mM GlcNAc-Ins as substrate with 0.1 mM metal ion.","The low metal ion content of the apoenzyme reduced the amidase activity to a greater extent than the deacetylase activity relative to the untreated purified protein.","The activity of the Ni2+ Sepharose purified (untreated) form of the MshB appears to be consistent with the Ni2+ form of the enzyme.","The Zn2+, Mn2+ and Ni2+ forms appear to have about the same activity for both the amidase and deacetylase reactions.","The MshB apoenzyme was not activated by the presence of Ca2+, Mg2+ or Cu2+ for either the amidase or deacetylase reactions.","The MshB deacetylase and amidase reactions were differently affected by the presence of the Cd2+ ion.","The amidase reaction was enhanced to the level of Zn2+ while the deacetylase reaction remained at the apoenzyme activity level.","Similarly anomalous activity with Cd2+ was observed for the peptidase and esterase activity of carboxypeptidase, a zinc metalloprotease (R. C. Davis et al Biochemistry 7, 1090-1099 (1968)).","The highest level of activation for both the amidase and deacetylase reactions was observed with Co2+, as has been reported for numerous zinc metalloproteases (D. S. Auld, Chapter 14, Methods in Enzymology, 248:229-243, 1995).","Some zinc metalloproteases have been reported to require the presence of Mg2+ for optimal activity (Vallee and Auld (1993) Biochemistry 32:6493-6500) so activity assays were conducted with 0.1 mM each Zn2+ and Mg2+.","No additional activation over that of Zn2+ alone for either the amidase or deacetylase reactions was observed.","Thus, the metal ion dependence of MshB is similar to that found for members of the metalloprotease superfamily and is therefore likely that MshB is a zinc metalloenzyme in its native state.","EXAMPLE 9 Kinetics and substrate specificity of His-tagged MshB.","The kinetic constants were determined for GlcNAc-Ins, the deacetylase substrate for MshB, and for MSmB, an amidase substrate.","The rates were determined with His-tagged MshB in 50 mM NaCl containing 50 mM HEPES pH 7.5 (assay buffer).","The enzyme (10 μg) was preincubated with 80 μl of assay buffer for 10 min at 37° C. and the reaction was initiated with the addition of the substrate.","The deacetylase substrate was assayed at 9 different concentrations between 10 μM and 2 mM.","Duplicate reactions were sampled at 3, 10, and 30 min by removing 20 μl of the sample, and mixing with 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.","The samples were quenched by incubation for 10 min at 60° C. The quenched sample was reacted with AccQ Fluor® reagent (Waters) and analyzed for GlcN-Ins by HPLC as described above.","The amidase reaction was assayed similarly using 10 concentrations of MSmB between 10 μM and 10 mM.","The amidase reaction was sampled at 3, 10, and 30 min by removing 20 μl of sample and mixing it with 60 μl of 40 mM methanesulfonic acid.","The mercapturate product, AcCySmB was analyzed by HPLC as described above.","Initial rates were obtained by extrapolation of the rates calculated at 3, 10, and 30 min to zero time.","The kinetic constants were obtained from an Eadie-Hofstee plot.","The amidase and deacetylase substrates have Km values near of 0.5 mM as shown below in Table 5 below.","TABLE 5 Kinetic characterization of M. tuberculosis MshB (Rv1170) Km Vmax kcat Substrate Activity μM nmol min−1mg−1 s−1 GlcNAc-Ins deacetylase 400 470 0.25 MSmB amidase 560 220 0.12 The intracellular concentration of GlcN-Ins is roughly twice the mycothiol level, or about 2-6 mM (Newton et al, J.","Bacteriol., 182: 6958-6963, 2000) and is sufficient to saturate the enzyme at physiological concentrations.","The substrate specificity of MshB was examined with various amidase and deacetylase substrates at a concentration of 0.1 mM.","The product analyzed will vary with the substrate used.","The deacetylase substrates tested were GlcNAc, GlcNAc-Ins, MSH, and MSmB, and the products assayed were GlcN, GlcN-Ins, Cys-GlcN-Ins, and CySmB-GlcN-Ins, respectively.","The amidase substrates tested were MSH, MSmB, AcCySmB-GlcN, CySmB-GlcN-Ins, MS-acetamide, and MS-acetophenone and the product assayed was GlcN-Ins, except for AcCySmB-GlcN were GlcN was assayed.","The amines were analyzed as the AccQ-derivative and thiols as the bimane derivative as described above.","The results of these substrate specificity studies are summarized in Table 6 below.","TABLE 6 Substrate specificity of M. tuberculosis MshB (Rv1170)a Amidase Specific Deacetylase Activityb Specific Activityb Relative Substratea nmol min−1mg−1 nmol min−1mg−1 Rate GlcNAc-Insc 70 ± 3 1.0 GlcNAcd 1.6 ± 1 0.023 MSHe <0.025 ± 0.012 <0.00036 MSHc 0.3 ± 0.1 0.0043 MSmBf <0.006 <0.00009 MSmBc 20 ± 2 0.29 AcCySmB-GlcNd <0.1 <0.001 CySmB-GlcN-Insc 265 ± 10 3.8 MS-acetamidec 0.14 ± 0.013 0.002 MS-acetophenonec 88 ± 8 1.3 aHis tagged MshB (Rv1170) in the Zn2+ form.","b0.1 mM substrate assayed in 50 mM NaCl + 50 mM HEPES pH 7.4 at 37° C. cReaction assayed for GlcN-Ins.","dReaction assayed for GlcN.","eReaction assayed for CyS-GlcN-Ins.","fReaction assayed for CySmB-GlcN-Ins.","As shown by the results in Table 6, the deacetylase activity of MshB is dependent on the presence of the inositol moiety as the GlcNAc substrate is 44 fold less active than GlcNAc-Ins.","Interestingly MSH and MSmB are not substrates for the deacetylase activity of MshB.","However, mycothiol S-conjugates with reasonably large groups on the cysteine sulfur, such as acetophenone and bimane S-conjugates, are good substrates for the amidase activity of the MshB.","The smaller mycothiol S-conjugate acetamide is a poor substrate for the MshB amidase activity, as was observed previously for mycothiol S-conjugate amidase from M. smegmatis (Newton et al Biochemistry, 39, 10739-10746, 2000).","The importance of the inositol moiety for the amidase activity of MshB is also apparent by the lack of activity with AcCySmB-GlcN.","One difference between the amidase activity of M. tuberculosis His-tagged MshB and the amidase from M. smegmatis is the ability to use CySmB-GlcN-Ins as substrate.","The M. smegmatis amidase required the acetylated amine on the cysteine moiety (or MSmB) for activity.","Another difference between MshB and the M. smegmatis amidase is that the amidase will not deacetylate GlcNAc-Ins (Newton et al J.","Bacteriol., 182, 6958-6963, 2000).","EXAMPLE 10 Inhibition of His-tagged MshB.","Naturally occurring inhibitors of the His-tagged MshB deacetylase and amidase activities have been examined.","The inhibition assays are similar to those described above in Example 9.For the amidase reaction of His-tagged MshB 10 μg enzyme was preincubated for 10 min at 37° C. in 80 μl of buffer containing 50 mM NaCl, 50 mM HEPES pH 7.5, 0.1 mM ZnCl2, 0.2 mM DTT and 0.1-1.0 mM inhibitor.","The inhibitors examined were mycothiol, glutathione and GlcN-Ins.","The purpose of the excess Zn+2 is to prevent depletion of the protein bound Zn2+ by thiols.","The DTT is added to limit the oxidation of the thiol inhibitors, which can be significant at the lower concentrations.","The uninhibited enzyme activity was determined using the same buffer and preincubation conditions without inhibitor.","The assays were initiated with the addition of MSmB to 0.55 mM and sampled at 3, 10, and 30 min by removing a 20 μl sample and quenching the reaction by mixing with 60 μl of 40 mM aqueous methanesulfonic acid.","This sample was assayed directly by HPLC for AcCySmB as described above.","The deacetylase reaction was assayed with GlcNAc-Ins using 0.1-1.0 mM glutathione and mycothiol as inhibitors.","The reaction was initiated by the addition of GlcNAc-Ins to 0.8 mM and was sampled at 3, 10, and 30 min.","The reaction was quenched and analyzed for GlcN-Ins as described in Example 9.Mycothiol appears to be an effective inhibitor of both the amidase (FIG.","8A) and the deacetylase (FIG.","8B) activities of His-Tagged MshB in the range of 0.1 to 1 mM.","This is not simply due to disruption of the enzyme metal binding site by a thiol as shown by the lack of inhibition by glutathione in this concentration range.","The lack of inhibition of the amidase reaction by GlcN-Ins (FIG.","8A) also shows that the immediate product of the reaction is not inhibitory.","Mycothiol is the terminal product in the biosynthesis scheme shown in FIG.","5 and it appears to completely inhibit the deacetylase (MshB) at mycothiol concentrations above 1 mM.","The estimated intracellular concentration of mycothiol in mycobacteria is about 1-3 mM and thus the deacetylase is in a substantially inhibited state until the mycothiol level falls below 1 mM, as may happen during cell division or cell stress.","Inhibition of the deacetylase reaction was analyzed at 0.8 mM GlcNAc-Ins, a concentration nearing saturation, but still below the estimated cellular concentration of 2-6 mM for M. smegmatis.","Thus, in M. smegmatis the deacetylase is exposed to saturating GlcNAc-Ins concentrations but is inhibited when the cellular mycothiol level is above 1 mM.","If mycothiol level falls substantially below 1 mM, the enzyme will catalyze the deacetylation of GlcNAc-Ins from a large cellular pool at near maximal rates to GlcN-Ins and ultimately to mycothiol.","It has previously been shown that mycothiol levels are quite constant from logarithmic through stationary phase growth of M. smegmatis (Newton et al, J.","Bacteriol., 178: 1990-1995, 1996).","It appears that the intracellular mycothiol level is regulated by feedback inhibition of the GlcNAc-Ins deacetylase by mycothiol itself.","The amidase substrate, MSmB, served as a useful model substrate for finding inhibitors of the GlcNAc-Ins deacetylase.","The amidase and deacetylase activities are both completely inhibited by mycothiol at concentrations over 1 mM.","These results identify mycothiol as an inhibitor for the deacetylase and amidase reactions with similar inhibitory concentrations.","Thus, the substrates for either the amidase or deacetylase activity of GlcNAc-Ins deacetylase can be used to screen for inhibitors of the enzyme.","EXAMPLE 11 Oxidant sensitivity of M. smegmatis MSH-deficient mutants.","The importance of mycothiol biosynthesis for the survival of mycobacteria under oxidative stress is illustrated by results obtained with chemical and transposon mutants deficient in mycothiol biosynthesis.","Chemical mutants were produced by mutagenesis of M. smegmatis mc2155 (˜2.5×109 colony forming units per ml Middlebrook 7H9 liquid medium) with N-methyl-N-nitro-N-nitrosoguanidine (4 μg per ml) for 30 min at 37° C. Cells were washed three times with 9 ml of fresh medium, resuspended in 10 ml of fresh medium, and incubated 8.5 h at 37° C. with shaking.","After dilution in fresh medium and passage through a 22-guage needle, the cells were plated on Middlebrook 7H9 agar containing 0.05% Tween 80 and 0.4% glucose.","After 9 days in a humidified incubator, the plates were scored and individual colonies replated in duplicate using a grid layout in 100 mm culture dishes.","After colony development, one plate was used to screen for MSH-deficient mutants using a membrane-based immunoassay specific for MSH (Unson, et al.","(1998) J. Immunol.","Methods 214:29-39).","MSH-deficient candidates were grown in bulk in liquid medium and analyzed for MSH content as described in Example 1.Two MSH-deficient mutants (strains 49 and 164) were identified and strain 49 was complemented using a M. tuberculosis library (pYUB412::H37Rv) of ˜18 kb DNA fragments cloned in the integrative shuttle plasmid pYUB412 to produce the strain 49::H37Rv A18 with partially restored MSH biosynthesis.","Transposon mutants of M. smegmatis mc2155 were prepared using the pCG79 plasmid carrying transposon Tn611 following the methods of Perez, et al.","(1998) Meth.","Molecular Biol.","101:187-198).","Plasmid pCG79 was introduced into M. smegmatis mc2155 by electroporation after which the cells were incubated 2 h at 32° C. in 7H9 medium without antibiotic.","They were then plated on Middlebrook 7H10 agar plates with ADC enrichment and 20 μg/mL of kanamycin and incubated 6 days at 32° C. Single colonies were restreaked on 7H10km plates and incubated 4 days at 32° C. A loopful of each streaked transformant was resuspended, serially diluted, and plated in duplicate on 7H10 Km plates.","One set of plates was incubated 36 h at 32° C. and then shifted to 42° C. for 5 days to select transposon mutants.","The other set of plates was incubated at 32° C. for 7 days and served as positive controls.","The transposon mutants were replated on 7H10Km plates at 1000-2000 colonies per plate and incubated 5d at 42° C. The cells were scraped into 10 ml of 7H9 medium, mixed with 1 mL of glycerol, and stored at −70° C. This library contained ˜10,000 mutants.","Screening of the library for MSH-deficient mutants was accomplished using as ELISA assay method to detect MSH production in cultures grown in microtiter plates (Unson, et al.","(1999) J. Clin.","Microbiol 37:2153-2157).","The first 1000 mutants screened produced three nearly identical MSH-deficient mutants (TN1, TN2 and TN3) that accumulated GlcN-Ins to high levels but failed to produce detectable levels of Cys-GlcN-Ins or MSH.","Since these clones seemed likely to be daughter cells, TN3 was selected for more detailed studies.","Results obtained with the parent and mutant strains of M. smegmatis are summarized in Table 7 below.","TABLE 7 Characterization of M. smegmatis mutant strains MSH Content H2O2 IC50a Strain μmol per g RDW Enzyme Deficiency mM mc2155 10 None 12 49b <0.004 no MshAc 1 49::H37Rv A18b 3.3 partial MshAd 1.8 I64 0.1 low MshCe 3.5 TN3 <0.004 no MshCe 0.5 aConcentration producing 50% loss of viability following 2 h incubation in Middlebrook 7H9 liquid medium containing H2O2 at 37° C., dilution and plating on 7H10 agar, incubation for 10 d, and counting of colony formation.","bData from Newton, et al.","(1999) Biochem.","Biophys.","Res.","Commun.","255: 239-244.cBased upon failure to produce GlcNAc-Ins or GlcN-Ins.","dBased upon restoration of ability to produce GlcN-Ins to ˜15% that of the parent strain.","eBased upon absence of MshC activity in cell extracts and accumulation of GlcN-Ins to 2-3 μmol per g RDW (˜25-fold above the level of the parent strain).","The results of these experiments show that the parent strain (mc2 155) has a high MSH content and exhibits a marked resistance to killing by hydrogen peroxide.","By contrast, strain 49, which is blocked in the first step of MSH biosynthesis and produces no detectable MSH, is 12-fold more sensitive to H2O2.Partial restoration of MSH biosynthesis in strain 49 through complementation (strain 49::H37Rv A18) reduced the peroxide sensitivity of mutant 49 by a factor of two.","Chemical mutant 164, blocked at the ligase biosynthetic enzyme MshC, has a low residual MSH content and is three-fold more sensitive to peroxide than the parent strain.","However, transposon mutant TN3, also blocked at MshC but producing no detectable MSH, was >20-fold more sensitive to H2O2 than the parent strain (FIG.","10).","These results show that blocking MSH biosynthesis before or after the deacetylase step (MshB) produces a dramatic increase in peroxide sensitivity.","It logically follows that blocking-MSH biosynthesis at MshB would produce a comparable increase in peroxide sensitivity in mycothiol-producing bacteria.","The sensitivity of strains mc2155, 164, and TN3 to superoxide was also examined (courtesy of Drs.","H. Buchmeier and D. Piddington).","Superoxide sensitivity was determined according to the method of De Groote, et al.","(1997) Proc.","Natl.","Acad.","Sci.","94: 13997-14001 using timed exposure of cells to 250 μM hypoxanthine in the presence of 0.1 unit/ml of xanthine oxidase followed by dilution and plating to determine survival.","Under these conditions, strain mc2155 exhibits no significant killing whereas mutant TN3 is >90% killed in 1 hour.","By contrast, chemical mutant 164, with a low residual MSH content, exhibited an intermediate level of killing.","Survival of mycothiol-deficient mutant in macrophage.","Transposon mutant TN3 was compared with the parent strain for survival in murine macrophages (data courtesy of Drs.","N. Buchmeier and D. Piddington).","Following the method of De Groote, et al.","(1997) Supra, periodate activated, γ-interferon treated murine peritoneal exudate cells were infected with TN3 and mc2155, and extracellular bacteria were washed free of the macrophages with medium.","At timed intervals the macrophages were resuspended in PBS and lysed with 0.5% deoxycholate; serial dilutions of the lysate were plated on Middlebrook 7H9 agar for determination of colony forming units.","The results of two independent experiments performed on different days (FIG.","11, experiments 1 and 2) show no loss of viability of the parent strain mc2155 in the macrophages over the period of 24 hours; whereas over 60% of the MSH-deficient mutant TN3 was killed by the macrophages within 6 hours.","These results suggest that mycobacteria deficient in MSH biosynthesis will exhibit significantly enhanced killing by mammalian macrophages.","While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed."]],"string":"[\n [\n \"Acetyl glucosaminyl inositol deacetylase, a mycothiol biosynthetic enzyme, and methods of use\",\n \"The present invention provides a family of bacterial acetyl glucosaminyl inositol deacetylases (MshB) with deacetylase activity against acyl glucosaminyl inositol and which play a key role in mycothiol biosynthesis.\",\n \"The invention deacetylases are characterized by a conserved 100 amino acid N-terminal region and three highly conserved histidine-containing regions and by having deacetylase activity as well as amide hydrolase activity.\",\n \"The invention further provides methods for using the invention deacetylases in drug screening assays to determine compounds that inhibit activity.\",\n \"The invention provides for treatment of actino-mycete infections in mammals using antibiotics that inhibit production or activity of MshB and thereby reduce the production of mycothiol and the virulence of the infecting bacteria.\"\n ],\n [\n \"1.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, b) three domains of conservation, wherein two of the domains contain conserved histidine residues, and c) deacetylase activity against acetyl glucosaminyl inositol.\",\n \"2.The purified deacetylase of claim 1, wherein the deacetylase hydrolyzes a C2-amide bond of the glucosaminyl inositol moiety.\",\n \"3.The purified deacetylase of claim 1, wherein the acetyl glucosaminyl inositol is a precursor of mycothiol.\",\n \"4.The purified deacetylase of claim 1, wherein the acetyl glucoasaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).\",\n \"5.The purified deacetylase of claim 1, wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6, conservative variations thereof, and any combination of two or more thereof.\",\n \"6.The purified deacetylase of claim 1, wherein the deacetylase is derived from an actinomycetes.\",\n \"7.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis.\",\n \"8.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. tuberculosis.\",\n \"9.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. leprae.\",\n \"10.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. bovis.\",\n \"11.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum.\",\n \"M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.\",\n \"12.The purified deacetylase of claim 6, wherein the bacterium is selected from the group consisting of Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.\",\n \"13.The purified deacetylase of claim 1, wherein the deacetylase has amidase activity against acyl glucosaminyl inositols.\",\n \"14.The purified deacetylase of claim 1, wherein the deacetylase has an amino acid sequence as set forth in SEQ ID NO:1 15.The purified deacetylase of claim 1, wherein the deacetylase is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO:7.16.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, b) one or more domains of conservation containing conserved metal chelating residues, and c) deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.\",\n \"17.The purified deacetylase of claim 16, wherein the metal ion is selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+.\",\n \"18.The purified deacetylase of claim 17 wherein the metal ion is Zn2+.\",\n \"19.The purified deacetylase of claim 16, wherein the metal chelating residues are selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.\",\n \"20.An antibody, or functional fragment thereof, that binds specifically to an deacetylase of claim 1.21.An isolated polynucleotide that encodes a deacetylase of claim 1.22.A vector containing a polynucleotide that encodes an deacetylase of claim 1.23.A cell transformed with a vector of claim 22.24.A method for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase, said method comprising: a) contacting a candidate compound with a deacetylase of claim 1 in the presence of an acyl glucosaminyl inositol under suitable conditions and b) determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity or amidase activity, wherein the substantial absence of the deacetylase activity or the amidase activity is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"25.The method of claim 24, wherein the deacetylase is an acetyl glucosaminyl inositol deacetylase.\",\n \"26.The method of claim 24, wherein a breakdown product is a free amine.\",\n \"27.The method of claim 26, wherein the free amine is GlcN-Ins.\",\n \"28.The method of claim 24, wherein a breakdown product is acetate 29.The method of claim 24, wherein a breakdown product is a derivative of cysteine.\",\n \"30.The method of claim 24, wherein the acyl glucosaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside.\",\n \"31.The method of claim 24, wherein the acyl glucosaminyl inositol is an S-conjugate of mycothiol.\",\n \"32.The method of claim 31, wherein the S-conjugate of mycothiol is the monobromobimane derivative of mycothiol (MSmB).\",\n \"33.The method of claim 24, wherein the three domains in the deacetylase that contain conserved histidine residues have amino acid sequences selected from SEQ ID NOs: 3, 5, 6 and any combination of two or more thereof.\",\n \"34.The method of claim 24, wherein the deacetylase is produced in an actinomycete.\",\n \"35.The method of claim 34, wherein the actinomycete is M. smegmatis.\",\n \"36.The method of claim 34, wherein the actinomycete is M. tuberculosis.\",\n \"37.The method of claim 34, wherein the actinomycete is M. leprae.\",\n \"38.The method of claim 34, wherein the actinomycete is M. bovis.\",\n \"39.The method of claim 34, wherein the actinomycete is M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.\",\n \"40.The method of claim 24, wherein candidate compound is a polypeptide, polynucleotide or small molecule.\",\n \"41.A high throughput screening method for identifying inhibitors of the deacetylase of claim 1, said method comprising: a) contacting each of a plurality of candidate compounds with a deacetylase of claim 1 in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and b) determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine, wherein the substantial absence of binding of the marker to a reaction mixture is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"42.The method of claim 41, wherein the plurality of reaction mixtures are formed in the wells of a microtiter plate.\",\n \"43.The method of claim 41, wherein the detectable marker is colorometric or fluorometric.\",\n \"44.The method of claim 43 wherein the fluorometric marker is fluorescamine or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.\",\n \"45.An inhibitor of the deacetylase of claim 1 wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.\",\n \"46.The inhibitor of claim 45 wherein the inhibitor additionally chelates one or more metal ions metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.\",\n \"47.A derivative of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.\",\n \"48.The derivative of claim 47, wherein the reactive residue is a nitroso residue.\",\n \"49.The derivative of claim 47, wherein the reactive residue is a nitroalkyl residue comprising 1 to 3 carbon atoms.\",\n \"50.The derivative of claim 47, wherein the reactive residue is a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms.\",\n \"51.The derivative of claim 47, wherein the reactive residue is a peroxyalkyl residue comprising 2 to 4 carbon atoms.\",\n \"52.A live mutant actinomycete, whose genome comprises a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein said disruption results in said mutant actinomycetes exhibiting transient survival in mammalian white blood cells for an immune response-raising period of time.\",\n \"53.The live mutant actinomycete of claim 52, wherein the period of time is from 1 to 30 days.\",\n \"54.The live mutant actinomycete of claim 52, wherein the survival of the mutant actinomycetes in mammalian white blood cells does not exceed 30 days.\",\n \"55.The live mutant actinomycete of claim 52, wherein the mutant actinomycete is derived from a pathogen selected from the group consisting of M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, and M. scrofulaceum.\",\n \"56.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes.\",\n \"is derived from M. smegmatis.\",\n \"57.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. tuberculosis.\",\n \"58.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. leprae.\",\n \"59.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. bovis and said mammal is bovine.\",\n \"60.A method for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells, said method comprising: introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity, wherein the intracellular presence of the inhibitor decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.\",\n \"61.The method of claim 60, wherein the inhibitor inhibits intracellular production of the deacetylase.\",\n \"62.The method of claim 60, wherein the inhibitor inhibits intracellular deacetylase activity of the deacetylase.\",\n \"63.The method of claim 60, wherein the introducing comprises culturing the bacterium in the presence of the inhibitor.\",\n \"64.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.\",\n \"65.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide that hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.66.The method of claim 60, wherein the bacterium is an actinomycete and the inhibitor inhibits intracellular production of mycothiol.\",\n \"67.The method of claim 60, wherein the bacterium is selected from the group consisting of the pathogenic bacteria M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M abscessus, and M. scrofulaceum.\",\n \"68.The method of claim 60, wherein the bacterium is an actinomycete.\",\n \"69.A method for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, said method comprising administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.\",\n \"70.The method of claim 69, wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates a metal ion selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+, or otherwise binds the enzyme active site in the deacetylase.\",\n \"71.The method of claim 70, wherein the moiety is ClCH2CONH—.\",\n \"72.The method of claim 70 wherein the moiety is HONHCONH—.\",\n \"73.The method of claim 70, wherein the moiety is HONHCOCH2—.\",\n \"74.The method of claim 70, wherein the moiety is HOPO(CH3)NH—.\",\n \"75.The method of claim 70, wherein the moiety is HOPO(CH3)nCH2—; wherein n=1-5.76.The method of claim 70 wherein the moiety is HSCH2(CH2)nNH—; wherein n=2-5.77.The method of claim 70 wherein the moiety is HS(CH2)nCONH—; wherein n=1-3.78.The method of claim 69 wherein the acetyl glucosaminyl inositol-producing bacterium is a mycothiol-producing bacterium.\",\n \"79.A process for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), said method comprising: contacting an acyl glucosaminyl inositol with an deacetylase of claim 1 under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.\",\n \"80.A method for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample, said method comprising: a) contacting a sample containing GlcNAc-Ins with a deacetylase of claim 1 under suitable conditions and b) determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.\",\n \"81.The method of claim 80, wherein the amount of GlcN-Ins is determined by HPLC after labeling thereof with a fluorometric or calorimetric reagent.\",\n \"82.The method of claim 81, wherein the fluorometric reagent is fluorescamine.\",\n \"83.The method of claim 81, wherein the fluorometric reagent is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.\"\n ],\n [\n \" BACKGROUND INFORMATION Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.\",\n \"However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.\",\n \"Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.\",\n \"Actinomycetes, including Streptomyces and Mycobacteria , do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).\",\n \"The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.\",\n \"However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.\",\n \"First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.\",\n \"(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)\",\n \"pp 517-551, John Wiley & Sons, et al.).\",\n \"An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.\",\n \"(1997) FEBS Lett.\",\n \"409:221-222).\",\n \"This enzyme has been sequenced (A. Norin, et al.\",\n \"(1997) Eur.\",\n \"J. Biochem.\",\n \"248:282-289).\",\n \"The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.\",\n \"(1999) J. Amer.\",\n \"Chem.\",\n \"Soc.\",\n \"120:11538-11539; M. P. Patel et al.\",\n \"(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).\",\n \"The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.\",\n \"Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.\",\n \"Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis , is a well-known problem faced by medical practitioners in treatment of bacterial diseases.\",\n \"Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals.\"\n ],\n [\n \" SUMMARY OF THE INVENTION The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.\",\n \"In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.\",\n \"Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.\",\n \"In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.\",\n \"Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.\",\n \"The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.\",\n \"Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.\",\n \"In still another embodiment, the present invention provides live mutant actinomycetes.\",\n \"The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.\",\n \"In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.\",\n \"The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.\",\n \"By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.\",\n \"For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.\",\n \"In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn 2+ , or otherwise binds the enzyme active site of the deacetylase.\",\n \"In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.\",\n \"In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.\"\n ],\n [\n \"FIELD OF THE INVENTION The present invention generally relates to a family of enzymatic compounds produced by bacteria and methods of their use in drug discovery and disease control, and more specifically to acetyl glucosaminyl inositol deacetylases and methods of their use.\",\n \"BACKGROUND INFORMATION Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.\",\n \"However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.\",\n \"Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.\",\n \"Actinomycetes, including Streptomyces and Mycobacteria, do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).\",\n \"The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.\",\n \"However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.\",\n \"First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.\",\n \"(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)\",\n \"pp 517-551, John Wiley & Sons, et al.).\",\n \"An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.\",\n \"(1997) FEBS Lett.\",\n \"409:221-222).\",\n \"This enzyme has been sequenced (A. Norin, et al.\",\n \"(1997) Eur.\",\n \"J. Biochem.\",\n \"248:282-289).\",\n \"The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.\",\n \"(1999) J. Amer.\",\n \"Chem.\",\n \"Soc.\",\n \"120:11538-11539; M. P. Patel et al.\",\n \"(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).\",\n \"The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.\",\n \"Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.\",\n \"Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases.\",\n \"Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals.\",\n \"SUMMARY OF THE INVENTION The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.\",\n \"In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.\",\n \"Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.\",\n \"In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.\",\n \"Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.\",\n \"The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.\",\n \"Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.\",\n \"In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.\",\n \"In still another embodiment, the present invention provides live mutant actinomycetes.\",\n \"The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.\",\n \"In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.\",\n \"The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.\",\n \"By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.\",\n \"For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.\",\n \"In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.\",\n \"In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.\",\n \"In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.\",\n \"BRIEF DESCRIPTION OF THE DRAWINGS FIGS.\",\n \"1A and B show the nucleotide sequence of M. tuberculosis deacetylase gene mshB (Rv1170) (SEQ ID NO:7).\",\n \"FIG.\",\n \"1C shows the deduced amino acid sequence of M. tuberculosis GlcNAc-Ins deacetylase (SEQ ID NO:1) encoded by mshB.\",\n \"FIG.\",\n \"2 is a schematic drawing showing a map of plasmid pYA1170b employed to express Rv1170 in E. coli.\",\n \"FIG.\",\n \"3 is a chart showing the chemical structures of the substrates for assaying deacetylase and amidase activities of the invention acetyl glucosaminyl inositol deacetylases (MshB), such as MshB Rv1170 and homologs.\",\n \"FIG.\",\n \"4 is a graph showing growth of a mutant M. smegmatis (mutant 49) in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins.\",\n \"Mycothiol (MSH) and MSH precursor contents in cells are expressed in micromoles per gram of residual dry weight (RDW).\",\n \"FIG.\",\n \"5 is a schematic representation of the proposed MSH biosynthesis pathway.\",\n \"FIG.\",\n \"6 is a chart showing alignment of the amino acid sequences of four homologs of MshB of M. tuberculosis H37Rv (Rv1170) GenBank accession number B70875 and M. leprae GenBank accession number CAC30445.Sequences for M. avium (The Institute for Genomic Research (TIGR)), and M. smegmatis (Sanger Center) are from unfinished genomes.\",\n \"FIG.\",\n \"7 is a graph showing MshB deacetylase activity with 0.1 mM GlcNAc-Ins in the presence of 1,10-phenanthroline.\",\n \"FIG.\",\n \"8A is a graph showing MshB deacetylase reaction specific activity with 0.8 mM GlcNAc-Ins as substrate in the presence of increasing concentrations of thiol inhibitors.\",\n \"FIG.\",\n \"8B is a graph showing MshB amidase reaction specific activity with 0.55 mM MSmB as substrate in the presence of increasing concentrations of inhibitors.\",\n \"FIG.\",\n \"9 is a map of the plasmid pYA1170C used to express the recombinant His-tagged acetyl glucosaminyl inositol deacetylase encoded by M. tuberculosis Rv1170.FIG.\",\n \"10 is a graph showing percent survival of strains of M. smegmatis versus hydrogen peroxide concentration after treatment as described in Example 11 herein.\",\n \"Open triangles=mutant TN3, which produces no MSH; open circles=chemical mutant 164, which produces a low residual MSH content; and darkened squares=high MSH content mc2 155.FIG.\",\n \"11 is a graph showing percent survival over time of strains of M. smegmatis in murine macrophages as described in Example 11 herein.\",\n \"Open circles=chemical mutant TN3 (0% MSH content), Experiment 1; open triangles=mutant TN3 (0% MSH content), Experiment 2; and darkened squares=mc2155 (100% MSH content).\",\n \"DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there are provided a family of purified acetyl glucosaminyl inositol deacetylase polypeptides with enzymatic deacetylase activity for acetyl glucosaminyl inositol.\",\n \"The invention acetyl glucosaminyl inositol deacetylases are characterized by having an N-terminal region with an amino acid sequence with 40% or greater sequence identity to an amino acid sequence as set forth in SEQ ID NO:2 and conservative variation thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.\",\n \"SEQ ID NO:2 corresponds to amino acid residues 1-100 of amino acids of SEQ ID NO:1 (FIG.\",\n \"1C).\",\n \"The members of the family of invention deacetylases hydrolyze a C2-amide bond of acetyl glucosaminyl inositol moiety, such as N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).\",\n \"Preferably the acetyl glucosaminyl inositol is a precursor of mycothiol (e.g., in a mycothiol producing bacterium).\",\n \"In preferred embodiments the three histidine-containing conserved domains in the invention deacetylases are selected from VHAHPDDE (SEQ ID NO:3), which corresponds to amino acids 10 through 17 of SEQ ID NO:1; VTCTLGEXGEV (SEQ ID NO:4), which corresponds to amino acids 38 through 49 of amino acid SEQ ID NO:1; YDPXGGYGHPDH (SEQ ID NO:5), which corresponds to amino acids 136 through 147 of SEQ ID NO:1; and AXXAHATQ (SEQ ID NO:6), which corresponds to amino acids 240 through 247 of amino acid SEQ ID NO:1 (FIG.\",\n \"1C) wherein X is any amino acid; and conservative variations thereof, or any combination of any two or more thereof (See FIG.\",\n \"6).\",\n \"Members of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides are metalloproteases.\",\n \"Accordingly, the invention further provides purified acetyl glucosaminyl inositol deacetylases that are characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.\",\n \"The invention deacetylases have been shown to contain an active site that contains a metal ion selected from the group consisting of Mn2+, Ni2, Cd2+, Co2+, and Zn2+, with the preferred metal ion being Zn2+.\",\n \"The metal chelating residues in the invention deacetylases are preferably selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.\",\n \"In preferred embodiments, the invention deacetylases have additional activity as amidases, catalyzing the hydrolysis of acyl glucosaminyl inositols.\",\n \"As used herein, the term “acyl glucosaminyl inositol” means substrate compounds for the invention family of deacetylases having the chemical formula R—CONH-GlcN-Ins wherein R═CH3(CH2)n— where n=0-6.Included in the group of substrate compounds is the natural substrate, acetyl-GlcN-Ins, where n=0.Members of this group of substrate compounds are derived from an alkanoic acid.\",\n \"In addition, compounds having the chemical structure R—CONH-GlcN-Ins wherein R=aryl-(CH2)n, where n=0-6 are encompassed by the term.\",\n \"Members of this group of substrate compounds are derived from an aryl alkanoic acid such as phenyl-(CH2)nCOOH.\",\n \"The acyl glucosaminyl inositols of the above chemical formula can also be derived from the reaction of GlcN-Ins with any commercial acid chloride in the form RCOCl, wherein R is: o-tolyl-, 4-ethylphenyl-, 4-propylphenyl-, 4-biphenyl-, 3,4-dimethoxyphenyl-, 3,4,5-trimethoxyphenyl-, 2-furyl-, and the like.\",\n \"The acyl glucosaminyl inositols of the above chemical formula can also be derived from an amino acid or an N-acetyl amino acid.\",\n \"Preferred amino acids include N-acetylcysteinyl-S—R′ or cysteinyl-S—R′, wherein R′ is an organic group attached to the cysteine sulfur, such as may be derived from commercial thiol labeling reagents like 2-bromoacetophenone, monobromobinane, N-ethylmaleimide, cerulenin, 7-diethylamino-3-(4′-maleimidyl phenyl)-4-methyl coumarin, 3(N-maleimidopropionyl)biocytin, and the like.\",\n \"The studies described herein further elaborate the pathway involved in MSH biosynthesis.\",\n \"The present studies demonstrate that GlcNAc-Ins is a major intracellular MSH component in M. smegmatis and is converted to GlcN-Ins by GlcNAc-Ins deacetylase.\",\n \"This conversion defines the second step in MSH biosynthesis.\",\n \"The discovery described herein that Rv1170 is the gene encoding the deacetylase in the M. tuberculosis genome represents the first gene of the MSH biosynthesis pathway to be identified.\",\n \"Assuming that a single enzyme produces GlcNAc-Ins, it is proposed that the genes for the MSH biosynthesis pathway be designated mshA, mshB, mshC, and mshD, with the corresponding enzymes labeled as shown in FIG.\",\n \"5.Identification of mshA, mshC, and mshD would be greatly simplified if they were clustered with mshB in a single operon, but inspection of the gene assignments and open reading frames surrounding mshB (Rv1170) in the M. tuberculosis genome indicates that this is not the case.\",\n \"All mycobacteria thus far examined have high MSH content (G. L. Newton et al.\",\n \"(1996) J. Bacteriol.\",\n \"178:1990-1995) and, based on the studies described herein, are expected to have an ortholog of the MSH biosynthesis enzyme MshB (Rv1170).\",\n \"The available unfinished mycobacterial genome databases yield homologs close to Rv1170 for Mycobacterium leprae (Sanger Centre), Mycobacterium bovis (Sanger Centre), M tuberculosis CDC1551 (The Institute for Genomic Research [TIGR]), M. smegmatis (TIGR), and Mycobacterium avium (TIGR).\",\n \"This sequence homology (FIG.\",\n \"6) indicates that MSH biosynthesis in these organisms utilizes a GlcNAc-Ins deacetylase (MshB) in the same manner as that described here for M. smegmatis.\",\n \"Other actinomycetes that produce MSH are also expected to have a GlcNAc-Ins deacetylase (MshB) gene homologous to Rv1170.Sequence BLAST searches to determine molecules with amino acid homology to the Rv1170 sequence of SEQ ID NO:1 (FIG.\",\n \"1C) also show M. tuberculosis homolog Rv1082 (mycothiol S-conjugate deacetylase) and the Rv1082 orthologs from M. leprae, M. bovis (Sanger Centre), M. tuberculosis CDC1551 (TIGR), M. smegmatis (TIGR), and M. avium (TIGR) to be close homologs.\",\n \"Sequences with partial homology to Rv1170 were also found in the yeast, rat, and human genome databases.\",\n \"These genes code for PIG-L, the second enzyme in glycosylphosphatidylinositol (GPI) anchor biosynthesis (Nakamura et al., (1997) J. Biol.\",\n \"Chem.\",\n \"272:15834-15840), and catalyze the deacetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI).\",\n \"The studies described in the Examples herein indicate that the invention deacetylase is rather specific for GlcNAc-Ins and is not a broad spectrum deacetylase.\",\n \"For example, removal of the inositol residue from the substrate GlcNAc-Ins resulted in a more than 300-fold decrease in reactivity (i.e., GlcNAc-Ins versus GlcNAc) (Table 1).\",\n \"Also, the studies described herein (Table 1) show that neither MSH nor MSmB is deacetylated by the invention deacetylases.\",\n \"Therefore, the AcCys moiety of MSH is inert to the deacetylase.\",\n \"This finding shows that the invention deacetylase has no significant activity for the degradation of MSH.\",\n \"The invention deacetylase appears to be the control point for MSH biosynthesis.\",\n \"In cells tested, a very large endogenous pool of its substrate GlcNAc-Ins (12 to 15 μmol per g of residual dry weight (RDW)) was found (Table 2), but quite low levels were found of its product GlcN-Ins (˜0.2 μmol per g of RDW) (Table 2) and of the final intermediate Cys-GlcN-Ins (<0.006 μmol per g of RDW) leading to MSH.\",\n \"This finding shows that substantial quantities of MSH can be produced upon demand from the endogenous pool of GlcNAc-Ins under control of the invention family of acetyl glucosaminyl inositol deacetylases.\",\n \"If a particular member of the invention family of polypeptide deacetylases hydrolyzes its substrate (GlcNAc-Ins), cleavage of the substrate molecule will form breakdown products wherein one product is a free amine (e.g., 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins)) and the other product is acetate.\",\n \"If a particular member of the invention family of polypeptide deacetylases has activity as an amide hydrolase (amidase), cleavage of the substrate acyl glucosaminyl inositol will form breakdown products wherein one product is a carboxylic acid, and the other product is a free amine (e.g., GlcN-Ins).\",\n \"If the substrate amide is a mycothiol-derived amide, one of the breakdown products will be GlcN-Ins and the other breakdown product will be a sulfur-containing carboxylic acid, such as an AcCys S-conjugate.\",\n \"AcCys S-conjugates are termed mercapturic acids, the final excreted product in the mercapturic acid pathway of glutathione-dependent detoxification in mammals.\",\n \"Mycothiol (1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside) (MSH) is present in a variety of actinomycetes and plays an essential role in a pathway of detoxification in such bacteria.\",\n \"Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes.\",\n \"In the mycothiol-dependent detoxification process in actinomycetes, an alkylating agent is converted to a S-conjugate of mycothiol, the latter is cleaved to release a mercapturic acid, and the mercapturic acid is excreted from the cell (Newton et al.\",\n \"(2000) Biochemistry 39:10739-10746).\",\n \"It has been discovered that invention acetyl glucosaminyl inositol deacetylases participate in the pathway of mycothiol biosynthesis in which substrate GlcNAc-Ins is an intermediate.\",\n \"In the mycothiol pathway as shown schematically in FIG.\",\n \"5, GlcNAc-Ins is converted to GlcN-Ins by the invention deacetylase.\",\n \"In fact, the studies disclosed herein have shown that the invention deacetylases are the critical step in the production of mycothiol in actinomycetes.\",\n \"For example, it has been shown by the studies described herein (Example 10) that MshB is inhibited by mycothiol in a manner consistent with a negative feedback loop dependent upon the level of mycothiol for control of mycothiol biosynthesis in the cell.\",\n \"Thus, a falling level of mycothiol in the cell available for detoxification of intracellular toxins increases activity of the deacetylase so that substrate GlcNAc-Ins will be cleaved by the expressed deacetylase, in turn, providing the GlcN-Ins moiety necessary for mycothiol production.\",\n \"A member of the invention family of polypeptide deacetylases shown to be responsible for cleavage of GlcNAc-Ins has been cloned from M. tuberculosis gene Rv1170 and has an amino acid sequence as set for in SEQ ID NO:1 (shown in FIG.\",\n \"1C; see also GenBank Accession No.\",\n \"B70875) and a nucleotide sequence as set forth in SEQ ID NO:7 (shown in FIG.\",\n \"1; see also nucleic acid residues 4222-5133 of GenBank Accession No.\",\n \"AL010186).\",\n \"The N-terminal region 100 residues of this newly discovered GlcNAc-Ins deacetylase has the amino acid sequence as shown in SEQ ID NO:2.Members of invention family of acetyl glucosaminyl inositol deacetylases are formed in vivo by bacteria as part of a mycothiol biosynthesis pathway, most usually in bacteria characterized by intracellular production of mycothiol.\",\n \"Additional bacteria from which the invention deacetylase polypeptides can be derived include actinomycetes, such as M. smegmatis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC) (Holzman, in Tuberculosis ed.\",\n \"by Rom and Gary (Little, Brown, and Company, 1996) Chapter 56), M. ulcerans, M. abscessus, or M. scrofulaceum, and the like.\",\n \"Homologous non-mycobacterial deacetylase proteins can also be derived from the antibiotic producers Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei, and the polyketide erythromycin antibiotic producer Saccharopolyspora erythraea.\",\n \"Inhibitors of the invention acetyl glucosaminyl inositol deacetylases are particularly well suited as antibiotics since mycothiol production will cease in the absence of the product GlcN-Ins produced by activity of the invention deacetylases.\",\n \"Accordingly, in another embodiment according to the present invention, there are provided methods for identifying an inhibitor of an invention acetyl glucosaminyl inositol deacetylase, MshB.\",\n \"The invention methods comprise contacting a candidate compound with a member of the invention family of deacetylases in the presence of a substrate for the enzyme under suitable conditions and determining the presence or absence of breakdown products of the substrate indicative of deacetylase (or optionally S-conjugate amidase) activity.\",\n \"For example, if the test compound is a putative inhibitor of amide hydrolase activity of the invention polypeptide deacetylase, the absence of acetate or other carboxylic acid and/or free amine, such as GlcN-Ins, as breakdown products indicates the candidate compound is an inhibitor of the activity of the invention polypeptide as a deacetylase.\",\n \"The preferred substrate for testing for deacetylase activity of the invention polypeptides is the acetyl glucosaminyl inositol (GlcNAc-Ins).\",\n \"On the other hand, if the test compound is a putative inhibitor of MshB, the appearance of free amine, such as GlcN-Ins, and/or acetate or carboxylate indicates that the candidate compound is not an inhibitor of the activity of the invention deacetylase as a deacetylase or as an amidase of an acyl glucosaminyl inositol.\",\n \"A preferred substrate for use in screening for inhibitors of deacetylase activity is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).\",\n \"Substantial absence of the breakdown products of enzymatic activity is indicative of a candidate compound that inhibits enzymatic activity of the deacetylase.\",\n \"In a preferred embodiment, the deacetylase used in the invention method is an acetyl glucosaminyl inositol deacetylase and the method is designed to assay for production of free amine.\",\n \"Such assays can be conducted en masse using a high throughput screening method for identifying inhibitors of the invention deacetylase.\",\n \"For example, a plurality of candidate compounds can be contacted with an invention deacetylase in the presence of a substrate for the enzyme (e.g.\",\n \"an acyl glucosaminyl inositol) under suitable conditions (e.g., as shown in the Examples herein) for activity of the deacetylase.\",\n \"Binding of a marker selected to bind to a breakdown product can then be assayed using any technique known in the art for high throughput screening.\",\n \"For example, a colorometric or fluorometric marker that binds to free amine can be used to screen a plurality of reaction mixtures formed in the wells of a microtitre plate (Wong, et al.\",\n \"(2001) Anal.\",\n \"Biochem.\",\n \"290:338-346).\",\n \"A preferred fluorometric marker for use in the invention high throughput screening methods is fluorescamine, which binds to free amine.\",\n \"In another embodiment of the invention, methods are provided for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample by contacting a sample containing GlcNAc-Ins with an invention deacetylase under suitable conditions and determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.\",\n \"The amount of GlcN-Ins in the sample can be determined by HPLC after labeling thereof with a fluorometric or colorimetric reagent, such as 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (sold as AccQFluor® reagent by Waters), and the like.\",\n \"In an alternative embodiment of the invention, methods are provided for decreasing the virulence in mammalian cells of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium, such as an actinomycete.\",\n \"In the invention method for decreasing the virulence of pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria, an inhibitor of acetyl glucosaminyl inositol deacetylase (for example, one identified by the above-described screening method) is introduced into the bacterium.\",\n \"Intracellular uptake by the treated bacterium of the inhibitor results in decreased activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.\",\n \"For example, for treatment of isolated mammalian cells, the introducing can comprise culturing the bacterium in the presence of the inhibitor.\",\n \"Alternatively, for treatment of mammalian cells contained in a living organism, the inhibitor may be administered systemically to the living organism.\",\n \"Pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria whose virulence can be reduced according to the invention methods include such actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis particularly in bovine subjects), M. intracellulare, M. africanum, and M. marinarum.\",\n \"M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.\",\n \"The inhibitors used in the invention methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium may either inhibit intracellular production of the deacetylase or inhibit intracellular deacetylase activity of the deacetylase.\",\n \"In one embodiment according to the present invention, there are provided inhibitors of the members of the invention family of deacetylase polypeptides.\",\n \"The invention inhibitors are characterized by being derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+ or otherwise binds the enzyme active site.\",\n \"The invention inhibitors may also chelate one or more metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.\",\n \"The following are examples of moieties that can substitute for the amino group in GlcN-Ins in the invention inhibitors: ClCH2CONH— HONHCONH— HONHCOCH2— HOPO(CH3)NH— HOPO(CH3)nCH2—; wherein n=1-5 HSCH2(CH2)nNH—; wherein n=2-5 HS(CH2)nCONH—; wherein n=1-3.In another embodiment according to the present invention, there are provided derivatives of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress (e.g., including nitrosative stress) so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.\",\n \"Examples of reactive residues that can be attached to the amino group of GlcN-Ins to cause the compound to be selectively toxic to mycothiol-producing actinomycetes by intracellular concentration include, but are not limited to a nitroso residue, a nitroalkyl residue comprising 1 to 3 carbon atoms, a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms, or a peroxyalkyl residue comprising 2 to 4 carbon atoms.\",\n \"In another embodiment according to the present invention, the invention inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or 80% or more sequence identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.\",\n \"In another embodiment the anti-sense oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.For example, in one embodiment, the candidate compound inhibits intracellular production or activity of the acetyl glucosaminyl inositol deacetylase.\",\n \"A presently preferred drug candidate for screening in live bacteria for activity that inhibits intracellular production or activity of acetyl glucosaminyl inositol deacetylase is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence with 40% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:2, or a conservative variation thereof, for example, 50%, 55%, 60% or 65% sequence identity.\",\n \"Suitable conditions for conducting invention drug screening methods are well known in the art and are described, for example, in the Examples hereinbelow.\",\n \"Alternatively, an anti-sense oligonucleotide can be designed to hybridize under in vivo conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2, or contains an amino acid segment as set forth in SEQ ID NOs:3, 4, 5, and 6, or a conservative variation thereof.\",\n \"The anti-sense oligonucleotide can comprise from about 10 to about 60 nucleic acid residues, for example from 10 to about 50, or from 10 to about 40, 30 or 20 nucleic acid residues.\",\n \"“Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing.\",\n \"Hybridization reactions can be sensitive and selective so that a particular sequence of interest will join with a complementary strand even in samples in which it is present at low concentrations.\",\n \"Suitable intracellular conditions for hybridization of an anti-sense oligonucleotide to messenger RNA will be determined by the particular bacterium used in the invention method.\",\n \"In general, the pH, temperature and salt concentration must be comparable to intracellular conditions in the test bacterium.\",\n \"In yet another embodiment according to the present invention, there are provided live mutant actinomycetes, whose genomes comprise a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene and thereby prevents mycothiol synthesis.\",\n \"Disruption of genes for mycothiol biosynthesis in mycobacteria reduces their survival in mammalian macrophages (FIG.\",\n \"11).\",\n \"Disruption of the endogenous acetyl glucosaminyl inositol deacetylase gene will prevent function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids should be substantially unaffected.\",\n \"As a result, invention live mutant actinomycetes exhibit the phenotype of transient survival in mammalian white blood cells, such as murine or human white blood cells, for an immune response raising period of time.\",\n \"Such genetically engineered live mutant actinomycetes will survive in mammalian white blood cells for a period of time from 1 to 30 days, for example from 4 to 25 days or from 5 to 20 days, but in no event for more than 30 days.\",\n \"Due to lack of intracellular acetyl glucosaminyl inositol deacetylase the invention live mutant bacterium will fail to produce mycothiol.\",\n \"Hence, the mutant live bacterium is unable to survive the oxidative stress inherent in the intracellular environment of mammalian white blood cells long enough to establish infection in the cells or to establish infection in an immunocompetent mammal containing such white blood cells.\",\n \"Thus, invention live mutant actinomycetes possess a combination of features desired for a vaccine effective in mammals against infection by such pathogenic actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.\",\n \"An individual (e.g., an animal, such as a mouse, a farm animal or a human) to which the live mutant is administered according to a protocol appropriate for inducing a protective immune response and who has not previously been infected by the counterpart wild type will mount an immune response to the vaccine, for example an immune response sufficient to protect the individual against future infection by the corresponding wild-type live pathogen.\",\n \"Alternatively, the invention live mutant actinomycetes are useful as a research tool to investigate the properties desirable in a live mutant vaccine.\",\n \"In still another embodiment according to the present invention, there are provided processes for preparation of GlcN-Ins by contacting an N-acyl glucosaminyl inositol under suitable conditions with an acetyl glucosaminyl inositol deacetylase so as to hydrolyze the amide bond therein to obtain stereochemically pure α(1-1) GlcN-Ins (1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside).\",\n \"For example, the N-acyl glucosaminyl inositol can be a mycothiol S-conjugate, such as the bimane derivative of mycothiol.\",\n \"The deacetylase cleaves the N-acyl glucosaminyl inositol, freeing GlcN-Ins as one of the cleavage breakdown products.\",\n \"GlcN-Ins has utility in conducting research regarding deacetylase activity and mycothiol biochemistry in bacteria, development of products and procedures for overcoming the antibiotic resistance of pathogenic bacteria, such as actinomycetes, and as a precursor for formation of acyl glucosaminyl inositol derivatives and inhibitors of deacetylases thereof as disclosed herein.\",\n \"A “conservative variation” in an amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.\",\n \"A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).\",\n \"One or more amino acids can be deleted, for example, from an deacetylase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.\",\n \"For example, carboxyl-terminal amino acids that are not required for deacetylase or deacetylase activity of the invention polypeptides can be removed.\",\n \"Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.\",\n \"Such vectors are used to incorporate DNA sequences of the invention.\",\n \"In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted eukaryotic genetic sequence are used in connection with the host.\",\n \"The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells.\",\n \"In addition to expression vectors known in the art such as bacterial, yeast and mammalian expression systems, baculovirus vectors may also be used.\",\n \"One advantage to expression of foreign genes in this invertebrate virus expression vector is that it is capable of expression of high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts.\",\n \"Baculovirus vectors and the appropriate insect host cells used in conjunction with the vectors will be known to those skilled in the art.\",\n \"The term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the invention acetyl glucosaminyl inositol deacetylase genetic sequences.\",\n \"Such expression vectors contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host.\",\n \"The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.\",\n \"Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol.\",\n \"Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.\",\n \"The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).\",\n \"The vector may include a phenotypically selectable marker to identify host cells which contain the expression vector.\",\n \"Examples of markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin (β-lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase).\",\n \"Examples of such markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and xanthine guanine phosphoribosyltransferse (XGPRT, gpt).\",\n \"The isolation and purification of host cell expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody.\",\n \"Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art.\",\n \"Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by electroporation or the CaCl2 method using procedures well known in the art.\",\n \"Alternatively, MgCl2 or RbCl could be used.\",\n \"Where the host used is a eukaryote, various methods of DNA transfer can be used.\",\n \"These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or the use of virus vectors.\",\n \"Eukaryotic cells can also be cotransformed with DNA sequences encoding the polypeptides of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.\",\n \"Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.\",\n \"(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).\",\n \"Examples of mammalian host cells include COS, BHK, 293, and CHO cells.\",\n \"Eukaryotic host cells may also include yeast.\",\n \"For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells.\",\n \"Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987).\",\n \"The invention provides antibodies that are specifically reactive with invention deacetylase polypeptides or fragments thereof.\",\n \"Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.\",\n \"Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).\",\n \"Monoclonal antibodies specific for acetyl glucosaminyl inositol deacetylase polypeptide can be selected, for example, by screening for hybridoma culture supernatants that react with acetyl glucosaminyl inositol deacetylase polypeptides, but do not react with other bacterial deacetylases.\",\n \"Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.\",\n \"Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).\",\n \"The term “antibody” as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2 and Fv, which are capable of binding the epitopic determinant.\",\n \"These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.\",\n \"Methods of making these fragments are known in the art.\",\n \"(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).\",\n \"As used in this invention, the term “epitope” means any antigenic determinant on an antigen to which the paratope of an antibody binds.\",\n \"Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.\",\n \"Antibodies that bind to acetyl glucosaminyl inositol deacetylase polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.\",\n \"The polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis and can be conjugated to a carrier protein, if desired.\",\n \"Such commonly used carriers, which are chemically coupled to the peptide, include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.\",\n \"The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).\",\n \"If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.\",\n \"Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated herein by reference).\",\n \"It is also possible to use the anti-idiotype technology to produce monoclonal antibodies that mimic an epitope.\",\n \"For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the “image” of the epitope bound by the first monoclonal antibody.\",\n \"In yet other preferred embodiments, the recombinant acetyl glucosaminyl inositol deacetylase polypeptide is a fusion protein further-comprising a second polypeptide portion having an amino acid sequence from a protein unrelated to the acetyl glucosaminyl inositol deacetylase.\",\n \"Such fusion proteins can be functional in a two-hybrid assay.\",\n \"Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an acetyl glucosaminyl inositol deacetylase polypeptide, or a fragment thereof, having an amino acid sequence at least 60% homologous to one of SEQ ID NOs:2, 3, 4 or 5.In a more preferred embodiment, the nucleic acid encodes a protein having an amino acid sequence 40% or more homologous to SEQ ID NO:2, more preferably at least 50% homologous to SEQ ID NO:2, and most preferably at least 65% homologous to SEQ ID NO:2.In another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 24 consecutive nucleotides encoding SEQ ID NO:3.In a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:4.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:5.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:6; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:6.Furthermore, in certain embodiments, the acetyl glucosaminyl inositol deacetylase nucleic acid will comprise a transcriptional regulatory sequence, e.g.\",\n \"at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the acetyl glucosaminyl inositol deacetylase-gene sequence so as to render the recombinant acetyl glucosaminyl inositol deacetylase gene sequence suitable for use as an expression vector.\",\n \"The present invention also features transgenic non-human organisms, e.g.\",\n \"live mutant actinomycetes, which either express a heterologous acetyl glucosaminyl inositol deacetylase gene, or in which expression of their own acetyl glucosaminyl inositol deacetylase gene is disrupted.\",\n \"In addition to the other utilities of such organisms disclosed herein, such a transgenic organism has utility for overproduction of acetyl glucosaminyl inositol needed for screening (particularly high throughput screening) for compounds that inhibit acetyl glucosaminyl inositol deacetylase activity in mycothiol-producing bacteria.\",\n \"The present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or anti-sense sequence encoding one of the amino acid sequences encompassed by SEQ ID NOs:2, 3, 4 5, or 6, or naturally occurring mutants thereof.\",\n \"Yet another aspect of the invention pertains to a peptidomimetic that binds to or interferes with an acetyl glucosaminyl inositol deacetylase polypeptide and inhibits its binding to or cleavage of substrate acyl glucosaminyl inositols.\",\n \"For example, a preferred peptidomimetic is an analog of a peptide having the sequence of one of the SEQ ID NOs.\",\n \"1, 2, 3, 4, 5, or 6.Non-hydrolyzable peptide analogs of such residues can be generated using, for example, benzodiazepine, azepine, substituted gama-lactam rings, keto-methylene pseudopeptides, beta-turn dipeptide cores, or beta-aminoalcohols.\",\n \"Other features and advantages of the invention will be apparent from the detailed description herein, and from the claims.\",\n \"The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.\",\n \"Such techniques are explained fully in the literature.\",\n \"See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.\",\n \"by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al.\",\n \"U.S. Pat.\",\n \"No.\",\n \"4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.\",\n \"1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.\",\n \"1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.\",\n \"154 and 155 (Wu et al.\",\n \"eds.\",\n \"), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).\",\n \"As used herein, the term “actinomycetes” and “an actinomycete” encompasses any bacterium of the order Actinomycetales.\",\n \"As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).\",\n \"The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or anti-sense) and double-stranded polynucleotides.\",\n \"As used herein, the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an open reading frame encoding an invention acetyl glucosaminyl inositol deacetylase, including both exon and (optionally) intron sequences.\",\n \"The term “intron” refers to a DNA sequence present in a given acetyl glucosaminyl inositol deacetylase gene which is not translated into protein and is generally found between exons.\",\n \"“Homology” refers to sequence similarity between two peptides or between two nucleic acid molecules.\",\n \"Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.\",\n \"When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position.\",\n \"Percent homology between sequences is a function of the number of matching or homologous positions shared by the sequences.\",\n \"The sequence data of a test clone is aligned to the sequences in the database or databases using algorithms designed to measure homology between two or more sequences.\",\n \"Sequence alignment methods include, for example, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), and FASTA (Person & Lipman, 1988).\",\n \"For example, optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.\",\n \"The term “transfection” or “transforming” and grammatical equivalents thereof, refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.\",\n \"“Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of one of the invention family of acetyl glucosaminyl inositol deacetylases.\",\n \"“Cells” or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context.\",\n \"These terms include the immediate subject cells that express the acetyl glucosaminyl inositol deacetylases of the present invention, and, of course, the progeny thereof.\",\n \"It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment.\",\n \"However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell.\",\n \"In the present case, such a characteristic might be the ability to produce a recombinant acetyl glucosaminyl inositol deacetylase polypeptide.\",\n \"As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.\",\n \"The term “expression vector” includes plasmids, cosmids or phages capable of synthesizing the subject acetyl glucosaminyl inositol deacetylase polypeptide encoded by the respective recombinant gene carried by the vector.\",\n \"Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.\",\n \"In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.\",\n \"Moreover, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.\",\n \"“Transcriptional regulatory sequence” is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, as well as polyadenylation sites, which induce or control transcription of protein coding sequences with which they are operably linked.\",\n \"In preferred embodiments, transcription of a recombinant acetyl glucosaminyl inositol deacetylase gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in a cell-type in which expression is intended.\",\n \"It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences that control transcription of the naturally occurring form of the regulatory protein.\",\n \"As used herein, a “transgenic organism” is any organism, preferably a bacteria in which one or more of the cells of the organism contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.\",\n \"The nucleic acid is introduced into the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or a vector.\",\n \"The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.\",\n \"This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.\",\n \"In the typical transgenic organisms described herein, the transgene causes cells to express a recombinant form of the subject acetyl glucosaminyl inositol deacetylase polypeptides.\",\n \"As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., an acetyl glucosaminyl inositol deacetylase polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic organism or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic organism or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the organism's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).\",\n \"A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.\",\n \"The term “evolutionarily related to”, with respect to nucleic acid sequences encoding acetyl glucosaminyl inositol deacetylase polypeptides, refers to nucleic acid sequences that have arisen naturally in an organism, including naturally occurring mutants.\",\n \"The term also refers to nucleic acid sequences which, while derived from a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide.\",\n \"One aspect of the present invention pertains to an isolated nucleic acid comprising the nucleotide sequence encoding an acetyl glucosaminyl inositol deacetylase polypeptide, fragments thereof encoding polypeptides having acetyl glucosaminyl inositol deacetylase activity, and/or equivalents of such nucleic acids.\",\n \"The term-nucleic acid as used herein is intended to include such fragments and equivalents.\",\n \"The term equivalent is understood to include nucleotide sequences encoding functionally equivalent acetyl glucosaminyl inositol deacetylase polypeptides or functionally equivalent peptides having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide such as described herein.\",\n \"Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will also include sequences that differ from the nucleotide sequence encoding native acetyl glucosaminyl inositol deacetylases due to the degeneracy of the genetic code.\",\n \"Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27° C. below the melting temperature of the DNA duplex formed in about 1 M salt) to the nucleotide sequence of an acetyl glucosaminyl inositol deacetylase gene, such as that as set forth in SEQ ID NO:7, particularly those segments encoding the polypeptides shown in one of SEQ ID NOs.\",\n \"2, 3, 4, 5 or 6, and conservative variations thereof.\",\n \"In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to such nucleotide sequences.\",\n \"The term “isolated” or “purified” as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.\",\n \"For example, an isolated nucleic acid encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the acetyl glucosaminyl inositol deacetylase gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.\",\n \"The term isolated or purified as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.\",\n \"Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state.\",\n \"In yet another embodiment, the nucleic acid of the invention encodes a peptide having an amino acid sequence as shown SEQ ID NO:1.Preferred nucleic acids encode a peptide having acetyl glucosaminyl inositol deacetylase polypeptide activity and being 40% or more homologous, more preferably 50% homologous and most preferably 65% homologous with an amino acid sequence as set forth in SEQ ID NO:2 (encoded by a nucleic acid sequence as set forth in SEQ ID NO:7).\",\n \"Nucleic acids that encode peptides having an activity of an invention acetyl glucosaminyl inositol deacetylase polypeptide are also within the scope of the invention.\",\n \"Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes an acetyl glucosaminyl inositol deacetylase polypeptide having all or a portion of an amino acid sequence shown in one of SEQ ID NOs.\",\n \"2, 3, 4, 5 or 6.Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Isolated nucleic acids that differ from the nucleotide sequences disclosed herein due to degeneracy in the genetic code are also within the scope of the invention.\",\n \"For example, more than one triplet designates a number of amino acids.\",\n \"Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of the protein.\",\n \"However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject acetyl glucosaminyl inositol deacetylase polypeptides will exist among prokaryotic cells.\",\n \"One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding a particular member of the acetyl glucosaminyl inositol deacetylase polypeptide family may exist among individuals of a given species due to natural allelic variation.\",\n \"Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.\",\n \"Fragments of the nucleic acid encoding a biologically active portion of the subject acetyl glucosaminyl inositol deacetylase polypeptides are also within the scope of the invention.\",\n \"As used herein, a fragment of the nucleic acid encoding an active portion of an acetyl glucosaminyl inositol deacetylase polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, for example, the deacetylase polypeptides represented in nucleic acid SEQ ID NO:1, and which encodes a peptide which retains at least a portion of the biological activity of the full-length protein (i.e., a peptide capable of acetyl glucosaminyl inositol deacetylase activity) as defined herein, or alternatively, which is functional as an antagonist of the deacetylase activity of the full-length protein.\",\n \"Nucleic acid fragments within the scope of the invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species, e.g.\",\n \"for use in screening protocols to detect homologs of the subject acetyl glucosaminyl inositol deacetylase polypeptides.\",\n \"Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant peptides.\",\n \"As indicated by the examples set out below, a nucleic acid encoding a peptide having an activity of an invention deacetylase polypeptide may be obtained from mRNA or genomic DNA present in any of a number of antibiotic-producing or pathogenic bacteria, particularly actinomycetes, in accordance with protocols described herein, as well as those generally known to those skilled in the art.\",\n \"A cDNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide, for example, can be obtained by isolating total mRNA from a bacterial cell.\",\n \"Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.\",\n \"A gene encoding an acetyl glucosaminyl inositol deacetylase polypeptide can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.\",\n \"Another aspect of the invention relates to the use of an “anti-sense” isolated nucleic acid.\",\n \"As used herein, an “anti-sense” inhibition of endogenous production of an acetyl glucosaminyl inositol deacetylase molecule is carried out by administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g.\",\n \"bind) under intracellular conditions, with the cellular mRNA and/or genomic DNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide so as to inhibit expression of that protein or a constituent thereof, e.g.\",\n \"by inhibiting transcription and/or translation.\",\n \"The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.\",\n \"In general, “anti-sense” therapy refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.\",\n \"An anti-sense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the transformed cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an acetyl glucosaminyl inositol deacetylase polypeptide.\",\n \"Alternatively, the anti-sense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one of the subject acetyl glucosaminyl inositol deacetylase proteins.\",\n \"Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g.\",\n \"exonucleases and/or endonucleases, and is therefore stable in vivo.\",\n \"Exemplary nucleic acid molecules for use as anti-sense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.\",\n \"Nos.\",\n \"5,176,996; 5,264,564; and 5,256,775).\",\n \"Additionally, general approaches to constructing oligomers useful in anti-sense techniques have been reviewed, for example, by van der Krol et al.\",\n \"(1988) Biotechniques 6:958-976; and Stein et al.\",\n \"(1988) Cancer Res 48:2659-2668.In addition, the oligomers of the invention may be used as reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind.\",\n \"Such diagnostic tests are described in further detail below.\",\n \"This invention also provides expression vectors comprising a nucleotide sequence encoding a member of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides and operably linked to at least one regulatory sequence.\",\n \"Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence.\",\n \"Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.\",\n \"Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements.\",\n \"Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).\",\n \"For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the acetyl glucosaminyl inositol deacetylase polypeptides of this invention.\",\n \"Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.\",\n \"It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.\",\n \"Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.\",\n \"As will be apparent, the subject gene constructs can be used to cause expression of the subject acetyl glucosaminyl inositol deacetylase polypeptides in cells propagated in culture, e.g.\",\n \"to produce proteins or peptides, including fusion proteins or peptides, for purification.\",\n \"This invention also pertains to a host cell transfected with a recombinant acetyl glucosaminyl inositol deacetylase gene in order to express a polypeptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.\",\n \"The host cell may be any prokaryotic or eukaryotic cell.\",\n \"For example, an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells.\",\n \"Other suitable host cells are known to those skilled in the art.\",\n \"Another aspect of the present invention concerns recombinant acetyl glucosaminyl inositol deacetylase polypeptides that have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide, or which are naturally occurring mutants thereof.\",\n \"The term “recombinant protein” refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the acetyl glucosaminyl inositol deacetylase polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.\",\n \"Moreover, the phrase “derived from”, with respect to a recombinant gene encoding the recombinant acetyl glucosaminyl inositol deacetylase polypeptide, is meant to include within the meaning of “recombinant protein” those proteins having an amino acid sequence of a native acetyl glucosaminyl inositol deacetylase polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions of a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide of a organism.\",\n \"The present invention further pertains to methods of producing the subject acetyl glucosaminyl inositol deacetylase polypeptides.\",\n \"For example, a host cell transfected with expression vector encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptide can be cultured under appropriate conditions to allow expression of the peptide to occur.\",\n \"The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide.\",\n \"Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.\",\n \"A cell culture includes host cells, media and other byproducts.\",\n \"Suitable media for cell culture are well known in the art.\",\n \"The peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the subject acetyl glucosaminyl inositol deacetylase polypeptides.\",\n \"Thus, a nucleotide sequence derived from the cloning of an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention, encoding all or a selected portion of the protein, can be used to produce a recombinant form of the protein via microbial cellular processes.\",\n \"The recombinant acetyl glucosaminyl inositol deacetylase polypeptide can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in bacterial cells.\",\n \"Expression vehicles for production of a recombinant acetyl glucosaminyl inositol deacetylase polypeptide include plasmids and other vectors.\",\n \"For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.\",\n \"A number of vectors exist for the expression of recombinant proteins in yeast.\",\n \"For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al.\",\n \"(1983) in Experimental Manipulation of Gene Expression, ed.\",\n \"M. Inouye Academic Press, p. 83, incorporated by reference herein).\",\n \"These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.\",\n \"In addition, drug resistance markers such as ampicillin can be used.\",\n \"The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.\",\n \"For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.\",\n \"by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.In some instances, it may be desirable to express the recombinant acetyl glucosaminyl inositol deacetylase polypeptide by the use of a baculovirus expression system.\",\n \"Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).\",\n \"This invention further contemplates a method of generating sets of combinatorial mutants of the present acetyl glucosaminyl inositol deacetylase polypeptides, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g.\",\n \"homologs) that are functional in cleaving acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide molecules.\",\n \"In a representative embodiment of this method, the amino acid sequences for a population of acetyl glucosaminyl inositol deacetylase polypeptide homologs are aligned, preferably to promote the highest homology possible.\",\n \"Such a population of variants can include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation.\",\n \"Amino acids that appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.\",\n \"The presence or absence of amino acids from an aligned sequence of a particular variant is relative to a chosen consensus length of a reference sequence, which can be real or artificial.\",\n \"In order to maintain the highest homology in alignment of sequences, deletions in the sequence of a variant relative to the reference sequence can be represented by an amino acid space (*), while insertional mutations in the variant relative to the reference sequence can be disregarded and left out of the sequence of the variant when aligned.\",\n \"Further expansion of the combinatorial library can be made by, for example, by including amino acids that would represent conservative mutations at one or more of the degenerate positions.\",\n \"Inclusion of such conservative mutations can give rise to a library of potential acetyl glucosaminyl inositol deacetylase sequences.\",\n \"Alternatively, amino acid replacement at degenerate positions can be based on steric criteria, e.g.\",\n \"isosteric replacement, without regard for polarity or charge of amino acid sidechains.\",\n \"Similarly, completely random mutagenesis of one or more of the variant positions can be carried out.\",\n \"In a preferred embodiment, the combinatorial acetyl glucosaminyl inositol deacetylase library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential acetyl glucosaminyl inositol deacetylase polypeptide sequences.\",\n \"For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential acetyl glucosaminyl inositol deacetylase nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g.\",\n \"for phage display) containing the set of acetyl glucosaminyl inositol deacetylase polypeptide sequences therein.\",\n \"There are many ways by which the library of potential acetyl glucosaminyl inositol deacetylase homologs can be generated from a degenerate oligonucleotide sequence.\",\n \"Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression.\",\n \"The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential acetyl glucosaminyl inositol deacetylase sequences.\",\n \"The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.\",\n \"(1981) Recombinant DNA, Proc 3rd Cleveland Sympos.\",\n \"Macromolecules, ed.\",\n \"A G Walton, Amsterdam: Elsevier pp.\",\n \"273-289; Itakura et al.\",\n \"(1984) Annu.\",\n \"Rev.\",\n \"Biochem.\",\n \"53:323; Itakura et al.\",\n \"(1984) Science 198:1056; Ike et al.\",\n \"(1983) Nucleic Acid Res.\",\n \"11:477.A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and, for that matter, for screening cDNA libraries for gene products having a certain property.\",\n \"Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of acetyl glucosaminyl inositol deacetylase homologs.\",\n \"The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene product was detected.\",\n \"Each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.\",\n \"The invention also provides for reduction of the subject acetyl glucosaminyl inositol deacetylase polypeptides to generate mimetics, e.g.\",\n \"peptide or non-peptide agents, which are able to mimic binding of the authentic acetyl glucosaminyl inositol deacetylase polypeptide to a substrate molecule.\",\n \"Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of an acetyl glucosaminyl inositol deacetylase polypeptide which participate in protein-protein interactions involved in, for example, binding of the subject acetyl glucosaminyl inositol deacetylase polypeptide to a substrate.\",\n \"To illustrate, the critical residues of a subject acetyl glucosaminyl inositol deacetylase polypeptide which are involved in molecular recognition of substrate can be determined and used to generate acetyl glucosaminyl inositol deacetylase-derived peptidomimetics which cleave acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide substrates and, like the authentic acetyl glucosaminyl inositol deacetylase polypeptide, cleave the substrate molecule, for example by amide hydrolase activity.\",\n \"By employing, for example, scanning mutagenesis to map the amino acid residues of a particular acetyl glucosaminyl inositol deacetylase polypeptide involved in binding to a substrate, peptidomimetic compounds (e.g.\",\n \"diazepine or isoquinoline derivatives) can be generated which mimic those residues cleaving substrate.\",\n \"For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al; in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al.\",\n \"in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al.\",\n \"in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.\",\n \"(1986) J Med Chem 29:295; and Ewenson et al.\",\n \"in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), beta-turn dipeptide cores (Nagai et al.\",\n \"(1985) Tetrahedron Lett 26:647; and Sato et al.\",\n \"(1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols (Gordon et al.\",\n \"(1985) Biochem Biophys Res Commun 126:419; and Dann et al.\",\n \"(1986) Biochem Biophys Res Commun 134:71).\",\n \"The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.\",\n \"EXAMPLE 1 Preparation of Reagents and Assay Methods Used.\",\n \"Mycothiol (MSH) was isolated from M. smegmatis and derivatized with monobromobimane to form the MSH-bimane derivative (MSmB).\",\n \"Mycothiol can be purified from Streptomyces clavuligerus, M. smegmatis, or from cells of any other mycothiol containing bacterium.\",\n \"Early stationary phase cells (25 gm wet weight) were extracted with 250 ml of 50% acetonitirile containing 25 mM methanesulfonic acid and 1 mM DTT at 60° C. for 15 min.\",\n \"The cell debris was removed by centrifugation for 30 min (4° C.) at 20,000×g.\",\n \"The excess acetonitrile was removed using a rotary evaporator.\",\n \"The aqueous extract was neutralized with Tris base to pH 8.0 and clarified by centrifugation for 30 min (4° C.) at 20,000×g The extract was loaded on a 25×70 mm propyl thiol Sepharose 6B resin activated with 2-mercaptopyridine with 1 mmole total thiol binding capacity.\",\n \"The column was washed with 10 column volumes of 20 mM Tris-HCl pH 8.0 to remove unbound extract components.\",\n \"The bound thiols were eluted with 200 ml of 20 mM Tris-HCl pH 8.0 containing 3 mM DTT.\",\n \"The eluate was extracted 5 times with an equal volume of ethyl acetate to remove excess DTT.\",\n \"The ethyl acetate washed aqueous extract was reduced to dryness by lyophilization.\",\n \"The extract was suspended in 5 ml of aqueous 0.1% trifluoroacetic acid (TFA).\",\n \"The mycothiol was purified by preparative HPLC using a 10×250 mm Vydac C-18 (Cat.\",\n \"# 218TP152022) reverse phase column (or equivalent).\",\n \"Mycothiol eluted at 26 min using an isocratic elution in aqueous 0.1% TFA at 5 ml per min.\",\n \"The volatile solvent was removed by lyophilization to yield pure mycothiol.\",\n \"Mycothiol is stable for years when stored as a mildly acidic aqueous solution of 30-100 mM at −70° C. The bimane derivative of mycothiol is readily made using monobromobimane (Molecular Probes) at 2 mM excess over the thiol concentration.\",\n \"The derivatization is carried out in aqueous 20 mM Tris-HCl pH 8.0 for 15 min in the dark at room temperature.\",\n \"The derivatization mixture was acidified to pH<3 with TFA and extracted 2 times with equal volumes of dichloromethane to remove excess monobromobimane.\",\n \"The derivatization mixture was purified on the preparative reverse phase column described above using a gradient elution.\",\n \"The A-buffer was 0.1% aqueous TFA and the B buffer was methanol.\",\n \"The linear gradient was 15-30% methanol over 30 min with a flow rate of 5 ml per min.\",\n \"The MSmB was detected at 385 nm and eluted as a symmetrical peak at 33 min.\",\n \"The volatile solvent was removed by lyophilization to yield>98% pure MSmB.\",\n \"This derivative was stored as a 10-30 mM solution in water at −70° C. The purified M. smegmatis mycothiol S-conjugate amidase was used to quantitatively hydrolyze MSMB to stereochemically pure GlcN-Ins, and the latter was purified from the other hydrolysis product, AcCySmB, using a Sep-Pak® C18 (Waters) cartridge.\",\n \"A 10 mM stock solution of GlcNAc-Ins was prepared by addition of a tenfold excess of acetic anhydride (Fisher) to 10 mM GlcN-Ins in 10 mM NaHCO3 over 20 min while adjusting the pH to 8.5 with NaOH.\",\n \"The reaction was monitored for GlcN-Ins loss as described below to insure that the reaction was complete and that no residual GlcN-Ins was present.\",\n \"This stock solution of GlcNAc-Ins was assayed as described below and used without further purification.\",\n \"Preparation of Mycothiol S-conjugates.\",\n \"Mycothiol S-conjugates were prepared by reaction of excess electrophile with mycothiol followed by removal of excess electrophile.\",\n \"Stock solutions (100 mM) of iodoacetamide (Sigma) or bromoacetophenone (Sigma) were prepared in acetonitrile.\",\n \"Reaction of these electrophiles at 2 mM in 25 mM HEPES chloride pH 7.5 was initiated by addition of mycothiol to 1 mM from a 32 mM stock solution in H2O (pH˜3) and was allowed to proceed 15 min at 22° C. Excess reagent was removed by extracting 3 times with an equal volume of H2O-saturated dichloromethane.\",\n \"Prior to extraction a 1 μl aliquot was withdrawn for reaction with mBBr and analysis of residual mycothiol by HPLC (see above) to verify complete reaction.\",\n \"This showed that>99% of the mycothiol had reacted.\",\n \"The MS-acetophenone was purified to homogeneity by reverse phase preparative HPLC as described above for MSmB, except that MS-acetophenone was detected at 260 nm.\",\n \"Assay of MSH and MSH precursors.\",\n \"Mycothiol and its precursors cysteine, and Cys-GlcN-Ins were assayed as the bimane derivative by high performance liquid chromatography (HPLC) on a Ultrasphere® ODS IP (Beckman) reversed phase column.\",\n \"The bimane derivatives were detected by fluorescence using a Fluoromonitor III® monitor (Thermo Separation products) with excitation filters 340-380 nm and emission filters 418-700 nm.\",\n \"The A buffer was 0.1% TFA and the B buffer was 7.5% methanol in acetonitrile.\",\n \"The separation was achieved at 22° C. and a flow rate of 1 ml/min with linear gradients as follows: time 0 min 0% B, 10 min 0% B, 30 min 20% B, 33 min 100% B, 36 min 100% B, 38 min 0% B, 50 min reinject.\",\n \"CySmB-GlcN-Ins, CySmB and mycothiol-mB (MSmB) eluted at 28, 31 and 33 min, respectively.\",\n \"GlcN-Ins was assayed by precolumn derivatization using AccQFluor® (Waters) reagent followed by HPLC analysis.\",\n \"A sample, 7.5 μl of 50% acetonitrile sample containing 5 mM NEM or 5 mM NEM and 1 mM 1,10 phenanthroline was mixed with 39.4 μl of buffer stock.\",\n \"The buffer stock was composed of 30% acetonitrile in aqueous 160 mM HEPES pH 7.5.This was mixed with 15.6 μl of AccQ-Fluor® reagent (Waters) and incubated for 1 min at 22° C. followed by 10 min at 60° C. The sample was diluted with 188 μl of water and the sample was analyzed by HPLC.\",\n \"The sample was centrifuged for 3 min 14,000×g in a microcentrifuge.\",\n \"The HPLC separation conditions were the same as described above for bimane derivatives, except the B buffer was 0.1% TFA in methanol.\",\n \"The product was detected by fluorescence using 254 nm excitation and 370-700 nm emission filters.\",\n \"AccQ-GlcN and AccQ-GlcN-Ins eluted at 12 and 24 min respectively.\",\n \"Mycothiol S-conjugate amidase assay.\",\n \"The enzymatic activity was routinely assayed by quantitation of the bimane derivative of N-acetylcysteine (AcCySmB) produced from the bimane derivative of mycothiol (MSmB), prepared from purified mycothiol (see above).\",\n \"To conduct an amidase assay a sample (2-10 μL) of extract was mixed with 40 μL of 30 μM MSmB in 3 mM 2-mercaptoethanol, 25 mM HEPES chloride pH 7.5 and reacted 10-30 min at 30° C. before quenching the reaction with 50 μL of 40 mM methanesulfonic acid on ice.\",\n \"The mixture was centrifuged for 3 min at 14,000×g in a microcentrifuge at room temperature and the supernatant analyzed by HPLC without dilution.\",\n \"Separation of MSmB and AcCySmB was achieved by HPLC on a 0.46×250 mM Ultrasphere®ODS column (Beckman) using a linear gradients: time 0 min 10% B, 15 min 18% B, 30 min 27% B, 32 min 100% B, 34 min 10% B, 45 min reinject.\",\n \"Buffer A (aqueous 0.25% acetic acid titrated to pH 3.6 with NaOH) and buffer B (methanol) were pumped at 1.2 ml/min at ambient temperature (22° C.).\",\n \"The bimane derivative of mycothiol (MSmB) eluted at 23.5 min and AcCySmB eluted at 27 min.\",\n \"EXAMPLE 2 Cloning and Expression of the Rv1170 gene from M. tuberculosis and demonstration of its GlcNAc-Ins deacetylase activity.\",\n \"Since the invention deacetylases and the mycothiol S-conjugate amidase both cleave an amide bond involving glucosamine, further studies were conducted to determine whether these proteins might be structurally related.\",\n \"A search was conducted of the Sanger Centre database for homologs of the 288 amino acid M. tuberculosis deacetylase, Rv1082.The closest homolog found was Rv1170, with a length of 304 residues and 36% identity to Rv1082 with homology throughout the sequence.\",\n \"To isolate the Rv1170 gene from M. tuberculosis H37Rv genomic DNA was prepared using known methods.\",\n \"The open reading frame Rv1170 was amplified from this DNA with the following primers: 5′-TAGCCATGGTGTCTGAGACGCCGCG-3′ (SEQ ID NO:8) and 5′-GGATCCCGGGGTGAAGCCCAGAC-3′ (SEQ ID NO:9) These primers contain NcoI and BamHI restriction sites, respectively.\",\n \"PCR was performed with Taq polymerase (Gibco BRL), using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.\",\n \"The 30 cycles of PCR included denaturation at 94° C. for 40 s, annealing at 55° C. for 1 min, and amplification at 72° C. The PCR products were separated on a 1% agarose gel.\",\n \"The appropriate PCR product was ligated into vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into Escherichia coli DH5α by standard chemical transformation procedures.\",\n \"Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml), and plasmid DNA was digested with the restriction endonucleases NcoI and BamHI (Fermentas).\",\n \"Restriction enzyme-digested plasmids were isolated with a QIAquick® gel extraction kit (Qiagen Ltd.).\",\n \"A corresponding digestion was applied to the plasmid pET-22b, and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170.In order to express Rv1170 in E. coli without the pelB leader sequence, the gene from pYA1170 was excised using NcoI and Bpu1102I (Fermentas) and ligated to an aliquot of pET16b cut with NcoI and Bpu1102I to generate the plasmid pYA1170b (FIG.\",\n \"2).\",\n \"This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg ampicillin/ml.\",\n \"Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).\",\n \"After overnight incubation at 37° C. with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37° C. with shaking.\",\n \"Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.4 mM when the A600 reached 0.6, and incubation was continued overnight at 25° C. before harvesting by centrifugation at 5000×g for 15 min at room temperature.\",\n \"The pellets were lysed by sonication.\",\n \"Proteins were separated by centrifugation (15,000×g, 4° C., 30 min) into soluble and insoluble fractions.\",\n \"Total proteins were separated by sodium dodecyl sulfate 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride membranes (Bio-Rad).\",\n \"The N-terminal amino acid sequence was verified using Edman degradation after separation of samples by SDS-PAGE and electroblotting to a polyvinylidene difluoride membrane.\",\n \"E. coli BL21 (DE3) was transformed with the blank cloning vector (pET16b) or with vector containing Rv1170 (pYA1170b).\",\n \"SDS-PAGE was used to separate desalted 20% to 50% saturated ammonium sulfate extracts of these isopropyl-β-D-thiogalactopyranoside induced E. coli.\",\n \"Bio-Rad Broad Range molecular mass standards and purified M. smegmatis mycothiol S-conjugate deacetylase were also assayed for controls.\",\n \"The results of the SDS-PAGE showed that the 20% to 50% saturated ammonium sulfate fraction from E. coli carrying the Rv 1170 gene contained elevated levels of a protein of the expected size (36 kDa) as compared to those from E. coli prepared with a blank cloning vector.\",\n \"Another, smaller protein (˜26 kDa) was also present at elevated levels and is believed to be a degradation product of the deacetylase.\",\n \"The identity of the 36-kDa protein was confirmed by determining the N-terminal amino acid sequence and finding it identical to that predicted for the protein encoded by open reading frame Rv1170.The amidase and deacetylase activity of the 20 to 50% saturated ammonium sulfate extracts of E. coli transformed with pYA1170b or with the blank cloning vector pET16b after desalting and concentrating was assayed as described above.\",\n \"The results of these assays are shown in Table 1 below.\",\n \"TABLE 1 Substrate specificity of crude deacetylase from E. coli transformed with pYA1170b Activity (nmol min−1 mg of protein−1)(n = 3) Substrate Rv1170 Blank vector MsmBa 1.2 ± 0.1 <0.002 MSmBb <10−4 NDf GlcNAc-Insc 27 ± 2 <0.002 GlcNAcc 0.087 ± 0.005 <0.005 MSHd 0.002 ± 0.001 ND MSHe <10−4 ND aAnalysis for AcCySmB produced by amidase cleavage.\",\n \"bAnalysis for production of the deacetylase product CySmB-GlcN-Ins.\",\n \"cAnalysis for corresponding amine resulting from deacetylase reaction.\",\n \"dAnalysis for AcCys produced by amidase cleavage.\",\n \"eAnalysis for production of the deacetylase product Cys-GlcN-Ins.\",\n \"fNot determined.\",\n \"As shown by the results summarized in Table 1, extract from cells expressing Rv1170 exhibited substantial deacetylase activity with GlcNAc-Ins as the substrate, over 300-fold greater than the deacetylase activity determined with GlcNAc and 23-fold greater than the amidase activity measured with MSMB (FIG.\",\n \"2).\",\n \"Thus, Rv1170 was putatively identified as the GlcNAc-Ins deacetylase gene in the MSH biosynthesis pathway of M. tuberculosis.\",\n \"M. smegmatis mycothiol S-conjugate amidase lacks deacetylase activity.\",\n \"Mycothiol S-conjugate amidase from M. smegmatis, a homolog of the protein encoded by M. tuberculosis Rv1082, cleaves the amide bond linking Cys to GlcN in MSH derivatives having an alkylated sulfur residue (G. L. Newton et al., Biochemistry 39:10739-10746, 2000).\",\n \"Tests were conducted to determine whether the mycothiol S-conjugate amidase also cleaves the corresponding bond in GlcNAc-Ins, functioning as a deacetylase (FIG.\",\n \"3).\",\n \"GlcNAc-Ins was prepared from GlcN-Ins and the ability of the amidase purified from M. smegmatis to hydrolyze GlcNAc-Ins (i.e., to function as a deacetylase) was compared with its ability to function as an amidase by cleaving the amide bond in the monobromobimane conjugate of MSH (MSmB).\",\n \"The assays utilized are described above.\",\n \"For a test with 100 μM substrate, the activity measured with deacetylase substrate MSmB was 4.5±1.0 μmol min−1 mg of protein−1, whereas that measured with the invention deacetylase substrate GlcNAc-Ins was <1 nmol min−1 mg of protein−1 (n=4).\",\n \"The comparative assays showed that the mycothiol S-conjugate deacetylase does not exhibit measurable deacetylase activity and cannot serve this function in the MSH biosynthesis pathway of M. smegmatis.\",\n \"EXAMPLE 3 Extracts of M. smegmatis and mutant 49 have deacetylase activity.\",\n \"If the deacetylase is involved in MSH biosynthesis and if mutant 49 is blocked at an earlier step in the MSH-production pathway, then strains mc2155 and 49 should both produce deacetylase activity.\",\n \"To test this hypothesis, centrifuged extracts of exponentially growing cells were assayed in duplicate for GlcN-Ins production with and without the addition of 100 μM GlcNAc-Ins.\",\n \"The background rate for production of GlcN-Ins measured without added GlcNAc-Ins was high for strain mc2155 (6.1±0.1 pmol min−1 mg of protein−1) presumably reflecting the substantial endogenous level of GlcNAc-Ins present in the undialyzed extract.\",\n \"Addition of 100 μM GlcNAc-Ins increased the rate to 19.7±0.4 pmol min−1 mg of protein−1, giving a net rate increase of 13.6±0.5 pmol min−1 mg of protein−1.For mutant 49, which does not contain GlcNAc-Ins, the background rate was <0.06 pmol min−1 mg of protein−1 and the rate with 100 μM GlcNAc-Ins was 16.4±1.4 pmol min−1 mg of protein−1.Thus, the deacetylase activity of strain 49 is essentially the same as that of the parent strain.\",\n \"EXAMPLE 4 Assay for GlcNAc-Ins.\",\n \"To confirm that GlcNAc-Ins is present in mycobacteria, an assay for this component was needed.\",\n \"The availability of cloned Rv1170 allowed the development of such an assay.\",\n \"Cell extracts were prepared in warm 50% acetonitrile containing NEM, as employed previously for an assay of GlcN-Ins (Anderberg et al.\",\n \"(1998) J. Biol.\",\n \"Chem.\",\n \"273:30391-30397).\",\n \"One portion of the extract was assayed directly for GlcN-Ins content while a second portion was assayed after treatment with a preparation of expressed Rv1170 under conditions sufficient to convert all GlcNAc-Ins to GlcN-Ins.\",\n \"The difference in these two assay results yields the GlcNAc-Ins content for the sample.\",\n \"In most cases, GlcN-Ins levels were at least an order of magnitude lower than those of GlcNAc-Ins, so this method gives reliable results.\",\n \"Recovery experiments for GlcNAc-Ins were conducted using a M. smegmatis MSH-deficient mutant (“mutant 49”), derived from mc2155 by chemical mutagenesis.\",\n \"Mutant 49 was cultured in Middlebrook 7H9 medium at 37° C. as previously described (Newton, et al.\",\n \"(1999) Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Comm.\",\n \"255:239-244).\",\n \"In the uptake and metabolism assay, 50% acetonitrile-NEM extracts were analyzed after the addition of 0.7 and 7.0 μM GlcNAc-Ins.\",\n \"Extracts of mutant 49 contained no detectable GlcN-Ins or MSH, so that any GlcN-Ins found in the extracts after treatment with the deacetylase preparation was derived solely from the deacetylation of GlcNAc-Ins.\",\n \"The relationship between A600 and RDW was determined for aliquots of mc2155 cells producing ˜30 mg of RDW and found to be 0.42±0.02 mg of RDW per ml of cells with A600 at 1.00.No variation of >15% in this value was seen between log- and stationary-phase cells.\",\n \"The method was tested with extracts prepared from log-phase cells of mutant 49 by spiking the extract with GlcNAc-Ins at levels equivalent to 0.13 and 0.013 μmol per g of RDW.\",\n \"The recoveries from triplicate determinations were 97%±5% and 132%±3%, respectively.\",\n \"At the lowest level of added GlcNAc-Ins, a significant correction had to be applied for background peaks present in unspiked controls that overlapped the GlcN-Ins peak.\",\n \"The greater than 100% recovery found for the samples spiked at lowest level indicates that this correction produces a systematic error and thus defines the lower limit of useful sensitivity for this assay.\",\n \"EXAMPLE 5 M. smegmatis mc2155 produces GlcNAc-Ins but MSH-deficient mutant 49 does not.\",\n \"M. smegmatis mc2155 cells were examined in exponential and stationary phases for their content of MSH and its precursors using the methods described above.\",\n \"The results of these studies are summarized in Table 2 below: TABLE 2 Levels of MSH and precursors in M. smegmatis mc2155 Content (μmol [g of RDW]−1) ( n = 3) Growth phase A600 MSH GlcNAc-Ins GlcN-Ins Log 0.54 7.8 ± 0.6 14.5 ± 1.2 0.2 ± 0.1a Stationary 3.3 6.6 ± 0.6 12.6 ± 0.6 <0.03 aBest estimate after correction for overlapping peak in HPLC analysis.\",\n \"These results show that M. smegmatis accumulates GlcNAc-Ins to a substantial level, almost twice the level of MSH content.\",\n \"The GlcN-Ins content was very much lower in log phase and declined further in stationary phase, as had been observed previously (Anderberg et al; (1998) Supra.\",\n \"EXAMPLE 6 MSH-deficient mutant 49 can import GlcNAc-Ins and utilize it to sythesize MSH.\",\n \"The foregoing results imply that mutant 49 should be able to synthesize MSH if supplied with GlcNAc-Ins.\",\n \"The concentration dependence of the uptake of GlcNAc-Ins was examined with cultures of M. smegmatis mutant 49.For this study mutant 49 was cultured at 37° C. for 23 hr in 5 ml of 7H9 Middlebrook medium as described below, supplemented with 0, 4, 8, 21 μM GlcNAc-Ins.\",\n \"The cells were collected by centrifugation and extracted for amine and thiol analysis as described above.\",\n \"The absence of GlcNAc-Ins in mutant 49 shows that it is defective in an initial step of MSH biosynthesis.\",\n \"When mutant 49 was grown in media supplemented with GlcNAc-Ins, the cells did show the presence of MSH and mycothiol precursors in increasing concentrations with the media content of GlcNAc-Ins.\",\n \"These results established that GlcNAc-Ins is indeed imported intact, deacetylated and utilized for the production of MSH in a concentration dependent manner (Table 3).\",\n \"TABLE 3 Levels of MSH and precursors in MSH-deficient strain 49 (M. smegmatis) Content (μmol [g of RDW]−1) [GlcNAc-Ins]a (μM) MSH GlcNAc-Ins GlcN-Ins 0 <0.04 <0.01 <0.02 4 0.47 0.39 <0.02 8 0.91 0.80 0.026 21 2.1 1.4 0.047 aIn Middlebrook 7H9 medium.\",\n \"; [GlcNAc-Ins] = 0).\",\n \"In another experiment mutant 49 was cultured in 7H9 Middlebrook medium with 0.4% glucose and 0.5% TWEEN 80 detergent to log phase (A600=0.6) and 10-ml aliquots transferred to sterile 25 ml erlenmeyer flasks.\",\n \"Duplicate flasks contained cells only (control), 20 μM inositol, 20 μM glucosamine, 20 μM N-acetylglucosamine, or 17 μM GlcNAc-Ins and were cultured at 37° C., shaking at 225 rpm.\",\n \"Samples (2.3×108 cells) were taken at 2, 6, 19 and 46 hr of culture.\",\n \"the cells were pelleted by centrifugation for 2 min at 14,000×g and were extracted in 50% acetonitrile containing 20 mM HEPES (pH 8.0) and either 2 mM monobromobimane for thiol analysis or 5 mM NEM for amine analysis and thiol control samples.\",\n \"Cells from duplicate cultures were analyzed for GlcN-Ins, GlcNAc-Ins, Cys-GlcN-Ins, cysteine, and MSH.\",\n \"No significant MSH was found in any sample except for the cultures supplemented with GlcNAc-Ins (FIG.\",\n \"4).\",\n \"Strain 49 cultured in 17 μM GlcNAc-Ins showed a cysteine level that remained unchanged at 0.36±0.08 μmol per gm of residual dry weight over the experiment.\",\n \"Cells contained 2.9 μmole per gm of residual dry weight of GlcNAc-Ins at the first measurement (2 hr), which corresponds to a level of GlcN-Ins approaching millimolar.\",\n \"This occurred prior to the appearance of significant GlcN-Ins in the cell, showing that strain 49 is able to import and concentrate GlcNAc-Ins intact prior to its deacetylation.\",\n \"The level of GlcNAc-Ins appeared to fall at 6 hr before rising to an even higher level in stationary phase.\",\n \"The MSH content was measurable as early as 2 hr, but was about 10-fold less than the GlcNAc-Ins content at that time.\",\n \"The MSH content increased thereafter, and the level at 48 hr was about 60% of that measured for the parent strain (Table 2).\",\n \"The level of GlcN-Ins ranged from 0.025 (2 hr) to 0.1 (48 hr) μmole per gm of residual dry weight, values in the range of those measured for mc2155 (Table 2).\",\n \"These results show that exogenous GlcNAc-Ins is efficiently imported by strain 49 and substantially restores its defective biosynthesis of MSH.\",\n \"The intracellular level of GlcN-Ins found in mutant 49 at 48 hours indicates that GlcNAc-Ins has been concentrated more than 100 fold from the supplemented medium.\",\n \"This implies that toxic analogs of GlcNAc-Ins, such as those with oxidizing or nitrosating moieties, will be concentrated inside pathogenic mycobacteria or other actinomycetes and function as antimicrobial agents.\",\n \"EXAMPLE 7 Cloning, expression and purification of M. tuberculosis His-tagged MshB.\",\n \"To facilitate the purification of the M. tuberculosis deacetylase, Rv1170 was cloned to contain a C-terminal HisTag permitting its easy isolation by affinity chromatography on a Ni2+ chelate resin.\",\n \"Genomic DNA of M. tuberculosis H37Rv was prepared as described previously (Av-Gay et al., Infect.\",\n \"Immun.\",\n \"67:5676-5682, 1999).\",\n \"The open reading frame Rv1170 was amplified from this DNA with the primers 5′-TTCATATGGTGTCTGAGACGCCG-3′; (SEQ ID NO:10 and 5′-ATAAGCTTGTAGCCGGACGCGGTG-3′ (SEQ ID NO:11) containing NdeI and HindIII restriction sites, respectively.\",\n \"PCR was performed with Taq polymerase obtained from Gibco BRL, using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.\",\n \"The 30 cycles of PCR included denaturation at 94° C. for 40 seconds, annealing at 55° C. for 1 min and amplification at 72° C. The PCR products were separated on a 1% agarose gel.\",\n \"The appropriate PCR product was ligated into the vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into E. coli DH5a by standard chemical transformation procedure.\",\n \"Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml) and plasmid DNA was digested with restriction endonucleases NdeI and HindIII (Fermentas).\",\n \"Restriction enzyme-digested plasmids were isolated with a QIAquick gel extraction kit (Qiagen Ltd.).\",\n \"A corresponding digestion was applied to the plasmid pET-22b and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170c (shown in FIG.\",\n \"9).\",\n \"This provides a recombinant protein without a pelB leader sequence, but with a c-terminal His tag.\",\n \"This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg/ml ampicillin.\",\n \"Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).\",\n \"After over-night incubation at 37° C. with shaking, 5 1-liter cultures were inoculated to 1% with the starter culture and incubated at 37° C. with shaking.\",\n \"Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM when the A600 reached 0.6 and incubation was continued overnight at 22° C. before harvesting by centrifugation at 5000×g for 20 minutes at 4° C. The pellets were lysed by sonication in 3 mM 2-mercaptoethanol with 50 mM HEPES pH 7.4 (lysis buffer) containing 35 μM each protease inhibitors (N-α-p-tosyl-L-lysinechloromethyl ketone and N-α-p-tosyl-L-phenylalaninechloromethyl ketone).\",\n \"Proteins were separated by centrifugation (1-5,000×g, 4° C., 30 min) into-soluble and insoluble-fractions.\",\n \"The soluble proteins were applied to a 2.5×11 cm Fast Flow® chelating resin (Pharmacia) in the Ni2+ form in lysis buffer containing 10 mM imidazole.\",\n \"The column was washed with 10 volumes of the loading buffer and then step eluted with lysis buffer containing 150 mM imidazole followed by lysis buffer containing 500 mM imidazole.\",\n \"Fractions were collected and the purity of the eluted protein was assayed by SDS PAGE gel electrophoresis.\",\n \"The early 150 mM imidazole fractions were contaminated with E. coli proteins, but the later fractions of the 150 mM and the early 500 mM imidazole fractions were judged>95% pure and pooled.\",\n \"The yield of pure recombinant MshB (Rv1170) was estimated to be ˜125 mg of protein.\",\n \"The protein was concentrated and the buffer exchanged repeatedly with 50 mM HEPES containing 50 mM NaCl (assay buffer) in a Centricon® 10 spin ultra filter, adjusted to 10% glycerol and frozen at −70° C. The enzyme as purified was assayed for deacetylase activity with 0.1 mM GlcNAc-Ins as substrate and was found to be sensitive to chelating agents such as 1,10-phenanthroline.\",\n \"The deacetylase reaction was substantially inhibited by the presence of 0.1 mM 1,10-phenanthroline in the reaction mixture (FIG.\",\n \"7).\",\n \"1 mM 1,10-phenanthroline completely inhibited the deacetylase reaction, indicating a dependence of the reaction on divalent metal ions.\",\n \"The apoenzyme was generated by treatment of the concentrated enzyme with 5 mM 1,10-phenanthroline in assay buffer for 30 min on ice.\",\n \"The chelator was removed by repeated dilution with assay buffer and concentration until the 1,10-phenanthroline concentration was estimated to be below 1 μM.\",\n \"All solutions were treated with Chelex-100® chelator to remove extraneous divalent cations.\",\n \"The apoenzyme was stored at 10 mg/ml in 50 mM NaCl+50 mM HEPES pH 7.5 with 10% glycerol at −70° C. The zinc form (Zn2+) of the deacetylase was prepared by treating the apoenzyme with 0.1 mM ZnCl2 in the assay buffer for 30 min on ice.\",\n \"The excess Zn2+ was removed by repeated dilution and concentration with assay buffer until the Zn2+ was estimated to be below 1 μM.\",\n \"The Zn2+ form of the enzyme was stored as described above for the apoenzyme and is herein referred to as “His-tagged MshB”.\",\n \"EXAMPLE 8 Metal ion dependence of the deacetylase.\",\n \"The metal ion dependence of the deacetylase activity of MshB was assayed using GlcNAc-Ins in the presence of 0.1 mM divalent metal ion.\",\n \"Previous studies with ZnCl2 have shown that excess Zn2+ is not inhibitory at 0.1 mM and only slightly inhibitory at 1 mM (see above).\",\n \"The metal salts tested, ZnCl2, CuSO4, MgCl2, MnCl2, CaCl2, COCl2, CdCl2 and NiCl2, were of reagent grade or higher purity and solutions of the metals were prepared as 10 mM stock solutions in water treated with Chelex-100 chelator.\",\n \"Enzymatic activity was assayed in Chelex-100® treated 50 mM NaCl with 50 mM HEPES pH 7.4 (assay buffer) containing 0.1 mM of the desired metal salt and 0.1 mM GlcNAc-Ins as substrate.\",\n \"Buffer and 10 μg of purified apoenzyme were preincubated the divalent metal ion in an 80 μl total reaction volume for 10 min at 37° C. and the assay initiated by the addition of the substrate.\",\n \"Duplicate reactions were sampled at 3, 10, and 30 min by removing a 20 μl sample of the reaction mixture and adding it to 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.\",\n \"The samples were incubated for 10 min at 60° C., placed on ice, and subsequently reacted with AccQ Fluor® (Waters) reagent as described above assayed for AccQ-GlcN-Ins by HPLC as described above.\",\n \"The MshB apoenzyme was able to catalyze both the deacetylase (GlcNAc-Ins as substrate) and the amidase (MSmB as substrate) reactions at substantially reduced rates, as shown in Table 4 below.\",\n \"TABLE 4 M. tuberculosis MshB (Rv1170) metal ion dependencea Amidase Specific Deacetylase Activityb Amidase Specific Deacetylase Metalloenzyme nmole/ Activity Activityc Activity Form min/mg relative rate nmole/min/mg Relative rate Apoenzyme 3.4 ± 0.5 1.0 35 ± 6 1.0 Untreated 15 ± 0.2 4.4 70 ± 19 2.0 Zn2+ 17 ± 0.7 5.0 81 ± 16 2.3 Ni2+ 13 ± 0.7 3.8 62 ± 23 1.8 Mn2+ 22 ± 0.7 6.5 69 ± 5 2.0 Ca2+ 3.3 ± 0.1 0.97 34 ± 5 0.97 Mg2+ 3.5 ± 0.2 1.0 41 ± 4 1.2 Cu2+ 3.2 ± 0.1 0.94 32 ± 10 0.9 Cd2+ 21 ± 1.5 6.2 34 ± 2 0.97 Co2+ 29 ± 0.3 8.5 236 ± 23 6.7 Zn2+ + Mg2+ ND — 75 ± 14 2.1 Ni2+ + Mg2+ ND — 64 ± 15 1.8 aRecombinant Rv1170 purified to homogeneity over Ni2+ Sepharose.\",\n \"b0.1 mM MSmB as substrate with 0.1 mM metal ion.\",\n \"c0.1 mM GlcNAc-Ins as substrate with 0.1 mM metal ion.\",\n \"The low metal ion content of the apoenzyme reduced the amidase activity to a greater extent than the deacetylase activity relative to the untreated purified protein.\",\n \"The activity of the Ni2+ Sepharose purified (untreated) form of the MshB appears to be consistent with the Ni2+ form of the enzyme.\",\n \"The Zn2+, Mn2+ and Ni2+ forms appear to have about the same activity for both the amidase and deacetylase reactions.\",\n \"The MshB apoenzyme was not activated by the presence of Ca2+, Mg2+ or Cu2+ for either the amidase or deacetylase reactions.\",\n \"The MshB deacetylase and amidase reactions were differently affected by the presence of the Cd2+ ion.\",\n \"The amidase reaction was enhanced to the level of Zn2+ while the deacetylase reaction remained at the apoenzyme activity level.\",\n \"Similarly anomalous activity with Cd2+ was observed for the peptidase and esterase activity of carboxypeptidase, a zinc metalloprotease (R. C. Davis et al Biochemistry 7, 1090-1099 (1968)).\",\n \"The highest level of activation for both the amidase and deacetylase reactions was observed with Co2+, as has been reported for numerous zinc metalloproteases (D. S. Auld, Chapter 14, Methods in Enzymology, 248:229-243, 1995).\",\n \"Some zinc metalloproteases have been reported to require the presence of Mg2+ for optimal activity (Vallee and Auld (1993) Biochemistry 32:6493-6500) so activity assays were conducted with 0.1 mM each Zn2+ and Mg2+.\",\n \"No additional activation over that of Zn2+ alone for either the amidase or deacetylase reactions was observed.\",\n \"Thus, the metal ion dependence of MshB is similar to that found for members of the metalloprotease superfamily and is therefore likely that MshB is a zinc metalloenzyme in its native state.\",\n \"EXAMPLE 9 Kinetics and substrate specificity of His-tagged MshB.\",\n \"The kinetic constants were determined for GlcNAc-Ins, the deacetylase substrate for MshB, and for MSmB, an amidase substrate.\",\n \"The rates were determined with His-tagged MshB in 50 mM NaCl containing 50 mM HEPES pH 7.5 (assay buffer).\",\n \"The enzyme (10 μg) was preincubated with 80 μl of assay buffer for 10 min at 37° C. and the reaction was initiated with the addition of the substrate.\",\n \"The deacetylase substrate was assayed at 9 different concentrations between 10 μM and 2 mM.\",\n \"Duplicate reactions were sampled at 3, 10, and 30 min by removing 20 μl of the sample, and mixing with 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.\",\n \"The samples were quenched by incubation for 10 min at 60° C. The quenched sample was reacted with AccQ Fluor® reagent (Waters) and analyzed for GlcN-Ins by HPLC as described above.\",\n \"The amidase reaction was assayed similarly using 10 concentrations of MSmB between 10 μM and 10 mM.\",\n \"The amidase reaction was sampled at 3, 10, and 30 min by removing 20 μl of sample and mixing it with 60 μl of 40 mM methanesulfonic acid.\",\n \"The mercapturate product, AcCySmB was analyzed by HPLC as described above.\",\n \"Initial rates were obtained by extrapolation of the rates calculated at 3, 10, and 30 min to zero time.\",\n \"The kinetic constants were obtained from an Eadie-Hofstee plot.\",\n \"The amidase and deacetylase substrates have Km values near of 0.5 mM as shown below in Table 5 below.\",\n \"TABLE 5 Kinetic characterization of M. tuberculosis MshB (Rv1170) Km Vmax kcat Substrate Activity μM nmol min−1mg−1 s−1 GlcNAc-Ins deacetylase 400 470 0.25 MSmB amidase 560 220 0.12 The intracellular concentration of GlcN-Ins is roughly twice the mycothiol level, or about 2-6 mM (Newton et al, J.\",\n \"Bacteriol., 182: 6958-6963, 2000) and is sufficient to saturate the enzyme at physiological concentrations.\",\n \"The substrate specificity of MshB was examined with various amidase and deacetylase substrates at a concentration of 0.1 mM.\",\n \"The product analyzed will vary with the substrate used.\",\n \"The deacetylase substrates tested were GlcNAc, GlcNAc-Ins, MSH, and MSmB, and the products assayed were GlcN, GlcN-Ins, Cys-GlcN-Ins, and CySmB-GlcN-Ins, respectively.\",\n \"The amidase substrates tested were MSH, MSmB, AcCySmB-GlcN, CySmB-GlcN-Ins, MS-acetamide, and MS-acetophenone and the product assayed was GlcN-Ins, except for AcCySmB-GlcN were GlcN was assayed.\",\n \"The amines were analyzed as the AccQ-derivative and thiols as the bimane derivative as described above.\",\n \"The results of these substrate specificity studies are summarized in Table 6 below.\",\n \"TABLE 6 Substrate specificity of M. tuberculosis MshB (Rv1170)a Amidase Specific Deacetylase Activityb Specific Activityb Relative Substratea nmol min−1mg−1 nmol min−1mg−1 Rate GlcNAc-Insc 70 ± 3 1.0 GlcNAcd 1.6 ± 1 0.023 MSHe <0.025 ± 0.012 <0.00036 MSHc 0.3 ± 0.1 0.0043 MSmBf <0.006 <0.00009 MSmBc 20 ± 2 0.29 AcCySmB-GlcNd <0.1 <0.001 CySmB-GlcN-Insc 265 ± 10 3.8 MS-acetamidec 0.14 ± 0.013 0.002 MS-acetophenonec 88 ± 8 1.3 aHis tagged MshB (Rv1170) in the Zn2+ form.\",\n \"b0.1 mM substrate assayed in 50 mM NaCl + 50 mM HEPES pH 7.4 at 37° C. cReaction assayed for GlcN-Ins.\",\n \"dReaction assayed for GlcN.\",\n \"eReaction assayed for CyS-GlcN-Ins.\",\n \"fReaction assayed for CySmB-GlcN-Ins.\",\n \"As shown by the results in Table 6, the deacetylase activity of MshB is dependent on the presence of the inositol moiety as the GlcNAc substrate is 44 fold less active than GlcNAc-Ins.\",\n \"Interestingly MSH and MSmB are not substrates for the deacetylase activity of MshB.\",\n \"However, mycothiol S-conjugates with reasonably large groups on the cysteine sulfur, such as acetophenone and bimane S-conjugates, are good substrates for the amidase activity of the MshB.\",\n \"The smaller mycothiol S-conjugate acetamide is a poor substrate for the MshB amidase activity, as was observed previously for mycothiol S-conjugate amidase from M. smegmatis (Newton et al Biochemistry, 39, 10739-10746, 2000).\",\n \"The importance of the inositol moiety for the amidase activity of MshB is also apparent by the lack of activity with AcCySmB-GlcN.\",\n \"One difference between the amidase activity of M. tuberculosis His-tagged MshB and the amidase from M. smegmatis is the ability to use CySmB-GlcN-Ins as substrate.\",\n \"The M. smegmatis amidase required the acetylated amine on the cysteine moiety (or MSmB) for activity.\",\n \"Another difference between MshB and the M. smegmatis amidase is that the amidase will not deacetylate GlcNAc-Ins (Newton et al J.\",\n \"Bacteriol., 182, 6958-6963, 2000).\",\n \"EXAMPLE 10 Inhibition of His-tagged MshB.\",\n \"Naturally occurring inhibitors of the His-tagged MshB deacetylase and amidase activities have been examined.\",\n \"The inhibition assays are similar to those described above in Example 9.For the amidase reaction of His-tagged MshB 10 μg enzyme was preincubated for 10 min at 37° C. in 80 μl of buffer containing 50 mM NaCl, 50 mM HEPES pH 7.5, 0.1 mM ZnCl2, 0.2 mM DTT and 0.1-1.0 mM inhibitor.\",\n \"The inhibitors examined were mycothiol, glutathione and GlcN-Ins.\",\n \"The purpose of the excess Zn+2 is to prevent depletion of the protein bound Zn2+ by thiols.\",\n \"The DTT is added to limit the oxidation of the thiol inhibitors, which can be significant at the lower concentrations.\",\n \"The uninhibited enzyme activity was determined using the same buffer and preincubation conditions without inhibitor.\",\n \"The assays were initiated with the addition of MSmB to 0.55 mM and sampled at 3, 10, and 30 min by removing a 20 μl sample and quenching the reaction by mixing with 60 μl of 40 mM aqueous methanesulfonic acid.\",\n \"This sample was assayed directly by HPLC for AcCySmB as described above.\",\n \"The deacetylase reaction was assayed with GlcNAc-Ins using 0.1-1.0 mM glutathione and mycothiol as inhibitors.\",\n \"The reaction was initiated by the addition of GlcNAc-Ins to 0.8 mM and was sampled at 3, 10, and 30 min.\",\n \"The reaction was quenched and analyzed for GlcN-Ins as described in Example 9.Mycothiol appears to be an effective inhibitor of both the amidase (FIG.\",\n \"8A) and the deacetylase (FIG.\",\n \"8B) activities of His-Tagged MshB in the range of 0.1 to 1 mM.\",\n \"This is not simply due to disruption of the enzyme metal binding site by a thiol as shown by the lack of inhibition by glutathione in this concentration range.\",\n \"The lack of inhibition of the amidase reaction by GlcN-Ins (FIG.\",\n \"8A) also shows that the immediate product of the reaction is not inhibitory.\",\n \"Mycothiol is the terminal product in the biosynthesis scheme shown in FIG.\",\n \"5 and it appears to completely inhibit the deacetylase (MshB) at mycothiol concentrations above 1 mM.\",\n \"The estimated intracellular concentration of mycothiol in mycobacteria is about 1-3 mM and thus the deacetylase is in a substantially inhibited state until the mycothiol level falls below 1 mM, as may happen during cell division or cell stress.\",\n \"Inhibition of the deacetylase reaction was analyzed at 0.8 mM GlcNAc-Ins, a concentration nearing saturation, but still below the estimated cellular concentration of 2-6 mM for M. smegmatis.\",\n \"Thus, in M. smegmatis the deacetylase is exposed to saturating GlcNAc-Ins concentrations but is inhibited when the cellular mycothiol level is above 1 mM.\",\n \"If mycothiol level falls substantially below 1 mM, the enzyme will catalyze the deacetylation of GlcNAc-Ins from a large cellular pool at near maximal rates to GlcN-Ins and ultimately to mycothiol.\",\n \"It has previously been shown that mycothiol levels are quite constant from logarithmic through stationary phase growth of M. smegmatis (Newton et al, J.\",\n \"Bacteriol., 178: 1990-1995, 1996).\",\n \"It appears that the intracellular mycothiol level is regulated by feedback inhibition of the GlcNAc-Ins deacetylase by mycothiol itself.\",\n \"The amidase substrate, MSmB, served as a useful model substrate for finding inhibitors of the GlcNAc-Ins deacetylase.\",\n \"The amidase and deacetylase activities are both completely inhibited by mycothiol at concentrations over 1 mM.\",\n \"These results identify mycothiol as an inhibitor for the deacetylase and amidase reactions with similar inhibitory concentrations.\",\n \"Thus, the substrates for either the amidase or deacetylase activity of GlcNAc-Ins deacetylase can be used to screen for inhibitors of the enzyme.\",\n \"EXAMPLE 11 Oxidant sensitivity of M. smegmatis MSH-deficient mutants.\",\n \"The importance of mycothiol biosynthesis for the survival of mycobacteria under oxidative stress is illustrated by results obtained with chemical and transposon mutants deficient in mycothiol biosynthesis.\",\n \"Chemical mutants were produced by mutagenesis of M. smegmatis mc2155 (˜2.5×109 colony forming units per ml Middlebrook 7H9 liquid medium) with N-methyl-N-nitro-N-nitrosoguanidine (4 μg per ml) for 30 min at 37° C. Cells were washed three times with 9 ml of fresh medium, resuspended in 10 ml of fresh medium, and incubated 8.5 h at 37° C. with shaking.\",\n \"After dilution in fresh medium and passage through a 22-guage needle, the cells were plated on Middlebrook 7H9 agar containing 0.05% Tween 80 and 0.4% glucose.\",\n \"After 9 days in a humidified incubator, the plates were scored and individual colonies replated in duplicate using a grid layout in 100 mm culture dishes.\",\n \"After colony development, one plate was used to screen for MSH-deficient mutants using a membrane-based immunoassay specific for MSH (Unson, et al.\",\n \"(1998) J. Immunol.\",\n \"Methods 214:29-39).\",\n \"MSH-deficient candidates were grown in bulk in liquid medium and analyzed for MSH content as described in Example 1.Two MSH-deficient mutants (strains 49 and 164) were identified and strain 49 was complemented using a M. tuberculosis library (pYUB412::H37Rv) of ˜18 kb DNA fragments cloned in the integrative shuttle plasmid pYUB412 to produce the strain 49::H37Rv A18 with partially restored MSH biosynthesis.\",\n \"Transposon mutants of M. smegmatis mc2155 were prepared using the pCG79 plasmid carrying transposon Tn611 following the methods of Perez, et al.\",\n \"(1998) Meth.\",\n \"Molecular Biol.\",\n \"101:187-198).\",\n \"Plasmid pCG79 was introduced into M. smegmatis mc2155 by electroporation after which the cells were incubated 2 h at 32° C. in 7H9 medium without antibiotic.\",\n \"They were then plated on Middlebrook 7H10 agar plates with ADC enrichment and 20 μg/mL of kanamycin and incubated 6 days at 32° C. Single colonies were restreaked on 7H10km plates and incubated 4 days at 32° C. A loopful of each streaked transformant was resuspended, serially diluted, and plated in duplicate on 7H10 Km plates.\",\n \"One set of plates was incubated 36 h at 32° C. and then shifted to 42° C. for 5 days to select transposon mutants.\",\n \"The other set of plates was incubated at 32° C. for 7 days and served as positive controls.\",\n \"The transposon mutants were replated on 7H10Km plates at 1000-2000 colonies per plate and incubated 5d at 42° C. The cells were scraped into 10 ml of 7H9 medium, mixed with 1 mL of glycerol, and stored at −70° C. This library contained ˜10,000 mutants.\",\n \"Screening of the library for MSH-deficient mutants was accomplished using as ELISA assay method to detect MSH production in cultures grown in microtiter plates (Unson, et al.\",\n \"(1999) J. Clin.\",\n \"Microbiol 37:2153-2157).\",\n \"The first 1000 mutants screened produced three nearly identical MSH-deficient mutants (TN1, TN2 and TN3) that accumulated GlcN-Ins to high levels but failed to produce detectable levels of Cys-GlcN-Ins or MSH.\",\n \"Since these clones seemed likely to be daughter cells, TN3 was selected for more detailed studies.\",\n \"Results obtained with the parent and mutant strains of M. smegmatis are summarized in Table 7 below.\",\n \"TABLE 7 Characterization of M. smegmatis mutant strains MSH Content H2O2 IC50a Strain μmol per g RDW Enzyme Deficiency mM mc2155 10 None 12 49b <0.004 no MshAc 1 49::H37Rv A18b 3.3 partial MshAd 1.8 I64 0.1 low MshCe 3.5 TN3 <0.004 no MshCe 0.5 aConcentration producing 50% loss of viability following 2 h incubation in Middlebrook 7H9 liquid medium containing H2O2 at 37° C., dilution and plating on 7H10 agar, incubation for 10 d, and counting of colony formation.\",\n \"bData from Newton, et al.\",\n \"(1999) Biochem.\",\n \"Biophys.\",\n \"Res.\",\n \"Commun.\",\n \"255: 239-244.cBased upon failure to produce GlcNAc-Ins or GlcN-Ins.\",\n \"dBased upon restoration of ability to produce GlcN-Ins to ˜15% that of the parent strain.\",\n \"eBased upon absence of MshC activity in cell extracts and accumulation of GlcN-Ins to 2-3 μmol per g RDW (˜25-fold above the level of the parent strain).\",\n \"The results of these experiments show that the parent strain (mc2 155) has a high MSH content and exhibits a marked resistance to killing by hydrogen peroxide.\",\n \"By contrast, strain 49, which is blocked in the first step of MSH biosynthesis and produces no detectable MSH, is 12-fold more sensitive to H2O2.Partial restoration of MSH biosynthesis in strain 49 through complementation (strain 49::H37Rv A18) reduced the peroxide sensitivity of mutant 49 by a factor of two.\",\n \"Chemical mutant 164, blocked at the ligase biosynthetic enzyme MshC, has a low residual MSH content and is three-fold more sensitive to peroxide than the parent strain.\",\n \"However, transposon mutant TN3, also blocked at MshC but producing no detectable MSH, was >20-fold more sensitive to H2O2 than the parent strain (FIG.\",\n \"10).\",\n \"These results show that blocking MSH biosynthesis before or after the deacetylase step (MshB) produces a dramatic increase in peroxide sensitivity.\",\n \"It logically follows that blocking-MSH biosynthesis at MshB would produce a comparable increase in peroxide sensitivity in mycothiol-producing bacteria.\",\n \"The sensitivity of strains mc2155, 164, and TN3 to superoxide was also examined (courtesy of Drs.\",\n \"H. Buchmeier and D. Piddington).\",\n \"Superoxide sensitivity was determined according to the method of De Groote, et al.\",\n \"(1997) Proc.\",\n \"Natl.\",\n \"Acad.\",\n \"Sci.\",\n \"94: 13997-14001 using timed exposure of cells to 250 μM hypoxanthine in the presence of 0.1 unit/ml of xanthine oxidase followed by dilution and plating to determine survival.\",\n \"Under these conditions, strain mc2155 exhibits no significant killing whereas mutant TN3 is >90% killed in 1 hour.\",\n \"By contrast, chemical mutant 164, with a low residual MSH content, exhibited an intermediate level of killing.\",\n \"Survival of mycothiol-deficient mutant in macrophage.\",\n \"Transposon mutant TN3 was compared with the parent strain for survival in murine macrophages (data courtesy of Drs.\",\n \"N. Buchmeier and D. Piddington).\",\n \"Following the method of De Groote, et al.\",\n \"(1997) Supra, periodate activated, γ-interferon treated murine peritoneal exudate cells were infected with TN3 and mc2155, and extracellular bacteria were washed free of the macrophages with medium.\",\n \"At timed intervals the macrophages were resuspended in PBS and lysed with 0.5% deoxycholate; serial dilutions of the lysate were plated on Middlebrook 7H9 agar for determination of colony forming units.\",\n \"The results of two independent experiments performed on different days (FIG.\",\n \"11, experiments 1 and 2) show no loss of viability of the parent strain mc2155 in the macrophages over the period of 24 hours; whereas over 60% of the MSH-deficient mutant TN3 was killed by the macrophages within 6 hours.\",\n \"These results suggest that mycobacteria deficient in MSH biosynthesis will exhibit significantly enhanced killing by mammalian macrophages.\",\n \"While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297512"}}},{"rowIdx":99,"cells":{"text":{"kind":"list like","value":[["Process for the electromagnetic modelling of electronic components and systems","Process for the electromagnetic modelling of electronic components and systems, for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes.","A test machine of the commercial type is used for these measurements, by generating stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer.","The measurement phase is followed by a simulation phase during which the electric parameters used for modelling electronic components and systems are extracted."],["1.Process for the electromagnetic modelling of electronic components and systems, in particular for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes of said electronic components and systems, to ports of these components and systems suitable stimulus signals are sent and the correlated signals are measured, reconstructing the respective time patterns, characterised in that a test machine of the commercial type is used for generating the stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer, in which case the test machine sends said stimulus signals to the component or system inputs or outputs, fitted on a suitable support and connected by means of suitable interconnection channels, and detects the reflection due to impedance offset, providing the respective time patterns, which are then compared with those provided by a simulated measurement system model, comprising the test machine signal generation and measurement part, the interconnection channels and the support, to verify the coincidence between the measure time pattern and that provided by the simulated model obtained by setting different values with respect to the electric parameters to be determined.","2.Process according to claim 1, characterised in that the model simulated by the measurement system is obtained by the reflectometer response of each channel of the test machine in the situation in which the support of the component or system is not connected to the machine.","3.Process according to claim 1, characterised in that the reflected signals are reconstructed by means of the periodical sampling method, by fine tuning the sampling pulse phase and operating on the comparison threshold of the measurement part of the test machine to identify the succession of values assumed in time by the reflected signal, so as to obtain the time pattern.","4.Process according to claim 1, characterised in that for measuring the electronic component and system input and output impedance, a variable load impedance is introduced in the simulated model, so as to obtain various patterns of the reflected signal, following the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated impedance value corresponding to the impedance of the input or output to be determined.","5.Process according to claim 1, characterised in that for measuring the switching time of the outputs or transition time of the protection diode, a variable time is introduced in the simulated model, so as to obtain different patterns of the reflected signal, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated switching or transition time corresponding to the value to be determined."],["This invention relates to measurement methods for evaluating electromagnetic compatibility, and particularly relates to a process for the electromagnetic modelling of electronic components and systems.","With the increased level of integration and operating frequency of electronic components and systems, the need to check the electromagnetic compatibility between different parts of any one system, or between different systems in conditions of mutual interference, and the integrity of signals exchanged in electronic systems between the various components is becoming increasingly important.","The integrity of signals is particularly important in digital systems, where the treated signals are essentially binary signals.","For the good operation of systems, these signals must satisfy certain minimum quality requirements.","Particularly, two voltage levels, corresponding to bit 1 and 0, and the transition times between the levels must be preserved.","Furthermore, interference between adjacent conductors, skin effect attenuation and spurious signals originated from reflection of the original signals in the presence of discontinuous impedance must be minimised.","In digital systems, the increase in signal switching front steepness and dock frequency increases the intensity of the electromagnetic field radiated by the various component parts, such as conductors and active and passive components, with the consequent possibility of exceeding the limits established in the emission standard.","As a consequence, the device may not be type-approved or, consequently, marketed.","These problems can be obviated by an accurate system design, which accounts for the electromagnetic performance of the various components employed.","The validity of design can be verified before system construction by exploiting the possibilities offered by simulation programs readily available on the market.","Electromagnetic compatibility data, which can be provided by such programs, will be closer to reality the more the component and system models employed for describing the entire device to be simulated are accurate.","Standard model definition processes have existed for several years.","These processes guide technicians in identifying the parameters of the model and describing the model Of such processes, the most common is that proposed by the IBIS (Input-output Buffer Information Specification) Association, which consists in describing the static V-I characteristics of all component inputs and outputs, the output transition times in particular conditions of load, the parasite parameters associated to all inputs and outputs, such as the capacitance with respect to a reference potential, and other parameters.","At this time, there are not many available electronic component models.","The few available models essentially refer to integrated circuits and are widely incomplete and not very accurate.","These models are obtained by simulation employing known methods (SPICE), using technological parameters, in some cases provided by the manufacturers, or by using the scarce information provided by component data sheets.","In other cases, the models are made by means of measurements using single instruments, with considerable waste of time for preparing the bench and for making settings and measurements.","The process illustrated in this invention obviates these disadvantages and solves the technical problem described above by allowing automatic electronic component and system modelling using normal electronic test machines in an original way, without requiring costly modifications.","All access points of the device under test can be characterised rapidly also when dealing with very complex stimulation signals.","The test machine can be used in a conventional way for conducting functional tests or, according to this invention, for modelling in the same test session, without the need for additional manual interventions on behalf of the operator.","Specifically, object of this invention is a process for the electromagnetic modelling of electronic components and systems the characteristics of which are described in claim 1.This invention will be better explained by the following detailed description with reference to the accompanying drawings as non-limiting example, wherein: FIG.","1 is a flow chart illustrating a typical measurement session; FIGS.","2 and 3 are flow charts illustrating the process for conducting the measurements by means of the test machine; FIG.","4 is a flow chart illustrating the process which is used to extract the output switching time and the capacitance of the input or output ports of the component or system under test.","The process for the electromagnetic modelling of electronic components and systems employs a test machine of the commercial type, designed for conducting parametric measurements in direct current, functional tests and digital integrated circuit timing.","The characteristics required for the machine to be used in this process are: the possibility of generating digital signals with sufficiently steep switching fronts, typically lower than 300 ps; impedance controlled along all interconnections in the device under test; possibility of monitoring all signals output by the device under test by sampling, with time resolution lower than 300 ps.","Various types of such machines are made by various manufacturers, e.g.","the machine identified by code ITS9000 IX, made by Schlumberger.","In normal use for integrated circuit functional tests, signals with a suitable pattern are sent to the device inputs and the output signals are measured, checking that such signals are as required in a precise instant of the clock cycle.","The sampling method is used to measure the signals.","Specifically, at input port level transition, the signal at the output port is compared with a pre-programmed reference threshold and the time instant in which the threshold was exceeded is recorded.","By using a sufficiently high sampling frequency, the signal can be closely reconstructed.","A subsequent study can be conducted to evaluate the response of the device, checking that the parametric, functional and timing characteristics, the output signal time delays and the input and output signal threshold values are within the tolerance range permitted in specifications.","The test machine according to this invention is used in a different way with respect to that described above: it is used as a time domain reflectometer, i.e.","the test machine is used to send specific signals to the component ports and the reflections due to impedance offset are measured.","This method is not exploited in normal test machine use.","Only in the initial machine channel calibration and characterisation phase, a signal is sent to the device support and the delay of the corresponding reflected signal is measured to determine the delay introduced by the device interconnection channel.","The consequent compensations can be implemented, if required.","However, the time pattern of the reflected signal is not studied in detail, to the extent that the integrated circuit under test is not present: in other words, the conditions are those of total reflection.","By extending the possibility of reconstructing signals, also to involve the signals reflected by the access ports of the device under test, the respective electric parameters can be obtained.","These are essential for the electromagnetic modelling of the entire device according to the IBIS standard process mentioned above.","In detail, the following parameters can be obtained: the static voltage-current (V-I) characteristics at inputs and outputs; the output switching times in the specific conditions of load; the input and output capacitance; the tripping times of the protection and clamp diode circuits, normally present in input ports; various parasite parameters.","For the use according to this invention, the stimulus signals generated by the test machine must be sufficiently steep and present a sufficient amplitude, so as to allow high frequency measurements, preserving a good signal/noise ratio.","For example, to measure the typical input capacitance of a digital integrated circuit, an amplitude of approximately 1 V is conveniently selected for obtaining a transition time of approximately 400 ps, which suitably adapts to the amplitude resolution of the test machine comparators.","The signal offset can be set at a suitable level for the device, e.g.","2.5 V for an integrated circuit powered at 5 V. Since impedance offset is measured, naturally the output impedance of the test machine must be controlled and fixed at a suitable value, e.g.","50 Ohms, as the impedance of all the interconnections between the generator and the access port of the device.","As mentioned above, the reflected signals are reconstructed using the periodical sampling method.","The sampling input phase can be very finely tuned, e.g.","with a resolution of 20 ps.","After stablising the sampling phase, the comparison threshold is used to identify the internal comparator switching value, by using a suitable search algorithm, eg.","dichotomy, linear etc., according to the morphology of the signal.","After reconstructing the pattern of the reflected signal, it is compared with the pattern obtained by simulating the measurement configuration in the time domain with a circuit simulator.","The simulated model must include the same stimulus signal, the same impedance and the same interconnection channel delay and a variable load impedance, which is the unknown quantity to be determined.","By varying the simulated impedance parameters (e.g.","capacitance), various reflected signal patterns are obtained, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation.","The value of the simulated impedance parameters thus corresponds to that associated to the device port.","Other parasite effects in the interconnection channel between generator and device, due to discontinuous impedance between support and connection cable, between support and device, etc., or loss due to skin effect in the cable and in the support, which are intrinsic to the test machine, can be accounted for in the model used for simulation and consequently compensated.","A similar process can be applied to the measurement of protection circuit and clamps on device access ports, by suitably arranging the voltage levels of the stimulus signal.","For example, to characterise the protection circuit at input of an integrated circuit to the earth terminal, a stimulus signal passing from 0 V to −1 V and then from −1 V to 0 V can be used.","By measuring the waveform reflected by the diode, which firstly conducts and then cuts off the stimulus signal, the characteristic transition time of the diode can be determined.","The subsequent approximation process herein described can also be applied to more conventional types of measurements, i.e.","measurements based on the use of the test machine as a stimulus signal generator and respective output signal sampler, instead of as a time domain reflectometer.","The component is programmed, by means of the test machine, so to generate repetitive output signals, which can be measured by means of the sampling procedure.","The measured waveforms are not those of the component under test but those effected by the distortion introduced in the entire measurement system, consisting of the machine, the interconnections, the physical component support, etc.","Consequently, a system model is required, for simulating the measurement providing the output waveform to be compared with those resulting from the measurements.","In this case, in the model used for the simulation, the unknown quantity to be identified consists of the intrinsic switching times of the device.","A sequence of increasing switching times within a predefined range is set in the model for each output port so that the simulated waveform output by the measurement system model better approximates the measured waveform.","The set switching time leading to the best approximation of the waveforms, is the unknown quantity sought.","The entire procedure, comprising the measurement phases, the simulation phases and the comparison phases, can be easily automated by implementing a specific test machine management and result processing application.","A typical measurement session according to the process of this invention is illustrated in the flow chart in FIG.","1.Test device 101, with respective data sheet 102 and JTAG data 103, where relevant, form the starting data of phase 100.As known, JTAG is a standard according to which some component pins are dedicated to functional tests.","For example, thanks to this standard, some parts of the device, deemed particularly critical, can be isolated and individually operated, during testing or trouble-shooting.","Consequently, the test support is prepared during phase 104.The test support is specific for the device under test and suitable to the test machine.","The device is fitted in phase 105.According to the type of parameters to be extracted, suitable stimulus signals 106, to be used in the test machine in phase 107 for the measurements, must be prepared.","According to the resulting measurements, the device input and output voltage-current static characteristics are extracted in phase 108, the output switching times in certain conditions of load are extracted in phase 109, and the input or output port capacitance reactance is extracted in phase 110.Particularly, the switching time measurements are made according to the measured waveforms and the capacitance on the basis of reflected signals.","During the subsequent phase 111, the IBIS model containing all the measurement parameters is finally created.","The measurement process implementing the test machine, indicated with numeral 107, is illustrated in greater detail in FIGS.","2 and 3.In FIG.","2, the various operations, indicated with numerals 201, .",".",".","206, consist in: measuring the input static characteristics; output conditioning by means of JOAG functions; measuring output static characteristics; generating and measuring input reflectometer signals; output conditioning by means of JTAG functions; measuring the output fronts.","The reflectometer and output waveform front measurement process is illustrated in FIG.","3.In phase 301 “Generate one input or output waveform front”, the test machine controls the device under test.","If measurements are carried out on the output signals, the machine programs the component to switch the outputs.","In the next phase 302, the starting point of the switching front is established.","This determines the start instant of the level measurement.","This measurement is conducted in phase 303, by comparing the signal with a variable threshold by means of a machine comparator to check correspondence of values.","Consequently, the measurement instant is increased by one time unit defined in phase 304 and, if the measurement time window is not over because the switching transient is not complete, the cycle is repeated to measure the new signal level in phase 305.The succession of levels obtained, which reproduces the pattern of the waveform, is finally stored in a file during phase 306.During reflectometer measurements, the output signal of the circuit under test is no longer considered in phase 301.In this case, the signal is reflected on an input port The process is similar and the result is the pattern of the reflected signal.","The process in which the output switching times and the input and output port capacitance, indicated with numerals 109 and 110, are extracted is illustrated in greater detail in FIG.","4.In phase 410 “setup model”, an electric simulator model representing the measurement configuration is created, specifically including the interconnection cables with respective loss and impedance discontinuity, where relevant, the physical support and the stimulus signals.","This model can be created on the basis of the reflectometer response obtained from the test machine in the configuration in which the support plate of the device under test is not inserted and is specific for each measurement channel available on the machine.","Said responses contain all the information (characteristics impedance levels, propagation delay, etc.)","required for the description by means of circuit simulation primitives.","The circuit model completed in phase 402 can be obtained by connecting the measurement system model to the device model containing one or more circuit parameters on circuit simulation level (for example capacitance, switching time, etc.)","which value is to be extracted.","In phase 402, the model is updated with a first hypothesis of parameter, for example the switching time, which is then simulated in phase 404 and compared with the measurements provided by the test machine in phase 405.If the error exceeds a predefined threshold, the parameter is changed in phase 403 and the approximation cycle is repeated to obtain the minimal discrepancy between the measurement and the simulation.","Finally, the resulting parameter is used for constructing the IBIS model.","The description herein is provided as a non-limiting example and obviously variations and changes are possible within the scope of protection of this invention."]],"string":"[\n [\n \"Process for the electromagnetic modelling of electronic components and systems\",\n \"Process for the electromagnetic modelling of electronic components and systems, for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes.\",\n \"A test machine of the commercial type is used for these measurements, by generating stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer.\",\n \"The measurement phase is followed by a simulation phase during which the electric parameters used for modelling electronic components and systems are extracted.\"\n ],\n [\n \"1.Process for the electromagnetic modelling of electronic components and systems, in particular for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes of said electronic components and systems, to ports of these components and systems suitable stimulus signals are sent and the correlated signals are measured, reconstructing the respective time patterns, characterised in that a test machine of the commercial type is used for generating the stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer, in which case the test machine sends said stimulus signals to the component or system inputs or outputs, fitted on a suitable support and connected by means of suitable interconnection channels, and detects the reflection due to impedance offset, providing the respective time patterns, which are then compared with those provided by a simulated measurement system model, comprising the test machine signal generation and measurement part, the interconnection channels and the support, to verify the coincidence between the measure time pattern and that provided by the simulated model obtained by setting different values with respect to the electric parameters to be determined.\",\n \"2.Process according to claim 1, characterised in that the model simulated by the measurement system is obtained by the reflectometer response of each channel of the test machine in the situation in which the support of the component or system is not connected to the machine.\",\n \"3.Process according to claim 1, characterised in that the reflected signals are reconstructed by means of the periodical sampling method, by fine tuning the sampling pulse phase and operating on the comparison threshold of the measurement part of the test machine to identify the succession of values assumed in time by the reflected signal, so as to obtain the time pattern.\",\n \"4.Process according to claim 1, characterised in that for measuring the electronic component and system input and output impedance, a variable load impedance is introduced in the simulated model, so as to obtain various patterns of the reflected signal, following the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated impedance value corresponding to the impedance of the input or output to be determined.\",\n \"5.Process according to claim 1, characterised in that for measuring the switching time of the outputs or transition time of the protection diode, a variable time is introduced in the simulated model, so as to obtain different patterns of the reflected signal, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated switching or transition time corresponding to the value to be determined.\"\n ],\n [\n \"This invention relates to measurement methods for evaluating electromagnetic compatibility, and particularly relates to a process for the electromagnetic modelling of electronic components and systems.\",\n \"With the increased level of integration and operating frequency of electronic components and systems, the need to check the electromagnetic compatibility between different parts of any one system, or between different systems in conditions of mutual interference, and the integrity of signals exchanged in electronic systems between the various components is becoming increasingly important.\",\n \"The integrity of signals is particularly important in digital systems, where the treated signals are essentially binary signals.\",\n \"For the good operation of systems, these signals must satisfy certain minimum quality requirements.\",\n \"Particularly, two voltage levels, corresponding to bit 1 and 0, and the transition times between the levels must be preserved.\",\n \"Furthermore, interference between adjacent conductors, skin effect attenuation and spurious signals originated from reflection of the original signals in the presence of discontinuous impedance must be minimised.\",\n \"In digital systems, the increase in signal switching front steepness and dock frequency increases the intensity of the electromagnetic field radiated by the various component parts, such as conductors and active and passive components, with the consequent possibility of exceeding the limits established in the emission standard.\",\n \"As a consequence, the device may not be type-approved or, consequently, marketed.\",\n \"These problems can be obviated by an accurate system design, which accounts for the electromagnetic performance of the various components employed.\",\n \"The validity of design can be verified before system construction by exploiting the possibilities offered by simulation programs readily available on the market.\",\n \"Electromagnetic compatibility data, which can be provided by such programs, will be closer to reality the more the component and system models employed for describing the entire device to be simulated are accurate.\",\n \"Standard model definition processes have existed for several years.\",\n \"These processes guide technicians in identifying the parameters of the model and describing the model Of such processes, the most common is that proposed by the IBIS (Input-output Buffer Information Specification) Association, which consists in describing the static V-I characteristics of all component inputs and outputs, the output transition times in particular conditions of load, the parasite parameters associated to all inputs and outputs, such as the capacitance with respect to a reference potential, and other parameters.\",\n \"At this time, there are not many available electronic component models.\",\n \"The few available models essentially refer to integrated circuits and are widely incomplete and not very accurate.\",\n \"These models are obtained by simulation employing known methods (SPICE), using technological parameters, in some cases provided by the manufacturers, or by using the scarce information provided by component data sheets.\",\n \"In other cases, the models are made by means of measurements using single instruments, with considerable waste of time for preparing the bench and for making settings and measurements.\",\n \"The process illustrated in this invention obviates these disadvantages and solves the technical problem described above by allowing automatic electronic component and system modelling using normal electronic test machines in an original way, without requiring costly modifications.\",\n \"All access points of the device under test can be characterised rapidly also when dealing with very complex stimulation signals.\",\n \"The test machine can be used in a conventional way for conducting functional tests or, according to this invention, for modelling in the same test session, without the need for additional manual interventions on behalf of the operator.\",\n \"Specifically, object of this invention is a process for the electromagnetic modelling of electronic components and systems the characteristics of which are described in claim 1.This invention will be better explained by the following detailed description with reference to the accompanying drawings as non-limiting example, wherein: FIG.\",\n \"1 is a flow chart illustrating a typical measurement session; FIGS.\",\n \"2 and 3 are flow charts illustrating the process for conducting the measurements by means of the test machine; FIG.\",\n \"4 is a flow chart illustrating the process which is used to extract the output switching time and the capacitance of the input or output ports of the component or system under test.\",\n \"The process for the electromagnetic modelling of electronic components and systems employs a test machine of the commercial type, designed for conducting parametric measurements in direct current, functional tests and digital integrated circuit timing.\",\n \"The characteristics required for the machine to be used in this process are: the possibility of generating digital signals with sufficiently steep switching fronts, typically lower than 300 ps; impedance controlled along all interconnections in the device under test; possibility of monitoring all signals output by the device under test by sampling, with time resolution lower than 300 ps.\",\n \"Various types of such machines are made by various manufacturers, e.g.\",\n \"the machine identified by code ITS9000 IX, made by Schlumberger.\",\n \"In normal use for integrated circuit functional tests, signals with a suitable pattern are sent to the device inputs and the output signals are measured, checking that such signals are as required in a precise instant of the clock cycle.\",\n \"The sampling method is used to measure the signals.\",\n \"Specifically, at input port level transition, the signal at the output port is compared with a pre-programmed reference threshold and the time instant in which the threshold was exceeded is recorded.\",\n \"By using a sufficiently high sampling frequency, the signal can be closely reconstructed.\",\n \"A subsequent study can be conducted to evaluate the response of the device, checking that the parametric, functional and timing characteristics, the output signal time delays and the input and output signal threshold values are within the tolerance range permitted in specifications.\",\n \"The test machine according to this invention is used in a different way with respect to that described above: it is used as a time domain reflectometer, i.e.\",\n \"the test machine is used to send specific signals to the component ports and the reflections due to impedance offset are measured.\",\n \"This method is not exploited in normal test machine use.\",\n \"Only in the initial machine channel calibration and characterisation phase, a signal is sent to the device support and the delay of the corresponding reflected signal is measured to determine the delay introduced by the device interconnection channel.\",\n \"The consequent compensations can be implemented, if required.\",\n \"However, the time pattern of the reflected signal is not studied in detail, to the extent that the integrated circuit under test is not present: in other words, the conditions are those of total reflection.\",\n \"By extending the possibility of reconstructing signals, also to involve the signals reflected by the access ports of the device under test, the respective electric parameters can be obtained.\",\n \"These are essential for the electromagnetic modelling of the entire device according to the IBIS standard process mentioned above.\",\n \"In detail, the following parameters can be obtained: the static voltage-current (V-I) characteristics at inputs and outputs; the output switching times in the specific conditions of load; the input and output capacitance; the tripping times of the protection and clamp diode circuits, normally present in input ports; various parasite parameters.\",\n \"For the use according to this invention, the stimulus signals generated by the test machine must be sufficiently steep and present a sufficient amplitude, so as to allow high frequency measurements, preserving a good signal/noise ratio.\",\n \"For example, to measure the typical input capacitance of a digital integrated circuit, an amplitude of approximately 1 V is conveniently selected for obtaining a transition time of approximately 400 ps, which suitably adapts to the amplitude resolution of the test machine comparators.\",\n \"The signal offset can be set at a suitable level for the device, e.g.\",\n \"2.5 V for an integrated circuit powered at 5 V. Since impedance offset is measured, naturally the output impedance of the test machine must be controlled and fixed at a suitable value, e.g.\",\n \"50 Ohms, as the impedance of all the interconnections between the generator and the access port of the device.\",\n \"As mentioned above, the reflected signals are reconstructed using the periodical sampling method.\",\n \"The sampling input phase can be very finely tuned, e.g.\",\n \"with a resolution of 20 ps.\",\n \"After stablising the sampling phase, the comparison threshold is used to identify the internal comparator switching value, by using a suitable search algorithm, eg.\",\n \"dichotomy, linear etc., according to the morphology of the signal.\",\n \"After reconstructing the pattern of the reflected signal, it is compared with the pattern obtained by simulating the measurement configuration in the time domain with a circuit simulator.\",\n \"The simulated model must include the same stimulus signal, the same impedance and the same interconnection channel delay and a variable load impedance, which is the unknown quantity to be determined.\",\n \"By varying the simulated impedance parameters (e.g.\",\n \"capacitance), various reflected signal patterns are obtained, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation.\",\n \"The value of the simulated impedance parameters thus corresponds to that associated to the device port.\",\n \"Other parasite effects in the interconnection channel between generator and device, due to discontinuous impedance between support and connection cable, between support and device, etc., or loss due to skin effect in the cable and in the support, which are intrinsic to the test machine, can be accounted for in the model used for simulation and consequently compensated.\",\n \"A similar process can be applied to the measurement of protection circuit and clamps on device access ports, by suitably arranging the voltage levels of the stimulus signal.\",\n \"For example, to characterise the protection circuit at input of an integrated circuit to the earth terminal, a stimulus signal passing from 0 V to −1 V and then from −1 V to 0 V can be used.\",\n \"By measuring the waveform reflected by the diode, which firstly conducts and then cuts off the stimulus signal, the characteristic transition time of the diode can be determined.\",\n \"The subsequent approximation process herein described can also be applied to more conventional types of measurements, i.e.\",\n \"measurements based on the use of the test machine as a stimulus signal generator and respective output signal sampler, instead of as a time domain reflectometer.\",\n \"The component is programmed, by means of the test machine, so to generate repetitive output signals, which can be measured by means of the sampling procedure.\",\n \"The measured waveforms are not those of the component under test but those effected by the distortion introduced in the entire measurement system, consisting of the machine, the interconnections, the physical component support, etc.\",\n \"Consequently, a system model is required, for simulating the measurement providing the output waveform to be compared with those resulting from the measurements.\",\n \"In this case, in the model used for the simulation, the unknown quantity to be identified consists of the intrinsic switching times of the device.\",\n \"A sequence of increasing switching times within a predefined range is set in the model for each output port so that the simulated waveform output by the measurement system model better approximates the measured waveform.\",\n \"The set switching time leading to the best approximation of the waveforms, is the unknown quantity sought.\",\n \"The entire procedure, comprising the measurement phases, the simulation phases and the comparison phases, can be easily automated by implementing a specific test machine management and result processing application.\",\n \"A typical measurement session according to the process of this invention is illustrated in the flow chart in FIG.\",\n \"1.Test device 101, with respective data sheet 102 and JTAG data 103, where relevant, form the starting data of phase 100.As known, JTAG is a standard according to which some component pins are dedicated to functional tests.\",\n \"For example, thanks to this standard, some parts of the device, deemed particularly critical, can be isolated and individually operated, during testing or trouble-shooting.\",\n \"Consequently, the test support is prepared during phase 104.The test support is specific for the device under test and suitable to the test machine.\",\n \"The device is fitted in phase 105.According to the type of parameters to be extracted, suitable stimulus signals 106, to be used in the test machine in phase 107 for the measurements, must be prepared.\",\n \"According to the resulting measurements, the device input and output voltage-current static characteristics are extracted in phase 108, the output switching times in certain conditions of load are extracted in phase 109, and the input or output port capacitance reactance is extracted in phase 110.Particularly, the switching time measurements are made according to the measured waveforms and the capacitance on the basis of reflected signals.\",\n \"During the subsequent phase 111, the IBIS model containing all the measurement parameters is finally created.\",\n \"The measurement process implementing the test machine, indicated with numeral 107, is illustrated in greater detail in FIGS.\",\n \"2 and 3.In FIG.\",\n \"2, the various operations, indicated with numerals 201, .\",\n \".\",\n \".\",\n \"206, consist in: measuring the input static characteristics; output conditioning by means of JOAG functions; measuring output static characteristics; generating and measuring input reflectometer signals; output conditioning by means of JTAG functions; measuring the output fronts.\",\n \"The reflectometer and output waveform front measurement process is illustrated in FIG.\",\n \"3.In phase 301 “Generate one input or output waveform front”, the test machine controls the device under test.\",\n \"If measurements are carried out on the output signals, the machine programs the component to switch the outputs.\",\n \"In the next phase 302, the starting point of the switching front is established.\",\n \"This determines the start instant of the level measurement.\",\n \"This measurement is conducted in phase 303, by comparing the signal with a variable threshold by means of a machine comparator to check correspondence of values.\",\n \"Consequently, the measurement instant is increased by one time unit defined in phase 304 and, if the measurement time window is not over because the switching transient is not complete, the cycle is repeated to measure the new signal level in phase 305.The succession of levels obtained, which reproduces the pattern of the waveform, is finally stored in a file during phase 306.During reflectometer measurements, the output signal of the circuit under test is no longer considered in phase 301.In this case, the signal is reflected on an input port The process is similar and the result is the pattern of the reflected signal.\",\n \"The process in which the output switching times and the input and output port capacitance, indicated with numerals 109 and 110, are extracted is illustrated in greater detail in FIG.\",\n \"4.In phase 410 “setup model”, an electric simulator model representing the measurement configuration is created, specifically including the interconnection cables with respective loss and impedance discontinuity, where relevant, the physical support and the stimulus signals.\",\n \"This model can be created on the basis of the reflectometer response obtained from the test machine in the configuration in which the support plate of the device under test is not inserted and is specific for each measurement channel available on the machine.\",\n \"Said responses contain all the information (characteristics impedance levels, propagation delay, etc.)\",\n \"required for the description by means of circuit simulation primitives.\",\n \"The circuit model completed in phase 402 can be obtained by connecting the measurement system model to the device model containing one or more circuit parameters on circuit simulation level (for example capacitance, switching time, etc.)\",\n \"which value is to be extracted.\",\n \"In phase 402, the model is updated with a first hypothesis of parameter, for example the switching time, which is then simulated in phase 404 and compared with the measurements provided by the test machine in phase 405.If the error exceeds a predefined threshold, the parameter is changed in phase 403 and the approximation cycle is repeated to obtain the minimal discrepancy between the measurement and the simulation.\",\n \"Finally, the resulting parameter is used for constructing the IBIS model.\",\n \"The description herein is provided as a non-limiting example and obviously variations and changes are possible within the scope of protection of this invention.\"\n ]\n]"},"source":{"kind":"string","value":"Patent_10297634"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":0,"numItemsPerPage":100,"numTotalItems":4495096,"offset":0,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTczNTAxMjAwNSwic3ViIjoiL2RhdGFzZXRzL2hvd2V5L2h1cGQiLCJleHAiOjE3MzUwMTU2MDUsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.oJdgmGZRFd_b4phTfw_D_5RpcGMzYNyHgw4PIczrgvyf3ghYyAfKt1Vkr2r1qvja_4gct6dZaPOh4sm5ru7FDA","displayUrls":true},"discussionsStats":{"closed":0,"open":0,"total":0},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false}">
text
sequencelengths 3
5
| source
stringlengths 15
15
|
|---|---|
[
[
"Stable expression of polymorphous forms of human cytochrome p450 2d6",
"The present invention relates to a test system containing cell lines expressing human cytochrome P450 2D6 as well as to the use of said test system for the study of pharmacological and toxicological aspects of the hCYP2D6 polymorphism.",
"The present invention further relates to methods for the detection of novel polymorphic forms of human cytochrome P450 2D6 using the test system according to the invention as well as to methods for the simple and exact quantification of the cytochrome P450 content using CO difference spectra."
],
[
"1.Test system consisting of cells expressing a cytochrome P450 2D6 (hCYP2D6) allele in a heterologous manner wherein at least three P450 2D6 alleles are expressed in said test system.",
"2.Test system according to claim 1 wherein said at least three P450 2D6 alleles correspond to the most frequent allele types in a population.",
"3.Test system according to claim 1 wherein said test system expresses at least 5 functional hCYP2D6 alleles in a heterologous manner.",
"4.Test system according to claim 3 wherein the alleles hCYP2D6*1, *2, *9, *10 and *17 are expressed.",
"5.Test system according to any one of claims 1 to 4 wherein said cells are Chinese hamster lung fibroblasts or cells derived therefrom.",
"6.Test system according to claim 5 wherein said cells are V79 cells.",
"7.Test system according to claim 6 wherein said cells are the cell lines V79MZh2D6*1, V79MZh2D6*2, V79MZh2D6*9, V79MZh2D6*10 and V79MZh2D6*17 deposited on Feb. 15, 2000, at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession numbers DSM ACC2446, DSM ACC2447, DSM ACC2448, DSM ACC2449 and DSM ACC2450.8.Test system according to any of the claims 1 to 7 wherein said cells express cDNA.",
"9.Kit comprising the test system according to any of the claims 1 to 8.10.Use of the test system according to any of the claims 1 to 8 for the study of the gene-dependent toxicity of metabolites.",
"11.Use according to claim 10 wherein said metabolites are drugs.",
"12.Use of the test system according to any of the claims 1 to 8 for determining a toxic, mutagenic or cancerogenous effect of compounds.",
"13.Use according to any one of claims 10 to 12 wherein the cells expressing human cytochrome P450 2D6 are contacted with the substance to be tested.",
"14.Method for screening of substances with respect to their metabolization by human cytochrome P450 2D6 wherein the cells of the test system according to any of the claims 1 to 8 are contacted with a substance and the metabolic product is measured.",
"15.Method for the detection of novel P450 2D6 alleles wherein said method comprises the heterologous expression of the allele in question in a cell, testing the cells expressing the allele in question with respect to the cytochrome P450 2D6-dependent metabolism of one or more compounds and comparison of the metabolism of the cells to the metabolism of cells of the test system according to any one of claims 1 to 8.16.Method for the quantification of the cytochrome P450 content wherein said method comprises the solubilization of cytochrome P450 by means of the non-ionic detergent emulgen 913, centrifuging the solubilizate and measurement using CO difference spectra.",
"17.Method according to claim 16 wherein said method comprises the following steps: (a) preparation of cell homogenate; (b) addition of emulgen 913 to the cell homogenate; (c) removing insoluble material; (d) determination of the reduced spectrum; (e) saturation with carbon monoxide; (f) measurement of the CO/reduced spectrum; (g) evaluation of the cytochrome P450 content by means of the spectra.",
"18.Method according to claim 16 or 17 wherein emulgen 913 is added in a final concentration of 0.25% (w/v)."
],
[
"The present invention relates to a test system containing cell lines expressing human cytochrome P450 2D6 as well as to the use of this test system for the study of pharmacological and toxicological aspects of the hCYP2D6 polymorphism.",
"Furthermore, the present invention relates to methods for detection of novel polymorphic forms of human cytochrome P450 2D6 using the test system according to the present invention as well as to methods for a simple and exact quantification of the cytochrome P450 content by means of CO difference spectra.",
"Day by the day, the human body takes up a plurality of foreign substances.",
"Harmful chemicals from the environment, ingredients of food and stimulants, and in some cases also medicaments.",
"All these foreign substances eventually have to be excreted to avoid damages to the organism.",
"Many of these compounds, however, have a poor solubility in water and therefore cannot be excreted easily.",
"Thus, in the course of evolution nearly all living organisms have developed a complex enzyme system capable of converting compounds into a hydrophilic excretable form.",
"This metabolic process is referred to as the metabolism of foreign substances and has been formally divided into two phases (Greim and Deml, 1996; Marquardt and Schäfer, 1997): In phase I the introduction of functional groups into the compound or a demasking of functional groups occurs, a process in which P450 cytochromes play a key role.",
"In phase II the functionalized metabolite is conjugated to substances with good solubility in water, such as sulfates, sugars, glutathione, carboxylic acids, or amino acids.",
"Thereby, the compound is rendered sufficiently hydrophilic to be excretable in the form of a non-reactive final product via the kidney or the intestine.",
"There is a risk, however, that reactive metabolites may react with the body's own structures such as DNA, RNA, proteins, and lipids and induce cytotoxic, cancerogenous or mutagenic effects.",
"In this case, the detoxification is converted into a toxification.",
"Drugs are metabolized and excreted by the same enzymatic system, a fact which may have an influence on the pharmacological efficacy in different ways: either the metabolites have a lower pharmacological effectiveness as the starting compounds or they may be completely ineffective, as in the case of barbiturates.",
"In other cases, the mother substance as well as the metabolites have an effect.",
"An example are the cough medicine codeine and its metabolite morphine.",
"In other cases, only the metabolite is effective for example the cleavage product of cyclophosphamnide in chemotherapy.",
"The efficacy of a drug may vary between different individual due to differences in its metabolization.",
"The metabolization is affected by parameters such as age, sex, physical condition, and diet.",
"Differences in the genes encoding foreign substance-metabolizing enzymes such as cytochrome P450 have been shown to be another important factor.",
"Thus, due to genetic differences some individuals can metabolize drugs faster, slower, in a different manner or not at all.",
"If the allelic sequences vary between different populations this will result in interethnical differences in the frequency of phenotypes.",
"Asians, Caucasians and Black Africans will therefore react very different to the same medicament.",
"These pharmacologically important inter-individual differences have been referred to as pharmacogenetic polymorphism (Meyer, 1991): “A pharmaco-genetic polymorphism is a monogenic feature caused by the presence within a population of more than one allele in the same locus and of more than one phenotype with respect to the efficacy of a drug in the organism.",
"The frequency of the rarest allele is >1%.” It has been agreed to set the frequency of the rarest allele to >1% since not every base difference will necessarily be relevant for a certain population group.",
"It has been estimated that up to 20% of all medicaments are subject to pharmaco-genetic polymorphism (Blech, 1999).",
"In the USA, more than 2 million people per year suffer from undesirable drug effects which take a lethal course in more than 100,000 cases.",
"Thus, they are among the six most frequent causes of death (Lazarou et al., 1998).",
"As a consequence, “personalized drug(s) (dosages)” are desired which can be specifically adapted to the individual pharmaco-genetics of a particular patient.",
"For example, this is the current practice in the therapy of children suffering from leukemia using azathioprine or 6-mercaptourine which otherwise would lead to life-threatening side effects in 3% of the cases.",
"Therefore, besides the development of efficient methods for detecting the individual pharmaco-genetic profile such as by DNA chip technology for maximal drug safety it will be required to gain a detailed knowledge of the drug metabolizing enzymes and their genetic polymorphisms.",
"The most important group of foreign substance metabolizing phase I enzymes are the cytochromes P450 (EC 1.14.14.1; unspecific monooxygenases).",
"In 1958, they were described for the first time idependently by Garfinkel (1958) and Klingenberg (1958) as “cell-coloring pigment” of the liver.",
"They were referred to as cytochrome P450 because cytochromes in their reduced state and after gassing with carbon monoxide have difference spectra showing a characteristic absorption peak at 450 nm.",
"A quantification may be carried out using the absorption together with the molar extinction coefficient (Omura and Sato, 1964a; Omura and Sato, 1964b).",
"At present, the cytochrome P450 superfamily includes 481 isoforms in 85 different eukaryotic and 20 prokaryotic species (Nelson et al., 1996).",
"The classification of cytochrome P450 enzymes is per conventionem based on their amino acid sequence homologies.",
"Their identity is more than 40% within a family and at least 55% within a subfamily.",
"Cytochrome P450 genes are abbreviated by “CYP” in italics while cDNAs, mRNAs, and proteins are abbreviated by “CYP” followed by an arabic numeral referring to the family as well as a latin capital letter for each subfamily.",
"Individual isoenzymes are numbered chronologically by another arabic numeral.",
"The whole symbol is preceded by a small latin letter indicating the species.",
"Thus, for example human cytochrome P450 2D6 is designated by hCYP2D6 for the gene and hCYP2D6 for cDNA, mRNA, and protein, respectively.",
"All foreign substance metabolizing P450 cytochromes are anchored in the endoplasmatic reticulum as well as in the nuclear membrane by means of a hydrophobic N-terminal sequence and are oriented towards the cytoplasm (Monier et al., 1988).",
"P450 cytochromes belong to the group of heme thiolate enzymes catalyzing the NADPH-dependent monooxygenation of their substrates with formation of the corresponding alcohols or epoxides as well as O- and N-dealkylations.",
"They form a multi-enzyme complex together with NADPH-dependent cytochrome P450 oxido-reductase (CYPOR) and cytochrome b5 catalyzing the transfer of electrons from NADPH to cytochrome P450.Differences in the ability of complex formation have been observed (Schenkman and Greim, 1993).",
"The activities of some isoforms such as hCYP3A4 is specifically dependent on CYPOR and cytochrome b5 (Buters et. al, 1994).",
"In the case of hCYP3A4 cytochrome b5 additionally increases the affinity to certain substrates (Schenkman et al., 1989).",
"The substrate specificity of individual P450 cytochromes is generally low and often overlapping thereby ensuring sufficient flexibility to deal with the enormous diversity of foreign substances to be metabolized.",
"On the other hand, besides organ-specific expression, polymorphism, and inducibility of many isoenzymes the overlapping substrate specificities substantially contribute to the complexity of the cytochrome P450-catalyzed metabolism of foreign substances and drugs, repectively.",
"The complexity may be further enhanced if the drug metabolism is affected by induction or inhibition of particular cytochrome P450 isoforms during simultaneous administration of several drugs.",
"Therefore, it is required to gain detailed knowledge about drug metabolizing P450 cytochromes on a genetic, regulatory and enzymatic level to avoid undesirable effects caused by metabolism.",
"Cytochrome P450 2D6 (EC 1.14.14.1; debrisoquine 4-hydroxylase) is one of the molecular species of cytochrome P450 characterized by a marked polymorphism.",
"In humans, CYP2D6 is the only functional isoenzyme of the 2D subfamily, and it was the first cytochrome P450 enzyme for which a genetic polymorphism has been described.",
"By the end of the seventies, the polymorphism of hCYP2D6 has been discovered independently for the antihypertensive drug debrisoquine (Evans et al., 1980; Mahgoub et al., 1977) and the antiarrhythmic drug sparteine (Eichelbaum et al., 1979a; Eichelbaum et al., 1979b).",
"In the next years, the polymorphic metabolic phenotype common to both substrates was demonstrated (Eichelbaum et al., 1982), and its genetic cause was discovered (Daly, 1995; Gonzalez et al., 1988a; Meyer and Zanger, 1997; Price-Evans, 1993; Steiner et al., 1985; Zanger et al., 1988).",
"Today, a plurality of important substrates (cf.",
"Table 1) and 17 alleles are known.",
"Thus, at present the “debrisoquine/sparteine” polymorphism is the most extensive pharmaco-genetic polymorphism which has the highest impact in practise (Bertilsson, 1995; Brosen and Gram, 1989b; Eichelbaum and Gross, 1990; Kroemer and Eichelbaum, 1995; Nebert, 1997; Tucker, 1998).",
"The expression of hCYP2D6 primarily occurs in the liver in a constitutive manner, and the enzyme is not inducible in contrast to all other drug metabolizing P450 cytochromes 1A1/2, 2B6, 2C8, 2C9, 2C18, 2C19, 2E1 and 3A4/5.During pregnancy, however, a slightly elevated metabolism of hCYP2D6 substrates was observed (Hogstedt et al., 1983; Wadelius et al., 1997).",
"The fraction of the total hepatic cytochrome P450 content is only about 2% and thus relatively low as compared to the two other important drug metabolizing isoforms hCYP3A4 with ≧30% and hCYP2C9 with ≧20% (Shimada et al., 1994).",
"There are dramatic inter-individual differences in the level of expression up to complete deficiency (Shimada et al., 1994).",
"In addition, an about 100 times lower expression of hCYP2D6 compared to the liver was detected in various extrahepatic tissues, and an association with different diseases has been discussed, in part controversially: in lung/lung cancer (Guidice et al., 1997; Kivisto et al., 1997), in brain/Parkinson (Fonne-Fister et al., 1987; Nebert and McKinnon, 1994; Sabbagh et al., 1999), in the gastrointestinal tract (Prueksaritanont et al., 1995), in breast and in mammary tumors (Huang et al., 1996; Huang et al., 1997), in the bladder mucosa and in tumor tissue (Romkes-Sparks et al., 1994), as well as in peripheral mononuclear blood cells (Carcillo et al., 1996).",
"Of particular interest is the expression in brain since hCYP2D6 metabolizes several pharmaceutics having a central-nervous activity and hydroxylates endogenous tryptamine to give the neurotransmitter dopamine (Hiroi et al., 1998).",
"hCYP2D6 is involved in phase I metabolism of about 30% of all clinically relevant drugs of different drug groups (cf.",
"Table 1; Alvan, 1991; Brosen and Gram, 1989a; Dahl and Bertilsson, 1993; Eichelbaum and Gross, 1992).",
"Thus, besides hCYP3A4 (55%) and hCYP2C9 (15%) it belongs to the most important drug metabolizing P450 cytochromes despite of its lower level of expression (Smith et al., 1998).",
"For example, the antihypertensive drugs debrisoquine and propafenone, the B blocker propanolol, the tricyclic antidepressant imipramine, etc.",
"have been described as specific substrates of CYP2D6 (Eichelbaum and Gross, 1990).",
"hCYP2D6 is selectively inhibited by quinidine or by specific inhibitory antibodies.",
"TABLE 1 Examples of drugs and other substrates at least partially metabolized by hCYP2D6.Drug Group Example Reaction Reference monoamineoxidase inhibitors amiflamine NDem (Alvan et al., 1984) β blockers bufuralol alH, arH (Boobis et al., 1985) analgetics codeine ODem (Mortimer et al., 1990) antihypertensives debrisoquine arH (Mahgoub et al., 1977) anorectics dexfenfluramine NDea (Gross et al., 1996) neuroleptics haloperidol NDea (Tyndale et al., 1991b) tricyclic antidepressants imipramine arH (Brosen et al., 1986) α1 adrenoceptor antagonists indoramine arH (Pierce et al., 1987) β2 adrenergic stimulants methoxyphenamine arH, NDem (Roy et al., 1985) SSRI paroxetine Dem (Bloomer et al., 1992) antianginal perhexiline alH (Cooper et al., 1987) antidiabetics phenformine arH (Oates et al., 1982) antiarrhythmics sparteine H (Eichelbaum et al., 1979b) anti-estrogenic compounds tamoxifen arH (Dehal und Kupfer, 1997) amphetamine (life-style drug) MDMA (ecstasy) alH (Tucker et al., 1994) endogenous neurotransmitter tryptamine arH (Hiroi et al., 1998) Abbreviations: alH: aliphatic hydroxylation; arH: aromatic hydroxylation; Dem: demethylation; MDMA: methylenedioxymethamphetamine; NDea: N-dealkylation; NDem: N-demethylation; ODem: O-demethylation; SSRI: selective serotonine reuptake inhibitor According to estimations of the WHO, 250,000 women worldwide die each year because of breast cancer (Logan, 1975).",
"In the USA and Western Europe, breast cancer is the cause of death in 4% of all cases in women (American Cancer Society).",
"In Germany alone about 42,000 women each year fall ill with breast cancer (Becker and Warendorf, 1981-1990).",
"Approx.",
"30% of the tumors are hormone-sensitive.",
"The non-steroidal selective estrogen receptor modulator tamoxifen (Novaldex®) is used for the treatment of estrogen-sensitive tumors (Furr and Jordan, 1984; Jordan, 1998; Osborne, 1998).",
"Tamoxifen binds to the estrogen receptor and thus blocks the stimulation of proliferation by estrogen binding.",
"In other organs, however, it shows a desired paradox partial estrogen effect in that it for example counteracts osteoporosis (McGregor and Jordan, 1998).",
"Studies to examine the preventive use of tamoxifen in risk groups are presently carried out (Jordan, 1997; Nayfield, 1995).",
"With respect to its pharmacology, metabolite profile, and DNS adduct formation, tamoxifen shows pronounced variations between different species (De Matteis et al., 1998; Glatt et al., 1998; Jordan and Chem, 1982; Jordan and Robinson, 1987; Lim et al., 1994).",
"The main metabolites are N-demethyl-tamoxifen, tamoxifen-N-oxide, and 4-hydroxy-tamoxifen; see FIG.",
"23.4-Hydroxy-tamoxifen is about 100 times more potent as an anti-estrogenic substance than the mother substance itself, and thus despite its lower plasma level is believed to contribute substantially to the pharmacological effect (Borgna and Rochefort, 1981; Furr and Jordan, 1984).",
"An involvement in the 4-hydroxylation of tamoxifen is discussed for the cytochrome P450 isoforms hCYP2C9, hCYP2D6, hCYP2E1 and hCYP3A4 (Crewe et al., 1997; Dehal and Kupfer, 1997; Styles et al., 1994).",
"It may be possible that it will be necessary to consider inter-individual differences with respect to the formation of 4-hydroxy-tamoxifen in establishing the therapeutical dose of tamoxifen.",
"Together with the pseudogenes CYP2D7P and CYP2D8P, CYP2D6 forms a gene cluster in the CYP2D locus at position q13.1 on the long arm of chromosome 22 (Eichelbaum et al, 1987; Gonzalez et al., 1988b; Gough et al., 1993; Kimura et al., 1989).",
"Similar to the two pseudogenes it consists of 9 exons and 8 introns.",
"Of the 17 known hCYP2D6 alleles only five, namely hCYP2D6*1, hCYP2D6*2, hCYP2D6*9, hCYP2D6*10 and hCYP2D6*17, encode an enzyme with (limited) functionality (Daly et al., 1996a).",
"At least another functional allele is postulated for the population of Ghana (Droll et al., 1998; Masimirembwa et al., 1996a).",
"The phenotypic classification is carried out using the ratio of test substrate/metabolite in urine per time unit.",
"The smaller this “metabolic ratio (MR)”, the faster will be the metabolism of the test substrate, e.g.",
"debrisoquine, dextromethorphane, metoprolol or sparteine.",
"Homozygous carriers of non-functional alleles are always deficient with respect to the 2D6 activity and show a so-called “poor metabolizer (PM)” phenotype characterized by a high MR.",
"Individuals who are homozygous for the wildtype allele hCYP2D6*1 phenotypically are “extensive metabolizers (EM)” having a low MR (Sachse et al., 1997).",
"Gene amplification of a functional allele results in a so-called “ultrarapid metabolizer (UM)” phenotype (Bertilsson et al., 1993; Johansson et al., 1993; Lundqvist et al., 1999).",
"Homozygous carriers of the allele hCYP2D6*10 showing a limited functionality have an increased MR compared to the EM phenotype and therefore are sometimes referred to as “intermediate metabolizers (IM)” (Armstrong et al., 1994; Yokota et al., 1993).",
"Furthermore, variations in the allele frequency between different populations result in inter-ethnical differences (Bertilsson, 1995): In Caucasians the frequencies of the functional alleles hCYP2D6*1 and hCYP2D6*2 are about 35% while hCYP2D6*2xN, hCYP2D6*9 and hCYP2D6*10 are rare with frequencies of 1-2% and hCYP2D6*1 7 has not been detected yet.",
"The non-functional alleles hCYP2D6*4 and hCYP2D6*5 have frequencies of about 20% and 5%, respectively, while the frequencies of the other allels is 0-2%.",
"Thus, the proportion of poor metabolizers in the Caucasian population is approx.",
"5-10% (Alvan et al., 1990; Evans et al., 1993; Griese et al., 1998; Sachse et al., 1997).",
"In contrast, in the Asian population the frequency for the non-functional allele hCYP2D6*4 is only 0.8% while the allele hCYP2D6*10 with limited functionality occurs in a frequency of 23-70%.",
"Accordingly, only about 1% of Asians are poor metabolizers although their mean MR is increased in comparison to Caucasians (Bertilsson et al., 1992; Dahl et al., 1995b; Horai et al., 1989; Roh et al., 1996).",
"The same applies to some Black African populations showing allele frequencies of about 4% for hCYP2D6*4, about 5% for hCYP2D6*10 , and 15-35% for hCYP2D6*17 (Evans et al., 1993; Masimirembwa et al., 1996b).",
"A heterologous expression of several hCYP2D6 alleles has been carried out in various systems: in E. coli (Gillam et al., 1995; Kempf et al., 1995; Pritchard et al., 1998), in yeast (Bichara et al., 1996; Ellis et al., 1992; Krynetski et al., 1993; Krynetski et al., 1995; Oscarson et al., 1997), in insect cells (Evert et al., 1997; Paine et al., 1996; Patten et al., 1996), in CHO cells (Patten et al., 1996), in COS-1 cells (Gonzalez et al., 1990; Johansson et al., 1994; Kagimoto et al., 1990; Oscarson et al., 1997), in Hep G2 cells (Aoyama et al., 1990; Tyndale et al., 1991a), in NIH3T3 cells (de Groene et al., 1996), and in human B lymphoblastoid cells AHH-1 TK± (Crespi et al., 1991; Crespi et al., 1995; Penman et al., 1993).",
"In addition, the wildtype allele hCYP2D6*1 has been already expressed in V79 Chinese hamster cells (Fischer et al., 1992; Rauschenbach et al., 1997).",
"Nevertheless, up to now no test system exists which is suitable for an examination of the pharmacological, toxicological, and other aspects of the hCYP2D6 polymorphism.",
"The technical object underlying the present invention is to provide a test system enabling a comparative examination of the metabolic activity of active forms of human cytochrome P450 2D6 with regard to a wide variety of substances.",
"Preferably, this system is intended to be suitable as an analytical tool in preclinical drug development and for in vitro analysis of the human metabolism of foreign substances in comparison to that of other species.",
"The system shall contribute to a replacement and completion of animal experiments in pharmacology and toxicology.",
"Particularly, the system is intended to be suitable for phase I studies of drug metabolism as well as to enable a reliable, simple and cost-effective identification of the enzymes involved in the metabolism of a candidate drug at a time point as early as possible in preclinical drug development.",
"Another technical object underlying the present invention is to provide methods for the study of pharmacological and toxicological aspects of hCYP2D6 polymorphism.",
"Preferably, these methods are contemplated for a use in the preclinical phase of drug development, they shall be carried out under standardized and reproducible conditions and are intended to have a high predictive value for humans.",
"Preferably, these methods are intended to replace animal experiments in this phase.",
"A method for the determination of the cellular content of cytochrome P450 by means of CO difference spectra is well-known.",
"According to this method, 1010 cells are required to record CO difference spectra in a cellular system.",
"This amount of cells can only be achieved with relatively high effort using special cell culture techniques such as cultivation on microcarriers while with an extinction.difference between 450 nm and 490 nm of about 0.001 the resulting spectra are still unsatisfactory (Onderwater et al., 1996).",
"Therefore, the amount of heterologously expressed cytochrome P450 has been often estimated by means of Western analyses (e.g.",
"Wolfel et al., 1991; Schneider et al., 1996).",
"This method, however, can only detect the content of cytochrome apoprotein while it is important to determine the holoenzyme as the functionally active cytochrome P450 including the prosthetic heme group.",
"Thus, another object underlying the present invention is to provide a method for the simple, sensitive and exact quantification of the cytochrome P450 content, particularly in cellular expression systems.",
"These objects are solved by the subject-matter of the claims.",
"In its first aspect, the present invention relates to a test system comprising cell lines each expressing different functional human cytochrome P450 2D6 alleles in a heterologous manner.",
"The test system contains three or more of said cell lines.",
"The cytochrome P450 2D6 alleles expressed may be selected with respect to the frequency of their presence in a population to be tested.",
"For example, if the most frequent alleles in a population are hCYP2D6 alleles *1, *10, and *17, the test system according to the present invention contains three cell lines each expressing one of said alleles.",
"In a preferred embodiment, the five hCYP2D6 alleles *1, *2, *9, *10, and *17 encoding functional enzymes will be heterologously expressed in a cellular system.",
"A preferred cell for expression generally lacks any cytochrome P450 activity.",
"The level of expression and the enzyme kinetic characteristics of the system expressing recombinant hCYP2D6 according to the present invention preferably are similar to both the physiological situation and to comparable expression systems.",
"A preferred expression system are eukaryotic cells, in particular mammalian cells, and most conveniently fibroblast cells.",
"In one embodiment the cells are derived from Chinese hamster and preferably are Chinese hamster lung fibroblasts or cells derived therefrom, particularly V79 cells.",
"In a preferred embodiment, cDNA is expressed.",
"Preferably, the test system according to the present invention is suitable for a comprehensive in vitro analysis of the human cytochrome P450 2D6 polymorphism.",
"In a particularly preferred embodiment, the test system enables testing and comparing the properties of the five known functional forms of human cytochrome P450 2D6 under standardized experimental conditions.",
"According to the present invention it is preferred to employ parental and mock-transfected cell lines, respectively, as the negative controls.",
"In the fifties, the cell line V79 was estasblished from morphologically and neoplastically transformed lung fibroblasts of an adult male Chinese hamster.",
"The establishment of the V79 cell line has not been published.",
"From the description of the experimental series for establishing cell lines of Chinese hamster lung tissue it is possible to infer the establishment of the V79 cell line by analogy (Ford and Yerganian, 1958).",
"Since then it has been widely used in toxicity and mutagenicity studies (Bradley et al., 1981; Chu and Malling, 1968; Doehmer, 1993; Sawada and Kamatki, 1998; Swierenga et al., 1991), and has been certified according to OECD Guidelines for the Testing of Chemicals.",
"The V79 cell lines employed in the test system according to the present invention may be already established V79 cell lines.",
"Some of the known V79 cell lines, however, show marked differences among each other.",
"Subclone V79MZ is particularly preferred.",
"In contrast to other V79 cells, this subclone grows adherent to a substrate and and is not released.",
"Furthermore, in contrast to V79NH cells, for example, this subclone has no acetyltransferase activity which might affect the measurements conducted with the test system according to the present invention.",
"Therefore, the test system according to the present invention in V79MZ cells bears significant advantages.",
"Preferred cytpchrome P450 2D6 expressing cells according to present invention therefore are cell lines V79MZh2D6*1, V79MZh2D6*2, V79MZh2D6*9, V79MZh2D6*10 and V79MZh2D6*17 deposited on Feb. 15, 2000, at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession numbers DSM ACC2446, DSM ACC2447, DSM ACC2448, DSM ACC2449 and DSM ACC2450.By providing the hCYP2C expressing test system of the present invention, there has been practically provided the completely human phase I drug metabolism in the form of an in vitro test system.",
"The cell lines according to the present invention are suitable for the identification of metabolically competent cytochrome P450 isoforms for a given substrate, for the detection of metabolite profiles and substrate binding mechanisms, for an examination of enzyme kinetics and drug interactions as well as for cytotoxicity and genotoxicity studies.",
"The test system according to the present invention in eukaryotic cells is suitable for metabolic studies of functional human cytochrome P450 2D6 isoforms.",
"In addition, it avoids the ethical problems associated with the use of organ material of human or animal origin.",
"Moreover, the test system has the advantage that the experimental conditions are exactly defined and may be standardized which cannot be achieved in the case of in vivo systems or native tissue samples due to the extraordinary complexity and inter-individual variability of these materials.",
"Thus, in complex systems the identification of metabolically competent P450cytochromes can only be performed in an indirect manner, for example by means of inhibition by inhibitory antibodies or by using chemicals with inhibitory effects following an induction of the isoform to be studied by chemicals such as dexamethasone, phenobarbital or dioxin.",
"An indirect identification has only an indicative value since inhibitory antibodies rarely achieve the complete inhibition or have an insufficient specificity.",
"Chemicals with inhibitory effects are not specific for cytochrome P450 at all.",
"Inducing chemicals may be misleading since a slight stimulation of other P450 cytochromes may mask the involvement of the cytochrome P450 which is induced in the first place.",
"In contrast, the heterologous expression according to the present invention enables a direct and unambiguous assignation of cytochrome P450 isoforms and metabolites.",
"The test system according to the present invention has the advantage in contrast to studies using purified enzyme that the cells may be used without laborious purification steps.",
"In addition, the system according to the present invention avoids an alteration of the substrate specificity of the cytochrome P450 2D6 forms tested or a contamination of the enzymes to be tested during a purification.",
"Moreover, the system in mammalian cells has the advantage as compared to e.g.",
"yeast cells that it provides relevant and important biological and metabolic endpoints as well as a metabolic competence which are comparable to those of an animal or of man.",
"Particularly, the system according to the present invention in V79 cells has the advantage that these cells provide a plurality of toxicological endpoints with a low and stable background and therefore are especially suitable for mutagenicity and toxicity studies (Bradley et al., 1981).",
"In contrast to all other mammalian cells including human cell lines V79 cells are particularly characterized by an extraordinarily stable pseudodiploid karyotype having a constant chromosome number which can be maintained also after transfection with foreign DNA (Doepker et al., 1998; Simi et al., 1999).",
"This is important regarding cytogenetic target points.",
"Furthermore, the stability of cells is important for a reliable examination according to the methods of the present invention.",
"With less than 12 h the doubling time of V79 cells is the shortest compared to all other cell lines studied up to now.",
"Most importantly, V79 cells do not express any endogenous cytochrome P450 (Kiefer and Wiebel, 1989; Onderwater et al., 1996).",
"Particularly for cell line V79MZ it was shown that no endogenous cytochrome P450 is expressed, and thus the cells are exactly defined for the cytochrome P450 isoenzyme transfected.",
"Moreover, it has been shown for cell line V79MZ that the cells exhibit adherent growth and show a stable phenotype in culture.",
"Furthermore, endogenous heme as well as cytochrome b5 are synthesized in sufficient amounts in V79 cells (Onderwater et al., 1996).",
"In contrast, in high-expressing systems such as baculovirus infected insect cells the endogenous heme synthesis is insufficient for a saturation of the expressed cytochrome with prosthetic heme (Asseffa et al., 1989; Barnes et al., 1994; Buters et al., 1994; Paine et al., 1996).",
"In the system according to the present invention using V79 cells a supplementation with or coexpression of cytochrome P450 NADPH oxido-reductase is necessary only in special cases (Schneider et al., 1996).",
"According to the present invention, however, there are also comprised test systems containing cell lines which coexpress functional forms of human cytochrome P450 2D6 and a cytochrome P450 NADPH oxido-reductase and/or cytochrome b5, preferably of human origin.",
"The system according to the present invention has the further advantage that it does not require adaption of the nucleic acid sequences, particularly cDNA sequences, as in the case of expression in E. coli (Sengstag et al., 1994).",
"Moreover, suitable intracellular membrane systems for an incorporation of cytochrome P450 are present so that no subsequent reconstitution is required (Gillam et al., 1995).",
"In contrast to yeast cells, for example, V79 cells are also permeable for many substrates.",
"Therefore, it is also possible to perform metabolic studies by utilizing the cell's own metabolism, e.g.",
"for the regeneration of NADPH, directly in cell culture.",
"According to the present invention there is provided a kit containing the test system of the present invention of cell lines expressing human cytochrome P450 2D6.The kit according to the invention may contain the cell lines expressing human cytochrome P450 2D6 according to the present invention and/or a lysate and/or a microsomal fraction thereof and/or a fraction enriched in human cytochrome P450 2D6 and/or purified human cytochrome P450 2D6.As further components, the kit according to the invention may comprise e.g.",
"growth media, control substances and cells, liver extract, liver homogenate and/or liver microsomes and/or metabolically active cells such as primary hepatocyte cultures and/or metabolically active enzymes such as cytochrome P450, etc.",
"In the following there will be described the different possibilities of use of the test system containing human cytochrome P450 2D6 expressing cells according to the present invention.",
"The methods of the present invention are either carried out with whole cells, a lysate, or a microsomal fraction thereof, a fraction enriched in cytochrome P450 2D6, or with cytochrome P450 2D6 purified from cytochrome P450 2D6 expressing cells according to the present invention.",
"In one embodiment, the cells of the present invention are contemplated for a use in combination with liver extract, homogenate and/or microsomes and/or metabolically active cells, such as primary hepatocyte cultures and/or metabolically active enzymes such as other P450 cytochromes, carboxylesterases, exoxide hydrolases, flavin containing monooxigenases, N-acetyltransferases, sulfotransferases, glutathione-S-transferase, methyltransferases, monoamino and diamino oxidases, etc.",
"In addition, the cells may be used in a combination with electrodes for cultivation on silicon or similar materials for a direct coupling of enzyme and data media and may be employed as metabolically competent Bio-Chips in pharmacology and toxicology.",
"The test system according to the present invention of human cytochrome P450 2D6 expressing cell lines may be employed in the study of gene-dependent toxicity of certain metabolites such as drugs.",
"Furthermore, the system of the present invention enables the examination and determination of the metabolic activation of cancerogenous substances and thus enables the determination of the cancerogenous effect of specific compounds, particularly depending on the form of human cytochrome P450 2D6 expressed.",
"Thus, the present invention relates to a method for the identification of mutagenic, cancerogenous, or toxic effects of substances wherein the cell lines expressing human cytochrome P450 2D6 according to the present invention are contacted with the substance to be tested.",
"For example, a mutagenic effect may be detected by a measurement of cells resistant to certain cell toxins, such as antibiotics, or cells with altered metabolism such as the appearance of a hypoxanthine guanine phosphoribosyl transferase (HGPRT) negative phenotype.",
"A toxic effect of the substance to be tested may be for example detected by a decrease in viability of the cell lines of the invention.",
"A cancerogenous effect may be examined by the appearance of a malign proliferation phenotype of the cell lines of the present invention, such as unlimited division, or due to the capability of the cell lines of the present invention to generate tumors in test animals.",
"Thereby, the test system according to the present invention provides a high experimental sensitivity and enables the examination of the metabolic functions of different polymorphic forms of functional human cytochrome P450 2D6.The suitability of the system of cell lines according to the present invention as an analytical tool in preclinical drug development has been demonstrated according to the invention using the pharmacologically and toxicologically important 4-hydroxylation of the breast cancer therapeutic tamoxifen as an example.",
"The correspondence of the catalytic properties of the novel polymorphic cell lines with the results of earlier in vitro and in vivo studies demonstrates the suitability of the novel cell battery for in vitro analysis of hCYP2D6 polymorphism.",
"Using the cell lines according to the present invention it will be possible for the first time to perform comparative studies of enzyme kinetics for the different polymorphic human hCYP2D6 forms.",
"The cell lines of the invention are particularly suitable for the identification of cytochrome P450 isoforms involved in complex metabolic situations and for the determination of their metabolic profiles because they are exactly defined for individual isoforms and allow for working under standardized, reproducible conditions.",
"In addition, in accordance with the results obtained from the hCYP2D6 specific hydroxylation of bufuralol it could be demonstrated according to the present invention that carriers of alleles hCYP2D6*17 and particularly of hCYP2D6*10 developed markedly lower plasma levels of 4-hydroxy-tamoxifen than carriers of the other functional alleles.",
"Because 4-hydroxy tamoxifen is at least 100 times more potent as an anti-estrogenic substance than tamoxifen itself, individual differences in the formation of this active metabolite may have a substantial clinical impact on the therapeutic effect.",
"Thus, another aspect of the present invention relates to a method of preclinical drug development using the test system according to the present invention.",
"For this purpose, in vitro studies shall indicate hCYP2D6 polymorphism-dependent differences in the in vivo metabolism of different compounds including drugs such as tamoxifen.",
"Presumably, marked inter-individual differences in metabolic processing which lead to the formation of pharmacologically less active or more potent metabolites such as the formation of 4-hydroxy tamoxifen from tamoxifen must be considered in establishing the therapeutical dose.",
"Furthermore, the test system according to the present invention enables the identification of individuals who belong to a risk group due to the metabolic activity or deficiency of the specifically expressed forms of cytochrome P450 2D6.Such individuals are for example characterized by a metabolic conversion of xenobiotics into toxic, mutagenic, or cancerogenous forms or by a lack of detoxification of drugs or other xenobiotics.",
"To measure the 4-hydroxylation of tamoxifen, the cells of the test system according to the present invention or e.g.",
"a homogenate thereof are reacted with tamoxifen.",
"The reaction product, 4-hydroxy-tamoxifen, may then be determined using well-known methods.",
"Differences in the amount of reaction product between the different cell lines of the test system of the present invention demonstrate whether tamoxifen is metabolized, poorly metabolized or not metabolized at all by the different forms of human cytochrome P450 2D6.According to the present invention there is provided a method for drug screening using the test system according to the present invention of cytochrome P450 2D6 expressing cell lines.",
"By using the method it will be possible to find substances which are metabolized or not metabolized by the various forms or by specific forms of human cytochrome P450 2D6.For this purpose, a substance to be tested is contacted successively with the different cell lines of the test system of the present invention, and a metabolic product is measured.",
"The presence of a metabolic product indicates that the respective form of human cytochrome P450 2D6 is capable of metabolizing the substance.",
"Thus, with this method according to the present invention it is possible to find new drugs which are derivatives of already known compounds and keep a pharmacological effect while being less metabolized or not metabolized at all.",
"Moreover, this method enables the discovery of substances which show better metabolization and thereby ensure a faster detoxification of the body.",
"For example, different modified forms of tamoxifen which preferably have a pharmacological activity may be contacted with the cells of the test system according to the present invention or e.g.",
"a homogenate thereof and may be reacted therewith.",
"The amount of specific metabolites being the reaction products, such as a 4-hydroxylated form, indicates whether a modified form of tamoxifen is metabolized well or poorly by the different or by specific forms of human cytochrome P450 2D6.Another aspect of the present invention relates to a method for the detection of novel alleles of hCYP2D6.According to this aspect of the present invention the heterologous expression of an allele in question is carried out.",
"A preferred cell for expression generally lacks any cytochrome P450 activity.",
"The level of expression and the enzyme kinetic properties of the expression system preferably correspond to both the physiological situation and to that of similar expression systems.",
"A preferred expression system are eukaryotic cells, particularly mammalian cells, and most conveniently fibroblast cells.",
"In one embodiment the cells are derived form Chinese hamster, and preferably are lung fibroblast cells of the Chinese hamster oder derived therefrom, particularly V79 cells and more preferably the subclone V79MZ.",
"In a preferred embodiment cDNA is expressed.",
"Subsequently, the cell line expressing the allele in question is tested with respect to the metabolism of one or more compounds including drugs such as tamoxifen and compared to the metabolism of the test system according to the present invention which preferably expresses three to five of the hCYP2D6 alleles *1, *2, *9, *10 and *17.Marked differences in the formation of specific metabolites such as the formation of 4-hydroxy-tamoxifen indicates that the allele in question is a novel hCYP2D6 allele.",
"Then, the allele in question or the novel allele and the expressed gene product may be further analyzed according to known methods including a determination of the nucleic acid sequence and the encoded amino acid sequence.",
"By comparison to the already known hCYP2D6 alleles the novel allele may be further characterized and differences in nucleic acid sequence such as mutations, insertions, or deletions, or in the amino acid sequence such as substitutions, insertions, or deletions may be determined.",
"Another aspect of the present invention relates to a method for the simple and exact quantification of the cytochrome P450 content, particularly of the hCYP2D6 content by means of CO difference spectra.",
"By solubilization of cytochrome P450 with the non-ionic detergent emulgen 913 and subsequent centrifugation to minimize the turbidity of the solubilisate the sensitivity of the measuring procedure is increased by a factor of 3000 as compared to known measuring procedures on the basis of CO difference spectra.",
"In a preferred embodiment the quantification of the cytochrome P450 content is carried out in a cellular expression system such as the expression system according to the present invention.",
"The method of quantification of the present invention preferably enables a direct comparison of polymorphic forms of hCYP2D6.Using the method according to the present invention it will be possible to take CO difference spectra in a cellular system while 100 times less cells than before may be used.",
"The exact quantification of cytochrome P450 using the method of the present invention enables a comparison of orthologous or polymorphic isoforms under defined conditions.",
"Preferably, the method according to the present invention comprises the following steps: (a) preparation of cell homogenate; (b) addition of emulgen 913 to the cell homogenate; (c) removing insoluble material; (d) determination of the reduced spectrum; (e) saturation with carbon monoxide; (f) measurement of the CO/reduced spectrum; (g) evaluation of the cytochrome P450 content by means of the spectra.",
"From the two spectra obtained the CO/reduced versus the reduced spectrum (CO difference spectrum) is evaluated as in step (g), and the concentration of cytochrome P450 and cytochrome P420 is derived therefrom.",
"Preparation of the cell homogenate is preferably performed by shock freezing and disrupting the cells in liquid nitrogen.",
"Preferably, protease inhibitors such as PMSF are added to the cell homogenate.",
"It is preferred to carry out the addition of emulgen 913 in step (b) together with a buffer such as 100 mM sodium hydrogenphosphate, pH 7.4, 10% (v/v) glycerol.",
"In a preferred embodiment emulgen 913 is added in a final concentration of 0.25% (w/v).",
"After addition of emulgen 213 the membrane-bound cytochrome P450 is solubilized preferably by stirring on ice, and the insoluble material in step (c) is removed by centrifugation.",
"Prior to measurement, the suspension without insoluble material may be reduced by sodium dithionite.",
"Preferably, the spectra are recorded between 400 and 500 nm, and extinction coefficients of 91 mM−1 cm−1 and 110 mM−1 cm−1 are used for the calculation of the concentrations of cytochrome P420 and cytochrome P450, respectively.",
"The abbreviations used herein have the following meanings: Besides abbreviations used according to Duden, the conventional codes for amino acids and nucleotides as well as the common abbreviations for restriction enzymes, polymerases etc.",
"were used.",
"A absorption APS ammoniumpersulfate bp base pair(s) BSA bovine serum albumine cDNA complementary deoxyribonucleic acid CMV cytomegalovirus Cyt b5 cytochrome b5 Cytc cytochrome c CYP cytochrome P450 protein, mRNA, cDNA CYP cytochrome P450 gene CYPOR NADPH-dependent cytochrome P450 oxido-reductase Da Dalton DEPC diethylpyrocarbonate DMEM Dulbecco's modified Eagle's medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate ECL enhanced chemiluminescence E. coli Escherichia coli EDTA ethylenediaminetetraacetate EM extensive metabolizer FCS fetal calf serum FITC fluoresceine isothiocyanate G418 geneticin 418 sulfate H homogenate HEPES N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid IM intermediate metabolizer kbp kilo base pairs kDa kilo Daltons KM Michaelis Menten constant LB Luria broth LMW low molecular weight LTR long terminal repeat MPSV myeloproliferative sarcoma virus MR metabolic ratio Mr relative molecular weight NADP+ nicotinamide adenine dinucleotide phosphate, oxidized NADPH+H+ nicotinamide adenine dinucleotide phosphate, reduced OD optical density P pellet PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline without Mg2+ and Ca2+ PCR polymerase chain reaction PEG polyethylene glycol pKB base exponent PMSF phenylmethylsulfonylfluoride PM poor metabolizer RSV rous sarcoma virus SDS sodiumdodecylsulfate SV40 simian virus 40 TAM tamoxifen TEMED N, N, N′, N′-tetramethylenediamine Tris tris-(hydroxymethyl)-aminomethane TRITC tetramethyl rhodamine isothiocyanate TSS transformation storage solution U unit(s) (enzyme unit(s)) UM ultrarapid metabolizer rpm rotations per minute V79 cells V79 Chinesische hamster fibroblasts V79MZ cells V79 Chinesische hamster fibroblasts, Mainz subclone V79MZCYP cells genetically engineered V79MZ cells heterologously expressing cytochrome P450 V79MZh2D6 cells genetically engineered V79MZ cells heterologously expressing human cytochrome P450 2D6 Vmax maximal reaction rate % (m/v) weight percent % (v/v) percent by volume Abbreviations of species: b bovine h human m murine r rat f fish (Stenotonus chrysops) The invention is further explained with respect to the following drawings: FIG.",
"1 shows the structure of hCYP2D6 cDNAs expressed according to the present invention.",
"The wildtype cDNA hCYP2D6*1 encodes native hCYP2D6 1 with Val374 (GenBank #g181349; SwissProt #P10635; Crespi et al, 1995; Gonzalez et al., 1988).",
"The mutations in cDNAs hCYP2D6*2, *9, *10 and *17 with respect to the wildtype cDNA hCYP2D6*1 are indicated.",
"The positions are numbered according to Kimura et al.",
"(1989).",
"FIG.",
"2 shows the structure of the original vector pSV450r2B1 (Doehmer et al., 1988).",
"The vector contains the following elements: fragment EcoR I-BamH I: from monkey virus SV40 (GenBank #J02400; Fiers et al.",
"1978); fragment BamH I-Pvu II: expression cassette which must be stably integrated into the genome of V79 cells; fragment BamH I-Bgl II: contains the early SV40 polyadenylation signal derived from SV40; fragment Bgl II-Hind III: inserted cDNA; fragment Hind lII-Pvu II: contains the early SV40 promoter derived from SV40; fragment Pvu II-EcoR I: from pBR322 (GenBank #J01749).",
"FIG.",
"3 shows the expression vector pSV450h2D6 for the expression of hCYP2D6 cDNAs: cf.",
"FIG.",
"1.Expression vector pSV450h2D6 was linearized with Sca I prior to transfection.",
"FIG.",
"4 shows expression vector pcDNA3.1Hygro(+)h2D6.The cDNA is under the control of the cytomegalovirus (CMV) promoter.",
"The hygromycin B resistance gene is regulated via the early SV40 promoter.",
"Both expression cassettes must be stably integrated into the genome of V79 cells.",
"The vector was linearized with Ssp I prior to transfection.",
"FIG.",
"5 shows the linker regions between the early SV40 promoter and the hCYP2D6 cDNA as well as between the hCYP2D6 cDNA and the early SV40 polyadenylation sequence in expression vector pSV450h2D6.The Kozak sequence is important for a high rate of translation of the mRNA (Kozak, 1987; Kozak, 1990).",
"FIG.",
"6 shows the relative positions and orientations of the primers used in the present invention with respect to plasmid pSV450h2D6.Primers 16298 and 16299 are complementary to the ends of template 16300, primers 19176 and 19177 to the ends of template 19183.Primers 18383 and 18384 are complementary to regions approx.",
"50 base pairs upstream and 30 base pairs downstream, respectively, of the polylinker of vector pcDNA3.1Hygro(+).",
"FIG.",
"7 shows a light micrograph of parental V79MZ cells using phase contrast at an enlargement of ×200 and a confluence of almost 50% (left) and 100% (right).",
"The cells grow as a flat layer adherent to the bottom and have numerous nucleoli.",
"Mitotic cells, in the ideal case about 3% of all cells, have temporary rounded shapes.",
"FIG.",
"8 illustrates the morphological alterations of V79MZ cells due to the transfection with respect to clones V79MZh2D6*9#C6 (a), V79MZf1A1#2 (b) and V79MZh2E1#13 (c) as examples in comparison to parental V79MZ cells (d) in phase contrast at an enlargement of ×200.Some of the cells were strongly enlarged, rounded and remarkably often polyploid (b and c).",
"A fraction of 2-3% of dead cells (light spots in a, b, and c) was present during the whole cultivation.",
"The doubling time was increased.",
"In some cases the confluence did not exceed approx 70% even during continuous cultivation (b and c).",
"Clones showing morphological alterations were discarded.",
"FIG.",
"9 shows an in situ immunofluorescence micrograph.",
"a) control mixture: V79MZh2D6*1-S cells show an intense color in contrast to parental V79MZ cells; b) homogenous clone V79MZh2D6*2; c) Confocal Laser Scanning Microscopy for detecting the localization of hCYP2D6 in the endoplasmic reticulum; d-f) double staining of the homogenous clone V79MZh2D6*1-hOR: hCYPOR shows a red color (d), hCYP2D6 1 is stained in green (e).",
"In double exposures of the film the two superimposed colors appear as yellow (f); g-h) control mixture for double staining: in double exposures V79MZh2D6*1-hOR cells appear yellow (i), V79MZh2D6*1 cells are red, and V79MzhOR cells are green.",
"FIG.",
"10 shows an acetonitrile gradient for the separation of tamoxifen and its metabolites.",
"The eluent A used was water, 1% acetic acid, and the eluent B was acetonitrile, 1% acetic acid.",
"FIG.",
"11 shows a Western blot of the V79MZh2D6 cell lines according to the invention.",
"5 μl of each cell homogenate corresponding to 5-15 μg of cellular protein were applied.",
"FIG.",
"12: Solubilization with emulgen 913.A.",
"CO difference spectrum of cell line V79MZh2D6*1 after solubilization in emulgen 913 compared to the negative control V79MZmockneo.",
"The characteristic peak at 450 nm was shifted to blue for about 1.8 nm by the solubilization.",
"B.",
"Western blot to confirm the quantitative solubilization of cytochrome P450.Applied were about 5 μg each of cellular protein of the solubilized cell homogenate prior to centrifugation (H), of the solubilisate after centrifugation (S), and of the pellet resuspended in the same volume (P).",
"FIG.",
"13 shows CO difference spectra of cell line V79MZh1A1 taken at 0, 5, 15, 30 and 60 min after saturation of the sample with carbon monoxide.",
"With increasing time cytochrome P450 is degraded to cytochrome P420 by reactive oxygen species.",
"FIG.",
"14: Effect of quinidine on the stability of hCYP2D6.A.",
"CO difference spectra of cell line V79MZh2D6*9 after cultivation with and without quinidine in culture medium.",
"B.",
"Western blot of solubilized cell homogenate prior to centrifugation.",
"For comparison purposes, 5 μg of cellular protein each of cells grown in culture medium without (−Q) and with quinidine (+Q) were applied.",
"FIG.",
"15 shows the hydroxylation of bufuralol.",
"A: structural formula of bufuralol.",
"(+)-bufuralol is hydroxylated at C1′, (−)-bufuralol is hydroxylated at C4.B: HPLC profile after incubation with a homogenate of cell line V79MZh2D6*1 (left) in comparison to the negative control V79MZmockneo (right).",
"The retention time was approx.",
"8 min for hydroxy-bufuralol and approx.",
"22 min for bufuralol.",
"FIG.",
"16 shows the inhibition of bufuralol hydroxylation by the hCYP2D6-specific inhibitor quinidine using hCYP2D6*1 as an example.",
"FIG.",
"17 shows the kinetics of the 1′-hydroxylation of (+)-bufuralol measured with homogenate of the cell lines V79MZh2D6*1, *2, *9, *10 and *17.FIG.",
"18 shows the structural formulas of the clinically administered Z-tamoxifen (left) and its configuration isomer E-tamoxifen (right).",
"The position of the 4-hydroxylation on Z-tamoxifen is indicated.",
"FIG.",
"19 shows an HPLC/MSD profile after incubation with homogenate of cell line V79MZh2D6*1 (left) in comparison to the negative control V79MZmockneo (right).",
"The retention times were about 5.5 min and 6.2 min for E-tamoxifen and Z-4-hydroxy-tamoxifen, respectively, about 8.4 min for tamoxifen-1,2-epoxide, and about 10.4 min for tamoxifen-N-oxide.",
"The peaks at 2.1 min and 6.8 min could not be assigned unambiguously since no appropriate standards were available.",
"FIG.",
"20 shows the relationship between the hCYP2D6-catalyzed 4-hydroxylation of tamoxifen and the concentrations of DMSO and tamoxifen.",
"The mean values and standard deviations from three independent measurements (2.5% DMSO) and the mean values of a single measurement series (10% DMSO), respectively, are given.",
"FIG.",
"21 shows the kinetics of the hCYP2D6-catalyzed 4-hydroxylation of tamoxifen measured with homogenates of cell lines V79MZh2D6*1, *2, *9, *10 and *17 using 2.5% DMSO as solubilizing agent.",
"The onset slope is linear up to the solubility limit of tamoxifen at 50 μM.",
"The slope of the regression line corresponds to Clint.",
"The mean values and standard deviations from three independent measurements are given.",
"FIG.",
"22 shows HPLC/MSD profiles after incubation with hCYP2C9*1-hOR and hCYP3A4-hOR “supersomes” as well as with homogenate of cell line V79MZh2D6*1 in comparison to the negative control V79MZmockneo.",
"FIG.",
"23 schematically shows a detail of the metabolism of Z-tamoxifen in human liver.",
"The 4-hydroxylation reaction of tamoxifen is highlighted horizontally while the different metabolites tested having modifications at the nitrogen are highlighted vertically.",
"Adapted according to (IARC, 1996).",
"FIG.",
"24 shows a 3D homology model of the binding of bufuralol and tamoxifen to the active site of hCYP2D6 according to-Lewis (1998).",
"Shown are the two substrates, the oxygen atom transferred to the substrate.",
"the water bridges stabilizing the substrate in the active site, and the characteristic ionic interaction between the protonated nitrogen of the substrate and Asp301 or Glu216, respectively, of the enzyme.",
"EXAMPLES The following Examples illustrate the present invention but should not be construed as limiting.",
"Example 1 Vector Construction for the Stable Expression of hCYP2D6 cDNAs in V79 Chinese Hamster Cells According to the present invention, three strategies were used for the stable expression of hCYP2D6 cDNA in V79 Chinese hamster cells: Cotransfection of the pSV450h2D6 expression vector and the neomycin resistance Plasmid pSV2neo (Clontech Laboratories, Inc., Palo Alto, Calif.) and selection with geneticin 418 (geneticin 418-sulfate; Calbiochem-Novabiochem Corp., La Jolla, Calif.) according to Doehmer et al.",
"(1988).",
"Transfection with plasmid pcDNA3.1Hygro(+)h2D6 and selection with hygromycin B (Boehringer Mannheim GmbH, Mannheim).",
"The cDNA and the hygromycin resistance were combined on one vector to reduce the amount of DNA necessary for transfection and to examine the effect on chromosomal integrity following transfection.",
"Furthermore, the direct coupling of cDNA and resistance gene was performed to increase the fraction of positive clones obtained after transfection.",
"Cotransfektion of expression vector pSV450h2D6 and plasmid pRc/RSVhCYPOR (Schneider et al., 1996) and selection with geneticin 418 according to Schneider et al.",
"(1996).",
"Vector pRc/RSVhCYPOR carries a neomycin resistance gene and a hCYPOR cDNA.",
"The coexpression of hCYP2D6 with hCYPOR was performed to demonstrate whether the endogenous amount of CYPOR in V79MZ cells is sufficient for maximal hCYP2D6 activity.",
"The cDNAs for the alleles hCYP2D6*1 in pBluescript SK(+) (Stratagene, Heidelberg), hCYP2D6*2 in pVL1393 (Invitrogen Corp., Carlsbad, Calif.), hCYP2D6*9 in M13mp19 (Stratagene, Heidelberg) and hCYP2D6*10A in M13mp19 were provided courtesy of Dr. U. M. Zanger (Dr. Margarete Fischer-Bosch-Institut, Stuttgart).",
"The alleles are shown in FIG.",
"1 and may be obtained also by means of standard methods.",
"A.",
"Construction of pSV450 Polylinker Vectors For a stable transfection of V79 Chinese hamster cells the expression vector pSV450 (Doehmer et al., 1988) was used.",
"To simplify the cloning of cDNAs into this vector, the pSV450 vectors were constructed with different polylinkers.",
"In a first step, the polylinkers for vectors pSV450HB and pSV450HK were generated by means of PCR (Saiki et al., 1988) according to PCR#1 and for vector pSV450HS by means of touch down PCR according to PCR#2.PCR#1 Sample: primer: 1.4 μl 16298 (25 mM), 1.4 μl 16299 (25 mM); template: 1 μl 16300 (40 μM); 1 μl dNTPs (20 mM); 0.4 μl Taq DNA polymerase (5 U/μl, QIAGEN, Hilden); 2.5 μl PCR buffer (10×); ad 25 μl with water PCR device: Gene Amp PCR System 2400 (Perkin Elmer, Norwalk Conn., USA) temperature program: 2 min 48° C., 1 min 72° C. (1×); 1 min 94° C., 1 min 55° C., 1 min 72° C. (30×); 10 min 72° C. (1×) amplificate: 84 bp PCR#2 Sample: primer: 1.4 μl 19176 (25 mM), 1.4 μl 19177 (25 mM); template: 1 μl 19183 (40 μM); 1 μl dNTPs (20 mM); 0.4 μl Taq DNA polymerase (5 U/μl, QIAGEN, Hilden); 2.5 μl PCR buffer (10×); 5 μl solution Q (5×); ad 25 μl with water PCR device: Gene Amp PCR System 2400 (Perkin Elmer, Norwalk Conn., USA) temperature program: 1 min 94° C. (1×); 1 min 94° C., 1 min 62° C.→52° C., 2 min 72° C. (10×, annealing temperature decreases in steps of 1° C.); 1 min 94° C., 1 min 52° C., 2 min 72° C. (35×); 10 min 72° C. (1×) amplificate: 114 bp Sequences of Primers and Templates: 16298: 5′-TAGACAAGCTTGGATCCATG-3′ 16299: 5′-GCTATAAGCTTAGATCTCGG-3′ 16300: 5′-TAGACAAGCTTGGATCCATGGTACCGAGCTCGAGTCGACTGCAGTTAACTC TAGATCGATGCGGCCGAGATCTAAGCTTATAGC-3′ 19176: 5′-GCATTAAGCTTAAGTCGACC-3′ 19177: 5′-CCGTATGATCACTAGTAGATC-3′ 19183: 5′-GCATTAAGCTTAAGTCGACCGGTACCGTACGCTAGCGAATTCCGGATATCG ATGGCGCGCCGCGGCCGCTCGAGCTCTAGACGCGTGGATCCAGATCTACTAGTG ATCATACGG-3′ From the original vector pSV450r2B1 (FIG.",
"2; Doehmer et al., 1988) the rCYP2B1 cDNA was cut out by Hind III and Bgl II and the respective polylinker (PCR#1 digested with Hind III and Bgl II; PCR#l digested with Hind III and BamH I; PCR#2 digested with Hind III and Bcl I) was inserted by ligation.",
"The novel vectors were named pSV450HB, pSV450HK and pSV450HS.",
"The polylinkers of vectors pSV450HB, pSV450HK and pSV450HS have the structures shown below.",
"All restriction sites mentioned are unique sites in the vectors.",
"pSV450HB: Hind III-Kpn I/Asp 718-Sac I/Ecl 136-Xho I/Ava I-Sal I-Hpa I-Xba I-Cla I-Xma III-Bgl II pSV450HK: Hind III-Bgl II-Xma III-Cla I-Xba I-Hpa I-Sal I-Xho I/Ava I-Sac I/Ecl 136-Kpn I/Asp 718 pSV450HS: Hind III-Afl II-Sal I-Age I-Kpn I/Asp 718-BspM II-EcoR V-Cla I-BssH I-Asc I-Sac I/-Xma III-Not I-Xho I/Ava I-Sac I/Ecl 136-Xba I-Mlu I-BsaB I-Bgl II-Spe I Thus, in combination with PCR which enables the addition of compatible ends to each cDNA the construction of novel pSV450 expression vectors can be performed in the future without time consuming intermediate cloning steps.",
"To ensure an optimal translation of the heterologous mRNA after transfection into V79MZ cells, the integrity of the Kozak sequence should be taken into account during cloning and this sequence should be optimized, respectively (Kozak, 1987; Kozak, 1990).",
"B.",
"Construction of the Vectors pSV450h2D6*1, *2, *9, *10 and *17 pSV450h2D6*1, *9 and *10 The cDNAs hCYP2D6*1 in pBluescript SK(+), hCYP2D6*9 in M13mp19 and hCYP2D6*10A in M13mp19 were excised from their original vectors by means of BamH I and EcoR I and subcloned into vector pIC19H (ATCC, Manassas, Va.) which previously was digested also with BamH I and EcoR I.",
"The novel vectors were named pICh2D6*1, *9 and *10.Restriction of these vectors with Hind III and Bgl II generated cDNAs with compatible ends for ligation into expression vector pSV450.The novel expression vectors were called pSV450h2D6*1, *9 and *10.According to the present invention, to confirm successful ligation of the cDNA into vector pSV450 the clones obtained after transformation of E. coli with pSV450 cDNA vectors were picked with a toothpick, resuspended in 10 μl of sterile water and subjected to the following PCR: PCR#4: Control PCR for Successful Ligation of a cDNA into Vectors pSV450, pSV450HB, pSV450HK and pSV450HS Sample: primer: 1.4 μl 20261 (25 mM), 1.4 μl 20262 (25 mM); template: 2 μl E. coli-suspension; 1 μl dNTPs (20 mM); 0.2 μl Taq DNA polymerase (5 U/μl, QIAGEN, Hilden); 2.5 μl PCR buffer (10×); ad 25 μl with water; 1 drop of silicone oil PCR device: Gene Amp PCR System 2400 (Perkin Elmer, Norwalk Conn., USA) temperature program: 3 min 94° C. (1×); 0.5 min 94° C., 1 min 56° C., 2 min 72° C. (30×); 10 min 72° C. (1×) Amplificate: If vector pSV450 contains an insert between the restriction sites for Hind III and Bgl II, the length of the amplificate will be 72 bp+insert bp.",
"If no insert is present, a fragment of 72 bp will be amplified.",
"For vectors pSV450HB, pSV450HK and pSV450HS which contain the polylinker the “72 bp fragment” is extended correspondingly.",
"Primer Sequences: 20261: 5′-TATTCCAGAAGTAGTGAGG-3′ 20262: 5′-ATCACCGAGCTGAGAAGC-3′ pSV450h2D6*2 hCYP2D6*2 cDNA was excised from vector pVL1393 (Invitrogen Corp., Carlsbad, Calif.) with Hind III and Kpn I and subcloned into vector pICh2D6*10 which previously was also digested with Hind III and Kpn I.",
"From the novel vector pICh2D6*2 the cDNA was excised again with Hind III and Bgl II and cloned into expression vector pSV450 (Doehrmer et al., 1988).",
"This vector was named pSV450h2D6*2.pSV450h2D6*17 Allele hCYP2D6*17 differs from allele hCYP2D6*2 only in the mutation C1111T.",
"This mutation is located immediately upstream of an Xho II restriction site (FIG.",
"1).",
"Therefore, the cDNA of hCYP2D6*17 could be obtained from the hCYP2D6*2-cDNA by site.",
"directed point mutagenesis: a cDNA fragment with a length of 362 bp carrying the mutation C1111T was synthesized according to PCR#3 and digested with Hind III and Sho II.",
"PCR#3 Sample: primer: 1.4 μl 16094 (25 mM), 1.4 μl 16095 (25 mM); template: 1 μl pSV450h2D6*1 (15.5 ng/μl); 1 μl dNTPs (20 mM); 0.125 μl Red Hot DNA Polymerase (5 U/μl, Advanced Biotechnologies Ltd., Surrey, England); 2.5 μl reaction buffer IV (10×); 1.5 μl magnesium chloride (25 mM); ad 25 μl with water; 1 drop of silicone oil PCR device: Genius (Techne Ltd., Duxford Cambridge, England) temperature program: 1 min 94° C. (1×); 1 min 94° C., 2 min 55° C., 3 min 72° C. (30×); 10 min 72° C. (1×) amplificate: 362 bp Primer Sequences: 16094: 5′-AGACGTGAAGCTTGCCGCCACCATGGGGCTA-3′ 16095: 5′-CAGGACGTAGAATGGATCTGGATGATGGGCAC-3′ The second cDNA fragment was obtained from plasmid pICh2D6*2 using Xho II and Bgl II.",
"In a three-component ligation, both cDNA fragments were cloned together with Hind III and Bgl II restricted expression vector pSV450.The novel vector was called pSV450h2D6*17.C.",
"Construction of Vectors pcDNA3.1Hygro(+)h2D6*1, *2, *9,*10 and *17 The cDNAs hCYP2D6*1, *2, *9, *10 and * 17 were excised from the respective pSV450h2D6 plasmid using Hind III and Bgl II and cloned into the expression vector pcDNA3.1Hygro(+) (Invitrogen Corp., Carlsbad, Calif.) digested with Hind III and BamH I.",
"The novel vectors were called pcDNA3.1Hygro(+)h2D6*1, *2, *9, *10 and *17.To confirm the successful ligation of the cDNA into vector pcDNA3.1Hygro(+) the clones obtained following transformation of E. coli with pcDNA3.1Hygro(+) cDNA vectors were picked with a toothpick, resuspended in 10 μl of sterile water and subjected to the following PCR: PCR#5: Control PCR for Successful Ligation of a cDNA into Vector pcDNA 3.1Hygro(+) Sample: primer: 1.4 μl 18383 (25 mM), 1.4 μl 18384 (25 mM); template: 2 μl E. coli suspension; 1 μl dNTPs (20 mM); 0.2 μl Taq DNA polymerase (5 U/μl, QIAGEN, Hilden); 2.5 μl PCR buffer (10×); ad 25 μl with water PCR device: Gene Amp PCR System 2400 (Perkin Elmer, Norwalk Conn., USA) temperature program: 3 min 94° C. (1×); 0.5 min 94° C., 1 min 56° C., 2 min 72° C. (30×); 10 min 72° C. (1×) amplificate: If the vector pcDNA3.1Hygro(+) contains an insert the length of the amplificate will be 200 bp+insert bp.",
"If no insert is present a fragment of 203 bp will be amplified.",
"Primer Sequences: 18383: 5′-CACTGCTTACTGGCTTATCG-3′ 18384: 5′-ACTAGAAGGCACAGTCGAGG-3′ Example 2 Transfection According to the present invention, parental V79MZ cells were transfected with recombinant expression vectors by potassium phosphate coprecipitation (Graham and Van der Eb, 1973; Parker and Stark, 1979).",
"According to the invention, V79MZ cells were cultured at 37° C., 7% CO2 and a humidity of 90% in tissue culture flasks (94/16 mm or 145/20 mm tissue culture dishes and 50 ml or 250 ml tissue culture flasks obtained from Greiner GmbH, (Frickenhausen); 24 well and 96 well tissue culture microtiter plates obtained from Nunc Inc. (Naperville, Ill.)) having a special coating in DMEM culture medium with increased glucose (4.5 g/l).",
"In addition, the DMEM culture medium was supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin (“complete medium”).",
"Following transfection, geneticin 418- or hygromycin B-resistant V79MZ cell clones were cultured in complete medium with 0.5 mg geneticin 418/ml, or complete medium with 0.4 mg hygromycin B/ml, respectively.",
"For transfections, the amounts of DNA used were added with 500 μl HEPES (Gibco BRL, Eggenstein) buffered saline (137 mM sodium chloride, 6 mM dextrose, 5 mM potassium chloride, 0.7 mM sodium hydrogen phosphate, 20 mM HEPES, pH 7.0).",
"By addition of 26 μl of 2.5 M calcium chloride solution and incubation for 30 min at room temperature the DNA was coprecipitated on calcium phosphate.",
"The precipitate was added dropwise to a culture of 1.5-2×106 parental V79MZ cells in a 145/20 mm tissue culture dish.",
"After careful mixing and incubation for 4 hours at 37° C., the cells were incubated for 2 min with 15% (v/v) glycerol in complete medium to increase the effectiveness of DNA uptake.",
"Subsequently, the glycerol was removed by aspiration of the medium and washing twice with 7 ml each of complete medium.",
"Since the cells have to pass through the cell cycle for stable integration of foreign DNA into their genome they were first incubated for about 36 h in complete medium without G418 or hygromycin B.",
"Afterwards, the cells were carefully trypsinized, suspended in 1 mg of 1 mg/ml G418 complete medium or 0.4 mg/ml hygromycin B complete medium, respectively, and distributed on three 96 well tissue culture microtiter plates.",
"Only cells which had taken up a resistance gene against G418 or hygromycin, respectively, during transfection with the vectors were able to survive (Mulligan and Berg, 1981).",
"After 10-14 days the resistant cell clones could be observed.",
"Individual clones were trypsinized directly in the well and half of the cells were removed for in situ immunofluorescence.",
"The clonality and stability was confirmed by repeated subcloning and passaging (the majority of the trypsinized cells was discarded or transferred to new culture flasks) of the novel cell lines as well as by repeated in situ immunofluorescence and determination of the enzymatic activity.",
"The different transfection samples T1-T14 performed according to the present invention as well as the cell lines are summarized in Table 2.Construction of Cell Lines V79MZh2D6*1, *2, *9, *10 and *17: 30 μg of Sca I linearized pSV450h2D6*1, *2, *9, *10 or *17 DNA and 1 μg of EcoR I linearized pSV2neo DNA were transfected.",
"In transfections of resistance vector pSV2neo, the expression vector pSV450h2D6 was used in a 30fold excess to increase the probability of obtaining a geneticin 418-resistant and at the same time hCYP2D6-expressing clone (T1-T5).",
"30-50 resistant clones were obtained per sample.",
"On average, one of 50 clones showed a homogenous expression of hCYP2D6 while about half of the clones were heterogenous in the in situ immunofluorescence and the rest did not express hCYP2D6 at all.",
"Cell lines V79MZh2D6*1, V79MZh2D6*2, V79MZh2D6*9, V79MZh2D6*10 and V79MZh2D6*17 were deposited on Feb. 15, 2000, at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession numbers DSM ACC2446, DSM ACC2447, DSM ACC2448, DSM ACC2449 and DSM ACC2450.Construction of Cell Lines V79MZh2D6*1-H, *2-H, *9-H, *10-H and *17-H: 3 μg of Ssp I linearized pcDNA3.1Hygro(+)h2D6*1, *2, *9, *10 or *17 DNA was transfected.",
"The combination of the cDNA and the resistance gene on a single vector enabled a transfection with only 3 μg of DNA (T6-T10).",
"10-30 resistant clones were obtained per sample.",
"On average, one of 10 clones showed a homogenous expression of hCYP2D6 while about two thirds of the clones were heterogenous in the in situ immunofluorescence, the rest did not express hCYP2D6 at all.",
"Thus, the amount of V79MZ clones showing a homogenous expression of hCYP2D6 could be enhanced by 5fold as compared to the cotransfection of pSV450h2D6 and pSV2neo.",
"This is important for the present method because the identification of clones showing a homogenous expression of the cDNA is the time-limiting step in the construction of novel cell lines.",
"Construction of Cell Line V79MZh2D6*1-hOR: 30 μg of Sca I linearized pSV450h2D6*1 DNA and 1 μg of Sca I linearized pRc/RSV-hCYPOR DNA were transfected.",
"In comparison to cell line V79MZh2D6*1 (T1) the coexpression of hCYP2D6*1 and hCYPOR (T11) should show whether the CYPOR content of V79MZ cells is sufficient for maximal hCYP2D6 activity.",
"As in the case of transfection samples T1-T5, the expression vector pSV450h2D6*1 was employed in a 30fold excess over resistance vector pRc/RSV-hCYPOR.",
"With this approach, 34 resistant clones were obtained one of which showed a homogenous expression of both hCYP2D6*1 and hCYPOR.",
"Construction of the Mock-transfected Cell Lines V79MZmockneo: The following transfections were carried out: a) 30 μg of EcoR I linearized pSV2neo DNA b) 1 μg of EcoR I linearized pSV2neo DNA c) 1 μg of EcoR I linearized pSV2neo DNA and 30 μg of Sca I linearized pSV450HB DNA Depending on the amount of DNA transfected, the chromosomal integrity of the recipient cell may be disturbed and chromosomal aberrations may be caused by recombination (Bradwell, 1989).",
"Therefore, in the context of the construction of mock-transfected V79MZ cells the transfection was performed with various amounts of DNA.",
"The karyotype of the resulting cell clones was characterized.",
"If the transfection was carried out with only 1 μg pSV2neo (T13) as well as with 1 μg pSV2neo and 30 μg pSV450HB (T14) the number of clones counted was the same as in samples (T1-T5).",
"Thus, the addition of pSV450HB as a “carrier DNA” (Graham and Van der Eb, 1973; Strain and Wylie, 1984) had no effect on the number of geneticin 418-resistant clones.",
"About 120 clones were obtained with 30 μg pSV2neo DNA, i.e.",
"a 30times higher amount of DNA yielded only 3-4times more clones.",
"Among 3×6 randomly selected clones, one clone with altered morphology was identified in the microscope.",
"A similar ratio was also observed for the other transfections.",
"Morphologically altered clones (FIG.",
"8) were excluded from further characterizations and were discarded.",
"The cell line V79MZmockneo130 was used as a negative control according to the present invention in addition to the parental V79 cells.",
"In the following it will be referred to as “V79MZmockneo”.",
"TABLE 2 Summary of transfection samples and novel cell lines.",
"Heterologous Expression Resistance Cell line expression Res.",
"vector (μg) vector (μg) T1 V79MZh2D6*1 hCYP2D6*1 G418 pSV450h2D6*1 (30) pSV2neo (1) T2 V79MZh2D6*2 hCYP2D6*2 G418 pSV450h2D6*2 (30) pSV2neo (1) T3 V79MZh2D6*9 hCYP2D6*9 G418 pSV450h2D6*9 (30) pSV2neo (1) T4 V79MZh2D6*10 hCYP2D6*10 G418 pSV450h2D6*10 (30) pSV2neo (1) T5 V79MZh2D6*17 hCYP2D6*17 G418 pSV450h2D6*17 (30) pSV2neo (1) T6 V79MZh2D6*1-H hCYP2D6*1 HyB pcDNA3.1Hygro(+)h2D6*1 (3) T7 V79MZh2D6*2-H hCYP2D6*2 HyB pcDNA3.1Hygro(+)h2D6*2 (3) T8 V79MZh2D6*9-H hCYP2D6*9 HyB pcDNA3.1Hygro(+)h2D6*9 (3) T9 V79MZh2D6*10-H hCYP2D6*10 HyB pcDNA3.1Hygro(+)h2D6*10 (3) T10 V79MZh2D6*17-H hCYP2D6*17 HyB pcDNA3.1Hygro(+)h2D6*17 (3) T11 V79MZh2D6*1-hOR hCYP2D6*1 G418 pSV450h2D6*1 (30) pRc/RSV hCYPOR -hCYPOR (1) T12 V79MZmockneo30 G418 pSV2neo (30) T13 V79MZmockneo1 G418 pSV2neo (1) T14 V79MZmockneo130 G418 pSV450HB (30) pSV2neo (1) Abbreviations: Res, resistance; G418, geneticin 418; HyB, hygromycin B Example 3 Characterization of the Novel Cell Lines In Situ Immunofluorescence For the detection of the heterologous expression of hCYP2D6 and/or hCYPOR the clones obtained after transfection were characterized by in situ immunofluorescence (FIG.",
"9).",
"Only homogenous clones in which all cells were stained homogenously and intensely were subjected to further cultivation.",
"Other criteria for the selection were a structured stain showing the subcellular localization of hCYP2D6 and hCYPOR in the endoplasmic reticulum (FIG.",
"9c) as well as a characteristic darker nuclear region.",
"For the in situ immunofluorescence, 104 cells of the clones to be tested were seeded on microchamber slides (Nunc Inc., Naperville, Ill.) and cultured for 24 h. Afterwards, the chambers were removed and the cells adhered on the slides were washed with PBS (Bio Whittaker, Verviers, Belgium) and incubated with icecold methanol/acetone (1:1) for 7 min for fixation, and then dried in air.",
"The cells fixed in this manner were covered with 150 μl of primary antibody solution (polyclonal anti-hCYP2D6 antiserum 637.2 from rabbits, diluted 1:200 in complete medium, provided courteously by Dr. U. M. Zanger, Dr. Margarete Fischer-Bosch-Institut, Stuttgart), covered with polyethylene foil and incubated for 90 min at room temperature.",
"Subsequently, they were washed 3times each for 10 min with PBS, 150 μl of secondary antibody solution (FITC-coupled anti-rabbit IgG antibody from goat, 1.5 mg/ml, diluted 1:125 in complete medium, Dianova, Hamburg) was applied, covered with polyethylene foil and incubated for 1 h at room temperature in the dark.",
"After washing three times with PBS for 10 min each 100 μl of “antifading” reagent (100 mg p-phenylene diamrmoniumdichloride in 10 ml PBS and 80 ml glycerol) was applied and a cover slip was placed on top without capture of air bubbles.",
"The samples were evaluated using a fluorescence microscope (Axioplan, Carl Zeiss, Oberkochen) with a set of standard filters at an excitation range of 450-490 nm.",
"To demonstrate the subcellular localization of cytochrome P450, confocal sections were observed using a Laser Scanning Microscope LSM 4.10 (Carl Zeiss, Oberkochen) with water immersion.",
"The fluorescence was excited at 488 nm and the emission detected at 515-565 nm.",
"Accordingly, the cytochrome expressed accordingly showed a green stain.",
"For detecting the coexpression of hCYPP2D6 and hCYPOR by double staining additionally anti-hCYPOR antibody from goat was added to the primary antibody solution (final dilution 1:500 in complete medium).",
"To avoid cross reactions a mixture of TRITC-coupled anti-rabbit IgG antibody from mouse (1.5 mg/ml, 1:125 dilution in complete medium, Pierce, Rockford, Ill.) and FITC coupled anti-goat IgG antibody from mouse (1.5 mg/ml, 1:125 dilution in complete medium, Sigma, Deisenhofen) was used as secondary antibody solution.",
"Subsequently in a double stain the oxidoreductase showed a green and the cytochrome P450 showed a red stain.",
"Selection of Representative Clones The in situ immunofluorescence identified 1-6 clones of each cell line which were homogenous with respect to their cDNA expression.",
"For a detailed characterization and later use, one representative clone for each transfection sample was selected from these clones.",
"Positive criteria for the selection were an unchanged morphology and a doubling time similar compared to that of the parental cell line V79MZ.",
"Eventually, clones were preferred which showed an intermediate hCYP2D6 activity.",
"For this purpose, the specific hydroxylation of (±)-bufuralol was measured for all clones.",
"Clones with an intermediate hCYP2D6 activity were selected to minimize distortions in the allele specific activities due to the site of cDNA integration or a (rare) multiple integration since in contrast to a homologous integration the site of cDNA integration in the V79MZ genome is to a certain extend random (Schulz et al., 1987).",
"So-called “chromatin effects” and adjacent sequence regions may affect the transcription of the integrated cDNA (Butner and Lo, 1986; Jaenisch and Jahner, 1984; Wahl et al., 1984).",
"Therefore, the amount of hCYP2D6 expressed in a heterologous manner and thus the enzyme activity per mg of cellular protein is dependent on the site of integration of the cDNA.",
"Accordingly, the bufuralol hydroxylase activities of different clones of one cell line differed up to threefold.",
"In the case of transfection samples T2 (cell line V79MZh2D6*2) and T11 (cell line V79MZh2D6*1-hOR) a selection with respect to the mean activity was impossible since only one clone in each sample fulfilled all other criteria.",
"The clones selected are identical to the novel cell lines, and only these clones were subjected to a detailed characterization.",
"Detection of the cDNAs Integrated into the Genome To confirm the genomic integration of the cDNAs transfected, the genomic DNA was isolated from.cell lines V79MZh2D6*1, *2, *9, *10 and *17 (deposited on Feb. 15, 2000, at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession numbers DSM ACC2446, DSM ACC2447, DSM ACC2448, DSM ACC2449 and DSM ACC2450), the hCYP2D6 expression cassette was amplified according to PCR#6 by means of touch down PCR in the form of an amplificate of 2198 and 2299 bp, respectively, and detected by means of gel electrophoresis.",
"For the isolation of genomic DNA from V79MZ cells the V79MZh2D6 cells were grown in 94/16 mm tissue culture dishes up to a confluence of almost 100%, the medium was removed and washed twice with 5 ml PBS.",
"Afterwards, the genomic DNA was isolated using the QIAamp Blood Midi kit (QIAGEN, Hilden) according to the protocol of the manufacturer.",
"PCR#6 Sample: primers: 1.4 μl 16014 (25 mM), 1.4 μl 15889 (25 mM); template: 1 μl of genomic DNA (approx.",
"0.2 μg/μl); 1 μl dNTPs (20 mM); 0.5 μl Taq DNA polymerase (5 U/μl, QIAGEN, Hilden); 2.5 μl PCR buffer (10×); 5 μl solution Q (5×); ad 25 μl with water PCR device: Gene Amp PCR System 2400 (Perkin Elmer, Norwalk Conn., USA) temperature program: 1 min 94° C. (1×); 1 min 94° C., 1 min 62° C.→52° C., 3 min 72° C. (10×, annealing temperature decreases in steps of 1° C.); 1 min 94° C., 1 min 52° C., 3 min 72° C. (35×); 10 min 72° C. (1×) Amplificate: Depending on the hCYP2D6 cDNA inserted an amplificate having a length of 2198 or 2299 bp, respectively, is obtained.",
"Without insert (mock transfection) the amplificate has a length of about 700 bp depending on the pSV450 vector integrated.",
"Primer Sequences: 16014: 5′-CAGAGGTTTTCACCGTCATC-3′ 15889: 5′-GGAATGTCCTCTCAAGTAGA-3′ To confirm the identity of the five alleles, the amplificates were sequenced using the primers 16094 (5′-AGACGTGAAGCTTGCCGCCACCATGGGGCTA-3′), 15887 (5′-AGCTGGATGAGCTGCTAA-3′) and 15888 (5′-ATCACCAACCTGTCATCGG-3′).",
"This also served to simultaneously confirm the integrity of the transfected DNA which is extremely susceptible to mutations, particularly deletions, until it is integrated into the genome (Bradwell, 1989; Calos et al., 1983).",
"Detection of the hCYP2D6 mRNA The transcription of the integrated hCYP2D6 cDNAs was verified on the level of mRNA by means of RT-PCR#1.For the isolation of total RNA from V79MZ cells, the V79MZh2D6 cells were grown in 94/16 mm tissue culture dishes until a confluence of about 90% was observed followed by removal of the medium and washing twice with 5 ml PBS.",
"Subsequently, the total RNA was isolated using 1.5 ml of peqGOLD TriFast™ solution (peqLab Biotechnologie GmbH, Erlangen) according to the protocol of the manufacturer.",
"RT-PCR#1 The PCR was performed using the Access RT-PCR system (Promega Corp., Madison, Wis.): Sample: primers: 2 μl 16093 (25 mM), 2 μl 17606 (25 mM); template: 0.5 μl of isolated total RNA (1-2 μg/μl); 1 μl dNTPs (10 mM); AMV reverse transcriptase (5 U/μl); 1 μl Tfl DNA polymerase (5 U/μl); 10 μl AMV/Tfl reaction buffer (5×); 2 μl magnesium sulfate (25 mM); ad 50 μl with DEPC (Sigma, Deisenhofen) treated water PCR device: Uno Thermoblock (Biometra biomedizinische Analytik GmbH, Göttingen) temperature program: 50 min 48° C. (1×); 2 min 94° C. (1×); 1 min 94° C., 1 min 60° C., 2 min 70° C. (40×); 10 min 70° C. (1×) Amplificate: A 410 bp fragment of reverse transcribed hCYP2D6 mRNA is amplified.",
"Primer Sequences: 16093: 5′-CATACTGCTTCGACCAGTTGCG-3′ 17606: 5′-GCAGGTGAGGGAGGCGATCAC-3′ Western Blot After the homogenous expression von hCYP2D6 in the V79MZh2D6 cell lines according to the invention was confirmed by means of in situ immunofluorescence, the molecular weight of heterologously expressed hCYP2D6 was confirmed by Western blotting and a qualitative indication as to the relative amounts of hCYP2D6 was obtained (FIG.",
"11).",
"For this purpose, the proteins of a cell homogenate (see Example 4) separated on SDS PAGE (Laemmli, 1970) were blotted from the polyacrylamide gel onto an Immobilon P membrane (Millipore, Dreieich) (Burnette, 1981) for 30 min using a “semi dry” method at 225 mA in a “Semi Dry Elektroblotter” device (Sartorius, Göttingen).",
"For the transfer, a pile was prepared starting from the graphite anode which consisted of two pieces of Whatman 3 MM filter papers soaked in buffer C (0.03 M Tris, 20% methanol, 0.04 M 6-aminohexanoic acid, ad pH 10 with aqueous sodium hydroxide), the gel which had been previously immersed for 15 min in buffer C, the membrane which was wetted previously with methanol and then soaked for 10 min in buffer B (0.03 M Tris, 20% methanol, ad pH 10 with aqueous sodium hydroxide), two pieces of Whatman 3 MM paper soaked in buffer B and two pieces of Whatman 3 MM paper soaked in buffer A (0.3 M Tris, 20% methanol, ad pH 10 with aqueous sodium hydroxide) without inclusion of air bubbles and then was fixed by the graphite cathode.",
"For the immunodetection of hCYP2D6, the blotted membrane was blocked over night in PBS, 7% skim milk powder (“Glücksklee” trademark, Nestlé Germany AG, Frankfurt) at 4° C., washed briefly in PBS and then shaken carefully for 1 h in a solution of the primary antibody (polyclonal anti-hCYP2D6 antiserum 637.2 from rabbits, diluted 1:100 in PBS), and afterwards washed three times for 15 min in PBS, 0.5% Tween 20, incubated for 30 min in the secondary antibody solution (POD-coupled anti-rabbit IgG from goat, 0.2 U/ml, diluted 1:10000 in PBS, Boehringer Mannheim, Mannheim) and washed again three times for 15 min in PBS, 0.5% Tween 20.Afterwards, hCYP2D6 was detected via the bound secondary antibody using the ECL kit (Enhanced Chemiluminescence, Amersham, Little Chalfont, England).",
"This assay detects the light emission during the peroxidase catalyzed oxidation of luminol in the presence of hydrogen peroxide using Hyperfilm ECL (Amersham, Little Chalfont, England).",
"With 56.0±0.6 kDa and 80.3±2.8 kDa, the experimental molecular weights of hCYP2D6 and hCYPOR closely corresponded to the calculated values of 55.8 kDa and 76.7 kDa, respectively.",
"Remarkable is the relatively low amount of hCYP2D6 10; see FIG.",
"11.Example 4 CO Difference Spektra For a comparison of the different hCYP2D6 1 expressing cell lines V79MZh2D6*1, -hOR, -H and -S as well as the various allelic variants, the amounts of cytochrome P450 was determined using CO difference spectra.",
"For the preparation of a cell homogenate, the V79MZ cells were cultured in at least three 250 ml tissue culture flasks up to a confluence of 80-100%.",
"The trypsin-treated cells were combined, distributed uniformly on three 145/20 mm tissue culture dishes each per 250 ml tissue culture flask and incubated in 20 ml of complete medium without G418 or hygromycin B up to a confluence of 90%.",
"The medium was discarded, followed by rinsing twice with 5 ml icecold buffer (100 mM potassium phosphate, pH 7.4).",
"The cells were loosened from the dish by means of a rubber scraper into 4 ml of icecold buffer, combined and pelleted by centrifugation for 10 min at 1500×g and 4° C. The supernatant was completely removed, the pellet was carefully resuspended in 1 ml buffer per nine 145/20-mm tissue culture dishes, and aliquots were taken as follows: 1 ml of the cell suspension was removed for CO difference spectra (1 ml aliquot), the remainder was diluted 1:1 with buffer, resuspended, and divided into aliquots of 100-300 μl for the determination of the protein content, enzymatic activity, measurements of enzyme kinetics, and for Western blotting (diluted aliquots).",
"All aliquots were shock frozen in liquid nitrogen to disrupt the cells and afterwards stored at −80° C. until use.",
"A 1 ml aliquot of cell homogenate was thawed on ice and after addition of 20 μl 100 mM PMSF (Boehringer Mannheim GmbH, Mannheim) in isopropanol was carefully resuspended in 1 ml of solubilization buffer (100 mM sodium hydrogenphosphate, pH 7.4, 10% (v/v) glycerol, 0.5% (w/v) emulgen 913 (Kao-Atlas, Tokyo, Japan) using a pipette.",
"For the measurement of the hCYP2D6 spectra there were also added 10 μl of 2 mM quinidine (hydrochloride, Sigma, Deisenhofen).",
"Membrane-bound cytochrome was solubilized by careful stirring for 15 min on ice, and insoluble material was pelleted by centrifugation for 10 min at 17,000×g at 4° C. to reduce the turbidity (Evert et al., 1997; Tyndale et al., 1991a).",
"The supernatant was introduced into a 2 ml glas-on-glas homogenizator, reduced by adding several crystals of sodium dithionite (Sigma, Deisenhofen) followed by 15 strokes, and split up between two quartz cuvettes.",
"After recording of the reduced spectrum between 400 and 500 nm by means of an Aminco DW-2000 UV/VIS spectrophotometer (SLM Instruments Inc., Urbana, Ill.) the solution in the test cuvette was saturated with about 60 bubbles of carbon monoxide and the CO/reduced spectrum was recorded immediately (Eastabrook et al., 1972).",
"From the two spectra the CO/reduced versus the reduced spectrum (CO difference spectrum) was evaluated, and using the extinction coefficients of 91 mM−1 cm−1 and 110 mM−1 cm−1 (Omura and Sato, 1964b) the concentrations of cytochrome P450 and cytochrome P420 were calculated.",
"In the absence of solubilization and centrifugation, the turbidity of the cell homogenate was to high for the measurement of CO spectra.",
"After centrifugation without previous solubilization, all of the cytochrome P450 was found in the pellet.",
"If the method according to the present invention using the non-ionic detergent emulgen 913 was employed, however, cytochrome P450 was almost completely solubilized (FIG.",
"12B).",
"After a centrifugation to remove the turbidity was carried out cytochrome P450 could be well measured in the solubilizate.",
"A final concentration of emulgen 913 of 0.25% was found to be optimal.",
"In addition, also a repeated treatment of the resuspended pellet with emulgen 913 did not solubilize any more cytochrome P450.The solubilization was independent of the incorporation of heme which becomes clear particularly from the solubilization of hCYP2D6 9 after culturing in the presence and absence of quinidine (FIGS.",
"12B and 14A).",
"If the expression was performed in baculovirus-infected insect cells, however, the solubilization with emulgen 913 yielded a maximum of about 50% of hCYP2D6 9 (Evert et al., 1997; Paine et al., 1996).",
"The variant hCYP2D6 7 which is unable to incorporate heme because of a His324Pro mutation and therefore is not functional remained practically unsolubilized (Evert et al., 1997).",
"Also in these two cases, no dependence on exogenous heme was observed.",
"For the solubilization of baculovirus-expressed bCYPc17, however, a marked dependence on heme incorporation has been reported (Barnes et al., 1994).",
"This indicates that also other factors besides the association with heme must affect the solubilization, for example the intracellular localization of the cytochrome P450 expressed in a heterologous manner, its tendency to form aggregates, the composition of the intracellular membrane systems of the expression system or the type of binding of cytochrome P450 within the membrane.",
"Aerobic Measurement A reduction of the solubilizate with sodium dithionite under aerobic conditions may weaken and destroy the prosthetic heme (Omura and Sato, 1964b).",
"Accordingly, recording a CO spectrum at different times after gassing of the sample with carbon monoxide showed a clear decrease of the absorption at 450 nm while the peak of cytochrome P420 increased.",
"At the same time it could be demonstrated, however, that under aerobic conditions the error was neglegible if the spectra were run only a few minutes after reduction of the sample (FIG.",
"13).",
"Effect of Quinidine on the Stability of hCYP2D6 Sometimes enzymes may be stabilized by their substrates or competitive inhibitors.",
"For hCYP2D6, a stabilization by the competitive inhibitor quinidine has been discussed (Gillam et al., 1995).",
"A more or less noticeable peak of cytochrome P420 was observed in recordings of the spectra of hCYP2D6 2, 9, 10 and 17 which was absent in the spectrum of hCYP2D6 1.Therefore, quinidine was added to the solubilization buffer to avoid a possible weakening caused by the solubilization.",
"The spectra, however, were more or less unchanged and particularly a cytochrome P420 peak still appeared.",
"This means that cytochrome P450 must have been degraded already prior to solubilization and reduction.",
"For the detection, the quinidine for recording the spectra was added directly to the culture medium during the cultivation of the cells.",
"A stabilizing effect was indeed observed which varied strongly with different hCYP2D6 alleles (Table 3).",
"The spectrum of hCYP2D6 1 (wildtype) remained unchanged.",
"The amount of cytochrome P450 was the same.",
"This has been expected because the enzyme has a high stability by itself.",
"In variant hCYP2D6 2 the cytochrome P420 peak was absent.",
"The calculated amount of cytochrome P450 exactly corresponded to the sum of the amounts of cytochrome P450 and cytochrome P420 determined previously (Table 3).",
"The observations in the case of hCYP2D6 17 were similar while the cytochrome P420 peak, however, did not completely disappear.",
"As expected from the results of the Western analysis, it was difficult to quantify the CO difference spectrum of hCYP2D6 10 because of the low amount of protein.",
"No cytochrome P450 peak could be observed.",
"If the incubation was performed in the presence of quinidine the cytochrome P420 content appeared to be slightly elevated.",
"A dramatic change was observed with variant hCYP2D6 9.The cytochrome P420 peak completely disappeared, and the amount of cytochrome P450 was increased by 4-6fold (FIG.",
"14A).",
"In contrast, as with all other variants a comparative Western blot indicated only a slight if at all increase in the amount of hCYP2D6 9 apoprotein (FIG.",
"14B).",
"Thus, quinidine is not important for the stability of the apoprotein but stabilizes the prosthetic heme in the cytochrome.",
"The solubilization of the hCYP2D6 variants examined was essentially unaffected by quinidine or heme incorporation, respectively (FIG.",
"12B).",
"Cytochrome P450 Content The amounts of cytochrome P450 determined are summarized in Table 3.TABLE 3 Cytochrome P450 content of different V79MZ cell lines.",
"The values given are either obtained from a single measurement or are the mean values and standard deviations of at least three independent measurements.",
"without quinidine with quinidine (pmol/mg cellular protein) (pmol/mg cellular protein) Cell line P450 P420 P450 + P420 P450 P420 P450 + P420 V79MZmockneo 0 0 0 — — — V79MZhOR 0 0 0 — — — V79MZh1A1 11.4 ± 1.8 0 11.4 ± 1.8 — — — V79MZr1A1 6.8 ± 1.9 5.6 ± 2.7 12.4 ± 4.7 — — — V79MZm1A1 7.6 ± 1.9 2.6 ± 0.8 10.2 ± 2.0 — — — V79MZf1A1 (scup) 2.5 ± 0.8 3.5 ± 1.6 6.1 ± 2.4 — — — V79MZh2E1 5.2 ± 0.6 0.9 ± 1.0 6.1 ± 0.5 — — — V79MZh3A4-hOR 8.0 ± 1.0 3.0 ± 0.9 11.0 ± 1.7 — — — V79MZh2D6*1-hOR 41.9 0 41.9 44.0 0 44.0 V79MZh2D6*1-S 17.4 ± 1.8 0 17.4 ± 1.8 14.41 0 14.41 V79MZh2D6*1-H 20.3 0 20.3 10.83 0 10.83 V79MZh2D6*1 24.9 ± 3.2 0 24.9 ± 3.2 30.9 0 30.9 V79MZh2D6*2 10.9 ± 4.3 2.5 ± 2.2 13.4 ± 4.7 12.2 0 12.2 V79MZh2D6*9 5.4 ± 2.4 1.0 ± 0.9 6.4 ± 1.9 35.7 ± 13.3 0 35.7 ± 13.3 V79MZh2D6*10 0 2.2 ± 0.2 2.2 ± 0.2 0 4.44 4.44 V79MZh2D6*17 4.3 ± 0.7 3.8 ± 0.7 8.2 ± 1.0 9.0 1.9 10.9 Abbreviations: —: not examined No cytochrome P450 could be detected in parental and mock transfected V79MZ cells.",
"The relative amounts of total cytochrome P450+cytochrome P420 corresponded well to the relative band intensities in the Western blots (FIGS.",
"12B and 14B).",
"This indicates that the amounts of cytochrome P450+cytochrome P420 determined spectrophotometrically correspond to the amounts of apoprotein and that, therefore, the endogenous heme sysnthesis in V79MZ cells is sufficient.",
"For a comparison of different cell lines and allelic variants either the amount of functional holoenzyme of cytochrome P450, the total amount of cytochrome P450+P420, the total amount of apoprotein or the level of expression, i.e.",
"the mRNA content, may be used as a basis.",
"Depending on the basis, the comparison may yield very different results, for example due to differences in heme incorporation or protein stability.",
"In the following, the total amounts of cytochrome P450+P420 will be used as a basis.",
"The corresponding values in Table 3 are printed in bold letters.",
"For this purpose it was assumed that the amount of P420 depended substantially on the preparation since although the total amount of P420+P450 remained nearly constant the fraction of P420 varied between different preparations.",
"Comparison to Previous Measurements By using a novel method of solubilization it was possible to record a CO difference spectrum with 100times less V79 cells than had to be used before.",
"The amounts of cytochrome P450 determined in this manner were somewhat higher than those published previously for V79MZCYP cell lines (Table 4).",
"TABLE 4 Comparison of the amounts of cytochrome P450 determined by means of CO difference spectra in V79MZCYP cell lines to previous results.",
"CO spectra with solubilizate pmol CYP/mg cellular protein Cell line P450 P420 Reference V79MZ parental 0 0 0 pmol/mg (Onderwater et al., 1996) microsomal CO difference spectrum V79MZh1A1 11.4 ± 1.8 0 14 pmol/mg (Onderwater et al., 1996) microsomal CO difference spectrum V79MZh3A4-hOR 8.0 ± 1.0 3.0 ± 0.9 5 pmol/mg (Schneider et al., 1996) Western analysis of cell homogenate Example 5 Hydroxylation of Bufuralol To confirm the functionality of cytochrome P450 2D6 expressed in a heterologous manner the hCYP2D6-specific hydroxylation of bufuralol (1′-hydroxylation of (+)-bufuralol or 4-hydroxylation of (−)-bufuralol) was determined (FIG.",
"15).",
"For this purpose, a diluted aliquot of cell homogenate was thawed on ice and resuspended carefully using a pipette.",
"The reaction was started by addition of homogenate to the reaction sample (in the final sample: 100 μl total reaction volume, 150 μg total protein (V79MZh2D6*10: 300 μg), 200 μM bufuralol, 2 mM NADPH (Boehringer Mannheim GmbH, Mannheim) in 0.1 M potassium phosphate buffer, pH 7.4) and after an incubation in a water bath at 37° C. for 30 min (V79MZh2D6*10: 90 min) was stopped by addition of 12 μl 60% (v/v) perchloric acid (Boehringer Mannheim GmbH, Mannheim; approx.",
"0.33 M).",
"The samples were incubated for several minutes on ice and the precipitate was collected by centrifugation (10 min, 17,000×g, 4° C.).",
"The substrate bufuralol and the product hydroxy-bufuralol contained in the supernatant were separated by means of HPLC and detected by fluorometry (Kronbach et al., 1987; Kronbach, 1991).",
"The chromatographic separation was performed in an isocratic manner using an aqueous-organic mobile phase (30% (v/v) acetonitrile (HPLC pure, Riedel-de Haen, Seelze), 40% (v/v) methanol, 30% (v/v) water, 2 mM perchloric acid) at a flow rate of 1 ml/min and 50° C. on a Hypersil ODS C18 “reversed phase” column (24 cm×4,6 mm, particle size 5 μm; Supelco, Bellefonte, Pa.).",
"A C8 column was connected ahead of the system.",
"The fluorescence signal (excitation at 252 nm, emission at 352 mn) was detected with a Fluorescence HPLC Monitor RF-530 (Shimadzu (Europe) GmbH, Düsseldorf), recorded by Chromatopac C-R3A 530 (Shimadzu (Europe) GmbH, Düsseldorf), and the peak area was integrated automatically.",
"The retention times were about 8 min for hydroxy-bufuralol and about 22 min for bufuralol.",
"The quantification was carried out using a standard curve prepared from 1′-hydroxy-bufuralol standards in the range of 0.5-20 μM final concentrations after incubation with homogenate of the mock transfected cell line V79MZmockneo.",
"The concentrations of the stock solutions prepared gravimetrically were checked spectrophotometrically.",
"Extinction coefficients of 16.3 mM−1 cm−1 for 1′-hydroxy-bufuralol at 245.5 nm (Gentest Corp., Woburn, Mass.)",
"and 15.1 mM−1 cm−1 for bufuralol at 248 nm (Ultrafine Chemicals, London, England) were used in the calculations.",
"At 37° C. the reaction was in the linear range for 30 min up to 150 μg total protein/100 μl.",
"To obtain a measurable signal in the case of variant hCYP2D6 10, 300 μg total protein/100 μl were incubated for 90 min.",
"Therefore, the activities given for hCYP2D6 10 underestimate the real values; see Table 6.Inhibition of the Bufuralol Hydroxylation For inhibition studies, to the bufuralol hydroxylation reaction sample was added quinidine in a final concentration of 0.01-2 μM (2 mM stock solution in methanol, afterwards diluted in 0.1 M potassium phosphate buffer, pH 7.4; the assay contained <0.1% methanol; control without quinidine with 0.1% methanol) or inhibitory anti-hCYP2D6 antiserum (LKM serum 2) and human control serum (provided courteously by Dr. U. M. Zanger, Dr. Margarete Fischer-Bosch-Institut, Stuttgart) in final dilutions of 1:100-1:1000.In all hCYP2D6 variants the hydroxylation of bufuralol was nearly completely inhibited with hCYP2D6-specific antiserum in a final dilution of 1:100.At a final concentration of 1 μM of the hCYP2D6-specific inhibitor quinidine the bufuralol hydroxylation was inhibited to about 90% independently of the allele (FIG.",
"16).",
"Example 6 Conditions for Culturing and Homogenization To exclude that the endogenous heme synthesis is limiting for the amount of functional cytochrome P450, the culture medium was supplemented with hemin chloride (Sigma, Deisenhofen) or with the limiting synthesis precursor δ-aminolevulinic acid (hydrochloride, Sigma, Deisenhofen) and ferric (III) citrate (Sigma, Deisenhofen), respectively.",
"Up to the cytotoxic limit at 10 μM for hemin chloride or 10 mM for 5-aminolevulinic acid/ferric (III) citrate, no increased bufuralol hydroxylase activity per mg total protein could be detected in the cell homogenate.",
"The bufuralol hydroxylase activity in the cell homogenate, however, was dependent on the cell density at the time of harvesting the cells, from the type of homogenization, and the number of freeze/thaw cycles.",
"Therefore, the method for preparing the cell homogenate was optimized: At cell densities of more than 90%, the bufuralol hydroxylase activity per mg total protein in the cell homogenate decreased by about 10-20%.",
"In overgrown cultures with cell densities of clearly more than 100% only about half of the maximal activity was measured.",
"To obtain cell homogenate for enzmyatic reactions the cells had to be disrupted.",
"The highest activities were determined after freezing in liquid nitrogen.",
"The use of a glas-on-glas homogenizer resulted in 10-20% lower activities.",
"The loss in activity could be avoided by the addition of 1 mM PMSF as a protease inhibitor.",
"Sonication either in the absence or presence of PMSF resulted in a loss of up to 70% of the activity.",
"Repeated freezing and thawing of the cell homogenate led to non-reproducible variations in the bufuralol hydroxylase activity.",
"Optimal conditions for the preparation of cell homogenate are cell densities of 90%, followed by harvesting of the cells by centrifugation, resuspending in buffer, aliquoting, shock freezing in liquid nitrogen and storage at −80° C. until use.",
"The aliquots were thawed only once and unused residues were discarded.",
"Example 7 Coexpression of hCYP2D6 and hCYPOR and Comparison of the Promoters For some of the heterologously expressed cytochrome P450 isoforms the endogenous CYPOR synthesis in V79 cells is insufficient for maximal enzyme activity.",
"For this reason, hCYP3A4 and hCYPOR, for example, were coexpressed (Schneider et al., 1996).",
"A similar approach was followed with hCYP2D6 1 (transfection sample T11); see Table 5.To detect the hCYPOR activity, the activity of the NADPH-dependent cytochrome c reductase was measured (Kubota et al., 1977).",
"For this purpose, a diluted aliquot of cell homogenate was thawed on ice and resuspended carefully by means of a pipette.",
"After preincubation of the reaction sample for 2 min at 37° C. (final sample: 800 μl total reaction volume, 40-100 μg total protein, 225 μM potassium cyanide (Sigma, Deisenhofen) to inhibit the reoxidation of reduced cytochrome c by cytochrome oxidase, 125 μM NADPH, 45 μM ferricytochrome c (Sigma, Deisenhofen) from bovine heart in 50 mM potassium phosphate buffer, pH 7.8) the reaction was started by addition of cytochrome c and the increase in the extinction at 550 nm and 37° C. was recorded using an Uvikam 941 Plus UV/VIS-spektrophotometer (Kontron Instruments Ltd., Watford, England).",
"For the calculation of the cytochrome c reductase activity from the initial slope an extinction coefficient of 19.1 mM−1 cm−1 was used (Chance, 1957).",
"In accordance with the results of Schneider et al.",
"(1996) the endogenous cytochrome c reductase activity of V79MZ cells was 9-14 nmol/min/mg cellular protein.",
"Clearly higher cytochrome c reductase activities of 31-182 nmol/mg/min were measured if hCYPOR was coexpressed.",
"TABLE 5 Cytochrome c reductase activities of CYPOR (cytc red.)",
"and bufuralol hydroxylase activities of hCYP2D6.The selectivity is calculated by the ratio (−)-bufuralol activity/(+)-bufuralol activity.",
"The mean values and standard deviations of at least three independent measurements are given.",
"bufuralol hydroxylation bufuralol hydroxylation pmol/pmol cytc red.",
"pmol/mg/min hCYP2D6/min selectivity Cell line Promoter nmol/mg/min (+) (+/−) (−) (+) (+/−) (−) (−)/(+) V79MZmockneo — 8.8 ± 1.3 0 0 0 — — — — V79MZhOR — 124.6 ± 24.9 — — — — — — — V79MZh3A4- — 31.5 ± 8.5 — — — — — — — hOR V79MZh2D6*1- SV40 182.2 ± 8.0 292 ± 34 143 ± 6 64 ± 6 7.0 ± 0.8 3.4 ± 0.2 1.5 ± 0.2 0.22 ± 0.05 hOR V79MZh2D6*1 SV40 13.5 ± 4.2 163 ± 32 108 ± 15 65 ± 14 6.5 ± 2.1 4.3 ± 1.2 2.6 ± 0.9 0.40 ± 0.16 V79MZh2D6*1-H CMVp 14.1 ± 1.6 139 ± 27 76 ± 5 60 ± 13 6.9 ± 1.3 3.8 ± 0.3 2.9 ± 0.7 0.43 ± 0.18 V79MZh2D6*1-S MPSV- 9.9 ± 1.1 110 ± 19 57 ± 5 45 ± 1 6.3 ± 1.7 3.3 ± 0.6 2.6 ± 0.3 0.41 ± 0.08 LTR + CMVe Abbreviations: (+): (+)-bufuralol; (−): (−)-bufuralol; (+/−): racemic bufuralol; SV40: SV40 early promoter; CMVp: cytomegalovirus promoter; MPSV-LTR + CMVe: myeloproliferative sarcoma virus long terminal repeat and cytomegalovirus enhancer If a coexpression of hCYP2D6 1 and hCYPOR was performed, markedly higher bufuralol hydroxylase activities were measured as in the case of a heterologous expression of hCYP2D6 1 alone (Table 5).",
"After normalization of the amounts of hCYP2D6 (see Table 4) nearly identical reaction rates were obtained for all hCYP2D6 1-expressing cell lines independently of the coexpression of hCYPOR.",
"Thus, in contrast to hCYP3A4 the activity of hCYP2D6 is not limited by the endogenous CYPOR activity.",
"The differences in activity between the cell lines V79MZh2D6*1-hOR and V79MZh2D6*1 therefore are due to different integration of the cDNA expression cassette into the V79MZ genome.",
"Furthermore, a comparison to the activities of cell lines V79MZh2D6*1-H and V79MZh2D6*1-S showed that the “integration effect” clearly has a greater influence on the differences in activity between different cell lines or homogenous clones of a transfection sample, respectively, than the promoter selected.",
"Particularly in the V79 system comparable expression rates are obtained with the SV40 promoter and the CMV promoter while in COS-1 cells 10fold higher expression rates are obtained with the CMV promoter (Clark and Waterman, 1991).",
"Similar results were obtained by Schneider et al.",
"(1996).",
"The good agreement of the reaction rates further demonstrates the reliability of both the bufuralol hydroxylase activity test and the quantification of the amount of cytochrome P450 by means of CO difference spectra of solubilized cell homogenate.",
"Example 8 Comparison of Cell Lines V79MZh2D6*1, *2, *9, *10 and *17 Bufuralol Hydroxylase Activity The polymorphic cell lines V79MZh2D6*1, *2, *9, *10 and *17 were compared with respect to the hCYP2D6-specific bufuralol hydroxylation.",
"While the selectivity towards (+)-bufuralol was practically identical for all cell homogenates, the activities were decreased compared to the wildtype cell homogenate V79MZh2D6*1 (Table 6).",
"Particularly the V79MZh2D6*10 cell homogenate exibited only about 2% of the activity of wildtype cell homogenate.",
"After normalization of the amounts of hCYP2D6 (see Table 4) comparable reaction rates were obtained for hCYP2D6 1 and hCYP2D6 2 while the reaction rate of hCYP2D6 9 was about twice as high and the reaction rate of hCYP2D6 17 was about half as high.",
"TABLE 6 Bufuralol hydroxylase activities of the polymorphic cell lines at 200 μM bufuralol.",
"The selectivity is the ratio of (−)-bufuralol activity/(+)-bufuralol activity.",
"The mean values and standard deviations of at least three independent meansurements are given.",
"Bufuralol hydroxylase activity bufuralol hydroxylase reaction rate pmol/mg/min pmol/pmol hCYP2D6/min selectivity Cell line (+) (+/−) (−) (+) (+/−) (−) (−)/(+) V79MZmockneo 0 0 0 — — — — V79MZh2D6*1 162.7 ± 31.6 108.0 ± 15.3 65.0 ± 13.5 6.53 ± 2.11 4.34 ± 1.17 2.61 ± 0.88 0.40 ± 0.16 V79MZh2D6*2 95.2 ± 28.6 59.7 ± 13.9 43.8 ± 14.7 7.11 ± 4.63 4.46 ± 2.60 3.27 ± 2.24 0.46 ± 0.29 V79MZh2D6*9 93.9 ± 11.5 61.8 ± 4.7 45.9 ± 3.1 14.67 ± 6.2 9.66 ± 3.60 7.17 ± 2.61 0.49 ± 0.16 V79MZh2D6*10 3.3 ± 0.4 2.3 ± 0.2 1.4 ± 0.3 1.48 ± 0.29 1.03 ± 0.17 0.63 ± 0.18 0.42 ± 0.14 V79MZh2D6*17 29.3 ± 1.9 21.6 ± 2.4 14.4 ± 3.3 3.57 ± 0.66 2.65 ± 0.61 1.77 ± 0.62 0.49 ± 0.14 Abbreviations: (+): (+)-bufuralol; (−): (−)-bufuralol; (+/−): racemic bufuralol Kinetic of the 1 -hydroxylation of (+)-bufuralol The allele specific kinetic parameters of the 1′-hydroxylation of (+)-bufuralol were determined assuming a monophasic Michaelis-Menten kinetic without inhibitor (V=Vmax* [S]/(KM+[S]) (Table 7).",
"The non-linear fitting of the curve to measuring points V was done by minimizing the sum of the error squares and weighting with a factor of 1/V (FIG.",
"17).",
"The mean values and standard deviations of three independent measurements were used.",
"TABLE 7 Kinetic parameters of the 1′-hydroxylation of (+)-bufuralol for hCYP2D6 1, 2, 9, 10 and 17.The mean values and standard deviations of three independent meansurements are given.",
"Based on mg of total protein Based on the amount of cytochrome P450 Vmax KM Clint reaction rate Clint Cell line pmol/mg/min μM ml/mg/min pmol/pmol P450/min ml/pmol P450/min V79MZh2D6*1 170.8 ± 15.5 13.8 ± 1.6 12400 ± 2580 6.9 ± 1.5 500 ± 170 V79MZh2D6*2 111.5 ± 2.1 22.6 ± 0.6 4940 ± 220 8.3 ± 3.1 370 ± 150 V79MZh2D6*9 100.0 ± 15.0 15.0 ± 2.9 6670 ± 1970 15.6 ± 7.0 1040 ± 670 V79MZh2D6*10 4.3 ± 0.1 53.3 ± 10.3 80 ± 17 1.9 ± 0.2 40 ± 10 V79MZh2D6*17 34.3 ± 2.8 28.2 ± 1.3 1220 ± 160 4.2 ± 0.9 150 ± 40 Abbreviations: Vmax: maximal reaction rate at substrate saturation of the enzyme; KM: Michaelis-Menten constant; Clint: intrinsic clearance (Clint = Vmax/KM) Example 9 Comparison to Other Expression Systems hCYP2D6 2 (Substitutions Arg296Cys and Ser486Thr) A slightly elevated KM value and a comparable reaction rate as compared to the wildtype enzyme hCYP2D6 1 were determined for V79MZh2D6*2 homogenate.",
"In contrast to wildtype enzyme, the CO difference spectrum shows low amounts of cytochrome P420.If the cDNA expression was carried out in COS-1 cells the absolute bufuralol hydroxylase activity of hCYP2D6 2 compared to the wildtype was only about 60% at a similar reaction rate which is in complete agreement with the V79 expression system (Oscarson et al., 1997).",
"It remained unclear whether this was just coincidence or whether an allele-dependent reason exisits.",
"If hCYP2D6 1 and hCYP2D6 2 are expressed in yeast (Oscarson et al., 1997) the (+)-bufuralol reaction rate at substrate saturation was similar and the amount of holoprotein of hCYP2D6 2 was slightly decreased.",
"It is discussed that this may be due to a decreased protein stability or also translation rate.",
"In addition, also the peak of cytochrome P420 in the CO difference spectrum of V79MZh2D6*2 indicates a decreased protein stability compared to the wild type enzyme.",
"hCYP2D6 9 (Deletion of Lys281) An identical KM value and a twice as high reaction rate compared to the wildtype enzyme hCYP2D6 1 were determined with V79MZh2D6*9 homogenate.",
"If hCYP2D6 9 was expressed in HepG2 cells using recombinant vaccinia virus and the (+)-bufuralol hydroxylation was examined, a 2.4fold higher KM and an about 4fold higher reaction rate were measured compared to the wildtype enzyme hCYP2D6 1 (Tyndale et al., 1991a).",
"I.e.",
"the intrinsic clearance of hCYP2D6 9 was twice as high compared to the wildtype in both in vitro expression systems.",
"hCYP2D6 10 (Substitutions Pro34Ser and Ser486Thr) A 4fold higher KM and a 3.5fold lower reaction rate were determined with V79MZh2D6*10 homogenate as compared to the wildtype enzyme hCYP2D6 1.Thus, the intrinsic clearance was about one twelfth of that of hCYP2D6 1.The normation, however, was carried out using the cytochrome P420 content since no peak could be quantified at 450 nm.",
"Moreover, the Western blots demonstrated that the amount of apoprotein was drastically decreased compared to all other variants.",
"This means that the reaction rate based on functional holoenzyme may correspond to that of the wildtype while the activity in the homogenate is only 2.5% of the activity of V79MZh2D6*1 homogenate.",
"Due to the low activity of the V79MZh2D6*10 homogenate the determination of the KM constant is also uncertain.",
"In vitro expression experiments in COS-1 cells showed that substitution Ser486Thr alone sligthly increases the amount of hCYP2D6 10 expressed as compared to the wildtype while the substitution Pro34Ser results in drastically reduced amounts of protein and in a 40fold reduced activity.",
"In the combination of both substitutions in hCYP2D6 10 the activity-lowering effect of the substitution Pro34Ser far predominates (Johansson et al., 1994; Kagimoto et al., 1990).",
"This is in close agreement to the findings of the Western analysis and the difference in activity beween the homogenates of V79MZh2D6*10 and V79MZh2D6*1.Thus, while 40fold differences in activity are found between hCYP2D6 1 and hCYP2D6 10 in vitro the difference in vivo is only one tenth (Droll et al., 1998).",
"hCYP2D6 17 (Substitutions Thr107Ile, Arg296Cys and Ser486Thr) A 2fold elevated KM and a 2fold lower reaction rate as compared to the wildtype enzyme hCYP2D6 1 were determined for V79MZh2D6*17 homogenate.",
"In contrast to the wildtype enzyme, the CO difference spectrum shows a fraction of cytochrome P420 of about 50%.",
"If the cDNA expression was carried out in COS-1 cells the absolute bufuralol hydroxylase activity of hCYP2D6 17 was only about 20% compared to the wildtype which is in agreement with the V79 expression system (Oscarsson et al., 1997): As observed with the substitutions Arg296Cys and Ser486Thr, also the substitution Thr107Ile had no substantial effect on the reaction rate of bufuralol hydroxylation.",
"The substitution Thr107Ile, however, resulted in elevated amounts of protein similar to the substitution Ser486Thr, while the combination of all three substitutions in hCYP2D6 17 led to decreased amounts of protein if the cDNA was expressed in COS-1 cells.",
"Decreased enzyme stability or translation rate is discussed as the reason.",
"Also the high proportion of cytochrome P420 in the CO difference spectrum of V79MZh2D6*17 indicates a decreased protein stability in comparison to the wildtype enzyme.",
"If the cDNA was expressed in yeast, the reaction rate for bufuralol hydroxylation at substrate saturation was similar for all mutations and combinations (Oscarsson et al., 1997).",
"The substitution of the hydrophilic Thr107 by a hydrophic Ile alone in the conserved region of the β′ helix which also is a part of the first substrate recognition region, however, resulted in an elevated KM for codeine O-demethylation (Oscarsson et al., 1997).",
"In contrast, the KM value for the hydroxylation of bufuralol remained unchanged.",
"For this reaction, only a combination of the substitutions Thr107Ile and Arg296Cys resulted in a 5fold increase in KM.",
"Thus, hCYP2D6 17 is the only known variant of hCYP2D6 in which a combination of different substitutions is responsible for an altered affinity of the enzyme to the substrate.",
"Example 10 4-hydroxylation of Tamoxifen by hCYP2D6 The cell lines according to the present invention were used to examine a possible effect of the hCYP2D6 polymorphism on the pharmacologically important 4-hydroxylation of tamoxifen (FIG.",
"18).",
"Substrate and metabolites were separated and detected by means of HPLC/MSD (FIG.",
"19).",
"A diluted aliquot of cell homogenate was thawed on ice and carefully resuspended using a pipette.",
"The reaction was started by addition of homogenate to the reaction sample (final sample: 100 μl total reaction volume, 150 μg total protein, 1-150 μM tamoxifen (2.5 μl stock solution in DMSO), 2 mM NADPH in 0.1 M potassium phosphate buffer, pH 7.4) and after an incubation for 30min (V79MZh2D6*10: 60 min) in a water bath at 37° C. was stopped by addition of 50 μl acetonitrile, 2% (v/v) acetic acid.",
"The samples were incubated for several minutes on ice and the precipitate was collected by centrifugation (10 min, 17,000×g, 4° C.).",
"The substrate tamoxifen and the reaction products including 4-hydroxy-tamoxifen contained in the supernatant were separated and detected by means of HPLC/ESI-MSD.",
"The chromatographic separation was performed in a gradient of acetonitrile (FIG.",
"10) at a flow rate of 0.5 ml/min (12th to 19th minute 0.8 ml/min) and 30° C. using a C8 (2) “reversed phase” column (Luna, 150 mm×2 mm, particle size 5 um; Phenomenex, Hosbach).",
"A C8 column (XDB-C8, narrow-bore column, 2.1 mm×12.5 mm; Zorbax HPLC Columns, Hewlett Packard, Waldbronn) was connected ahead of the system.",
"The detection of tamoxifen and its metabolites was performed using a HP Series 1100 MSD (Hewlett Packard, Waldbronn) in the single ion monitoring modus at m/z 344.2 (N-didemethyl-tamoxifen), m/z 358.3 (N-demethyl-tamoxifen), m/z 360.2 (monooxygenated metabolites of N-didemethyl-tamoxifen), mn/z 372.3 (tamoxifen), m/z 374.3 (monooxygenated metabolites of N-demethyl-tamoxifen), m/z 388.3 (monooxygenated metabolites of tamoxifen) and m/z 404.3 (monooxygenated metabolites of tamoxifen-N-oxide).",
"The retention times were 2.1 min for Z-tamoxifen-3,4-epoxide, approx.",
"5.5 and 6.2 min for E- and Z-4-hydroxy-tamoxifen, about 7.2 and 7.9 min for E- and Z-4-hydroxy-tamoxifen-N-oxide, about 8.4 min for Z-tamoxifen-1,2-epoxide, 9.9 min for Z-N-didemethyl-tamoxifen, 10.0 min for Z-N-demethyl-tamoxifen, 10.4 min for Z-tamoxifen-N-oxide and approx.",
"9.7 and 10.1 min for E- and Z-tamoxifen.",
"The peaks of E- and Z-4-hydroxy-tamoxifen, Z-tamoxifen-N-oxide, Z-tamoxifen-1,2-epoxide, Z-N-demethyl-tamoxifen and Z-N-didemethyl-tamoxifen were verified and externally standardized using the corresponding pure substances.",
"The calibration was performed for a final concentration range of 0.039-20 μM after incubation with homogenate of the mock transfected cell line of V79MZmockneo.",
"To identify the isoforms of cytochrome P450 involved in the 4-hydroxylation of tamoxifen incubations were performed with homogenates of cell lines V79MZh2EI and V79MZh3A4-hOR as well as with microsomes of insect cells coexpressing hCYP3A4 and hCYPOR and hCYP2C9*1 and hCYPOR, respectively, so-called “supersomes” (Gentest, Woburn, Mass., Produkt-Nr.",
"P207 und P218).",
"With respect to the incubations with V79MZh2D6 homogenate the following parameters were varied: 150 μg V79MZh2E1 homogenate were incubated up to 45 min, up to 500 μg V79MZh3A4-hOR homogenate and up to 25 μl hCYP3A4-hOR “supersomes” corresponding to 50 pmol hCYP3A4 were incubated up to 60 min with 100 μM magnesium chloride and 10 μM EDTA, and 12.5 μl hCYP2C9*1-hOR “supersomes” corresponding to 25 pmol hCYP2C9*1 were incubated for 30 min with 100 μM magnesium chloride and 10 μM EDTA.",
"The reaction at 37° C. was in a linear range for 30 min up to 150 μg of total protein/100 μl.",
"To be able to measure a signal which could be quantified in the case of hCYP2D6 10, the reaction sample had to be incubated for 60 min.",
"Therefore, the activities indicated sligthly underestimate the real values.",
"Effect of DMSO as a Solubilizing Agent for Tamoxifen Since tamoxifen has a poor water-solubility, DMSO was added as solubilizing agent.",
"Acetonitrile and methanol did not improve the solubility.",
"In addition, they were not as suitable as DMSO due to other undesired properties.",
"Up to a DMSO concentration of 2.5% the tamoxifen-4-hydroxylase activity was only sligthly affected.",
"At a concentration of 10% DMSO the tamoxifen-4-hydroxylase activity was only about 20% of that obtained at 2.5% (FIG.",
"20).",
"On the other hand, the onset slope of the kinetic at 10% DMSO was linear up to about 75 μM tamoxifen while at 2.5% DMSO it was linear only up to 50 μM.",
"This behaviour exactly correponded to the maximum solubility of tamoxifen at 10% and 2.5% DMSO, respectively.",
"Accordingly, the lower slope of the kinetik was not caused by the enzyme kinetic but was only dependent on the solubility limit of tamoxifen.",
"To be able to measure a signal of all hCYP2D6 variants which could be quantified, all other measurements were performed with 2.5% DMSO.",
"An slight decrease in hCYP2D6 activity up to 2.5% DMSO has also been published for the O-demethylation of dextromethorphane (Chauret et al., 1998; Hickman et al., 1998).",
"In contrast, a drastic decrease already at low concentrations of DMSO was found for the (±)-bufuralol hydroxylase activity (Busby et al., 1999).",
"Possibly, the inhibitory effect of the solvents is dependent on the substrate.",
"Kinetics of the 4-hydroxylation of Tamoxifen The linearity in the kinetic of the hCYP2D6-catalyzed 4-hydroxylation of tamoxifen was not limited by enzyme kinetics but was dependent only on the solubility limit of tamoxifen.",
"Therefore, it was impossible to determine the Vmax and KM indepedently of each other.",
"Thus, assuming a monophasic Michaelis-Menten kinetic without inhibitor (V=Vmax*[S]/(KM+[S])) the intrinsic clearance (Clint=Vmax/KM) was calculated from the linear onset slope ([S]→0) (Table 8).",
"Fitting of the line to measuring points V was performed by linear regression (FIG.",
"21).",
"An analogous kinetic was recorded for the 4-hydroxylation of tamoxifen by hCYP2C9 1 (not shown).",
"TABLE 8 Kinetic parameters of the 4-hydroxylation of tamoxifen for hCYP3A4, hCYP2C9 1 and hCYP2D6 1, 2, 9, 10 and 17.The values for hCYP3A4 and hCYP2C9 are based on a single measurement series.",
"All other values are mean values and standard deviations of three independent measurement series.",
"Based on mg of total protein Based on the amount of CYP450 V50 μM reaction rate50 μM Cell line pmol/mg/min Clint ml/mg/min pmol/pmol/min Clint ml/pmol/min V79MZmockneo 0 0 0 0 h3A4 (supers.)",
"approx.",
"1.95 — approx.",
"0.10 — h2C9 1 (supers.)",
"24.08 482 0.96 19.3 V79MZh2D6*1 58.3 ± 8.2 1166 ± 165 2.34 ± 0.63 46.8 ± 12.6 V79MZh2D6*2 21.2 ± 3.3 424 ± 66 1.58 ± 0.80 31.6 ± 16.0 V79MZh2D6*9 26.2 ± 6.5 524 ± 131 4.09 ± 2.23 81.9 ± 44.8 V79MZh2D6*10 0.41 ± 0.06 8.2 ± 1.2 0.18 ± 0.04 3.7 ± 0.8 V79MZh2D6*17 6.0 ± 1.3 119 ± 27 0.73 ± 0.25 14.6 ± 5.0 Abbreviations: V50 μM: reaction rate at 50 μM tamoxifen; Clint: intrinsic clearance; supers.",
": “supersomes” To date, no comparative data for the kinetics of the hCYP2D6-catalyzed tamoxifen-4-hydroxylation have been available.",
"At a substrate concentration of 1 μM, tamoxifen-4-hydroxylation activities of 0.7-0.8 pmol/min/mg protein and of 1.1-3 pmol/min/mg protein were detected with human liver microsomes of poor metabolizers and extensive metabolizers, respectively (Crewe et al., 1997).",
"A tamoxifen concentration of 1 μM is in the range of the plasma concentration of the therapeutical dosage (Buckley and Goa, 1989).",
"At a substrate concentration of 18 μM, the activities were between 6 and 8 pmol/min/mg protein for poor metabolizers and between 12 and 25 pmol/min/mg protein for extensive metabolizers (Crewe et al., 1997).",
"With 1.2 and 21 pmol/min/mg cellular protein, respectively, the activities of V79MZh2D6*1 homogenate were in the same ranges.",
"As in the case of the bufuralol hydroxylase activity (see Table 11) an agreement in the activities between V79MZh2D6*1 cell homogenate and human liver microsomes was observed.",
"The substrate dependence, however, of the tamoxifen-4-hydroxylase activity of recombinant hCYP2D6 was more pronounced as in the case of human liver microsomes.",
"Furthermore, activities in the ranges of those of V79MZh2D6*2 and *9 homogenate and higher than those of V79MZh2D6*10 and *17 homogenate were measured in liver microsomes of poor metabolizers.",
"Therefore, besides hCYP2D6 other isoforms with higher affinity to tamoxifen, possibly hCYP2C9, must be involved in tamoxifen-4-hydroxylation in vivo so that the effect of the hCYP2D6 polymorphism in vivo may be masked at the low therapeutic concentrations of tamoxifen.",
"Nevertheless, the comparison between poor and extensive metabolizers shows that there is indeed a relationship between the rate of tamoxifen-4-hydroxylation and the hCYP2D6 phenotype.",
"The relative intrinsic clearances of the hCYP2D6 catalyzed hydroxylation of tamoxifen and bufuralol are identical (Table 9).",
"The absolute values for the 1′-hydroxylation of (+)-bufuralol are about 10fold higher than those of the 4-hydroxylation of tamoxifen.",
"This difference can be explained by the very different structures and binding of the two substrates in the active site of hCYP2D6 (see FIG.",
"24).",
"TABLE 9 Comparison of the intrinsic clearances of the hydroxylation of tamoxifen and bufuralol using the novel polymorphic cell lines V79MZh2D6*1, *2, *9, *10 and *17.cell line V79MZh2D6 intrinsic clearance *1 *2 *9 *10 *17 4-hydroxylation [ml/min/mg protein] 1166 ± 165 424 ± 66 524 ± 131 8.2 ± 1.2 119 ± 27 of tamoxifen [ml/min/pmol P450] 47 ± 13 32 ± 16 82 ± 45 4 ± 1 15 ± 5 1′-hydroxylation [ml/min/mg protein] 12400 ± 2580 4940 ± 220 6670 ± 1970 80 ± 17 1220 ± 160 of (+)-bufuralol [ml/min/pmol P450] 500 ± 170 370 ± 150 1040 ± 670 40 ± 10 150 ± 40 Isoforms Involved in the 4-hydroxylation of Tamoxifen An involvement of the isoforms hCYP3A4, hCYP2C9 and hCYP2E1 besides hCYP2D6 in the 4-hydroxylation of tamoxifen is discussed (Crewe et al., 1997; Dehal and Kupfer, 1997; Styles et al., 1994).",
"To check the ambiguous results and to get an indication on the hCYP2D6-catalyzed fraction of the total tamoxifen 4-hydroxylation, incubations were carried out with homogenates of the cell lines V79MZh2E1 and V79MZh3A4-hOR as well as hCYP3A4-hOR and hCYP2C9*1-hOR “supersomes” (FIG.",
"22).",
"In this case, the conditions for the incubations were not optimized.",
"Therefore, the values given should only be construed as guide values (Table 8).",
"The 4-hydroxylation of tamoxifen was catalyzed by isoforms hCYP2D6, hCYP2C9 and hCYP3A4.Metabolites having masses of 388.3 (monooxygenated metabolites, FIG.",
"22) and 358.3 (demethylated metabolites, not shown) were detected.",
"As expected, the predominating metabolites were 4-hydroxy-tamoxifen in the incubation with hCYP2D6 and hCYP2C9 and N-demethyl-tamoxifen in the incubation with hCYP3A4.The 4-hydroxylation of tamoxifen by hCYP3A4, however, was negligible.",
"In contrast to the other isoforms, the peak areas of E- and Z-4-hydroxy-tamoxifen were about identical and the peak at 2.2 min (tamoxifen-3,4-epoxide?)",
"was strikingly high.",
"If cell line V79MZh2E1 was incubated with homogenate, no metabolites were detected which had not also been observed previously after incubation with homogenate of the mock transfected cell line V79Mzmockneo, i.e.",
"tamoxifen-N-oxide and N-demethyl-tamoxifen.",
"Both metabolites were contained as contaminations in tamoxifen though in a smaller amount than after the incubation.",
"Tamoxifen-N-oxide was detected in all samples independently of hCYP2D6 or other enzymes and also with heat-inactivated homogenate of mock-transfected cells.",
"The amount was practically independent of the amount of total protein and the concentration of tamoxifen, and varied up to a factor of 20 between the different measurement series.",
"The standard curve for tamoxifen-N-oxide for worked up standard solutions in negative control samples with tamoxifen was shifted in parallel as compared to standard solutions which had not been worked up.",
"The reason for these findings was assumed to be a mere chemical oxidation of the dissolved tamoxifen, presumably by oxygen in air.",
"Thus, the interval between incubation and sample measurement would provide a clue for the differing amounts in different measurement series.",
"N-Demethyl-tamoxifen was also detected in all samples independently of hCYP2D6 or other enzymes and also with heat-inactivated homogenate of mock-transfected cells.",
"In contrast to tamoxifen-N-oxide, however, the amount of N-demethyl-tamoxifen depended both on the tamoxifen concentration and on the amount of total protein in the sample and varied only by a factor of 2 between different measurement series.",
"It was impossible to carry out an exact quantification.",
"TABLE 10 Comparison of the contribution of different cytochrome P450 isoforms to the 4- hydroxylation of tamoxifen in different expression systems.",
"Microsomes of recombinant human B “supersomes” lymphoblastoid cells human liver V79MZhCYP of recombinant insect (Dehal & Kupfer, (Styles et al., (Crewe et al., microsomes Isoform homogenate cells 1997) 1994) 1997) (Crewe et al., 1997) hCYP2E1 − n.e.",
"− + − − hCYP3A4 − − − − + + hCYP2C9*1 n.e.",
"+ − − + + hCYP2D6*1 + n.e.",
"+ + + + +: unambiguous contribution to the 4-hydroxylation of tamoxifen; −: no or only negligible tamoxifen 4-hydroxylation; n.e.",
": not examined Table 10 compares the results of the present and of previous studies.",
"Considering all results, it seems that hCYP2E1 does not contribute to tamoxifen 4-hydroxylation.",
"Whether tamoxifen-N-oxide and N-demethyl-tamoxifen were formed in the incubation with V79MZh2E1 as reported by Dehal and Kupfer (1997) remained unclear because of the high background of these two metabolites.",
"Human cytochrome P450 2D6 catalyzed the 4-hydroxylation of tamoxifen in all expression systems.",
"In contrast, the role of hCYP3A4 remained unclear: Although a substantial contribution of hCYP3A4 to the formation of 4-hydroxy-tamoxifen was found in inhibition studies in human liver microsomes (Crewe et al., 1997), the tamoxifen 4-hydroxylation was practically negligible in all other expression systems.",
"In accordance with Dehal and Kupfer (1997) and Styles et al.",
"(1994) both V79MZh3A4-hOR homogenate and hCYP3A4-hOR “supersomes” catalyzed the N-demethylation of tamoxifen.",
"Baculovirus-expressed hCYP2C9*1 (“supersomes”) clearly catalyzed the 4-hydroxylation of tamoxifen.",
"The intrinsic clearance was about half that of hCYP2D6 1 expressed in V79MZ cells.",
"With about 20% the fraction of hCYP2C9 on the total amount of cytochrome P450 in the liver, however, is about 10fold higher than that of hCYP2D6.Therefore, although the studies with microsomes of recombinant human B lymphoblastoid cells yielded ambiguous results a substantial contribution of hCYP2C9 to the 4-hydroxylation of tamoxifen in vivo may be assumed.",
"Presumably, these discrepancies may be explained by different (concentrations of) solvent(s) during incubation.",
"Metabolites of Tamoxifen as Substrates of hCYP2D6 To examine the effect of certain modifications of the substrate, tamoxifen, and particularly of the nitrogen which is important for substrate binding, on hCYP2D6-catalyzed hydroxylation, incubations were carried out with various derivatives of tamoxifen.",
"For all experimental series the relative reaction rates of the allelic variants were about the same.",
"For this purpose, the relative peak areas were evaluated for non-standardized metabolites.",
"Configuration Isomers In contrast to Z-tamoxifen, the configuration isomer E-tamoxifen was practically not metabolized by V79MZh2D6 homogenate.",
"Z-4-Hydroxy-tamoxifen The predominant metabolite of the hCYP2D6-catalyzed tamoxifen metabolism, Z-4-hydroxy-tamoxifen, was metabolized by V79MZh2D6 homogenate.",
"The metabolites were identified as dihydroxy derivatives with respect to their mass spectra.",
"For example, the hCYP2D6-catalyzed ortho-hydroxylation of 4-hydroxy-tamoxifen forming the catechol has been described (Dehal and Kupfer, 1999).",
"A detailed identification of the metabolites was impossible since no appropriate standards were available.",
"Although this reaction should be less important quantitatively due to the low in vivo concentration of 4-hydroxy-tamoxifen it is of interest in view of toxicology because of the formation of catechols which may form protein adducts (Dehal and Kupfer, 1999).",
"Modifications of the Tamoxifen Nitrogen Z-Tamoxifen-N-oxide Z-Tamoxifen-N-oxide was reacted to Z-4-hydroxy-tamoxifen-N-oxide, although only to a minor extent.",
"A corresponding standard was synthesized by N-oxidation of 4-hydroxy-tamoxifen with hydrogen peroxide.",
"Z-N-Demethyl-tamoxifen The reaction of Z-N-demethyl-tamoxifen proceeded well.",
"The main metabolite had about the same retention time as Z-4-hydroxy-tamoxifen, and the chromatogram was similar to that obtained after incubation with Z-tamoxifen.",
"Presumably, Z-4-hydroxy-N-demethyl-tamoxifen and all other metabolites typical for Z-tamoxifen were generated in the Z-N-demethyl form.",
"Z-N-Didemethyl-tamoxifen The reaction of Z-N-didemethyl-tamoxifen was negligible.",
"Example 11 Comparison to Human Liver Microsomes and Purified Native hCYP2D6 The enzyme kinetic characteristics of recombinant hCYP2D6 were in the physiological range as demonstrated by a comparison to liver microsomes and purified native hCYP2D6 (Table 11).",
"With 171±16 pmol/mg/min the (+)-bufuralol hydroxylase activity of V79MZh2D6*1 cell homogenate was in good agreement with published values for human liver microsomes of 167±43 (Kronbach et al., 1987) and 199±80 (Dayer et al., 1987), respectively.",
"TABLE 11 Comparison of the kinetic parameters of the hCYP2D6 1-catalyzed bufuralol hydroxylation in different expression systems.",
"If the evaluation was performed assuming a biphasic Michaelis-Menten kinetic, the values for the isoform with high affinity and stereoselectivity are given.",
"The selectivity is the ratio of (−)-bufuralol activity/(+)-bufuralol activity.",
"KM Vmax (+) Reaction rate Expression system Work up μM pmol/mg/min 1/min selectivity (Reference) electron source (+) (+) (+/−) (+) (+/−) (−)/(+) V79MZh2D6*1 Homogenate 13.8 ± 1.6 171 ± 16 108 ± 15 6.9 ± 1.5 4.3 ± 1.2 0.40 ± 0.16 NADPH V79MZh2D6*1 Homogenate 153 ± 26 (Bogni, 1999) NADPH V79MZh2D6*1 in culture 7-8 170 ± 10 (Appel, 1999) Human liver*1 microsomes 4.7 ± 2.2 167 ± 43 0.56 ± 0.17 (Kronbach et al., 1987) NADPH Human liver*2 microsomes 17.9 ± 6.3 199 ± 80 0.49 ± 0.09 (Dayer et al., 1987) NADPH Human liver*2 microsomes 715 0.46 (Zanger et al., 1988) NADPH Human liver*1 microsomes 50-2400 (Gonzalez et al., 1988a) NADPH Human liver*2 microsomes 47.3 ± 8.1 0.6 ± 0.2 0.48 ± 0.15 (Gut et al., 1986) NADPH Human liver*1 purified 53.6 ± 27.4 3.4 ± 0.2 0.15 ± 0.02 (Gut et al., 1986) CYPOR/NADPH Human liver*1 purified 16.8 66333 25.9 0.17 (Zanger et al., 1988) CYPOR/NADPH Human liver*1 purified 3.7-9.5 4.4-6.4 0.14-0.16 (Distlerath et al., 1985) CYPOR/NADPH E. coli purified 39 ± 5 1.2 ± 0.1 (Gillam et al., 1995) CYPOR/NADPH (+/−) AHH-1 TK+/− cell lysate 68 ± 0 (2D6Met/Hol) NADPH (Crespi et al., 1991) AHH-1 TK+/− cell lysate 126 ± 1 (h2D6Metv2) NADPH (Penman et al., 1993) AHH-1 TK+/− microsomes 5.3 723 ± 35 4.5 0.42 (h2D6Metv2) NADPH (Penman et al., 1993) AHH-1 TK+/− microsomes 6.7 ± 0.2 18.3 ± 0.1 (h2D6Val/OR) CYPOR/NADPH (Crespi et al., 1995) AHH-1 TK+/− microsomes 550 10.38 (h2D6Val/OR) CYPOR/NADPH (Gentest, produkt # M117r) COS-1 cells homogenate 20-50 (Kagimoto et al., 1990) NADPH COS-1 cells homogenate 3.4 (Johansson et al., 1994) CYPOR/NADPH Hep G2 cell lysate 4.0 34.1 2.3 (Tyndale et al., 1991a) CuOOH S. cerevisiae W(R) microsomes 2 116 10 (Oscarson et al., 1997) Yred/NADPH Sf9 insect cells membranes 18.5 ± 7.3 17500 ± 2700 26.2 ± 0.4 0.16 ± 0.02 (Evert et al., 1997) CYPOR/NADPH Sf9 insect cells membranes 55 500-1000 0.96 (Patten et al., 1996) CYPOR/NADPH Sf9 insect cells cell extract 4.7 370 12.23 (Paine et al., 1996) CYPOR/NADPH (+/−) Abbreviations: (+): (+)-bufuralol; (−): (−)-bufuralol; (+/−): racemic bufuralol; CuOOH: cumene hydroperoxide; Yred: yeast reductase *1No indication of the hCYP2D6 genotype or phenotype.",
"*2Phenotyped as EM using sparteine and/or debrisoquine in vivo.",
"In validation studies, a good reproducibility of the bufuralol hydroxylase activities for the polymorphic cell lines could be demonstrated (Table 11) although the experiments were performed according to different protocols and both in culture and in cell homogenate.",
"Thus, the reproducibility and standardization of the novel cell lines was confirmed which is of critical importance for a future use in preclinical drug development.",
"With up to about 25 pmol/mg of cellular protein the amounts of cytochrome P450 in the V79MZh2D6 cell lines were in the physiological range of 8-115 pmol/mg in human liver microsomes (Distlerath et al., 1985).",
"Similar values have been achieved in other mammalian cell systems such as COS-1 cells (Clark and Waterman, 1991) or human B lymphoblastoid cells (Crespi, 1991).",
"With different expression systems such as baculovirus-infected insect cells substantially higher amounts of cytochrome P450 of up to 800 pmol/mg cell protein have been achieved (Evert et al., 1997).",
"The level of expression, however, is only one criterion among many others in the selection of the suitable expression system.",
"Substantially more important for most questions are the experimental possibilities provided by the expression system due to its biology.",
"References Aklillu, E.; Persson, I.; Bertilsson, L.; Johansson, I.; Rodrigues, F.; Ingelman-Sundberg, M. (1996) Frequent distribution of ultrarapid metabolizers of debrisoquine in an Ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles.",
"J. Pharmacol.",
"Exp.",
"Ther.",
"278: 441-446.Alvan, G. (1991) Clinical consequences of polymorphic drug oxidation.",
"Fundam.",
"Clin.",
"Pharmacol.",
"5: 209-228.Alvan, G.; Bechtel, P.; Iselius, L.; Gundert-Remy, U.",
"(1990) Hydroxylation polymorphisms of debrisoquine and mephenytoin in European populations.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"39: 533-537.Alvan, G.; Grind, M.; Graffner, C.; Sjoqvist, F. (1984) Relationship of N-demethylation of amiflamine and its metabolites to debrisoquine hydroxylation polymorphism.",
"Clin.",
"Pharmacol.",
"Ther.",
"36: 515-519.Aoyama, T.; Yamano, S.; Guzelian, P. S.; Gelboin, H. V.; Gonzalez, F. J.",
"(1990) Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1.Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 87: 4790-4793.Appel, K. (1999) PHARMBIODYN GmbH, Denzlingen.",
"persönliche Mitteilung.",
"Armstrong, M.; Fairbrother, K.; Idle, J. R.; Daly, A. K. (1994) The cytochrome P450 CYP2D6 allelic variant CYP2D6J and related polymorphisms in a European population.",
"Pharmacogenetics 4: 73-81.Asseffa, A.; Smith, S. J.; Nagata, K.; Gillette, J.; Gelboin, H. V.; Gonzalez, F. J.",
"(1989) Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus.",
"Arch.",
"Biochem.",
"Biophys.",
"274: 481-490.Barnes, H. J.; Jenkins, C. M.; Waterman, M. R. (1994) Baculovirus expression of bovine cytochlrome P450c17 in Sf9 cells and comparison with expression in yeast, mammalian cells, and E. coli.",
"Arch.",
"Biochem.",
"Biophys.",
"315: 489-494.Becker, N. und Wahrendorf, J.",
"(1981-1990).",
"Brust.",
"Krebsatlas der Bundesrepublik Deutschland.",
"3.Ausgabe.",
"Heidelberg: Springer Verlag, 366-371.Bertilsson, L. (1995) Geographical/interracial differences in polymorphic drug oxidation.",
"Current state of knowledge of cytochromes P450 (CYP) 2D6 and 2C19.Clin.",
"Pharmacokin.",
"29: 192-209.Bertilsson, L.; Dahl, M. L.; Sjoqvist, F.; Aberg-Wistedt, A.; Humble, M.; Johansson, I.; Lundqvist, E.; Ingelman-Sundberg, M. (1993) Molecular basis for rational megaprescribing in ultrarapid hydroxylators of debrisoquine.",
"The Lancet 341: 363.Bertilsson, L.; Lou, Y. Q.; Du, Y. L.; Liu, Y.; Kuang, T. Y.; Liao, X. M.; Wang, K. Y.; Reviriego, J.; Iselius, L.; Sjoqvist, F. (1992) Pronounced differences between native Chinese and Swedish populations in the polymorphic hydroxylations of debrisoquin and S-mephenytoin.",
"Clin.",
"Pharmacol.",
"Ther.",
"51: 388-397.Bichara, N.; Ching, M. S.; Blake, C. L.; Ghabrial, H.; Smallwood, R. A.",
"(1996) Propranolol hydroxylation and N-desisopropylation by cytochrome P4502D6.Drug Metab.",
"Dispos.",
"24: 112-118.Blech, J.",
"(1999) Die ganz private Pille.",
"Die Zeit 22: 42.Bloomer, J. C.; Woods, F. R.; Haddock, R. E.; Lennard, M. S.; Tucker, G. T. (1992) The role of cytochrome P4502D6 in the metabolism of paroxetine by human liver microsomes.",
"Clin.",
"Pharmacol.",
"33: 521-523.Bogni, A.",
"(1999) European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italien.",
"persönliche Mitteilung.",
"Boobis, A. R.; Murray, S.; Hampden, C. E.; Davies, D. S. (1985) Genetic polymorphism in drug oxidation: in vitro studies of human debrisoquine 4-hydroxylase and bufuralol 1′-hydroxylase activities.",
"Biochem.",
"Pharmacol.",
"34: 65-71.Borgna, J.-L. and Rochefort, H. (1981) Hydroxylated metabolites of tamoxifene are formed in vivo and bound to estrogen receptor target tissues.",
"J. Biol.",
"Chem.",
"256: 859-868.Bradley, M. O.; Bhuyan, B.; Francis, M. C.; Langenbach, R.; Peterson, A.; Huberman, E. (1981) Mutagenesis by chemical agents in V79 Chinese hamster cells.",
"Mutat.",
"Res.",
"87: 81-142.Bradwell, L. (1989) The mutagenic and carcinogenic effects of gene transfer.",
"Mutagenesis 4: 245-253.Broly, F.; Marez, D.; Lo Guidice, J. M.; Sabbagh, N.; Legrand, M.; Boone, P.; Meyer, U.",
"A.",
"(1995a) A nonsense mutation in the cytochrome P450 CYP2D6 gene identified in a Caucasian with an enzyme deficiency.",
"Hum.",
"Genet.",
"96: 601-603.Broly, F.; Marez, D.; Sabbagh, N.; Legrand, M.; Millecamps, S.; Lo Guidice, J.-M.; Boone, P.; Meyer, U.",
"A.",
"(1995b) An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.",
"Pharmacogenetics 5: 373-384.Broly, F. and Meyer, U.",
"A.",
"(1993) Debrisoquine oxidation polymorphism: phenotypic consequences of a 3-base-paar deletion in exon 5 of the CYP2D6 gene.",
"Pharmacogenetics 3: 123-130.Brosen, K. and Gram, L. F. (1989a) Clinical significance of the sparteine/debrisoquine oxidation polymorphism.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"36: 537-547.Brosen, K. and Gram, L. F. (1989b) Clinical significance of the sparteine/debrisoquine oxidation polymorphism.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"36: 537-547.Brosen, K.; Otton, V.; Gram, L. F. (1986) Imipramine demethylation and hydroxylation: Impact of the sparteine oxidation phenotype.",
"Clin.",
"Pharmacol.",
"Ther.",
"40: 543-549.Buckley, M. T. and Goa, K. L. (1989) Tamoxifen: a reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic use.",
"Clin.",
"Pharmacol.",
"37: 451-490.Burnette, W. N. (1981) Western-Blotting.",
"Analyt.",
"Biochem.",
"112: 195-203.Busby, W. F. J.; Ackermann, J. M.; Crespi, C. L. (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450.Drug Metab.",
"Dispos.",
"27: 246-249.Buters, J. T. M.; Korzekwa, K. R.; Kunze, K. L.; Omata, Y.; Hardwick, J. P.; Gonzalez, F. J.",
"(1994) cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus.",
"Drug Metab.",
"Dispos.",
"22(5): 688-692.Butner, K. A. and Lo, C. W. (1986) High frequency rearrangements associated with mouse centromeric satellite DNA.",
"J. Mol.",
"Biol.",
"187: 547-556.Calos, M. P.; Lebkowski, J. S.; Botchan, M. R. (1983) High mutation frequency in DNA transfected into mammalian cells.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 80: 3015-3019.Carcillo, J.",
"A.; Parise, R. A.; Adedoyin, A.; Frye, R.; Branch, R. A.; Romkes, M. (1996) CYP2D6 mRNA expression in circulating peripheral blood mononuclear cells correlates with in vivo debrisoquine hydroxylase activity in extensive metabolizers.",
"Res.",
"Commun.",
"Mol.",
"Pathol.",
"Pharmacol.",
"91: 149-159.Chance, B.",
"(1957).",
"Techniques for the Assay of the Respiratory Enzymes.",
"In: Methods in Enzymology, Colowick, S. P. and Kaplan, N. O.",
"(eds.).",
"New York: Academic Press, IV: 273-329.Chauret, N.; Gauthier, A.; Nicoll-Griffith, D. A.",
"(1998) Effect of common solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes.",
"Drug Metab.",
"Dispos.",
"26: 1-4.Chu, E. H. Y. and Malling, H. V. (1968) Mammalian cell genetics II.Chemical induction of specific locus mutations in Chinese hamster cells in vitro.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"61: 1306-1312.Clark, B. J. and Waterman, M. R. (1991).",
"Heterologous expression of mammalian P450 in COS cells.",
"In: Methods in Enzymology, Waterman, M. R. and Johnson, E. F., (eds.).",
"New York: Academic Press, 206: 100-108.Cooper, R. G.; Evans, D. A. P.; Price, A. H. (1987) Studies on the metabolism of perhexiline in man.",
"Eur.",
"J Clin.",
"Pharmacol.",
"32: 569-576.Crespi, C. L. (1991).",
"Expression of cytochrome P450 cDNAs in human B lymphoblastoid cells: applications to toxicology and metabolite analysis.",
"In: Methods in Enzymology, Waterman, M. R. and Johnson, E. F., (eds.).",
"New York: Academic Press, 206: 123-129.Crespi, C. L.; Penman, B. W.; Gelboin, H. V.; Gonzalez, F. J.",
"(1991) A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6.Carcinogenesis 12: 1197-1201.Crespi, C. L.; Steimel, D. T.; Penman, B. W.; Korzekwa, K. R.; Femandezsalguero, P.; Buters, J. T. M.; Gelboin, H. V.; Gonzalez, F. J.; Idle, J. R.; Daly, A. K. (1995) Comparison of substrate metabolism by wild type CYP2D6 protein and a variant containing methionine, not valine, at position 374.Pharmacogenetics 5: 234-243.Crewe, H. K.; Ellis, S. W.; Lennard, M. S.; Tucker, G. T. (1997) Variable contribution of cytochrome P450 2D6, 2C9 and 3A4 to the 4-hydroxylation of tamoxifen by human liver microsomes.",
"Biochem.",
"Pharmacol.",
"53: 171-178.Dahl, M.; Johansson, I.; Bertilsson, L.; Ingelman-Sundberg, M.; Sjoqvist, F. (1995a) Ultrarapid hydroxylation of debrisoquine in a Swedish population.",
"Analysis of the molecular genetic basis.",
"J. Pharmacol.",
"Exp.Ther.",
"274: 516-520.Dahl, M. L. and Bertilsson, L. (1993) Genetically variable metabolism of antidepressants and neuroleptic drugs in man.",
"Pharmacogenetics 3: 61-70.Dahl, M. L.; Yue, Q. Y.; Roh, H. K.; Johansson, I.; Sawe, J.; Sjoqvist, F.; Bertilsson, L. (1995b) Genetic analysis of the CYP2D locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects.",
"Pharmacogenetics 5: 159-164.Daly, A. K. (1995) Molecular basis of polymorphic drug metabolism.",
"J. Mol.",
"Med.",
"173: 539-553.Daly, A. K.; Brockmoller, J.; Broly, F.; Eichelbaum, M.; Evans, W. E.; Gonzalez, F. J.; Huang, J.-D.; Idle, J. R.; Ingelman-Sundberg, M.; Ishizaki, T.; Jacqz-Aigrain, E.; Meyer, U.",
"A.; Nebert, D. W.; Steen, V. M.; Wolf, C. R.; Zanger, U. M. (1996a) Nomenclature for human CYP2D6 alleles.",
"Pharmacogenetics 6: 193-201.Daly, A. K.; Fairbrother, K. S.; Andreasson, 0.A.",
"; London, S. J.; Idle, J. R.; Steen, V. M. (1996b) Characterization and PCR-based detection of two different hybrid CYP2D7/CYP2D6 alleles associated with the poor metabolizer phenotype.",
"Pharmacogenetics 6: 231-240.Daly, A. K.; Leathart, J.",
"B. S.; London, S. J.; Idle, J. R. (1995) An inactive cytochrome P450 CYP2D6 allele containing a deletion and a base substitution.",
"Human Genet.",
"95: 337-341.Dayer, P.; Kronbach, T.; Eichelbaum, M.; Meyer, U.",
"A.",
"(1987) Enzymatic basis of the debrisoquine/sparteine-type genetic polymorphism of drug oxidation: characterization of bufuralol 1′-hydroxylation in liver microsomes of in vivo phenotyped carriers of the genetic deficiency.",
"Biochem.",
"Pharmacol.",
"260: 4145-4152.De Groene, E. M.; Hassing, I. G. M.; Blom, M. J.; Seinen, W.; Fink-Gremmels, J.; Horbach, G. J.",
"(1996) Development of human cytochrome P450-expressing cell lines: application in mutagenicity of ochratoxin A.",
"Cancer Res.",
"56: 299-304.De Groot, M. J. and Vermeulen, N. P. E. (1997) Modeling the active sites of cytochrome P450s and glutathione S-transferases, two of the most important biotransformation enzymes.",
"Drug Metab.",
"Rev.",
"29: 747-799.De Matteis, F.; White, I. N. H.; Smith, L. L. (1998) Species differences in the metabolic activation of tamoxifen into genotoxic derivatives: risk assessment in woman.",
"Eur.",
"J.",
"Drug Metab.",
"Pharmacokin.",
"23: 425-428.Dehal, S. S. and Kupfer, D. (1996) Evidence that the catechol 3,4-dihydroxy tamoxifen is a proximate intermediate to the reactive species binding covalently to proteins.",
"Cancer Res.",
"56: 1283-1290.Dehal, S. S. and Kupfer, D. (1997) CYP2D6 catalyzes tamoxifen-4-hydroxylation in human liver.",
"Cancer Res.",
"57: 3402-3406.Dehal, S. S. and Kupfer, D. (1999) Cytochrome P-450 3A and 2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and 3-hydroxytamoxifen (droloxifen) yielding tamoxifen catechol: involvement of catechols in covalent binding to hepatic proteins.",
"Drug Metab.",
"Dispos.",
"27: 681-688.Distlerath, L. M.; Reilly, P. E. B.; Martin, M. V.; Davis, G. G.; Wilkinson, G. R.; Guengerich, F. P. (1985) Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphisms in oxidative drug metabolism.",
"J. Biol.",
"Chem.",
"260: 9057-9067.Doehmer, J.",
"(1993) V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies.",
"Toxicology 82: 105-118.Doehmer, J.; Dogra, S.; Friedberg, T.; Monier, S.; Adesnik, M.; Glatt, H.; Oesch, F. (1988) Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.Proc.",
"iVatl.",
"Acad.",
"Sci.",
"USA 85: 5769-5773.Doepker, C. L.; Livingston, G. K.; Schumann, B. L.; Srivastava, A. K. (1998) Structural and numerical chromosomal aberrations in a metabolically competent human lymphoblast cell line (MCL-5).",
"Mutagenesis 13(3): 275-280.Dogra, S.; Doehmer, J.; Glatt, H.; Molders, H.; Siegert, P.; Friedberg, T.; Seidel, A.; Oesch, F. (1990) Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing.",
"Mol.",
"Pharmacol.",
"37: 608-613.Droll, K.; Bruce-Mensah, K.; Otton, S. V.; Gaedigk, A.; Sellers, E. M.; Tyndale, R. F. (1998) Comparison of three CYP2D6 probe substrates and genotype in Ghanians, Chinese and Caucasians.",
"Pharmacogenetics 8: 325-333.Eichelbaum, M.; Baur, M. P.; Dengler, H. J.; Osikowska-Evers, B. O.; Tieves, G.; Zekom, C.; Rittner, C. (1987) Chromosomal assignment of human cytochrome P-450 (debrisoquine/sparteine type) to chromosome 22.Br.",
"J Clin.",
"Pharmacol.",
"23: 455-458.Eichelbaum, M.; Bertilsson, L.; Sawe, J.; Zekorn, C. (1982) Polymorphic oxidation of sparteine and debrisoquine: related pharmacogenetic entities.",
"Clin.",
"Pharmacol.",
"Ther.",
"31: 184-186.Eichelbaum, M. and Gross, A. S. (1990) The genetic polymorphism of debrisoquine/sparteine metabolism—clinical aspects.",
"Pharmac.",
"Ther.",
"46: 377-394.Eichelbaum, M. and Gross, A. S. (1992).",
"In: Pharmacogenetics of drug metabolism, Kalow, W., (ed.).",
"New York: Pergamon Press, Inc.. Eichelbaum, M.; Spannbrucker, N.; Dengler, H. J.",
"(1979a) Influence of the defective metabolism of sparteine on its pharrnacokinetics.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"16: 189-194.Eichelbaum, M.; Spannbrucker, N.; Steincke, B.; Dengler, H. J.",
"(1979b) Defective N-oxidation of sparteine in man: a new pharmacogenetic defect.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"16: 183-187.Eichelbaum, M. and Woolhouse, N. M. (1985) Interethnic differences in sparteine oxidation among Ghanaians and Germans.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"28: 79-83.Ellis, S. W.; Ching, M. S.; Watson, P. F.; Hendersson, C. J.; Simula, A. P.; Lennard, M. S.; Tucker, G. T.; Woods, H. F. (1992) Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast.",
"Biochem.",
"Pharmacol.",
"44: 617-620.Ellis, S. W.; Hayhurst, G. P.; Smith, G.; Lightfoot, T.; Wong, M. M. S.; Simula, A. P.; Ackland, M. J.; Sternberg, M. J. E.; Lennard, M. S.; Tucker, G. T.; Wolf, C. R. (1995) Evidence that aspartic acid 301 is a critical substrate-contact residue in the active site of cytochrome P450 2D6.J.",
"Biol.",
"Chem.",
"270: 29055-29058.Estabrook, R. W.; Peterson, J.; Baron, J.; Hildebrandt, A.",
"(1972).",
"The spectrophotometric measurement of turbid suspensions of cytochromes associated with drug metabolism.",
"In: Methods in Pharmacology, Chigell, C. F., (ed.).",
"New York (u.a.",
"): Plenum Press, 2: 303-350.Evans, D. A.; Mahgoub, A.; Sloan, T. P.; Idle, J. R.; Smith, R. L. (1980) A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population.",
"J. Med Genet.",
"17: 102-105.Evans, W. E.; Relling, M. V.; Rahman, A.; McLeod, H. L.; Scott, E. P.; Lin, J. S. (1993) Genetic basis for a lower prevalence of deficient CYP2D6 oxidative drug metabolism phenotypes in black Americans.",
"J. Clin.",
"Invest.",
"91: 2150-2154.Evert, B.; Eichelbaum, M.; Haubruck, H.; Zanger, U. M. (1997) Functional properties of CYP2D6 1 (wildtype) and CYP2D6 7 (His324Pro) expressed by recombinant baculovirus in insect cells.",
"Naunyn-Schmiedeberg's Arch.",
"Pharmacol.",
"355: 309-318.Evert, B.; Griese, E.-U.",
"; Eichelbaum, M. (1994a) Cloning and sequencing of a new non-functional CYP2D6 allele: deletion of T1795 in exon 3 generates a premature stop codon.",
"Pharmacogenetics 4: 271-274.Evert, B.; Griese, E.-U.",
"; Eichelbaum, M. (1994b) A missense mutation in exon 6 of the CYP2D6 gene leading to a histidine to proline exchange is associated with the poor metabolizer phaenotype of sparteine.",
"Naunyn-Schmiedeberg's Arch.",
"Pharmacol.",
"350: 434-439.Fiers, W.; Contreras, R. R.; Haegeman, G.; Rogiers, R.; De Voorde, A.; van Heuverswyn, H.; Van Herreweghe, J.; G., V.; M., Y.",
"(1978) Complete nucleotide sequence of SV40 DNA.",
"Nature 273: 113-120.Fischer, V.; Vogels, B.; Maurer, G.; Tynes, R. E. (1992) The antipsychotic clozapine is metabolized by the polymorphic human microsomal and recombinant cytochrome P450 2D6.J.",
"Pharmacol.",
"Exp.",
"Ther.",
"260: 1355-1360.Fonne-Fister, R.; Bargetzi, M. J.; Meyer, U.",
"A.",
"(1987) MPTP, the neurotoxin inducing Parkinson's disease, is a potent competitive inhibitor of human and rat cytochrome P450 isozymes (P450bufI, P450dbl) catalyzing debrisoquine 4-hydroxylation.",
"Biochem.",
"Biophys.",
"Res.",
"148: 1144-1150.Ford, D. K. and Yerganian, G. (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture.",
"J. Natl.",
"Cancer Inst.",
"21: 393-425.Furr, B. J.",
"A. and Jordan, V. C. (1984) The pharmacology and clinical uses of tamoxifen.",
"Pharmac.",
"Ther.",
"25: 127-205.Gaedigk, A.; Blum, M.; Gaedigk, R.; Eichelbaum, M.; Meyer, U.",
"A.",
"(1991) Deletion of the entire cytochrome P450 gene as a cause of impaired drug metabolism on poor metabolizers of the debrisoquine/sparteine polymorphism.",
"Am.",
"J. Hum.",
"Genet.",
"48: 943-950.Garfinkel, D. (1958) Isolation and properties of cytochrome b5 from pig liver.",
"Arch.",
"Biochem.",
"Biophys.",
"77: 111-120.Gillam, E. M. J.; Guo, Z.; Martin, M. V. M.; Jenkins, C. M.; Guengerich, F. P. (1995) Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization.",
"Arch.",
"Biochem.",
"Biophys.",
"319: 540-550.Glatt, H.; Davis, W.; Meinl, W.; Hermersdorfer, H.; Venitt, S.; Phillips, D. H. (1998) Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.",
"Carcinogenesis 19: 1709-1713.Glatt, H.; Gemperlein, I.; Turchi, G.; Heinritz, H.; Doehmer, J.; Oesch, F. (1987) Search for cell culture systems with diverse xenobiotic-metabolizing activities and their use in toxicological studies.",
"Mol.",
"Toxicol.",
"1: 313-334.Glatt, H. R.; Gemperlein, I.; Setiabudi, F.; Platt, K. L.; Oesch, F. (1990) Expression of xenobiotic metabolizing enzymes in propagable cell cultures and induction of micronuclei by 13 compounds.",
"Mutagenesis 5: 241-249.Gonzalez, F. J. and Korzekwa, K. R. (1995) Cytochromes P450 expression systems.",
"Annu.",
"Rev.",
"Pharmocol.",
"Toxicol.",
"35: 369-390.Gonzalez, F. J.; Matsunaga, T.; Nagata, K.; Meyer, U.; Nebert, D. W.; Pastewka, J.; Kozak, C. A.; Gillette, J.; Gelboin, H. V.; Hardwick, J. P. (1987) Debrisoquine 4-hydroxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis in the DA rat polymorphism.",
"DNA 6: 149-161.Gonzalez, F. J.; Skoda, R. C.; Kimura, S.; Umeno, M.; Zanger, U. M.; Nebert, D. W.; Gelboin, H. V.; Hardwick, J. P.; Meyer, U.",
"A.",
"(1988a) Characterization of the common genetic defect in humans deficient in debrisoquine metabolism.",
"Nature 331: 442-446.Gonzalez, F. J.; Skoda, R. C.; Kimura, S.; Umeno, M.; Zanger, U. M.; Nebert, D. W.; Gelboin, H. V.; Hardwick, J. P.; Meyer, U.",
"A.",
"(1990) Molecular characterization of the common human deficiency in metabolism of debrisoquine and other drugs.",
"Nature 331: 442-446.Gonzalez, F. J.; Vilbois, F.; Hardwick, J. P.; McBride, O. W.; Nebert, D. W.; Gelboin, H. V.; Meyer, U.",
"A.",
"(1988b) Human debrisoquine 4-hydroxylase (P4501ID6): cDNA and deduced amino acid sequence and assignment of the CYP2D6 locus to chromosome 22.Genomics 2: 174-179.Gotoh, O.",
"(1991) Substrate recognition sites in cytochrome P450 family 2 (CYP2).",
"Proteins inferred from comparative analysis of amino acid and encoding nucleotide sequences.",
"J. Biol.",
"Chem.",
"267: 83-90.Gough, A. C.; Miles, J. S.; Spurr, N. K.; Moss, J. E.; Gaedigk, A.; Eichelbaum, M.; Wolf, C. R. (1990) Identification of the primary gene defect at the cytochrome P450 CYP2D6 locus.",
"Nature 347: 773-776.Gough, A. C.; Smith, C. A.; Howell, S. M.; Wolf, C. R.; Bryant, S. P.; Spurr, N. K. (1993) Localization of the CYP2D gene locus to human chromosome 22q13.1 by polymerase chain reaction, in situ hybridization, and linkage analysis.",
"Genomics 15: 430-432.Graham, F. L. and Van der Eb, A. J.",
"(1973) A new technique for the assay of infectivity of human adenovirus 5 DNA.",
"Virology 52: 456-467.Greim, H. und Deml, E. H.",
"(Hrsg.)",
"(1996).",
"Toxikologie.",
"Eine Einführung für Naturwissenschaftler und Mediziner.",
"Weinheim: VCH.",
"Griese, E.-U.",
"; Zanger, U. M.; Brudermanns, U.; Gaedigk, A.; Mikus, G.; Morike, K.; Stuven, T.; Eichelbaum, M. (1998) Assessment of the predictive power of genotypes for the in-vivo catalytic function of CYP2D6 in a German population.",
"Pharmacogenetics 8: 15-26.Gross, A. S.; Phillips, A. C.; Rieutord, A.; Shenfield, G. M. (1996) The influence of the sparteine/debrisoquine genetic polymorphism on the disposition of dexfenfluramine.",
"Br.",
"J. Clin.",
"Pharmacol.",
"41: 311-317.Guidice, J. M.; Marez, D.; Sabbagh, N.; Legrand-Andreoletti, M.; Spire, C.; Alcaide, E.; Lifitte, J. J.; Broly, F. (1997) Evidence for CYP2D6 expression in human lung.",
"Biochem.",
"Biophys.",
"Res.",
"241: 79-85.Gut, J.; Catin, T.; Dayer, P.; Kronbach, T.; Zanger, U.; Meyer, U.",
"A.",
"(1986) Debrisoquine/sparteine-type polymorphism of drug oxidation: purification and characterization of two functionally different human liver cytochrome P-450 isoenzymes involved in impaired hydroxylation of the prototype substrate bufuralol.",
"J. Biol.",
"Chem.",
"261: 11734-11743.Halliday, R. C.; Jones, B. C.; Park, B. K.; Smith, D. A.",
"(1997) Synthetic strategies to lower affinity for CYP2D6.Eur.",
"J Drug Metab.",
"Pharmacokin.",
"22: 291-294.Hickman, D.; Wang, J.-P.; Wang, Y.; Unadkat, J. D. (1998) Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities.",
"Drug Metab.",
"Dispos.",
"26: 207-215.Higashi, Y.; Hiromasa, T.; Tanae, A.; Miki, T.; Nakura, J.; Kondo, T.; Ohura, T.; Ogawa, E.; Nakayama, K.; Fujii-Kuriyama, Y.",
"(1991) Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency.",
"J. Biochem.",
"(Tokyo) 109: 638-644.Hiroi, T.; Imaoka, S.; Funae, Y.",
"(1998) Dopamine formation from tyramine by CYP2D6.Biochem.",
"Biophys.",
"Res.",
"Commun.",
"249: 838-843.Hogstedt, S.; Lindberg, B.; Rane, A.",
"(1983) Increased oral clearance of metoprolol in pregnancy.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"24: 217-220.Horai, Y.; Nakano, M.; Ishizaki, T.; Ishikawa, K.; Zhou, H. H.; Zhou, B. I.; Liao, C. L.; Zhang, L. M. (1989) Metoprolol and mephenytoin oxidation polymorphisms in Far Eastern Oriental subjects: Japanese versus mainland Chinese.",
"Clin.",
"Pharmacol.",
"Ther.",
"46: 198-207.Huang, Z.; Fasco, M. J.; Figge, H. L.; Keyomarsi, K.; Kaminsky, L. S. (1996) Expression of cytochromes P450 in human breast tissue and tumors.",
"Drug Metab.",
"Dispos.",
"24: 899-905.Huang, Z.; Fasco, M. J.; Kaminsky, L. S. (1997) Alternative splicing of CYP2D mRNA in human breast tissue.",
"Arch.",
"Biochem.",
"Biophys.",
"343: 101-108.IARC (1996).",
"Some pharmaceutical drugs: anti-estrogenic compounds.",
"Tamoxifen.",
"IARC monographs on the evaluation of carcinogenic risks to humans.",
"WHO, 66: 253-365.Ingelman-Sundberg, M.; Oscarson, M.; Persson, I.; Masimirembwa, C. M.; Dahl, M.-L.; Bertilsson, L.; Sjoqvist, F.; Johansson, I.",
"(1995).",
"Genetic polymorphism of human drug metabolizing enzymes.",
"Recent aspects on polymorphic forms of cytochromes P450.European conference on specificity and variability in drug metabolism, Luxembourg, EU.",
"Jaenisch, R. and Jahner, D. (1984) Methylation, expression and chromosomal position of genes in mammals.",
"Biochem.",
"Biophys.",
"Acta 782: 179-189.Johansson, I.; Lundqvist, E.; Bertilsson, L.; Dahl, M.-L.; Sjoqvist, F.; Ingelman-Sundberg, M. (1993) Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 90: 11825-11829.Johansson, I.; Oscarson, M.; Yue, Q.-Y.",
"; Bertilsson, L.; Sjoqvist, F.; Ingelman-Sundberg, M. (1994) Genetic analysis of the Chinese cytochrome P4502D locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity for debrisoquine hydroxylation.",
"Mol.",
"Pharmacol.",
"46: 452-459.Jordan, V. C. (1997) Tamoxifen: the herald of a new era of preventive therapeutics.",
"J. Nat.",
"Cancer Inst.",
"89: 747-749.Jordan, V. C. (1998) Designer estrogens.",
"Scientific American Okt.",
": 60-67.Jordan, V. C. and Allen, K. E. (1980) Evaluation of the antitumor activity of the non-steroidal anti-estrogen monohydroxytamoxifen in the DMBA-induced rat carcinoma model.",
"Eur.",
"J.",
"Cancer Clin.",
"Oncol.",
"16: 239-251.Jordan, V. C. and Chem, C. (1982) Metabolites of tamoxifen in animals and man: identification, pharmacology, and significance.",
"Breast Cancer Res.",
"and Treatment 2: 123-138.Jordan, V. C. and Robinson, S. P. (1987) Species-specific pharmacology of antiestrogens: role of metabolism.",
"Federation proceedings 46: 1870-1874.Kagimoto, M.; Heim, M.; Kagimoto, K.; Zeugin, T.; Meyer, U.",
"A.",
"(1990) Multiple mutations of the human cytochrome IID6 gene (CYP2D6) in poor metabolisers of debrisoquine.",
"J. Biol.",
"Chem.",
"265: 17209-17214.Kempf, A. C.; Zanger, U. M.; Meyer, U.",
"A.",
"(1995) Truncated human P450 2D6: expression in Escherichia coli, Ni(2+)-chelate affinity purification, and characterization of solubility and aggregation.",
"Arch.",
"Biochem.",
"Biophys.",
"321: 277-288.Kiefer, F. and Wiebel, F. J.",
"(1989) V79 Chinese hamster cells express cytochrome P-450 activity after simultaneous exposure to polycyclic aromatic hydrocarbons and aminophylline.",
"Toxicol.",
"Letters 48: 265-273.Kimura, S.; Umeno, M.; Skoda, R. C.; Meyer, U.",
"A.; Gonzalez, F. J.",
"(1989) The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene.",
"Am.",
"J. Hum.",
"Genet.",
"45: 889-904.Kivisto, K. T.; Griese, E.-U.",
"; Stuven, T.; Fritz, P.; Friedel, G.; Kroemer, H. K.; Zanger, U. M. (1997) Analysis of CYP2D6 expression in human lung: implications for the association between CYP2D6 activity and susceptibility to lung cancer.",
"Pharmacogenetics 7: 295-302.Klingenberg, M. (1958) Pigments of rat liver microsomes.",
"Arch.",
"Biochem.",
"Biophys.",
"75: 376-386.Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.",
"J. Mol.",
"Biol.",
"196: 947-950.Kozak, M. (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes.",
"Proc.",
"Natl.",
"Acad .Sci.",
"USA(87): 8301-8305.Kroemer, H. K. and Eichelbaum, M. (1995) “It's the genes, stupid”.",
"Molecular bases and clinical consequences of genetic cytochrome P450 2D6 polymorphism.",
"Life Sci.",
"56: 2285-2298.Kronbach, T. (1991) Bufuralol, dextromethorphan, and debrisoquine as prototype substrates for human P450IID6.Methods in Enzymology 206: 509-517.Kronbach, T.; Mathys, D.; Gut, J.; Catin, T.; Meyer, U.",
"A.",
"(1987) High-performance liquid chromatographic assay fot bufuralol 1′-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylation in microsomes and purified cytochrome P-450 isozymes of human liver.",
"Analytical Biochemistry 162: 24-32.Krynetski, E. Y.; Drutsa, V. L.; Kovaleva, I. E.; Luzikov, V. N. (1995) High yield expression of functionally active human liver CYP2D6 in yeast cells.",
"Pharmacogenetics 5: 103-109.Krynetski, E. Y.; Drutsa, V. L.; Kovaleva, I. E.; Luzikov, V. N.; Uvarov, V. Y.",
"(1993) Effects of amino-terminus truncation in human cytochrome P450IID6 on its insertion into the endoplasmatic reticulum membrane of Saccharomyces cerevisiae.",
"FEBS Lett.",
"336: 87-89.Kubota, S.; Yoshida, Y.; Kumaoka, H.; Furumichi, A.",
"(1977) Studies on the microsomal electron-transport system of anaerobically grown yeast.",
"J. Biochem.",
"81: 197-205.Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680-685.Lazarou, J.; Pomeranz, B. H.; Corey, P. N. (1998) Incidence of adverse drug reactions in hospitalized patients.",
"JAMA 4 279: 1200-1205.Lewis, D. F. V. (1998) The CYP2 family: models, mutants and interactions.",
"Xenobiotica 28: 617-661.Lim, C. K.; Yuan, Z.-X.",
"; Lamb, J. H.; White, I. N. H.; De Matteis, F.; Smith, L. L. (1994) A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes.",
"Carcinogenesis 15: 589-593.Lodwick, R.; McConkey, B.; Brown, A. M.; Beeley, L. (1987) Life threatening interaction between tamoxifen and warfarin.",
"Br.",
"Med.",
"J.",
"295: 1141.Logan, W. P. D. (1975) World Health Organisation Statistics Report.",
"WHO, 28: 232.Lowry, O. H.; Rosebrough, A. L.; Farr, A. L.; Randell, R. J.",
"(1951) Protein measurement with the folin phenol reagent.",
"J. Biol.",
"Chem.",
"214: 741.Lundqvist, E.; Johansson, I.; Ingelman-Sundberg, M. (1999) Genetic mechanisms for duplication and multiplication of the human CYP2D6 gene and methods for detection of duplicated CYP2D6 genes.",
"Gene 226: 327-338.MacGregor, J. and Jordan, V. C. (1998) Basic guide to the mechanisms of antiestrogen action.",
"Pharmacol.",
"Rev.",
"50: 151-196.Mahgoub, A.; Idle, J. R.; Dring, L. G.; Lancaster, R.; Smith, R. L. (1977) Polymorphic hydroxylation of debrisoquine in man.",
"Lancet 2: 584-586.Marez, D.; Legrand, M.; Sabbagh, N.; Lo Guidice, J. M.; Boone, P.; Broly, F. (1996) An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine.",
"Human Genetics 97: 668-670.Marez, D.; Sabbagh, N.; Legrand, M.; Lo Guidice, J. M.; Boone, P.; Broly, F. (1995) A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype.",
"Pharmacogenetics 5: 305-311.Marquardt, H. und Schäfer, S. G.",
"(Hrsg.)",
"(1997).",
"Lehrbuch der Toxikologie.",
"1.Ausgabe.",
"Heidelberg, Berlin: Spektrum Akademischer Verlag.",
"Masimirembwa, C.; Hasler, J.; Bertilssons, L.; Johansson, I.; Ekberg, O.; Ingelman-Sundberg, M. (1996a) Phenotype and genotype analysis of debrisoquine hydroxylase (CYP2D6) in a black Zimbabwean polulation.",
"Reduced enzyme activity and evaluation of metabolic correlation of CYP2D6 probe drugs.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"51: 117-122.Masimirembwa, C.; Persson, I.; Bertilsson, L.; Hasler, J.; Ingelman-Sundberg, M. (1996b) A novel mutant variant of the CYP2D6 gene (CYP2D6*17) common in black African population: association with diminished debrisoquine hydroxylase activity.",
"Br.",
"J. Clin.",
"Pharmacol.",
"42: 713-719.Matsunaga, E.; Umeno, M.; Gonzalez, F. J.",
"(1990) The rat P450 IID subfamily: complete sequences of four closely linked genes and evidence that gene conversions maintained sequence homogeneity at the heme-binding region of the cytochrome P450 active site.",
"J. Mol.",
"Evol.",
"30: 155-169.Meyer, U.",
"A.",
"(1991) Genotype or phenotype: the definition of a pharmacogenetic polymorphism.",
"Pharmacogenetics 1: 66-67.Meyer, U.",
"A. and Zanger, U. M. (1997) Molecular mechanisms of genetic polymorphisms of drug metabolising enzymes.",
"Annu.",
"Rev.",
"Pharmacol.",
"Toxicol.",
"37: 269-296.Monier, S.; Van Luc, P.; Adesnik, M. (1988) Signals for the incorporation and orientation of cytochrome P450 in the endoplasmatic reticulum membrane.",
"J.",
"Cell Biol.",
"107: 457-570.Moorthy, B.; Sriram, P.; Pathak, D. N.; Bodell, W. J.; Randerath, K. (1996) Tamoxifen metabolic activation: comparison of DNA adducts formed by microsomal and chemical activation of tamoxifen and 4-hydroxy-tamoxifen with DNA adducts formed in vivo.",
"Cancer Res.",
"56: 53-57.Mortimer, O.; Persson, K.; Ladona, M. G.; Spalding, D.; Zanger, U. M.; Meyer, U.",
"A.; Rane, A.",
"(1990) Polymorphic formation of morphine from codeine in poor and extensive metabolizers of dextromethorphan: relationship to the presence of immunoidentified cytochrome P-450IID1.Clin.",
"Pharmacol.",
"Ther.",
"47: 27-35.Mulligan, R. C. and Berg, P. (1981) Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 78: 2972-2976.Nayfield, S. G. (1995) Tamoxifen's role in chemoprevention of breast cancer: an update.",
"J.",
"Cell.",
"Biol.",
"Suppl.",
"22: 42-50.Nebert, D. W. (1997) Polymorphisms in drug-metabolizing enzymes: what is their clinical significance and why do they exist?",
"Am.",
"J. Hum.",
"Genet.",
"60: 265-271.Nebert, D. W. and McKinnon, R. A.",
"(1994) Cytochrome P450: evolution and functional diversity.",
"Prog.",
"Liv.",
"Dis.",
"12: 63-97.Nelson, D. R.; Koymans, L.; Kamataki, T.; Stegeman, J. J.; Feyereisen, R.; Waxman, D. J.; Gotoh, O., Coon, M. J.; Estabrook, R. W.; Gunsalus, I. C.; Nebert, D. W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.",
"Pharmacogenetics 6: 1-42.Nelson, D. R. and Stobel, H. W. (1988) On the membrane topolgy of vertebrate cytochrome P-450 proteins.",
"J. Biol.",
"Chem.",
"263: 6038-6050.Oates, N. S.; Shah, R. R.; Idle, J. R.; Smith, R. L. (1982) Genetic polymorphism of phenformin 4-hydroxy-lation.",
"Clin.",
"Pharmacol.",
"Ther.",
"32: 81-89.Ohgiya, S.; Komori, M.; Ohi, H.; Shiramatsu, K.; Shinriki, N.; Kamataki, T. (1992) Six-base deletion occurring in messages of human cytochrome P-450 in the CYP2C subfamily results in reduction of tolbutamide hydroxylase activity.",
"Biochemistry International 27: 1073-1081.Omura, T. and Sato, R. (1964a) The carbon monoxide-binding pigment of liver microsomes.",
"I.",
"Evidence for its hemoprotein nature.",
"J. Biol.",
"Chem.",
"239: 2370-2385.Omura, T. and Sato, R. (1964b) The carbon monoxide-binding pigment of liver microsomes.",
"II.",
"Solubilization, purification, and properties.",
"J. Biol.",
"Chem.",
"239: 2379-2385.Onderwater, R. C. A.; Goeptar, A. R.; Levering, P. R.; Vos, R. M. E.; Konings, P. N. M.; Doehmer, J.; Commandeur, J. N. M.; Vermeulen, N. P. E. (1996) The use of macroporous microcarriers for the large-scale growth of V79 cells genetically designed to express single human cytochrome P450 isoenzymes and for the characterization of the expressed cytochrome P450.Prot.",
"Expr.",
"Pur.",
"8: 439-446.Osborne, C. K. (1998) Tamoxifen in the treatment of breast cancer.",
"Drug Therapy 339: 1609-1618.Osborne, M. R.; Davis, W. D.; Hewer, A. J.; Hardcastle, I. R.; Phillips, D. H. (1999) 4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells.",
"Chem.",
"Res.",
"Toxicol.",
"12: 151-158.Oscarson, M.; Hidestrand, M.; Johansson, I.; Ingelman-Sundberg, M. (1997) A combination of mutations in the CYP2D6*17 (CYP2D6Z) allele causes alterations in enzyme function.",
"Mol.",
"Pharmacol.",
"52: 1034-1040.Paine, M. J. I.; Gilham, D.; Roberts, G. C. K.; Wolf, C. R. (1996) Functional high level expression of cytochrome P450 CYP2D6 using baculovirus expression systems.",
"Arch.",
"Biochem.",
"Biophys.",
"328: 143-150.Panserat, S.; Mura, C.; Gerard, N.; Vincent-Viry, M.; Galteau, M. M.; Jacqz-Aigrain, E.; Krishnamorthy, R. (1994) DNA haplotype-dependent differences in the amino acid sequence of debrisoquine 4-hydroxylase (CYP2D6): evidence for two major allozymes in extensive metabolisers.",
"Hum.",
"Genet.",
"94: 401-406.Panserat, S.; Mura, C.; Gerard, N.; Vincent-Viry, M.; Galteau, M. M.; Jacqz-Aigrain, E.; Krishnamorthy, R. (1995) An unequal cross-over event within the CYP2D6 gene cluster generates a chimeric CYP2D71CYP2D6 gene which is associated with the poor metabolizer phenotype.",
"Br.",
"J. Clin.",
"Pharmacol.",
"40: 361-367.Parker, B.",
"A. and Stark, F. R. (1979) Regulation of simian virus 40 transcription.",
"J. Virol.",
"31: 360.Pathak, D. N.; Pongracz, K.; Bodell, W. J.",
"(1995) Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen.",
"Carcinogenesis (Lond.)",
"16: 11-15.Patten, C. J.; Smith, T. J.; Murphy, S. E.; Wang, M.-H.; Lee, J.; Tynes, R. E.; Koch, P.; Yang, C. S. (1996) Kinetic analysis of the activation of 4-(Methylnitroso)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes.",
"Arch.",
"Biochem.",
"Biophys.",
"333: 127-138.Penman, B. W.; Reece, J.; Smith, T.; Yang, C. S.; Gelboin, H. V.; Gonzalez, F. J.; Crespi, C. L. (1993) Characterization of a human cell line expressing high levels of cDNA-derived CYP2D6.Pharmacogenetics 3: 28-39.Pierce, D. M.; Smith, S. E.; Franklin, R. A.",
"(1987) The pharmacogenetics of indoramin in poor and excessive hydroxylators of debrisoquine.",
"Eur.",
"J. Clin.",
"Pharmacol.",
"33: 59-65.Poon, G. K.; Chui, Y. C.; McCague, R.; Lonning, P. E.; Feng, R.; Rowlands, M. G.; Jarman, M. (1993) Analysis of phase I and phase II metabolites of tamoxifen in breast cancer patients.",
"Drug Metab.",
"Dispos.",
"21: 1119-1124.Poulos, T. L.; Finzel, B. C.; Howard, A. J.",
"(1987) High-resolution crystal structure of cytochrome P450cam.",
"J. Mol.",
"Biol.",
"195: 687-700.Price-Evans, D. A. P. (1993).",
"Genetic factors in drug therapy.",
"Clinical and molecular pharmacogenetics.",
"Cambridge: Cambridge University Press.",
"Pritchard, M. P.; Glancey, M. J.; Blake, J.",
"A. R.; Gilham, D. E.; Burchell, B.; Wolf, C. R.; Friedberg, T. (1998) Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.",
"Pharmacogenetics 8: 33-42.Prueksaritanont, T.; Dwyer, L. M.; Cribb, A. E. (1995) (+)-Bufuralol 1′-hydroxylation activity in human and rhesus monkey intestine and liver.",
"Biochem.",
"Pharmacol.",
"50: 1521-1525.Randerath, K.; Moorthy, B.; Mabon, N.; Sriram, P. (1994) Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite.",
"Carcinogenesis (Lond.)",
"15: 2987-2094.Rauschenbach, R.; Gieschen, H.; Salomon, B.; Kraus, C.; Kuhne, G.; Hildebrand, M. (1997) Development of a V79 cell line expressing human cytochrome P450 2D6 and its application as a metabolic screening tool.",
"Environm.",
"Toxicol.",
"Pharmacol.",
"3: 31-39.Rettie, A. E.; Korzekwa, K. R.; Kunze, K. L.; Laurence, R. F.; Eddy, A. C.; Ayoma, T.; Gelboin, H. V.; Gonzalez, F. J.; Trager, W. F. (1992) Hydroxylation of warfarin by human cDNA expressed cytochrome P450: a role for P4502C9 in the etiology of (S)-warfarin drug interactions.",
"Chem.",
"Res.",
"Toxicol.",
"5: 54-59.Roh, H. K.; Dahl, M. L.; Johansson, I.; Ingelman-Sundberg, M.; Cha, Y. N.; Bertilsson, L. (1996) Debrisoquine and S-mephenytoin hydroxylation phenotypes and genotypes in a Korean population.",
"Pharmacogenetics 6: 441-447.Romkes-Sparks, M.; Mnuskin, A.; Chem, H. D.; Persad, R.; Fleming, C.; Sibley, G. N.; Smith, P.; Wilkinson, G. R.; Branch, R. A.",
"(1994) Correlation of polymorphic expression of CYP2D6 mRNA in bladder mucosa and tumor tissue to in vivo debrisoquine hydroxylase activity.",
"Cacinogenesis 15: 1955-1961.Roy, S. D.; Hawes, E. M.; McKay, G.; Korchinski, E. D.; Midha, K. K. (1985) Metabolism of methoxyphenamine in extensive and poor metabolisers of debrisoquine.",
"Clin.",
"Pharmacol.",
"Ther.",
"38: 128-133.Sabbagh, N.; Brice, A.; Marez, D.; Durr, A.; Legrand, M.; Lo Guidice, J.-M.; Korzekwa, K. R.; Destee, A.; Agid, Y.; Broly, F. (1997) CYP2D6 polymorphism in familial and sporadic Parkinson's disease.",
"unveröffentlicht.",
"Sabbagh, N.; Brice, A.; Marez, D.; Durr, A.; Legrand, M.; Lo Guidice, J.-M.; Korzekwa, K. R.; Destee, A.; Agid, Y.; Broly, F. (1999) CYP2D6 polymorphism and Parkinson's disease susceptibility.",
"Mov.",
"Disord.",
"14: 230-236.Sachse, C.; Brockmöller, J.; Bauer, S.; Reum, T.; Roots, I.",
"(1996) A rare insertion of T226 in exon 1 of CYP2D6 causes a frameshift and is associated with the poor metabolizer phenotype: CYP2D6*15.Pharmacogenetics 6: 269-272.Sachse, C.; Brockmöller, J.; Bauer, S.; Roots, I.",
"(1997) Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.",
"Am.",
"J. Hum.",
"Genet.",
"60: 284-95.Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A.",
"(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.",
"Science 239: 487-491.Sambrook, J.; Fritsch, E. F.; Maniatis, T. (1989).",
"Molecular Cloning.",
"2nd Edition.",
"Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.",
"Sawada, M. and Karnataki, T. (1998) Genetically engineered cells stably expressing cytochrome P450 and their application to mutagen assays.",
"Mutation Research 411: 19-43.Saxena, R.; Shaw, G. L.; Relling, M. V.; Frame, J. N.; Moir, D. T.; Evans, W. E.; Caporaso, N.; Weiffenbach, B.",
"(1994) Identification of a new variant CYP2D6 single base pair deletion in exon 3 and its association with the poor metabolizer phenotype.",
"Hum.",
"Mol.",
"Genet.",
"3: 923-926.Schenkman, J.",
"B. und Greim, H. (1993).",
"Cytochrome P450, 105.Berlin: Springer.",
"Schenkman, J.",
"B.; Tamburini, P. P.; Jansson, I.; Epstein, P. M. (1989).",
"Functional aspects of protein-protein interactions of cytochrome b5, cytochrome P-450 reductase and cytochrome P-450.In: Book of abstracts and lecture notes.",
"NATO Advanced Study Institute on Molecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds.",
"Schmalix, W. A.; Barrenscheen, M.; Landsiedel, R.; Janzowski, C.; Eisenbrand, G.; Gonzalez, F.; Eliasson, E.; Ingelman-Sundberg, M.; Perchermeier, M.; Greim, H.; Doehmer, J.",
"(1995) Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells.",
"Eur.",
"J. Pharmacol.",
"293: 123-1131.Schmalix, W. A.; Maeser, H.; Kiefer, F.; Reen, R.; Wiebel, F. J.; Gonzalez, F. J.; Seidel, A.; Glatt, H. R.; Doehmer, J.",
"(1993) Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene.",
"Eur.",
"J. Pharmacol.",
"248: 251-261.Schneider, A.; Schmalix, W. A.; Siruguri, V.; de Groene, E. M.; Horbach, G. J.; Kleingeist, B.; Lang, D.; Bocker, R.; Belloc, C.; Beaune, P.; Greim, H.; Doehmer, J.",
"(1996) Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 hamster cells.",
"Arch.",
"Biochem.",
"Biophys.",
"332: 295-304.Schulz, M.; Freisem-Rabien, U.; Jessberger, R.; Doerfler, W. (1987) Transcriptional activities of mammalian genomes at active sites of recombination with foreign DNA.",
"J. Virol.",
"61: 344-353.Schulz-Utermoehl, T.; Bennett, A.; Ellis, S.; Tucker, G. B.",
"A.; Edwards, R. (1999) Polymorphic debrisoquine 4-hydroxylase activity in the rat is due to differences in CYP2D2 expression.",
"Pharmacogenetics in press.",
"Sengstag, C.; Eugster, H. P.; Wurgler, F. E. (1994) High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase.",
"Carcinogenesis 15: 837-843.Sharp, P. A.; Sugden, B.; Sambrook, J.",
"(1973) Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis.",
"Biochemistry 12: 3055-3063.Shimada, T.; Yamazaki, H.; Mimura, M.; Inui, Y.; Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians.",
"J. Pharmacol.",
"Exp.",
"Ther.",
"270: 414-423.Simi, S.; Xiao, Y.; Campagna, M.; Doehmer, J.; Darroudi, F. (1999) Dual-colour FISH analysis to characterize a marker chromosome in cytochrome P450 2B1 recombinant V79 Chinese hamster cells.",
"Mutagenesis 14: 101-105.Smith, D. A.; Abel, S. M.; Hyland, R.; Jones, B. C. (1998) Human cytochrome P450s: selectivity and measurement in vivo.",
"Xenobiotica 28: 1095-1128.Smith, L. L. and White, I. N. H. (1998) Antiestrogen therapy: uncertainties and risk assessment.",
"Oncology 12 Suppl.",
"5: 14-22.Stearns, V. and Gelmann, E. P. (1998) Does Tamoxifen cause cancer in humans?",
"J. Clin.",
"Oncol.",
"16: 779-792.Steen, V. M.; Molven, A.; Aarskog, N. K.; Gulbrandsen, A.-K. (1995) Homologous unequal cross-over involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of the human cytochrome P450 CYP2D6 gene.",
"Hum.",
"Mol.",
"Genet.",
"4: 2251-2257.Steiner, E.; Iselius, L.; Alvan, G.; Lindsten, J.; Sjoqvist, F. (1985) A family study of genetic and environmental factors determining polymorphic hydroxylation of debrisoquin.",
"Clin.",
"Pharmacol.",
"Ther.",
"38: 394-401.Strain, A. J. and Wylie, A. H. (1984) Uptake and stability of SV40 DNA after calcium phosphate transfection of CV-1 cells.",
"Biochem.",
"J.",
"218: 475-482.Styles, J.",
"A.; Davies, A.; Lim, C. K.; De Matteis, F.; Stanley, L. A.; White, I. N. H.; Yuan, Z.-X.",
"; Smith, L. L. (1994) Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s.",
"Carcinogenesis 15: 5-9.Swierenga, S. H.; Heddle, J.",
"A.; Sigal, E. A.; Gilman, J. P.; Brillinger, R. L.; Douglas, G. R.; Nestmann, E. R. (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV.",
"Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures.",
"Mutat.",
"Res.",
"246: 301-322.Szczesna-Skorupa, E.; Straub, P.; Kemper, B.",
"(1993) Deletion of a conserved tetrapeptide, PPGP, in P450 2C2 results in loss of enzymatic activity without a change in its cellular location.",
"Arch.",
"Biochem.",
"Biophys.",
"304: 170-175.Tenni, P.; Lalaich, D. L.; Byrne, M. J.",
"(1989) Life threatening interaction between tamoxifen and warfarin.",
"Br.",
"Med.",
"J.",
"298: 93.Tucker, G. T. (1998).",
"Clinical aspects of polymorphic drug metabolism.",
"In: Drug metabolism.",
"towards the next millenium, Gooderman, N.",
"(ed.).",
"Amsterdam, Berlin, Oxford, Tokyo, Washington, D.C.: IOS Press, 109-118.Tucker, G. T.; Lennard, M. S.; Ellis, S. W.; Woods, H. F.; Cho, A. K.; Lin, L. Y.; Hiratsuka, A.; Schmitz, D. A.; Chu, T. Y.",
"(1994) The demethylenation of methylenedioxymethamphetamine (“ecstasy”) by debrisoquine hydroxylase (CYP2D6).",
"Biochem.",
"Pharmacol.",
"47: 1151-1156.Tyndale, R.; Aoyama, T.; Broly, F.; Matsunaga, T.; Inaba, T.; Kalow, W.; Gelboin, H. V.; Meyer, U.",
"A.; Gonzalez, F. J.",
"(1991a) Identification of a new CYP2D6 allele lacking the codon encoding Lys-281: possible association with the poor metabolizer phenotype.",
"Pharmacogenetics 1: 26-32.Tyndale, R. F.; Kalow, W.; Inaba, T. (1991b) Oxidation of reduced haloperidol to haloperidol: involvement of human P450IID6 (sparteine/debrisoquine monooxygenase).",
"Br.",
"J. Clin.",
"Pharmacol.",
"31: 655-660.Vorhees, C. V.; Reed, T. M.; Schilling, M. A.; Fisher, J. E.; Moran, M. S.; Cappon, G. D.; Nebert, D. W. (1998) CYP2D1 polymorphism in methamphetamine-treated rats: genetic differences in neonatal mortality and effects on spatial learning and acoustic startle.",
"Neurotoxicology and Teratology 20: 263-273.Wadelius, M.; Darj, E.; Frenne, G.; Rane, A.",
"(1997) Induction of CYP2D6 in pregnancy.",
"Clin.",
"Pharmacol.",
"Ther.",
"62: 400-407.Wahl, G. M.; de Saint Vincent, B. R.; DeRose, M. L. (1984) Effect of chromosomal position on amplification of transfected genes in animal cells.",
"Nature 307: 516-520.Wang, S. (1992).",
"Phenotypes and genotypes of debrisoquine hydroxylation polymorphism in Chinese.",
"Master's thesis.",
"National Cheng Kung University, Tainan, Taiwan.",
"Wilking, N.; Isaksson, E.; von Schoultz, A.",
"(1997) Tamoxifen and secondary tumors.",
"Drug Safety 16: 104-117.Wolfel, C.; Platt, K. L.; Dogra, S.; Glatt, H.; Wachter, F.; Doehmer, J.",
"(1991) Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.",
"Mol.",
"Carcinog.",
"4: 489-498.Yamamoto, Y.; Tasaki, T.; Nakamura, A.; Iwata, K.; Kazusaka, A.; Gonzalez, F. J.; Fujita, S. (1998) Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2.Pharmacogenetics 8: 73-82.Yokota, H.; Tamura, S.; Furuya, H.; Kimura, S.; Watanabe, M.; Kanazawa, I.; Kondo, I.; Gonzalez, F. J.",
"(1993) Evidence for a new variant allele CYP2D6J in a Japanese population associated with lower in vivo rates of sparteine metabolism.",
"Pharmacogenetics 3: 256-263.Zanger, U. M.; Vilbois, F.; Hardwick, J. P.; Meyer, U.",
"A.",
"(1988) Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoquine oxidation in man.",
"Biochemistry 27: 5447-5454."
]
] | Patent_10018320 |
[
[
"Connecting device for fluids",
"A connection means for two base bodies (3a and 3b) of a subassembly conducting a fluid.",
"On each base body (3a and 3b) holding means are provided, which respectively have at least one holding pin (16) projecting toward the respectively other base body (3a and 3b), such holding pin fitting between two coupling bodies (17 and 18) and being at the same time peripherally acted upon by the working faces (22 and 23) of both coupling bodies (17 and 18).",
"The coupling bodies (17 and 18) are able to be clamped together athwart the connection direction (5) of the two base bodies (3a and 3b) and by virtue of oblique faces are able to exert a connection force (Fv) acting to produce a movement together of the base bodies (3a and 3b)."
],
[
"1.A connection means for two base bodies of a fluid conducting subassembly and more particularly a modularly designed device for treating compressed air, comprising holding means provided on the mutually facing connection faces (9a and 9b) of the base bodies (3a and 3b) to be connected, and furthermore a coupling unit (7) fitting between the base bodies to be connected, said coupling unit (7) having two coupling bodies (17 and 18), said coupling bodies (17 and 18) being able to be clamped athwart the connection direction (5) of the two base bodies (3a and 3b) and thereby by virtue of inclined faces extending obliquely in relation to the connection direction (5) exerting a connection force (Fv) on the holding means, said connection force acting to provide a movement together of the base bodies (3a and 3b), characterized in that the holding means provided on each respective base body (3a and 3b) each possess at least one holding pin (16) extending toward the respectively other base body (3a and 3b), said pin fitting between the two coupling bodies (17 and 18) and simultaneously being acted upon peripherally by working faces (22 and 23) of both coupling bodies (17 and 18).",
"2.The connection means as set forth in claim 1, characterized in that on the connection faces (9a and 9b) of both base bodies (3a and 3b) respectively two mutually spaced apart holding pins (16) are provided, which respectively are able to cooperate with working faces (22 and 23) of both coupling bodies (17 and 18).",
"3.The connection means as set forth in claim 2, characterized in that the two holding pins (16) on the associated connection face (9a and 9b) are arranged on mutually diametrally opposite sides of the opening (8a and 8b) of a fluid duct (4a and 4b) opening toward the connection face (9a and 9b).",
"4.The connection means as set forth in any one of the claims 1 through 3, characterized in that the holding pins (16) are placed within periphery of the associated connection face (9a and 9b) in contact with the coupling unit (7), such holding pins being located preferably near the edge of the respective connection face (9a and 9b).",
"5.The connection means as set forth in any one of the claims 1 through 4, characterized in that the holding pins (16) are in the form of components separate from the associated base body (3a and 3b), which components are attached to the respective base body (3a and 3b) more particularly in a detachable manner.",
"6.The connection means as set forth in claim 5, characterized in that the holding pins (16) are screwed to the associated base body (3a and 3b).",
"7.The connection means as set forth in any one of the claims 1 through 6, characterized in that the holding pins (16) on the base bodies (3a and 3b) to be connected together are coaxially opposite to one another in pairs.",
"8.The connection means as set forth in any one of the claim 1 through 7, characterized in that for the mutual clamping together of the two coupling bodies (17 and 18) clamping means (25) engaging same are provided which are preferably in the form of clamping means (25) designed in the form of screw connection means.",
"9.The connection means as set forth in claim 8 in conjunction with claim 7, characterized in that the clamping means (25) are provided with clamping screws (38) one respective clamping screw fitting through an intermediate space (37) present between two holding pins (16) associated with each other in a pair.",
"10.The connection means as set forth in any one of the claims 1 through 9, characterized in that both the working faces (22 and 23) of the coupling bodies (17 and 18) and also the mating working faces (24) cooperating with the same, of the holding pins (16) are designed in the form of oblique faces.",
"11.The connection means as set forth in claim 10, characterized in that, as related to the connection direction (5) of the two base bodies (3a and 3b), the working faces (22 and 23), have the same angles (w) of inclination as the mating working faces (24).",
"12.The connection means as set forth in any one of the claims 1 through 11, characterized in that the mating working faces (22 and 23,) cooperating with the working faces (22 and 23), of the holding pins (16) have a conical form.",
"13.The connection means as set forth in any one of the claims 1 through 11, characterized in that the mating working faces (22 and 23,) cooperating with the working faces (22 and 23), of the holding pins (16) are provided on a surrounding radial projection (31), which preferably is formed by a head portion (33) of the respective holding pin (16).",
"14.The connection means as set forth in any one of the claims 1 through 13, characterized in that each holding pin (16) possesses a mating working face (24) facing the connection face (9a and 9b) of the base body (3a and 3b) bearing it and cooperating with the associated working faces (22 and 23) of the coupling bodies (17 and 18), the coupling bodies (17 and 18) fitting, in the clamped together condition thereof, between a respective mating working face (24) and the associated connection face (9a and 9b) and acting on both the mating working face (24) and the connection face (9a and 9b).",
"15.The connection means as set forth in any one of the claims 1 through 14, characterized in that the coupling bodies (17 and 18) fit round the holding pins (16) like clips in the clamped together state, each coupling body (17 and 18) possessing, for each holding pin (16), a recess partly receiving it.",
"16.The connection means as set forth in any one of the claim 1 through 15, characterized in that the coupling bodies (17 and 18) engage each other in the direction of the biasing force when in the clamped together state.",
"17.The connection means as set forth in any one of the claims 1 through 16, characterized in that the coupling unit (7) has a through duct (13) flush with the duct openings (8a and 8b) provided on the connection faces (9a and 9b) in the fitted state, such through duct being completely formed in one of the two coupling bodies.",
"18.The connection means as set forth in claim 17, characterized in that the through duct (13) provided on the one coupling body (18) is delimited, on the side facing the other body (17), by a wall bulging out toward the other coupling body (17), such wall fitting into a complementary recess in the other coupling body (17).",
"19.The connection means as set forth in claim 17 or in claim 18, characterized in that an annular seal (15) is fitted between the coupling body (18) having the opening and the two base bodies (3a and 3b) to be connected, said seal being coaxial in relation to the through duct (13)."
],
[
"The invention relates to a connection means for two base bodies of a fluid conducting subassembly and more particularly to a modularly designed device for treating compressed air, comprising holding means provided on the mutually facing connection faces of the base bodies to be connected, and furthermore a coupling unit fitting between the base bodies to be connected, said coupling unit having two coupling bodies, said coupling bodies being able to be clamped athwart the connection direction of the two base bodies and thereby by virtue of inclined faces extending obliquely in relation to the connection direction exerting a connection force on the holding means, said connection force acting to provide a movement together of the base bodies.",
"Such a connection means is for instance described in the patent publication WO 95/02149 in connection with a device for treating compressed air.",
"The latter comprises a plurality of components, whose housing-like base bodies are connected together firmly and at the same time in a fluid-tight fashion with a coupling unit between them.",
"The coupling unit comprises a slide-like first coupling body able to be introduced between the two base bodies, said first coupling body being clamped against a second coupling body mounted from the opposite side, so that inclined working faces provided on the coupling bodies cooperate with correspondingly inclined oblique faces on the base bodies and acting on the inclined plane principle ensure that the base bodies are clamped together.",
"The oblique faces of the base bodies are provided on holding means, which are formed by integral marginal extensions of the base bodies.",
"In the case of a similar connection means disclosed in the U.S. Pat.",
"No.",
"5,372,392 after the putting base the bodies together clamp-like coupling bodies are applied from the outside.",
"In order to create a pressure-tight connection a separate perforated intermediate plate is inserted between the base bodies to be connected.",
"Unlike this known design the German patent publication 19,707,630 C1 discloses a connection means, in the case of which the base bodies are in direct contact at their connection faces, coupling bodies, which are clamped together, being mounted on the side faces of the base bodies.",
"One object of the present invention is to provide a connection means of the type initially mentioned rendering possible easy handling and a reliable connection of two base bodies.",
"On the basis of a connection means of the type initially mentioned this object is to be achieved by the invention because the holding means provided on each respective base body each possess at least one holding pin extending toward the other base body, said pin fitting between the two coupling bodies and simultaneously being acted upon peripherally by the working faces of both coupling bodies.",
"It is in this manner that the connection force clamping the base bodies held together may be caused to act at the relevant points in order to systematically clamp the base bodies together at those positions which are best from the point of view of design and function.",
"There is for instance the possibility of so applying the connection forces that in the peripheral part of communicating fluid ducts of the base bodies there is an optimum sealing pressure.",
"On the contrary in the prior art the application of the forces necessarily takes place at the edge of the base bodies, something which can not always ensure the desired area related pressure.",
"Furthermore, the connection means may be manufactured extremely simply and it is extremely easy to use.",
"Further advantageous developments of the invention are defined in the dependent claims.",
"It would in principle be possible, for the connection of the coupling unit with a respective base body, to provide only one holding pin.",
"However, for reasons of symmetry and optimum application of force, recourse will generally be had to a plurality of holding pins for each base body, it being best more particularly to have two spaced holding pins in each case, which again may cooperate with working faces of both coupling bodies simultaneously.",
"If the connection means is at the same time to provide a fluid-tight between fluid ducts in the two base bodies, it is an advantage for the two holding pins to be arranged on the associated connection faces on diametrally opposite sides of the respective duct opening.",
"The two holding pins are preferably located respectively within the periphery of the connection face in contact with the coupling unit, of the associated base body, while at the same time being more particularly placed near the edge of the respective connection face render possible a large flow cross section for the fluid ducts to be connected.",
"For the sake of improving even application of the connection force it is furthermore an advantage for the holding pins of the base bodies to be joined to be coaxially opposite each other in pairs.",
"If screw connection means are provided for mutually clamping the two coupling bodies together, which have clamping screws acting on the two couple bodies, the arrangement will preferably be such that respectively one of the clamping screws extends through the axial intermediate space between the two associated paired holding pins.",
"Accordingly the forces applied by the clamping screws are directed along the shortest path and applied to the holding pins.",
"When the coupling bodies are clamped the working faces provided on them will cooperate with the mating working faces provided on the holding pins.",
"Although respectively merely one of such types of faces can have an inclined form, it is an advantage if in both cases the design is in the form of oblique faces, whose angle of inclination is preferably identical in relation to the connection means so that they extend in parallelism to one another.",
"Dependent on the particular design the oblique faces can be straight or curved in shape.",
"The holding pins may in some cases be an integral part of the associated base body, but however for reasons of convenience of manufacture it is preferred to have a separate design for firmly fixing to the associated base body.",
"For anchoring it is more particularly possible to use a screw connection, the holding pins being able to have a threaded shank by means of which they are firmly screwed in the associated base body.",
"It is furthermore an advantage for the mating working faces of the holding pins to be respectively provided on a peripheral radial projection on the respective holding pin, which may be constituted by a head of the holding pin.",
"The mating working face then will preferably have a conical shape becoming narrower toward the associated connection face.",
"When the connection has been produced the coupling bodies are preferably clamped together and also respectively braced against the holding pins and furthermore with the connection faces of the base bodies.",
"For this purpose it is possible for each holding pin to possess a mating working face facing the connection face of the base body bearing it and cooperating with the associated faces of the coupling bodies, the coupling bodies fitting between a respective mating working face and the connection face facing same and acting on both the mating working face and also the connection face.",
"It is convenient for the coupling bodies to engage each other in the clamped condition so that a fixed abutment is formed.",
"If a fluid connection is to be produced through the coupling unit between fluid ducts in the two base bodies, it is advantageous for a corresponding through duct of the coupling unit to be formed completely in only one of the two coupling bodies so that between the two coupling bodies no sealing means are necessary.",
"In order nevertheless to have compact dimensions of the coupling unit, the through duct may be delimited at the side facing the other coupling body by an outwardly curved wall fitting into a complementary recess in the other coupling body.",
"In what follows the invention will be described with reference to the accompanying drawings.",
"FIG.",
"1 shows a device for treating compressed air in the form of a subassembly and having two functional units, which are connected firmly or permanently together by the intermediary of a preferred embodiment of the connection means of the invention.",
"FIG.",
"2 shows part of the arrangement of FIG.",
"1 as sectioned on the line II - II, the coupling unit being illustrated during mounting on the two base bodies of the functional units while the coupling units are still separate from each other.",
"FIG.",
"3 is a plan view of the arrangement of FIG.",
"1 looking in the direction of the arrow III, the coupling unit being depicted with the top coupling body removed.",
"FIG.",
"4 shows the part marked IV on a larger scale with the as yet not completely mounted, unclamped coupling unit.",
"FIG.",
"5 is a view similar to that of FIG.",
"4 with the coupling unit completely mounted and clamped.",
"By way of example FIG.",
"1 shows a device 1 serving for the conditioning or treatment of compressed air, such device being in the operational state placed on a compressed air line, not illustrated in detail.",
"The device 1 could also be termed a compressed air servicing device.",
"It is a modular subassembly through which a fluid flows during operation and which in the working example comprises two diagrammatically indicated functional units 2a and 2b which could for example be a pressure regulating unit, a filter unit and/or an oiler unit.",
"The individual functional units each comprise a body which in the example here is a block- or cube-like in shape, and which will as a rule be like a housing in structure and in the present case are referred to as base bodies 2a and 2b.",
"Each of the base bodies 3a and 3b respectively comprises at least one internal fluid duct 4a and 4b, which is indicated in chained lines in FIG.",
"3 respectively opening at opposite end faces of the respective base body 3a and 3b.",
"The base bodies 3a and 3b may be arranged in any desired number along a connection direction 5 indicated in chained lines, immediately following base bodies being able to be detachably connected together using the connection means 6 in accordance with the invention.",
"In the present case the fluid ducts 4a and 4b of adjacent base bodies 3a and 3b are also connected together so that a common flow duct is produced extending through the entire device 1, such duct communicating with the connectable fluid ducts.",
"Within each functional unit 2a and 2b the respective section of the fluid duct 4a and 4b constituting the corresponding section of the flow duct will have a form specific to its function and may for instance act so that the fluid pressure medium is passed through a filter means or a pressure regulating unit.",
"The connection means 6 comprises a coupling unit 7, which is placed and fits,between the base bodies 3a and 3b to be connected.",
"In the mounted state the base bodies 3a and 3b have their facing connection faces 9a and 9b in engagement with a respectively facing coupling face 12a and 12b of the intermediately placed coupling unit 7.The connection faces 9a and 9b are provided here on those side faces of the base bodies 3a and 3b, at which a duct opening 8a and 8b of the associated fluid duct 4a and 4b is located.",
"The coupling faces 12a and 12b are located on mutually opposite sides of the coupling unit 7.By the intermediary of the connection means 6 the base bodies 3a and 3b may be detachably clamped together to form a component group, able to be handled as a single body with the coupling unit 7 in between.",
"The coupling unit 7 possesses a through duct 12, which preferably extends through the coupling unit 7 in the direction 5 of connection and opens at both coupling faces 12a and 12b.",
"Its openings 14 are in this case flush with the respectively associated duct opening 8a and 8b, there being in the joint between the coupling unit 7 and a respective base body 3a and 3b an intermediately fitted annular seal 15, which is concentric to the above mentioned openings, such seal 15 providing for a transfer passage, sealed off from the outside, between the fluid ducts 4a and 4b and the through duct 13.The annular seal 15 will as a rule consist of elastically resilient material and is axially clamped between on the one hand the connection faces 9a and 9b on the other hand the coupling faces 12a and 12b.",
"In addition to the coupling unit 7 the connection means 6 comprises holding means, arranged on the base bodies 3 to be connected, in the form of separate holding pins 16, which are located on the connection faces 9a and 9b, same being preferably placed within the periphery, which in the working example is rectangular, of the respectively associated connection face 9a and 9b.",
"In relation to two base bodies 3a and 3b to be connected together there is a provision such that each base body 3a and 3b has at least one holding pin 16 on the connection face 9a and 9b facing the other base body 3a and 3b, such pin extending like projection past the associated connection face 9a and 9b and extending away from same toward the oppositely placed connection face of the other base body.",
"The holding pins 16 cooperate with the coupling unit 7, which, which possesses two first and second coupling bodies 17 and 18 to be clamped or braced together athwart the connection direction 5.The direction 19 of clamping is indicated in the drawings by a chained line.",
"Such line runs in parallelism to the plane of the coupling faces 12a and 12b, which are also mutually parallel and thus at a right angle to the connection direction 5.The holding pins 16 fit between the two coupling bodies 17 and 18, such pins peripherally respectively having a mating working face 24, which at the same time is able to be acted upon by the first and the second working faces 22 and 23 of the two coupling bodies 17 and 18.Either both working faces 22 and 23 or the mating working face 24, but preferably both working faces 22 and 23 and also the mating working face 24—this being the case with the embodiment illustrated—are oblique faces and inclined in relation to the connection direction 5, the angle of inclination being indicated in the figure as “w”.",
"Here the angle of inclination of the working faces 22 and 23 and of the mating working face 24 is preferably identical.",
"As shown in FIGS.",
"4 and 5, the mating working face 24 is arranged some distance in front of the base bodies 3a and 3b bearing the respective holding pin 16, while at the same time facing the above mentioned connection line.",
"The working faces 22 and 23 are on the contrary so arranged on the two coupling faces 17 and 18 that they face away from that connection face 9a and 9b, which has the holding pin 16, with which they individually cooperate.",
"In this respect both the working faces 22 and 23 and also mating working face 24 extend, with an increasing distance form the connection face 9a and 9b, obliquely athwart the connection direction 5 in an outward direction.",
"In order to ensure a firm connection between the base bodies 3a and 3b the two coupling bodies 17 and 18 (which are preferably in the form of separate components) are introduced from opposite sides in the clamping direction between the base bodies 3a and 3b and mounted on the holding pins 16.This phase is illustrated in FIGS.",
"2 and 4.Following this the two coupling bodies 17 and 18 are braced together by tightening or loading means 25 in the clamping direction 19 toward one another and clamped together with a clamping force Fs firmly, the first and the second working faces 22 and 23 coming into engagement with the mating working face 24 of the holding pins 16 and acting on same.",
"During this clamping or bracing operation by means of the working faces and mating working faces, sliding on each other the mutually facing coupling faces 12a and 12b and the connection faces 9a and 9b are simultaneously moved together and urged onto one another.",
"All in all this inclined plane action means that from a connection force Fs, applied in the connection direction 19, a connection force Fv—effective in the connection direction 5—and by such connection force Fv the base bodies 3a and 3b, firmly connected with the holding pins 16, are drawn and braced firmly together with the coupling unit 7 in between.",
"In the working embodiment the mating working faces 24 possess a conical configuration.",
"Accordingly it is possible for the first and the second working faces 22 and 23 to be respectively constituted by a peripheral section of a conical face possessing a suitable cone angle.",
"In principle a flat oblique face would also be possible.",
"Generally it is to be pointed out that the oblique faces do not necessarily have to have a straight form and it may be a question of curved or vault-like faces.",
"The holding pins 16 could possibly be in the form of integral components of the base bodies 3a and 3b, but it is preferred to adopt the structure employed in the working example in the form of separate components, which are attached or anchored to the respective base body 3a and 3b by suitable means.",
"More particularly, a detachable anchoring means may be provided for, each base body 3a and 3b having suitable attachment means 26 at mutually opposite connection faces, such means 26 allowing customized anchoring of a holding pin 16.This means that it is possible for the base bodies 3a and 3b to be fitted, if required, with holding pins 16 on those side faces, at which connection with an other base body is to take place.",
"In the working embodiment illustrated the holding pins 16 are screwed to the associated base body 3a and 3b.",
"For this purpose it is possible for the attachment means 26, as illustrated, to be in the form of threaded holes in the base bodies 3a and 3b at the connection faces 9a and 9b, into which holes a threaded shank 27 of the holding pins may be screwed.",
"The holding pins 16 are accordingly special-purpose or customized screws and preferably have a specially formed tool engaging portion 28—for instance a polygonal portion—for engagement with a wrench.",
"The mating working face 24 is preferably located on a radial projection 31 like a sort of surrounding annular collar, which by means of a shank-like intermediate portion 32—preferably with the tool engagement portion 28 in between—is connected with the screw shank 27 and projects beyond shank-like intermediate portion 32 radially.",
"Preferably, it is possible for the radial projection 31, as illustrated, to be constituted by a head section 33 of the respective holding pin 16.As clearly shown in FIGS.",
"2 and 3, in each case several and preferably two holding pins 16 are arranged on the two connection faces 9a and 9b, such holding pins being at a distance apart from each other.",
"As regards the central duct opening 8a and 8b it is more particularly possible to make a provision such that the two holding pins 16 are located on diametrally opposite sides of the associated duct opening 8a and 8b, the latter thus being flanked on diametrally opposite sides by same.",
"The centers of each respective duct opening 8a and 8b and of the holding pins 16 flanking same in this case preferably lie on a common imagery connection line 34, which runs at a right angle to the clamping direction 19.The coupling bodies 17 and 18, which are braced together, fit around the holding pins 16 preferably like cleats or curved clips, each of the coupling bodies having a recess 35 and 36 and a first and a second working face 22 and 23 for each holding pin 16 to partly receive same.",
"Accordingly in the working example each coupling body 17 and 18 has four working faces 22 and 23 with associated recesses 35 and 36, preferably completely surrounding the respective holding pin 16 in the state in which the parts are clamped together.",
"Because the holding pins 16 are placed within the periphery of the connection faces 9a and 9b, they may be placed as close as possible to the duct transition between the through duct 13 and the fluid ducts 4a and 4b so that the resulting connection forces Fv take effect in the immediate vicinity of the seals 15 and a reliable seal specific surface pressure is established.",
"However, the holding pins 16 may be placed near the edge of the respective connection face in order to render possible large flow cross sections of the through duct 13 and of the fluid ducts 4a and 4b.",
"Owing to the use of holding pins it is furthermore possible for extremely compact transverse dimensions to be designed for.",
"The holding pins 16 on the connection faces 9a and 9b on either side are preferably so arranged that same coaxially opposite each other in pairs.",
"Each holding pin 16 of the one base body 3a is therefore in line in the connection direction 5 with a holding pin 16 on the other base body 3b.",
"This ensures a symmetrical application of forces is such that the holding pins 16 of each aligned holding pin pair do not engage each other even when coupling bodies 17 and 18 are clamped so that the clamping operation is not interfered with.",
"On the contrary, it is even an advantage for the width, as measured in the connection direction 5, of the two coupling bodies 17 and 18 to be so set in relation to the length of the holding pins 16 that in the clamped state as well an intermediate space 27 will remain between the respectively mutually aligned holding pins 16, it being possible for clamping means to extend through the space 27.These clamping means 25 are constituted by screw connection means in the working embodiment, a clamping screw 38 being associated with pair of holding pins in alignment with each other, such screw 38 clamping together the two coupling bodies 17 and 18 and extending respectively through one of the intermediate spaces 37.This means that the lines of introduction or application of the connection force Fv and of the clamping force Fs may be in the direct vicinity of each other and preferably, as in the example, even intersect, something which ensures an optimum redirection of forces without the danger of action on the skew.",
"In the working embodiment illustrated there is a provision such that for the placement of a respective clamping screw 38 the one coupling body 18 possesses a through hole 41, which is in line with a threaded hole 42 in the other coupling body 17 so that the clamping screw 38 can be screwed into the threaded hole 42 after slipping it through the hole 41, such screw coming at its head 45 into engagement with the coupling body 18 having the through hole 41.The head 45 preferably fits into a counter-sunk recess at the end of the through hole 41.As shown clearly in FIG.",
"5 the two coupling bodies 17 and 18 are preferably so designed that in the braced together state they fit between a respective mating working face 24 and the connection face 9a and 9b facing same.",
"There is here a provision such that the coupling bodies 17 and 18 engage each other in the clamped state so that it is possible to achieve a limitation of the end connection force Fv in order to forestall danger owing to overloads.",
"As shown in FIG.",
"2 the through duct 13 preferably extends in only one of the coupling bodies, in the present case this being the second coupling body 18.This means that there is an uninterrupted, continuous duct wall surrounding and defining the through duct 13 and it is possible to do without seal means between the braced together coupling bodies 17 and 18.In order nevertheless to have a through duct 13 with maximum flow cross section, the through duct 13 is delimited at the side facing the first coupling body 17 by an outwardly bulging wall 43 in the second coupling body 18, which fits into a complementary recess in the first coupling body 17, which in this case may have an essentially U-like configuration.",
"Thus it is possible to ensure extremely compact transverse dimensions of the coupling unit 7 when the coupling bodies 17 and 18 are fitted together.",
"When the coupling unit 7 is mounted the joint zone, defined between the two coupling bodies 17 and 18 extends in the outer portion, associated with the holding pins 16, generally along the imaginary connection line 34 in order to depart from the said connection line 34 between the holding pins 16 in the direction 19 of clamping, for example along an arc around the through duct 13.It is furthermore to be noted that the connection means may also be employed for joining together bodies of other subassemblies besides devices serving for the treatment of compressed air, as for example valve bodies or module bodies in fluid distributing plates."
]
] | Patent_10018639 |
[
[
"Process for preparing optically active 2-[6-(hydroxy-methyl)-1,3-dioxan-4-yl] acetic acid derivatives",
"The present invention is to provide a production technology by which an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative, which are of value as pharmaceutical intermediates, can be produced from inexpensive and readily available starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor.",
"The present invention is a production process of an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative which comprises reacting an enolate, prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative with (S)-β-hydroxy-γ-butyrolactone at a temperature not lower than −30° C. to give a dihydroxyoxohexanoic acid derivative, treating the same with an acylating agent in the presence of a base to produce a dihydroxyoxohexanoic acid monoacyl derivative, reducing this compound with a microorganism to produce a trihydroxyhexanoic acid monoacyl derivative, treating this compound with an acetal-forming reagent in the presence of an acid catalyst to produce an acyloxymethyldioxanylacetic acid derivative, and finally, subjecting this compound to solvolysis in the presence of a base."
],
[
"1.A production process of a following compound (I); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R2 and R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, and R2 and R3 may jointly form a ring, which comprises (1) reacting an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative represented by the following formula (II); X1CH2CO2R1 (II) in the formula, R1 is as defined above; and X1 represents a hydrogen or a halogen atom, with (S)-β-hydroxy-γ-butyrolactone represented by the following formula (III); at a temperature not lower than −30° C. to produce a compound represented by the following formula (IV); in the formula, R1 is as defined above, (2) treating this compound with an acylating agent in the presence of a base to produce a compound represented by the following formula (V); in the formula, R1 is as defined above; and R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, (3) reducing this compound with a microorganism to produce a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, (4) treating this compound with an acetal-forming reagent in the presence of an acid catalyst to produce a compound represented by the following formula (VII); in the formula, R1, R2, R3, and R4 are as defined above, and (5) subjecting this compound to solvolysis in the presence of a base.",
"2.The production process according to claim 1 wherein X1 of the acetic acid ester derivative (II) is a hydrogen atom and a magnesium amide represented by the following formula (VIII); in the formula, R5 and R6 each independently represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group; and X2 represents a halogen atom, is used as the base in preparing the enolate.",
"3.The production process according to claim 2 wherein, of the magnesium amide (VIII), each of R5 and R6 is an isopropyl group and X2 is a chlorine atom.",
"4.The production process according to claim 1 wherein X1 of the acetic acid ester derivative (II) is a halogen atom and magnesium or zinc is used as the 0-valent metal in preparing the enolate.",
"wherein the reaction of the enolate with (S)-β-hydroxy-γ-butyrolactone (III) is carried out in the presence of a polyether.",
"6.The production process according to claim 5 wherein dimethoxyethane is used as the polyether.",
"7.The production process according to claim 1 wherein (S)-β-hydroxy-γ-butyrolactone represented by the following formula (III); is treated, in advance, with a Grignard reagent represented by the following formula (IX); R7—Mg—X3 (IX) in the formula, R7 represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and X3 represents a halogen atom, and reacted, at a temperature not lower than −30° C., with the enolate prepared by permitting the base or the 0-valent metal to act on the acetic acid ester derivative represented by the following formula (II); X1CH2CO2R1 (II) in the formula, R1 and X1 are as defined above, to produce the compound represented by the following formula (IV); in the formula, R1 is as defined above.",
"8.The production process according to claim 7 wherein, of the Grignard reagent (IX), R7 is a tert-butyl group and X3 is a chlorine atom.",
"9.The production process according to claim 1 wherein (S)-β-hydroxy-butyrolactone (III) is treated with a base and a magnesium compound in advance and reacted, at a temperature not lower than −30° C., with the enolate prepared by permitting the base or the 0-valent metal to act on the acetic acid ester derivative (II) to produce the compound represented by the above formula (IV).",
"10.The production process according to claim 9 wherein the base is sodium hydride, lithium diisopropylamide, or chloromagnesium diisopropylamide.",
"11.The production process according to claim 9 wherein the magnesium compound is magnesium chloride or magnesium bromide.",
"12.The production process according to claim 7 wherein X1 of the acetic acid ester derivative (II) is a hydrogen atom and a lithium amide represented by the following formula (X); in the formula, R8 and R9 each independently represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group, is used as the base in preparing the enolate.",
"13.The production process according to claim 12 wherein, of the lithium amide (X), each of R0 and R9 is an isopropyl group.",
"14.The production process according to claim 7 wherein X1 of the acetic acid ester derivative (II) is a halogen atom and magnesium or zinc is used as the 0-valent metal in preparing the enolate.",
"15.The production process according to claim 1 wherein, in the acylation step, a compound represented by the following formula (XI); or a compound represented by the following formula (XVI); in the above formulas, R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and Q represents a leaving group, is used as the acylating agent.",
"16.The production process according to claim 15 wherein Q of the acylating agent (XI) is a halogen atom.",
"17.The production process according to claim 16 wherein the halogen atom is a chlorine atom.",
"18.The production process according to claim 1 wherein an amine is used as the base in the acylation step.",
"19.The production process according to claim 18 wherein triethylamine or pyridine is used as the amine.",
"20.The production process according to claim 1 wherein, in the acylation step, the compound contaminated with an impurity and represented by the following formula (V); in the formula, R1 and R4 are as defined above, is treated with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (V) and the compound represented by the above formula (V) is obtained in a crystal form.",
"21.The production process according to claim 20 wherein the impurity contaminating the compound represented by the above formula (V) is a compound represented by the following formula (XII); in the formula, R1 and R4 are as defined above.",
"22.The production process according to claim 20 wherein the aliphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane, heptane, octane, or isooctane.",
"23.The production process according to claim 20 wherein the crystallization is carried out with additional use of an auxiliary solvent, said solvent being used for a purpose of improving at least one of a solubility, yield, treatment concentration, effect of purification, and physical properties of obtainable crystals of the compound represented by the above formula (V).",
"24.The production process according to claim 23 wherein the auxiliary solvent is used in such an amount that the weight ratio of said auxiliary solvent and the aliphatic hydrocarbon solvent (said auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization.",
"25.The production process according to claim 23 wherein the auxiliary solvent is at least one species selected from the group consisting of toluene, ethyl acetate, methyl tert-butyl ether and methylene chloride.",
"26.The production process according to claim 1 wherein a culture broth, cells or processed cells of the microorganism is used in the reduction step using the microorganism, said microorganism for use being selected from among microorganisms belonging to the genera Ashbya, Botryoascus, Brettanomyces, Candida, Citeromyces, Clavispora, Cryptococcus, Debaryomyces, Dekkera, Dipodascus, Galactomyces, Geotrichum, Hanseniaspora, Hansenula, Hormoascus, Hyphopichia, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Metschnikowia.",
"Nakazawaea, Ogataea, Pachysolen, Pichia, Rhodotorula, Rhodsporidium, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Torulaspora, Torulopsis, Trichosporon, Trigonopsis, Willopsis, Yamadazyma, Zygosaccharomyces, Acidiphilium, Aerobacter, Alcaligenes, Arthrobacter, Aureobacterium, Bacillus, Brevibacterium, Buttiauxella, Cedecea, Cellulomonas.",
"Citrobacter, Clostridium, Comamonas, Corynebacterium, Enterobacter, Erwinia.",
"Escherichia, Flavobacterium, Klebsiella, Luteococcus, Microbacterium, Micrococcus, Ochrobactrum, Proteus, Providencia, Pseudomonas, Rhodococcus, Sarcina, Serratia, Sphingobacterium, Tsukamurella, Absidia, Acremonium, Aegerita, Agrocybe, Amylostereum, Aspergillus, Byssochlamys, Chaetomidium, Chaetosartorya, Cladosporium, Coprinus, Crinipellis, Endophragmia, Flavolus, Fomitopsis, Fusarium, Ganoderma, Glomerella, Laetiporus, Lentinus, Lenzites, Macrophoma, Monascus, Mortierella, Paecilomyces, Penicillium, Phialophora, Pholiota, Pleurotus, Scopulariopsis, Sehizophyllum, Sporotrichum, Zygorhynchus, Microtetraspora, and Streptomyces.",
"27.The production process according to claim 1 wherein the microorganism for use in the reduction step using the microorganism is selected from the group consisting of Ashbya gossypii, Botryoascus synnaedendrus, Brettanomyces custersianus, Candida arborea, Candida catenulata, Candida fennica, Candida galucla, Candida haemulonii, Candida magnoliae, Candida musae, Candida nitratophila, Candida parapsilosis, Candida pararugosa, Candida stellata, Citeromyces matritensis, Clavispora lusitaniae, Cryptococcus laurentii, Debaryomyces carsonii, Debaryomyces hansenii var.",
"fabryi, Debaryomyces hansenii var.",
"hansenii, Debaryomyces kloeckeri, Debaryomyces marama, Debaryomyces pseudopolymorphus, Debaryomyces robertsiae, Debaryomyces sp., Dekkera anomala, Dipodascus armillariae, Dipodascus ovetensis, Dipodascus tetrasperma, Galactomyces reessii, Geotrichum candidum, Geotrichum fermentans, Geotrichum fragrans, Geotrichum loubieri, Hanseniaspora guilliermondii, Hansenula methanolosa, Hansenula polymorpha.",
"Hormoascus philentomus, Hormoascus platypodis, Hyphopichia burtonii, Issatchenkia orientalis, Issatchenkia terricola, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi, Metschnikowia bicuspidata, Metschnikowia pulcherrima, Nakazawaea holstii, Ogataea minuta var.",
"minuta, Ogataea pini, Ogataea polymorpha, Ogataea wickerhamii, Pachysolen tannophilus, Pichia canadensis, Pichia farinose, Pichia jandinii, Pichia saitoi, Pichia toletana, Pichia triangularis, Pichia wickerhamii, Rhodotorula graminis, Rhodotorula minula, Rhodsporidium dlobovatum, Rhodsporidium toruloides, Saccharomyces bayanus, Saccharomyces pastorianus.",
"Saccharomyces rosei, Saccharomyces sake, Saccharomyces steineri, Saccaromyces unisporus, Saccharomycodes ludwigii, Saccharomycopsis capsularis, Saccharomycopsis malanga, Saturnospora dispora, Schizoblastosporion kobayasii, Schizosaccharomyces pombe, Schwanniomyces occidentalis var.",
"occidentalis, Sporidiobolus johnsonii, Sporobolomyces pararoseus, Sporobolomyces salmonicolor, Torulaspora delbrueckii, Torulopsis methanolevescens, Torulopsis osboenis, Torulopsis sp., Torulopsis uvae, Trichosporon pullulans, Trichosporon sp.",
"Trigonopsis variabilis, Willopsis saturnus var.",
"mrakii, Willopsis saturnus var.",
"saturnus, Yamadazyma farinosa, Yamadazyma haplophila, Zygosaccharomyces naniwensis, Zygosaccharomyces sp., Acidiphilium cryptum, Aerobacter cloacae, Alcaligenes xylosoxidans, Alcaligenes xylosoxidans subsp.",
"denitrificans, Arthrobacter globiformis, Arthrobacter protophormiae, Aureobacterium esteraromaticum, Bacillus badius, Bacillus sphaericus, Brevibacterium ammomiagenes, Buttiauxella agrestis, Cedecea davisiae, Cellulomonas sp., Cellulomonas turbata, Citrobacter freundii, Clostridium cylindrosporum, Comamonas testosteroni, Corynebacterium acectoacidophilum, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Corynebacterium glutamicus, Enterobacter aerogenes, Enterobacter cloacae, Erwinia carotovora subsp.",
"carotovora, Escherichia coli, Flavobacterium flavesceus, Klebsiella planticola, Luteococcus japonicus, Microbacterium arborescens, Micrococcus flavus, Micrococcus luteus, Ochrobactrum sp., Proteus inconstans, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Rhodococcus equi, Sarcina lutea, Serratia plymuthicum, Serratia proteamaculans subsp.",
"proteamaculans, Sphingobacterium spiritivorum, Tsukamurella paurometabolum, Absidia orchidis, Acremonium bacillisporum, Aegerita candida, Agrocybe cylindracea, Amylostereum areolatum, Aspergillus parasiticus, Aspergillus phoenicis, Byssochlamys fulva, Chaetomidium fimeti, Chaetosartorya stromatoides, Cladosporium resinae F. avellaneum, Coprinus cinereus, Coprinus lagopus, Coprinus sp., Crinipellis stipitaria, Endophragmia alternata, Flavolus arcularius, Fomitopsis pubertatis, Fusarium merismoides, Ganoderma lucidum, Glomerella cingulata, Laetiporus sulphureus, Lentinus lepideus, Lenzites betulina, Macrophoma commelinae, Monascus purpureus, Mortierella isabellina, Paecilomyces varioti, Penicillium chermesinum, Penicillium chrysogenum, Penicillium expansum, Penicillium lilacinium, Phialophora fastigiata, Pholiota aurivella, Pholiota limonella, Pleurotus dryinus, Pleurotus ostreatus, Pleurotus porrigens, Scopulariopsis brevicaulis, Sehizophyllum commune, Sporotrichum aurantiacum, Zygorhynchus moelleri, Microtetraspora roseoviolacea, Streptomyces achromogenes subsp.",
"rubradiris, Streptomyces sp.",
"and Streptomyces aureus.",
"28.The production process according to claim 1 wherein an amine salt composed of an acid and an amine is used as the acid catalyst in the acetal-forming step.",
"29.The production process according to claim 28 wherein the amine salt is prepared and used in situ.",
"30.The production process according to claim 28 wherein the acid is hydrogen chloride, hydrogen bromide, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or trifluoroacetic acid.",
"31.The production process according to claim 28 wherein the amine is a tertiary amine.",
"32.The production process according to claim 31 wherein the tertiary amine is triethylamine, N-methylmorpholine, diisopropylethylamine, pyridine, 2-methylpyridine, 3-methylpyridine, or imidazole.",
"33.The production process according to claim 28 wherein the amine is used in an excess amount relative to the acid.",
"34.The production process according to claim 1 wherein the acetal-forming reagent is 2,2-dimethoxypropane.",
"35.The production process according to claim 1 wherein a compound contaminated with an impurity and represented by the following formula (VII); in the formula, R1, R2, R3 and R4 are as defined above, is treated with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (VII) and the compound represented by the above formula (VII) is obtained in a crystal form.",
"36.The production process according to claim 35 wherein the impurity contaminating the compound represented by the above formula (VII) is at least one compound selected from the group consisting of a compound represented by the following formula (XIII); in the formula, R2, R3 and R4 are as defined above; and R10 represents a lower alkyl group and is different from R1, a diastereomer represented by the following formula (XIV); in the formula, R1, R2, R3, and R4 are as defined above, a compound represented by the following formula (XV); in the formula, R4 is as defined above, and a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above.",
"37.The production process according to claim 36 wherein R10 of the compound (XIII) is a methyl group.",
"38.The production process according to claim 35 wherein the aliphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane, heptane, octane, or isooctane.",
"39.The production process according to claim 35 wherein the crystallization is carried out with additional use of an auxiliary solvent, said solvent being used for a purpose of improving at least one of a solubility, yield, treatment concentration, effect of purification, and physical properties of obtainable crystals of the compound represented by the above formula (VII).",
"40.The production process according to claim 39 wherein the auxiliary solvent is used in such an amount that the weight ratio of said auxiliary solvent and the aliphatic hydrocarbon solvent (said auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization.",
"41.The production process according to claim 39 wherein the auxiliary solvent is at least one species selected from the group consisting of toluene, ethyl acetate, methyl tert-butyl ether and methylene chloride.",
"42.The production process according to claim 1 wherein R1 is a tert-butyl group.",
"43.The production process according to claim 1 wherein each of R2 and R3 is a methyl group.",
"44.The production process according to claim 1 wherein 14 is a phenyl group.",
"45.An isolation/purification process which comprises treating a compound contaminated with an impurity and represented by the following formula (V); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (V) and obtaining the compound represented by the above formula (V) in a crystal form.",
"46.The isolation/purification process according to claim 45 wherein the impurity contaminating the compound of the above formula (V) is a compound represented by the following formula (XII); in the formula.",
"R1 and R4 are as defined above.",
"47.The isolation/purification process according to claim 45 wherein the aliphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane, heptane, octane, or isooctane 48.The isolation/purification process according to claim 45 wherein the crystallization is carried out with additional use of an auxiliary solvent, said solvent being used for a purpose of improving at least one of a solubility, yield, treatment concentration, effect of purification, and physical properties of obtainable crystals of the compound represented by the above formula (V).",
"49.The isolation/purification process according to claim 48 wherein the auxiliary solvent is used in such an amount that the weight ratio of said auxiliary solvent and the aliphatic hydrocarbon solvent (said auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization.",
"50.The isolation/purification process according to claim 48 wherein the auxiliary solvent is at least one species selected from the group consisting of toluene, ethyl acetate, methyl tert-butyl ether and methylene chloride.",
"51.The isolation/purification process according to claim 45 wherein the compound represented by the above formula (V) is used, said compound being produced by treating a compound represented by the following formula (IV); in the formula, R1 is as defined above, with an acylating agent in the presence of a base.",
"52.The isolation/purification process according to claim 51 wherein a compound represented by the following formula (XI); or a compound represented by the following formula (XVI); in the above formulas, R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and Q represents a leaving group, is used as the acylating agent.",
"53.The isolation/purification process according to claim 52 wherein Q of the acylating agent (XI) is a halogen atom.",
"54.The isolation/purification process according to claim 53 wherein the halogen atom is a chlorine atom.",
"55.The isolation/purification process according to claim 51 wherein an amine is used as the base.",
"56.The isolation/purification process according to claim 55 wherein triethylamine or pyridine is used as the amine used in the acylation step.",
"57.The isolation/purification process according to claim 51 wherein the compound represented by the above formula (IV) is used, said compound being produced by reacting an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative represented by the following formula (II); X1CH2CO2R1 (II) in the formula, R1 is an defined above; and X1 represents a hydrogen or a halogen atom, with (S)-γ-hydroxy-γ-butyrolactone represented by the formula (III); at a temperature not lower than −30° C. 58.The isolation/purification process according to claim 57 wherein X1 of the acetic acid ester derivative (IT) is a hydrogen atom and a magnesium amide represented by the following formula (VIII); in the formula, R1 and R6 each independently represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group; and X2 represents a halogen atom, is used as the base in preparing the enolate.",
"59.The isolation/purification process according to claim 58 wherein, of the magnesium amide (VIII), each of R5 and R6 is an isopropyl group and X2 is a chlorine atom.",
"60.The isolation/purification process according to claim 57 wherein X1 of the acetic acid ester derivative (II) is a halogen atom and magnesium or zinc is used as the 0-valent metal in preparing the enolate.",
"61.The isolation/purification process according to claim 57 wherein the reaction of the enolate with (S)-β-hydroxy-γ-butyrolactone (Ill) is carried out in the presence of a polyether.",
"62.The isolation/purification process according to claim 61 wherein dimethoxyethane is used as the polyether.",
"63.The isolation/purification process according to claim 51 wherein the compound represented by the above formula (IV) is used, said compound being produced by treating, in advance, (S)-β-hydroxy-γ-butyrolactone (III) with a Grignard reagent represented by the following formula (IX); R7—Mg—X3 (IX) in the formula, R7 represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and X3 represents a halogen atom, and reacting, at a temperature not lower than −30° C., with an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative (II).",
"64.The isolation/purification process according to claim 63 wherein, of the Grignard reagent (IX), R7 is a tert-butyl group and X3 is a chlorine atom.",
"65.The isolation/purification process according to claim 51 wherein the compound represented by the above formula (IV) is used, said compound being produced by treating, in advance, (S)-β-hydroxy-γ-butyrolactone (III) with a base and a magnesium compound, and reacting, at a temperature not lower than −30° C., with an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative (II).",
"66.The isolation/purification process according to claim 65 wherein the base is sodium hydride, lithium diisopropylamide, or chloromagnesium diisopropylamide.",
"67.The isolation/purification process according to claim 65 wherein the magnesium compound is magnesium chloride or magnesium bromide.",
"68.The isolation/purification process according to claim 63 wherein X1 of the acetic acid ester derivative (II) is a hydrogen atom and a lithium amide represented by the following formula (X); in the formula, R8 and R9 each independently represents an alkyl group of 1 to 12 carbon atoms, tin aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group, is used as the base in preparing the enolate.",
"69.The isolation/purification process according to claim 68 wherein, of the lithium amide (X), each of R8 and R9 represents an isopropyl group.",
"70.The isolation/purification process according to claim 63 wherein X1 of the acetic acid ester derivative (II) is a halogen atom and magnesium or zinc is used as the 0-valent metal in preparing the enolate.",
"71.The isolation/purification process according to claim 45 wherein R1 is a tert-butyl group.",
"72.The isolation/purification process according to claim 45 wherein R1 is a phenyl group.",
"73.A production process of a compound represented by the following formula (VI); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, which comprises reducing a compound represented by the following formula (V); in the formula, R1 and R4 are as defined above, with a microorganism.",
"74.The production process according to claim 73 wherein a culture broth, cells or processed cells of the microorganism is used, said microorganism being selected from among microorganisms belonging to the genera Ashbya, Botryoascus, Brettanomyces, Candida, Citeromyces, Clavispora, Cryptococcus, Debaryomyces, Dekkera, Dipodascus, Galactomyces, Geotrichum, Hanseniaspora, Hansenula, Hormoascus, Hyphopichia, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Metschnikowia, Nakazawaea, Ogataea, Pachysolen, Pichia, Rhodotorula, Rhodsporidium, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Torulaspora, Torulopsis, Trichosporon, Trigonopsis, Willopsis, Yamadazyma, Zygosaccharomyces, Acidiphilium, Aerobacter, Alcaligenes, Arthrobacter, Aureobacterium, Bacillus, Brevibacterium, Buttiauxella, Cedecea, Cellulomonas, Citrobacter, Clostridium, Comamonas, Corynebacterium, Enterobacter, Erwinia, Escherichia, Flavobacterium, Klebsiella, Luteococcus, Microbacterium, Micrococcus, Ochrobactrum, Proteus, Providencia, Pseudomonas, Rhodococcus, Sarcina, Serratia, Sphingobacterium, Tsukamurella, Absidia, Acremonium, Aegerita, Agrocybe, Amylostereum, Aspergillus, Byssochlamys, Chaetomidium, Chaetosartorya, Cladosporium, Coprinus, Crinipellis, Endophragmia, Flavolus.",
"Fomitopsis, Fusarium, Ganoderma, Glomerella, Laetiporus, Lentinus, Lenzites, Macrophoma, Monascus, Mortierella, Paecilomyces, Penicillium, Phialophora, Pholiota, Pleurotus, Scopulariopsis, Sehizophyllum, Sporotrichum, Zygorhynchus, Microtetraspora, and Streptomyces.",
"75.The production process according to claim 73 wherein the microorganism is selected from the group consisting of Ashbya gossypii, Botryoascus synnaedendrus, Brettanomyces custersianus, Candida arborea, Candida catenulata, Candida fennica, Candida galacta, Candida haemulonii, Candida magnoliae, Candida musae, Candida nitratophila, Candida parapsilosis, Candida pararugosa, Candida stellata, Citeromyces matritensis, Clavispora lusitaniae, Cryptococcus laurentii, Debaryomyces carsonii, Debaryomyces hansenii var.",
"fabryi, Debaryomyces hansenii var.",
"hansenii, Debaryomyces kloeckeri, Debaryomyces marama, Debaryomyces pseudopolymorphus, Debaryomyces robertsiae, Debaryomyces sp., Dekkera anomala, Dipodascus armillariae, Dipodascus ovetensis, Dipodascus tetrasperma, Galactomyces reessii, Geotrichum candidum, Geotrichum fermentans, Geotrichum fragrans, Geotrichum loubieri, Hanseniaspora guilliermondii, Hansenula methanolosa, Hansenula polymorpha, Hormoascus philentomus, Hormoascus platypodis, Hyphopichia burtonii, Issatchenkia orientalis, Issatchenkia terricola, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi, Metschnikowia bicuspidata, Metschnikowia pulcherrima, Nakazawaea holstii, Ogataea minuta var.",
"minuta, Ogataea pini, Ogataea polymorpha, Ogataea wickerhamii, Pachysolen tannophilus, Pichia canadensis, Pichia farinose, Pichia jandinii, Pichia saitoi, Pichia toletana, Pichia triangularis, Pichia wickerhamii, Rhodotorula graminis, Rhodotorula minuta, Rhodsporidium diobavatum, Rhodsporidium toruloides, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces rosei.",
"Saccharomyces sake, Saccharomyces steineri, Saccaromyces unisporus, Saccharomycodes ludwigii, Saccharomycopsis capsularis, Saccharomycopsis malanga, Saturnospora dispora, Schizoblastosporion kobayasii, Schizosaccharomyces pombe, Schwanniomyces occidentalis var.",
"occidentalis, Sporidiobolus johnsonii, Sporobolomyces pararoseus, Sporobolomyces salmonicolor, Torulaspora delbrueckii, Torulopsis methanolevescens, Torulopsis osboenis, Torulopsis sp.",
"Torulopsis uvae, Trichosporon pullulans, Trichosporon sp.",
"Trigonopsis variabilis, Willopsis saturnus var.",
"mrakii, Willopsis saturnus var.",
"saturnus, Yamadazyma farinosa, Yamadazyma haplophila, Zygosaccharomyces naniwensis, Zygosaccharomyces sp., Acidiphilium cryptum, Aerobacter cloacae, Alcaligenes xylosoxidans, Alcaligenes xylosoxidans subsp.",
"denitrificans, Arthrobacter globiformis, Arthrobacter protophormiae, Aureobacterium esteraromaticum, Bacillus badius, Bacillus sphaericus, Brevibacterium ammomiagenes, Buttiauxella agrestis, Cedecea davisiae, Cellulomonas sp., Cellulomonas turbata, Citrobacter freundii, Clostridium cylindrosporum, Comamonas testosteroni, Corynebacterium acectoacidophilum, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Corynebacterium glutamicus, Enterobacter aerogenes, Enterobacter cloacae, Erwinia carotovora subsp.",
"carotovora, Escherichia coli, Flavobacterium flavesceus, Klebsiella planticola, Luteococcus japonicus, Microbacterium arborescens, Micrococcus flavus, Micrococcus luteus, Ochrobactrum sp., Proteus inconstans, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Providencia stuartii, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Rhodococcus equi, Sarcina lutea, Serratia plymuthicum, Serratia proteamaculans subsp.",
"proteamaculans, Sphingobacterium spiritivorum, Tsukamurella paurometabolum, Absidia orchidis, Acremonium bacillisporum, Aegerita candida, Agrocybe cylindracea, Amylostereum areolatum, Aspergillus parasiticus, Aspergillus phoenicis, Byssochlamys fulva, Chaetomidium fimeti, Chaetosartorya stromatoides, Cladosporium resinae F. avellaneum, Coprinus cinereus, Coprinus lagopus, Coprinus sp., Crinipellis stipitaria, Endophragmia alternata, Flavolus arcularius, Fomitopsis pubertatis, Fusarium merismoides, Ganoderma lucidum, Glomerella cingulata, Laetiporus sulphureus, Lentinus lepideus, Lenzites betulina, Macrophoma commelinae, Monascus purpureus, Mortierella isabellina, Paecilomyces varioti, Penicillium chermesinum, Penicillium chrysogenum, Penicillium expansum, Penicillium lilacinium, Phialophora fastigiata, Pholiota aurivella, Pholiota limonella, Pleurotus dryinus, Pleurotus ostreatus, Pleurotus porrigens, Scopulariopsis brevicaulis, Sehizophyllum commune, Sporotrichum aurantiacum, Zygorhynchus moelleri, Microtetraspora roseoviolacea, Streptomyces achromogenes subsp.",
"rubradiris, Streptomyces sp.",
"and Streptomyces aureus.",
"76.The production process according to claim 73 wherein R1 is a tert-butyl group.",
"77.The production process according to claim 73 wherein R4 is a phenyl group.",
"78.A production process of a compound represented by the following formula (VII); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R2 and R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R2 and R3 may jointly form a ring, which comprises treating a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, with an acetal-forming reagent using an amine salt composed of an acid and an amine as a catalyst.",
"79.The production process according to claim 78 wherein the amine salt is prepared and used in situ.",
"80.The production process according to claim 78 wherein the acid is hydrogen chloride, hydrogen bromide, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or trifluoroacetic acid.",
"81.The production process according to claim 78 wherein the amine is a tertiary amine.",
"82.The production process according to claim 81 wherein the tertiary amine is triethylamine, N-methylmorpholine, diisopropylethylamine, pyridine, 2-methylpyridine, 3-methylpyridine, or imidazole.",
"83.The production process according to claim 78 wherein the amine is used in an excess amount relative to the acid.",
"84.The production process according to claim 78 wherein the acetal-forming reagent is 2,2-dimethoxypropane.",
"85.The production process according to claim 78 wherein R1 is a tert-butyl group.",
"86.The production process according to claim 78 wherein R4 is a phenyl group.",
"87.The production process according to claim 78 wherein each of R2 and R3 is a methyl group.",
"88.An isolation/purification process which comprises treating a compound represented by the following formula (VI); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, with an acetal-forming reagent in the presence of an acid catalyst to thereby convert the same to a compound represented by the following formula (VII); in the formula, R1 and R4 are as defined above; R2 mid R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R2 and R3 may jointly form a ring, treating the compound contaminated with an impurity and represented by the above formula (VII) with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (VII) and obtaining the compound represented by the above formula (VII) in a crystal form.",
"89.The isolation/purification process according to claim 88 wherein the impurity contaminating the compound represented by the above formula (VII) is at least one compound selected from the group consisting of a compound represented by the following formula (XIII); in the formula, R2, R3 and R4 are as defined above; and R10 represents a lower alkyl group and is different from R1, a diastereomer represented by the following formula (XIV); in the formula, R1, R2, R3, and R4 are as defined above, a compound represented by the following formula (XV); in the formula R4 is as defined above, and a compound represented by the following formula (VT); in the formula, R1 and R4 are as defined above.",
"90.The isolation/purification process according to claim 88 wherein the aliphatic hydrocarbon solvent is pentane, hexane, methylcyclohexane, heptane, octane, or isooctane.",
"91.The isolation/purification process according to claim 88 wherein the crystallization is carried out with additional use of an auxiliary solvent, said solvent being used for a purpose of improving at least one of a solubility, yield, treatment concentration, effect of purification, and physical properties of obtainable crystals of the compound represented by the above formula (VII).",
"92.The isolation/purification process according to claim 91 wherein the auxiliary solvent is used in such an amount that the weight ratio of said auxiliary solvent and the aliphatic hydrocarbon solvent (said auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization.",
"93.The isolation/purification process according to claim 91 wherein the auxiliary solvent is at least one species selected from the group consisting of toluene, ethyl acetate, methyl tert-butyl ether and methylene chloride.",
"94.The isolation/purification process according to claim 88 wherein an amine salt composed of an acid and an amine is used as the acid catalyst.",
"95.The isolation/purification process according to claim 94 wherein the amine salt is prepared and used in situ.",
"96.The isolation/purification process according to claim 94 wherein the acid is hydrogen chloride, hydrogen bromide, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or trifluoroacetic acid.",
"97.The isolation/purification process according to claim 94 wherein the amine is a tertiary amine.",
"98.The isolation/purification process according to claim 97 wherein the tertiary amine is triethylamine, N-methylmorpholine, diisopropylethylamine, pyridine, 2-methylpyridine, 3-methylpyridine, or imidazole.",
"99.The isolation/purification process according to claim 94 wherein the amine is used in an excess amount relative to the acid.",
"100.The isolation/purification process according to claim 88 wherein the acetal-forming reagent is 2,2-dimethoxypropane.",
"101.The isolation/purification process according to claim 88 wherein R1 is a tert-butyl group and R10 is a methyl group.",
"102.The isolation/purification process according to claim 88 wherein R4 is a phenyl group.",
"103.The isolation/purification process according to claim 88 wherein each of R2 and R3 is a methyl group."
],
[
"<SOH> BACKGROUND ART <EOH>The hitherto-known technology for producing 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives includes the following processes.",
"(1) A process starting with 3-hydroxy-γ-butyrolactone to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 3,5-dihydroxyhexanoic ester derivative (JP-A-04-173767).",
"(2) A process starting with 3,4-dihydroxybutyronitrile acetonide to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 3,5-dihydroxyhexanoic ester derivative (JP-A-O 2 -262537).",
"(3) A process starting with a 4-chloroacetoacetic acid ester to synthesize a 3,5,6-trihydroxyhexanoic ester derivative through benzyloxylation, reduction, chain extension and like steps.",
"(JP-A-06-65226).",
"(4) A process starting with a 4-chloro-3-hydroxybutyric ester to synthesize a 3,5,6-trihydroxyhexanoic ester derivative through chain extension, reduction and like steps.",
"(U.S. Pat.",
"No.",
"5,278,313).",
"(5) A process starting with malic acid to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 2,4-dihydroxyadipic acid derivative (JP-A-04-69355).",
"However, these processes involve ultra-low temperature reactions around −80° C. in some stage or other of the respective production processes (1,2,4 and 5) or a high-pressure hydrogenation reaction requiring a pressure of as high as 100 kg/cm 2 (3), thus invariably requiring extraordinary reaction equipment.",
"Moreover, expensive starting materials are used in some or other stages, so that none of the processes are efficient enough for industrial-scale production."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In the above state of the art, the object of the present invention is to provide a production technology by which an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative represented by the following formula (I), which are of value as pharmaceutical intermediates, can be produced with ease and high efficiency from inexpensive starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor; in the formula, R 1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R 2 and R 3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R 2 and R 3 may jointly form a ring.",
"Under the circumstances, the inventors of the present invention carried out intensive investigations and, as a consequence, developed an expedient technology for producing optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives of the following formula (I) from inexpensive, readily available starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor; in the formula, R 1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R 2 and R 3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R 2 and R 3 may jointly form a ring.",
"The present invention, therefore, is directed to a production process of an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative represented by the general formula (I); in the formula, R 1 , R 2 and R 3 are as defined above, which comprises (1) reacting an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative represented by the following formula (II); in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"X 1 CH 2 CO 2 R 1 (II) in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"in the formula, R 1 is as defined above; and X 1 represents a hydrogen or a halogen atom, with (S)-β-hydroxy-γ-butyrolactone represented by the following formula (III); at a temperature not lower than −30° C. to produce a compound represented by the following formula (IV); in the formula, R 1 is as defined above, (2) treating this compound with an acylating agent in the presence of a base to produce a compound represented by the following formula (V); in the formula, R 1 is as defined above; and R 4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, (3) reducing this compound with a microorganism to produce a compound represented by the following formula (VI); in the formula, R 1 and R 4 are as defined above, (4) treating this compound with an acetal-forming reagent in the presence of an acid catalyst to produce a compound represented by the following formula (VII); in the formula, R 1 , R 2 , R 3 , and R 4 are as defined above, and (5) subjecting this compound to solvolysis in the presence of a base.",
"The present invention is also directed to an isolation/purification process which comprises treating a compound contaminated with an impurity and represented by the following formula (V); in the formula, R 1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R 4 represents a hydrogen, an alkyl group of 1 and 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 and 12 carbon atoms, with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (V) and obtaining the compound represented by the above formula (V) in a crystal form.",
"Furthermore, the present invention is directed to a production process of a compound represented by the following formula (VI); in the formula, R 1 and R 4 are as defined above, which comprises reducing a compound represented by the following formula (V); in the formula, R 1 and R 4 are as defined above, with a microorganism.",
"In another aspect, the present invention is directed to a production process of a compound represented by the following formula (VII); in the formula, R 1 , R 2 , R 3 and R 4 are as defined above, which comprises treating a compound represented by the following formula (VI); in the formula, R 1 and R 4 are as defined above, with an acetal-forming reagent using an amine salt composed of an acid and an amine as a catalyst.",
"In still another aspect, the present invention is directed to an isolation/purification process which comprises treating a compound represented by the following formula (VI); in the formula, R 1 and R 4 are as defined above, with an acetal-forming reagent in the presence of an acid catalyst to thereby convert the same to a compound represented by the following formula (VII); in the formula, R 1 , R 2 , R 3 and R 4 are as defined above, treating the compound contaminated with an impurity and represented by the above formula (VII) with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (VII) and obtaining the compound represented by the above formula (VII) in a crystal form."
],
[
"TECHNICAL FIELD The present invention relates to pharmaceutical intermediates, particularly optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives of value as intermediates of HMG-CoA reductase inhibitors.",
"BACKGROUND ART The hitherto-known technology for producing 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives includes the following processes.",
"(1) A process starting with 3-hydroxy-γ-butyrolactone to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 3,5-dihydroxyhexanoic ester derivative (JP-A-04-173767).",
"(2) A process starting with 3,4-dihydroxybutyronitrile acetonide to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 3,5-dihydroxyhexanoic ester derivative (JP-A-O2-262537).",
"(3) A process starting with a 4-chloroacetoacetic acid ester to synthesize a 3,5,6-trihydroxyhexanoic ester derivative through benzyloxylation, reduction, chain extension and like steps.",
"(JP-A-06-65226).",
"(4) A process starting with a 4-chloro-3-hydroxybutyric ester to synthesize a 3,5,6-trihydroxyhexanoic ester derivative through chain extension, reduction and like steps.",
"(U.S. Pat.",
"No.",
"5,278,313).",
"(5) A process starting with malic acid to synthesize a 3,5,6-trihydroxyhexanoic ester derivative via a 2,4-dihydroxyadipic acid derivative (JP-A-04-69355).",
"However, these processes involve ultra-low temperature reactions around −80° C. in some stage or other of the respective production processes (1,2,4 and 5) or a high-pressure hydrogenation reaction requiring a pressure of as high as 100 kg/cm2 (3), thus invariably requiring extraordinary reaction equipment.",
"Moreover, expensive starting materials are used in some or other stages, so that none of the processes are efficient enough for industrial-scale production.",
"SUMMARY OF THE INVENTION In the above state of the art, the object of the present invention is to provide a production technology by which an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative represented by the following formula (I), which are of value as pharmaceutical intermediates, can be produced with ease and high efficiency from inexpensive starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor; in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R2 and R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R2 and R3 may jointly form a ring.",
"Under the circumstances, the inventors of the present invention carried out intensive investigations and, as a consequence, developed an expedient technology for producing optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives of the following formula (I) from inexpensive, readily available starting materials without using any extraordinary equipment such as an ultra-low-temperature reactor; in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; R2 and R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R2 and R3 may jointly form a ring.",
"The present invention, therefore, is directed to a production process of an optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivative represented by the general formula (I); in the formula, R1, R2 and R3 are as defined above, which comprises (1) reacting an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative represented by the following formula (II); X1CH2CO2R1 (II) in the formula, R1 is as defined above; and X1 represents a hydrogen or a halogen atom, with (S)-β-hydroxy-γ-butyrolactone represented by the following formula (III); at a temperature not lower than −30° C. to produce a compound represented by the following formula (IV); in the formula, R1 is as defined above, (2) treating this compound with an acylating agent in the presence of a base to produce a compound represented by the following formula (V); in the formula, R1 is as defined above; and R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, (3) reducing this compound with a microorganism to produce a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, (4) treating this compound with an acetal-forming reagent in the presence of an acid catalyst to produce a compound represented by the following formula (VII); in the formula, R1, R2, R3, and R4 are as defined above, and (5) subjecting this compound to solvolysis in the presence of a base.",
"The present invention is also directed to an isolation/purification process which comprises treating a compound contaminated with an impurity and represented by the following formula (V); in the formula, R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms; and R4 represents a hydrogen, an alkyl group of 1 and 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 and 12 carbon atoms, with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (V) and obtaining the compound represented by the above formula (V) in a crystal form.",
"Furthermore, the present invention is directed to a production process of a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, which comprises reducing a compound represented by the following formula (V); in the formula, R1 and R4 are as defined above, with a microorganism.",
"In another aspect, the present invention is directed to a production process of a compound represented by the following formula (VII); in the formula, R1, R2, R3 and R4 are as defined above, which comprises treating a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, with an acetal-forming reagent using an amine salt composed of an acid and an amine as a catalyst.",
"In still another aspect, the present invention is directed to an isolation/purification process which comprises treating a compound represented by the following formula (VI); in the formula, R1 and R4 are as defined above, with an acetal-forming reagent in the presence of an acid catalyst to thereby convert the same to a compound represented by the following formula (VII); in the formula, R1, R2, R3 and R4 are as defined above, treating the compound contaminated with an impurity and represented by the above formula (VII) with an aliphatic hydrocarbon solvent to remove the impurity contaminating the compound represented by the above formula (VII) and obtaining the compound represented by the above formula (VII) in a crystal form.",
"DISCLOSURE OF INVENTION The present invention is now described in detail.",
"As illustrated in the following reaction scheme, the present invention comprises five non-ultra-low temperature reaction steps (1) through (5).",
"The present invention is now described, step by step, in detail.",
"Step (1) In this step, an enolate prepared by permitting a base or a 0-valent metal to act on an acetic acid ester derivative represented by the following formula (II); X1CH2CO2R1 (II) is reacted with (S)-β-hydroxy-γ-butyrolactone represented by the following compound (III); at a temperature not lower than −30° C. to produce a (5S)-configured dihydroxyoxohexanoic acid derivative represented by the following formula (IV); Generally when a reaction involving the enolate of an acetic acid ester or the like is carried out in a non-ultra-low temperature reaction over, for example, not lower than −30° C., the self-condensation of the enolate proceeds preferentially to markedly sacrifice the conversion rate of the objective reaction.",
"However, it was found that according to the following technique developed by the present inventors, the self-condensation of the acetic acid enolate can be minimized and the objective reaction can be carried through with good product yield.",
"(S)-β-hydroxy-γ-butyrolactone, which is used in the step (1), can be produced in a large scale by the known technology (e.g.",
"SYNTHETIC COMMUNICATION, 1986, 16, 183.).",
"Referring to the acetic acid ester derivative for use in the step (1), R1 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms, and more specifically, including a hydrogen, methyl, ethyl, i-propyl, tert-butyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl, and like groups.",
"The preferred is a tert-butyl group.",
"X1 represents a hydrogen or a halogen atom, and more specifically including a hydrogen, a chlorine, a bromine, and an iodine, preferably a hydrogen and a bromine.",
"The level of use of the acetic acid ester derivative relative to (S)-β-hydroxy-γ-butyrolactone is 1 molar equivalent to 10 molar equivalents, preferably 1 molar equivalent to 5 molar equivalents.",
"In the step (1), a base or a 0-valent metal is permitted to act on the acetic acid ester derivative to prepare an enolate.",
"Generally speaking, in preparing the enolate, a base is used when X1 of the acetic acid ester is a hydrogen; a 0-valent metal is used in preparing the enolate when X1 is a halogen atom.",
"The base which is used in preparing the enolate includes, for example, lithium amides such as lithium amide, lithium diisopropylamide, lithium dicyclohexylamide, lithium hexamethyldisilazide, etc.",
"; magnesium amides such as chloromagnesium diisopropylamide, bromomagnesium diisopropylamide, iodomagnesium diisopropylamide, chloromagnesium dicyclohexylamide, etc.",
"; sodium amides such as sodium amide, sodium diisopropylamide, etc.",
"; potassium amides such as potassium amide, potassium diisopropylamide, etc.",
"; alkyllithiums such as methyllithium, n-butyllithium, phenyllithium, tert-butyllithium, etc.",
"; Grignard reagents such as methylmagnesium bromide, phenylmagnesium chloride, iso-propylmagnesium chloride, tert-butylmagnesium chloride, etc.",
"; metal alkoxides such as sodium methoxide, magnesium ethoxide, potassium tert-butoxide, etc.",
"; and metal hydrides such as lithium hydride, sodium hydride, potassium hydride, calcium hydride, and so forth.",
"The preferred are metal hydrides, magnesium amides, lithium amides and Grignard reagents.",
"These bases can be used each independently or in combination.",
"For example, a lithium amide or a metal hydride is effective when used in combination with a magnesium-containing base such as a Grignard reagent or a magnesium amide.",
"Moreover, said magnesium-containing base may be prepared in situ from a base and a magnesium compound such as magnesium chloride, magnesium bromide, or the like.",
"The magnesium amide is represented by the general formula (VIII); in the above formula, R5 and R6 each independently represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group.",
"Specifically, there can be mentioned methyl, ethyl, i-propyl, tert-butyl, cyclohexyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl, and like groups.",
"The preferred is an isopropyl group.",
"X2 represents a halogen atom and is preferably a chlorine, a bromine, or an iodine.",
"The more preferred is a chlorine.",
"It is to be understood that the magnesium amide can be prepared from an inexpensive, readily available secondary amine and a Grignard reagent by the known technology (for example, JP-A-08-523420).",
"As an alternative, it can be prepared from lithium amide and a magnesium halide by the known technology (e.g.",
"J. Org.",
"Chem.",
"1991, 56, 5978-5980).",
"The Grignard reagent is represented by the following formula (IX); R—Mg—X3 (IX) in the above formula, R7 represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms.",
"Specifically, there can be mentioned methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl and like groups.",
"The preferred groups include methyl, ethyl, i-propyl, n-butyl, tert-butyl and so forth.",
"X3 represents a halogen atom and preferably is a chlorine, a bromine, or an iodine.",
"The more preferred is a chlorine.",
"The lithium amide is represented by the general formula (X); in the above formula, R8 and R9 each independently represents an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, an aralkyl group of 7 to 12 carbon atoms, or a silyl group.",
"Specifically, there can be mentioned methyl, ethyl, i-propyl, tert-butyl, cyclohexyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl, trimethylsilyl, triethylsilyl, phenyldimethylsilyl and like groups.",
"The preferred is an isopropyl group.",
"The level of use of the base in the step (1), relative to (S)-β-hydroxy-γ-butyrolactone, is 1 molar equivalent to 20 molar equivalents, preferably 2 molar equivalent to 8 molar equivalents.",
"The 0-valent metal usable in preparing the enolate of the step (1) includes zinc, magnesium, tin, etc., although zinc and magnesium are preferred.",
"The level of use of the 0-valent metal relative to (S)-β-hydroxy-γ-butyrolactone is 1 molar equivalent to 20 molar equivalents, preferably 2 molar equivalent to 8 molar equivalents.",
"The solvent usable in the step (1) includes, for example, aprotic organic solvents.",
"As such organic solvents, there can be mentioned, for example, hydrocarbon solvents such as benzene, toluene, n-hexane, cyclohexane, etc.",
"; ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, ethylene glycol dimethyl ether, etc.",
"; halogenated hydrocarbon solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, etc.",
"; and aprotic polar solvents such as dimethylformamide, N-methylpyrrolidone, hexamethylphosphoric triamide, and so forth.",
"The above solvents may be used each independently or in a combination of two or more species.",
"The preferred, among the above-mentioned solvents, are hydrocarbon solvents such benzene, toluene, n-hexane, cyclohexane, etc.",
"; and ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, diethylene glycol dimethyl ether, and so forth.",
"The more preferred are polyether solvents such as dimethoxyethane, diethylene glycol dimethyl ether, and so forth.",
"These polyether solvents may be used each as a sole solvent or added, as an addendum, to another reaction solvent, at the level of about 1 molar equivalent to about 10 molar equivalents relative to (S)-β-hydroxy-γ-butyrolactone.",
"The reaction temperature to be used in the step (1) is preferably −30° C. to 100° C., more preferably −10° C. to 60° C. In the step (1), the order of mixing the reactants may be random but it is preferred to treat (S)-β-hydroxy-γ-butyrolactone with a base, more preferably with a base and a magnesium compound in advance.",
"The preferred base includes metal hydrides and lithium amides.",
"The preferred magnesium compound includes magnesium chloride, magnesium bromide, magnesium sulfate, and so forth.",
"A magnesium-containing base may be used so that it will double as the base and the magnesium compound.",
"The magnesium-containing base includes, for example, Grignard reagents such as methylmagnesium bromide, iso-propylmagnesium chloride, phenylmagnesium chloride, tert-butylmagnesium chloride, etc.",
"and magnesium amides such as chloromagnesium diisopropylamide, bromomagnesium diisopropylamide, iodomagnesium diisopropylamide, chloromagnesium dicyclohexylamide and so forth.",
"The preferred is tert-butylmagnesium chloride.",
"The level of use of the base in this pretreatment, relative to (S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent through 3 molar equivalents, preferably 0.5 molar equivalent to 1.5 molar equivalents.",
"The level of use of the magnesium compound in the pretreatment, relative to (S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent to 3 molar equivalents, preferably 0.5 molar equivalent to 1.5 molar equivalents.",
"The level of use of the magnesium-containing base in the pretreatment, relative to (S)-β-hydroxy-γ-butyrolactone, is 0.01 molar equivalent to 3 molar equivalents, preferably 0.5 molar equivalent to 1.5 molar equivalents.",
"The pretreatment of (S)-β-hydroxy-γ-butyrolactone with the base may be carried out on a mixed solution of (S)-β-hydroxy-γ-butyrolactone and an acetic acid ester derivative.",
"After the pretreatment, the reaction may be conducted under dropwise addition of a base, such as a lithium amide, e.g.",
"lithium amide, lithium diisopropylamide, lithium dicyclohexylamide, lithium hexamethyldisilazide or the like or a magnesium amide, e.g.",
"diisopropylmagnesium chloride, diisopropylmagnesium bromide or the like, or a solution of the base.",
"The level of the base to be reacted after the pretreatment, relative to (S)-β-hydroxy-γ-butyrolactone, is 1 molar equivalent to 20 molar equivalents, preferably 2 molar equivalents to 8 molar equivalents.",
"Thus, the step (1) can be carried out with advantage by pretreating (S)-β-hydroxy-γ-butyrolactone with the base and the magnesium compound in advance followed by permitting the base to act in the presence of the acetic acid ester derivative.",
"As an alternative, (S)-β-hydroxy-γ-butyrolactone may be treated with the base in advance and reacted with the enolate prepared by permitting a 0-valent metal to act on the acetic acid ester derivative.",
"The after-treatment following the step (1) may be an after-treatment which is generally carried out for recovery of the product from a reaction mixture.",
"For example, the reaction mixture available on completion of the reaction is mixed with the common inorganic or organic solvent, e.g.",
"hydrochloric acid, sulfuric acid, nitric acid, acetic acid or citric acid followed by extraction with the common extractant solvent, e.g.",
"ethyl acetate, diethyl ether, methylene chloride, toluene, or hexane.",
"From the extract thus obtained, the reaction solvent and the extractant solvent are removed by heating under reduced pressure or the like procedure to give the objective compound.",
"The objective compound thus obtained may further be purified by the routine technique such as crystallization, fractional distillation or column chromatography but it can be transmitted directly to the next step without isolation.",
"Step (2) In this step, the dihydroxyoxohexanoic acid derivative represented by the following formula (IV); as obtained in the step (1), is treated with an acylating agent in the presence of a base to produce a dihydroxyoxohexanoic acid monoacyl derivative represented by the following formula (V); As the acylating agent usable in the step (2), any of a compound represented by the following formula (XI); and a compound represented by the following formula (XVI); can be used.",
"In the above formulas, R4 represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms.",
"Specifically, there may be mentioned a hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, n-octyl, phenyl, naphthyl, p-methoxyphenyl, p-nitrobenzyl, and like groups.",
"The preferred are methyl, ethyl, i-propyl, tert-butyl, and phenyl groups, with a phenyl group being particularly preferred.",
"Q represents a leaving group.",
"Specifically, there can be mentioned a halogen atom, e.g.",
"chlorine, bromine, iodine, etc.",
"; an alkoxycarbonyloxy group, e.g.",
"methoxycarbonyloxy, ethoxycarbonyloxy, tert-butoxycarbonyloxy, etc.",
"; a cyano group; an imidazolido group, and so forth.",
"The preferred is a chlorine.",
"The level of use of the acylating agent relative to the dihydroxyoxohexanoic acid derivative is preferably 0.5 to 2 molar equivalents, more preferably 0.8 to 1.5 molar equivalents.",
"The base which can be used in the step (2) includes inorganic bases such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, barium hydroxide, magnesium hydroxide, etc.",
"; ammonia and amides such as triethylamine, pyridine, N-methylmorpholine, diisopropylethylamine, N,N-dimethylaminopyridine, etc., with triethylamine or pyridine being preferred.",
"The level of use of the base relative to the dihydroxyoxohexanoic acid derivative is preferably 1 to 10 molar equivalents, more preferably 1 to 3 molar equivalents.",
"The reaction solvent which can be used in the step (2) includes hydrocarbon solvents such as benzene, toluene, cyclohexane, etc.",
"; ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, dimethoxyethane, etc.",
"; ester solvents such as ethyl acetate, butyl acetate, etc.",
"; ketone solvents such as acetone, methyl ethyl ketone, etc.",
"; halogenated hydrocarbon solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, etc.",
"; nitrogen-containing solvents such as dimethylformamide, acetamide, formamide, acetonitrile, etc.",
"; and aprotic polar solvents such as dimethyl sulfoxide, N-methylpyrrolidone, hexamethylphosphoric triamide, and so forth.",
"The above-mentioned organic solvents may be used each independently or in a combination of two or more species.",
"The preferred are toluene, ethyl acetate, acetone, methylene chloride, methyl tert-butyl ether, tetrahydrofuran, dimethylformamide, acetonitrile, and so forth.",
"The reaction temperature in the step (2) is −30° C. to 80° C., preferably −10° C. to 40° C. The after-treatment following the step (2) may be an after-treatment which is generally carried out for recovery of the product from the reaction mixture on completion of the reaction.",
"For example, the reaction mixture available on completion of the reaction is added with water and extraction procedure is carried out with the common extractant solvent, such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane or the like.",
"From the extract thus obtained, the reaction solvent and extractant solvent are removed by heating under reduced pressure or the like procedure to obtain the objective compound.",
"The objective compound thus obtained tends to contain various impurities originating from various decompositions and side reactions which take place in the course of production.",
"Particularly, the dihydroxyoxohexanoic acid diacyl derivative of the following general formula (XII); (R1 and R4 are as defined hereinbefore) tends to be by-produced as a major impurity and in order that the objective compound of high quality grade may be isolated, such impurities must be somehow removed.",
"Generally, however, an impurity structurally resembling the objective compound (structural analog) is not easy to remove, and in order that such impurities may be removed to give the objective compound of high purity grade, a good protocol for purification and isolation is required.",
"The inventors of the present invention found that said impurities can be efficiently removed by carrying out a crystallization procedure under the conditions described below.",
"The crystallization solvent for use in the present invention is preferably an aliphatic hydrocarbon solvent.",
"Specifically, aliphatic hydrocarbons containing 5-20 carbon atoms, such as pentane, petroleum ether, neopentane, hexane, cyclohexane, methylcyclohexane, heptane, cycloheptane, octane, isooctane, nonane, decane, undecane, dodecane, etc.",
"can be mentioned.",
"Among these, pentane, hexane, methylcyclohexane, heptane, octane, and isooctane are preferred.",
"These may be used each independently or in a combination of two or more species.",
"Particularly in terms of the ease of removal of the solvent from wet crystals by desiccation or the recovery and reuse of the solvent (distillative recovery), the use of a solvent having a comparatively low boiling point is preferred.",
"As such solvents, solvents having a boiling point of not higher than about 100° C. at atmospheric or subatmospheric pressure can generally be mentioned.",
"More particularly, aliphatic hydrocarbon solvents of 5-8 carbon atoms, such as pentane, hexane, methylcyclohexane, heptane, octane and isooctane, etc.",
"can be mentioned, and when the cost of the solvent, ease of handling, and other factors are globally taken into consideration, hexane and methylcyclohexane are particularly preferred.",
"The use of the above aliphatic hydrocarbon solvent provides for the stabilization and assurance of a high yield of the objective compound as well as a high degree of purification, that is to say effective removal of various impurities, particularly said compound (XII).",
"The level of use of said aliphatic hydrocarbon solvent is preferably such that, at completion of the procedure for crystallization of said compound (V), the fluidity of the obtained product can be retained, and may, for example, be about 5 to 20 parts by weight, or even more in some cases, relative to said compound (V).",
"For the crystallization of said compound (V) in the present invention, crystallization by cooling, crystallization by concentration, and other methods for crystallization can be used each independently or in combination.",
"The crystallization by concentration, mentioned above, may be a crystallization procedure in which a solution composed of a solvent other than said aliphatic hydrocarbon solvent is converted to a solution composed of said aliphatic hydrocarbon solvent.",
"Moreover, seed crystals may be added in this crystallization procedure.",
"In the present invention, for the purpose of improving at least one of a solubility, yield, treatment concentration, purification effect (efficiency of impurity removal), and physical properties of obtainable crystals of the above compound (V), an auxiliary solvent can be used in addition to said aliphatic hydrocarbon solvent in conducting the crystallization procedure.",
"The above auxiliary solvent may be added to said aliphatic hydrocarbon solvent as necessary or the compound (V) may be dissolved in the auxiliary solvent in advance and the solution added to said aliphatic hydrocarbon solvent.",
"The auxiliary solvent mentioned above is not particularly restricted but includes, for example, acetone, methyl ethyl ketone, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, isopropyl acetate, tert-butyl acetate, ethanol, isopropanol, toluene, benzene, xylene, chlorobenzene, methylene chloride, chloroform, and 1,2-dichloroethane, etc.",
"These may be used each independently or in a combination of two or more species.",
"Among these, ethyl acetate, toluene, methyl tert-butyl ether, methylene chloride, etc.",
"can contribute to increased solubility and improved treatment parameters such as treatment concentration and purification effect.",
"The auxiliary solvent mentioned above expresses its effect more prominently when used in a suitable amount combined with said aliphatic hydrocarbon solvent, which suitable amount being established according to the characteristics of the auxiliary solvent in relation to the desired effect and other factors.",
"The optimal level of use of said auxiliary solvent can be found by simple experimentation.",
"From the standpoint of yield and purification effect, the level of use of the above auxiliary solvent is preferably such that the weight ratio of said auxiliary solvent and said aliphatic hydrocarbon solvent (auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization of said compound (V).",
"The more preferred level is such that e ratio id 0.5 or less.",
"The purification/isolation process according to the present invention can be carried out in the neighborhood of room temperature.",
"Where necessary, warming or cooling can be carried out, for example at a temperature not over about 60° C., usually at −30° C. to 50° C. The above compound (V) thus obtained is separated by a solid-liquid separation technique, optionally further followed by cake washing and drying.",
"The solid-liquid separation technique is not particularly restricted but includes, for example, filtration under pressure, suction filtration, centrifugation, and so forth.",
"The above drying is preferably carried out under reduced pressure (drying in vacuo) at a temperature not exceeding about 60° C. in order to avoid pyrolysis or fusion, for instance.",
"Step (3) In this step, the (5S)-configured dihydroxyoxohexanoic acid monoacyl derivative represented by the following formula (V); as obtained in said step (2) is reduced using a microorganism to give a (3R,5S)-configured trihydroxyhexanoic acid monoacyl derivative represented by the following formula (VI); Generally for the highly stereoselective reduction of the carbonyl group of a dihydroxyoxohexanoic acid monoacyl derivative such as the above compound, the reduction process using a hydride series reducing agent, such as sodium borohydride, in the presence of an alkylborane at an ultra-low temperature is employed (e.g.",
"JP-A-02-262537).",
"For the purpose of reducing a dihydroxyoxohexanoic acid monoacyl derivative stereoselectively under non-ultra-low temperature conditions at low cost, the inventors of the present invention developed a reduction method using a microorganism.",
"The microorganism capable of reducing dihydroxyoxohexanoic acid monoacyl derivatives to the corresponding trihydroxyhexanoic acid monoacyl derivatives, which is to be used in the step (3), can be found by the methods described below.",
"Taking a yeast as an example, there can be used, for example, the method which comprises charging a large test tube with 1 ml of Medium A (pH 7.0) of the composition: glucose 3%, yeast extract 0.3%, potassium dihydrogen phosphate 0.7%, diammonium hydrogen phosphate 1.3%, magnesium sulfate.7H2O 0.08%, zinc sulfate.7H2O 0.007%, iron sulfate.7H2O 0.009%, copper sulfate-5H2O 0.0005%, manganese sulfate.4H2O 0.001%, and sodium chloride 0.01%, sterilizing the medium, inoculating a yeast, carrying out a shake culture at 27° C. for 2 to 3 days, harvesting the grown cells by centrifugation, suspending the cells in 0.5 ml of phosphate buffer containing 0.01 to 1% of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic acid tert-butyl ester and 8% of glucose, and shaking the suspension in a large test tube at 27° C. for 1 to 3 days.",
"For the identification of the objective reducing ability, the reaction mixture after shake culture is extracted with ethyl acetate and the organic phase is analyzed by high performance liquid chromatography [column: Dovelosil ODS-HG-3 (4.6 mm×150 mm) (product of Nomura Chemical), eluent: 0.1% trifluoroacetic acid/acetonitrile=6/4, flow rate: 0.8 ml/min, detection: 210 nm, column temperature: room temperature, elution time: (3S,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic acid tert-butyl ester; 10.1 min, (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoic acid tert-butyl ester; 11.0 min, and (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic acid tert-butyl ester; 16.7 min; it is to be understood that these conditions are for illustrative purposes only].",
"When the candidate microorganism is a bacterial strain, Medium B (pH 7.0) of the composition: glycerol 1.5%, Proextract 1.0%, and yeast extract 0.5%, for instance, is employed, for instance.",
"In the case of a fungal strain, Medium C (pH 6.0) of the composition: glucose 5% and corn steep liquor 5% is employed, for instance.",
"In the case of an actinomycete, Medium D (pH 7.2) of the composition: Difco-tryptic soy broth 3% and soluble starch 1% is employed.",
"Using these media, the respective strains of microorganisms are cultured, and the microorganisms having the objective ability are selected by the same procedure as described above.",
"The microorganism which can be used in the practice of the present invention includes microorganisms belonging to the genera: Ashbya, Botryoascus, Brettanomyces, Candida, Citeromyces, Clavispora, Cryptococcus, Debaryomyces, Dekkera, Dipodascus, Galactomyces, Geotrichum, Hanseniaspora, Hansenula, Hormoascus, Hyphopichia, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Metschnikowia, Nakazawaea, Ogataea, Pachysolen, Pichia, Rhodotorula, Rhodsporidium, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Torulaspora, Torulopsis, Trichosporon, Trigonopsis, Willopsis, Yamadazyma, Zygosaccharomyces, Acidiphilium, Aerobacter, Alcaligenes, Arthrobacter, Aureobacterium, Bacillus, Brevibacterium, Buttiauxella, Cedecea, Cellulomonas, Citrobacter, Clostridium, Comamonas, Corynebacterium, Enterobacter, Erwinia, Escherichia, Flavobacterium, Klebsiella, Luteococcus, Microbacterium, Micrococcus, Ochrobactrum, Proteus, Providencia, Pseudomonas, Rhodococcus, Sarcina, Serratia, Sphingobacterium, Tsukamurella, Absidia, Acremonium, Aegerita, Agrocybe, Amylostereum, Aspergillus, Byssochlamys, Chaetomidium, Chaetosartorya, Cladosporium, Coprinus, Crinipellis, Endophragmia, Flavolus, Fomitopsis, Fusarium, Ganoderma, Glomerella, Laetiporus, Lentinus, Lenzites, Macrophoma, Monascus, Mortierella, Paecilomyces, Penicillium, Phialophora, Pholiota, Pleurotus, Scopulariopsis, Sehizophyllum, Sporotrichum, Zygorhynchus, Microtetraspora, and Streptomyces.",
"More particularly, the usable are, for example, Ashbya gossypii IFO 0560, Botryoascus synnaedendrus IFO 1604, Brettanomyces custersianus IFO 1585, Candida arborea IAM 4147, Candida catenulata IFO 0745, Candida fennica CBS 6028, Candida galacta IFO 10031, Candida haemulonii IFO 10001, Candida magnoliae IFO 0705, Candida musae IFO 1582, Candida nitratophila IFO 10004, Candida parapsilosis IFO 0585, Candida pararugosa IFO 0966, Candida stellata IFO 0701, Citeromyces matritensis IFO 0651, Clavispora lusitaniae IFO 1019, Cryptococcus laurentii IFO 0609, Debaryomyces carsonii IFO 0795, Debaryomyces hansenii var.",
"fabryi IFO 0794, Debaryomyces hansenii var.",
"hansenii IFO 0032, Debaryomyces hansenii var.",
"hansenii IFO 0047, Debaryomyces hansenii var.",
"hansenii IFO 0018, Debaryomyces kloeckeri, Debaryomyces marama IFO 0668, Debaryomyces pseudopolymorphus IFO 1026, Debaryomyces robertsiae IFO 1277, Debaryomyces sp.",
"IFO 0025, Dekkera anomala IFO 0627, Dipodascus armillariae IFO 0102, Dipodascus ovetensis IFO 1201, Dipodascus tetrasperma CBS 765.70, Galactomyces reessii CBS 179.60, Geotrichum candidum CBS 187.67, Geotrichum fermentans IFO 1199, Geotrichum fragrans CBS 164.32, Geotrichum loubieri CBS 252.61, Hanseniaspora guilliermondii IAM 4972, Hansenula methanolosa, Hansenula polymorpha DL1 AKU4752, Hormoascus philentomus IFO 1847, Hormoascus platypodis IFO 1471, Hyphopichia burtonii IFO 0844, Issatchenkia orientalis IFO 1279, Issatchenkia terricola IFO 0933, Kluyveromyces lactis IFO 1012, Kluyveromyces marxianus IFO 0541, Kluyveromyces marxianus IFO 0288, Kluyveromyces polysporus IFO 0996, Kluyveromyces thermotolerans IFO 0662, Komagataella pastoris IFO 1013, Lipomyces starkeyi IFO 0678, Metschnikowia bicuspidata IFO 1408, Metschnikowia pulcherrima IFO 0561, Nakazawaea holstii IFO 0980, Ogataea minuta var.",
"minuta IFO 0975, Ogataea pini IFO 1342, Ogataea polymorpha IFO 0799, Ogataea polymorpha IFO 1475, Ogataea wickerhamii IFO 1706, Pachysolen tannophilus IFO 1007, Pichia canadensis IFO 0976, Pichia farinose IAM 4369, Pichia jandinii IFO 0987, Pichia saitoi IAM 4945, Pichia toletana IFO 0950, Pichia triangularis IFO 0836, Pichia wickerhamii IFO 1278, Rhodotorula graminis IFO 0190, Rhodotorula minuta IFO 0387, Rhodotorula minuta IFO 0715, Rhodsporidium diobovatum IFO 0688, Rhodsporidium toruloides IFO 0413, Saccharomyces bayanus IFO 0251, Saccharomyces pastorianus IFO 1265, Saccharomyces pastorianus ATCC 9080, Saccharomyces rosei IFO 0252, Saccharomyces sake, Saccharomyces steineri IAM 4608, Saccaromyces unisporus IFO 0215, Saccharomycodes ludwigii IFO 0339, Saccharomycopsis capsularis IFO 0672, Saccharomycopsis malanga IFO 1710, Saturnospora dispora IFO 0035, Schizoblastosporion kobayasii IFO 1644, Schizosaccharomyces pombe IFO 0347, Schizosaccharomyces pombe IFO 0362, Schwanniomyces occidentalis var.",
"occidentalis IFO 1840, Sporidiobolus johnsonii IFO 6903, Sporobolomyces pararoseus IFO 0471, Sporobolomyces salmonicolor IFO 1038, Torulaspora delbrueckii IFO 0381, Torulopsis methanolevescens, Torulopsis osboenis IFO 0646, Torulopsis sp., Torulopsis uvae IFO 0649, Trichosporon pullulans, Trichosporon sp.",
"Trigonopsis variabilis IFO 0671, Willopsis saturnus var.",
"mrakii IFO 0895, Willopsis saturnus var.",
"saturnus IFO 0992, Yamadazyma farinosa IFO 0459, Yamadazyma farinosa IFO 0602, Yamadazyma haplophila IFO 0947, Zygosaccharomyces naniwensis IFO 0524, Zygosaccharomyces sp.",
"IFO 0522, Acidiphilium cryptum IFO 14242, Aerobacter cloacae IAM 1221, Alcaligenes xylosoxidans IFO 13495, Alcaligenes xylosoxidans subsp.",
"denitrificans IFO 12669, Alcaligenes xylosoxidans subsp.",
"denitrificans ATCC 15173, Arthrobacter globiformis ATCC 8010, Arthrobacter protophormiae IFO 12128, Aureobacterium esteraromaticum IFO 3752, Bacillus badius IAM 11059, Bacillus sphaericus IFO 3525, Brevibacterium ammomiagenes IFO 12071, Buttiauxella agrestis JCM 1090, Cedecea davisiae JCM 1685, Cellulomonas sp.",
"JCM 2471, Cellulomonas turbata IFO 15015, Citrobacter freundii IFO 12681, Clostridium cylindrosporum IFO 13695, Comamonas testosteroni IFO 12047, Corynebacterium acectoacidophilum ATCC 21476, Corynebacterium ammoniagenes IFO 12072, Corynebacterium glutamicum ATCC 21269, Corynebacterium glutamicus ATCC 13287, Enterobacter aerogenes IFO 13534, Enterobacter cloacae IFO 12935, Erwinia carotovora subsp.",
"carotovora IFO 3830, Escherichia coli IFO 12734, Flavobacterium flavesceus, Klebsiella planticola IFO 3317, Luteococcus japonicus IFO 12422, Microbacterium arborescens IFO 3750, Micrococcus flavus, Micrococcus luteus IFO 13867, Ochrobactrum sp.",
"IFO 12950, Proteus inconstans IFO 12931, Proteus mirabilis IFO 3849, Proteus rettgeri IFO 1350, Proteus vulgaris IFO 3167, Providencia stuartii IFO 12930, Pseudomonas aeruginosa IAM 1007, Pseudomonas putida IFO 14164, Pseudomonas stutzeri IFO 13596, Rhodococcus equi JCM 1313, Sarcina lutea, Serratia plymuthicum IFO 3055, Serratia proteamaculans subsp.",
"proteamaculans IFO 12979, Sphingobacterium spiritivorum JCM 1277, Tsukamurella paurometabolum IFO 12160, Absidia orchidis HUT 1036, Acremonium bacillisporum IFO 9387, Aegerita candida IFO 6988, Agrocybe cylindracea IFO 30299, Amylostereum areolatum IFO 9221, Aspergillus parasiticus IFO 4403, Aspergillus phoenicis IFO 6670, Byssochlamys fulva IFO 6307, Chaetomidium fimeti IFO 30419, Chaetosartorya stromatoides IFO 9652, Cladosporium resinae F. avellaneum IFO 6367, Coprinus cinereus TD-822, Coprinus lagopus IFO 9533, Coprinus sp., Crinipellis stipitaria IFO 30259, Endophragmia alternata IFO 30204, Flavolus arcularius, Fomitopsis pubertatis, Fusarium merismoides IFO 30040, Ganoderma lucidum IFO 31863, Glomerella cingulata IFO 5257, Laetiporus sulphureus, Lentinus lepideus, Lenzites betulina IFO 8715, Macrophoma commelinae IFO 9569, Monascus purpureus IFO 5965, Mortierella isabellina IFO 7829, Paecilomyces varioti HUT 4028, Penicillium chermesinum IFO 5800, Penicillium chrysogenum IFO 4640, Penicillium expansum IFO 5854, Penicillium lilacinium IFO 31914, Phialophora fastigiata IFO 6850, Pholiota aurivella IFO 30265, Pholiota limonella IFO 31868, Pleurotus dryinus, Pleurotus ostreatus, Pleurotus porrigens, Scopulariopsis brevicaulis IFO 4843, Sehizophyllum commune IFO 6503, Sehizophyllum commune IFO 6504, Sporotrichum aurantiacum IFO 9381, Zygorhynchus moelleri HUT 1305, Microtetraspora roseoviolacea IFO 14098, Streptomyces achromogenes subsp.",
"rubradiris IFO 14000, Streptomyces sp.",
"and Streptomyces aureus NIHJ 122.These microorganisms can be obtained generally from stock cultures which are readily available at no cost or at cost.",
"These may also be isolated from the natural kingdom.",
"In this connection, these microorganisms may optionally be subjected to induced mutagenesis to obtain strains having characteristics more beneficial to the intended reaction.",
"Furthermore, the strains derived from these microorganisms by genetic engineering or biotechnological processes, such as recombinant DNA technology or cell fusion may also be employed.",
"As an example of such microorganism, there can be mentioned Escherichia coli HB101 (pNTCRG) FERM BP-6898 (PCT/JP00/08321) harboring the reductase gene derived from Candida magnoliae IFO 0705.For the cultivation of these microorganisms, any nutrient sources that these microorganisms are generally able to utilize can be employed.",
"For example, as carbon sources, saccharides such as glucose, sucrose, maltose, etc.",
"; organic acids such as lactic acid, acetic acid, citric acid, propionic acid, etc.",
"; alcohols such as ethanol, glycerol, etc.",
"; hydrocarbons inclusive of paraffin; oils such as soybean oil, rapeseed oil, etc.",
"; and mixtures thereof, and as nitrogen sources, ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, corn steep liquor, etc.",
"can be admixed.",
"Furthermore, inorganic salts, vitamins, and other nutrients can also be properly admixed.",
"The above microorganisms can be cultured under the conventional conditions.",
"For example, they are cultured aerobically under the pH of 4.0 to 9.5 in the temperature range of 20° C. to 45° C. for 10 to 96 hours.",
"To permit a microorganism to act on a dihydroxyoxohexanoic acid monoacyl derivative, usually a culture of the above microorganism can be used as such for the reaction but a concentrate of the culture broth can likewise be employed.",
"Moreover, in cases where some ingredients in the culture broth might adversely affect the reaction, it is preferred to use the cells or processed cells as obtainable by centrifugation and/or other treatments on the culture broth.",
"The processed cells mentioned above are not particularly restricted but include, for example, dried cells obtainable by dehydration with acetone or diphosphorus pentoxide or by drying with a desiccator or a fan, surfactant-treated cells, an enzymatic digest of cells, immobilized cells, or a cell-free extract obtainable by disruption of cells.",
"Furthermore, from the culture broth, an enzyme catalyzing asymmetric reduction may be purified and used.",
"In conducting the reduction reaction, the substrate dihydroxyoxohexanoic acid monoacyl derivative can be added all at once in the initial stage of the reaction or serially in portions with the progress of reaction.",
"The temperature during the reaction is generally 10 to 60° C., preferably 20 to 40° C., and the pH during the reaction is 2.5 to 9, preferably 5 to 9.The concentration of the microorganism in the reaction mixture can be properly selected according to its ability to reduce the substrate.",
"The concentration of the substrate in the reaction mixture is preferably 0.01 to 50% (w/v), more preferably 0.1 to 30%.",
"The reaction is usually carried out under shaking or stirring with aeration.",
"The reaction time is properly selected according to the concentration of the substrate, the concentration of the microorganism, and other reaction conditions.",
"Generally, the various conditions are preferably selected so as to insure that the reaction may be completed in 2 to 168 hours.",
"To promote the reduction reaction, it is preferred to add an energy source, such as glucose, ethanol, and/or the like, in a proportion of 1 to 30% to the reaction mixture, for it will lead to a more satisfactory result.",
"The reaction can also be accelerated by adding a coenzyme, such as reduced nicotinamide-adenine dinucleotide (NADH) or reduced nicotinamide-adenine dinucleotide phosphate (NADPH), which is generally considered to be necessary for reduction reactions by biological techniques.",
"More particularly, these may be directly added to the reaction mixture or a reaction system producing NADH or NADPH may be added together with the oxidized coenzyme to the reaction mixture.",
"For example, the reaction system which reduces NAD to NADH as a formate dehydrogenase produces carbon dioxide and water from formic acid or the reaction system which reduces NAD or NADP to NADH or NADPH respectively, as a glucose dehydrogenase produces gluconolactone from glucose can be exploited.",
"Furthermore, it is also effective to add a surfactant such as Triton (product of Nakalai Tesque), Span (product of Kanto Chemical) or Tween (product of Nakalai Tesque) to the reaction mixture.",
"Moreover, for the purpose of avoiding inhibition of the reaction by the substrate and/or alcohol, which is the byproduct of the reduction reaction, a water-insoluble organic solvent such as ethyl acetate, butyl acetate, isopropyl ether, toluene or the like may be added to the reaction mixture.",
"For the purpose of enhancing the solubility of the substrate, a water-soluble organic solvent, such as methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, or the like, may also be added.",
"The trihydroxyhexanoic acid monoacyl derivative produced by the reduction reaction can be recovered, directly from the reaction mixture or after removal of the cells and others, extraction with a solvent, such as ethyl acetate, toluene, or the like, and subsequent removal of the solvent.",
"Moreover, the trihydroxyhexanoic acid monoacyl derivative can be obtained in a highly pure grade by a purification procedure such as recrystallization, silica gel column chromatography or the like.",
"Step (4) In this step, the (3R,5S)-configured trihydroxyhexanoic acid monoacyl derivative represented by the following formula (VI); as obtained in the step (3), is treated with an acetal-forming reagent in the presence of an acid catalyst to produce a (4R,6S)-configured acyloxymethyldioxanylacetic acid derivative represented by the following formula (VII); In the step (4), the acetal-forming reagent which can be used includes, for example, a ketone, an aldehyde, an alkoxyalkane, and an alkoxyalkene.",
"As specific examples of said ketone, aldehyde, alkoxyalkane and alkoxyalkene, there can be mentioned, for example, acetone, cyclohexanone, formaldehyde, benzaldehyde, dimethoxymethane, 2,2-dimethoxypropane, 2-methoxypropene, and 1,1-dimethoxycyclohexane, etc.",
"The preferred are acetone, 2-methoxypropene, 2,2-dimethoxypropane, etc.",
"and more preferred is 2,2-dimethoxypropane.",
"The level of use of the acetal-forming reagent relative to the trihydroxyhexanoic acid monoacyl derivative is preferably 1 to 10 molar equivalents, more preferably 1 to 5 molar equivalents.",
"For promoting the reaction, the acetal-forming reagent can be used as the reaction solvent as well.",
"The acid catalyst which can be used in the step (4) includes Lewis acids or Brönsted acids.",
"As said Lewis acids and Brönsted acids, there can be mentioned, for example, Lewis acids such as aluminum trichloride, boron trifluoride, zinc dichloride, tin tetrachloride, etc.",
"; carboxylic acids such as oxalic acid, formic acid, acetic acid, benzoic acid, trifluoroacetic acid, etc.",
"; sulfonic acids such as methanesulfonic acid, benzensulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, etc.",
"; and inorganic acids such as hydrogen chloride, hydrogen bromide, sulfuric acid, nitric acid, boric acid, etc.",
"The level of use of the acid catalyst in the step (4), relative to the trihydroxyhexanoic acid monoacyl derivative, is preferably 0.001 to 0.5 molar equivalent, more preferably 0.005 to 0.1 molar equivalent.",
"The acetal-forming reaction using said acid catalyst is a side reaction involving the intramolecular hydroxyl groups, and it has heretofore been difficult to let the desired reaction proceed with good efficiency.",
"Regarding side reactions, a cyclization reaction involving the intramolecular hydroxyl groups and the ester group, for instance, produces an acyloxymethylhydroxylacetone represented by the following formula (XV); (in the formula, R4 is as defined hereinbefore) as a byproduct.",
"When an alkoxyalkane or alkoxyalkene is used as the acetal-forming reagent, the byproduct alcohol reacts with the above compound (XV) to give an analog compound (having different ester group) of said acyloxymethyldioxalylacetic acid derivative represented by the following formula (XIII); (in the formula, R2, R3 and R4 are as defined hereinbefore; and R10 represents a lower alkyl group (preferably containing 1 to 4 carbon atoms) which is different from R1) as a byproduct.",
"When 2,2-dimethoxypropane, for instance, is used as the acetal-forming reagent, the byproduct methanol takes part in the reaction to give an acyloxymethyldioxalylacetic acid methyl ester of the above formula (XIII) in which R10 represents a methyl group as a byproduct.",
"By-production of these impurities detract from the yield and quality of the objective product compound, so that when an acetal-forming reaction is to be conducted using said acid catalyst, the reaction conditions such as reaction temperature, reaction time, amounts of reagents, etc.",
"are necessary to be critically selected and controlled.",
"The inventors of the present invention developed a method of conducting the acetal-forming reaction in the presence of an amine salt composed of an acid and an amine as a catalyst, whereby the formation of said impurities (XIII) and (XV) and several other trace byproduct impurities, which have not been structurally established, can be suppressed to a minimum without detracting from the yield.",
"As the acid, any of the acids mentioned hereinbefore can be employed, and the preferred include hydrogen chloride, hydrogen bromide, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.",
"The level of use of the acid relative to the trihydroxyhexanoic acid monoacyl derivative is preferably 0.001 to 0.5 molar equivalent, more preferably 0.005 to 0.1 molar equivalent.",
"The amine includes ammonia; primary amines such as methylamine, ethylamine, butylamine, aniline, etc.",
"; secondary amines such as diethylamine, diisopropylamine, diphenylamine, piperidine, morpholine, etc.",
"; and tertiary amines such as triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2-methylpyridine, 3-methylpyridine, imidazole, N,N-dimethylaminopyridine, 1,7-diazabicyclo[5,4,0]-undec-7-ene, and so forth.",
"The preferred are tertiary amines, with triethylamine, N-methylmorpholine, diisopropylethylamine, pyridine, 2-methylpyridine, 3-methylpyridine or imidazole being more preferred.",
"The level of use of the amine relative to the acid is preferably 1 to 10 molar equivalents, more preferably 1 to 3 molar equivalents.",
"This reaction may be carried out using an amine salt, as a catalyst, prepared from an acid and an amine and isolated in advance.",
"The amine salt includes, for example, pyridinium hydrochloride, pyridinium hydrobromide, pyridinium sulfate, pyridinium trifluoroacetate, pyridinium methanesulfonate, pyridinium p-toluenesulfonate, triethylammonium hydrochloride, triethylammonium sulfate, 3-methylpyridinium p-toluenesulfonate, N-methylmorpholine p-toluenesulfonate salt, N,N-dimethylaminopyridinium benzenesulfonate, diisopropylammonium hydrochloride, ammonium hydrochloride, ammonium sulfate, ammonium nitrate, and ammonium methyl p-toluenesulfonate.",
"The preferred is pyridinium p-toluenesulfonate or triethylammonium p-toluenesulfonate.",
"The level of use of the amine salt relative to the trihydroxyhexanoic acid monoacyl derivative is preferably 0.001 to 0.5 molar equivalent, more preferably 0.005 to 0.1 molar equivalent.",
"For conducting the reaction according to the step (4), various organic solvents can be used as the reaction solvent.",
"As such organic solvents, there can be mentioned, for example, hydrocarbon solvents such as benzene, toluene, cyclohexane, etc.",
"; ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, etc.",
"; ester solvents such as ethyl acetate, butyl acetate, etc.",
"; ketone solvents such as acetone, methyl ethyl ketone, etc.",
"; halogenated hydrocarbon solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, etc.",
"; nitrogen-containing solvents such as N,N-dimethylformamide, acetamide, formamide, acetonitrile, etc.",
"; and aprotic polar solvents such as dimethyl sulfoxide, N-methylpyrrolidone, hexamethylphosphoric triamide, etc.",
"These organic solvents can be used each independently or two or more of them may be used in combination.",
"The preferred are toluene, acetone, ethyl acetate, methylene chloride, tetrahydrofuran, methyl tert-butyl ether, dimethylformamide, and acetonitrile, with acetone being more preferred.",
"The reaction temperature in the step (4) is −20° C. to 100° C., preferably 0° C. to 50° C. The after-treatment following the step (4) may be carried out by the routine after-treatment for recovery of the product from a reaction mixture.",
"For example, water is added to the reaction mixture after completion of the reaction and the extraction is carried out with an ordinary extractant solvent, for example, ethyl acetate, diethyl ether, methylene chloride, toluene, or hexane.",
"From the extract thus obtained, the reaction solvent and extractant solvent are removed by heating under reduced pressure or the like procedure to give the objective compound.",
"The objective compound thus obtained tends to contain various impurities originating from various decompositions and side reactions which take place in the course of production.",
"Particularly, it tends to contain at least one member of the group consisting of an acyloxyhydroxylactone represented by the following general formula (XV); (in the formula, R4 is as defined above), an analog compound (having different ester group) of an acyloxymethyldioxalylacetic acid derivative represented by the following general formula (XIII); (in the formula, R2, R3 and R4 are respectively as defined above; R10 represents a lower alkyl group which is different from R1), a diastereomer represented by the following formula (XIV); (in the formula, R1, R2, R3 and R4 are respectively as defined above), and the reaction substrate represented by the formula (VI), and in order to obtain the objective compound of high quality, these impurities need to be removed.",
"Generally, however, any impurity structurally resembling the objective compound (structural analog) is not easy to remove, and in order that such impurities may be removed to give the objective compound of high quality, a valid purification/isolation process is required.",
"The inventors of the present invention found that said impurities can be efficiently removed by carrying out a crystallization under the conditions described below.",
"The crystallization solvent for use in the present invention is preferably an aliphatic hydrocarbon solvent.",
"Specifically, there can be mentioned, for example, aliphatic hydrocarbons of 5 to 20 carbon atoms, such as pentane, petroleum ether, neopentane, hexane, cyclohexane, methylcyclohexane, heptane, cycloheptane, octane, isooctane, nonane, decane, undecane, dodecane, etc.",
"Among these, pentane, hexane, methylcyclohexane, heptane, octane, and isooctane are preferred.",
"These may be used each independently or in a combination of two or more species.",
"Particularly in terms of removal of the solvent from wet crystals by desiccation or the recovery and reuse of the solvent (distillative recovery), the use of a solvent having a comparatively low boiling point is preferred.",
"As such solvents, there can generally be mentioned solvents having a boiling point of not higher than about 100° C. at atmospheric or subatmospheric pressure.",
"More particularly, for example, aliphatic hydrocarbon solvents of 5 to 8 carbon atoms, such as pentane, hexane, methylcyclohexane, heptane, octane, isooctane, etc.",
"can be mentioned, and when the cost of the solvent, ease of handling, and other factors are globally taken into consideration, hexane and methylcyclohexane are more preferred.",
"The use of the above aliphatic hydrocarbon solvent provides for the stabilization and assurance of a high yield of the above compound as well as a high degree of purification, that is to say effective removal of various impurities, particularly said compounds (XIII), (XIV), (XV) and (VI).",
"The level of use of said aliphatic hydrocarbon solvent is preferably such that the obtainable product at completion of crystallization of said compound (VII) retains sufficient fluidity, and may, for example, be about 5 to 20 parts by weight, or even more in some cases, relative to said compound (VII).",
"For the crystallization of said compound (VII) in the present invention, crystallization by cooling, crystallization by concentration, and other processes for crystallization can be used each independently or in combination.",
"The crystallization by concentration, mentioned above, may be a crystallization process in which a solution composed of a solvent other than the aliphatic hydrocarbon solvent is converted to a solution composed of said aliphatic hydrocarbon solvent.",
"Moreover, seed crystals may be added in this crystallization.",
"In the present invention, for the purpose of improving at least one parameter among the solubility, yield, treatment concentration, purification effect (efficiency of impurity removal), and physical properties of obtainable crystals of the above compound (VII), an auxiliary solvent can be used in addition to said aliphatic hydrocarbon solvent in conducting the crystallization.",
"The above auxiliary solvent may be added to said aliphatic hydrocarbon solvent as necessary or the above compound (VII) may be dissolved in the auxiliary solvent in advance and the solution added to said aliphatic hydrocarbon solvent.",
"The auxiliary solvent mentioned above is not particularly restricted but includes, for example, acetone, methyl ethyl ketone, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, isopropanol, tert-butyl acetate, ethanol, isopropyl alcohol, toluene, benzene, xylene, chlorobenzene, methylene chloride, chloroform, and 1,2-dichloroethane, etc.",
"These may be used each independently or in a combination of two or more species.",
"Among these, ethyl acetate, toluene, methyl tert-butyl ether, methylene chloride, etc.",
"contribute to increased solubility and improved treatment effects such as treatment concentration and purification effect.",
"The auxiliary solvent mentioned above expresses its effect more prominently when used in a suitable amount in combination with said aliphatic hydrocarbon solvent, which suitable amount is established according to the characteristics of auxiliary solvent in relation to the desired effect and other factors.",
"The optimal level of use of said auxiliary solvent can be found by simple experimentation.",
"From the standpoint of yield and purification effect, the level of use of the above auxiliary solvent is preferably such that the weight ratio of said auxiliary solvent and said aliphatic hydrocarbon solvent (auxiliary solvent/aliphatic hydrocarbon solvent) is not greater than 1 at completion of the procedure for crystallization of said compound (VII).",
"The more preferred level is such that said ratio will be 0.5 or less.",
"The purification/isolation method according to the present invention can be carried out in the neighborhood of room temperature.",
"Where necessary, the method can be practiced under warming or cooling, for example at a temperature not over about 60° C., usually at 50° C. to −30° C. The above compound (VII) thus obtained can be separated by a solid-liquid separation technique, optionally followed by cake washing and drying.",
"The above solid-liquid separation technique is not particularly restricted but includes, for example, filtration under pressure, suction filtration, centrifugation, and so forth.",
"The above-mentioned drying is preferably carried out under reduced pressure (drying in vacuo) at a temperature not exceeding about 60° C. in order to avoid pyrolysis or fusion.",
"In the acyloxymethyldioxanylacetic acid derivative of the following formula (VII); as obtained in the step (4), R2 and R3 each independently represents a hydrogen, an alkyl group of 1 to 12 carbon atoms, an aryl group of 6 to 12 carbon atoms, or an aralkyl group of 7 to 12 carbon atoms.",
"Specifically, methyl, ethyl, tert-butyl, hexyl, phenyl, benzyl, p-methoxybenzyl and like groups can be mentioned.",
"The preferred is a methyl group.",
"Moreover, R2 and R3 may jointly form a ring.",
"For example, R2 and R3 may form a cyclopentane ring, a cyclohexane ring, a cycloheptane ring, a benzocyclopentane ring or the like to thereby constitute a spiro structure with the 1,3-dioxane ring.",
"Step (5) In this step, the (4R,6S)-configured acyloxymethyldioxanylacetic acid derivative represented by the following formula (VII); as obtained in the step (4), is subjected to solvolysis in the presence of a base by the known method or the like, to give a (4R,6S)-configured hydroxymethyldioxanylacetic acid derivative of the general formula (I); The base which can be used for the solvolysis in the step (5) includes inorganic and organic bases, such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, barium hydroxide, magnesium hydroxide, sodium acetate, potassium acetate, ammonia, triethylamine, pyridine, piperidine, N,N-dimethylaminopyridine, and so forth.",
"The preferred is potassium carbonate.",
"The level of use of the base in this step, relative to the acyloxymethyldioxanylacetic acid derivative, is 0001 equivalent to 5 equivalents, preferably 0.01 equivalent to 1.0 equivalent.",
"In step (5), for effecting the solvolysis, the reaction is carried out in water or in a protic organic solvent, or in a mixture of water or a protic organic solvent and an aprotic organic solvent.",
"The protic organic solvent mentioned above includes, for example, alcohol solvents, such as methanol, ethanol, butanol, isopropyl alcohol, ethylene glycol, methoxyethanol, etc.",
"; and amine solvents, such as diethylamine, pyrrolidine, piperidine, and so forth.",
"The aprotic organic solvent mentioned above includes, for example, hydrocarbon solvents such as benzene, toluene, cyclohexane, etc.",
"; ether solvents such as diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl t-butyl ether, dimethoxyethane, etc.",
"; ester solvents such as ethyl acetate, butyl acetate, etc.",
"; ketone solvents such as acetone, methyl ethyl ketone, etc.",
"; halogenated hydrocarbon solvents such as methylene chloride, chloroform, 1,1,1-trichloroethane, etc.",
"; nitrogen-containing solvents such as N,N-dimethylformamide, acetonitrile, etc.",
"; and aprotic polar solvents such as dimethyl sulfoxide, N-methylpyrrolidone, hexamethylphosphoric triamide, and so forth.",
"The preferred are water, methanol, and ethanol.",
"The reaction temperature for the step (5) is −20° C. to 100° C., preferably −10° C. to 50° C. The after-treatment following completion of the reaction may be the after-treatment which is generally carried out for recovery of the product from the reaction mixture.",
"For example, the reaction mixture available on completion of the reaction is added with water and extracted with the common extractant solvent, such as ethyl acetate, diethyl ether, methylene chloride, toluene, hexane or the like.",
"From the extract thus obtained, the reaction solvent and extractant solvent are removed by heating under reduced pressure or the like procedure, to isolate the objective compound.",
"As an alternative, after completion of the reaction, the reaction solvent may be immediately distilled off by heating under reduced pressure or the like procedure, and then the same procedure as above be carried out.",
"The objective product thus obtained may be purified to a still higher purity by the routine method such as purification by crystallization, fractional distillation, column chromatography, and/or the like.",
"BEST MODE FOR CARRYING OUT THE INVENTION The following examples are intended to illustrate the present invention in further detail without defining the scope of the invention.",
"EXAMPLE 1 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester To 21.3 mL (35 mmol) of a solution of n-butyllithium in hexane (1.5 mol/L) was added a solution of diisopropylamine (3.54 g, 35 mmol)-tetrahydrofuran (10 mL) dropwise under stirring at 5° C., and the mixture was stirred under argon for 1 hour to prepare a lithium diisopropylamide solution.",
"After this solution was cooled to −70° C., 4.06 (35 mmol) of tert-butyl acetate was added dropwise thereto and the mixture was stirred at the same temperature for 1 hour.",
"Then, 1 mL of a solution of (S)-β-hydroxy-γ-butyrolactone (1.02 g, 10 mmol) in THF was added dropwise and the whole mixture was stirred at −70° C. for 2 hours, at the end of which time the temperature was increased to −10° C. In a separate vessel, 60 mL of 1N-hydrochloric acid and 60 ml of diethyl ether were stirred to mix and the above reaction mixture was poured therein.",
"The aqueous phase was adjusted to pH 6.5 with 1N-hydrochloric acid and, after standing, the organic layer was separated.",
"The aqueous layer was further extracted with 3 portions of ethyl acetate, 50 mL each, and the organic layers were pooled and dehydrated over anhydrous magnesium sulfate.",
"The solvent was then distilled off under reduced pressure and the residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane: ethyl acetate=2:1).",
"By this procedure, 1.56 g of (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil) was obtained.",
"Yield: 71%.",
"1H-NMR (CDCl3, 400 MHz/ppm); 1.48 (9H, s), 2.68-2.83 (2H, m), 3.05-3.80 (2H, bs), 3.42 (2H, s), 4.02-4.17 (2H, m), 4.40 (1H, m) 13C-NMR (CDCl3, 400 MHz/ppm); 27.8, 45.7, 51.0, 65.6, 68.0, 82.3, 166.4, 203.4 IR (neat); 3425, 3000, 1710, 850 cm−1 [α]D20=−17.25 (c=2.14, MeOH).",
"EXAMPLE 2 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester To 30 mL (45 mmol) of a solution of n-butyllithium in hexane (1.5 mol/L) was added a solution of diisopropylamine (5.01 g, 49.5 mmol)tetrahydrofuran (5 mL) dropwise under stirring at 5° C., and the mixture was stirred under argon for 1 hour to prepare a lithium diisopropylamide solution.",
"In a separate vessel, 1.02 g (10 mmol) of (S)-β-hydroxy-γ-butyrolactone and 2.32 g (20 mmol) of tert-butyl acetate were dissolved in 8.0 mL of tetrahydrofuran and the mixture was stirred under argon at 0 to 5° C. To this solution, the lithium diisopropylamide solution prepared above was added dropwise over 30 minutes, and the mixture was further stirred at 5 to 20° C. for 16 hours.",
"In a separate vessel, 35 mL of 3N-hydrochloric acid and 30 ml of ethyl acetate were stirred to mix and the above reaction mixture was poured therein.",
"After standing, the organic layer was taken, washed with saturated aqueous sodium chloride solution, and dehydrated over anhydrous magnesium sulfate.",
"The solvent was then distilled off under reduced pressure and the residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane:ethyl acetate=2:1) to give 124 mg of (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil).",
"Yield 6%.",
"EXAMPLE 3 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester To 22.9 mL (35 mmol) of a solution of n-butyllithium in hexane (1.5 mol/L) was added a solution of diisopropylamine (3.90 g, 38.5 mmol)-tetrahydrofuran (3 mL) dropwise under stirring at 5° C., and the mixture was stirred under argon for 1 hour to prepare a lithium diisopropylamide solution.",
"In a separate vessel, 1.02 g (10 mmol) of (S)-β-hydroxy-γ-butyrolactone and 2.32 g (20 mmol) of tert-butyl acetate were dissolved in 3.0 mL of tetrahydrofuran and the mixture was stirred under argon at 0 to 5° C. To this solution, 5.7 g (10 mmol) of a solution (1.75 mol/kg) of tert-butylmagnesium chloride in toluene/tetrahydrofuran (1:2.5 by weight) was added dropwise over 10 minutes, and the mixture was further stirred at 5° C. for 50 minutes.",
"To this mixture was added the above-prepared lithium diisopropylamide solution dropwise over 30 minutes, followed by 16 hours of stirring at 5 to 20° C. In a separate vessel, 30 mL of 3N-hydrochloric acid and 30 ml of ethyl acetate were stirred to mix and the above reaction mixture was poured therein.",
"After standing, the organic layer was taken, washed with saturated aqueous sodium chloride solution, and dehydrated over anhydrous magnesium sulfate.",
"The solvent was then distilled off under reduced pressure and the residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane:ethyl acetate=2:1) to give 980 mg of (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (red oil).",
"Yield 48%.",
"EXAMPLE 4 (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester To a suspension of 9.82 g (150 mmol) of zinc dust in 40 mL of tetrahydrofuran was added 1.9 mL (15 mmol) of trimethylsilyl chloride at room temperature, and the mixture was stirred for 30 minutes.",
"To this mixture were added 2.4 mL (10.5 mmol) of tert-butyl α-bromoacetate and 4.27 g (42 mmol) of (S)-β-hydroxy-γ-butyrolactone, and the temperature was increased to 65° C. At the same temperature, 15.3 mL (94.5 mmol) of tert-butyl α-bromoacetate was further added gradually over 30 minutes.",
"After completion of addition, the mixture was further stirred at 65° C. for 30 minutes, at the end of which time the reaction mixture was cooled to room temperature and diluted with 50 mL of water.",
"The reaction mixture was then adjusted to pH 6.8 with 20% aqueous NaOH solution and the precipitated solid was filtered off.",
"The filtrate was extracted with 3 portions of ethyl acetate, 100 mL each, and the organic layers were combined and dehydrated over anhydrous sodium sulfate.",
"The solvent was then distilled off under reduced pressure to give a yellow oil.",
"This residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane: acetone=5:1) to give 2.66 g of (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester (yellow oil).",
"Yield 29%.",
"EXAMPLE 5 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester To a solution of 16.8 g (77 mmol) of the (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester produced in Example 1 in 120 mL of methylene chloride were added 11.2 mL of pyridine and 10.2 mL of benzoyl chloride at 0° C., and the mixture was stirred at 0° C. for 2 hours.",
"After completion of the reaction, the reaction mixture was diluted with 38 mL of water and adjusted to pH 7 with 20% aqueous NaOH solution.",
"The aqueous layer was separated and further extracted with 2 portions of methylene chloride, 120 mL each.",
"The organic layers were combined and dehydrated over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to give an oil.",
"This residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane: acetone=5:1) to give 19.3 g of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester (white solid).",
"Yield 78%.",
"1H-NMR (CDCl3, 400 MHz/ppm); 1.46 (9H, s), 2.85 (2H, d), 3.09 (1H, d), 3.42 (2H, s), 4.36 (2H, m), 4.50 (1H, m), 7.45 (2H, dd), 7.56 (1H, dd), 8.05 (2H, d) IR (KBr); 3495, 1730, 1700, 1335, 1290, 1150, 720 cm−1 m.p.",
"67 to 68° C. EXAMPLE 6 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester To 109 mL (175 mmol) of a solution of n-butyllithium in hexane (1.5 mol/L) was added a solution of diisopropylamine (19.48 g, 195 mmol)-tetrahydrofuran (30 mL) dropwise under stirring at 5° C., and the mixture was stirred under argon for 1 hour to prepare a lithium diisopropylamide solution.",
"In a separate vessel, 5.10 g (50 mmol) of (S)-β-hydroxy-γ-butyrolactone and 14.5 g (125 mmol) of tert-butyl acetate were dissolved in 60 ml of tetrahydrofuran and the solution was stirred under argon at 0 to 5° C. To this solution, 27.8 g (50 mmol) of a mixed solution of tert-butylmagnesium chloride in toluene/tetrahydrofuran (1:2.5, by weight) (1.8 mol/kg) was added dropwise over 30 minutes and the mixture was further stirred at 5° C. for 30 minutes.",
"To this mixture was added the above-prepared lithium diisopropylamide solution dropwise over 3 hours, and the mixture was further stirred at 5 to 20° C. for 16 hours.",
"In a separate vessel, 25.05 g of acetic acid, 75 mL of water, and 150 mL of ethyl acetate were stirred to mix and the above reaction mixture was poured therein.",
"After standing, the aqueous layer was separated and further extracted with 2 portions of ethyl acetate, 150 mL each.",
"The organic layers were combined, diluted with 20 mL of saturated aqueous sodium chloride solution, and adjusted to pH 3 with 3N-hydrochloric acid.",
"After the aqueous layer was separated, the organic layer was further washed with 20 mL of saturated aqueous sodium hydrogen carbonate solution and dehydrated over anhydrous magnesium sulfate.",
"The solvent was distilled off under reduced pressure to give 18.18 g of a yellow oil containing (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester.",
"To the above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene were added, and the mixture was cooled to 5° C. To this mixture was added 6.32 g (45 m=ol) of benzoyl chloride, and the whole mixture was stirred at 5° C. for 1.5 hours.",
"Then, 25 mL of water and 15 mL of 3N-hydrochloric acid were added.",
"This mixture was extracted with 100 mL of ethyl acetate and the organic layer was washed with 30 mL of saturated sodium hydrogen carbonate solution and 50 mL of water twice.",
"The solvent was then distilled off under reduced pressure to give 18.43 g of a yellow oil.",
"This oil was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The analysis showed that the reaction yield of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was 55%.",
"EXAMPLE 7 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester To 125 mL (225 mmol) of a solution of n-butylmagnesium chloride in tetrahydrofuran (1.8 mol/L) was added 25.04 g (247.5 mmol) of diisopropylamine dropwise under stirring at 40° C., and the mixture was further stirred under argon at 40° C. for 2 hours to prepare a white slurry of chloromagnesium diisopropylamide.",
"In a separate vessel, 5.10 g (50 mmol) of (S)-β-hydroxy-γ-butyrolactone and 1.45 g (125 mmol) of tert-butyl acetate were dissolved in 30 mL of dimethoxyethane and the solution was stirred at 0 to 5° C. under argon.",
"To this solution was added the above-prepared chloromagnesium diisopropylamide slurry dropwise over 3 hours, and the mixture was further stirred at 5 to 20° C. for 16 hours.",
"In a separate vessel, 28.4 g of acetic acid, 100 mL of water, and 150 ml of ethyl acetate were stirred to mix and the above reaction mixture was poured therein.",
"After standing, the aqueous layer was separated and further extracted with 2 portions of ethyl acetate, 150 ml each.",
"The organic layers were combined, diluted with 20 mL of saturated aqueous sodium chloride solution, adjusted to pH 3 with 3N-hydrochloric acid, and the aqueous layer was separated.",
"The organic layer was further washed with 20 mL of saturated aqueous sodium hydrogen carbonate solution and dehydrated over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to give 14.24 g of a red oil containing (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester.",
"To the above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene were added, and the mixture was cooled to S° C. To this mixture was added 5.62 g (40 mmol) of benzoyl chloride, and the whole mixture was stirred at 5° C. for 1 hour.",
"Then, 25 mL of water and 15 mL of 3N-hydrochloric acid were added.",
"This mixture was extracted with 150 mL of ethyl acetate and the organic layer was washed with 30 mL of saturated sodium hydrogen carbonate solution and 30 mL of water twice.",
"The solvent was then distilled off under reduced pressure to give 18.12 g of a yellow oil.",
"This oil was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"As a result, the reaction yield of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was found to be 53%.",
"EXAMPLE 8 (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester To 141 mL (225 mmol) of a solution of n-butyllithium in hexane (1.6 mol/L) was added a solution of diisopropylamine (25.04 g, 247.5 mmol)-tetrahydrofuran (30 mL) dropwise under stirring at 5° C., and the mixture was stirred under argon for 1 hour to prepare a lithium diisopropylamide solution.",
"In a separate vessel, 5.10 g (50 mmol) of (S)-β-hydroxy-γ-butyrolactone, 14.5 g (125 mmol) of tert-butyl acetate and 9.52 g (100 mmol) of anhydrous magnesium chloride were dissolved in 30 ml of tetrahydrofuran and the solution was stirred under argon at 0 to 5° C. To this solution was added the above-prepared a lithium diisopropylamide solution dropwise over 3 hours, and the mixture was further stirred at 5 to 20° C. for 16 hours.",
"In a separate vessel, 28.4 g of acetic acid, 100 mL of water, and 150 mL of ethyl acetate were stirred to mix and the above reaction mixture was poured therein.",
"After standing, the aqueous layer was separated and further extracted with 2 portions of ethyl acetate, 150 ml each.",
"The organic layers were combined, diluted with 20 mL of saturated aqueous sodium chloride solution, adjusted to pH 3 with 3N-hydrochloric acid, and the aqueous layer was separated.",
"The organic layer was further washed with 20 mL of saturated aqueous sodium hydrogen carbonate solution and dehydrated over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to give 12.74 g of a red oil containing (5S)-5,6-dihydroxy-3-oxohexanoic tert-butyl ester.",
"To the above oil, 6.32 g (80 mmol) of pyridine and 50 mL of toluene were added, and the mixture was cooled to S° C. To this mixture was added 5.62 g (40 mmol) of benzoyl chloride, and the whole mixture was stirred at 5° C. for 1 hour.",
"Then, 25 mL of water and 15 mL of 3N-hydrochloric acid were added.",
"This mixture was extracted with 150 mL of ethyl acetate and the organic layer was washed with 30 mL of saturated sodium hydrogen carbonate solution and 30 mL of water twice.",
"The solvent was then distilled off under reduced pressure to give 17.92 g of a red oil.",
"This oil was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"As a result, the reaction yield of (55)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was found to be 55%.",
"EXAMPLE 9 Purification of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester The oil containing (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester as produced in Example 6 was analyzed high-performance liquid chromatography (the conditions are described in Example 6).",
"Purity: 48.2 weight % (58.1 area %).",
"As an impurity, the oil contained 4.8 weight % (6.5 area %) of (5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester.",
"To 18.43 g of this oil ((55)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester: 8.88 g) was added 30 mL of toluene to make a homogeneous solution, followed by addition of 80 mL of hexane, and the mixture was cooled to 5° C. (treatment concentration: 8% (substrate weight/solution volume)).",
"To this opaque solution, about 10 mg of seed crystals were added, and the mixture was further stirred vigorously at the same temperature for 1 hour.",
"The resulting crystals were collected by suction filtration, drained thoroughly, washed with 50 mL of hexane, and dried in vacuo (ca 1-5 mmHg, 20 to 40° C., 2 hours), whereby 6.12 g of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester was obtained as crystals (crystallization recovery rate 67%).",
"Analysis: purity 97.7 weight % (95.4 area %); (5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester content: 0.7 weight % (0.7 area %).",
"EXAMPLE 10 Purification of (5S)-6-benzoyloxy-S-hydroxy-3-oxohexanoic tert-butyl ester The oil containing (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester as produced in Example 7 was analyzed by high-performance column chromatography (the conditions are described in Example 6).",
"The purity was 45.0 weight % (47.2 area %) and the impurity (5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester content: 4.7 weight % (4.9 area %).",
"To 18.12 g of this oil ((5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester: 8.15 g) was added 20 mL of toluene to prepare a homogeneous solution.",
"To this solution was added 80 mL of hexane, and the mixture was cooled to −30° C. (treatment concentration: 8% (substrate weight/solution volume)).",
"To the resulting opaque solution was added about 10 mg of seed crystals, and the mixture was stirred vigorously at the same temperature for 1 hour.",
"The crystals separating out were collected by suction filtration, drained thoroughly, and washed with 50 mL of hexane.",
"This crystal crop was dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2 hours) to obtain 6.68 g of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester crystals (crystallization recovery rate 77%).",
"Analysis of the crystals showed a purity of 95.8 weight % (94.9 area %) and a (5S)-5,6-dibenzyloxy-3-oxohexanoic tert-butyl ester content of 1.6 weight % (1.6 area %).",
"EXAMPLE 11 (3R, 5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl ester A large test tube was charged with 5 ml of Medium A described hereinbefore and, after sterilization, inoculated with one of the microorganisms indicated in Table 1 and Table 2.Aerobic shake culture was carried out at 27° C. for 2 to 3 days.",
"From a 1.5 ml portion of the resulting culture, the cells were harvested by centrifugation and suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 0.05% of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester and 8% of glucose.",
"The suspension was put in a test tube equipped with a threaded stopper and the reaction was carried out under shaking at 27° C. for 20 hours.",
"After the reaction, 4 volumes of ethyl acetate were added to the reaction mixture and after thorough mixing, the cells were centrifugally removed.",
"The supernatant was analyzed by high-performance liquid chromatography for the amount of (3R,5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced per ml of the reaction mixture and the diastereomer ratio (=(3R,5S)/(3S,5S) ratio).",
"The results are shown in Table 1 and Table 2.TABLE 1 Output D.E.",
"Microorganism (ug/ml) (%) Ashbya gossypii IFO 0560 500.0 61.1 Botryoascus synnaedendrus IFO 1604 330.6 63.7 Brettanomyces custersianus IFO 1585 59.7 95.1 Candida arborea IAM 4147 77.3 72.4 Candida catenulata IFO 0745 46.5 10.1 Candida fennica CBS 6028 135.1 87.8 Candida galacta IFO 10031 121.0 84.3 Candida haemulonii IFO 10001 133.7 94.3 Candida magnoliae IFO 0705 175.0 27.1 Candida musae IFO 1582 39.8 33.5 Candida nitratophila IFO 10004 20.1 48.3 Candida parapsilosis IFO 0585 118.4 65.7 Candida pararugosa IFO 0966 26.6 56.0 Candida stellata IFO 0701 27.4 100.0 Citeromyces matritensis IFO 0651 446.9 26.2 Clavispora lusitaniae IFO 1019 342.1 53.7 Cryptococcus laurentii IFO 0609 37.5 14.5 Debaryomyces carsonii IFO 0795 449.1 97.3 Debaryomyces hansenii var.",
"fabryi IFO 0794 55.4 78.8 Debaryomyces hansenii var.",
"hansenii IFO 0032 130.1 93.6 Debaryomyces hansenii var.",
"hansenii IFO 0047 135.4 95.4 Debaryomyces hansenii var.",
"hansenii IFO 0018 103.1 95.5 Debaryomyces kloeckeri 140.7 95.4 Debaryomyces marama IFO 0668 161.5 95.4 Debaryomyces pseudopolymorphus IFO 1026 75.4 90.9 Debaryomyces robertsiae IFO 1277 278.7 66.1 Debaryomyces sp.",
"IFO 0025 32.7 74.7 Dekkera anomala IFO 0627 115.8 94.9 Dipodascus armillariae IFO 0102 154.6 76.8 Dipodascus ovetensis IFO 1201 134.9 96.9 Dipodascus tetrasperma CBS 765.70 203.0 33.1 Galactomyces reessii CBS 179.60 378.8 25.0 Geotrichum candidum CBS 187.67 148.6 51.8 Geotrichum fermentans IFO 1199 98.4 93.9 Geotrichum fragrans CBS 164.32 29.1 94.1 Geotrichum loubieri CBS 252.61 81.4 14.8 Hanseniaspora guilliermondii IAM 4972 35.8 4.0 Hansenula methanolosa 93.7 99.2 Hansenula polymorpha DL1 AKU 4752 21.6 100.0 Hormoascus philentomus IFO 1847 176.6 84.2 Hormoascus platypodis IFO 1471 260.8 36.6 Hyphopichia burtonii IFO 0844 228.8 77.7 Issatchenkia orientalis IFO 1279 443.0 36.4 Issatchenkia terricola IFO 0933 258.4 100.0 Kluyveromyces lactis IFO 1012 324.6 48.8 Kluyveromyces marxianus IFO 0541 102.7 85.9 Kluyveromyces marxianus IFO 0288 419.3 27.1 Kluyveromyces polysporus IFO 0996 132.6 5.1 Kluyveromyces thermotolerans IFO 0662 500.0 100.0 Komagataella pastoris IFO 1013 246.0 66.4 Lipomyces starkcyi IFO 0678 28.6 100.0 Metschnikowia bicuspidata IFO 1408 381.0 39.6 Metschnikowia pulcherrima IFO 0561 359.4 48.0 TABLE 2 Output D.E.",
"Microorganism (ug/ml) (%) Nakazawaea holstii IFO 0980 0.5 100.0 Ogataea minuta var.",
"minuta IFO 0975 18.2 9.4 Ogataea pini IFO 1342 268.3 43.1 Ogataea polymorpha IFO 0799 500.0 67.3 Ogataea polymorpha IFO 1475 275.0 6.0 Ogataea wickerhamii IFO 1706 144.8 73.6 Pachysolen tannophilus IFO 1007 488.2 5.4 Pichia canadensis IFO 0976 17.5 86.9 Pichia farinosa IAM 4369 208.9 71.4 Pichia jandinii IFO 0987 296.4 96.9 Pichia saitoi IAM 4945 97.4 22.5 Pichia toletana IFO 0950 300.0 13.1 Pichia triangularis IFO 0836 328.8 24.6 Pichia wickerhamii IFO 1278 175.8 81.7 Rhodotorula graminis IFO 0190 96.5 3.3 Rhodotorula minuta IFO 0387 108.3 12.0 Rhodotorula minuta IFO 0715 0.5 100.0 Rhodsporidium diobovatum IFO 0688 1.8 17.4 Rhodsporidium toruloides IFO 0413 10.2 46.7 Saccharomyces bayanus IFO 0251 375.2 18.3 Saccharomyces pastorianus IFO 1265 442.5 80.5 Saccharomyces pastorianus ATCC 9080 83.1 72.9 Saccharomyces rosei IFO 0252 456.8 83.1 Saccharomyces sake 349.5 92.6 Saccharomyces steineri IAM 4608 98.3 100.0 Saccharomyces unisporus IFO 0215 97.0 84.5 Saccharomycodes ludwigii IFO 0339 99.0 43.8 Saccharomycopsis capsularis IFO 0672 112.5 76.8 Saccharomycopsis malanga IFO 1710 0.3 100.0 Saturnospora dispora IFO 0035 16.6 16.8 Schizoblastosporion kobayasii IFO 1644 207.0 54.6 Schizosaccharomyces pombe IFO 0347 119.5 55.2 Schizosaccharomyces pombe IFO 0362 96.3 56.0 Schwanniomyces occidentalis var.",
"occidentalis IFO 1840 219.7 46.9 Sporidiobolus johnsonii IFO 6903 2.7 100.0 Sporobolomyces pararoseus IFO 0471 66.0 67.8 Sporobolomyces salmonicolor IFO 1038 8.8 100.0 Torulaspora delbrueckii IFO 0381 186.4 95.9 Torulopsis methanolcycscens 337.0 33.2 Torulopsis osboenis IFO 0646 58.5 16.9 Torulopsis sp.",
"99.7 84.9 Torulopsis uvae IFO 0649 287.1 88.8 Trichosporon pullulans 20.9 51.0 Trichosporon sp.",
"4.6 19.2 Trigonopsis variabilis IFO 0671 126.4 13.4 Willopsis saturnus var.",
"mrakii IFO 0895 445.3 3.6 Willopsis saturnus var.",
"saturnus IFO 0992 394.4 6.1 Yamadazyma farinosa IFO 0459 472.7 86.7 Yamadazyma farinosa IFO 0602 97.0 55.0 Yamadazyma haplophila IFO 0947 7.2 66.4 Zygosaccharomyces naniwensis IFO 0524 263.1 43.0 Zygosaccharomyces sp.",
"IFO 0522 282.8 9.4 EXAMPLE 12 (3R, 5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl ester Using 5 ml of Medium B described hereinbefore, the microorganisms indicated in Table 3 were cultured in the same manner as in Example 11.Thereafter, the reaction was carried out in the same manner.",
"The results are shown in Table 3.TABLE 3 Output D.E.",
"Microorganism (ug/ml) (%) Acidiphilium cryptum IFO 14242 2.7 12.2 Aerobacter cloacae IAM 1221 29.2 79.0 Alcaligenes xylosoxidans IFO 13495 13.2 38.9 Alcaligenes xylosoxidans subsp.",
"denitrificans IFO 12669 3.9 22.9 Alcaligenes xylosoxidans subsp.",
"denitrificans ATCC 15173 11.2 90.3 Arthrobacter globiformis ATCC 8010 0.4 100.0 Arthrobacter protophormiae IFO 12128 28.1 100.0 Aureobacterium esteraromaticum IFO 3752 245.9 24.1 Bacillus badius IAM 11059 0.3 100.0 Bacillus sphaericus IFO 3525 0.7 20.2 Brevibacterium ammomiagenes IFO 12071 193.1 98.8 Buttiauxella agrestis JCM 1090 25.7 40.3 Cedecea davisiae JCM 1685 3.2 37.0 Cellulomonas sp.",
"JCM 2471 119.8 95.9 Cellulomonas turbata IFO 15015 88.8 24.3 Citrobacter freundii IFO 12681 75.2 68.9 Clostridium cylindrosporum IFO 13695 4.8 50.6 Comamonas testosteroni IFO 12047 1.1 48.8 Corynebacterium acectoacidophilum ATCC 21476 211.3 63.4 Corynebacterium ammoniagenes IFO 12072 1.8 66.0 Corynebacterium glutamicum ATCC 21269 269.3 92.2 Corynebacterium glutamicus ATCC 13287 276.9 98.3 Enterobacter aerogenes IFO 13534 54.5 91.4 Enterobacter cloacae IFO 12935 490.7 88.9 Erwinia carotovora subsp.",
"carotovora IFO 3830 1.1 100.0 Escherichia coli IFO 12734 23.2 53.9 Flavobacterium flavesceus 17.3 25.1 Klebsiella planticola IFO 3317 127.3 61.6 Luteococcus japonicus IFO 12422 0.2 100.0 Microbacterium arborescens IFO 3750 7.6 90.8 Micrococcus flavus 4.0 30.7 Micrococcus luteus IFO 13867 500.0 13.5 Ochrobactrum sp.",
"IFO 12950 12.5 51.4 Proteus inconstans IFO 12931 5.6 87.6 Proteus mirabilis IFO 3849 1.1 100.0 Proteus rettgeri IFO 13501 0.3 100.0 Proteus vulgaris IFO 3167 0.5 100.0 Providencia stuartii IFO 12930 2.9 76.9 Pseudomonas aeruginosa IAM 1007 2.8 100.0 Pseudomonas putida IFO 14164 11.2 31.1 Pseudomonas stutzeri IFO 13596 8.0 32.5 Rhodococcus equi JCM 1313 48.4 72.7 Sarcina lutea 369.2 86.8 Serratia plymuthicum IFO 3055 2.7 100.0 Serratia proteamaculans subsp.",
"proteamaculans IFO 12979 38.5 47.3 Sphingobacterium spiritivorum JCM 1277 61.9 33.6 Tsukamurella paurometabolum IFO 12160 40.6 8.2 EXAMPLE 13 (3R,5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl ester Using 5 ml of Medium C described hereinbefore, the microorganisms indicated in Table 4 were cultured in the same manner as in Example 11.From 5 ml of each culture, the cells were centrifugally harvested, suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 0.05% of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester and 8% of glucose, and the reaction was carried out in the same manner.",
"The results are shown in Table 4.TABLE 4 Output D.E.",
"Microorganism (ug/ml) (%) Absidia orchidis HUT 1036 4.4 25.2 Acremonium bacillisporum IFO 9387 1.0 100.0 Aegerita candida IFO 6988 500.0 92.8 Agrocybe cylindracea IFO 30299 96.2 59.5 Amylostereum areolatum IFO 9221 71.7 32.9 Aspergillus parasiticus IFO 4403 2.6 100.0 Aspergillus phoenicis IFO 6670 1.4 32.0 Byssochlamys fulva IFO 6307 164.6 99.2 Chaetomidium fimeti IFO 30419 0.5 100.0 Chaetosartorya stromatoides IFO 9652 1.6 100.0 Cladosporium resinae F. avellaneum IFO 6367 1.4 100.0 Coprinus cinereus 401.3 76.0 Coprinus lagopus IFO 9533 37.9 93.7 Coprinus sp.",
"1.9 100.0 Crinipellis stipitaria IFO 30259 16.6 40.6 Endophragmia alternata IFO 30204 10.4 54.9 Flavolus arcularius 217.0 8.8 Fomitopsis pubertatis 102.0 6.6 Fusarium merismoides IFO 30040 125.7 16.2 Ganoderma lucidum IFO 31863 2.1 31.8 Glomerella cingulata IFO 5257 47.8 78.7 Laetiporus sulphureusII 66.9 32.8 Lentinus lepideus TD-832 165.2 35.1 Lenzites betulina IFO 8715 155.9 34.5 Macrophoma commelinae IFO 9569 210.2 99.3 Monascus purpureus IFO 5965 135.7 13.7 Mortierella isabellina IFO 7829 8.7 100.0 Paecilomyces varioti HUT 4028 34.6 100.0 Penicillium chermesinum IFO 5800 39.9 86.7 Penicillium chrysogenum IFO 4640 133.8 97.4 Penicillium expansum IFO 5854 4.5 51.1 Penicillium lilacinium IFO31914 47.2 95.1 Phialophora fastigiata IFO 6850 38.4 89.4 Pholiota aurivella IFO 30265 74.5 100.0 Pholiota limonella IFO 31868 0.8 100.0 Pleurotus dryinus 123.9 26.0 Pleurotus ostreatus 159.1 22.4 Pleurotus porrigens 247.7 87.3 Scopulariopsis brevicaulis IFO 4843 88.6 23.9 Sehizophyllum commune IFO 6503 119.9 43.7 Sporotrichum aurantiacum IFO 9381 84.4 8.9 Zygorhynchus moelleri HUT 1305 167.6 93.5 EXAMPLE 14 (3R,5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl Using 5 ml of Medium D described hereinbefore, the microorganisms indicated in Table 5 were cultured in the same manner as in Example 13.Then, the same reaction was carried out.",
"The results are shown in Table 5.TABLE 5 Output D.E.",
"Microorganism (μg/ml) (%) Microtetraspora roseoviolacea IFO 14098 27.8 15.4 Streptomyces achromogenes subsp.",
"IFO 14000 1.6 19.3 rubradiris Streptomyces sp.",
"29.4 42 Streptomyces aureus NIHJ 122 1 100 EXAMPLE 15 (3R,5S)-6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl A Sakaguchi flask of 500 ml capacity was charged with 100 ml of a medium composed of Bacto-tryptone 1.6%, Bacto-yeast extract 1%, and sodium chloride 1% (pH 7.0) and, after sterilization, inoculated with Escherichia coli HB101 (pNTCRG); FERM BP-6898 (deposited with National Institute of Bioscience and Human-Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) as of Sep. 28, 1999).",
"Shake culture was carried out at 37° C. for 12 hours.",
"After completion of cultivation, 1 g of (5S)-6-benzyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester, 610 mg of glucose, and 3 mg of oxidized nicotinamide-adenine dinucleotide phosphate were added and the reaction was conducted for 24 hours, during which the pH was maintained at 6.5 with sodium hydroxide.",
"After completion of the reaction, the cells were centrifugally removed and the supernatant was extracted with 2 portions of ethyl acetate, 100 mL each.",
"The organic phase obtained was dehydrated over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to give 900 mg of 6-benzyloxy-3,5-dihydroxyhexanoic tert-butyl ester as oil.",
"As analyzed by the method described in Example 11, the diastereomer ratio of this product was (3R,5S)/(3S,5S)=99.5/0.5.1H-NMR (CDCl3, 400 MHz/ppm); 1.47 (9H, s), 1.63-1.82 (2H, m), 2.45 (2H, d), 4.1-4.3 (4H, m), 7.32-7.7 (3H, m), 8.0-8.22 (2H, m) IR (neat); 3450, 3000, 1730, 1040, 850, 720 cm−1.EXAMPLE 16 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 8.94 g (27.6 mmol) of (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 35.8 mL of 2,2-dimethoxypropane, and 2.5 mL of methylene chloride was added 269 mg (1.4 mmol) of p-toluenesulfonic acid.1H2O, and the mixture was stirred at 20° C. for 4 hours, after which 500 mL of saturated sodium hydrogen carbonate solution was added.",
"The aqueous layer was separated and further extracted with 2 portions of methylene chloride, 20 mL each, and the organic layers were combined.",
"The combined solution was dehydrated over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to give a colorless oil.",
"This residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane: acetone=10:1) to give 7.24 g of (5S)-6-benzoyloxy-5-hydroxy-3-oxohexanoic tert-butyl ester (white solid).",
"Yield: 72%.",
"1H-NMR (CDCl3, 400 MHz/ppm); 1.44 (9H, s), 1.45 (6H, d), 1.55-1.59 (2H, m), 2.35-2.46 (2H, m), 4.22-4.37 (4H, m), 7.43-7.59 (3H, m), 8.0-8.1 (2H, m) IR (neat); 2975, 1720, 1270, 1150, 1100, 718 cm−1 m.p.",
"S5 to 56° C. EXAMPLE 17 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone was added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H2O, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 78.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 7.5%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 5.9%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 3.0%.",
"EXAMPLE 18 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H2O and 11.9 mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 93.1%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 3.4%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 0.1%.",
"EXAMPLE 19 2-[(4R, 6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H2O and 15.2 mg (0.15 mmol) of triethylamine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 93.3%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 3.0%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 0.1%.",
"EXAMPLE 20 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H2O and 10.2 mg (0.15 mmol) of imidazole, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 93.9%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 3.0%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 0.1%.",
"EXAMPLE 21 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 5.7mg (0.03 mmol) of p-toluenesulfonic acid.1H2O and 14.0 mg (0.15 mmol) of 3-methylpyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 93.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 2.8%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 0.1%.",
"EXAMPLE 22 2-[(4R, 6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 5.7 mg (0.03 mmol) of p-toluenesulfonic acid.1H2O and 18.3 mg (0.15 mmol) of N,N-dimethylaminopyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 93.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 2.3%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 0.1%.",
"EXAMPLE 23 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 3.0 mg (0.03 mmol) of methanesulfonic acid and 11.9 mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 95.6%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 2.6%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: undetected.",
"EXAMPLE 24 2-[(4R, 6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 3.5 mg (0.03 mmol) of trifluoroacetic acid and 11.9 mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 89.3%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 8.8%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: undetected.",
"EXAMPLE 25 2-[(4R, 6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 108 mg (90.2 weight %, 0.3 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 62.4 mg (0.6 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone were added 1.5 mg (0.015 mmol) of sulfuric acid and 11.9 mg (0.15 mmol) of pyridine, and the mixture was stirred at 40° C. for 16 hours.",
"This reaction mixture was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 95.8%; (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 2.3%; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1%; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: undetected.",
"EXAMPLE 26 Purification of 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 3.24 g (90.2 weight %, 9.0 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 2.12 g (18.0 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone was added 95 mg (0.45 mmol) of p-toluenesulfonic acid 1H2O, and the mixture was stirred at 40° C. for 4 hours.",
"The solvent was distilled off under reduced pressure and the residue was extracted using 25 mL of ethyl acetate and 10 mL of saturated aqueous sodium hydrogen carbonate.",
"After separation of the aqueous layer, the organic layer was further washed with 10 mL of water.",
"The solvent was then distilled off under reduced pressure to give 3.764 g of a colorless oil.",
"This oil was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 79.7 weight %.",
"As impurities, (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 0.1 weight %; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1 weight %; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 5.0 weight %; 2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester:0.2 weight % were contained.",
"To the above oil was added 30 mL of hexane, and the mixture was cooled to −30° C. (treatment concentration: 10% (substrate weight/solution volume)).",
"About 10 mg of seed crystals were added and the mixture was further stirred vigorously at the same temperature for 1 hour.",
"The crystals separating out were collected by suction filtration, drained thoroughly, and washed with 10 mL of cold hexane.",
"The crystal crop was then dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2 hours) to obtain 2.46 g of 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester as crystals (crystallization recovery rate 81%).",
"Analysis of the above crystals showed a purity of 97.2 weight % (96.8 area %).",
"As impurities, the crystal crop contained: 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 2.8 weight % (2.8 area %); (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: undetected; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: undetected; 2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: undetected.",
"EXAMPLE 27 Purification of 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution composed of 3.24 g (90.2 weight %, 9.0 mmol) of the (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester produced in Example 15, 2.12 g (18.0 mmol) of 2,2-dimethoxypropane, and 5 mL of acetone was added 95 mg (0.45 mmol) of p-toluenesulfonic acid.1H2O, and the mixture was stirred at 40° C. for 4 hours.",
"The solvent was then distilled off under reduced pressure and the residue was extracted using 25 mL of ethyl acetate and 10 mL of saturated aqueous sodium hydrogen carbonate.",
"After separation of the aqueous layer, the organic layer was further washed with 10 mL of water.",
"The solvent was then distilled off under reduced pressure to give 3.764 g of a colorless oil.",
"This oil was analyzed by high-performance liquid chromatography (column: Develosil ODS-HG-3 4.6×250 mm, product of Nomura Chemical, eluent: water/acetonitrile=50/50, flow rate: 1.0 mL/min, detector: UV 220 nm, column temperature: 40° C.).",
"The compositional yield values were as follows.",
"2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 86.0 weight %.",
"As impurities, (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: 0.1 weight %; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: 0.1 weight %; 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 5.4 weight %; 2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: 0.2 weight % were contained.",
"To the above oil was added 30 mL of methylcyclohexane, and the mixture was cooled to −30° C. (treatment concentration: 10% (substrate weight/solution volume)).",
"About 10 mg of seed crystals were added and the mixture was further stirred vigorously at the same temperature for 1 hour.",
"The crystals separating out were collected by suction filtration, drained thoroughly, and washed with 10 mL of cold methylcyclohexane.",
"The crystal crop was then dried in vacuo (ca 1 to 5 mmHg, 20 to 40° C., 2 hours) to obtain 2.24 g of 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester as crystals (crystallization recovery rate 74%).",
"Analysis of the above crystals showed a purity of 97.2 weight % (96.8 area %).",
"As impurities, the crystal crop contained: 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic methyl ester: 2.8 weight % (2.8 area %); (5S)-6-benzoyloxy-3,5-dihydroxyhexanoic tert-butyl ester: undetected; (2S,4R)-4-hydroxy-6-oxo-2-[(benzoyloxy)methyl]tetrahydro-2H-pyran: undetected; 2-[(4S,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester: undetected.",
"EXAMPLE 28 2-[4R,6S]-6-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-4-yl]acetic tert-butyl ester To a solution of 3.64 g (10 mmol) of the 2-[(4R,6S)-2,2-dimethyl-6-benzoyloxymethyl-1,3-dioxan-4-yl]acetic tert-butyl ester produced in Example 26 in methanol (36 mL) was added 10 mL of 1N-aqueous sodium hydroxide solution, and the mixture was stirred at room temperature for 2 hours.",
"This reaction mixture was adjusted to pH 7 by gradual addition of 1N-hydrochoric acid under ice-cooling.",
"The methanol was then distilled off under reduced pressure and the residual aqueous solution was extracted with 2 portions of methylene chloride, 70 mL each.",
"The organic layer was dehydrated over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure to give a colorless oil.",
"This residue was purified by silica gel column chromatography (Kieselgel 60, product of Merck; hexane: acetone=5:1) to give 2.34 g of 2-[(4R,6S)-6-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-4-yl]acetic tert-butyl ester (white solid).",
"Yield 90%.",
"1H-NMR (CDCl3, 400 MHz/ppm); 1.29-1.52 (2H, m), 1.39 (3H, m), 1.45 (9H, s), 1.47 (3H, s), 2.05 (1H, bs), 2.33 (1H, dd), 2.44 (1H, dd), 3.47-3.53 (1H, m), 3.99-4.04 (1H, m), 4.27-4.33 (1H, m) IR (neat); 2980, 1720, 1365, 1200, 1150, 1020 cm−1.INDUSTRIAL APPLICABILITY In accordance with the present invention constituted as above, pharmaceutical intermediates, particularly optically active 2-[6-(hydroxymethyl)-1,3-dioxan-4-yl]acetic acid derivatives which are of value as intermediates of HMG-CoA reductase inhibitors, can be produced from inexpensive, readily available starting materials without using any extraordinary equipment such as a low-temperature reactor."
]
] | Patent_10048553 |
[
[
"Method for working and processing materials",
"FIELD: techniques for processing different materials, mainly elastomers, possibly in the different manufacturing processes.",
"SUBSTANCE: method comprises steps of acting upon material by means of moving cutting tools for creating in working zone ultrasonic-frequency oscillations of material; setting power of drive units at working and processing materials no less than 100-300 kW; selecting revolution number of tool in range 3000-12000rev/min; selecting quantity of cutting edges of tool according to relation ωn, is less than 800, where ω is equal to the angular velocity of tool, n is equal to the quantity of cutting edges of tool; setting attach angles of tool in range 85-95 degrees; removing separate particles of material embedded to cutting portion of tool my means of fluid flow containing gas and (or) liquid; feeding fluid flow by excess pressure material is fed to tool or vise versa in mode of reciprocation motion or start-stop motion.",
"EFFECT: enhanced efficiency of method, increased disposition degree and uniformity of ready product structures."
],
[
"1.This method for the working and processing of materials, during which a moving working cutting tool acts on the material being worked and ultrasound-frequency oscillations are created in the material working area, is distinctive in that the output of the drive mechanism is set at not less than 100-300 kW, the rotational speed of the tool is set at 3,000-12,000 rpm, the number of tool cutting edges is set based on the correlation ω×n>8,000, where ω is the angular rotational speed of the tool and n is the number of tool cutting edges, the angle of incidence of the tool is set at 85-95°, and the separated particles of the material embedded in the cutting section of the working tool are removed by a medium flow that consists of a gas, or a fluid, or a combination thereof.",
"Here, this medium flow is delivered at gauge pressure and the material being worked is conveyed to and from the tool in the reciprocating or start-stop mode.",
"2.A method for processing materials, the method comprising the steps of: providing a housing including an inlet for introducing source materials to be processed and an outlet for discharging finished product, the housing having a working tool having a plurality of cutting edges and a plurality of channels adjacent said plurality of cutting edges, the working tool being rotationally mounted within the housing, means for rotating the working tool and means for delivering a pressurized fluid medium to said plurality of cavities; and operating the apparatus such that ω×n>8,000, where ω is representative of the angular rotational speed of the working tool and n is a number representative of said plurality of cutting blades.",
"3.The method as recited in claim 2, wherein the working tool is rotated at an angular rotational speed of about 3,000 rpm to about 12,000 rpm.",
"4.The method as recited in claim 2, further comprising the steps of inputting tires into the inlet and operating the working tool such that the finished product has a crumb size of about 10-100 microns.",
"5.A method for grinding vehicle tires, the method comprising the steps of: providing a housing including an inlet for introducing the vehicle tires therein and an outlet for discharging ground tire material, rotatably mounting a cylindrical working tool in the housing, the cylindrical working tool having a plurality of cutting edges each having an angle of incidence of about 85°-95° on an outer surface thereof and a plurality of channels between said cutting edges and the working tool having a header in fluid communication with each of said plurality of channels; rotating the cylindrical working tool at an angular speed of rotation of about 3,000-12,000 rpm; providing a pressurized fluid to the header such that pressurized fluid is introduced into said plurality of channels; and grinding the tire material to a crumb size of about 10-100 microns."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The invention at hand falls into the category of techniques for the working of different types of materials, primarily elastomers, and can be employed in various technological processes.",
"2.Description of the Prior Art This method can be used to process worn automobile and aircraft tires following their disposal.",
"A method already exists for the working of materials that contain magnetostrictive components in metallic-phase and nonmetallic-phase aggregates, which includes their exposure to a variable magnetic field and to a mechanical load, during which exposure to the magnetic field is accomplished over a range of audio frequencies, with intensity in the region where the magnetostrictive effect appears, and immediately after which the materials are exposed to a mechanical load (Inventor's Certificate 1811421, B 02 C 18/18).",
"The shortcomings of this method consist of a low level of efficiency during the working of plastic materials and the impossibility of its use during the processing of worn tires.",
"A method also already exists for the working and processing of materials during which the material being worked is exposed to a moving cutting tool and ultrasound-frequency oscillations are created in the material working area (see English Patent 2004200, Mar.",
"28, 1979).",
"Because it has the greatest number of similar features and the most similar result is achieved during its use, this latter preexisting engineering solution was selected as the closest analog of the invention at hand.",
"The shortcomings of this analog consist of a low level of efficiency during the working of elastomers due to their high plasticity and high friction coefficient.",
"The disintegration of elastomers is a complex technical task that requires a high frequency and a high working speed, which ensures the observance of the condition wherein the relaxation rate of the elastomer being worked, is less than its disintegration rate."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention at hand is based on the resolution of the problem of creating an efficient technology for the working, primarily the disintegration, of different materials, especially elastomers, by means of moving the oscillations that inevitably occur in a “tool-material” system to higher resonance frequencies, which ensures the existence of cumulative jets in the area where the cutting edges exert an influence on the material being worked, as well as a cumulative jet energy density that is high enough to facilitate the formation of running cracks in the body of the material being worked.",
"In this instance, conditions are created that ensure the disintegration of elastomers during their exposure to the cutting tool and the enhancement of surface smoothness during the working of metals.",
"This stated objective is achieved by virtue of the fact that in the subject method for the working and processing of materials, which consists of exposing the material being worked to a moving cutting tool and creating ultrasound-frequency oscillations in the material working area, the output of the drive mechanism is fixed at 100 to 300 kilowatts (kW) during the working and processing of materials, the rotational speed of the tool is set at 3,000-12,000 revolutions per minute (rpm), and the number of tool cutting edges is set based on the correlation ω×n>8,000, where ω is the angular rotational speed of the tool, n is the number of tool cutting edges, and the angle of incidence of the tool is set at 85-95°.",
"Here, the loosened particles of the material embedded in the cutting section of the working tool are carried away by a medium flow that consists of a gas, or a fluid, or a combination thereof.",
"This medium flow is delivered at gauge pressure, while the material being worked is conveyed to and from the tool in the reciprocating or “start-stop” mode."
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention The invention at hand falls into the category of techniques for the working of different types of materials, primarily elastomers, and can be employed in various technological processes.",
"2.Description of the Prior Art This method can be used to process worn automobile and aircraft tires following their disposal.",
"A method already exists for the working of materials that contain magnetostrictive components in metallic-phase and nonmetallic-phase aggregates, which includes their exposure to a variable magnetic field and to a mechanical load, during which exposure to the magnetic field is accomplished over a range of audio frequencies, with intensity in the region where the magnetostrictive effect appears, and immediately after which the materials are exposed to a mechanical load (Inventor's Certificate 1811421, B 02 C 18/18).",
"The shortcomings of this method consist of a low level of efficiency during the working of plastic materials and the impossibility of its use during the processing of worn tires.",
"A method also already exists for the working and processing of materials during which the material being worked is exposed to a moving cutting tool and ultrasound-frequency oscillations are created in the material working area (see English Patent 2004200, Mar.",
"28, 1979).",
"Because it has the greatest number of similar features and the most similar result is achieved during its use, this latter preexisting engineering solution was selected as the closest analog of the invention at hand.",
"The shortcomings of this analog consist of a low level of efficiency during the working of elastomers due to their high plasticity and high friction coefficient.",
"The disintegration of elastomers is a complex technical task that requires a high frequency and a high working speed, which ensures the observance of the condition wherein the relaxation rate of the elastomer being worked, is less than its disintegration rate.",
"SUMMARY OF THE INVENTION The invention at hand is based on the resolution of the problem of creating an efficient technology for the working, primarily the disintegration, of different materials, especially elastomers, by means of moving the oscillations that inevitably occur in a “tool-material” system to higher resonance frequencies, which ensures the existence of cumulative jets in the area where the cutting edges exert an influence on the material being worked, as well as a cumulative jet energy density that is high enough to facilitate the formation of running cracks in the body of the material being worked.",
"In this instance, conditions are created that ensure the disintegration of elastomers during their exposure to the cutting tool and the enhancement of surface smoothness during the working of metals.",
"This stated objective is achieved by virtue of the fact that in the subject method for the working and processing of materials, which consists of exposing the material being worked to a moving cutting tool and creating ultrasound-frequency oscillations in the material working area, the output of the drive mechanism is fixed at 100 to 300 kilowatts (kW) during the working and processing of materials, the rotational speed of the tool is set at 3,000-12,000 revolutions per minute (rpm), and the number of tool cutting edges is set based on the correlation ω×n>8,000, where ω is the angular rotational speed of the tool, n is the number of tool cutting edges, and the angle of incidence of the tool is set at 85-95°.",
"Here, the loosened particles of the material embedded in the cutting section of the working tool are carried away by a medium flow that consists of a gas, or a fluid, or a combination thereof.",
"This medium flow is delivered at gauge pressure, while the material being worked is conveyed to and from the tool in the reciprocating or “start-stop” mode.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a graphical representation of the dependence of change in frequency of the tool-material system on the angle of incidence of a working tool cutting edge; FIG.",
"2 is a graphical representation of the dependence upon the rate of advance of the working tool; FIG.",
"3 is a graphical representation of the dependence on the number of cutting edges on the working tool; FIG.",
"4 is a graphical representation of the dependence on the rotational speed of the working tool; and FIG.",
"5 is a schematic representation of an apparatus operating according to the method of the present invention.",
"DETAILED DESCRIPTION OF THE INVENTION The essence of the invention for which this patent is pending is expressed in the following complement of indispensable features, which is adequate for achieving the technical result described above.",
"Using the subject invention, the previously specified objective is achieved by virtue of the fact that the method for the working of materials, which includes the exposure of the material being worked to a moving, for example, a rotating, working tool, is characterized by the fact that the speed of movement of the working tool and its rate of advance, as well as the number of working tool cutting edges, are set based on the condition of creating ultrasound-frequency oscillations in the working area, during which the removal of the separated particles is accomplished by means of injecting the set of channels cut into the working tool's body with a medium flow that consists of at least one gas and/or at least one fluid.",
"This constitutes of a set of indispensable features that ensure the achievement of the desired technical result in all instances when the proposed method is used.",
"In addition, the solution for which this patent is pending is characterized by specific parameters for the technological mode, to wit: the medium flow is delivered to the channels at gauge pressure; the static pressure of the medium flow within a channel is selected with allowance for the geometric dimensions of the separated particles of the material being worked and accordingly their sail effect; the geometry of the cutting edges, particularly the angle of incidence, is selected based on the assurance of the existence of cumulative jets in the area where the cutting edges exert an influence on the material being worked, as well as a cumulative jet energy density that is high enough to facilitate the formation of running cracks in the body of the material being worked; the conveyance of the material being worked to and from the working tool is accomplished in the reciprocating or “start-stop” mode, and; the gas that is a part of the pressurized medium is treated beforehand, for example, it is ionized or ozonized.",
"The realization of the distinctive features of the subject invention (together with the features listed in the condensed patent claims section) culminates in the achievement of important new object properties.",
"In the proposed engineering solution, the efficiency of the working of materials, especially elastomers, is considerably enhanced, in addition to which the homogeneity of the finished product is improved by virtue of the previously described combination of working conditions, parameters, and factors.",
"The dependence of a change in the frequency of the “tool-material” system on the angle of incidence of a working tool cutting edge is depicted in FIG.",
"1, the dependence upon the rate of advance of the working tool is shown in FIG.",
"2, the dependence upon the number of cutting edges is reflected in FIG.",
"3, the dependence upon rotational speed—in FIG.",
"4, and the device that facilitates the use of the subject method is pictured in FIG.",
"5.The proposed parameters were selected based on the condition of the development of oscillations in the “material-tool” system, the frequency of which lies in the ultrasound oscillation region and which ensure the production of disperse structures.",
"The method at hand is realized in the following manner (FIG.",
"5).",
"The device contains a rotating cylindrical working tool, 1.The surface of the working tool, 1, is equipped with a sufficient number of cutting edges, 2.Channels, 3, cut into the working tool's housing are fashioned between the adjacent cutting edges, 2.The cavities of these channels, 3, are connected to a pressurized medium source by means of shaped conduits, 4.This medium is delivered to the device through a central.",
"header, 5.The working tool, 1, is situated inside a housing, 6, that has pipes for introducing the source material, 7, and discharging the finished product, 8.Here, the working tool is mounted on a shaft, 9.The angle of incidence of the cutting edges is set within limits of 85-95°.",
"The output of the drive mechanism is 100-300 kW and the rotational speed of the tool is 3,000-12,000 rpm.",
"The shaft, 9, is mounted in the housing, 6, through the use of bearings, 10.The finished product proceeds to a receiving tank, 11.A filter, 12, is installed in order to facilitate fluid drainage.",
"For the purpose of reducing the likelihood of working tool setting, helical guides, 13, are fashioned inside the housing, 6.A high-speed (3,000-12,000 rpm) electric motor, 14, serves to set the working tool into rotation.",
"The speed of movement, for example, the rotation, of the working tool and its rate of advance, as well as the number of working tool cutting edges, are selected based on the condition of the creation of ultrasound oscillations in the working zone using the correlation ω n>8,000, where ω is the angular rotational speed of the tool and n is the number of tool cutting edges.",
"The large number of cutting edges on the working tool increases the number of individual impacts by the working tool on the material being worked.",
"By acting on the material being worked in the working area, the ultrasound oscillations significantly reduce power consumption for working by means of lowering the elastomer vitrification temperature, as well as by means of energy release at the boundaries and within the structural defects of the material being worked.",
"These oscillations also take part in superimposing an energy effect on the working area.",
"A set of channels cut into the working tool's body is created between the adjacent cutting edges for the purpose of removing the separated particles and diminishing the thermal load in the working areas.",
"These channels are oriented along the surface of the working tool and are connected to the medium source by lines.",
"The injection of the subject channels is accomplished using a medium flow that consists of at least one gas and/or at least one fluid, which is delivered through the conduits at a gauge pressure of several atmospheres as necessary.",
"The flow of this pressurized medium through the channels simultaneously performs a number of functions that are crucial to the enhancement of working efficiency, to wit: it takes part in detaching particles of the material being worked by acting on the root of a chip by virtue of its sail effect; removes the heat released during working; it creates a pseudofluidized bed of detached particles of the material being work by removing them from the working area, thereby precluding their participation in the process of heat formation as a result of friction, and; it alters the friction coefficient in the “cutting edge-material” system, thereby ensuring the absence of slip in the presence of high working speeds.",
"Gases and/or fluids in different combinations and with different parameters are selected as the pressurized medium depending upon the nature and properties of the material being worked.",
"This medium may be comprised of compressed air, an air-water mixture, inert gases, and active gases.",
"The prior ozonization or ionization of a gas that is a part of a pressurized medium can appreciably enhance working efficiency by means of improving the oxidative or active properties of the gas.",
"The geometry of the cutting edges, particularly the angle of incidence, is selected based on the assurance of the existence of cumulative jets in the area where the cutting edges exert an influence on the material being worked, as well as a cumulative jet energy density that is high enough to facilitate the formation of running cracks in the body of the material being worked, which leads to the appearance of shear planes and the efficient disintegration of the material being worked (the angle of incidence equals 85-95°).",
"In order to reduce the temperature load in the working area, the material being worked can be conveyed to and from the working tool in the reciprocating or “start-stop” mode, during which the supplemental cooling of the working zone occurs at those moments in time when the conveyance of the material being worked is halted.",
"As compared to all existing tools with similar applications, the use of the invention at hand ensures a significant increase in material working efficiency.",
"During the working of elastomers, a high degree of finished product homogeneity and fineness is achieved.",
"During the working of wood, a considerable increase in the speed of cutter rotation is possible without scorching the wood.",
"Here, productive capacity is increased, the quality of the surface being worked is enhanced, and the generation of disperse particles instead of the traditional chips is ensured."
]
] | Patent_10048576 |
[
[
"System and method for creating a searchable word index of a scanned document including multiple interpretations of a word at a given document location",
"Multiple recognition engines (110) provide different interpretations (116) of a word at a given location within a scanned document (108).",
"A word node corresponding to each unique interpretation is stored within a word index (102), with each word node being linked to word nodes of previously and subsequently recognized words."
],
[
"1.A method in a computer system for creating a searchable word index of a scanned document, the method comprising: generating a first interpretation of a word at a given location within the scanned document using a first recognition engine; generating a second interpretation of the word using a second recognition engine, wherein the second interpretation is different from the first interpretation; storing a first word node in the searchable word index associating the first interpretation of the word and the location of the word within the scanned document; and storing a second word node in the searchable word index associating the second interpretation of the word and the location of the word within the scanned document.",
"2.The method of claim 1, wherein the first and second recognition engines employ different optical character recognition (OCR) techniques.",
"3.The method of claim 1, wherein the location of the word is defined by a bounding rectangle.",
"4.The method of claim 3, wherein the bounding rectangle is defined by at least two coordinates, each coordinate comprising a percentage of a width and a height of the scanned document.",
"5.The method of claim 1, further comprising: linking the first and second word nodes to at least one word node of a previously recognized word from the scanned document.",
"6.The method of claim 1, further comprising: linking the first and second word nodes to at least one word node of a subsequently recognized word from the scanned document.",
"7.The method of claim 1, further comprising: generating a third interpretation of the word using a third recognition engine; determining whether the third interpretation of the word is contained within a word list; and storing a third word node in the searchable word index when the third interpretation of the word is contained within the dictionary, the third word node associating the third interpretation of the word and the location of the word within the scanned document.",
"8.The method of claim 7, wherein the word list comprises a dictionary.",
"9.The method of claim 1, further comprising: generating a third interpretation of the word using a third recognition engine; determining whether the third interpretation of the word contains an improbable character triplet; storing a third word node in the searchable word index when the third interpretation of the word does not contain an improbable character triplet, the third word node associating the third interpretation of the word and the location of the word within the scanned document, 10.The method of claim 9, wherein an improbable character triplet comprises three consecutive characters not found within a word of a dictionary.",
"11.A system for creating a searchable word index of a scanned document, the system comprising: a first recognition engine configured to generate a first interpretation of a word at a given location within the scanned document; a second recognition engine configured to generate a second interpretation of the word, wherein the second interpretation is different from the first interpretation; an index creation component configured to store first and second word nodes in the searchable word index, the first word node associating the first interpretation of the word and the location of the word within the scanned document and the second word node associating the second interpretation of the word and the location of the word within the scanned document.",
"12.The system of claim 11, wherein the first and second recognition engines employ different optical character recognition (OCR) techniques.",
"13.The system of claim 11, wherein the location of the word is defined by a bounding rectangle.",
"14.The system of claim 13, wherein the bounding rectangle is defined by at least two coordinates, each coordinate comprising a percentage of a width and a height of the scanned document.",
"15.The system of claim 11, further comprising: a linking component configured to link the first and second word nodes to a word node of a previously recognized word from the scanned document.",
"16.The system of claim 11, further comprising: a linking component configured to link the first and second word nodes to a word node of a subsequently recognized word from the scanned document.",
"17.The system of claim 11, further comprising: a third recognition engine configured to generate a third interpretation of the word using a third recognition engine; and a word filter configured to determine whether the third interpretation of the word is contained within a word list; wherein the index creation component is further configured to store a third word node in the searchable word index when the third interpretation of the word is contained within the dictionary, the third word node associating the third interpretation of the word and the location of the word within the scanned document.",
"18.The system of claim 17, wherein the word list comprises a dictionary.",
"19.The system of claim 11, further comprising: a third recognition engine configured to generate a third interpretation of the word using a third recognition engine; and a word filter configured to determine whether the third interpretation of the word contains an improbable character triplet; wherein the index creation component is further configured to store a third word node in the searchable word index when the third interpretation of the word does not contain an improbable character triplet, the third word node associating the third interpretation of the word and the location of the word within the scanned document, 20.The system of claim 19, wherein an improbable character triplet comprises at three consecutive characters not found within a word of a dictionary.",
"21.A computer program product on a computer-readable medium for creating a searchable word index of a scanned document, the computer program product comprising: program code for generating a first interpretation of a word at a given location within the scanned document using a first recognition engine; program code for generating a second interpretation of the word using a second recognition engine, wherein the second interpretation is different from the first interpretation; program code for storing a first word node in the searchable word index associating the first interpretation of the word and the location of the word within the scanned document; and program code for storing a second word node in the searchable word index associating the second interpretation of the word and the location of the word within the scanned document.",
"22.The computer program product of claim 21, wherein the first and second recognition engines employ different optical character recognition (OCR) techniques.",
"23.The computer program product of claim 21, wherein the location of the word is defined by a bounding rectangle.",
"24.The computer program product of claim 23, wherein the bounding rectangle is defined by at least two coordinates, each coordinate comprising a percentage of a width and a height of the scanned document.",
"25.The computer program product of claim 21, further comprising: program code for linking the first and second word nodes to at least one word node of a previously recognized word from the scanned document.",
"26.The computer program product of claim 21, further comprising: program code for linking the first and second word nodes to at least one word node of a subsequently recognized word from the scanned document.",
"27.The computer program product of claim 21, further comprising: program code for generating a third interpretation of the word using a third recognition engine; program code for determining whether the third interpretation of the word is contained within a word list; and program code for storing a third word node in the searchable word index when the third interpretation of the word is contained within the dictionary, the third word node associating the third interpretation of the word and the location of the word within the scanned document.",
"28.The computer program product of claim 7, wherein the word list comprises a dictionary.",
"29.The computer program product of claim 21, further comprising: program code for generating a third interpretation of the word using a third recognition engine; program code for determining whether the third interpretation of the word contains an improbable character triplet; and program code for storing a third word node in the searchable word index when the third interpretation of the word does not contain an improbable character triplet, the third word node associating the third interpretation of the word and the location of the word within the scanned document, 30.The computer program product of claim 9, wherein an improbable character triplet comprises three consecutive characters not found within a word of a dictionary."
],
[
"<SOH> TECHNICAL BACKGROUND <EOH>In the field of optical character recognition (OCR), analog documents (e.g., paper, microfilm, etc.)",
"are digitally scanned, segmented, and converted into text that may be read, searched, and edited by means of a computer.",
"In order to provide for rapid searching, each recognized word is typically stored in a searchable word index with links to the location (e.g., page number and page coordinates) at which the word may be found within the scanned document.",
"In some conventional OCR systems, multiple recognition engines are used to recognize each word in the document.",
"The use of multiple recognition engines generally increases overall recognition accuracy, since the recognition engines typically use different OCR techniques, each having different strengths and weaknesses.",
"When the recognition engines produce differing interpretations of the same image of a word in the scanned document, one interpretation is typically selected as the “correct” interpretation.",
"Often, the OCR system rely on a “voting” (winner takes all) strategy with the majority interpretation being selected as the correct one.",
"Alternatively, or in addition, confidence scores may be used.",
"For example, suppose two recognition engines correctly recognize the word “may” with confidence scores of 80% and 70%, respectively, while another recognition engine interprets the same input data as “way” with a 90% confidence score, while yet another recognition engine recognizes the input data as “uuav” with a 60% confidence score.",
"In such an example, a combination of voting and confidence scores may lead to a selection of “may” as the preferred interpretation.",
"Unfortunately, by selecting a single interpretation and discarding the rest, the objectively correct interpretation is also frequently discarded.",
"Often, image noise and other effects confuse a majority of the recognition engines, with only a minority of the recognition engines arriving at the correct interpretation.",
"In the above example, the correct interpretation could have been “way,” which would have been discarded using standard methods.",
"Accordingly, conventional OCR systems have never been able to approach total accuracy, no matter how many recognition engines are employed.",
"What is needed, then, is a system and method for creating a searchable word index of a scanned document including multiple interpretations of a word at a given location within the document.",
"What is also needed is a system and method for creating a searchable word index that selectively reduces the size of the index by eliminating interpretations that are not found in a dictionary or word list.",
"In addition, what is needed is a system and method for creating a searchable word index that permits rescaling of a scanned document without requiring modification of location data within the word index."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Non-exhaustive embodiments of the invention are described with reference to the figures, in which: FIG.",
"1 is a block diagram of a conventional system for creating a searchable word index of a scanned document; FIG.",
"2 is a block diagram of a system for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document; FIG.",
"3 is block diagram of linked word nodes; FIG.",
"4 is a block diagram of a system for creating a searchable word index including a word filter in communication with a dictionary; FIG.",
"5 is a physical block diagram of a computer system for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document; and FIG.",
"6 is a flowchart of a method for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"RELATED APPLICATIONS The present application is related to and claims priority from U.S.",
"Provisional Application No.",
"60/187,362, filed Mar.",
"6, 2000, for System and Method for Converting Archived Data into Searchable Text,” with inventors G. Bret Millar, Timothy L. Andersen, and E. Derek Rowley, which is incorporated herein by reference in its entirety.",
"THE FIELD OF THE INVENTION The present invention relates generally to the field of optical character recognition (OCR).",
"More specifically, the present invention relates to a system and method for creating a searchable word index of a scanned document including multiple interpretations of a word at a given location within the document.",
"TECHNICAL BACKGROUND In the field of optical character recognition (OCR), analog documents (e.g., paper, microfilm, etc.)",
"are digitally scanned, segmented, and converted into text that may be read, searched, and edited by means of a computer.",
"In order to provide for rapid searching, each recognized word is typically stored in a searchable word index with links to the location (e.g., page number and page coordinates) at which the word may be found within the scanned document.",
"In some conventional OCR systems, multiple recognition engines are used to recognize each word in the document.",
"The use of multiple recognition engines generally increases overall recognition accuracy, since the recognition engines typically use different OCR techniques, each having different strengths and weaknesses.",
"When the recognition engines produce differing interpretations of the same image of a word in the scanned document, one interpretation is typically selected as the “correct” interpretation.",
"Often, the OCR system rely on a “voting” (winner takes all) strategy with the majority interpretation being selected as the correct one.",
"Alternatively, or in addition, confidence scores may be used.",
"For example, suppose two recognition engines correctly recognize the word “may” with confidence scores of 80% and 70%, respectively, while another recognition engine interprets the same input data as “way” with a 90% confidence score, while yet another recognition engine recognizes the input data as “uuav” with a 60% confidence score.",
"In such an example, a combination of voting and confidence scores may lead to a selection of “may” as the preferred interpretation.",
"Unfortunately, by selecting a single interpretation and discarding the rest, the objectively correct interpretation is also frequently discarded.",
"Often, image noise and other effects confuse a majority of the recognition engines, with only a minority of the recognition engines arriving at the correct interpretation.",
"In the above example, the correct interpretation could have been “way,” which would have been discarded using standard methods.",
"Accordingly, conventional OCR systems have never been able to approach total accuracy, no matter how many recognition engines are employed.",
"What is needed, then, is a system and method for creating a searchable word index of a scanned document including multiple interpretations of a word at a given location within the document.",
"What is also needed is a system and method for creating a searchable word index that selectively reduces the size of the index by eliminating interpretations that are not found in a dictionary or word list.",
"In addition, what is needed is a system and method for creating a searchable word index that permits rescaling of a scanned document without requiring modification of location data within the word index.",
"BRIEF DESCRIPTION OF THE DRAWINGS Non-exhaustive embodiments of the invention are described with reference to the figures, in which: FIG.",
"1 is a block diagram of a conventional system for creating a searchable word index of a scanned document; FIG.",
"2 is a block diagram of a system for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document; FIG.",
"3 is block diagram of linked word nodes; FIG.",
"4 is a block diagram of a system for creating a searchable word index including a word filter in communication with a dictionary; FIG.",
"5 is a physical block diagram of a computer system for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document; and FIG.",
"6 is a flowchart of a method for creating a searchable word index of a scanned document including multiple interpretations for a word at a given location within the document.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.",
"Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.",
"Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.",
"In the following description, numerous specific details are provided, such as examples of programming, user selections, network transactions, database queries, database structures, etc., to provide a thorough understanding of embodiments of the invention.",
"One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc.",
"In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.",
"Referring now to FIG.",
"1, there is shown a conventional optical character recognition (OCR) system 100 that produces a searchable word index 102 from an analog document 104 (such as a paper or microfilm document).",
"Initially, the analog document 104 is scanned by a digital scanner 106.Digital scanners 106 are well known in the art, such as the Hewlett Packard 9100C® digital sender, which is a high-speed, multi-page, networkable scanning device.",
"The resolution of the digital scanner 106 is generally in excess of 300 dpi (dots per inch) in order to provide accurate recognition.",
"The output of the digital scanner 106 is a scanned document 108, also referred to herein as a document image.",
"The scanned document 108 typically includes one or more bi-level bitmap images, each corresponding to a page of the analog document 104.In the depicted embodiment, the OCR system 100 includes a plurality of recognition engines 110.Examples of standard recognition engines 110 include Finereader®, available from Abbyy USA of Fremont, Calif., and Omnipage®, available from Scansoft, Inc. of Peabody, Mass.",
"As noted above, the use of multiple recognition engines 110 generally increases overall recognition accuracy, since the recognition engines 110 typically use different OCR techniques, each having different strengths and weaknesses.",
"For example, one recognition engine 110 may use a neural net-based OCR technique, while another recognition engine 110 may use a template-matching technique.",
"After the scanned document 108 is obtained, a segmentation module (not shown) reduces the document 108 into image segments corresponding to individual words (and other objects).",
"Each image segment is marked by a bounding rectangle 112.Typically, a bounding rectangle is defined by a pair of coordinates 114 expressed in image pixels (e.g., x pixels down, y pixels across).",
"In some cases, each recognition engine 110 may include a separate segmentation module, providing different segmentations of the same document 108.After the scanned document 108 is segmented, a particular image segment marked by a bounding rectangle 112 is selected for recognition.",
"Thereafter, each recognition engine 110 attempts to recognize the word contained within the selected image segment and provides its own interpretation 116.In some cases, the interpretation 116 may be accompanied by a confidence score.",
"For example, a confidence score of 90% may indicate that a recognition engine 110 is 90% confident that its interpretation is correct.",
"The confidence score is influenced by numerous factors, which are beyond the scope of the present discussion, but well known to those of skill in the art.",
"In a conventional OCR system 100, each interpretation 116, including any confidence score, is provided to a conflict resolution module 118, which selects a single preferred interpretation 120 for storage in the word index 102.Various techniques may be used to select the preferred interpretation 120.Typically, a voting technique is used, where a majority of the recognition engines 110 agree on a preferred interpretation 120.In other cases, the confidence score may be used to more heavily weight the “vote” of a particular recognition engine 110.Normally, the non-selected interpretations 116 are discarded, while the preferred interpretation 120 is inserted into the word index 102.The word index 102 typically associates the preferred interpretation 120 with the location of the corresponding word in the scanned document 108 (as indicated by the bounding rectangle 112).",
"Where a scanned document 108 includes multiple pages, a page number may also be included with the location data.",
"The implementation details of a word index 102 may vary from system to system.",
"For example, a word index 102 may be implemented in the context of a relational database.",
"In alternative embodiments, hashing techniques may be used.",
"The precise structure and organization of a word index 102 is not crucial to the invention.",
"After each of word of the scanned document 108 has been recognized and entered into the word index 102, a search engine (not shown) may use the word index 102 to rapidly locate a given word within the scanned document 108.For example, a user may enter the word “maximum,” after which the search engine returns a location on page 3, with a bounding rectangle of “(150,125)(190,140).” As previously noted, one disadvantage of conventional OCR systems 100 is the fact that a majority of the recognition engines 110 can sometimes be wrong.",
"Thus, by selecting a single preferred interpretation 120 and discarding the rest, the objectively correct interpretation is also frequently discarded.",
"Accordingly, conventional OCR systems 100 have never been able to approach total accuracy, no matter how many recognition engines 110 are employed.",
"Referring now to FIG.",
"2, there is shown a system 200 for creating a searchable word index 102 of a scanned document 108 including multiple interpretations for a word at a given location within the document 108.As described above, a plurality of recognition engines 110 arrive at independent interpretations 116 of a word within a bounding rectangle 112.In one embodiment, the recognition engines 110 may operate in parallel using a multi-threaded operating system.",
"Alternatively, the recognition engines 110 may operate serially on the same input data.",
"Unlike the conventional OCR system 100, however, each unique interpretation 116 is stored in the word index 102 with an indication of the corresponding word's location (e.g., bounding rectangle 112).",
"For example, if three recognition engines 110 interpret a word as “may,” while one recognition engine 110 interprets the same word as “way,” both “may” and “way” are added to the word index 102.Thus, the system 200 does not rely on a conflict resolution module 118 to select a single preferred interpretation 120.Moreover, unlike conventional approaches, the coordinates 114 of the bounding rectangle 112 are represented, in one embodiment, as percentages of the length or width, as appropriate, of the scanned document 108.This allows for simplified re-scaling of the scanned document 108 without requiring modification of location data within the word index 102.As used herein, an association between the interpretation 116 of a word and its location is called a “word node” 202.Thus, for each interpretation 116, a word node 202 is inserted into the word index 102.A word node 202 may be embodied as any suitable data structure or combination of data structures.",
"The above-described approach has a significant impact on keyword search accuracy when compared to conventional approaches.",
"On the assumption that OCR errors between recognition engines 110 are uncorrelated, the probability that a correct interpretation 116 of a word is recognized (and thus returned by a user search on that word) by at least one recognition engine 110 is 1−((1−A1)·(1−A2)·.",
".",
".",
"·(1−An)) Eq.",
"1 where Ai is the word accuracy of recognition engine i; and n is the number of recognition engines 110 applied to the scanned document 108.This probability asymptotically approaches 100 percent as the number of recognition engines 100 increases.",
"For example, if there are two recognition engines 110, each of which has only a 60% probability of recognizing the correct word, the probability that at least one of them will correctly identify the word is 1−(1−0.60)2=84% If a 3rd, 60% accurate recognition engine 110 is added, the probability goes to 1−(1−0.60)3=93.4% This compares with the 60% probability of correctly recognizing the word and returning the document on a phrase search on that word if the output of only one of the engines 110 is selected.",
"In one embodiment, as shown in FIG.",
"3, each word node 202 is linked to the word node(s) 202 corresponding to each interpretation 116 of the previous and next word in the scanned document 108.For example, the word node 202e corresponding to “cost” is linked bi-directionally to the word nodes 202b-d corresponding to “maximum, “maximal,” and “maxwzm.” Likewise, the word nodes 202b-d are linked bi-directionally to the word node 202a corresponding to “The.” In alternative embodiments, unidirectional linking may be used.",
"Links may be implemented using any suitable technique, such as pointers, key fields, and the like, which may or may not be embedded within the word nodes 202.In one embodiment, the bi-directional linking is used to facilitate phrase searching.",
"As shown in FIG.",
"3, the insertion of multiple word nodes 202 for different interpretations 116 of a word results in multiple phrase paths, which provides for increased accuracy during phrase searching.",
"For example, in a conventional approach where an incorrect interpretation 116 is inserted into the word index 102, e.g.",
"“maximal” instead of “maximum,” a phrase search for “the maximum cost” would not result in a hit.",
"By contrast, using the word index 102 of the present invention, a phrase search for “the maximum cost” would be successful.",
"FIG.",
"4 illustrates an alternative embodiment of a system 400 in accordance with the present invention in which a word filter 402 eliminates one or more of the interpretations 116 generated by the recognition engines 110.Unlike standard approaches, multiple word nodes 202 are still inserted into the word index 102 for different interpretations 116 of the same word.",
"However, where a particular interpretation 116 is not found within a dictionary 404 or other word list, a word node 202 is not inserted into the word index 102 in one embodiment.",
"In general, where an interpretation 116 is not found within the dictionary 404, the likelihood that the interpretation 116 will be correct is relatively low.",
"By eliminating improbable interpretations 116, the size of the word index 102 is reduced and response time is increased.",
"Accuracy is not diminished, however, since it is unlikely that a user would search for a word that is not in the dictionary 404.Of course, certain interpretations 116 may still be indexed despite not being found in the dictionary 404.For example, acronyms, proper nouns, and technical words may still be inserted into the word index 102 regardless of whether they are found in the dictionary 404 or other word list.",
"In one embodiment, interpretations 116 containing improbable character triplets are also eliminated.",
"An improbable character triplet is a series of three characters that does not exist in a dictionary 404.For example, the interpretation 116 generated by the third recognition engine 100 of FIG.",
"4, i.e.",
"“maxwzm,” contains an improbable character triplet, i.e.",
"“xwz.” FIG.",
"5 is a schematic block diagram of a hardware architecture for the systems 200 and 400 of FIGS.",
"2 and 4, respectively.",
"In one embodiment, a central processing unit (CPU) 502 executes instructions stored in a memory 504, such as a random access memory (RAM) and/or read only memory (ROM).",
"The CPU 502 may be in electrical communication with one or more input devices 506, such as a mouse and/or keyboard.",
"The CPU 502 may be coupled to the input devices 506, as well as the other illustrated components, via a bus 503.Likewise, the CPU 502 may be in electrical communication with one or more output devices 508, such as a monitor and/or printer.",
"In various embodiments, the CPU 502 may also be coupled to one or more ports 510, such as an RS-232, printer, and/or USB port.",
"Similarly, the CPU 502 may be coupled to a network interface 512, such as an Ethernet adapter.",
"In one embodiment, the CPU 502 is in electrical communication with a storage device 514, such as a hard disk drive, CD-ROM, and/or DVD-ROM.",
"The storage device 514 may be used to store the dictionary 404, the word index 102, and various software modules to be loaded into the memory 504 during operation of the systems 200 and 400.In one embodiment, the memory 504 stores a plurality of recognition engines 110.In addition, the memory 504 stores an index creation module 516, which receives the interpretations 116 of the recognition engines 110 and stores corresponding word nodes 202 in the word index 102 using the techniques described with reference to FIG.",
"2.In alternative embodiments, an index creation module 516 may be incorporated into one or more of the recognition engines 110.The memory 504 may also store a linking module 518, which links each word node 202 to the word node(s) 202 corresponding to each interpretation 116 of the previous and next word in the scanned document 108, as described in connection with FIG.",
"3.The linking module 518 may be integrated with the index creation module 516 in certain embodiments.",
"The memory 504 may also store an operating system (OS) 520, such as Windows 2000® or Linux®, which manages and provides resources to the above-described software modules.",
"In alternative embodiments, the software modules depicted within the memory 504 may be implemented as hardware or firmware.",
"Of course, the hardware architecture illustrated in FIG.",
"5 may be embodied in various configurations without departing from the spirit and scope of the invention.",
"In addition, certain standard components known to those of skill in the art are not illustrated in order to avoid obscuring aspects of the invention.",
"Referring now to FIG.",
"6, there is shown a flowchart of method 600 for creating a searchable word index 102 of a scanned document 108 including multiple interpretations of a word at a given location within the document 108.The method 600 begins by segmenting 602 a scanned document 108 generated by a digital scanner 106.Any conventional segmentation process may be used to reduce the scanned document 108 into a plurality of image segments marked by bounding rectangles 112.Thereafter, a next bounding rectangle 112 is selected 604 for recognition.",
"In one embodiment, a first interpretation 116 of a word within the selected bounding rectangle 112 is generated 606 by a first recognition engine 110.Thereafter, a second interpretation 116 of the word is generated 608 by a second recognition engine 110.Any number of additional recognition engines 110 may be used to generate additional interpretations 116.Next, a first word node 202 is stored 610 in the word index 102.In one embodiment, the first word node 202 associates the first interpretation 116 of the word with the location (e.g., bounding rectangle 112) of the word within the scanned document 108.Similarly, a second word node 202 is stored 612 in the word index 102.In one configuration, the second word node 202 associates the second interpretation 116 of the word with the location (e.g., bounding rectangle 112) of the word within the scanned document 108.In certain embodiments, the method 600 continues by linking 614 the first and second word nodes 202 to one or more word nodes 202 corresponding to interpretations 116 the previously recognized word from the scanned document 108.As noted above, the linking may be bi-directional and is used to facilitate phrase searching.",
"A determination 616 is then made whether additional bounding rectangles 112 within the scanned document 108 need to be recognized.",
"If so, the method 600 returns to step 604 to select the next bounding rectangle 112.Otherwise, the method 600 is complete.",
"Based upon the foregoing, the present invention offers a number of advantages not found in conventional approaches.",
"By storing word nodes 202 corresponding to all of the unique interpretations 116 of a word, accuracy in a keyword search is significantly enhanced.",
"In addition, by eliminating interpretations 116 not found in a dictionary 404, index size and search time is reduced, without impacting accuracy.",
"Moreover, by defining bounding rectangles 112 using percentage-based coordinates 114, the scanned document 108 may be easily rescaled without the requirement for modifying locations within the index 102.While specific embodiments and applications of the present invention have been illustrated and described, it is to be understood that the invention is not limited to the precise configuration and components disclosed herein.",
"Various modifications, changes, and variations which will be apparent to those skilled in the art may be made in the arrangement, operation, and details of the methods and systems of the present invention disclosed herein without departing from the spirit and scope of the invention."
]
] | Patent_10049016 |
[
[
"Terry tile board",
"The board provides protection against damage on a slate shingle and tile roof.",
"The upper side of board with plywood serves as the solid foundation for standing on as tool is laid upon the roof.",
"The opposite or under side has plywood and foam rubber.",
"This side is placed directly onto the roof.",
"An added feature is the board is good on a steep roof."
],
[
"1.A tool for protection of roofs, the tool consisting of one piece of plywood, having wood slats and a non-slip surface on one side with other side bearing an adhesive and foam rubber."
],
[
"This invention is a tool that provides protection against breakage or damage of slate shingles and roof tiles.",
"When roof maintenance or new roof installation is necessary the traditional roof access is via foot traffic.",
"This board eliminates actual foot traffic on a roof.",
"This board is also good for safety on a steep roof.",
"The board distributes one's weight proportionally throughout entire size of board as opposed to the traditional confined area where one would step.",
"The upper side of board is plywood material.",
"Bolts and nuts attach wood ladder slats to this side of board.",
"The ladder slats provide traction.",
"This is the side on which one would stand.",
"The opposite or under side of board is plywood material.",
"A medium density foam rubber is attached to this side with an adhesive.",
"The foam rubber side is placed directly onto the roof.",
"The foam rubber grips and conforms to the roofing material.",
"The board weighs about twenty one pounds and can be easily handled.",
"This invention is meant to be used by a roofer.",
"However, it will be useful to persons in other trades needing to access a roof; such as sheet metal workers, plumbers, window washers, and exterior painters."
]
] | Patent_10066553 |
[
[
"Maintenance system for an equipment set",
"The invention relates to the maintenance of a rapidly installable and rapidly removable set of equipment (1-6, 11) which can be replaced by standard exchanges, furnished with individual electronic circuits for monitoring proper operation (1a-6a, 20a-23a, 30a-32a, 11a) assuming, at their level, a BITE function (Build [sic] In Test Equipment) for testing, for fault diagnosis and for issuing fault messages sent by data transmission to a central maintenance computer (7) itself formulating a report regarding the overall state of operation of the set of equipment.",
"It relates more especially to the hardware parts of the electronic circuits for monitoring proper operation which are integral with the equipment (1-6) or with equipment parts (20-23, 30-32) and which are furnished with a nonvolatile memory (405) and with means of detection, selection and capture (403, 413, 423) into their nonvolatile memories (405), of the report formulated by the central maintenance computer (7).",
"Since these hardware parts of circuits for monitoring proper operation track the tribulations of the equipment parts or pieces of equipment with which they are integral, they make it possible for the latest report regarding the overall state of operation of the set of equipment often causing the removal, to be always available, in a repair center, with an item removed for repair, thereby facilitating the drawing up of a repair diagnosis."
],
[
"1.A maintenance system for a set of equipment comprising: electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"of each piece of equipment (1, 2, 3, .",
".",
".",
"), each furnished with means for formulating tests of proper operation and for issuing fault messages should the tests (400, 401) fail, as well as a nonvolatile memory (405) integral with the monitored piece of equipment, a central maintenance computer (7) allied with said electronic circuits for monitoring proper operation (1, 2a, 3a, .",
".",
".",
"), provided with means of diagnosis of the state of operation of the set of equipment (1, 2, 3, .",
".",
".",
"), functioning on the basis of the fault messages of said electronic circuits for monitoring proper operation (1, 2a, 3a, .",
".",
". )",
"and formulating a report regarding the overall state of operation of the set of equipment (1, 2, 3, .",
".",
".",
"), one or more data transmission links (10) linking said electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"to the central maintenance computer (7), said maintenance system for a set of equipment (1, 2, 3, .",
".",
". )",
"being characterized in that the central maintenance computer (7) comprises means for making its report regarding the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available on the data transmission link or links (10) linking it to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"and in that the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"comprise means of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment, of the report regarding the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"formulated by the central maintenance computer (7) when this report travels over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (1a, 2a, 3a, etc.)",
"to the central maintenance computer (7).",
"2.The system as claimed in claim 1, in which pieces of equipment (3, 4, 5, 6) are grouped together in subsets (11) themselves furnished, at their upper level of assemblage, with electronic circuits for monitoring proper operation (11a) generating fault messages relating to said subsets (3, 4, 5, 6) destined for the central maintenance computer (7), characterized in that the electronic circuits for monitoring proper operation (3a, 4a, 5a, 6a) of these pieces of equipment (3, 4, 5, 6) grouped into one and the same subset (11) are also furnished with means of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment, of the fault messages issued by the (11a) or the electronic circuits for monitoring proper operation of the subset(s) (11) to which the pieces of equipment (3, 4, 5, 6) belong, when these messages travel over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (3a, 4a, 5a, 6a, 11a) to the central maintenance computer (7).",
"3.The system as claimed in claim 1, in which pieces of equipment (2, 3) comprise parts (20, 21, 22, 23, 30, 31, 32) themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation (20a, 21a, 22a, 23a, 30a, 31a, 32a) generating fault messages relating to said equipment parts (20, 21, 22, 23, 30, 31, 32) destined for the central maintenance computer (7), characterized in that the electronic circuits for monitoring proper operation (20a, 21a, 22a, 23a, 30a, 31a, 32a) of these equipment parts (20, 21, 22, 23, 30, 31, 32) are also furnished with means of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment parts, of fault messages issued by the circuit or circuits for monitoring proper operation (2a, 3a) of the piece or pieces of equipment (2, 3) to which the equipment parts (20, 21, 22, 23, 30, 31, 32) belong when they travel over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (1a, 2a, 3a, etc.)",
"to the central maintenance computer (7).",
"4.The system as claimed in claim 1, in which pieces of equipment (3) comprise parts (30, 31, 32) themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation (30a, 31a, 32a) generating fault messages relating to said equipment parts (30, 31, 32) destined for the central maintenance computer (7), and where the pieces of equipment (3) which comprise these equipment parts (30, 31, 32) are grouped together with other pieces of equipment (4, 5, 6) in subsets (11) themselves furnished, at their upper level of assemblage, with electronic circuits for monitoring proper operation (11a) generating fault messages relating to said subsets (11) destined for the central maintenance computer (7), characterized in that the electronic circuits for monitoring proper operation (30a, 31a, 32a) of the parts (30, 31, 32) of these pieces of equipment (3) are also furnished [lacuna] means (403) of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment parts, of fault messages issued by the circuit or circuits for monitoring proper operation (3a, 11a) of the piece or pieces of equipment (3) and of the subset(s) of equipment (11) to which the equipment parts (30, 31, 32) belong, when these messages travel over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (1a, 2a, 3a, etc.)",
"to the central maintenance computer (7).",
"5.The system as claimed in claim 1, in which pieces of equipment (3) comprise parts (30, 31, 32) themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation (30a, 31a, 32) generating fault messages relating to said equipment parts (30, 31, 32) destined for the central maintenance computer (7), characterized in that the electronic circuits for monitoring proper operation (3a, 11a) of these pieces of equipment (3) are also furnished with means (403) of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment, of the fault messages issued at the lower level of assemblage by the circuit or circuits for monitoring proper operation (30a, 31a, 32a, 3a, 4a, 5a, 6a) of the equipment parts (30, 31, 32) of which they are composed when these messages travel over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (1a, 2a, 3a, etc.)",
"to the central maintenance computer (7).",
"6.The system as claimed in claim 1, in which pieces of equipment (3) comprise parts (30, 31, 32) themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation (30a, 31a, 32) generating fault messages relating to said equipment parts (30, 31, 32) destined for the central maintenance computer (7), and where the pieces of equipment (3) which comprise these equipment parts (30, 31, 32) are grouped together with others (4, 5, 6) in subsets (11) themselves furnished, at their upper level of assemblage, with electronic circuits for monitoring proper operation (11a) generating fault messages relating to said subsets destined for the central maintenance computer (7), characterized in that the electronic circuits for monitoring proper operation (3a, 11a) of these pieces of equipment (3) are also furnished with means (403) of detection, capture and transfer (403, 413, 423) into their nonvolatile memories (405) integral with the equipment, of the fault messages issued at the lower level of assemblage by the circuit or circuits for monitoring proper operation (30a, 31a, 32a, 3a, 4a, 5a, 6a) of their equipment parts (30, 31, 32) and, at the upper level of assemblage, by the circuit or circuits for monitoring proper operation of the pieces of equipment (3, 4, 5, 6) belonging to the same subset, when these messages travel over said transmission link or links (10) linking the electronic circuits for monitoring proper operation (1a, 2a, 3a, etc.)",
"to the central maintenance computer (7).",
"7.The system as claimed in claim 1, comprising a printer (9) linked to the central maintenance computer (7) by the or one of said data transmission links (10) attaching the central maintenance computer (7) to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
".",
"), characterized in that the means of the central maintenance computer (7) which make its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available on the data transmission link or links (10) linking it to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"are also the means of means of making its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available to the printer (9).",
"8.The system as claimed in claim 1, comprising a keyboard/screen interface (8) linked to the central maintenance computer (7) by the or one of said data transmission links (10) attaching the central maintenance computer (7) to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
".",
"), characterized in that the means of the central maintenance computer (7) which make its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available on the data transmission link or links (10) linking it to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"are also the means of means of making its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available to the keyboard/screen interface (8).",
"9.The system as claimed in claim 1, associated with an airborne external telecommunication network linked to the central maintenance computer (7) by the or one of said data transmission links (10) attaching the central maintenance computer (7) to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
".",
"), characterized in that the means of the central maintenance computer (7) which make its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available on the data transmission link or links (10) linking it to the electronic circuits for monitoring proper operation (1a, 2a, 3a, .",
".",
". )",
"are also the means of means of making its report on the overall state of operation of the set of equipment (1, 2, 3, .",
".",
". )",
"available to the airborne external telecommunication network."
],
[
"The present invention relates to the maintenance of a set of equipment, such as the set of avionic equipment of an aircraft which fulfill the various functions required for accomplishing a flight.",
"An aircraft comprises a large number of pieces of equipment, of diverse kinds, mechanical, hydraulic, electrical or electronic, whose proper operation is essential during a flight.",
"To improve the degree of confidence accorded to these pieces of equipment, a monitoring of their proper operation is carried out for each of them as often as possible, consisting of a monitoring of the fundamental parameters and within automatic or semiautomatic tests of proper operation, followed by a fault diagnosis which may lead to the issuing of fault messages.",
"This monitoring of proper operation, associated with a piece of equipment, is known as the BITE function, stemming from the abbreviation for the expression “Built In Test Equipment”.",
"The BITE function of a piece of equipment is assumed by a piece of electronics which may be specific or shared with other functions of the relevant piece of equipment.",
"This piece of electronics performs the software processing required by the BITE function.",
"It comprises a greater or lesser hardware part integral with the piece of equipment, with, as a minimum, in this hardware part, a nonvolatile memory in which are stored the violations of specification by the monitored parameters, the results of the tests, the fault diagnosis when it exists as well as the fault messages issued.",
"The fault messages of the BITE functions of the monitored pieces of equipment of an aircraft are addressed, via an airplane data transmission link, to a centralizing piece of equipment placed on board the aircraft so as to gather the various fault messages issued.",
"On board recent aircraft, the fault messages originating from the BITE functions of the various pieces of equipment are consultable from the flight deck.",
"They are furthermore preprocessed, with a view to easing the task of the crews and of the maintenance personnel, by a special-purpose central computer known by various titles such as CMC standing for the expression “Central Maintenance Computer” or else CFDIU standing for “Centralized Fault Display Interface Unit”.",
"This central maintenance computer is accessible to the crew via an interface with keyboard and screen which may be that known by the abbreviation MCDU stemming from the expression “Multipurpose Control Display Unit” but which may also be a portable computer of the PC type attached via a disconnectable data link which may or may not utilize the airplane bus.",
"Its main function is to make, in real time or at the end of the flight, a diagnosis of the general situation of the aircraft on the basis of a summary of the fault messages received from the various pieces of equipment of the aircraft.",
"It also fulfills other functions such as the correlating of the fault messages received with the alarms received at the flight deck level, the conducting of particular tests on the equipment, undertaken on request, by an operator intervening from the keyboard/screen interface affording access to the central maintenance computer or the compiling of a “post-flight” report, known by various titles such as PFR or LLR standing for “Post Flight Report” or “Last Leg Report”, destined for the ground maintenance teams, encompassing a log of the fault messages issued by the various pieces of equipment of the aircraft and of the alarms presented to the crew as well as the summary of the fault messages made in the last resort and more generally, all the information about the states of operation of the equipment, capable of easing the work of the ground maintenance team, whether this information results from automatic exploitation of the equipment fault messages or from remarks by the crew.",
"To reduce the time for which an aircraft is grounded, its equipment, be it mechanical such as valves, pumps etc., electrical such as switches, relays, batteries etc., or electronic such as automatic pilot computers, navigation computers etc., is, as often as possible, designed in such a way as to be able to be easily dismantled and replaced rapidly by standard exchange.",
"One then speaks of LRU equipment standing for the expression “Line Replaceable Unit”.",
"The concept of items which can easily be dismantled and replaced by standard exchange is even extended to a lower tier of assemblage, within the pieces of equipment themselves, by use of modular architectures with modules which can easily be dismantled and replaced by standard exchange, some of them possibly being multifunction, that is to say usable in several different pieces of equipment.",
"One then speaks of LRM modules standing for “Line Replaceable Module”.",
"The BITE function for testing for proper operation exists at each of the two possible levels of standard exchange of items within an aircraft: LRU equipment level and LRM equipment module level.",
"It is referred to as the resource BITE function when it is concerned with a hardware setup or with the first-level software used (such as the operating system) and as the application BITE function when it is concerned with higher-level software.",
"It is ensured by a piece of electronics, of which a greater or lesser hardware part tracks the fate of the item capable of a standard exchange.",
"The BITE function can also exist at a third level of assemblage grouping together several LRM modules placed in one and the same cabinet or rack.",
"It is then referred to as the overall BITE function and consists of a prediagnosis easing that of the central maintenance computer.",
"An exemplary system for maintenance of the equipment of an aircraft by means of BITE functions integrated into the equipment and of a central maintenance computer of the aforesaid kind is described in American patent U.S. Pat.",
"No.",
"4,943,919.Once an equipment part or a piece of equipment has been tagged as defective and removed from an aircraft, it has to be overhauled in a repair station.",
"To ease this overhauling, it is known practice for the nonvolatile memory of the hardware part of the electronics ensuring the BITE function of the removed item, which remains integral with the item even after its removal, to be made to play an overhaul help function.",
"Specifically, this memory which can be consulted while the item is still installed on board the aircraft, by way of the MCDU screen/keyboard interface ensuring the interface with the central maintenance computer, is also consultable in the repair station by way of a test rig specially suited to this consultation function and used to diagnose the fault.",
"This overhaul help resulting from the storing in memory of the diagnosis of the BITE function of the item examined is sometimes insufficient, in particular in the case of faults occurring only in a particular context.",
"The repair technician is then required to take account of the reasons for and conditions of removal.",
"Hitherto, these reasons for and conditions of removal have appeared in a written note drafted by the operator managing the aircraft, usually an airline company, on the basis of the notes made by the ground maintenance personnel in the aircraft's maintenance logbook (Technical/Maintenance Logbook), from indications provided by the central maintenance computer, including the “post-flight” PFR/LLR report.",
"This entirely manual process for advising as to the reasons for and conditions of removal of an equipment part or of a piece of equipment involving several successive intervening parties often produces retranscription errors and exaggerated simplifications in the information transmitted to the repair station, or even a complete absence of information so that the help afforded to the fault diagnosis by the note accompanying the removed item and explaining the reasons for and conditions of removal is often less than that which one would be entitled to expect.",
"The aim of the present invention is to reduce the manual interventions in the process for advising a repair station as to the reasons for and conditions of removal of an equipment part or of a piece of equipment to be repaired by profiting from the presence, within the removed item, of a nonvolatile memory dedicated to the BITE function of this item, so as to make this nonvolatile memory play a role of storage, not only of the diagnosis of the BITE function of the removed item, but also of the “post-flight” PFR/LLR report drawn up by the central maintenance computer and, possibly, of the diagnoses of the BITE functions of other levels of assemblage encompassing that of the removed item: BITE function of the piece of equipment encompassing the removed item when the latter is an LRM module and when this piece of equipment is also furnished at its level with a BITE function, and/or an overall BITE function when the piece of equipment to which the removed item belongs is grouped together with other pieces of equipment in a cabinet or rack furnished at its level with an overall BITE function.",
"By virtue of this reducing of the manual interventions in the process for advising a repair station as to the reasons for and conditions of removal of an item to be repaired, the accuracy and the reliability of the information reaching the repair station, with the item removed, regarding the anomalies noted while operational and regarding the circumstances of the fault are improved.",
"Numerous benefits may be expected from an improvement in the reliability and in the accuracy of the information given to the repair station, including: better quality of repair related to better knowledge of the circumstances of the fault, reduction in the repair time, likewise related to better knowledge of the circumstances of the fault, possibility of continuous improvement of items and equipment stemming from better knowledge of their anomalies noted while operational, possibility of prompting and training actions on the part of the maintenance personnel with regard to the operators managing the aircraft with a view to reducing the incorrect removals of items or of pieces of equipment following poor interpretation of the fault messages, better traceability of incorrect removals, referred to as NFF (the abbreviation standing for “No Fault Found”).",
"The subject of the invention is a maintenance system for a set of equipment comprising: electronic circuits for monitoring proper operation of each piece of equipment, each furnished with means for formulating tests of proper operation and for issuing fault messages should the tests fail, as well as a nonvolatile memory integral with the monitored piece of equipment, a central maintenance computer allied with said electronic circuits for monitoring proper operation, provided with means of diagnosis of the state of operation of the set of equipment, functioning on the basis of the fault messages of said electronic circuits for monitoring proper operation and formulating a report regarding the overall state of operation of the set of equipment, one or more data transmission links linking said electronic circuits for monitoring proper operation to the central maintenance computer, said maintenance system for a set of equipment being characterized in that the central computer comprises means for making its report regarding the overall state of operation of the set of equipment available on the data transmission link or links linking it to the electronic circuits for monitoring proper operation and in that the electronic circuits for monitoring proper operation comprise means of detection, capture and transfer into their nonvolatile memories integral with the equipment, of the report regarding the overall state of operation of the set of equipment formulated by the central maintenance computer when this report travels over the transmission link or links linking the electronic circuits for monitoring proper operation to the central maintenance computer.",
"Advantageously, when pieces of equipment are grouped together in subsets themselves furnished, at their upper level of assemblage, with electronic circuits for monitoring proper operation generating fault messages relating to said subsets destined for the central maintenance computer, the electronic circuits for monitoring proper operation of these pieces of equipment are also furnished with means of detection, capture and transfer into their nonvolatile memories integral with the equipment, of fault messages issued by the electronic circuit or circuits for monitoring proper operation of the subset(s) to which the monitored pieces of equipment belong, when these messages travel over said data transmission link or links linking the circuits for monitoring proper operation to the central maintenance computer.",
"Advantageously, when, pieces of equipment comprise parts themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation generating fault messages relating to said equipment parts destined for the central maintenance computer, characterized in that the electronic circuits for monitoring proper operation of these equipment parts are also furnished with means of detection, capture and transfer into their nonvolatile memories integral with the equipment parts, of fault messages issued by the circuit or circuits for monitoring proper operation of the piece or pieces of equipment to which the monitored equipment parts belong when these messages travel over the data transmission link or links linking the circuits for monitoring proper operation to the central maintenance computer.",
"Advantageously, when pieces of equipment comprise parts themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation generating fault messages relating to said equipment parts destined for the central maintenance computer, and when the pieces of equipment which comprise these equipment parts are grouped together in subsets themselves furnished, at their upper level of assemblage, with electronic circuits for monitoring proper operation generating fault messages relating to said subsets destined for the central maintenance computer, the electronic circuits for monitoring proper operation of the parts of these pieces of equipment are also furnished with means of detection, capture and transfer into their nonvolatile memories integral with the equipment parts, of fault messages issued by the circuit or circuits for monitoring proper operation of the piece or pieces of equipment and of the subgroup(s) of equipment to which the equipment parts which they are monitoring belong, when these messages travel over the data transmission link or links linking the circuits for monitoring proper operation to the central maintenance computer.",
"Advantageously, when pieces of equipment comprise parts themselves furnished, at their lower level of assemblage, with electronic circuits for monitoring proper operation generating fault messages relating to said equipment parts destined for the central maintenance computer, the electronic circuits for monitoring proper operation of these pieces of equipment are also furnished with means of detection, capture and transfer into their nonvolatile memories integral with the equipment, of fault messages issued at the lower levels of assemblage by the circuit or circuits for monitoring proper operation of the equipment parts of which they are composed when these messages travel over the data transmission link or links linking the circuits for monitoring proper operation to the central maintenance computer.",
"Other characteristics and advantages of the invention will emerge from the description hereinbelow of an embodiment given by way of example.",
"This description will be given in conjunction with the drawing in which: a FIG.",
"1 represents, diagrammatically, an exemplary architecture of a centralized maintenance system for equipment of an aircraft, and a FIG.",
"2 details an exemplary electronic circuit for monitoring proper operation according to the invention, intended to be mounted on a removable item of equipment or piece of equipment whose proper operation is monitored by means of a centralized maintenance system such as that of FIG.",
"1.The main pieces of equipment of a modern aircraft contributing the accomplishment of the flight, such as the engines, the motors actuating the rudder and elevators as well as the various flaps, the landing gear, etc., the interfaces for controlling these various engines and motors, the measurement apparatus for the aerodynamic parameters, for the heading, for the altitude, for position, for velocity, the automatic pilot, the flight management computer, the fuel management computer, the radio communication apparatus, etc.",
"are furnished with individual devices for monitoring proper operation carrying out a BITE function, that is to say the checking of the main operating parameters of this apparatus as well as the conducting of automatic or semiautomatic tests when necessary and the issuing of fault messages when the measured parameters depart from the permitted value ranges or when the results of a test are not as expected.",
"The diversity of the possible fault messages as well as of the possibilities of propagation of the initial fault from one piece of equipment to other dependent pieces of equipment makes it difficult for the crew or the maintenance personnel to draw up in real time and in all possible situations a diagnosis of the general state of the aircraft.",
"This is why modern aircraft are equipped with a central maintenance computer responsible for the most thorough possible automatic processing of the fault messages so as to inform the crew or the maintenance personnel only of the actual fault or faults and provide them with indications regarding the conduct to be taken in order to alleviate the consequences of these faults.",
"To ease the maintenance of an aircraft and reduce the time periods for which it is grounded, there are ever greater endeavors to generalize the standard exchange, both at the level of certain items of equipment, and at the level of certain complete pieces of equipment.",
"These easily dismantleable items of equipment or pieces of equipment are then furnished, at their respective levels of assemblage, with individual electronic circuits for monitoring proper operation ensuring a BITE function for checking the main parameters, for formulating automatic and semiautomatic tests of proper operation, and for fault diagnosis, and issuing, when necessary, fault messages destined for the central maintenance computer so that the latter can draw up its diagnosis regarding the general state of the aircraft and pinpoint the defective item of equipment or piece of equipment.",
"FIG.",
"1 shows, diagrammatically, a centralized architecture of a maintenance system as may be found in a modern aircraft.",
"Depicted therein is a set of equipment 1 to 6 geographically dispersed within the airframe and linked to one another and to a central maintenance computer CMC 7, to a keyboard/screen interface MCDU 8 and to a printer 9, by way of an airplane data transmission link 10.All the pieces of equipment 1 to 6 are furnished with individual electronic circuits for monitoring proper operation assuming a BITE function at their levels.",
"These individual electronic circuits for monitoring proper operation have a hardware part 1a, 2a, 3a, 4a, 5a, 6a integral with the monitored piece of equipment and communicate their fault messages to the central maintenance computer CMC 7 by way of the airplane data transmission link 10.Certain pieces of equipment, such as the pieces of equipment 1, 4, 5, 6 are indivisible and comprise, individually, only one electronic circuit for monitoring proper operation ensuring a BITE function at the overall level of the piece of equipment itself.",
"This electronic circuit for monitoring proper operation comprises a hardware part 1a, 4a, 5a, 6a integral with the monitored piece of equipment.",
"Other pieces of equipment, such as the pieces of equipment 2, 3, are designed according to a modular architecture and enclose various modules 20, 21, 22, 23 or 30, 31, 32 which can be easily dismantled and replaced by standard exchange.",
"These pieces of equipment 2, 3, are then each furnished with an electronic circuit for monitoring proper operation ensuring a BITE function at the overall level of the piece of equipment, and with a set of electronic circuits for monitoring proper operation individually ensuring a BITE function at the level of their modules.",
"The electronic circuits for monitoring proper operation functioning at the overall level of a piece of equipment have a hardware part 2a, 3a integral with the piece of equipment or with one of its modules when the piece of equipment is organized in modules, while the electronic circuits for monitoring proper operation functioning at the level of the modules 20, 21, 22, 23, 30, 31, 32 each comprise a hardware part 20a, 21a, 22a, 23a, or 30a, 31a, 32a integral with the monitored module.",
"Certain finally of the pieces of equipment 1, 2 are geographically isolated while others 3, 4, 5, 6 are grouped together in cabinets or racks 11, themselves individually equipped with an electronic circuit for monitoring proper operation, with a hardware part 11a, assuming a BITE function at the level of the equipment grouping, integral with the cabinets or racks or even with one of the pieces of equipment or with one of the equipment modules of the grouping.",
"The set of hardware parts of the electronic circuits for monitoring proper operation, integral with equipment modules such as the parts 20a, 21a, 22a, 23a, or 30a, 31a, 32a, with pieces of equipment such as the parts 1a, 2a, 3a, 4a, 5a, 6a, or with groupings of equipment in a cabinet or rack, such as the part 11a, are connected to the airplane data transmission link 10 which may for example be a bus of ARINC 429, 629 or Ethernet type to which are also connected the central maintenance computer 7, the keyboard/screen interface MCDU 8 and a printer 9.The central maintenance computer 7, the makeup of which is well known to the person skilled in the art and an example of which is described in American patent U.S. Pat.",
"No.",
"4,943,919, performs, in real time or at the end of the flight, a diagnosis of the general state of the aircraft, pinpoints the defective piece or pieces of equipment from which the fault messages originated, the latter reaching it from the various BITE functions assumed by the electronic circuits for monitoring proper operation distributed over equipment modules, pieces of equipment and groupings of equipment of the aircraft, informs the crew or the maintenance personnel, of the piece or pieces of equipment which are actually defective, saves in a nonvolatile part of its memory, a log of the fault messages received, of the alarms issued destined for the crew and draws up, destined for the team ensuring the ground maintenance of the aircraft, a “post-flight” PFR/LLR report which comprises a summary of the fault messages, alarms and information of a general context (date, time, flight phase, etc.)",
"useful for the interpretation of this information.",
"This “post-flight” PFR/LLR report available just after landing can be printed by means of the printer 9 installed on board the aircraft, automatically at the end of each flight or when asked for by an operator requesting same from the keyboard/screen interface MCDU 8 placed in the flight deck of the aircraft.",
"It may also be sent to a fleet management center belonging for example to the operator of the aircraft, by way of an airborne external telecommunication network such as the ACARS network, the abbreviation standing for “Aircraft Communication Addressing and Reporting System”, using a transmission link of VHF, UHF or other type.",
"The keyboard/screen interface MCDU 8, which allows an exchange of commands and of information between an operator and the central maintenance computer 7, is the interface used moreover, and this is its main function, to allow the crew to exchange orders and information with the automatic pilot AP, the flight management computer FMS (Flight Management System) and the fuel management computer.",
"The functions of automatic pilot, flight management, fuel management and centralized maintenance may be assumed by one and the same computer or by several different computers often grouped together in one and the same cabinet or rack.",
"This keyboard/screen interface MCDU 8 can, as far as dialog with the central maintenance computer 7 is concerned, be twinned or replaced by a portable computer of the PC kind attached to the central maintenance computer 7 by a disconnectable data link which may or may not utilize the airplane data transmission bus 10.The printer 9 is placed in the flight deck at an easily accessible location and is linked, by the airplane data transmission bus 10, to various pieces of equipment, including the central maintenance computer 7.When it has carried out a standard exchange of an equipment module or of a complete piece of equipment pursuant to the indications contained in the “post-flight” PFR/LLR report delivered by the central maintenance computer 7, which indications may possibly be specified by additional operating tests programmed from the screen/keyboard interface MCDU 8, the aircraft's ground maintenance team sends the removed item: module or complete piece of equipment, to a repair station for overhaul with an accompanying note explaining the reasons for the removal.",
"Having arrived at the repair station, the removed item is subjected to tests so as to draw up a repair diagnosis, repaired and checked before being declared fit for service once again.",
"In a certain number of cases, the repair diagnosis turns out to be difficult to draw up.",
"To help the repair diagnosis of an item furnished with an electronic circuit for monitoring proper operation, thought has been given to harnessing the log of the fault messages formulated during the item's commissioning period preceding its removal, by making provision to equip the hardware part of the electronic circuit for monitoring proper operation, which remains integral with the item, with a nonvolatile memory, that is to say one which retains the data stored should the power supply be disabled, in which this log of the fault messages is stored.",
"Thus, it is possible, upon the arrival of this item at the repair station, to consult the log of the fault messages which is stored in the nonvolatile memory of the hardware part of the electronic circuit integral therewith, thereby facilitating repair diagnosis.",
"However, in a certain number of cases, help from the log of the fault messages of the BITE function associated with the item undergoing repair diagnosis is not sufficient to arrive at a safe diagnosis.",
"Hence, only the explanatory removal note accompanying the item then remains as diagnostic aid.",
"However, this note, the drafting of which results from an entirely manual process involving several intervening parties, often comprises retranscription errors and exaggerated simplifications of the grounds for and circumstances of the removal, or even sometimes a complete absence of these grounds.",
"Since the motivation for the removal of an item by the aircraft's ground maintenance team results in the great majority of cases from the consulting of the “post-flight” PFR/LLR report drawn up by the central maintenance computer, it is proposed that not only the log of the fault messages generated by the BITE function of the item but also the latest “post-flight” PFR/LLR report or reports drawn up by the central maintenance computer be stored in the nonvolatile memory of the hardware part, integral with the item, of the electronic circuit for monitoring proper operation ensuring the BITE function of the item.",
"This “post-flight” PFR/LLR report storage operation is made easier by the fact that a “post-flight” PFR/LLR report is often available, at one moment or another on the airplane data transmission link.",
"Thus, in the exemplary configuration of the system for centralized monitoring illustrated in the above figure, the “post-flight” PFR/LLR reports necessarily utilize the airplane data transmission link 10 for their printing since the printer 9 is connected by the latter to the central maintenance computer 7.Under these conditions, it is sufficient to furnish the various electronic circuits ensuring BITE functions, with means for detecting the presence of a “post-flight” PFR/LLR report on the airplane data transmission link, and for capturing and for transferring this “post-flight” PFR/LLR report into their nonvolatile memories integral with the items liable to be removed.",
"Even if the printer 9 is connected to the central maintenance computer 7 by a specific link, these means may be effective since the “post-flight” PFR/LLR reports utilize the airplane transmission link 10 on other occasions, in particular for their consultation via the keyboard/screen interface 8 or for their transmission to the ground by way of an airborne external telecommunication network.",
"It is also possible to envision by a suitable programming of the central maintenance computer 7, a specific operation for making its “post-flight” PFR/LLR report available on the airplane data transmission link 10 executed at the end of each flight or upon the intervention of an operator.",
"It is also proposed, more generally, that not only the log of their fault messages and the “post-flight” PFR/LLR report but also the logs of the fault messages of the BITE functions associated with higher levels of assemblage, that is to say of the piece or pieces of equipment to which a module belong [sic] when the item in question is a module, or of the possible grouping of equipment to which a piece of equipment belongs when the item in question is a piece of equipment or one of its modules, be stored in the nonvolatile memories of the hardware parts, which remain integral with the removed items, of the electronic circuits for monitoring proper operation ensuring the BITE functions.",
"Thus, a fairly complete summary of the in-situ behavior of the item which led the aircraft's ground maintenance team to replace it will be available at the repair station.",
"As earlier in respect of the “post-flight” PFR/LLR report, the operation is made easier by the fact that the fault messages generated by the electronic circuits for monitoring proper operation assuming the BITE functions are often available at one moment or another on the airplane data transmission link 10.If such is not the case, the BITE functions of the higher levels of assemblage being linked to the central maintenance computer 7 by a specific data transmission link, it is sufficient to have these BITE functions of higher level of assemblage make available, by means of suitable programming, the log of their fault messages on the airplane data transmission bus, at the end of each flight or upon the intervention of an operator.",
"It is even proposed that in addition to the latest “post-flight” PFR/LLR report or reports, the logs of the fault messages of the BITE functions associated with lower levels of assemblage be stored in the nonvolatile memories of the hardware parts, which remain integral with the removed items, of the electronic circuits for monitoring proper operation ensuring the BITE functions, the electronic circuit for monitoring proper operation ensuring the BITE function of a piece of equipment storing in the nonvolatile memory of its hardware part remaining integral with the removed item, the logs of the fault messages of the BITE functions associated with its modules, these logs being able to aid the tagging of a fault caused by the defectiveness of an internal element of the removed item, which element is itself furnished with a BITE function.",
"FIG.",
"2 gives one possible exemplary configuration of the hardware part, which remains integral with the removed item, of an electronic circuit for monitoring proper operation 40a assuming a BITE function, allowing local storage, at its level, not only of the log of the BITE function of the item but also of the “post-flight” PFR/LLR report of the central maintenance computer and optionally, of logs of other BITE functions assumed by other electronic circuits of proper operation.",
"This hardware part of an electronic circuit for monitoring proper operation 40a is integral with an item 40 whose tribulations during installation and removal it tracks and which may, as was envisioned earlier, be an equipment module (20, 21, 22, 23, 30, 31, 32 FIG.",
"1) or a piece of equipment (1, 2, 3, 4, FIG.",
"1).",
"It comprises a circuit 400 for bidirectional access to the bus of the airplane data transmission link 10, an automated test facility 401, a clock 402, a circuit for 403 for detection, capture and transfer of the messages traveling over the bus of the airplane data transmission link 10 with a first stage for detecting messages 413 and a second stage for selecting messages 423, a nonvolatile memory 405 and a nonvolatile memory management circuit 406.The circuit 400 for bidirectional access to the bus of the airplane data transmission link 10 is a special-purpose circuit which is found in any piece of equipment attached to a bus of the airplane data transmission link 10.It constitutes the interface between a piece of equipment and the bus of the airplane data transmission link, that is to say on the one hand, the transition, in both directions, between a data organization suited to a piece of equipment and a data organization suited to the airplane data transmission link and, on the other hand, the switch, in both directions, from a signal conveying the data having the physical characteristics suited to a piece of equipment, to a signal conveying the data having the physical characteristics suited to the bus of the transmission link.",
"It will not be detailed since it does not constitute part of the invention.",
"It is sufficient to know that it serves as interface for the bus of the data transmission link for which it provides locally, at the level of a piece of equipment, access 400e on transmission and access 400r on reception.",
"The automated test facility 401 is connected to elements, sensors, actuators, etc.",
"dispersed within the item 40, to the transmission access 400e and reception access 400r of the circuit 400 for access to the airplane data transmission bus 10, to the clock circuit 402 and to the memory management circuit 406.It assumes the BITE function of the item 40.Its connections to sensors and actuators dispersed within the item 40 allow it to monitor operating parameters of the item 40 and also to perform tests of proper operation on this item.",
"Its connection to the clock circuit 402 allows it to datestamp its messages and also to undertake tests or measurements with fixed periodicities or dates.",
"Its connections to the transmission access 400e and reception access 400r of the circuit 400 for bidirectional access to the airplane data transmission bus 10 allow it to communicate with the central maintenance computer 7 for its parametrization, the conducting of tests of proper operation and the transmitting of the fault messages.",
"Its design will not be detailed since it does not constitute part of the invention and it is furthermore highly dependent on the nature of the item 40 whose operation is monitored.",
"The clock circuit 402 can be reset to time from the central maintenance computer 7 by virtue of a branchoff from the reception access 400r of the circuit 400 for bidirectional access to the bus of the airplane data transmission link 10.It delivers a time reference to the various elements of the circuit for monitoring proper operation 40a.",
"The nonvolatile memory 405 is, for example, an electrically erasable read-only memory or so-called EEPROM (Electrically Erasable/Programmable Read-Only Memory).",
"The memory management circuit 406 which is associated with the nonvolatile memory 405 makes it possible, for example, given the limited capacity of the latter, to use it as a register of first-in first-out type known by the acronym FIFO in such a way that the most recent part of the log is always in memory.",
"It receives the fault messages issued by the automated test facility 401 traveling over the transmission access 400e of the circuit 400 for bidirectional access to the bus of the airplane data transmission link 10 as well as the “post-flight” PFR/LLR reports of the central maintenance computer 7 and the fault messages of other circuits for monitoring proper operation assuming BITE functions, available as output from the circuit 423 for selecting messages and datestamps them before entering them into the nonvolatile memory 405.Like the automated test facility 401 and the clock circuit 402, it has a control input connected to the reception access 400r of the circuit 400 for bidirectional access to the bus of the airplane data transmission link 10 which makes it possible to control it remotely by way of this bidirectional access circuit 400.The first detection stage 413 of the circuit [lacuna] detection, capture and transfer of messages 403 which is connected to the reception access 400r of the circuit 400 for bidirectional access to the bus of the airplane data transmission link 10 isolates each message traveling over the bus of the airplane transmission link while the second stage 423 of the detection, capture and transfer circuit 403 analyzes the labels of the messages traveling over the airplane transmission link 10 which are made available to it by the first stage for detecting messages 413 so as to retain only those issued by the central maintenance computer 7 and belonging to a “post-flight” PFR/LLR report, and the fault messages originating from certain electronic circuits for monitoring proper operation assuming BITE functions, duly referenced at the level of the hardware part 40a of an electronic circuit for monitoring proper operation.",
"Like the automated test facility 401, the clock circuit 402 and the memory management circuit 406, it comprises a control input connected to the reception access 400r of the circuit 400 for bidirectional access to the bus of the airplane data transmission link 10 which makes it possible to control it remotely by way of this bidirectional access circuit 400, so as in particular to provide it with the identities of the electronic circuits for monitoring proper operation assuming BITE functions, whose messages it must capture.",
"To facilitate understanding, the hardware part, represented in FIG.",
"2, of the electronic circuit for monitoring proper operation is itself sufficient for carrying out a complete BITE function.",
"There are however cases where it is more advantageous to simplify this hardware part to the point at which it can now assume, by itself, only part of a BITE function, the responsibility for the remaining part of the BITE function being off-loaded onto the central maintenance computer or onto a prediagnosis computer.",
"Likewise, with the aim of facilitating comprehension, the hardware part of an electronic circuit for monitoring proper operation has been represented in FIG.",
"2, in the form of an assemblage of separate boxes dedicated to distinct functions.",
"It should nonetheless not be concluded that this assemblage implies that such boxes necessarily exist in an embodiment, it being possible for the functions carried out by different boxes to be so by means of one or more microprocessors driven by software and operating in multitask mode.",
"The electronic circuits for monitoring proper operation which have just been proposed for assuming the BITE function of an equipment part or of a piece of equipment which can be dismantled, capable of standard exchanges, within a maintenance system with a centralized computer, allow a repair station to obtain, from the removed item itself, a sufficiently complete overview of the progress of the latest campaign of use of the item so as to: note the conditions under which the fault motivating the removal of the item was declared (concomitance of faults with other pieces of equipment or equipment parts, possible disturbances of the electrical or hydraulic networks, etc.",
"), assess whether the removal of the item did indeed follow an announcement incriminating the item concerned."
]
] | Patent_10089089 |
[
[
"Acid-sensitive compounds, preparation and use thereof",
"Novel acid-sensitive compounds comprising at least one hydrophilic substituent and a cyclic ortho-ester which is acid-sensitive, and their salts.",
"These compounds are useful for forming conjugates (liposomes, complexes, nanoparticles and the like) with biologically active substances and releasing them into cellular tissues or compartments whose pH is acidic, or as nonionic surfactant for stabilizing particles encapsulating a biologically active substance and then destabilizing them in acid medium, or alternatively as a vector covalently linked to a therapeutic molecule so as to release said therapeutic molecule into the cellular tissues or compartments whose pH is acidic."
],
[
"1.Acid-sensitive compounds characterized in that they comprise a cyclic ortho-ester and at least one hydrophilic substituent chosen from polyalkylene glycols, mono- or polysaccharides, hydrophilic therapeutic molecules, or alternatively radicals of the polyamine type, as well as their salts.",
"2.Acid-sensitive compounds according to claim 1, characterized in that they have the general formula: in which: g is an integer which may take the values 0, 1, 2, 3 or 4, G represents a hydrogen atom, an alkyl radical containing 1 to 6 carbon atoms in the form of a saturated or unsaturated, straight or branched chain, or an aryl radical, G1 and G2 represent: (a) one a hydrophilic substituent chosen from radicals of the polyamine type, and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives or hydrophobic dendrimers, or alternatively (b) one a hydrophobic linear alkyl group comprising 10 to 24 carbon atoms and optionally comprising one or more unsaturations, and the other a group of general formula: in which i is an integer ranging from 1 to 4 and j is an integer ranging from 9 to 23, and the hydrophilic substituent is chosen from radicals of the polyamine type, or alternatively (c) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a substituent chosen from polyalkylene imines, or alternatively (d) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives, hydrophobic dendrimers, or the covalent conjugates between a single- or double-chain alkyl, a steroid derivative, or a hydrophobic dendrimer and a polyalkylene glycol molecule comprising 1 to 20 monomeric units, or alternatively (e) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a therapeutic molecule, or alternatively (f) one a therapeutic molecule of a hydrophilic nature and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives or hydrophobic dendrimers, as well as their salts.",
"3.Acid-sensitive compounds according to claim 2, characterized in that G is chosen from hydrogen, methyl, ethyl or phenyl.",
"4.Acid-sensitive compounds according to claim 2, characterized in that the single- or double-chain alkyls consist of one or two linear alkyl chains comprising 10 to 24 carbon atoms and optionally comprising one or more unsaturations.",
"5.Acid-sensitive compounds according to claim 2, characterized in that the steroid derivative is chosen from sterols, steroids and steroid hormones.",
"6.Acid-sensitive compounds according to claim 2, characterized in that the hydrophobic dendrimer is poly(benzyl ether).",
"7.Acid-sensitive compounds according to claim 2, characterized in that the polyalkylene glycols are chosen from polyalkylene glycols having an average molecular weight of between 102 and 105 Daltons.",
"8.Acid-sensitive compounds according to claim 7, characterized in that the polyalkylene glycols are chosen from polyethylene glycols (PEG) having an average molecular weight of between 102 and 105 Daltons.",
"9.Acid-sensitive compounds according to claim 2, characterized in that the mono- or polysaccharides are chosen from pyranoses, furanoses, dextrans, α-amylose, amylopectin, fructans, mannans, xylans and arabinans.",
"10.Acid-sensitive compounds according to claim 2, characterized in that the polyalkylene glycol or the mono- or polysaccharide is covalently linked to a targeting element.",
"11.Acid-sensitive compounds according to claim 10, characterized in that the targeting element is chosen from sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives.",
"12.Acid-sensitive compounds according to claim 2, characterized in that the polyalkyleneimines are chosen from the polymers comprising the monomeric units of general formula: in which R may be a hydrogen atom or a group of formula: and n is an integer of between 2 and 10, p and q are integers chosen such that the sum p+q is such that the average molecular weight of the polymer is between 100 and 107 Da, it being understood that the value of n may vary between the different units —NR—(CH2)n-.",
"13.Acid-sensitive compounds according to claim 2, characterized in that each of the substituents G1 and G2 is indirectly linked to the cyclic ortho-ester via a “spacer” molecule.",
"14.Acid-sensitive compounds according to claim 13, characterized in that said “spacer” molecule is chosen from alkyls (1 to 6 carbon atoms), carbonyl, ester, ether, amide, carbamate or thiocarbamate bonds, glycerol, urea, thiourea or a combination of several of these groups.",
"15.Acid-sensitive compounds according to claim 2, characterized in that the therapeutic molecules are chosen from peptides, oligopeptides, proteins, antigens and their antibodies, enzymes and their inhibitors, hormones, antibiotics, analgesics, bronchodilators, antimicrobials, antihypertensive agents, cardiovascular agents, agents acting on the central nervous system, antihistamines, antidepressants, tranquilizers, anticonvulsants, anti-inflammatory substances, stimulants, antiemetics, diuretics, antispasmodics, antiischemics, agents limiting cell death, or anticancer agents.",
"16.Compositions characterized in that they comprise at least one acid-sensitive compound as defined in claims 1 to 15.17.Compositions characterized in that they comprise at least one biologically active substance and an acid-sensitive compound as defined in claim 2 and for which G1 and G2 have the definitions indicated under (a), (b), (c) or (d).",
"18.Compositions according to claim 17, characterized in that said biologically active substance is either a therapeutic molecule as defined in claim 15, or a nucleic acid.",
"19.Compositions according to either of claims 16 and 17, characterized in that they comprise, in addition, one or more adjuvants.",
"20.Compositions according to claim 19, characterized in that said adjuvant is one or more neutral lipids.",
"21.Compositions according to claim 20, characterized in that said adjuvant is chosen from natural or synthetic, zwitterionic lipids or lipids lacking an ionic charge under physiological conditions.",
"22.Compositions according to claim 21, characterized in that said adjuvant is chosen from dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine, dipalmitoylphosphatidyl-ethanolamine, dimirystoylphosphatidylethanolamine as well as their derivative which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or alternatively asialogangliosides (such as in particular asialoGM1 and GM2).",
"23.Compositions according to one of claims 16 to 22, characterized in that they comprise, in addition, a pharmaceutically acceptable vehicle for an injectable formulation.",
"24.Compositions according to one of claims 16 to 22, characterized in that they comprise, in addition, a pharmaceutically acceptable vehicle for administration to the skin and/or the mucous membranes.",
"25.Use of an acid-sensitive compound as defined in claims 1 to 15 for the manufacture of a medicament intended for treating diseases.",
"26.Use of an acid-sensitive compound as defined as in claim 2 and for which G1 and G2 have the definitions indicated under (a), (b), (c) or (d), for the manufacture of a medicament intended for the transfection of nucleic acids.",
"27.Acid-sensitive compounds as defined in claim 2 and for which G1 and G2 have the definitions indicated under (e) or (f) for use as a medicament."
],
[
"The present invention relates to acid-sensitive compounds and their preparation.",
"These compounds comprise at least one hydrophilic substituent and a cyclic ortho-ester which is acid-sensitive.",
"These compounds are useful for forming conjugates (liposomes, complexes, nanoparticles and the like) with biologically active substances and releasing them into cellular tissues or compartments whose pH is acidic, or as nonionic surfactant for stabilizing particles encapsulating a biologically active substance and then destabilizing them in acid medium, or alternatively as a vector covalently linked to a therapeutic molecule so as to release it into the cellular tissues or compartments whose pH is acidic.",
"The release of biologically active substances into tissues or cells having an increased acidity relative to what is physiologically normal is a known problem which has been the subject of numerous studies, without as a result giving completely satisfactory results up until now.",
"Thus, many pH-sensitive liposomes have been designed so as to release biologically active substances by taking advantage of the acidification of certain tissues or of the endosome.",
"For example, Yatvin et al.",
"(Science, Vol.",
"210, 1980, pp.",
"1253-4) designed pH-sensitive lipids capable of becoming inserted into the lipid bilayer of conventional liposomes, of formula: The homocysteine present in this lipid is in its open form at neutral or alkaline pH and resembles, in this case, a fatty acid which becomes perfectly inserted into the bilayer of the liposomes.",
"At this pH, it exists in its closed form: it then forms a cyclic thiolactone, thus resembling a neutral lipid which destabilizes the liposomal bilayer and thus allows the release of the active substance.",
"Such a molecule allows the release of medicinal molecules in the regions of the body in which the pH is less than the physiological pH, for example in primary tumors, metastases, or alternatively sites of inflammation and of infection.",
"American patent U.S. Pat.",
"No.",
"5,965,434 proposes amphiphatic lipids comprising a cationic pH-sensitive hydrophilic part of formula: in which R1 and R2 represent, independently of each other, CH3(CH2)14, CH3(CH2)12, CH3(CH2)7CHCH(CH2)7, and R3 represents a substituent 1-methylimidazole, imidazole, 4,9-dioxo-1,12-dodecanediamine, cysteamine, 1-(3-aminopropyl)imidazole, morpholine, 4-aminopyridine, pyridine, guanidine, hydrazine, thiouronium or piperazine.",
"These compounds have the characteristic feature of carrying an overall positive charge (at the level of the compound R3) which increases when the pH decreases from 8.0 to 4.5.This modification of the charge induces a conformational transformation of the liposome, allowing it to release its content.",
"These lipids thus allow the release of medicinal molecules or of nucleic acids into acidic media whose pH varies up to 4.5.Moreover, application WO 97/31624 proposes pH-sensitive phospholipids (“triggerable lipids”) which comprise a vinyl ether function which may be degraded in the cytoplasm, and which have the general formula: in which p and q are equal to 0 or 1, at least one of the two being equal to 1, R1 and R2 represent, independently of each other, an alkyl or an alkene containing 12 to 24 carbon atoms, and R represents a group chosen from 2-aminoethyl, 2-(trimethylamino)-ethyl, 2-(N,N-dimethylamino)ethyl, 2-(trimethyl-ammonium)ethyl, 2-carboxy-2-aminoethyl, succinamido-ethyl or inosityl.",
"These phospholipids are mixed with other phospholipids, which are themselves complexed with cell receptor ligands, so as to form liposomes capable of undergoing conformational changes at acidic pH.",
"Such liposomes allow the encapsulation of numerous medicinal substances and also of nucleic acids for gene therapy.",
"Another approach has also been described (Kratz et al., Crit.",
"Rev.",
"Ther.",
"Drug Carrier Syst.",
"1999, 16(3), pp.",
"245-88) in which a therapeutic molecule is covalently linked to a polymer via an acid-sensitive bond so as to ensure the release of said therapeutic molecule into weakly acidic tumor tissues or alternatively into the endosomes and lysosomes after cellular internalization of the polymeric conjugate.",
"Numerous possible bonds have thus been described, for example acetal, disulfide, hydrazone, cis-aconitrile, trityl or alternatively silylated ether bonds.",
"However, all the pH-sensitive compounds developed up until now have the disadvantage of not being modulable as regards their sensitivity.",
"Thus, it would be highly advantageous to be able to have acid-labile compounds whose sensitivity could be modulated according to, for example, the tissues or cells targeted, the biologically active substance to be released or alternatively the applications envisaged.",
"More generally, it would also be advantageous to be able to have new pH-sensitive compounds which would be easy to prepare and effective for the transfection of nucleic acids in particular.",
"To solve this problem, the Applicant has thus developed a novel family of acid-sensitive compounds characterized in that they comprise a cyclic ortho-ester and at least one hydrophilic substituent chosen from polyalkylene glycols, mono- or polysaccharides, hydrophilic therapeutic molecules, or radicals of the polyamine type.",
"Such compounds are useful for the vectorization and the release of biologically active substances into the acidic regions of the body by virtue of the cyclic ortho-ester function which is acid-sensitive.",
"They are most particularly advantageous because the pH-sensitivity of the compound may be modulated according to the choice of the substituent present on the central carbon and the size of the ortho-ester ring.",
"It is thus possible to broadly vary the kinetics of hydrolysis of these compounds and therefore to modulate the time necessary for the release of the biologically active substance.",
"In addition, the acid-sensitive compounds according to the present invention have the additional advantage of becoming degraded in acidic medium in an autocatalytic manner.",
"Indeed, the partial degradation of the acid-sensitive compounds according to the invention causes the gradual release of an acid (for example formic acid when the starting compound is derived from an ortho-formate, or alternatively acetic acid when the starting compound is derived from an ortho-acetate, or alternatively benzoic acid when the starting compound is derived from an ortho-benzoate) which induces a decrease in the pH, further promoting their degradation.",
"More particularly, the acid-sensitive compounds according to the present invention have the general formula: in which: g is an integer which may take the values 0, 1, 2, 3 or 4, G represents a hydrogen atom, an alkyl radical containing 1 to 6 carbon atoms in the form of a saturated or unsaturated, a straight or branched chain, or an aryl radical, G1 and G2represent: (a) one a hydrophilic substituent chosen from radicals of the polyamine type, and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives or hydrophobic dendrimers, or alternatively (b) one a hydrophobic linear alkyl group comprising 10 to 24 carbon atoms and optionally comprising one or more unsaturations, and the other a group of general formula: in which i is an integer chosen from between 1 and 4 inclusive and j is an integer chosen from between 9 and 23 inclusive, and the hydrophilic substituent is chosen from radicals of the polyamine type, or alternatively (c) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a substituent chosen from polyalkylene imines, or alternatively (d) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives, hydrophobic dendrimers, or the covalent conjugates between a single- or double-chain alkyl, a steroid derivative, or a hydrophobic dendrimer and a polyalkylene glycol molecule comprising 1 to 20 monomeric units, or alternatively (e) one a hydrophilic substituent chosen from polyalkylene glycols or mono- or polysaccharides and the other a therapeutic molecule, or alternatively (f) one a therapeutic molecule of a hydrophilic nature and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives or hydrophobic dendrimers.",
"The substituent G placed on the central carbon of the ortho-ester is chosen so as to modulate the sensitivity of the acid-sensitive compound according to the present invention.",
"Thus, the more electron-donating the group G, the more acid-sensitive the compound, and the more electron-attracting the group G, the less acid-sensitive the compound.",
"It seems, therefore, that the choice of the radical G is particularly important for determining and modulating the properties of the acid-sensitive compounds of general formula (I).",
"According to a preferred aspect of the invention, G is chosen from the hydrogen atom, the alkyl radicals comprising 1 to 6 carbon atoms in the form of a saturated or unsaturated, straight or branched chain, or aryl radicals.",
"In a more particularly advantageous manner, G is chosen from hydrogen, methyl, ethyl or phenyl.",
"For the purposes of the present invention, the expression “aryl radicals” is understood to mean univalent aromatic hydrocarbon radicals.",
"The aryl radicals according to the present invention generally contain between 6 and 14 carbon atoms.",
"Preferably, the aryl radicals according to the present invention are chosen from phenyl, naphthyl, for example 1-naphthyl or 2-naphthyl, biphenylyl, for example 2-biphenylyl, 3-biphenylyl or 4-biphenylyl, anthryl or fluorenyl.",
"The phenyl is more particularly preferred.",
"The aryl radicals, in particular the phenyl, may be substituted or otherwise, for example monosubstituted, disubstituted, trisubstituted or tetrasubstituted, the substituents being identical or different.",
"Preferably, said substituents are chosen from halogen atoms, (C1-C8)alkyl or (C1-C8)alkoxy radicals.",
"In the case of monosubstituted phenyl radicals, said substituent may be substituted at position 2, at position 3 or at position 4.In the case of disubstited phenyl radicals, said substituents may be situated at position 2,3, at position 2,4, at position 2,5, at position 2,6, at position 3,4 or at position 3,5.In the case of trisubstituted phenyl radicals, said substituents may be situated, for example, at position 2,3,4, at position 2,3,5, at position 2,4,5, at position 2,4,6, at position 2,3,6 or at position 3,4,5.Depending on the cases, each of the substituents G1 and G2 is either directly linked to the cyclic ortho-ester, or indirectly via a “spacer” molecule chosen from those known to persons skilled in the art.",
"Such a “spacer” molecule makes it possible both to ensure the binding and to move the substituent(s) in question away from the cyclic ortho-ester in order to reduce any undesirable interaction between the acid-sensitive cyclic ortho-ester and its subsituent(s).",
"Preferred spacer molecules may be chosen for example according to the nature of the substituents G1 or G2 from alkyls (1 to 6 carbon atoms), carbonyl, ester, ether, amide, carbamate or thiocarbamate bonds, glycerol, urea, thiourea or a combination of several of these groups.",
"For example, when the hydrophobic substituent is a steroid derivative, the spacer molecule may be a bond of the carbamate —N—C(O)—O— type, or alternatively when the hydrophobic substituent is a double-chain alkyl, the spacer molecule may be chosen from the groups of formula -alkyl-C(O)—N, the two alkyl chains then being fixed to the nitrogen atom.",
"According to a preferred aspect of the invention, the radicals of the polyamine type may be defined as being linear or branched alkyls comprise at least 3 carbon atoms and in which at least one of the methylene groups may be replaced with an amino group which is optionally substituted (with a methyl group for example) and the terminal methyl(s) is(are) substituted with one or more groups chosen from (primary, secondary, tertiary or quaternary) amines, guanidines or cyclic guanidines.",
"These radicals of the polyamine type are preferably chosen from the polyamine radicals which are already known and described in the literature for the vectorization of nucleic acids, for example in the publications WO 96/17823, WO 97/18185, WO 98/54130 or alternatively WO 99/51581.In particular, this may include for example polyamines of general formula: in which R represents a hydrogen atom with the exception of only one of the groups R which is absent and therefore represents the covalent bond with the cyclic ortho-ester n is an integer of between 1 and 9 inclusive m is an integer of between 2 and 6 inclusive, it being possible for the values of m to be identical or different within the different groups —(CH)m—NH—.",
"According to another alternative, this may also include a radical of the polyamine type of general formula: in which: R1, R2, and R3 represent, independently of each other, a hydrogen atom or a group —(CH2)q—NRR═, it being possible for q to vary between 1, 2, 3, 4, 5 and 6, this being in an independent manner between the different groups R1, R2 and R3, and R and R═ representing, independently of each other, a hydrogen atom or a group —(CH2)q=—NH2, it being possible for q= to vary between 1, 2, 3, 4, 5 and 6, this being in an independent manner between the different groups R and R═, m and p represent, independently of each other, an integer which may vary between 1 and 6, and n represents an integer which may vary between 0 and 6, with when n is greater than 1, it being possible for m to take different values and R3 different meanings in the general formula, and with, when n is equal to 0, at least one of R1 and R2 which is different from hydrogen.",
"According to another aspect of the present invention, the radical of the polyamine type may also be represented by a substituent with a general formula identical to the preceding one, but with R and R═ representing, independently of each other, a hydrogen atom or a group of formula (1): in which r is an integer which may vary from 0 to 6 inclusive, and the groups R5 represent, independently of each other, a hydrogen atom, an alkyl, carbamate or aliphatic or aromatic acyl substituent, which is optionally halogenated, it being understood that at least one of the groups R1, R2 and R3 comprises at least one group of formula (1).",
"A radical of the polyamine type is thus obtained which comprises one or more terminal guanidine functions.",
"According to another variant of the invention, the radical of the polyamine type may also represent a polyamine such as those described above but with a terminal group of the cyclic guanidine type (instead of an amine or a guanidine) of general formula (2): for which: m and n are integers, independent of each other, of between 0 and 3 inclusive and such that m+n is greater than or equal to 1, R1 represents a group of general formula (3): (CH2)p—Yq(*) (3) for which p and q are integers, independent of each other, of between 0 and 10 inclusive, Y represents a carbonyl, amino, methylamino or alternatively methylene group, it being possible for Y to have different meanings in the different groups [(CH2)p—Y], and (*) represents either a hydrogen atom or a covalent bond, it being understood that R1 may be linked to any atom of the general formula (2), including Z, and that there is a single group R1 in the formula (2), X represents a group NR2 or alternatively CHR2, R2 being either a hydrogen atom or the bond with the group R1 as defined above, the group represents: 1st case: a group of general formula (4): for which W═ represents CHR═══ or alternatively NR═══, and R══ and R═══ represent, independently of each other, a hydrogen atom, a methyl, or the bond with the group R1 as defined above, or alternatively 2nd case: a group of general formula (5): for which W═ represents CHR═══ or alternatively NR═══, and R═ and R═══ represent, independently of each other, a hydrogen atom, a methyl, or the bond with the group R1 as defined above.",
"In general, any other radical of the polyamine type known to persons skilled in the art for combining with nucleic acids, in particular via electrostatic interactions, may also be suitable.",
"For the purposes of the present invention, there is understood by single- or double-chain alkyls the hydrophobic radicals consisting of one or two linear alkyl chains comprising 10 to 24 carbon atoms and optionally comprising one or more unsaturations.",
"In the case of the double-chain alkyls, this may include for example two alkyl chains bonded to a nitrogen atom so as to form a dialkylamino substituent the two alkyl chains being, for example, linear and comprising 10 to 24 carbon atoms and optionally one or more unsaturations.",
"This may also include saturated or unsaturated fatty acids such as, for example palmitic acid, oleic acid, stearic acid or alternatively myristic acid.",
"Preferably, the single- or double-chain alkyls possess 12 to 18 carbon atoms, and more preferably still, they are chosen from the groups possessing 12, 14, 16 or 18 carbon atoms (for each alkyl chain).",
"There is understood by “steroid derivative” for the purposes of the present invention the substituents chosen for example from sterols, steroids and steroid hormones.",
"More preferably, the steroid derivatives are chosen from cholesterol, cholestanol, 3-αa-5-cyclo-5-αa-cholestan-6-βb-ol, cholic acid, cholesteryl formate, cholestanyl formate, 3αa,5-cyclo-5αa-cholestan-6βb-yl formate, cholesterylamine, 6-(1,5-dimethylhexyl)-3a,5a-dimethylhexadecahydrocyclopenta-[a]cyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-yl-amine, cholestanylamine or alternatively dexamethasone.",
"The hydrophobic dendrimers according to the present invention are preferably chosen from hydrophobic poly(alkyl ethers) or alternatively hydrophobic poly(aryl ethers).",
"In a particularly advantageous manner, the hydrophobic dendrimers according to the present invention are chosen from poly(benzyl ethers).",
"For the purposes of the present invention, the polyalkylene glycols are preferably chosen from polyalkylene glycols having an average molecular weight of between 102 and 105 Daltons (Da), and optionally covalently linked to a targeting element.",
"In a particularly advantageous manner, the polyalkylene glycols according to the present invention are chosen from polyethylene glycols (PEG) having an average molecular weight of between 102 and 105 Da, and more preferably between 500 and 105 Da.",
"For the purposes of the present invention, there is understood by “mono- or polysaccharide” the molecules consisting of one or more saccharides, optionally covalently linked to a targeting element.",
"There may be mentioned by way of example pyranoses and furanoses, for example glucose, mannose, rhamnose, galactose, fructose or alternatively maltose, lactose, saccharose, sucrose, fucose, cellobiose, allose, laminarobiose, gentiobiose, sophorose, melibiose and the like.",
"Furthermore, this may also include so-called “complex” saccharides, that is to say several saccharides which are covalently coupled to each other, each sugar being preferably chosen from the list cited above.",
"By way of suitable polysaccharides, there may be mentioned dextrans, α-amylose, amylopectin, fructans, mannans, xylans and arabinans.",
"Preferably, the mono- or polysaccharides according to the present invention are chosen from natural or commercial derivatives which are compatible with pharmacological applications such as natural sugars, cyclodextrins or alternatively dextrans.",
"According to another alternative of the present invention, the polyalkylene glycol or the mono- or polysaccharide may optionally be covalently linked to a targeting element.",
"In this case, this may include either an extracellular targeting element which makes it possible to orient the acid-sensitive compounds according to the present invention or the compositions containing them toward certain cell types or certain desired tissues (tumor cells, hepatic cells, hematopoietic cells and the like), or alternatively this may include an intracellular targeting element which allows orientation toward certain preferred cellular compartments (mitochondria, nucleus and the like).",
"Among the targeting elements which can be used in the context of the invention, there may be mentioned sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or derivatives thereof.",
"Preferably, this includes sugars, peptides, vitamins or proteins such as for example antibodies or antibody fragments, ligands for cellular receptors or fragments thereof, receptors or alternatively receptor fragments.",
"For example, this may include ligands for growth factor receptors, cytokine receptors, receptors of the cellular lectin type, folate receptors, or ligands having the sequence RGD with affinity for the receptors for adhesion proteins such as integrins.",
"There may also be mentioned the receptors for transferrin, HDLs and LDLs, or the folate transporter.",
"The targeting element may also be a sugar which makes it possible to target lectins such as the receptors for the asialoglycoproteins or for the syalydes such as Sialyl Lewis X, or alternatively an antibody Fab fragment, or a single-chain antibody (ScFv).",
"For the purposes of the present invention, there is understood by “polyalkyleneimines” the polymers described in the publication WO 96/02655, namely the polymers comprising the monomeric units of general formula: in which R may be a hydrogen atom or a group of formula: and n is an integer of between 2 and 10, p and q are integers chosen such that the sum p+q is such that the average molecular weight of the polymer is between 100 and 107 Da.",
"It is understood that, in this formula, the value of n may vary between the different units —NR—(CH2)n—.",
"Thus, this formula groups together both the homopolymers and the heteropolymers.",
"Commercial polyalkyleneimines constitute an advantageous alternative.",
"The polyethyleneimines (PEI) are most particularly preferred, and more specifically PEI 25K (PEI having an average molecular weight of 25 KDa), PEI 50K, PEI 100K or alternatively PEI 200K.",
"According to the present invention, there is understood by “therapeutic molecule” the molecules which make it possible to prevent or cure a pathology which manifests itself in the regions of the body producing an increased acidity compared with what is physiologically normal.",
"Such regions are more specifically, but not solely: tumors, in particular tumor cells and also normal cells in the vicinity of these tumors (for example the endothelial cells of the tumors), which exhibit a higher local acidity than what is physiologically normal (N. Raghunand et al., Drug Resistance Updates, 2000, 3, pp.",
"30-38), muscles affected by ischemia, for example the cardiac muscle, in which the acidosis partly results from the lactic acid produced by the anaerobic fermentation of hydrocarbons of the sugar type or of the fatty acids, the inflammation areas where the production of superoxide ions by the macrophages consumes a lot of oxygen, or alternatively the tissues where a metabolic, infectious or inflammatory disorder produces local acidosis.",
"According to another alternative, the “therapeutic molecules” according to the present invention make it possible to prevent or cure a pathology by their release into an acidic cellular compartment, for example into the endosome of the cells which is acidic.",
"The therapeutic molecules may thus be chosen for example from peptides, oligopeptides, proteins, antigens and their antibodies, enzymes and their inhibitors, hormones, antibiotics, analgesics, bronchodilators, antimicrobials, antihypertensive agents, cardiovascular agents, agents acting on the central nervous system, antihistamines, antidepressants, tranquilizers, anticonvulsants, anti-inflammatory substances, stimulants, antiemetics, diuretics, antispasmodics, antiischemics, agents limiting cell death, or alternatively anticancer agents.",
"In addition, there is understood by “biologically active substance” the substances chosen either from the therapeutic molecules as defined above, or from nucleic acids.",
"For the purposes of the invention, there is understood by “nucleic acid” both a deoxyribonucleic acid and a ribonucleic acid.",
"This may include natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or synthetic or semisynthetic sequences, oligonucleotides which are modified or otherwise.",
"These nucleic acids may be for example of human, animal, plant, bacterial, viral or alternatively synthetic origin.",
"They may be obtained by any technique known to persons skilled in the art, and in particular by screening libraries, by chemical synthesis, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by screening libraries.",
"They may be modified chemically.",
"As regards more particularly the deoxyribonucleic acids, they may be single or double-stranded as well as short oligonucleotides or longer sequences.",
"In particular, the nucleic acids advantageously consist, for example, of plasmids, vectors, episomes or expression cassettes.",
"These deoxyribonucleic acids may in particular carry a replication origin which is functional or otherwise in the target cell, one or more marker genes, sequences for regulation of transcription or of replication, genes of therapeutic interest, antisense sequences which are modified or otherwise, or alternatively regions for binding to other cellular components.",
"Preferably, the nucleic acid comprises an expression cassette consisting of one or more genes of therapeutic interest under the control of one or more promoters and of a transcriptional terminator which are active in the target cells.",
"For the purposes of the invention, there is understood by “expression cassette for a gene of interest” a DNA fragment which may be inserted into a vector at specific restriction sites.",
"The DNA fragment comprises a nucleic acid sequence encoding an RNA or nucleic peptide of interest and comprises, in addition, the sequences necessary for the expression (activator(s), promoter(s), polyadenylation sequences and the like) of said sequence.",
"The cassette and the restriction sites are designed to ensure insertion of the expression cassette into an appropriate reading frame for transcription and translation.",
"This generally includes a plasmid or an episome carrying one or more genes of therapeutic interest.",
"By way of example, there may be mentioned the plasmids described in patent applications WO 96/26270 and WO 97/10343 incorporated into the present by reference.",
"For the purposes of the invention, there is understood by gene of therapeutic interest in particular any gene encoding a protein product having a therapeutic effect.",
"The protein product thus encoded may be in particular a protein or a peptide.",
"This protein product may be exogenous, homologous or endogenous with respect to the target cell, that is to say a product which is normally expressed in the target cell when the latter exhibits no pathology.",
"In this case, the expression of a protein makes it possible, for example, to compensate for an inadequate expression in the cell or the expression of an inactive or a weakly active protein because of a modification, or alternatively to overexpress said protein.",
"The gene of therapeutic interest may also encode a mutant of a cellular protein, which has for example increased stability or a modified activity.",
"The protein product may also be heterologous with respect to the target cell.",
"In this case, an expressed protein can for example supplement or provide an activity which is deficient in the cell, allowing it to combat a pathology, or to stimulate an immune response.",
"Among the therapeutic products for the purposes of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines, interleukins, interferons or TNF for example: FR 92/03120), growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors (for example BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, or alternatively HARP/pleiotrophin), apolipoproteins (for example ApoAI, ApoAIV, or ApoE: FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), cystic fibrosis associated protein CFTR, tumor suppresser genes (for example p53, Rb, Rap1A, DCC, or k-rev: FR 93/04745), genes encoding factors involved in coagulation (factors VII, VIII, IX), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), the genes for hemoglobin or other protein carriers, metabolic enzymes, catabolic enzymes and the like.",
"The nucleic acid of therapeutic interest may also be a gene or an antisense sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.",
"Such sequences can, for example, be transcribed in the target cell into RNAs which are complementary to cellular mRNAs and thus block their translation to protein, according to the technique described in patent EP 140 308.The therapeutic genes also comprise the sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).",
"As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide, capable of generating an immune response in humans or animals.",
"In this particular embodiment, the invention allows the production either of vaccines or of immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancer.",
"This may include in particular antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the syncitia forming virus, other viruses or alternatively antigenic peptides specific for tumors (EP 259,212).",
"Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and/or of the gene encoding the antigenic peptide in the desired cell or organ.",
"This may include sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell.",
"This may also include sequences of a different origin (responsible for the expression of other proteins, or even synthetic).",
"In particular, this may include promoter sequences of eukaryotic or viral genes.",
"For example, this may include promoter sequences derived from the genome of the cell which it is desired to infect.",
"Likewise, this may include promoter sequences derived from the genome of a virus.",
"In this regard, there may be mentioned for example the promoters of the E1A, MLP, CMV and RSV genes and the like.",
"In addition, these expression sequences may be modified by addition of activating or regulatory sequences and the like.",
"This may also include an inducible or repressible promoter.",
"Moreover, the nucleic acid may also comprise, in particular upstream of the therapeutic gene of interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell.",
"This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.",
"The nucleic acid may also comprise a signal sequence directing the therapeutic product synthesized toward a particular compartment of the cell.",
"According to a preferred aspect of the invention, the acid-sensitive compounds are more specifically chosen from the compounds of general formula: in which: g is an integer equal to 0 or 1 G represents a substituent chosen from the hydrogen atom, the alkyl substituents comprising 1 to 6 carbon atoms in the form of a saturated or unsaturated, straight or branched chain, or aryls, and G1 and G2 represent: (a) one a hydrophilic substituent chosen from radicals of the polyamine type, and the other a hydrophobic substituent chosen from single- or double-chain alkyls or steroid derivatives, or alternatively (b) one a hydrophobic linear alkyl group comprising 10 to 24 carbon atoms and optionally comprising one or more unsaturations, and the other a group of general formula: in which i is an integer ranging from 1 to 4 and j is an integer ranging from 9 to 23, and the hydrophilic substituent is chosen from radicals of the polyamine type, or alternatively (c) one a hydrophilic substituent chosen from polyalkylene glycols and the other a substituent chosen from polyalkyleneimines, or alternatively (d) one a hydrophilic substituent chosen from polyalkylene glycols and the other a hydrophobic substituent chosen from single- or double-chain alkyls, steroid derivatives or the covalent conjugates between a single- or double-chain alkyl or a steroid derivative and a polyalkylene glycol molecule comprising 1 to 20 monomeric units, or alternatively (e) one a hydrophilic substituent chosen from polyalkylene glycols and the other a therapeutic molecule.",
"More preferably still, the acid-sensitive compounds of the invention are chosen from the compounds of general formula: in which: g is an integer equal to 0 or 1 G represents a substituent chosen from the hydrogen atom, the alkyl radicals comprising 1 to 6 carbon atoms in the form of a saturated or unsaturated, straight or branched chain, or phenyl, and G1 and G2 represent: (a) one a hydrophilic substituent chosen from the radicals of the polyamine type, and the other a hydrophobic substituent chosen from single- or double-chain alkyls or steroid derivatives, or alternatively (d) one a hydrophilic substituent chosen from polyalkylene glycols and the other a hydrophobic substituent chosen from single- or double-chain alkyls or steroid derivatives.",
"The novel acid-sensitive compounds of general formula (I) may be provided in the form of nontoxic and pharmaceutically acceptable salts.",
"These nontoxic salts comprise the salts with inorganic acids (hydrochloric, sulfuric, hydrobromic, phosphoric or nitric acids) or with organic acids (acetic, propionic, succinic, maleic, hydroxymaleic, benzoic, fumaric, methanesulfonic or oxalic acids).",
"The acid-sensitive compounds according to the present invention may be prepared according to many methods chosen from those described in the literature for the synthesis of molecules containing a cyclic ortho-ester group (for example, reference may be made to the examples given in the review Synthesis, Robert H. DeWolfe, 1974, pp.",
"153-172).",
"According to an alternative which can be envisaged, the acid-sensitive compounds of general formula (I) may for example be obtained by reacting an alcohol of formula G1OH with an ortho-ester of general formula: in which g, G, G1 and G2 are as defined for the general formula (I), and Z represents a linear or branched alkyl group containing 1 to 4 carbon atoms.",
"This substitution may be carried out in the presence of an acid catalyst and/or may be heat activated at a temperature of between 50 and 150° C., with or without solvent.",
"If it is chosen to carry out the procedure in the presence of a solvent, the latter is chosen from conventional organic chemistry solvents such as for example the organochlorinated solvents, aromatic solvents or alternatively ethers.",
"When a catalyst is used, it may be an inorganic or organic acid, a Lewis or Brönsted acid.",
"For example, the catalyst may be chosen from hydrochloric acid, sulfuric acid, para-toluenesulfonic acid, camphorsulfonic acid, pyridinium para-toluenesulfonate, or alternatively magnesium chloride.",
"This substitution can also be favored by distilling the alcohol ZOH produced during the reaction if it is more volatile than the alcohol G1OH.",
"This continuous distillation may be carried out by heating at atmospheric pressure or under reduced pressure.",
"The starting alcohol G1OH is either commercially available, or it can be synthesized by any method known to persons skilled in the art, for example by hydration of the corresponding alkene, by hydrolysis of the corresponding halogenated derivative, or alternatively by reducing the corresponding carbonyl-containing derivative.",
"According to another variant of the invention, the group Z may already represent the group G1 and in this case, the step for the reaction between the ortho-ester of general formula (III) and the alcohol G1OH is not necessary.",
"The compound of general formula (III) may be obtained by the action of a trialkyl ortho-ester of general formula (IV): in which Z and G are as defined above, and Z1 and Z2, which are identical or different, represent linear or branched alkyl groups containing 1 to 4 carbon atoms, on a diol of general formula (V): in which g and G2 are as defined above.",
"The reaction may be carried out according to conventional methods for protecting diols as ortho-ester, for example according to the methods indicated by T. W. Greene and P. G. M. Wuts in “Protective Groups in Organic Synthesis” (2nd Ed., Wiley-Interscience, pp.",
"135-136).",
"The procedure is generally carried out in a conventional organic solvent (for example organochlorinated solvents, aromatic solvents, ethers and the like) in the presence of an acid catalyst.",
"The catalyst may be chosen from inorganic or organic acids, Lewis or Brönsted acids.",
"For example, it is possible to use hydrochloric acid, sulfuric acid, para-toluenesulfonic acid, camphorsulfonic acid, pyridinium para-toluenesulfonate, or alternatively magnesium chloride.",
"The trialkyl ortho-ester of general formula (IV) is either commercially available, or it can be synthesized according to conventional methods known to persons skilled in the art, for example from the corresponding ester, or alternatively by substitution of the alkoxy groups starting with another commercial trialkyl ortho-ester.",
"The diol of general formula (V) is either commercially available, or can be obtained by the reaction between a commercial diol and G2, or alternatively it can be obtained by direct functionalization of G2 to a diol.",
"This functionalization may for example consist in an oxidation of the corresponding alkene, or alternatively in the opening of a corresponding epoxide, according to methodologies well known to persons skilled in the art.",
"When either of G1 and G2 represents a radical of the polyamine type, it is either commercially available, or it is obtained according to conventional methods known to persons skilled in the art, for example according to the methods described in the prior art (for example in the publications WO 96/17823, WO 97/18185, WO 98/54130 or alternatively WO 99/51581), or according to analogous methods.",
"When either of G1 and G2 represents a hydrophobic substituent chosen from the single- or double-chain alkyls, the latter is either commercially available, or it is obtained according to conventional methods known to persons skilled in the art.",
"For example, when this includes a dialkylamino substituent with a long carbon chain, it can be prepared from the corresponding primary amine by alkylation (monosubstitution of a halogenated alkyl), by alkylative reduction (from an aldehyde), or alternatively by condensation/reduction (formation of an amide function from an acid and then reduction).",
"When either of G1 and G2 represents a hydrophobic substituent chosen from the steroid derivatives or the hydrophobic dendrimers, it is preferably chosen from commercially available products.",
"When either of G1 and G2 represents a substituent chosen from polyalkylene glycols or mono- or polysaccharides, the latter is either commercially available, or it is obtained by conventional methods known to persons skilled in the art, in particular by polymerization.",
"In the case or this substituent is covalently linked to a targeting element, the synthesis of the acid-sensitive compounds according to the present invention described above can be carried out before or after the binding, by the conventional methods of persons skilled in the art, of said targeting element to this substituent.",
"When either of G1 and G2 represents a substituent chosen from polyalkyleneimines, the latter is either commercially available, or it is obtained according to the conventional methods known to a person skilled in the art or according to the methods described in the prior art, for example in the publication WO 96/02655.The method of preparation indicated above constitutes only a method given by way of illustration, and any other equivalent method of preparation can naturally also be used.",
"For example, it is possible to carry out the reactions starting with a diol of general formula (V) which does not possess the group G2 but, in place, a functional group which is optionally protected (for example a protected amine), by carrying out an additional final step for the binding of the group G2 (for example, deprotection of the amine and then condensation of the acid of formula G2COOH).",
"Another subject of the invention relates to the compositions comprising at least one acid-sensitive compound of general formula (I) as defined above.",
"According to a variant of the invention, said compositions comprise at least one biologically active substance and an acid-sensitive compound of general formula (I) in which G1 and G2 have the definitions indicated under (a), (b), (c) or (d).",
"The compositions according to the invention may, in addition, comprise one or more adjuvants capable of binding with the complexes formed between the acid-sensitive compound according to the invention and the biologically active substance.",
"In another embodiment, the present invention therefore relates to the compositions comprising at least one biologically active substance, an acid-sensitive compound of formula (I) in which G1 and G2 have the definitions indicated under (a), (b), (c) or (d), and one or more adjuvants.",
"The presence of this type of adjuvants (lipids, peptides or proteins for example) can advantageously make it possible to increase the transfecting power of the compounds in the cases where the biologically active substance is a nucleic acid to be transfected.",
"In this perspective, the compositions according to the present invention may comprise, as adjuvant, one or more neutral lipids.",
"It has indeed been shown that the addition of a neutral lipid makes it possible to improve the formation of the nucleolipid particles (in the case where the biologically active substance is a nucleic acid), and to promote the penetration of the particle into the cell by destabilizing its membrane.",
"More preferably, said neutral lipids are lipids with two fatty chains.",
"In a particularly advantageous manner, natural or synthetic, zwitterionic lipids or lipids free of ionic charge under physiological conditions, are used.",
"They may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, dimirystoylphosphatidyl-ethanolamine as well as their derivatives which are N-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingomyelins) or alternatively asialogangliosides (such as in particular asialoGM1 and GM2).",
"These various lipids may be obtained either by synthesis, or by extraction from organs (example: the brain) or from eggs, by conventional techniques well known to persons skilled in the art.",
"In particular, the extraction of the natural lipids may be carried out by means of organic solvents (see also Lehninger, Biochemistry).",
"Preferably, in the case where the biologically active substance is a nucleic acid, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant(s) for one equivalent of nucleic acids in mol/mol and, more preferably, from 0.5 to 5.The acid-sensitive compounds according to the invention may have various uses depending on the substituents G1 and G2 situated on either side of the cyclic ortho-ester.",
"In the case where the substituents G1 and G2 have the definitions indicated in (a), (b) or (c) in the general formula (I), the acid-sensitive compounds according to the invention can form conjugates (for example of the type including liposomes, complexes or alternatively nanoparticles) directly with biologically active substances which may then be released into the tissues or cellular compartments, which are more acidic than what is physiologically normal.",
"These acid-sensitive compounds are in particular more particularly useful for the transfection of nucleic acids.",
"In the case where the substituents G1 and G2 have the definitions indicated in (d) in the general formula (I), the acid-sensitive compounds according to the invention constitute nonionic surfactants which make it possible both to stabilize particles encapsulating a biologically active substance and to release said biologically active substance by degradation in the regions which are very weakly acidic to acidic in the body, in particular regions where the pH is acidic and is between about 4 and about 7.In addition, the polysaccharide or polyalkylene glycol substituents, and more specifically polyethylene glycol (PEG), are known to confer a sort of “furtiveness” on the particles with which they are associated by inhibiting the nonspecific adsorption by the serum proteins, and consequently the recognition of said particles by the microphages (see for example Torchilin et al., Biochim.",
"Biophys.",
"Acta 1994, 1195, pp.",
"11-20 or Papahadjopoulos et al., PNAS 1991, 88, p. 11460-4).",
"Thus, the acid-sensitive compounds comprising a PEG molecule according to the invention have an advantage from the safety point of view and also an additional advantage in the sense that they reduce the risk of interference with other proteins.",
"At the level of the acidic regions in the body, the degradation of the ortho-ester present in the compounds according to the invention allows the separation of the PEG molecules from the rest of the particle, making the biologically active substance again “available” (there is in fact “disappearance of the furtiveness”).",
"A selective transfer can thus be expected with respect to the acidic tissues.",
"Finally, in the case where the substituents G1 and G2 have the definitions indicated in (e) or (f) in the general formula (I), the acid-sensitive compounds according to the invention constitute covalent conjugates with a therapeutic molecule, thereby allowing its vectorization and then its release in the acidic regions of the body.",
"These covalent conjugates are of the same type as those described by Kratz et al., but with a novel acid-sensitive bond between the therapeutic molecule and the “vector” part which has the advantage of having a modulable sensitivity compared with the pH-sensitive bonds used up until now.",
"Thus, the subject of the present invention is also the use of the acid-sensitive compounds of general formula (I) as defined above for the manufacture of a medicament intended for treating diseases.",
"In this case, the disease targeted determines the choice of the biologically active substance.",
"According to a particularly advantageous variant, when the biologically active substance is a nucleic acid, the acid-sensitive compounds of general formula (I) in which G1 and G2 have the definitions indicated under (a), (b) or (c) can be used for the manufacture of a medicament intended for the in vitro, ex vivo or in vivo transfection of nucleic acids, in particular into primary cells or into established lines.",
"This may include for example fibroblast cells, muscle cells, nerve cells (neurones, astrocytes, glyal cells), hepatic cells, cells of the hematopoietic line (lymphocytes, CD34, dendritic cells and the like), or alternatively epithelial cells, in differentiated or pluripotent form (precursors).",
"Finally, according to another alternative of the invention, the acid-sensitive compounds of general formula (I) in which G1 and G2 have the definitions indicated under (e) or (f) can be used as a medicament.",
"In the preceding text, the acid-sensitive compounds according to the present invention become degraded in the tissues or cellular compartments whose pH is more acidic than what is physiologically normal.",
"However, according to another alternative, it is possible to induce or to increase the acidity in the target region of the body by a general or local treatment known to persons skilled in the art.",
"There may be mentioned by way of example, without limitation, the injection of an acidic product into the region to be treated or alternatively the intravenous injection of glucose which causes specific acidification of tumor tissues (T. Volk et al.",
"; Br.",
"J.",
"Cancer; 1993, 68 (3), 492-500).",
"Thus, the acid-sensitive compounds according to the present invention may also be used in regions of the body which are a priori nonacidic and which have been made acidic by treatments known to persons skilled in the art.",
"For all the uses of the acid-sensitive compounds according to the present invention indicated above, the compositions according to the invention comprising: either an acid-sensitive compound of general formula (I) in which G1 and G2 have the definitions indicated under (e) or (f), or an acid-sensitive compound of general formula (I) in which G1 and G2 have the definitions indicated under (a), (b), (c) or (d) and a biologically active substance, can be formulated for administration, for example, by the topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal route.",
"Preferably, the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for administration by the topical route (to the skin and/or mucous membrane).",
"This may include in particular isotonic, sterile solutions or dry, in particular freeze-dried compositions which, upon addition, depending on the case, of sterilized water or physiological saline, allow the preparation of injectable solutions.",
"The doses of biologically active substances used for the injection as well as the number of administrations may be adjusted according to various parameters, and in particular according to the mode of administration used, the relevant pathology, the chain to be expressed when the biologically active substance is a nucleic acid, or alternatively the desired duration of treatment.",
"As regards more particularly the mode of administration, this may include either a direct injection into the tissues, for example at the level of the tumors, or the circulatory pathways, or a treatment of cells in culture followed by their reimplantation in vivo, by injection or transplantation.",
"The relevant tissues in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, blood, the bones, the cartilages, the pancreas, the kidneys, the bladder, the stomach, the intestines, the testicles, the ovaries, the rectum, the nervous system, the eyes, the glands or alternatively the connective tissues.",
"In addition to the preceding arrangements, the present invention also comprises other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting the scope thereof.",
"In particular, the Applicant proposes, without limitation, various operating protocols as well as reaction intermediates which can be used to prepare the compounds of general formula (I).",
"Of course, it is within the capability of persons skilled in the art to draw inspiration from these protocols and/or intermediate products in order to develop similar procedures so as to arrive at other compounds of general formula (I) according to the invention.",
"FIGURES FIG.",
"1: Variation of the level of fluorescence as a function of time at pH 5 of complexes formed between DNA and a control cationic lipid or alternatively the acid-sensitive compounds A Syn or Trans, in 3 different ratios: 0.4 or 1.7 or 6.0 nmol of cationic lipid or of acid-sensitive compound/μg of DNA.",
"FIG.",
"2: Efficiency of transfection in vitro into HeLa cells of complexes formed between DNA and compound A Syn or Trans or a control cationic lipid, at different charge ratios, with or without serum.",
"The y-axis represents the expression of luciferase in pg/well/μg of protein.",
"The x-axis indicates the compound A Syn or Trans or control cationic lipid/DNA charge ratio.",
"FIG.",
"3: Variation of the size (in nm) of control cationic lipid/DNA nucleolipid particles as a function of the quantity of compound C or compound D or of Brij 700 or of a non-acid-sensitive analog of compound D (Analog D) used relative to the quantity of DNA (weight/weight).",
"A small size indicates that the nucleolipid particles are stabilized.",
"A very large size indicates on the contrary destabilization of the nucleolipid particles which then tend to aggregate.",
"FIG.",
"4: Variation of the size (in nm) of control cationic lipid/DNA/compound or analog D nucleolipid particles as a function of time, at different ratios (weight/weight), when the pH is 5.A small size of the nucleolipid particles indicates that they are stabilized.",
"A very large size indicates on the contrary destabilization of the nucleolipid particles which then tend to aggregate.",
"FIG.",
"5: Variation of the size (in nm) of control cationic lipid/DNA/compound C or compound E nucleolipid particles as a function of time, at various pH values (pH 4, pH 5, pH 6 and pH 7.4).",
"The compound C or compound E/DNA ratio is set at 1 (in nmol/μg of DNA).",
"FIG.",
"6: Schematic representation of the plasmid pXL3031.FIG.",
"7: Dose/response curve for compound D on the in vivo gene transfer activity mediated by the cationic lipid/DOPE/DNA (5/5/1) complexes.",
"The compound “Analog D” is used as a negative control.",
"The data are averages (lines) and individual values (dots) for 4 Balb/c mice caryying subcutaneous M109 tumors.",
"EXAMPLES The usual reagents and catalysts such as triethylamine, trifluoroacetic acid, trifluoroacetic anhydride, tert-butyl bromoacetate, butyrolactone, 3-aminopropan-1,2-diol, serinol (2-aminopropan-1,3-diol), trimethyl ortho-formate, trimethyl ortho-acetate, para-toluenesulfonic acid, pyridinium para-toluenesulfonate or alternatively benzotriazol-1-yloxytris(dimethyl-amino)phosphonium (BOP) hexafluorophosphate, are commercially available.",
"The washings are performed with aqueous solutions saturated with sodium chloride, saturated with sodium hydrogen carbonate and with a concentrated solution of potassium hydrogen sulfate at 0.5 mol/l.",
"The hydrophilic polymers (polyethylene glycols of different sizes) are commercially available.",
"The hydrophilic substituents of the polyamine type are also commercially available or alternatively they can be synthesized by conventional methods known to a person skilled in the art as indicated in particular in the examples which follow.",
"The hydrophobic substituents (single- or double-chain dialkylamines, fatty alcohols and the like) are commercially available or alternatively synthesized according to conventional methods known to a person skilled in the art.",
"For example, the single- or double-chain dialkylamines may be synthesized from primary amines and the corresponding halogenated alkyl derivatives as indicated in the examples which follow.",
"The Proton Nuclear Magnetic Resonance (1H NMR) spectra were recorded on Bruker 300, 400 and 600 MHz spectrometers.",
"The clinical shifts are expressed in ppm (part per million) and the multiplicities by the usual abbreviations.",
"The plasmid used is pXL3031 described in the publication Gene Therapy (1999) 6, pp, 1482-1488, which contains the luc gene encoding luciferase under the control of the cytomegalovirus CMV E/P promoter.",
"This plasmid is represented in FIG.",
"6.Its size is 3671 bp.",
"The plasmid solution used is diluted to 1.262 g/l in water for injection.",
"Example 1 Synthesis of 2,2,2-trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (“Ortho 1”) 2,2,2-Trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (“Ortho 1”) Has the Formula: It can be obtained in two steps from 3-aminopropan-1,2-diol: 1) Preparation of N-(2,3-dihydroxypropyl)-2,2,2-trifluoroacetamide 15 g of 3-aminopropan-1,2-diol (164.6 mmol) are solubilized in 100 ml of tetrahydrofuran in a round-bottomed flask provided with a magnetic bar.",
"The reaction mixture is then cooled to 0° C. on an ice bath and 21.5 ml of ethyl trifluoroacetate (181.1 mmol) are gradually added.",
"The reaction mixture is stirred for 2 hours at room temperature.",
"The crude reaction product is then evaporated to dryness.",
"29 g of a pure colorless oil are thus obtained (yield: 95%), which product is used without further purification.",
"2) Preparation of 2,2,2-trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (ortho 1) The 29 g of N-(2,3-dihydroxypropyl)-2,2,2-trifluoroacetamide obtained in the preceding step (155 mmol) are solubilized in 75 ml of dichloromethane supplemented with 75 ml of trimethyl ortho-formate (685 mmol).",
"300 mg of para-toluenesulfonic acid (1.7 mmol) are then added and the reaction mixture is stirred for 2 hours at room temperature.",
"This crude product is then diluted in 500 ml of dichloromethane, washed with 3 times 200 ml of saturated sodium hydrogen carbonate and then 3 times 200 ml of saturated sodium chloride.",
"The organic phase is dried over magnesium sulfate, filtered and concentrated to dryness.",
"30 g of a pure oily product are thus obtained (yield: 85%) without further purification.",
"1H NMR (300 MHz, CDCl3, δd in ppm) .",
"A mixture of two diastereoisomers in the proportions 50/50 is observed.",
"* 3.33 and 3.37 (2s: 3H in total); from 3.35 to 3.80 (mts: 3H); from 4.10 to 4.25 (mt: 1H); 4.50 (mt: 1H); 5.73 and 5.78 (2s: 1H in total); 6.66 and 7.55 (2 unresolved complexes: 1H in total).",
"Example 2 Synthesis of 2,2,2-trifluoro-N-(2-methoxy-2-methyl-[1,3]dioxan-5-yl)acetamide (“Ortho 2) 2,2,2-Trifluoro-N-(2-methoxy-2-methyl-[1,3]dioxan-5-yl)acetamide (“Ortho 2”) has the formula: It can be obtained in two steps from 2-aminopropan-1,3-diol (serinol): 1) Preparation of 2,2,2-trifluoro-N-(2-hydroxy-1-hydroxymethylethyl)acetamide 4 g of 2-aminopropan-1,3-diol (43.9 mmol) are supplemented with 20 ml of tetrahydrofuran.",
"The reaction mixture is then cooled to 0° C. on an ice bath and 5.8 ml of ethyl trifluoroacetate (48.3 mmol) are gradually added.",
"This solution is stirred for 2 hours at room temperature.",
"The reaction mixture is then evaporated to dryness, taken up 3 times in dichloromethane so as to completely evaporate the tetrahydrofuran.",
"8.1 g of white powder (yield: 99%) are obtained pure and used in the next step without further purification.",
"2) Preparation of 2,2,2-trifluoro-N-(2-methoxy-2-methyl-[1,3]dioxan-5-yl)acetamide (Ortho 2) 7.9 g of 2,2,2-trifluoro-N-(2-hydroxy-1-hydroxymethylethyl)acetamide obtained in the preceding step (42.2 mmol) are solubilized in 30 ml of dichloromethane supplemented with 16.1 ml of trimethyl ortho-acetate (126.7 mmol).",
"73 mg of para-toluenesulfonic acid (0.42 mmol) are then added and the reaction mixture is stirred for 3 hours at room temperature.",
"The crude reaction product is then diluted with 150 ml of dichloromethane, washed with 3 times 50 ml of a saturated sodium hydrogen carbonate solution, and then 3 times 50 ml of a saturated sodium chloride solution.",
"The organic phase is dried over magnesium sulphate, filtered and concentrated to dryness.",
"9.9 g of a white solid are obtained pure without further purification (yield: 96%).",
"1H NMR (300 MHz, CDCl3, δd in ppm).",
"A mixture of two diastereoisomers in the approximate proportions 75/25 is observed.",
"* 1.49 and 1.50 (2 s: 3H in total); 3.34 and 3.35 (2 s: 3H in total); 3.66 and 3.82 (respectively dmt, J=12 Hz and dd, J=11 and 8 Hz: 2H in total); from 3.90 to 4.00 and from 4.20 to 4.35 (2 mts: 1H in total); 3.97 and 4.33 (respectively dd, J=11 and 5 Hz and dmt, J=12 Hz: 2H in total); 6.38 and 7.04 (2 broad unresolved complexes: 1H in total).",
"Example 3 Synthesis of the Syn and Trans Compounds 4-{4-[(2-{3-[4-(3-amino-propylamino)butylamino]propyl-amino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-dioctadecylbutyramide tetraacetate This example describes the route for the synthesis of the acid-sensitive compound A in the form of its two distinct diastereoisomeric forms Syn and Trans of formula:} a—Synthesis of the Syn and Trans 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-dioctadecylbutyramide (lipid portion-O-ortho 1-NH2) This synthesis is carried out in three stages: functionalization of the dioctadecylamine to an alcohol and attachment onto the group Ortho 1 whose protecting group is then cleaved.",
"1) Preparation of 4-hydroxy-N,N-dioctadecylbutyramide 4.6 g of aluminium chloride (34 mmol) are supplemented with 25 ml of chloroform, the whole being cooled to about 10° C. on a thermostated bath.",
"6.4 ml of triethylamine (46 mmol) in 15 ml of chloroform are added dropwise and then the reaction mixture is allowed to return to room temperature.",
"6 g of dioctadecylamine (11.5 mmol), mixed with 1 ml of butyrolactone (13.8 mmol) in 110 ml of chloroform, are gradually added to the mixture using a dropping funnel.",
"The reaction no longer changes after magnetical stirring for 2 hours at room temperature.",
"75 ml of water are then added and the reaction mixture is stirred for 30 minutes.",
"The crude product is filtered on Celite and then washed with chloroform.",
"The filtrate is separated by settling out and the organic phase is washed with 3 times 50 ml of a saturated sodium chloride solution.",
"The chloroformic solution is dried over magnesium sulfate, filtered and concentrated.",
"4.9 g of a white powder are obtained after chromatography on silica (yield: 70%).",
"2) Preparation of the Syn and Trans N,N-dioctadecyl-4-{4-[2,2,2-trifluoroacetylamino)methyl]-[1,3]dioxolan-2-yloxy}butyramides 2.8 g of 4-hydroxy-N,N-dioctadecylbutyramide obtained in the preceding step (4.6 mmol) are mixed with 2.6 g of 2,2,2-trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (Ortho 1, 11.5 mmol) and 90 mg of magnesium chloride (0.92 mmol).",
"The whole is heated without solvent at 80° C. for two hours.",
"The crude reaction product is then dissolved in 150 ml of cyclohexane and washed with 3 times 30 ml of a saturated sodium hydrogen carbonate solution, and then with 3 times 30 ml of a saturated sodium chloride solution.",
"The organic phase is dried over magnesium sulfate, filtered and concentrated.",
"The purification is carried out by chromatography on silica.",
"1.2 g and 1.1 g of the two expected diastereoisomers Syn and Trans are thus isolated in the form of a white powder (yield: 62%).",
"1H NMR for the compound SYN (300 MHz, CDCl3, δ in ppm): 0.89 (t, J=7 Hz: 6H); from 1.15 to 1.40 (mt: 60H); 1.52 (mt: 4H); 1.94 (mt: 2H); 2.39 (t, J=7 Hz: 2H); 3.20 (broad t, J=8 Hz: 2H); 3.29 (mt: 2H); 3.61 (t, J=5 Hz: 2H); 3.66 (mt: 2H); 3.79 (dd, J=8 and 7 Hz: 1H); 4.11 (t, J=8 Hz: 1H); 4.50 (mt: 1H); 5.82 (s: 1H); 8.13 (unresolved complex: 1H).",
"1H NMR for the compound TRANS (300 MHz, CDCl3, δ in ppm): 0.89 (t, J=7 Hz: 6H); from 1.15 to 1.40 (mt: 60H); 1.52 (mt: 4H); 1.95 (mt: 2H); 2.38 (t, J=7 Hz: 2H); 3.21 (broad t, J=8 Hz: 2H); 3.29 (broad t, J=8 Hz: 2H); 3.44 (mt, 1H); from 3.55 to 3.75 (mt: 1H); 3.60 (t, J=6 Hz: 2H); 3.69 (dd, J=8.5 and 5 Hz: 1H); 4.19 (dd, J=8.5 and 7 Hz: 1H); 4.50 (mt: 1H); 5.87 (s, 1H); 6.70 (unresolved complex: 1H).",
"3) Preparation of the Syn and Trans 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-dioctadecylbutyramide 1.1 g of N,N-dioctadecyl-4-{4-[(2,2,2-trifluoroacetylamino)methyl]-[1,3]dioxolan-2-yloxy}butyramide obtained in the preceding step (1.37 mmol, Syn or Trans) are dissolved in 10 ml of tetrahydrofuran and 10 ml of molar sodium hydroxide at 4% are added, with vigorous stirring.",
"The reaction is left overnight at room temperature.",
"The tetrahydrofuran is then concentrated and then the product is extracted with three times 150 ml of diethyl ether.",
"The organic phase is dried over calcium chloride, filtered and evaporated.",
"840 mg of the expected products are thus isolated (yield: 86%), and used as they are for the next step.",
"b—Synthesis of (trifluoroacetyl-{3-[trifluoroacetyl-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)-propyl]amino}butyl)amino]propyl}amino)acetic acid (hydrophilic part polyamine-COOH) This synthesis is performed in two steps: protection of the four amines of the spermine and then substitution of one of the primary amines with the protected bromoacetic acid.",
"1) Synthesis of 2,2,2-trifluoro-N-[3-(2,2,2-trifluoroacetylamino)propyl]-N-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)propyl]amino}-butyl)acetamide 8 g of spermine (39.5 mmol) are solubilized in 75 ml of dichloromethane.",
"33 ml of triethylamine (237 mmol) are added and then the reaction mixture is cooled to 0° C. on an ice bath.",
"41.5 g of trifluoroacetic anhydride diluted in 100 ml of dichloromethane are then added dropwise over 1 hour using a dropping funnel.",
"The reaction mixture is then allowed to return to room temperature and the reaction is left overnight with stirring.",
"75 ml of a 5% sodium hydrogen carbonate solution are then added to the reaction mixture and the solution is stirred for 15 minutes at room temperature.",
"The aqueous phase is extracted with 3 times 150 ml of dichloromethane.",
"The organic phases are combined and washed with 3 times 100 ml of a concentrated potassium hydrogen sulfate solution at 0.5 M, and then 3 times 100 ml of a saturated sodium chloride solution.",
"The organic phase is then dried over magnesium sulfate, filtered and concentrated to dryness.",
"22.5 g of a pure yellow powder are isolated without further purification (yield: 97%).",
"2) Preparation of (trifluoroacetyl-{3-[trifluoroacetyl-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)-propyl]amino}butyl)amino]propyl}amino)acetic acid 1 g of sodium hydride (60% in oil, that is 25.6 mmol) are supplemented with 60 ml of dry dimethylformamide.",
"The reaction mixture, under an argon stream, is cooled on a water bath and then 10 g of the 2,2,2-rifluoro-N-[3-(2,2,2-trifluoroacetylamino)-propyl]-N-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)propyl]amino}butyl)acetamide obtained above (17 mmol), solubilized in 40 ml of dry dimethylformamide, are added dropwise.",
"The reaction mixture is left for 1 hour at room temperature and then is again cooled on an ice bath and 3.66 g of tert-butyl bromoacetate (18.7 mmol) are added.",
"The reaction mixture is stirred overnight at room temperature.",
"500 ml of ethyl acetate are then added and then the mixture is washed with three times 100 ml of a saturated sodium hydrogen carbonate solution, and three times 100 ml of a saturated sodium chloride solution.",
"The organic phase is then dried over magnesium sulfate, filtered and concentrated to dryness.",
"A yellow oil containing the expected impure product is thus isolated in the form of a tert-butyl ester.",
"This crude reaction product is diluted in 50 ml of dichloromethane and 50 ml of trifluoroacetic acid are added.",
"The solution is stirred for 3 hours at room temperature.",
"The reaction mixture is then evaporated to dryness and then diluted in 50 ml of dichloromethane.",
"The product is then extracted with 3 times 150 ml of a saturated sodium hydrogen carbonate solution.",
"The aqueous phase obtained is washed with 3 times 30 ml of dichloromethane and is then acidified by addition of concentrated hydrochloric acid.",
"The product is then extracted with 3 times 300 ml of dichloromethane.",
"The organic phase is dried over magnesium sulfate, filtered and concentrated to dryness.",
"The purification is continued by chromatography on silica (elution: dichloromethane/methanol 8/2).",
"3.5 g of a yellow powder are thus recovered (total yield over the two steps: 32%).",
"1H NMR (400 MHz, (CD3)2SO d6 at a temperature of 373K, δd in ppm): 1.62 (mt: 4H); from 1.80 to 2.00 (mt: 4H); 3.28 (mt: 2H); from 3.30 to 3.60 (mt: 10H); 4.01 (s: 2H); from 9.15 to 9.35 (unresolved complex: 1H).",
"c—Synthesis of the Syn and Trans 4-{4-[(2-{3-[4-(3-aminopropylamino)butylamino]propylamino}acetylamino)-methyl]-[1,3]dioxolan-2-yloxy}-N,N-dioctadecyl-butyramides tetraacetate (acid-sensitive compound A) This synthesis is performed in three steps: condensation of the two molecules whose synthesis has just been described in a and b, and then deprotection of the polyamine and finally salification.",
"The same protocol was used for the products Syn and Trans.",
"1) Synthesis of the Syn and Trans N,N-dioctadecyl-4-(4-{[2-(trifluoroacetyl-{3-[trifluoroacetyl-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)propyl]amino}-butyl)amino]propyl}amino)acetylamino]methyl}-[1,3]-dioxolan-2-yloxy)butyramides 800 mg of 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-dioctadecylbutyramide (1.13 mmol Syn or Trans) obtained above (step a) dissolved in 10 ml of dichloromethane are successively supplemented with 390 μl of triethylamine (2.8 mmol), 800 mg of (trifluoroacetyl-{3-[trifluoroacetyl-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)propyl]amino}-butyl)amino]propyl}amino)acetic acid (1.24 mmol) obtained above in step b and 600 mg of BOP.",
"The solution is stirred for 2 hours at room temperature.",
"The crude reaction product is then concentrated, taken up in 150 ml of ethyl acetate, washed with three times 40 ml of a saturated sodium hydrogen carbonate solution and then three times 40 ml of a saturated sodium chloride solution.",
"After drying over magnesium sulfate, filtration and evaporation, the product is purified by chromatography on silica (elution: ethyl acetate).",
"1.1 g of white powder are thus isolated (yield:73%).",
"2) Preparation of the Syn and Trans 4-{4-[(2-{3-[4-(3-aminopropylamino)butylamino]propylamino}acetylamino)-methyl]-[1,3]dioxolan-2-yloxy}-N,N-dioctadecyl-butyramides 290 mg of N,N-dioctadecyl-4-(4-{[2-(trifluoroacetyl-{3-[trifluoroacetyl-(4-{trifluoroacetyl-[3-(2,2,2-trifluoroacetylamino)propyl]amino}-butyl)amino]propyl}amino)acetylamino]methyl}-[1,3]-dioxolan-2-yloxy)butyramide (0.22 mmol, Syn or Trans) which were obtained above are dissolved in 3 ml of tetrahydrofuran, and 3 ml of molar sodium hydroxide at 4% are added with vigorous stirring.",
"The reaction is left overnight at room temperature.",
"The solvent is then concentrated and then the crude product is taken up in a dichloromethane/methanol 1/1 mixture.",
"This crude solution is purified by chromatography on silica (dichloromethane/methanol/ammonia, 45/45/10).",
"The product is concentrated and then freeze-dried after addition of water.",
"180 mg of white freeze-dried product are thus obtained (yield: 87%).",
"3) Preparation of the Diastereoisomers Syn and Trans of 4-{4-[(2-{3-[4-(3-aminopropylamino)butylamino]propyl-amino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-dioctadecylbutyramide (compound A) The product obtained in the preceding step, in the form of a free base, is then quantitatively salified on an ion-exchange resin: it is solubilized in a water/ethanol mixture and is eluted in a column containing a large excess of acetate resin (BIO-RAD; AG 1-X2 Resin).",
"1H NMR for the compound SYN (400 MHz, CDCl3, δd in ppm): 0.89 (t, J=7 Hz: 6H); from 1.20 to 1.40 (mt: 60H); 1.52 (mt: 4H); from 1.65 to 1.90 (mt: 8H); 1.93 (mt: 2H); 1.97 (s: 3H); 2.37 (t, J=7 Hz: 2H); 2.73 (mt: 4H); 2.81 (t, J=6.5 Hz: 2H); 2.89 (mt: 4H); 2.95 (t, J=6.5 Hz: 2H); from 3.15 to 3.30 (mt: 4H); 3.32 (AB, J=17 Hz: 2H); 3.45 (dt, J=14 and 6.5 Hz: 1H); from 3.55 to 3.65 (mt: 1H); 3.61 (split t, J=7 and 2 Hz: 2H); 3.74 (t, J=8 Hz: 1H); 4.06 (t, J=8 Hz: 1H); 4.31 (mt: 1H); 5.79 (s : 1H), 7.81 (t, J=5.5 Hz: 1H).",
"1H NMR for the compound TRANS (400 MHz, CDCl3, δd in ppm): 0.88 (t, J=7 Hz: 6H); from 1.05 to 1.45 (mt: 60H); 1.51 (mt: 4H); from 1.65 to 1.90 (mt: 8H); 1.92 (mt: 2H); 1.97 (s: 3H); 2.37 (t, J=7 Hz: 2H); 2.73 (mt: 4H); 2.80 (t, J=6 Hz: 2H); 2.88 (mt: 4H); 2.96 (t, J=6 Hz: 2H); from 3.15 to 3.55 (mt: 8H); 3.57 (broad t, J=6 Hz: 2H); 3.69 (dd, J=7.5 and 5.5 Hz: 1H); 4.11 (t, J=7.5 Hz: 1H); 4.43 (mt: 1H); 5.85 (s, 1H); 7.73 (broad t, J=5.5 Hz: 1H).",
"Example 4 Synthesis of 4-{4-[(2-{3-[bis(3-aminopropyl)amino]propylamino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-ditetradecylbutyramide tetrachlorohydride This example describes a route of synthesis of the acid-sensitive compound B, in the form of an equimolar mixture of the two diastereoisomers Syn and Trans, of formula: a—Synthesis of 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-ditetradecylbutyramide (lipid part-O-Ortho 1-NH2) This synthesis is performed in four steps: synthesis of the ditetradecylamine which is then functionalized to an alcohol and then attached to the group Ortho 1 whose protecting group is then cleaved.",
"1) Ditetradecylamine Hydrochloride 74 g of bromotetradecane (267.1 mmol) are supplemented with 400 ml of ethanol and 57 g of tetradecylamine (267.1 mmol).",
"70.8 g of sodium carbonate (667 mmol) are then placed in suspension and the reaction mixture is heated under reflux overnight.",
"The reaction mixture is then evaporated to dryness, taken up in 1.5 l of dichloromethane and washed with 3 times 200 ml of water and then once 400 ml of a saturated sodium chloride solution.",
"The organic phase is dried over calcium chloride and concentrated.",
"The salification is carried out by solubilization of the crude product in the hot state in 600 ml of isopropanol supplemented with 300 ml of 5 N hydrochloric acid in isopropanol.",
"The clear solution thus obtained is allowed to cool, which induces crystallization of the expected product.",
"48.4 g of flocculant white powder are obtained after filtration and washing with isopropanol (yield: 41%).",
"2) Preparation of 4-hydroxy-N,N-ditetradecylbutyramide 22.4 g of aluminum chloride (168.1 mmol) are supplemented with 75 ml of chloroform, the whole being cooled to about 10° C. on an ice-cold water bath.",
"39 ml of triethylamine (280.1 mmol) in 100 ml of chloroform are added dropwise and then the reaction mixture is allowed to return to room temperature.",
"25 g of ditetradecylamine hydrochloride (56 mmol) mixed with 5.2 ml of butyrolactone (67.2 mmol) in 350 ml of chloroform are gradually added to the mixture, with mechanical stirring.",
"The reaction no longer changes after 2 hours at room temperature.",
"200 ml of water are then added and the reaction mixture is stirred for 30 minutes.",
"The crude product is filtered on Celite and then washed with chloroform.",
"The filtrate is separated after settling out and the organic phase is washed with three times 150 ml of a saturated sodium chloride solution.",
"The chloroformic solution is dried over magnesium sulfate, filtered and concentrated.",
"21.2 g of white powder are obtained with a yield of 76% after chromatography on silica (ethyl acetate/cyclohexane 1/1).",
"3) Preparation of N,N-ditetradecyl-4-{4-[(2,2,2-trifluoroacetylamino)methyl]-[1,3]dioxolan-2-yloxy}butyramide 3.5 g of 4-hydroxy-N,N-ditetradecylbutyramide (7.1 mmol) obtained in the preceding step are mixed with 1.8 g of 2,2,2-trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (Ortho 1, 7.8 mmol) and 18 mg of pyridinium para-toluenesulfonate (PPTS, 0.071 mmol).",
"The whole is heated without solvent at 80° C. for 3 hours.",
"The crude reaction product is then dissolved in 200 ml of heptane, washed with 3 times 50 ml of a saturated sodium hydrogen carbonate solution, with 3 times 50 ml of acetonitrile and is then concentrated to dryness.",
"A small fraction of the crude product is purified on silica in order to characterize this intermediate, the remainder being used as it is for the next step.",
"The chromatography on silica (cyclohexane/ethyl acetate 7/3 V/V) allows us to isolate a few mg of oily product.",
"4) Preparation of 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-ditetradecylbutyramide The crude product obtained in the preceding step is solubilized in 20 ml of tetrahydrofuran supplemented with 20 ml of sodium hydroxide at 4%.",
"The reaction mixture is left overnight with vigorous stirring at room temperature.",
"The tetrahydrofuran is then concentrated and then the product is extracted with 3 times 200 ml of diethyl ether.",
"The organic phase is dried over calcium chloride, filtered and evaporated.",
"Chromatography on silica (dichloromethane/methanol 9/1 V/V) makes it possible to isolate 1.6 g of a colorless oil (yield on the two steps: 38%).",
"1H NMR (300 MHz, CDCl3, δd in ppm): a mixture of two diastereoisomers in the proportions 50/50 is observed.",
"* 0.89 (t, J=7 Hz: 6H); from 1.15 to 1.45 (mt: 44H); 1.52 (mt: 4H); 1.58 (unresolved complex: 2H); 1.95 (quintuplet, J=6.5 Hz: 2H); 2.39 (t, J=6.5 Hz: 2H); from 2.75 to 3.00 (mt: 2H); 2.31 (mt: 2H); 3.29 (mt: 2H); 3.60 (mt: 2H); 3.71 and 3.80 (respectively dd, J=7.5 Hz and 6 Hz and t, J=7.5 Hz: 1H in total); 4.06 and 4.14 (2 t, J=7.5 Hz: 1H in total); 4.21 and 4.33 (2 mts: 1H in total); 5.82 and 5.85 (2 s: 1H in total).",
"b—Synthesis of [(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}propyl)trifluoroacetylamino]acetic acid in the form of a trifluoroacetate salt (hydrophilic part of the polyamine-COOH type) The proposed synthesis is performed in 6 steps starting with 3-aminopropanol and 3,3′-iminobispropylamine.",
"1) Preparation of 2,2,2-trifluoro-N-{3-[3-(2,2,2-trifluoroacetylamino)propylamino]propyl}acetamide 35 g of 3,3′-iminobispropylamine (266.7 mmol) are solubilized in 150 ml of anhydrous tetrahydrofuran under an argon stream.",
"The reaction mixture is then cooled to 0° C. on an ice bath and 65 ml of ethyl trifluoroacetate (546.8 mmol) are added dropwise (very slowly) using a dropping funnel.",
"At the end of the addition (3 hours later), the reaction mixture is allowed to return to room temperature and the stirring is maintained for a few hours under argon.",
"The reaction mixture is then filtered on paper.",
"The filtrate is concentrated to dryness, taken up several times in dichloromethane, the final drying being carried out in an oven at 40° C. under the vacuum produced by a slide vane rotary vacuum pump for 16 hours.",
"85.3 g of a white powder are isolated pure without further purification (yield: 99%).",
"2) Preparation of tert-butyl (3-hydroxypropylamino)acetate 196 ml of 3-aminopropanol (2.56 mol) are diluted in 250 ml of dichloromethane and the whole is cooled to 0° C. on an ice bath.",
"20 g of tert-butyl bromoacetate (102.5 mmol), solubilized in 200 ml of dichloromethane, are then added dropwise while the reaction mixture is maintained at 0° C. At the end of the addition (2 hours later), the reaction mixture is left at room temperature for 3 hours.",
"This crude product is then washed with 3 times 150 ml of a saturated sodium hydrogen carbonate solution, and then 3 times 150 ml of a saturated sodium chloride solution.",
"The organic phase is dried over calcium chloride, filtered and concentrated.",
"17.8 g of a colorless oil are thus isolated (yield: 92%).",
"3) Preparation of tert-Butyl [(3-hydroxypropyl)-trifluoroacetylamino]acetate 17.65 g of tert-butyl (3-hydroxypropylamino)acetate (93.3 mmol) are solubilized in 100 ml of dichloromethane and the whole is cooled to 0° C. on an ice bath.",
"26 ml of triethylamine (186.6 mmol) are added, followed by a dropwise addition of 21.5 g of trifluoroacetic anhydride (102.6 mmol) using a dropping funnel.",
"At the end of the addition, the reaction mixture is left at room temperature overnight, with magnetic stirring.",
"This solution is then washed with 3 times 50 ml of a saturated sodium hydrogen carbonate solution, 3 times 50 ml of a 0.5 M potassium hydrogen sulfate solution, and then 3 times 50 ml of a saturated sodium chloride solution.",
"The organic phase is dried over magnesium sulfate, filtered and concentrated.",
"24.5 g of a pale yellow oil are thus isolated (yield: 92%).",
"4) Preparation of tert-butyl [(3-bromopropyl)trifluoroacetylamino]acetate 10 g of tert-butyl [(3-hydroxypropyl)-trifluoroacetylamino]acetate (35 mmol) are solubilized in 150 ml of tetrahydrofuran.",
"12.4 g of triphenylphosphine (47.3 mmol) are added and the reaction mixture is thermostated at 15-20° C. 15.1 g of carbon tetrabromide (45.6 mmol), dissolved in 60 ml of acetonitrile, are added dropwise using a dropping funnel and the whole is stirred at room temperature for 4 hours.",
"The reaction mixture is then concentrated to dryness, taken up in ethyl acetate and filtered on paper.",
"The filtrate is concentrated to dryness, taken up in cyclohexane and filtered on sintered material No.",
"3.The filtrate is again concentrated and purified by chromatography on silica (cyclohexane/ethyl acetate 8/2 V/V).",
"10.4 g of a pale yellow oil are thus isolated (yield: 85%).",
"5) Preparation of tert-butyl [(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}propyl)trifluoroacetylamino]acetate 26 g of tert-butyl [(3-bromopropyl)trifluoroacetylamino]acetate (74.7 mmol) and 24.1 g of 2,2,2-trifluoro-N-{3-[3-(2,2,2-trifluoroacetylamino)propylamino]propyl}acetamide (74.7 mmol) are solubilized in 130 ml of acetonitrile.",
"30 g of potassium carbonate (224 mmol) are then placed in suspension and the whole is heated under reflux for 6 hours.",
"The reaction mixture is then filtered on paper and concentrated to dryness.",
"The crude product is then purified by chromatography on silica (cyclohexane/ethyl acetate 2/8 V/V).",
"16.6 g of a pale yellow oil (yield: 38%) are thus isolated.",
"6) Preparation of the trifluoroacetate salt of [(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}-propyl)trifluoroacetylamino]acetic acid 15.8 g of tert-butyl [(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}propyl)trifluoroacetylamino]acetate (28.76 mmol) are supplemented with 50 ml of dichloromethane and then with 50 ml of trifluoroacetic acid.",
"This mixture is stirred for a few hours at room temperature.",
"The reaction mixture is then concentrated to dryness.",
"18.7 g of pale yellow honey are thus isolated (yield: 100%).",
"1H NMR (300 MHz, CDCl3, δd in ppm): at room temperature, a mixture of rotamers is observed.",
"* from 1.80 to 2.10 (mt: 6H); 3.12 (mt: 6H); 3.29 (mt: 4H); 3.50 (mt: 2H); 4.13 and 4.29 (respectively broad s and mt: 2H in total); from 9.50 to 9.75 (mt: 2H) c—Synthesis of 4-{4-[(2-{3-[bis(3-aminopropyl)amino]-propylamino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-ditetradecylbutyramide tetrahydrochloride (compound B) This synthesis is performed in three steps: condensation of the two molecules obtained in parts a and b above, and then deprotection of the polyamine and salification.",
"1) Preparation of 4-[4-({2-[(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}propyl)trifluoroacetylamino]-acetylamino}methyl)-[1,3]dioxolan-2-yloxy]-N,N-ditetradecylbutyramide 1.9 ml of triethylamine (13.8 mmol), 2 g of [(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}-propyl)trifluoroacetylamino]acetic acid (trifluoroacetate salt, 3.04 mmol) and 1.8 g of BOP (4.14 mmol) are successively added to 1.65 g of 4-(4-aminomethyl-[1,3]dioxolan-2-yloxy)-N,N-ditetradecylbutyramide (2.76 mmol) dissolved in 30 ml of dichloromethane.",
"The solution is stirred for 1 hour at room temperature.",
"The crude reaction product is then concentrated to dryness, taken up in 200 ml of ethyl acetate, washed with 40 ml of a saturated sodium chloride solution, and then 3 times 40 ml of a saturated sodium hydrogen carbonate solution, and then 3 times 40 ml of a saturated sodium chloride solution.",
"The product is then purified by chromatography on silica (elution: ethyl acetate).",
"2.2 g of pale yellow honey are thus isolated (yield: 72%).",
"2) Preparation of 4-{4-[(2-{3-[bis(3-aminopropyl)-amino]propylamino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-ditetradecylbutyramide 2.1 g of 4-[4-({2-[(3-{bis[3-(2,2,2-trifluoroacetylamino)propyl]amino}propyl)trifluoroacetylamino]acetylamino}methyl)-[1,3]dioxolan-2-yloxy]-N,N-ditetradecylbutyramide (1.88 mmol) are dissolved in 30 ml of tetrahydrofuran, and 30 ml of molar sodium hydroxide at 4% are added, with vigorous stirring.",
"The reaction is left overnight at room temperature.",
"The solvent is then concentrated and then the crude product is taken up in a dichloromethane/methanol 1/1 mixture.",
"This crude solution is purified by chromatography on silica (dichloromethane/methanol/ammonia, 45/45/10, V/V/V).",
"The product is concentrated and then freeze-dried after addition of water.",
"1.3 g of white freeze-dried product are thus obtained (yield: 84%).",
"3) Preparation of 4-{4-[(2-{3-[bis(3-aminopropyl)amino]propylamino}acetylamino)methyl]-[1,3]dioxolan-2-yloxy}-N,N-ditetradecylbutyramide tetrahydrochloride (compound B) The product obtained in the preceding step in the form of a free base is then quantitatively salified on an ion-exchange resin: it is solubilized in water, and eluted in a column containing a large excess of chloride resin (FLUKA; DOWEX 21K).",
"The structure of the white freeze-dried product obtained is confirmed by 1H NMR.",
"1H NMR (300 MHz, (CD3)2SO d6, δd in ppm): a mixture of two diastereoisomers in the proportions 50/50 is observed.",
"* 0.89 (t, J=7 Hz: 6H); from 1.15 to 1.40 (mt: 44H); from 1.40 to 1.60 (mt: 4H); 1.72 (mt: 8H); 2.31 (mt: 2H); from 2.40 to 2.55 (mt: 6H); from 2.70 to 2.90 (mt: 6H); from 3.15 to 3.75-from 4.00 to 4.40 (2 series of mt: 13H in total); 5.83 and 5.86 (2 s: 1H in total).",
"Example 5 Synthesis of the PEGoylated Lipids C and D This example describes a route of synthesis of the pegoylated lipids Octadecanol-Ortho 1-PEG5000-OMe and Cholesterol-Ortho 1-PEG5000-OMe which differ from each other only in their lipid portion: octadecanol for compound C and cholesterol for compound D. These two acid-sensitive compounds have the general formula: a—Synthesis of C-(2-octadecyloxy-[1,3]dioxolan-4-yl)methylamine and of C-{2-[17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-[1,3]dioxolan-4-yl}methylamine (lipid parts-O-Ortho 1-NH2) This synthesis is performed in two steps starting with the compound Ortho 1 by substitution of the exocyclic methoxy group with the fatty alcohol (cholesterol or octadecanol) and then deprotection of the amine.",
"1) Preparation of 2,2,2-trifluoro-N-(2-octadecyloxy-[1,3]dioxolan-4-ylmethyl)acetamide 3 g of 2,2,2-trifluoro-N-(2-methoxy-[1,3]dioxolan-4-ylmethyl)acetamide (Ortho 1, 13.09 mmol) are mixed with 3.54 g of octadecanol (13.09 mmol).",
"The mixture is melted at 80° C. and left for 2 hours after the addition of 32 mg of pyridinium para-toluenesulfonate (0.13 mmol).",
"The crude reaction product is then dissolved in cyclohexane, washed with a saturated sodium hydrogen carbonate solution and then with a saturated sodium chloride solution, dried over magnesium sulfate and then concentrated to dryness.",
"This crude product is used as it is for the next step.",
"1′) Preparation of N-{2-[17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-[1,3]dioxolan-4-ylmethyl)-2,2,2-trifluoroacetamide The protocol is identical to that described above, it being possible for the reaction to also be performed without catalyst.",
"2) Preparation of C-(2-octadecyloxy-[1,3]dioxolan-4-yl)methylamine 6.12 g of crude reaction product of the preceding step 1 (13.09 mmol) are dissolved in 20 ml of tetrahydrofuran.",
"The reaction mixture is cooled on an ice bath, and 30 ml of sodium hydroxide at 4% are added.",
"The mixture is stirred at room temperature until complete disappearance of the reagent is obtained (over a period of 4 hours).",
"Next, the solvent is concentrated in part and then-extracted with 3 times 200 ml of diethyl ether.",
"The organic phase is dried over calcium chloride, filtered and evaporated.",
"The crude reaction product is purified by chromatography on silica (dichloromethane/methanol 9/1, V/V).",
"2.6 g of a white powder are thus recovered (yield: 53% on the two consecutive steps 1 and 2).",
"1H NMR (300 MHz, CDCl3, δd in ppm).",
"A mixture of two diastereoisomers in the approximate proportions 50/50 is observed.",
"* 0.89 (t, J=7 Hz: 3H); from 1.20 to 1.45 and 1.58 (2 mts: 32H in total); from 2.75 to 3.00 (mt: 2H); 3.53 (mt: 2H); from 3.65 to 3.85 (mt: 1H); from 4.00 to 4.40 (mt: 2H); 5.81 and 5.84 (2 s: 1H in total).",
"2′) Preparation of C-{2-[17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy]-[1,3]dioxolan-4-yl}methylamine The protocol is identical to that of step 2).",
"1.4 g of white powder are thus recovered (yield: 22% on the two consecutive steps 1′ and 2″).",
"1H NMR (400 MHz, CDCl3, δd in ppm).",
"A mixture of two diastereoisomers in the approximate proportions 50/50 is observed.",
"* 0.69 (s: 3H); from 0.85 to 1.75-1.86 and 2.00 (mts: 26H in total); 0.88 (mt: 6H); 0.93 (d, J=7 Hz: 3H); 1.01 (s: 3H); 2.33 (mt: 2H); from 2.75 to 3.00 (mts: 2H); 3.50 (mt: 1H); 3.71-3.85-4.05 and 4.16 (4 mts: 2H in total); 4.20 and 4.35 (2 mts: 1H in total); 5.36 (mt: 1H); 5.93 and 5.96 (2 s: 1H in total).",
"b—Synthesis of the acid of methoxypolyethylene glycol 5000 (hydrophilic part MeO-PEG5000-COOH) A single step is necessary: oxidation of the terminal hydroxyl group of the commercial methoxypolyethylene glycol.",
"20 g of MeO-PEG5000-OH (4 mmol) are dissolved in 100 ml of an equal volume water/acetonitrile mixture.",
"312 mg of 2,2,6,6-tetramethylpiperidinyloxyl (2 mmol) and then 6.4 g of [bis(acetoxy)iodo]benzene (20 mmol) are added and the reaction mixture is left stirring for 16 hours at room temperature.",
"This crude reaction product is then evaporated to dryness, taken up in 40 ml of a dichloromethane/ethanol (1/1; V/V) mixture, and then precipitated by addition of 500 ml of diethyl ether.",
"19 g of a white powder are thus isolated by filtration and washing with ether (yield: 95%).",
"1H NMR (300 MHz, CDCl3, δd in ppm): 3.39 (s: 3H); from 3.40 to 3.95 (mts: 404H); 4.15 (s: 2H).",
"c—Synthesis of methoxy-(polyethylene glycol 5000)-(N-(2-octadecyloxy-[1,3]dioxolan-4-ylmethyl)amide (compound C) and of methoxy-(polyethylene glycol 5000)-N-{2-[17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]-phenanthren-3-yloxy]-[1,3]dioxolan-4-ylmethyl}amide (compound D) Compound C: 1.2 g of MeO-PEG5000-COOH (0.24 mmol) obtained in the preceding steps are dissolved in 5 ml of dichloromethane.",
"188 μl of triethylamine (1.34 mmol) are added and then 100 mg of C-(2-octadecyloxy-[1,3]dioxolan-4-yl)methylamine (0.27 mmol).",
"143 mg of BOP (0.32 mmol, 1.2 eq) are then added and the reaction is left stirring at room temperature for one hour.",
"The reaction mixture is precipitated by addition of diethyl ether (60 ml), centrifuged, washed with ether and then injected into preparative high-performance liquid chromatography (HPLC).",
"By isolating the purest fractions, 415 mg of white freeze-dried product are thus obtained (yield: 32%).",
"1H NMR (300 MHz, CDCl3, δd in ppm): a mixture of two diastereoisomers in the approximate proportions 50/50 is observed.",
"* 0.90 (t, J=7 Hz: 3H); from 1.20 to 1.40 and from 1.50 to 1.75 (mts: 32H); 3.39 (s: 3H); from 3.40 to 3.95 (mts: 448H); 4.02 and 4.03 (2s: 2H in total); from 4.00 to 4.25 (mts: 3H in total); 5.80 and 5.84 (2s: 1H in total).",
"Compound D: The protocol is identical to that presented for the preparation of compound C. By isolating the purest fractions, 395 mg of white freeze-dried product are thus obtained (yield: 30%).",
"1H NMR (400 MHz, CDCl3, δd in ppm): a mixture of two diastereoisomers in the approximate proportions 50/50 is observed.",
"* 0.68 (s: 3H); from 0.85 to 1.75-1.85 and 2.00 (mts: 26H in total); 0.87 (mt: 6H); 0.92 (d, J=7 Hz: 3H); 1.00 (s: 3H); 2.33 (mt: 2H); 3.39 (s: 3H); from 3.40 to 3.90 (mts: 448H); from 4.00 to 4.20 (mts: 1H in total); 4.01 and 4.04 (2s: 2H in total); 4.28 and 4.46 (2 mts: 1H in total); 5.35 (mt: 1H); 5.90 and 5.95 (2s: 1H in total).",
"Example 6 Synthesis of the PEGoylated lipid E This example describes a route of synthesis of the pegoylated lipid octadecanol-Ortho 2-PEG5000-OMe which has the general formula: a—Synthesis of 2-methyl-2-octadecyloxy-[1,3]dioxan-5-ylamine (hydrophobic lipid part-O-Ortho2-NH2) This synthesis if performed in two steps starting with the substrate Ortho 2 whose synthesis is described above, by substitution of the exocyclic methoxy group with octadecanol and then deprotection of the amine.",
"1) Preparation of 2,2,2-trifluoro-N-(2-methyl-2-octadecyloxy-[1,3]dioxan-5-yl)acetamide 3 g of 2,2,2-trifluoro-N-(2-methoxy-2-methyl-[1,3]dioxan-5-yl)acetamide (12.34 mmol) are mixed with 3 g of octadecanol (11.1 mmol).",
"The mixture is melted at 80° C. and left for 2 hours in order to evaporate all the methanol produced during the alcohol exchange.",
"The molten reaction mixture is then poured into 50 ml of acetonitrile, which induces its precipitation.",
"The expected product is purified by recrystallization from acetonitrile.",
"2.85 g of white powder are thus isolated (yield: 53% after recrystallization).",
"2) Preparation of 2-methyl-2-octadecyloxy-[1,3]dioxan-5-ylamine 1 g of the trifluoroacetamide derivative of the preceding step (2.08 mmol) is dissolved in 10 ml of tetrahydrofuran, to which 10 ml of molar sodium hydroxide at 4% are added.",
"The mixture is vigorously stirred at room temperature until complete disappearance of the reagent is obtained (over a period of 4 hours).",
"Next, the solvent is concentrated in part and then extracted with 3 times 100 ml of diethyl ether.",
"The organic phase is dried over calcium chloride, filtered and evaporated to dryness.",
"810 mg of white powder are thus isolated without purification (yield: 100%).",
"1H NMR (400 MHz, CDCl3, δd in ppm): 0.89 (t, J=7 Hz: 3H); from 1.20 to 1.50 (mt: 30H); 1.49 (s: 3H); 1.62 (mt: 2H); 1.67 (broad s: 2H); 2.71 (mt: 1H); 3.46 (t, J=7 Hz: 2H); 3.54 (broad d, J=10 Hz: 2H); 4.30 (dd, J=10 and 1.5 Hz: 2H).",
"b—Synthesis of methoxy-(polyethylene glycol 5000)-N-(2-methyl-2-octadecyloxy-[1,3]dioxan-5-yl)amide (Compound E) The last step consists in condensing the lipid amine with the acid of PEG (whose synthesis is described in example 5).",
"1.15 g of MeO-PEG5000-COOH (0.23 mmol) are dissolved in 5 ml of dichloromethane.",
"181 μl of triethylamine (1.30 mmol) are added and then 100 mg of 2-methyl-2-octadecyloxy-[1,3]dioxan-5-ylamine (0.26 mmol).",
"172 mg of BOP (0.39 mmol) are then added and the reaction is left stirring at room temperature for one hour.",
"The reaction mixture is precipitated by addition of diethyl ether (60 ml), centrifuged, washed with ether and then injected into preparative HPLC.",
"By isolating the purest fractions, 420 mg of white freeze-dried product are thus obtained (yield: 34%).",
"1H NMR (400 MHz, CDCl3, δd in ppm); 0.89 (t, J=7 Hz: 3H); from 1.20 to 1.50 (mt: 30H); 1.48 (s: 3H); from 1.55 to 1.75 (mt: 2H); 3.39 (s: 3H); from 3.40 to 3.90 (mt: 448H); 3.89 (broad d, J=8.5 Hz: 1H); 4.05 (s: 2H); 4.30 (broad d, J=12 Hz: 2H); 7.57 (d, J=8.5 Hz: 1H).",
"Example 7 Study of the Compaction of DNA by the Acid-Sensitive Compounds A Syn and Trans The acid-sensitive compounds A forms Syn and Trans prepared above have a structure analogous to the cationic lipids conventionally used for the nonviral transfection of DNA, and they possess, inter alia, in their structure a cyclic ortho-ester function which contributes to making them acid-sensitive.",
"The aim of this example is therefore to demonstrate that the acid-sensitive compounds A Syn and Trans preserve the power to compact DNA to be transfected specific to the cationic lipids, while having the capacity to become degraded in acidic medium and therefore to release the compacted DNA.",
"This can be easily shown by a fluorescence test with ethidium bromide (EtBr): the absence of fluorescence reflects the absence of free DNA, which means that the DNA is compacted.",
"In the text which follows, the two forms Syn and Trans of the acid-sensitive compound A as prepared in example 3 were used and the non-acid-sensitive analog described in the publication WO 97/18185, and which has the formula: H2N—(CH2)3—NH—(CH2)4—NH—(CH2)3—NH—CH2—CO-Gly-N[(CH2)17—CH3]2 was used as control.",
"This non-acid-sensitive analog is called in the text which follows “control cationic lipid”.",
"The DNA is brought into contact with increasing quantities of control cationic lipid or of acid-sensitive compound A Syn or Trans, by mixing an equal volume of lipid solutions of different titers with the DNA solutions.",
"Samples of 800 μl of DNA complexes having a concentration of 10 μg/ml are thus prepared in a sodium chloride solution at 150 mM with increasing quantities of control cationic lipid or of acid-sensitive compound A Syn or Trans.",
"At time t=0, 200 μl of buffer having a concentration of 0.1 mol/l at pH 5 are added to these samples and the samples are stored in an oven at 37° C. The fluorescence with ethidium bromide (EtBr) is measured over time (measurement at 20° C.) using a FluoroMax-2 (Jobin Yvon-Spex), with excitation and emission wavelengths of 260 nm and 590 nm respectively.",
"The slit widths for excitation and for emission are set at 5 nm.",
"The fluorescence value is recorded after addition of 3 μl of ethidium bromide at 1 g/l per ml of DNA/cationic lipid or DNA/acid-sensitive compound solution (at 0.01 mg of DNA/ml).",
"The results are summarized in FIG.",
"1.At pH 5, when the quantity of acid-sensitive compound A Syn or Trans or of control cationic lipid used to compact the DNA is too low (0.4 nmol lipid/μg of DNA) and when the DNA is therefore not completely compacted, no significant variation in fluorescence is measurable over time.",
"On the other hand, a different behavior of the acid-sensitive compounds A Syn and Trans is observed with respect to the (non-acid-sensitive) control cationic lipid for larger quantities (1.7 nmol lipid/μg of DNA and 6.0 nmol lipid/μg of DNA) which allow complete compaction of the DNA.",
"Indeed, the acid-sensitive compounds A Syn and Trans release the DNA over time as demonstrated by the increase in fluorescence, which is not the case with the control cationic lipid which is not acid-sensitive.",
"It is observed, in addition, that this release of DNA occurs a few hours after the addition of acid (at pH 5 and 37° C.).",
"A shift is also observed in the kinetics of release of the DNA according to the quantity of acid-sensitive compound used: the lower the quantity of acid-sensitive compound A used, the more rapid the release of the DNA.",
"This study demonstrates the remarkable properties of the acid-sensitive compounds A Syn and Trans: they are capable of forming complexes with DNA by compacting it, and their degradation in acidic medium causes a degradation of the complexes formed with the DNA, and therefore the release of the DNA.",
"These acid-sensitive compounds are therefore particularly useful in the context of the nonviral transfection of DNA into cells.",
"Example 8 Study of the Transfecting Power of the Acid-Sensitive Compounds A Syn and Trans in Vitro This example illustrates the in vitro transfecting power of the acid-sensitive compounds A Syn and Trans, compared with their non-acid-sensitive analog described in the preceding example (the control cationic lipid).",
"This study was carried out at 4 different charge ratios: 1.0 or 4.0 or 6.0 or 10.0 nmol of lipid/μg of DNA.",
"Each of these conditions was tested with and without fetal calf serum (“+ or − Serum”) Culture of the cells: HeLa cells (American type Culture Collection (ATCC) Rockville, Md., USA) derived from a human cervical epithelium carcinoma, are cultured in the presence of a medium of the MEM (“minimum essential medium”) type with addition of 2 mM L-glutamine, 50 units/ml of penicillin, and 50 units/ml of streptomycin.",
"The medium and the additives are obtained from Gibco/BRL life Technologies (Gaithersburg, Md., USA).",
"The cells are cultured in flasks at 37° C. and at 5% carbon dioxide in an incubator.",
"Transfection: a day before the transfection, the HeLa cells are transferred into 24-well plates with a cell number of 30,000 to 50,000 per well.",
"These dilutions represent approximately 80% confluence after 24 hours.",
"For the transfection, the cells are washed twice and incubated at 37° C. with 500 μl of medium with serum (10% FCS v/v) or without serum.",
"50 μl of complexes containing 0.5 μg of plasmid DNA are added to each well (the complexes are prepared at least 30 minutes before the addition to the well).",
"After two hours at 37° C., the plates without serum are supplemented with 10% (v/v) of FCS (“Fetal Calf Serum”).",
"All the plates are placed for 36 hours at 37° C. and at 5% carbon dioxide.",
"Determination of the luciferase activity: Briefly, the transfected cells are washed twice with 500 μl of PBS (phosphate buffer) and then lysed with 250 μl of reagent (Promega cell culture lysis reagent, from the Luciferase Assay System kit).",
"An aliquot of 10 μl of supernatant of the lysate centrifuged (12,000×g) for 5 minutes at 4° C. is measured with the Wallac Victor2 luminometer (1420 Multilabel couter).",
"The luciferase activity is assayed by the emission of light in the presence of luciferin, of coenzyme A and of ATP for 10 seconds and expressed relative to 2000 treated cells.",
"The luciferase activity is thus expressed as Relative Light Unit (“RLU”: “Relative light unit”) and normalized with the concentration of proteins in the sample obtained using a Pierce BCA kit (Rockford, Ill., USA).",
"The results, summarized in FIG.",
"2, show a high transfection activity for the three compounds tested (the acid-sensitive compounds A Syn and Trans and the control cationic lipid).",
"No significant difference is observed between them.",
"In the absence of serum, the level of transfection is high in all the cases (105 to 107 RLU/μg of protein) and the transfecting power increases with the quantity of acid-sensitive compound or of control cationic lipid used.",
"The presence of serum induces inhibition of transfection in all cases.",
"This example therefore shows that the transfecting power of the acid-sensitive compound A in its Syn and Trans forms is preserved compared with its non-acid-sensitive analog (the control cationic lipid).",
"More generally, the introduction of an acid-sensitive cyclic ortho-ester function into molecules of the cationic lipid type which are known to be useful in nonviral transfection does not destroy the capacity of these compounds to efficiently transfect DNA.",
"Example 9 Use of the Acid-Sensitive Compounds C and D as Nonionic Surfactants for the Colloidal Stabilization of DNA/cationic Lipid Transfecting Complexes This example illustrates the fact that the acid-sensitive pegoylated lipids of the type defined under (d) in the present application can be used as nonionic surfactants which play a colloidal stabilizing role with respect to the DNA/cationic lipid transfecting particles.",
"In the present example, the cationic lipid used is that already used in examples 7 and 8 and described in the publication WO 97/18185 under the formula: H2N—(CH2)3—NH—(CH2)4—NH—(CH2)3—NH—CH2—CO-Gly-N[(CH2)17—CH3]2 (control cationic lipid).",
"Compounds C and D prepared in example 5 are used as acid-sensitive pegoylated lipids.",
"As controls, there are used BRIJ 700 (SIGMA) and the pegoylated lipid of formula: which are non-acid-sensitive analogs of compounds C and D, respectively, and which are known as nonionic surfactants (see for example the publication WO 98/34648).",
"The two controls are used at 10 g/l in water.",
"1 ml samples of nucleolipid complexes (DNA/control cationic lipid) are prepared from DNA at 10 μg/ml in 75 mM of a sodium chloride solution, by mixing in equal volume the solution containing the control cationic lipid and one of the pegoylated lipids (compound C or compound D or Brij 700 or the analog D) with the DNA solution.",
"All these samples have a control cationic lipid/DNA ratio of 1.5 (in nmol of lipid per μg of DNA) and contain increasing quantities of pegoylated lipid (expressed as polymer/DNA weight/weight ratio).",
"The measurement of the size of the particles obtained is made 30 minutes after the mixing and makes it possible in particular to study the influence of the quantity of acid-sensitive pegoylated lipid (compound C or D) or of the non-acid-sensitive pegoylated lipid (Brij 700 or analog D) on the stabilization of the control cationic lipid/DNA complexes.",
"The measurement of the hydrodynamic diameter is made with a Coulter N4Plus apparatus using plastic cuvettes (four transparent sides) filled with 800 μl of the different solutions containing 0.01 mg of DNA/ml, the measurement being carried out at 90° in unimodal mode.",
"The results are presented in FIG.",
"3 which describes the variation in the size of the control cationic lipid/DNA particles as a function of the quantity of compound C or D or of Brij 700 or of analog D used.",
"It is observed that the acid-sensitive pegoylated lipids, as well as their stable controls, allow the formation of small-sized (less than 100 nm) control cationic lipid/DNA particles when a minimal quantity is reached, whereas these same control cationic lipid/DNA particles spontaneously aggregate (size greater than 1 μm) in the absence of acid-sensitive or non-acid-sensitive pegoylated lipid or when the quantity of the latter is too low.",
"This example thus demonstrates that compounds C and D, and more generally acid-sensitive pegoylated lipids of the type defined under (d) in the general formula (I) of the present application, can be used as nonionic surfactants, and their colloidal stabilizing power is comparable to that of the stable nonionic surfactants (that is to say non-acid-sensitive such as Brij 700 for example) which are conventionally used for the colloidal stabilization of nucleolipid particles.",
"Example 10 Influence of the pH on the Colloidal Stabilization of DNA/Cationic Vector Complexes by Compounds C and D This example illustrates the fact that acid-sensitive pegoylated lipids of the type defined under (d) in the general formula (I) of the present application, and which are used as nonionic surfactants for stabilizing DNA/cationic lipid nucleolipid complexes, can be degraded at acidic pH and thus release said nucleolipid complexes.",
"The remarkable property which is used here is the absence of colloidal stabilization when a pegoylated lipid is replaced with a PEG without a lipid portion.",
"Indeed, the formulation of the DNA in the presence of a cationic vector and of an acid-sensitive pegoylated lipid leads, in a non-buffered medium, to small particles (see example 9 above).",
"The same study with a PEG alone (that is to say not coupled to a lipid portion) does not, on the other hand, give any stabilization.",
"The acid-sensitive pegoylated lipid used in this example is the compound D prepared in example 5, as well as its non-acid-sensitive analog called “Analog D” in the preceding example and in the text which follows.",
"1 ml samples of nucleolipid complexes (DNA/control cationic lipid) are prepared from DNA at 10 μg/ml in 75 mM of a sodium chloride solution, by mixing in equal volume the solution containing the control cationic lipid and compound D or the non-acid-sensitive analog D with the DNA solution.",
"All these samples have a control cationic lipid/DNA ratio of 1.5 (in nmol of lipid per μg of DNA) and contain increasing quantities of pegoylated lipid (expressed as polymer/DNA weight/weight ratio).",
"100 μl of acetic acid/sodium acetate buffer at pH 5 (0.1 mol/l) are added to these samples thermostated at 37° C. in a ventilated oven.",
"The size of the particles is measured as a function of time.",
"The results obtained are represented in FIG.",
"4 which represents the size of the nucleolipid particles as a function of time, and also according to the acid-sensitive or non-acid-sensitive pegoylated lipid/DNA ratio (3 weight/weight ratios tested: 0.5 or 0.75 or 1) In the case of analog D (non-acid-sensitive pegoylated lipid), the pH has no influence on the colloidal stability of the particles: the particles formed have a small size, of the order of 100 nm, regardless of the analog D/DNA ratio used.",
"At time t=0, the same stability is observed with compound D, regardless of the compound D/DNA ratio.",
"On the other hand, an increase in the size of the nucleolipid particles as a function of time is observed when compound D is used as nonionic surfactant at acidic pH (pH 5).",
"The lower the compound D/DNA ratio, the more rapid this increase in size of the nucleolipid particles.",
"Thus, after 4 hours, the particles have completely formed into aggregates in the case of a low ratio (of 0.5) and not at all or only slightly when a large excess of compound D is used (ratio of 1).",
"It is thus possible to deduce therefrom that compound D is sensitive to the pH value.",
"Indeed, the increase in the size of the nucleolipid particles as a function of time reflects the degradation of the acid-sensitive pegoylated lipid (compound D) when an acidic medium is used (there is in fact “ungrafting” of the lipid portion at the level of the acid-sensitive portion of the compound).",
"In addition, by increasing the acid-sensitive pegoylated lipid/DNA ratio, the time necessary for the aggregation is also increased, which tends to show that the more the acid-sensitive pegoylated lipid is used, the more of it there is to be degraded before crossing the threshold beyond which aggregation of the nucleolipid particles occurs.",
"It is thus possible, by adjusting the quantity of acid-sensitive colloidal stabilizer, to program the time necessary for the release of the active ingredient at a given pH.",
"Example 11 Transfection in Vivo of DNA Formulated with a Cationic Lipid/Neutral Co-Lipid/Compound D Mixture This example illustrates the impact of a nonionic surfactant such as compound D on the efficiency of DNA transfer formulated with a cationic lipid-based liposome.",
"1) Preparation of the Liposomes The cationic lipid of formula: and the neutral lipid DOPE (obtained from Avanti Polar Lipids, Birmingham, Ala.) where it is dissolved in chloroform and then mixed in equimolar quantities.",
"Appropriate quantities of compound D were dissolved in chloroform and added to the mixture.",
"The quantities of compound D used are given in mol % of the total quantity of lipid.",
"The organic solvent is then evaporated under an argon stream so that a thin lipid film forms at the bottom of the tube.",
"This film is dried under vacuum for at least 1 hour and then rehydrated with a 20 mM HEPES buffer at pH 7.4 and 5% dextrose, at 4° C. for 2 hours.",
"The lipid suspension thus obtained is heated at 50° C. for 30 minutes, and then sonicated for 5 minutes so as to form a homogeneous suspension of liposomes of about 100 nm.",
"The DNA used is plasmid DNA containing the CAT gene under the control of a CMV promoter.",
"The plasmid DNA used possesses the following criteria: endotoxin level of less than 20 U/mg, quantity of supercoiled DNA greater than 90%, contamination with E. coli DNA less than 5%, contamination with RNA less than 5% and contamination with proteins less than 1%.",
"2) Preparation of the DNA/Lipid Complexes Equivalent volumes of liposome suspension (in appropriate concentration) are rapidly added to a DNA solution while mixing rapidly and vigorously.",
"Overall, 1 μg of DNA forms a complex with 5 nmol of cationic lipid.",
"The complexes formed have a diameter of approximately 100 to 240 nm.",
"3) Administration in Vivo About 6 week old female Balb/C mice are used in all the experiments.",
"Each mouse receives a subcutaneous injection with 106 M109 cells in the right flank.",
"When the tumours reach a size of about 300 mm3, the mice receive 200 μl of DNA/lipid complexes containing 50 μg of DNA, by intravenous injection.",
"The tissues are removed 24 hours after injection and stored at −70° C. until used.",
"4) CAT Assay The tissues are homogenized using a “FastPrep Cell Disrupter FP120” apparatus (Bio 101/Savant).",
"The samples are then centrifuged at 1 000 revolutions/minute for 5 minutes.",
"The quantity of CAT transgene expressed is determined using a standard CAT ELISA procedure (Roche, Ind.).",
"5) Results The dose-response curve for compound D on the gene transfer activity of DNA/cationic lipid/DOPE complexes was studied.",
"The compound “Analog D” was used as negative control.",
"As expected, the highest gene transfer occurs in the lungs.",
"It was observed that the use of the compound “Analog D” inhibits the transfection activity in the lungs and the tumors in a dose-dependent manner.",
"By increasing the quantity of compound D, the transfection activity is also inhibited in the lungs.",
"On the other hand, gene transfer does not appear to be affected in the tumors.",
"These results are consistent with the hypothesis that the acidic environment of the tumors leads to a separation of the PEG parts from the rest of the particle after extravasation.",
"The results obtained and presented in FIG.",
"7 thus demonstrate that compound D makes it possible to increase the gene transfer activity specific to the tumors relative to the lungs by a factor of 40.Example 12 Study of the Stability of the Acid-Sensitive Compounds as a Function of the pH This example illustrates the fact that the acid-sensitive compounds according to the present invention have an acid-sensitivity which is modulable according to the nature of the ortho-ester ring present (5- or 6-membered ring).",
"To this effect, the variation of the size of nucleolipid complexes (identical to those used in examples 9 and 10) is measured as a function of the pH and time, for various acid-sensitive pegoylated lipids, which makes it possible to cover different ranges of sensitivity.",
"These studies are carried out at fixed control cationic lipid/DNA and acid-sensitive pegoylated lipid/DNA ratios.",
"1 ml samples of DNA/control cationic lipid/acid-sensitive pegoylated lipid complex are prepared such that: the control cationic lipid/DNA ratio is 1.5 nmol/μg, the acid-sensitive pegoylated lipid/DNA ratio is 0.5 or 1, and the DNA concentration is 20 μg/ml in 75 mM of a sodium chloride solution.",
"After 30 minutes, 500 μl of a buffer solution at 0.05 mol/l and 500 μl of a sodium chloride solution at 150 mM are added to these samples thermostated at 37° C. in a ventilated oven.",
"An acidic pH is thus established and the size of the particles is measured as a function of time.",
"The final concentration of the samples is 10 μg of DNA/ml in 75 mM of a sodium chloride solution.",
"The buffers used are citric acid/sodium citrate buffers at pH 4, pH 5 and pH 6 and a Hepes/sodium hydroxide buffer at pH 7.4.The results obtained are represented in FIG.",
"5 which represents the variation in the size of the nucleolipid particles as a function of the pH and of time for the acid-sensitive compounds C and E prepared in the preceding examples, and which differ only in the nature of the ortho-ester ring used (5- or 6-membered ring).",
"For these two compounds, an increase is observed in the size of the nucleolipid particles as a function of time when the pH is acidic, which reflects their degradation.",
"On the other hand, an increase in the size of the nucleolipid particles as a function of time is not observed when the pH is 7.4.In addition, the lower the pH, the more rapid the aggregation of the nucleolipid particles.",
"Finally, a large difference in kinetics is observed between the two compounds C and E tested: at pH 6, the aggregation of the nucleolipid particles starts about 1 hour after the acidification in the case of the use of compound E, whereas the use of the compound C allows stabilization for at least 4 hours.",
"These results demonstrate several remarkable properties of the acid-sensitive compounds C and E, and more generally of the acid-sensitive compounds of this type according to the present application: they both exhibit high sensitivity at acidic pH values, and in all cases, it is observed that the lower the pH, the more rapid their destabilization, they are both relatively stable at physiological pH (pH of 7.4), and their kinetics of degradation is very different depending on the ortho-ester group used (5- or 6-membered ring)."
]
] | Patent_10129262 |
[
[
"Jaw orthopedic device",
"The invention comprises a jaw orthopedic device for the movement and/or correction of teeth in the tooth arch with a synthetic resin element and/or at least one mature element or tooth contacting or tooth movement, whereby the synthetic resin element is comprised of a hard synthetic resin material and the synthetic resin element (1) has at least one soft plastic element (3) integrated therewith for tooth contacting or tooth movement."
],
[
"1.A jaw orthopedic device for the movement and/or correction of teeth in the tooth arch having a synthetic resin element and/or at least one wire element for tooth contacting or tooth movement, whereby the synthetic resin element is comprised of a hard synthetic resin material, characterized in that the synthetic resin element (1) has at least one soft plastic element (3) integrated therein for tooth contacting or tooth movement.",
"2.The jaw orthopedic device according to claim 1, characterized in that the hard synthetic resin material for the synthetic resin element (1) is comprised of methylmethacrylate or polymethylmethacrylate.",
"3.The jaw orthopedic device according to claim 1 or 2, characterized in that the soft plastic element (3) is comprised of a silicone material, e.g.",
"from vinylpolysiloxane.",
"4.The jaw orthopedic device according to one of claims 1 to 3, characterized in that the soft plastic element (3) matches the contour of at least one tooth (4).",
"5.The jaw orthopedic device according to one of claims 1 to 4, characterized in that the soft plastic element (3) contacts against at least one additional element (5) of the tooth (4) for extrusion, intrusion or rotation of the tooth (4).",
"6.The jaw orthopedic device according to one of claims 1 to 5, characterized in that the soft plastic element (3) contacts flat against at least one tooth (4).",
"7.The jaw orthopedic device according to one of claims 1 to 6, characterized in that the soft plastic element (3) serves for positioning at least one tooth (4).",
"8.The jaw orthopedic device according to one of claims 1 to 7, characterized in that the synthetic resin element (4) serves to guide or anchor the jaw orthopedic device in the tooth arch.",
"9.The jaw orthopedic device according to one of claims 1 to 8, characterized in that at least one spring (6) is provided for tooth contacting.",
"10.The jaw orthopedic device according to one of claims 1 to 9, characterized in that at least one screw (7) is provided for tooth contacting.",
"11.The jaw orthopedic device according to one of claims 9 or 10 characterized in that the spring (6) directly contacts a tooth (4).",
"12.The jaw orthopedic device according to one of claims 9 to 11, characterized in that the spring (6) contacts at least one tooth (4) indirectly via the soft plastic element (3).",
"13.The jaw orthopedic device according to one of claims 10 to 12 characterized in that the screw (7) contacts at least one tooth (4) directly.",
"14.The jaw orthopedic device according to one of claims 10 to 13, characterized in that the screw (7) contacts at least one tooth (4) indirectly via the soft plastic element (3).",
"15.The jaw orthopedic device according to one of claims 1 to 14, characterized in that the synthetic resin element (1) of the jaws orthopedic device is composed of locally different hard synthetic resin materials.",
"16.The jaw orthopedic device according to one of claims 1 to 15, characterized in that a plurality of soft plastic elements (3) of different materials is provided.",
"17.The jaw orthopedic device according to one of claims 1 to 16, characterized in that at least one wire element (2) is incorporated in or on the soft plastic element (3).",
"18.The jaw orthopedic device according to one of claims 1 to 17, characterized in that at least one wire element (2) is incorporated on the one hand on the synthetic resin element (3) and on the other hand on the soft plastic element (3)."
],
[
"The invention relates to a jaw orthopedic device for the movement and/or correction of teeth with the features of the preamble of patent claim 1.As the state of the art, colloquially also known as tooth correction, jaw orthopedic devices are known which have, apart from hard synthetic resin materials and wire elements, springs or screws for tooth contacting.",
"A tooth movement is possible with such springs or screws only in a limited manner.",
"The locations of attack of the springs or screws are effective by limited points and the respective effective forces can be controlled only to a limited degree.",
"In addition, such jaw orthopedic devices with springs or screws, because of their bulky construction, can only be accommodated uncomfortably in the mouth space.",
"The invention has as its object the provision of a jaw orthopedic device for movement or correction of teeth which, for example, affords improved possibilities for effecting tooth movement.",
"The object is achieved, by the features in the characterizing part of patent claim 1 in combination with the features in the preamble.",
"Advantageous embodiments of the invention are described in patent claims 2-18.Via the soft plastic [synthetic resin] element permanently applied and integrated in the synthetic resin element of the jaw orthopedic device according to the invention, new possibilities of tooth contacting and tooth movement are afforded by comparison with the state of the art.",
"By contrast with conventional jaw orthopedic devices with springs or screws, integrated soft plastic elements enable a yieldable contacting of the respective tooth or plurality of teeth, whereby the effective force can be uniformly and thus more pleasantly applied to the teeth.",
"The synthetic resin element can be composed of various hard synthetic resin materials, for example of methylmethacrylate or polymethylmethacrylate which are known per se from prior jaw orthopedic appliances.",
"The soft plastic element can be composed especially of silicone material, for example, of vinylpolysiloxane.",
"In sum, the jaw orthopedic device according to the invention enables regions of the synthetic resin element to be equipped with conventional hard synthetic resin material when a guide and anchoring of the jaw orthopedic device is desired.",
"In those regions of the tooth arch in which individual teeth or groups of teeth are to be moved and newly positioned, soft plastic elements can be used which are integrated in and bonded permanently with the hard plastic material (e.g.",
"cross-linked or adhesively bonded) so that the synthetic resin element is configured as a two-component or multicomponent unit.",
"Because of the use of soft plastic elements, tooth movements can be achieved which are controllable in a targeted manner and a number of variations are enabled like, for example, intrusion, extrusion or rotation.",
"Especially it is possible above all in the front tooth region for the soft plastic element to engage around the teeth and thereby transmit a tensile stress to the teeth.",
"The soft plastic element can also be used in fields in which no tooth movement is to be achieved but rather only an especially comfortable guidance for anchoring of the jaw orthopedic appliance is desired.",
"The soft plastic element can be effective especially for intrusion or extrusion of a tooth on the respective attachment which is to be applied to the particular tooth.",
"As an additional point or as an alternative, a flat contact of the soft element with the tooth can be produced in order to distribute the forces which may arise over large areas of the tooth.",
"In addition or alternatively, the jaw orthopedic device according to the invention can also be provided with conventional springs or screws which are either effective directly on the tooth in the conventional way or bear upon the tooth indirectly in that the springs or the screws initially act upon that plastic element which then conducts the pressure to the tooth or the teeth.",
"According to a further advantageous embodiment, the jaw orthopedic device of the invention can also have local synthetic resin elements of different hard synthetic resin materials.",
"To obtain the desired pressure distribution or pressing forces, for example in different regions of the tooth arch, the soft plastic elements can also be integrated locally and can include different materials or be of different thicknesses and/or different dimensions.",
"According to a further embodiment, at least one wire element can be incorporated in the yieldable jaw orthopedic appliance according to the invention or in a soft plastic element.",
"The wire element can be incorporated in another application also on a synthetic resin element.",
"As a whole, through alternative anchoring points of the wire element on the synthetic resin or soft plastic element, desirable loading forces for the tooth arch can be controllable and metered out with precision.",
"The invention is described in greater detail in conjunction with exemplary embodiments in the drawing Figures.",
"They show: FIG.",
"1 a schematic illustration of a jaw with a jaw orthopedic appliance set in place before the desired tooth movement, FIG.",
"2 an illustration of the jaw with the jaw orthopedic appliance set in place after the desired tooth movement as well as FIG.",
"3 a section A-A according to FIG.",
"1.From FIG.",
"1 a schematic illustration of the jaw (tooth arch) can be seen, with a tooth 4 which is for example dislocated and is to be repositioned in the tooth arch through the use of the jaw orthopedic appliance (for example as a dental brace).",
"The jaw orthopedic appliance is comprised of a removable synthetic resin element 1 with wire elements 2 which engage through and around teeth 4 for guiding and anchoring.",
"In the region of the tooth 4a, a soft plastic element 3a is integrated in the synthetic resin element 1 with a permanent connection and contacts the tooth 4a in order to apply a continuous pressure from the jaw orthopedic appliance to move it into its outwardly shifted position according to FIG.",
"2.FIG.",
"2 shows a jaw orthopedic appliance with a synthetic resin element 1 and a first soft plastic element 3a which bears upon the tooth 4a and a further soft plastic element 3b which bears upon the tooth 4b.",
"Behind the soft plastic element 3b a schematically shown spring 6 or screw 7 is incorporated which is effective against the tooth 4b via the soft plastic element 3b.",
"By means of the intervening action of the soft plastic element 3b, the pressure of the spring 6 or the screw 7 can be applied to the tooth 4b with damping and in a measured manner.",
"According to the sectional illustration of FIG.",
"3, the soft plastic element 3a, which is permanently bonded with the synthetic resin element 1, acts upon the additional element 5 applied to the tooth 4a and upon the latter so that it applies pressure to the latter by way of intrusion in the direction 8.In the case of an extrusion, the soft plastic element 3 can engage beneath the additional element 5.Furthermore, the soft plastic element 3 can also engage around the additional element 5, for example, to effect a rotation (not illustrated).",
"In summary, the jaw orthopedic appliance of the invention can be locally configured and detailed so that for each individual tooth of the tooth arch, a respective engagement can be achieved whether by a region of hard synthetic resin material or an integrated soft plastic element 3 with or without backup stress by means of a spring 6 or a screw 7."
]
] | Patent_10129429 |
[
[
"Device for the dosing of a reducing agent",
"An apparatus for metering a urea or a urea-water solution for delivery to a catalytic converter assembly for removing nitrogen oxides from the exhaust gases of a Diesel engine, includes a housing block supporting function components communicating via a line, formed by recesses in the housing block, for transporting the reducing agent, and the walls of the line are formed by the housing block.",
"This apparatus assures a simple line layout for reducing agent with a minimum number of sealing points that is accordingly appropriate for large-scale mass production."
],
[
"1-14.",
"(Canceled) 15.An apparatus for metering a reducing agent, in particular urea or a urea-water solution, comprising a housing block, means (4, 7, 11), secured to the housing block for delivering the reducing agent to a catalytic converter assembly (30) for removing nitrogen oxides from the exhaust gases of a Diesel engine, said means communicating via a line (12; 80, 81, 82, 83), formed by recesses in the housing block (400), for transporting the reducing agent, and the walls of the line (12) being formed by the housing block.",
"16.The apparatus of claim 15, wherein at least one recess (80) rectilinearly traverses the entire housing block (400).",
"17.The apparatus of claim 15, further comprising a heating element extending generally parallel to at least one recess, said heating element being secured to the housing block or embedded in the housing block.",
"18.The apparatus of claim 16, further comprising a heating element extending generally parallel to at least one recess, said heating element being secured to the housing block or embedded in the housing block.",
"19.The apparatus of claim 15, wherein the recesses are embodied as bores.",
"20.The apparatus of claim 17, wherein the recesses are embodied as bores.",
"21.The apparatus of claim 19, wherein the housing block is injection-molded.",
"22.The apparatus of claim 15, wherein the housing block is of plastic.",
"23.The apparatus of claim 22, wherein the plastic has a low modulus of elasticity in the range from approximately 1000 N/mm2 to approximately 7000 N/mm2.24.The apparatus of claim 15, wherein all the ends of the line (12) communicate with the means (4, 7, 11) and further function components (1, 14, 50, 110), so that no separate closure elements are required.",
"25.The apparatus of claim 21, wherein all the ends of the line (12) communicate with the means (4, 7, 11) and further function components (1, 14, 50, 110), so that no separate closure elements are required.",
"26.The apparatus of claim 15, wherein one end of the line (12) is closed by a compensation element embodied as a spring-loaded flange (110).",
"27.The apparatus of claim 24, wherein one end of the line (12) is closed by a compensation element embodied as a spring-loaded flange (110).",
"28.The apparatus of claim 15, wherein the means or further function components are at least in part secured to the housing and connected to the line (12) with the aid of elastic elements (61, 51) in such a way that the applicable means or function components can execute a compensatory motion if ice forms in the line.",
"29.The apparatus of claim 26, wherein the means or further function components are at least in part secured to the housing and connected to the line (12) with the aid of elastic elements (61, 51) in such a way that the applicable means or function components can execute a compensatory motion if ice forms in the line.",
"30.The apparatus of claim 28, wherein said means includes a pump (4), and wherein the elastic element that secures the pump is embodied as an elastic sheet-metal angle piece (61) secured to the housing block.",
"31.The apparatus of claim 28, wherein the function components include a pressure sensor (50), which is secured resiliently to the housing and closes the line (12) in a tightly displaceable manner.",
"32.The apparatus of claim 15, wherein means include a pressure regulator (11) that has a diaphragm acting to compensate for pressure in the event of ice formation.",
"33.The apparatus of claim 15, wherein at least one volumetrically elastic component, in particular an air-filled element (63), is disposed in the line."
],
[
"PRIOR ART The invention is based on an apparatus for metering a reducing agent, in particular a urea or a urea-water solution, in the context of catalytic posttreatment of exhaust gases, as generically defined by the preamble to the main claim.",
"To achieve a reduction in NOx components in exhaust gases, reduction catalytic converters have been developed, especially for Diesel engines, and are typically classified as either so-called SCR catalytic converters (for Selective Catalytic Reduction) or reservoir-type catalytic converters.",
"The so-called SCR catalytic converters are regenerated by delivering a reducing agent comprising urea and/or ammonia, while the so-called reservoir-type catalytic converters are regenerated in so-called rich exhaust gas phases with hydrocarbons from the entrained internal combustion engine fuel.",
"It is known for the various components of a metering system to be made to communicate via hoses.",
"From German Patent Application 199 46 900.8, an apparatus is known which for removing nitrogen oxides from exhaust gases, for instance from a Diesel engine, meters in urea as a reducing agent.",
"Means intended for this purpose are sometimes secured to a plastic or metal block or integrated with such a block.",
"The metering system described is relatively large and complicated to produce, since it comprises a plurality of components located in line with one another.",
"ADVANTAGES OF THE INVENTION The metering apparatus of the invention, having the characteristics of the body of the main claim, has the advantage over the prior art of a simple, sturdy line layout with a minimum number of sealing points, which can be produced economically and in large-scale mass production.",
"Since there are only a few sealing points, there is less risk of leaks and therefore less risk of failure.",
"Hoses and separate screw fastenings for lines can be omitted.",
"Because of the smaller number of required components and the smaller structural size, the effort and expense of assembly is less, the overall structural volume is decreased, and the production and system costs are thus lowered.",
"The structural unit can be checked for tightness, for instance, after preassembly, which means reduced rejection costs compared to finding defects upon final system checking.",
"The recesses can be disposed in various ways, for instance in the form of bores; additional bores make it possible to expand the basic functions of the metering apparatus by mounting additional components.",
"The provision of recesses in a housing block makes it possible to attach the metering means and other function components to the housing block; the length of the line filled with reducing agent is kept short as a result, so that the liquid can be rapidly thawed again after ice forms.",
"Short line layouts are also fast to fill, and the requisite pressure for operation can be built up quickly.",
"By the provisions recited in the dependent claims, advantageous refinements of and improvements to the metering apparatus defined by the main claim are possible.",
"It is especially advantageous for all the means to be connected to one another via at least one rectilinear supply line that traverses the entire housing block.",
"This line layout is simple to produce and makes possible an adroit arrangement of components that have to be connected to one another.",
"Moreover, it can be embodied in a simple way as an injection-molded bore, for instance in a plastic block that receives the various system components.",
"The open ends of the line can advantageously be closed by function components, so that separate closure elements such as closure screws are unnecessary.",
"A heating element, introduced in particular axially parallel to the recess, such as an electric heating rod embodied as an electrical resistor, can advantageously heat a central line quickly in order to thaw a frozen fluid or to protect the apparatus from freezing.",
"Alternatively, or in combination with the electric heating rod, the housing block can also comprise an electrically conductive material, in particular an electrically conductive plastic, which as described in German Patent Application 199 46 900.8, is provided with electrodes that can be subjected to an electrical voltage, in order to achieve an electric current for heating the entire assembly by way of the housing block.",
"The material comprising the housing block is advantageously selected such that because of a low modulus of elasticity of the material, this material can contribute to volumetric compensation if ice forms in the line.",
"It is also advantageous to integrate compensatory or resilient elements in the assembly, so that a freezing-resistant metering system can be furnished that remains intact after freezing and thawing cycles and that protects integrated components against destruction from ice formation.",
"The individual components themselves need not be embodied as completely freeze-resistant.",
"Moreover, materials that are not resistant to high pressure can also be used, in particular for the housing block, since the buildup of excessive pressure forces in such extreme situations as freezing is averted.",
"DRAWING Exemplary embodiments of the invention are shown in the drawing and explained in further detail in the ensuing description.",
"Shown are FIG.",
"1, the functional layout of a metering apparatus; FIG.",
"2, components of a metering apparatus that are integrated into a housing block provided with recesses; FIG.",
"3, the detail view of a further embodiment of the invention; and FIG.",
"4, a further detail view.",
"DESCRIPTION OF THE EXEMPLARY EMBODIMENTS In FIG.",
"1, reference numeral 1 indicates the inlet to the metering apparatus, by way of which a urea-water solution is supplied to the apparatus.",
"A metering pump 4 aspirates the fluid.",
"The pump 4 is rpm-controlled via a stepping motor 4a.",
"A pressure regulator 11 carries any excess pumped quantity of fluid via the outlet 11a of the pressure regulator either back to the inlet of the metering apparatus or to the metering pump or to a urea tank, not shown in detail, from which the metering pump 4 is supplied via the inlet 1.The line 12 connecting the inlet 1, pump 4 and pressure regulator 11 carries the pumped fluid onward to a metering valve 7.A pressure sensor 50 for measuring the pressure in the line 12 is mounted upstream of the metering valve.",
"The metering valve is electrically triggerable and dispenses the fluid in accordance with the electrical triggering to components connected to the outlet 14.This is for example a mixing chamber, not shown in detail but already described in the aforementioned German application 199 46 900.8, to which compressed air from a compressed air container can be delivered in order to form an aerosol from the urea-water solution, which can then be injected into the inlet region, in particular of a motor vehicle exhaust gas catalytic converter.",
"The metering pump 4 meters the requisite quantity of urea-water solution in accordance with the reducing method employed.",
"A control unit, not shown in detail, acquires data for this purpose pertaining to the engine operating state, which are received from a higher-ranking engine control unit via a CAN data line, along with the signals of various pressure, temperature and fill level sensors, not described in detail here.",
"From the sensor information and the information from the engine control unit, the control unit calculates a urea metering quantity and triggers the metering valve accordingly.",
"In an alternative embodiment, the reducing agent can also be injected by the injection valve 7 directly into the inlet region of the catalytic converter, in other words without reinforcement with compressed air or without having to provide a mixing chamber.",
"FIG.",
"2 is a cross-sectional view through a metering apparatus of the invention, which has a housing block 400, in particular of electrically conductive plastic, with a modulus of elasticity between approximately 1000 N/mm2 and approximately 7000 N/mm2.The housing block has recesses in the form of bores 80, 81, 82 and 83, which form the reducing agent line 12 shown in FIG.",
"1.The bore 80 traverses the entire block.",
"The pump lines 60 of the metering pump 40 are connected via O-rings to the ends of the bores 80 and 82, and the pump is secured to the surface of the housing block via an elastic sheet-metal angle piece 61.A pressure regulator 11, acting to compensate for pressure if ice forms and having a diaphragm not shown in detail, is flanged to the surface of the housing block, and two O-rings seal off the head of the pressure regulator that protrudes into the housing block.",
"Analogously, a metering valve 7 is secured to the housing block.",
"On the end of the housing block opposite the metering pump 4, the bore 80 merges with a region of larger cross section, where a pressure sensor 50 is accommodated.",
"The pressure sensor is secured to the surface of the housing block via a flexible, elastic flange 51.Once again, O-rings assure sealing of the bore that can be filled with a fluid.",
"Via the bore 81, the metering valve 7 communicates with the flanged-on outlet 140 of the metering apparatus.",
"The inlet 1 flanged on next to it communicates with the bores 83 and 82 and serves to supply the reducing agent from a reservoir to the metering pump 4.In the bore 80, between the pressure regulator 11 and the metering valve 7, there is an air-filled elastic hose 63, which is secured to the bore wall, for instance by means of an adhesive.",
"The electric pump motor, which is also secured to the housing block 400, is disposed above the metering pump.",
"A control unit, not shown in detail, is connected electrically, in a manner not shown in detail, to both the metering valve and the pressure sensor and also to other sensors, not shown in detail, such as a fill level sensor for the urea tank, and from the engine control unit this control unit receives data on the operating state of the engine whose exhaust gases are to be chemically reduced with the aid of the metering apparatus in the exhaust system.",
"The housing block 400 serves to receive and secure various means for supply reducing agent and further function components, such as the pressure sensor 50, metering valve 7, pressure regulator 11 and metering pump 4.The rectilinear bore 80 traverses the housing block from one end to the other and can be designed in a way appropriate for manufacture, for instance conically or stepped in the case of a plastic block, or cylindrically for the sake of metal-cutting machining in the case of a metal housing block.",
"In addition, further bores 81 through 83 are provided, some of which extend parallel and others perpendicular to the through bore 80 and assure the attachment of the assembly to a reducing agent reservoir and to the catalytic converter as well as assuring pressure compensation via the pressure regulator 11.The pressure regulator 11 and the metering valve 7 protrude with their line connections, not visible in the drawings, into the bore 80, so that they each communicate with the bore; simultaneously, they close off the bore from the outside.",
"The assembly has a plurality of structural characteristics for compensating for volumetric fluctuations resulting from freezing or melting of the reducing agent during cold weather.",
"Because the metering pump 4 is secured to the housing block 400 by means of the elastic sheet-metal angle piece 61, a compensation capability in the event of severe pressure fluctuations caused by a phase transition is assured because the pump lines 60 together with the metering pump 4 all move relative to the bores 80 and 82, and thus the volume in the line system that carries the reducing agent can adapt automatically when otherwise the housing block could burst, or such components as the metering valve or pressure sensor could become damaged.",
"O-ring seals continue to keep the line tightly closed.",
"The pressure sensor 50, secured axially resiliently to the housing block via the flexible elastic flange 51, is likewise pressed outward by a volumetric expansion in the event of ice formation.",
"If the ice melts again, the pressure sensor and the metering pump move reversibly back to their outset position.",
"The pressure regulator 11, which is known per se and is commercially available, has a built-in elastic diaphragm, which is relieved to the ambient air.",
"This diaphragm can yield elastically if ice forms and can thus also help to compensate for the increase in volume if ice forms.",
"Moreover, because of its low modulus of elasticity, the housing block can to a certain extent absorb the ice pressure by expanding.",
"In addition, the air-filled elastic hose 63 serves to reduce the circumferential tension in the bore wall, because upon freezing of a urea-water solution, for instance, it is compressed and thus can absorb some of the line pressure building up at the time.",
"In an alternative embodiment, still other bores may be provided, which connect the components for feeding and metering compressed air to one another, so that if a compressed air-supported development of an aerosol is intended for injection into a catalytic converter assembly, once again a compact, integrated assembly can be furnished.",
"In that case, instead of the metering valve 7, a metering valve together with a mixing chamber is secured to the housing block, into which chamber the metering valve can be metered and which chamber can be subjected to compressed air.",
"In this case, the outlet 140 forms the outlet of the mixing chamber.",
"The bores both for the reducing agent lines and for the supply of compressed air can also be made by injection molding, especially when plastic is used.",
"Instead of an air-filled elastic hose 63, other volumetrically elastic elements can be used, such as gas-filled plastic microballoons; hollow, gas-filled fibers closed on the ends; or gas-filled hoses, wound spirally in a manner similar to a compression spring.",
"FIG.",
"3, in cross section, shows a detail of an alternative metering apparatus, in which a bore discharging into a cavern 100 is provided.",
"This bore is for instance a bore that is in communication with the bore 80 via a corner connection.",
"The cavern is embodied as a bore that is conical toward the outside and is closed with a flange 110; O-ring seals assure sealing even if the position of the flange changes.",
"The flange is in fact supported axially movably via compression springs 111 and screws 112.In the event of freezing, the fluid in the line, that is, in the bores and thus also in the bore discharging into the cavern 100, expands and presses against the housing block.",
"To a limited extent, the bore walls yield, as already noted above.",
"Any further increase in the pressure could cause the housing to burst.",
"The support of the flange 110 embodied as a resilient element is now dimensioned such that in good time before any risk of bursting, it yields to the ice pressure by moving axially and thus limits the pressure in the bore in the event of ice formation.",
"The conical embodiment of the bore here results in an amplified axial direction of action of the ice pressure and carries this pressure to the resilient flange.",
"Given suitable dimensioning of the components, this process can be repeated cyclically as often as desired.",
"The cavern 100 can also be embodied as a cylinder, or as a cylinder with a multiply graduated inside diameter, or in other words can be formed by the peripheral region of the bore itself or by a cylindrical hollow chamber with a different diameter, in particular a larger diameter, than the bore.",
"The cavern can also have any feasible geometry.",
"FIG.",
"4 illustrates an alternative, axially movable securing of the pressure sensor 50.A compression spring 120 presses it against a bore step 131 of the sensor bore 130.The compression spring 120 is braced here on a threaded ring 121 that is secured to the housing block 400.An O-ring seal 140 in a radial indentation in the pressure sensor assures sealing off of the fluid volume formed by the bore 80 and the sensor bore 130.If the urea-water solution freezes, then as a consequence of a volumetric expansion the pressure in the bore 80 and in the widened-diameter sensor bore 130 increases, until the spring force of the compression spring 120 is reached.",
"Then the pressure sensor 50 is displaced counter to the spring force and increases the volume in the sensor bore 130.By this variant installation, both the pressure sensor 50 and the block 400 are protected against excessively high ice pressure loads."
]
] | Patent_10130697 |
[
[
"Starting device",
"A starter device for internal combustion engines is proposed that comprises a drive mechanism (16) and a gearing (22) having a variable gear ratio, which said gearing is situated after the drive mechanism (16).",
"The starter device (10) is characterized by the fact that the gear ratio of the gearing (22) is infinitely variable."
],
[
"1.A starter device for internal combustion engines comprising a drive mechanism (16) and a gearing (22) having a variable gear ratio, which said gearing is situated after the drive (16), wherein the gear ratio is infinitely variable.",
"2.The starter device according to claim 1, wherein the gearing (22) is self-regulating.",
"3.The starter device according to claim 1, wherein the gearing (22) is capable of being regulated according to a input torque of the drive mechanism (16).",
"4.The starter device according to claim 1, wherein the gearing (22) is capable of being regulated according to a drive speed of the drive mechanism (16).",
"5.The starter device according to claim 1, wherein the gearing (22) is a planetary-gear set with a center gear (34) having at least one planet gear (46) and a internal gear (49), and wherein a radial actuating force acting on the at least one planet gear (46) can be achieved by means of the input torque.",
"6.The starter device according to claim 1, wherein the gearing (22) is a planetary-gear set with a center gear (34) having at least one planet gear (46) and a internal gear (49), and wherein a radial actuating force acting on the at least one planet gear (46) can be achieved by means of the drive speed."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention concerns a starter device for internal combustion engines according to the general class in the independent claim.",
"A starter device is made known in DE 199 27 905 A1, which said starter device comprises a gearing between a drive element and a pinion, which said gearing has a variable gear ratio.",
"The gearing is a planetary-gear set, the sun gear of which is capable of being driven by the drive element.",
"The output of the planetary-gear set takes place via the planet gears and, therefore, via the planetary carrier.",
"The planetary-gear set makes two different gear ratios possible: in the case of the first gear ratio at low speeds, gear reduction takes place via the internal ring gear, which is held stationary by means of an overrunning clutch.",
"When the drive mechanism reaches a certain speed, a plurality of centrifugal clutch elements attached to the planetary carrier cause the internal ring gear to be held stationary with the planetary carrier; this causes the planetary-gear set to be shifted from gear reduction to a one-to-one gear ratio.",
"The disadvantage of this embodiment is the fact that the assistance provided to run the internal combustion engine up to speed is adjusted optimally at only two operating points."
],
[
"<SOH> SUMMARY OF THE DRAWINGS <EOH>Exemplary embodiments of a starter device according to the invention are shown in the drawings.",
"FIG.",
"1 shows a schematic view of a starter device according to the invention.",
"FIG.",
"2 shows a spacial sectional detail view of the torque-controlled gearing, FIG.",
"3 shows a spacial sectional detail view of the speed-controlled gearing, FIG.",
"4 shows a spacial view of the coupled planet gears.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"BACKGROUND OF THE INVENTION The invention concerns a starter device for internal combustion engines according to the general class in the independent claim.",
"A starter device is made known in DE 199 27 905 A1, which said starter device comprises a gearing between a drive element and a pinion, which said gearing has a variable gear ratio.",
"The gearing is a planetary-gear set, the sun gear of which is capable of being driven by the drive element.",
"The output of the planetary-gear set takes place via the planet gears and, therefore, via the planetary carrier.",
"The planetary-gear set makes two different gear ratios possible: in the case of the first gear ratio at low speeds, gear reduction takes place via the internal ring gear, which is held stationary by means of an overrunning clutch.",
"When the drive mechanism reaches a certain speed, a plurality of centrifugal clutch elements attached to the planetary carrier cause the internal ring gear to be held stationary with the planetary carrier; this causes the planetary-gear set to be shifted from gear reduction to a one-to-one gear ratio.",
"The disadvantage of this embodiment is the fact that the assistance provided to run the internal combustion engine up to speed is adjusted optimally at only two operating points.",
"ADVANTAGES OF THE INVENTION The starter device according to the invention having the feature of the main claim has the advantage that the starter device can be adjusted as favorably as possible for the operating states of the internal combustion engine during start-up by means of the variable gear ratio of the gearing.",
"As a result, the assistance provided by the starter device to run the internal combustion engine up to speed is optimal.",
"The continuously-variable design of the gearing leads to reduced wear and reduced noise in the gearing, because no force or load peaks occur here when the gear ratio is changed.",
"Advantageous further developments of the starter device according to the main claim are possible due to the measures listed in the dependent claims.",
"The expense required to realize the variable gear ratio in the case of the starter device is particularly low when the gearing is self-regulating.",
"Sensors, adjusting devices, and expensive control technology can be eliminated.",
"SUMMARY OF THE DRAWINGS Exemplary embodiments of a starter device according to the invention are shown in the drawings.",
"FIG.",
"1 shows a schematic view of a starter device according to the invention.",
"FIG.",
"2 shows a spacial sectional detail view of the torque-controlled gearing, FIG.",
"3 shows a spacial sectional detail view of the speed-controlled gearing, FIG.",
"4 shows a spacial view of the coupled planet gears.",
"DESCRIPTION OF THE PREFERRED EMBODIMENTS A starter device 10 is shown FIG.",
"1, which said starter device accommodates a plurality of assemblies in a housing 13.The assemblies include the drive mechanism 16 that drives an output shaft 25 via an input shaft 19, and a gearing 22.A pinion-engaging drive mechanism 28 is supported on the output shaft 25 in rotating-slideable fashion.",
"The pinion-engaging drive mechanism 28 is capable of being engaged with a not shown ring gear of an internal combustion engine by means of a solenoid switch 31.A first exemplary embodiment of the gearing 22 is shown in FIG.",
"2.This first exemplary embodiment is a gearing having a variable gear ratio, and the gear ratio is continuously-variable.",
"The gearing 22 is designed as a planetary-gear set.",
"A center gear 34 having a double-component design is driven via the input shaft 19.The center gear 34 is composed of a first side gear 37 and a second side gear 40.The first side gear 37 and the second side gear 40 each comprise side surfaces 43 that face each other, between which said side surfaces at least one planet gear 46 can be held tightly.",
"A further gear element is the internal gear 49, which also has a double-component design.",
"The internal gear 49 is composed of a first side internal gear 52 and a second side internal gear 55.Both the first and second side internal gears 52 and 55 have side surfaces 53 that face each other.",
"One flange element is situated on each side of the planet gears 46, and an output flange 58 is integrally connected to the output shaft 25 on the output end.",
"An annular input flange 61 is arranged on the input end.",
"The output flange 58 and the input flange 61 are interconnected in torsion-resistant fashion.",
"This torsion-resistant connection is obtained by arranging opposing bores 64 in the output flange 58 and the input flange 61.Bolts 67 are inserted through said bores for connecting purposes.",
"The output flange 58 and the input flange 61 are interconnected in torsion-resistant and axially non-displaceable fashion.",
"The input flange 61 as well as the output flange 58 comprise radially outwardly extending grooves 70 on their circumferences.",
"These grooves 70 allow the axles 73—on which the planet gears 46 are securely arranged—to slide radially outwardly or radially inwardly in them.",
"The input shaft 19 comprises a shaft section 76 in which a helical spline 77 is machined.",
"The helical spline 77 is limited on both ends by two circlips.",
"The first circlip acts as a stop for the first side gear, and the other circlip acts as a stop for the second side gear.",
"The first side gear 37 comprises a center bore in which a helical spline 77 is machined as well.",
"The first side gear 37 further comprises a cylindrical outer section 80 in which straight teeth are machined.",
"The outer section 80 lies radially within the planet gears 46.The second side gear 40 is placed on the outer section 80 having the straight teeth 81, which said side gear comprises an internal straight toothing that matches the straight teeth 81 and meshes with them.",
"In a first variant, the second side gear 40 comprises an axially extending, cylindrical section 84 facing the drive mechanism, which said section can be displaceably supported with its exterior in a bore 86 of the input flange 61.In a second variant, the cylindrical section 84 can extend only into the input flange 61—due to spacial considerations—without transferring bearing forces here.",
"The motion of the second side gear is hindered by means of the other circlip via a plate washer arranged at the end of the cylindrical section 84.The internal gear 49 encompasses—with the first side internal gear 52 and the second side internal gear 55—the at least one planet gear 46.The second side internal gear 55 is thereby supported in moveable fashion within the first side internal gear 52.The second side internal gear thereby bears against an internal retainer 95 by means of a spring element 93.The first side internal gear 52 is supported in displaceable fashion within the housing 13.The at least one planet gear 46 has a biconical design overall, and the planet gear 46 comprises an outwardly directed conical surface 47 on each of its rotational-axial ends.",
"The function of the starter device 10—particularly the gearing 22—will be explained hereinbelow.",
"The starting position of the gearing 22 is as follows: The drive mechanism 16 does not yet apply input torque, so the planet gears 46 assume their radially innermost positions.",
"This is due to the spring element 93, which presses on the second side internal gear 55, by way of which the first side internal gear 52 comes to bear against the planet gears 46.Due to the conical surfaces 47 and the forces acting on the conical surfaces 47 by the side internal gears 52 and 55, this leads to a radially inwardly directed force on the planet gears 46; the planet gears 46 assume their radially innermost positions.",
"After the starter device 10 is started, the input shaft 19 begins to rotate anticlockwise.",
"Due to the helical spline 77, the first side gear 37 is drawn in the direction toward the drive mechanism 16.The first side gear 37 thereby presses with its side surface 43 against the conical surface 47 of the planet gear 46 and moves it in the direction toward the side gear 40 until said side gear comes to bear against one of the side surfaces 43 there.",
"Since the sliding motion on the helical spline 77 has now stopped, input torque from the drive mechanism 16 is transferred to the center gear 34, which said center gear now drives the planet gears 46.The planet gears 46 thereby roll with their conical surfaces 47 on the side surfaces 43 of the first and second side internal gears 52 and 55 as well.",
"The side surfaces do not rotate therewith; instead, they are axially displaceable.",
"The planet gears 46 thereby drive the output flange 58 and the input flange 61 via the axles 73 and the grooves 70, so that output torque is transferred to the pinion-engaging drive mechanism 28 via the output shaft 25.Due to the very high initial input torque in the case of drive mechanisms 16 designed as electric motors, this leads to high clamping forces between the first and second side gears 37 and 40.These clamping forces ultimately lead to the forces exceeding the spring forces of the spring element 93, so that the first and second side internal gears 52 and 55 are forced apart.",
"The planet gears 46 walk radially outwardly.",
"This leads to a situation in which, based on the axles 73 and [numeral missing?",
"], the planet gears 46 cover an ever-increasing rolling circle, which finally reaches a maximum.",
"The reverse applies for the pitch lines between the first and second side internal gears 52 and 55 and the planet gear 46.Here, the radius, i.e., the distance between the center line of the axles 73 and the pitch lines, becomes smaller and smaller.",
"The significance of this for the speed ratios that occur between the input shaft 19 and the output shaft 25 is that, when the planet gears 46 initially lie radially inwardly, the output shaft 25 rotates rapidly compared to the input shaft 19.When the planet gears 46 move further outwardly, the kinematic relationships then change in such a fashion that the speed of the output shaft 25 decreases compared to the initial situation.",
"Assuming a constant drive power of the drive mechanism 16, the significance of this for torque output by the output shaft 25 is that the torque at the output shaft 25 increases as the planet gears 46 wander further outwardly.",
"This is favorable in terms of starting internal combustion engines, because said internal combustion engines require a particularly high amount of torque at the beginning of the starting procedure.",
"Once the internal combustion engine has broken away, the torque demand of the internal combustion engine drops continuously.",
"As a result, the drive mechanism 16 need not deliver as much input torque via the input shaft 19 to the gearing 22, either.",
"Consequently, this leads to a reduced force in the helical spline 77 and, therefore, to lesser forces between side faces 43 of the first side gear and the second side gear 37 and 40.The force of the spring element 93 now becomes greater than the force acting on the spring element 93 from the helical spline 77, so that the side faces 43 of the first side internal gear 52 and the second side internal gear 55 press on the conical surfaces 47 of the planet gears 46 with greater force than the force between the side surfaces 43 of the first and second side gears 37 and 40.As a result, the planet gears 46 move radially inwardly along the grooves 70 once more, so that, on the one hand, the torque at the output shaft 25 decreases and, on the other hand, the speed of the output shaft 25 increases.",
"The second exemplary embodiment of the gearing 22 is shown in FIG.",
"3.In contrast to the gearing 22 shown in FIG.",
"2, this exemplary embodiment is regulated according to the drive speed of the drive mechanism 16.The input shaft 19 has a positive contour via the shaft section on which the center gear 34 is arranged, which said contour meshes with a matching positive counter-contour of the center gear 34.These two positive contours make an axial displacement of the center gear 34 on the input shaft 19 possible.",
"The center gear 34 also comprises a first side gear 37 with the side surface 43.The side surface 43 of the first side gear 37 cooperates with a second side surface 43 that is part of a surface of a center ring 98.The center ring 98 is held on the center gear 35 by the fact that said center gear 34 bears against a retainer 104 secured to the first side gear 37 via a spring element 101.The side surfaces 43 of the center ring 98 and the first side gear 37 encompass a plurality of planet gears 46 which are encompassed by the side surfaces 43 of an internal gear 49 on their radial exterior, as is the case with the first exemplary embodiment.",
"The internal gear 49 is designed analogously to the first exemplary embodiment.",
"A plurality of bores 64 is machined in the output flange 58, in which bores a plurality of bolts 67 is secured.",
"The bolts 67 carry a planetary gear carrier 107 which is arranged on the side of the center gear 34 facing the drive mechanism 16.The planetary gear carrier 107 carries a multiple-component swivel arm 109 on the bolts 67, refer to FIG.",
"4 as well.",
"The swivel arm 109 is arranged between the output flange 58 and the planetary gear carrier 107.The swivel arm comprises two individual arms 111, each of which comprises three individual openings along their length.",
"The openings are aligned with each other in each case.",
"The center openings serve to support the planetary gear carrier 107 on the bolt 67.Two lower openings accommodate an axle 113, to which a counterweight 114 is secured.",
"Two upper openings accommodate the axle 73, on which the planet gear 46 is situated.",
"The planet gear 46 is held between the two individual arms 111.The function of the second exemplary embodiment will be explained using FIGS.",
"3 and 4.If the input shaft 19 of the drive mechanism 16 is driven, the center gear 34 is rotated simultaneously.",
"The planet gears 46 are driven via the side surfaces 43 of the first side gear and the center gear 98 via frictional forces.",
"The planet gears 46 thereby roll on the side surfaces 43 of the internal gear 49 in an already-known fashion.",
"If the speed of the output shaft 25 is relatively low at first, the side surfaces of the center gear 34 initially force the planet gears 46 radially outwardly with assistance from the spring element 101.As a result, the spring element 93 of the internal gear 49 is loaded.",
"The output torque at the output shaft 25 is therefore relatively high initially and serves to break away or start the internal combustion engine.",
"If the speed of the output shaft 25 then increases, this means an increase in the angular speed of the planetary gear carrier 107 and, therefore, an increase in the angular speed of the counterweights 114 as well.",
"Due to the kinematic relationships at the swivel arm 109, the increasing centrifugal force of the counterweights 114 leads to an increased axial force in the center gear 34, so that the side surfaces 43 of the center gear 34 are forced apart.",
"The kinematic relationships in the gearing 22 therefore change as well, so that planet gear 46 therefore moves radially inwardly once more, and the speed of the output shaft 25 increases over-proportionally to the speed of the input shaft 19."
]
] | Patent_10130806 |
[
[
"Force -transmision unit comprising speed -dependent hydraulic clutch and centrifugal force compensation",
"A power transmission unit with a hydraulic coupling dependent on a rotational-speed difference, in which, when a rotational-speed difference occurs between the input member (1) and the output member (6), a hydrostatic displacement machine (20) produces in a pressure space (34) a pressure that acts on a piston (27) acting on a friction clutch (31), has a housing (4).",
"To compensate for the centrifugal force acting on the working fluid in the pressure space (34), at least one centrifugal-force element (11, 12, 13) is provided in the housing, exerting on the piston (27) a force counter to the pressure produced by the centrifugal force in the pressure chamber (34)."
],
[
"1.A power transmission unit with an input member and an output member and a hydraulic coupling dependent on a rotational-speed difference, in which, when a rotational-speed difference occurs between the input member (1; 6) and the output member (6; 1), a hydrostatic displacement machine (20) produces in a pressure space (34) a pressure that acts on a piston (27) acting on a friction clutch (31), the friction clutch having first and second disks connected in terms of drive to the input member and the output member respectively, and one of the members (1; 6) forming a housing (4) that contains the displacement machine, wherein at least one centrifugal-force element (11, 12, 13; 41, 44; 50, 55) is provided in the housing, exerting on the piston (27) a force counter to the pressure produced by the centrifugal force in the pressure chamber (34).",
"2.The power transmission unit as claimed in claim 1, wherein the at least one centrifugal-force element is a flyweight (11).",
"3.The power transmission unit as claimed in claim 2, wherein the centrifugal-force element is a two-armed lever (12), one leg of which forms the flyweight (11) and the other lever of which forms a pressure finger (13).",
"4.The power transmission unit as claimed in claim 1, wherein the centrifugal-force element is an annular space (44; 50) that contains an operating fluid and rotates with the housing (4).",
"5.The power transmission unit as claimed in claim 4, wherein the rotating annular space (44) is formed by a cylindrical sleeve (41) surrounding the housing (4) and having a wall (42) in the form of a circular ring normal to the axis and by a wall (43), normal to the axis, of the housing (4), and wherein the sleeve (41) is connected to the piston (27) and can be displaced in an axial direction.",
"6.The power transmission unit as claimed in claim 4, wherein the radially outermost zone of the rotating annular space (50) is connected via a passage (54) to a compensation pressure space (57) on the opposite side of the piston (27) from the pressure space (34).",
"7.The power transmission unit as claimed in claim 6, wherein the compensation pressure space (57) is formed by an annular cylinder (55) in the housing (4) and by an annular continuation (56) on the opposite side of the piston (27) from the pressure space (34)."
],
[
"The invention relates to a power transmission unit with an input member and an output member and a hydraulic coupling dependent on a rotational-speed difference, in which, when a rotational-speed difference occurs between the input member and the output member, a hydrostatic displacement machine produces in a pressure space a pressure that acts on a piston acting on a friction clutch, the friction clutch having first and second disks connected in terms of drive to the input member and the output member respectively, and one of the members forming a housing that contains the displacement machine.",
"Power transmission units of this kind are used especially in drive trains of motor vehicles, preferably all-wheel-drive vehicles; either together with a differential, the hydraulic coupling limiting the differential action, or to drive the second driven axle, the torque transmitted depending on the difference between the wheel speed and the drive shaft connected to the wheels of the other axle.",
"The pressure produced by the displacement machine acts on a clutch, preferably a multi-plate clutch.",
"This action can be influenced by means of various valves, whether these are automatically acting valves or valves actuated by an external control system.",
"U.S. Pat.",
"No.",
"5,536,215 has disclosed a power transmission unit of this kind, as has Austrian Utility Model 2964.In these and all such power transmission units, the pressure space in which the pressure acting on the piston is built up is in the rotating housing.",
"As a result, the operating fluid contained in this housing is subject to a centrifugal force, which increases and thus distorts the pressure prevailing in the pressure chamber as a function of the rotational speed.",
"This is particularly disruptive if the pressure is dependent on a rotational-speed difference and is supplied by a hydrostatic displacement machine, and this applies in both possible cases: if, in the first case—that of an unregulated coupling—there are no control valves, compensation is impossible; and if, in the second case, control valves intended to depressurize the pressure space for disengagement are provided, this is not possible at higher absolute rotational speeds because the discharge line adjoining the control valve has to end in a smaller radius.",
"However, the pressure there is always less than in the pressure chamber, owing to centrifugal force.",
"It is therefore the object of the invention to eliminate these disadvantages corresponding to the special features of couplings of the generic type.",
"The influence of centrifugal force should be at least partially compensated for to a necessary extent.",
"According to the invention, at least one centrifugal-force element is provided for this purpose in the housing, exerting on the piston a force that is the square of the rotational speed and acts counter to the pressure acting on the piston.",
"By virtue of the fact that it is likewise situated in the housing, compensation to a specifiable extent is possible in all rotational-speed ranges without any outlay on regulation systems, given appropriate design.",
"It is thereby possible to establish a speed dependence of the transmitted torque corresponding to the requirements as regards driving dynamics.",
"The extent of compensation ranges from partial compensation and full compensation to overcompensation.",
"In this arrangement, the transmitted torque falls as the speed increases, giving better traction at low speed and improved interaction with electronic brake systems (e.g.",
"ABS) at high speed.",
"In an advantageous design, the at least one centrifugal-force element is a flyweight (claim 1).",
"Compensation of centrifugal force is thus performed in a purely mechanical way, and, in a preferred embodiment, the centrifugal-force element is part of a two-armed lever, one leg of which is the flyweight and the other lever of which is a pressure finger (claim 3).",
"The levers, of which there are three for example, are very simple and can be accommodated in the housing with only slight design changes.",
"This is the simplest solution and can even be retrofitted to existing couplings.",
"The other design comprises the centrifugal-force element being an annular space that contains an operating fluid and rotates with the housing (claim 4).",
"This is a hydraulic method of compensating for centrifugal force.",
"Since there is sufficient operating fluid in and around the coupling, there is no problem with supplying it.",
"In a first advantageous embodiment of this other design, the rotating annular space is formed by a cylindrical sleeve surrounding the housing and having a wall in the form of a circular ring normal to the axis and by a wall, normal to the axis, of the housing, and the sleeve is connected to the piston and can be displaced in an axial direction (claim 5).",
"In this way, the annular space is bounded on one side by a displaceable wall and on the other side by a nondisplaceable wall of the housing.",
"The liquid level in the annular space is determined by the inner radius of the wall in the form of a circular ring normal to the axis.",
"The centrifugal force acting on the working medium in the annular space pushes apart the walls normal to the axis.",
"This compensating force is transmitted to the piston by the displaceable sleeve.",
"In a second advantageous embodiment of this other design, the radially outermost zone of the rotating annular space is connected via a passage to a compensation pressure space on the opposite side of the piston from the pressure space (claim 6).",
"The annular space and the passage can also be provided within the housing.",
"It is even possible, by means of valves associated with the passage, to achieve special effects in terms of driving dynamics.",
"A particularly elegant solution is for the compensation pressure space to be formed by an annular cylinder in the housing and by an annular continuation on the opposite side of the piston from the pressure space (claim 7).",
"The invention is described and explained below with reference to figures, in which: FIG.",
"1 shows a longitudinal section through a device according to the invention in a first embodiment, FIG.",
"2 shows a longitudinal section through a device according to the invention in a second embodiment, and FIG.",
"3 shows a longitudinal section through a device according to the invention in a third embodiment.",
"In FIG.",
"1, the input member is denoted by 1 but it could also be the output member, to which a shaft 2, indicated in broken lines, is flanged by means of bolts, which are merely indicated.",
"It comprises a front plate 3, an essentially cylindrical housing 4, which is connected integrally or in a fixed manner to the front plate 3, and an end plate 5, which is connected releasably to the housing 4 for the purpose of assembly, though in a leaktight way.",
"The output member 6 (it could also be the input member) is a hollow shaft, into which a shaft that is merely indicated is introduced by means of splines; it is supported in bearings 7 in the front plate 3 and the end plate 5 of the input member 1 and can be sealed off relative to the latter by means of seals 8.Simple sealing rings are sufficient because the rotational-speed difference is very small on average.",
"9 denotes the axis of rotation or center line.",
"Within the housing 4, there is a hydrostatic displacement machine 20, which comprises an inner part 21 and an outer part 22.The first of these is connected in a rotationally fixed manner to the output member 6, while the second is connected to the input member 1 and, more specifically, to the housing 4.The corresponding coupling teeth are merely indicated.",
"Extending between the inner part 21 and the outer part 22 is a working space 23, which is supplied via an intake passage 24 in a manner that is not shown.",
"Adjoining the hydrostatic displacement machine 20 on the other side is an insert 25, which contains a pressure passage 26 and a piston 27, which is acted upon by the pressurized fluid supplied via the pressure passage 26 and, with the insert 25, delimits a pressure space 34.Some of this pressurized fluid can be directed into the space, which contains a clutch 31, via a throttle valve 28 by a piston 27, a number of inner plates 29 and outer plates 30 being arranged in said space.",
"The first of these are connected to the output member 6 in a way that prevents relative rotation but allows translation, while the second are connected in the same way to the housing 4 of the input member 1.For the purpose of mounting a device for compensating the force exerted on the piston 27 by the centrifugal force in the pressure chamber 34, the housing 4 here has a plurality of apertures 10, which are distributed around the circumference and through which two-armed angled levers 12 reach.",
"One leg of such a lever is constructed as a flyweight 11, while the other is constructed as a pressure finger 13, which engages in a recess 14 on the opposite side of the piston 27 from the pressure space 34.Instead of a pivot passing through the two-armed lever 12, a bearing edge 15, on which a bearing shoulder 16 on the rear side of the pressure finger 13 is supported, is provided here on the aperture 10 in the housing 4.This ensures that the lever 12 does not fly off.",
"A projection 18, which is held by an end stop 17 when the outermost permitted position of the flyweight 11 is reached, can be provided on the outermost end of the flyweight 11.FIG.",
"2 shows a different design.",
"Here too, the housing 4 has a plurality of apertures 10 distributed around the circumference, through which radial pins 40 inserted into the piston 27 extend outward and are connected to a cylindrical sleeve 41 surrounding the housing 4 all the way round.",
"They can transmit a force in the axial direction between the sleeve 41 and the piston 27.The cylindrical sleeve 41 extends toward the left in the figure, projects beyond the housing 4 and ends in a wall 42 in the form of a circular ring normal to the axis.",
"An annular space 44 is thus formed between this wall and a wall 43, normal to the axis, of the housing 4.This annular space is sealed off by means of a sealing ring 45 between the housing 4 and the sleeve 41 and contains working fluid to a level determined by the inside diameter of the wall 42.When the housing 4 is rotated, this liquid surface 46 becomes a cylindrical surface.",
"During rotation, the centrifugal force in this annular space 44 gives rise to a pressure that pulls the wall 43 of the sleeve 41 to the left in the exemplary embodiment illustrated and thus, in turn, exerts on the piston 27, via the pins 40, a force that compensates for the centrifugal force in the pressure space 34.The design and position of the annular space 44 can also be modified.",
"The essential point is that an axial force counter to the force acting on the piston 27 in the pressure space 34 arises.",
"According to the variant in FIG.",
"3, the connection between the annular space and the piston can also be established hydraulically.",
"For this purpose, an annular space 50 is again provided, on the opposite side of the piston 27 from the pressure space 34 and within the housing 4 in the exemplary embodiment shown.",
"The annular space 50 is kept filled from the interior of the clutch space via a feed hole 51, a drain hole 52 ensuring that a constant (cylindrical) liquid surface 53 is maintained.",
"The pressure produced by the centrifugal force in the annular space 50 acts via an axial passage 54 (or a plurality of such passages) on an annular cylinder 55.This is likewise formed in the housing 4 and accepts an annular continuation 56 of the piston 27 in a sealing manner.",
"With the annular cylinder 55, it forms a compensation pressure space 57.There, the pressure acts on the surface 58 in the form of a circular ring and thus compensates for the action of the pressure prevailing in the pressure chamber 34.It is possible to modify many details of the exemplary embodiments illustrated while remaining within the scope of the invention.",
"Thus the construction of the hydrostatic displacement machine can vary very widely, both as regards the shape of its rotors and as regards their arrangement in the housing 4.Finally, the power transmission unit can be arranged at various points within the drive train, in particular ahead of or after the axle differential in the power flow.",
"It can also be arranged within a housing containing the axle differential."
]
] | Patent_10149078 |
[
[
"Encasing arrangement for a semicoductor component",
"A semiconductor component package configuration includes a semiconductor chip mounted to a printed circuit board, and a substrate arranged between the semiconductor chip and the printed circuit board.",
"The substrate is for routing the wiring terminals of the semiconductor chip to the printed circuit board.",
"The substrate is connected to the printed circuit board by solder joints.",
"A filler between the semiconductor chip and the substrate mechanically isolates the semiconductor chip and the solder joints.",
"A metal layer, which is connected to solder joints, is applied to the substrate.",
"At least one molded element of heat-dissipating material is applied to the metal layer and is connected in a heat-conducting manner to the metal layer.",
"This provides the package configuration with an improved capability of conducting the lost power that is dissipated from the installed semiconductor chip, and the desired mechanical properties of the package arrangement are retained."
],
[
"1-13.Cancel 14.A semiconductor component package configuration, comprising: a semiconductor chip having wiring terminals; a printed circuit board having said semiconductor component mounted thereon; a substrate configured between said semiconductor chip and said printed circuit board, said substrate for routing said wiring terminals of said semiconductor chip to said printed circuit board; solder joints connecting said substrate to said printed circuit board; a filler configured between said semiconductor chip and said substrate, said filler for mechanically isolating said semiconductor chip and said soldered joints; a metal layer applied to said substrate, said metal layer being connected to at least one of said solder joints; and at least one molded element of heat-dissipating material applied to said metal layer, said molded element connected in a heat-conducting manner to said metal layer, said molded element not directly contacting said semiconductor chip.",
"15.The configuration according to claim 14, wherein: said molded element is configured between said substrate and said semiconductor chip and protrudes into said filler; and said molded element is at a distance of about 10-20 μm from said semiconductor chip.",
"16.The configuration according to claim 15, wherein: said molded element is formed as a cylinder.",
"17.The configuration according to claim 14, wherein: said molded element is formed from metal.",
"18.The configuration according to claim 17, wherein: said molded element has been electrodeposited onto said metal layer.",
"19.The configuration according to claim 17, wherein: said molded element has been applied to said metal layer by a mask etching process.",
"20.The configuration according to claim 14, wherein: said substrate has a side facing said semiconductor chip; said semiconductor chip has a side; and said molded element is configured on said side of said substrate facing said semiconductor chip and to said side of said semiconductor chip.",
"21.The configuration according to claim 20, wherein: said molded element is formed as an electrically conductive, metallic frame surrounding said semiconductor chip; and said molded element is connected to ground potential.",
"22.The configuration according to claim 20, comprising: a heat-conducting adhesive connecting said molded element to said metal layer.",
"23.The configuration according to claim 20, wherein: said molded element is bonded to said metal layer.",
"24.The configuration according to claim 20, comprising: an additional, electrically non-conducting, thermally conductive connection configured between said semiconductor chip and said molded element."
],
[
"The present invention relates to a semiconductor component package configuration which includes a printed circuit board, a semiconductor chip and a substrate lying in between for routing the wiring terminals of the semiconductor chip to the printed circuit board.",
"The substrate is bonded to the printed circuit board by solder joints and the solder joints are mechanically isolated from the semiconductor chip by a filler.",
"Integrated semiconductor circuits are used in various applications.",
"The semiconductor chip is generally housed in a package and mounted on a printed circuit board.",
"For example, a semiconductor component can be arranged in a package arrangement according to what is known as an FBGA package arrangement (FBGA: Fine Pitch Ball Grid Array), which is based on what is known as the beam-lead bonding technique.",
"This type of package arrangement is characterized by a special package design with respect to the arrangement of the solder balls.",
"A package arrangement such as the FBGA package arrangement, also referred to as an FBGA package, usually includes a semiconductor chip with terminals for electrically connecting to terminals of the printed circuit board and a substrate that acts as a kind of wiring plane.",
"The substrate is in this case arranged between the semiconductor chip and the printed circuit board.",
"The substrate, as a wiring plane, has electrically conducting connections to the terminals of the semiconductor chip.",
"These interconnects of the substrate are connected in turn to the printed circuit board by solder joints.",
"Since the semiconductor chip and the solder joints or solder balls generally have different coefficients of expansion under changing temperatures, it is necessary for the semiconductor chip and the solder balls to be mechanically isolated from one another.",
"For this purpose, a filler, also referred to as an encapsulate or cushioning material, is provided, for example, between the semiconductor chip and the substrate.",
"During the operation of an integrated semiconductor circuit there is generally a certain loss of power, which, to avoid potential damage, for example, from overheating or accelerated aging, must be dissipated.",
"To eliminate the possibility of overheating a semiconductor component, it is provided with heat sinks or heat spreaders.",
"Heat sinks are usually cooled externally and consequently assume a constant temperature.",
"Heat spreaders are used, for example, to create a larger surface area, which better dissipates the heat to the outside by convection.",
"They are formed, for example, by metal plates that are fastened on the package or introduced directly into the package.",
"U.S. Pat.",
"No.",
"5,814,894 shows a semiconductor component in which blind leads, which are connected to the chip and lead to contact bumps, are provided for heat dissipation.",
"However, only little heat can be dissipated in this way, and a heat sink is additionally provided.",
"Problems also arise because of the different coefficients of expansion, since the arrangement is formed without any filling material.",
"The main heat path in the case of the described type of package arrangements for dissipating the lost power is the path from the semiconductor chip via the filling material and the solder balls to the printed circuit board.",
"The relatively poor thermal conductivity of the filling material or the cushioning material in this case limits the heat resistance of the package.",
"With limited thermal conductivity of the package, the lost power that can be dissipated is limited, as a result of which the performance of an integrated semiconductor circuit may likewise be limited.",
"A semiconductor component with such a filling material is presented, for example, in U.S. Pat.",
"No.",
"5,843,810, where the space between the semiconductor chip and the wiring elements is filled with a buffer material that is in contact, via a seal, with an outer ring serving as reinforcement.",
"The reference does not concern itself with the problem of how the lost heat of the chip will be dissipated.",
"An improvement in the thermal conductivity of the main heat path is possible by using thermally conductive filling material or cushioning material.",
"However, there are limits to this method, since with the thermally conductive filler, the elastic properties and the desired processing properties of the filling material are generally lost.",
"This has the consequence of reduced mechanical isolation of the semiconductor chip and the solder balls.",
"The object of the present invention is to specify an arrangement of a semiconductor component in the described package arrangement which has improved conductivity of the lost power that will be dissipated from an installed semiconductor chip and with which the mechanical properties of the package arrangement are retained.",
"The object of the invention is obtained by providing a semiconductor component package configuration including: a semiconductor chip with wiring terminals; a printed circuit board, on which the semiconductor component is mounted; a substrate, which is arranged between the semiconductor chip and the printed circuit board, for routing the wiring terminals of the semiconductor chip to the printed circuit board; in which the substrate is connected to the printed circuit board by solder joints; in which a filler is arranged between the semiconductor chip and the substrate for mechanically isolating the semiconductor chip and the solder joints; in which there is applied to the substrate, a metal layer, which is connected to at least one of the solder joints; and in which at least one molded element of heat-dissipating material is applied to the metal layer; in which the molded element is connected in a heat-conducting manner to the metal layer; and in which the molded element of heat-dissipating material is not in direct contact with the semiconductor chip.",
"The arrangement has a semiconductor chip with wiring terminals, a printed circuit board, onto which the semiconductor component is mounted, and a substrate that is arranged between the semiconductor chip and the printed circuit board.",
"The substrate serves for routing the wiring terminals of the semiconductor chip to the printed circuit board and is connected to the printed circuit board by solder joints.",
"Arranged between the semiconductor chip and the substrate is a filler, which serves for mechanically isolating the semiconductor chip and the solder joints.",
"Also applied on the substrate, in addition to the interconnects to the terminals of the semiconductor chip, is a metal layer that is connected to at least one of the solder joints.",
"Furthermore, at least one molded element of heat-dissipating material is applied to the metal layer and is connected to it in a heat-conducting manner.",
"The provision of the heat-conducting molded element improves the capability of conducting the lost power of the package arrangement to be dissipated.",
"The previously used filling material with the desired properties may be used as the filler.",
"The molded element serves, for example, for reducing at certain points, the distance between the substrate, also referred to as the interposer, and the semiconductor chip, and consequently for bridging the relatively poor thermal conductivity of the filling material.",
"Similarly, a further possible heat path can be created by an appropriate locational arrangement of the molded element, contributing to the removal of the prodced lost power.",
"The transported heat is transferred to the metal layer of the substrate and conducted from it via the solder joints into the metal areas of the printed circuit board.",
"Accordingly, the heat-dissipating molded element performs the function of a heat sink with respect to the semiconductor chip.",
"So that the mechanical properties of the package arrangement are not influenced by the molded element, the molded element is not in direct contact with the semiconductor chip.",
"The described structure of the package arrangement is used, in particular, in the case of FBGA package arrangements.",
"To shorten the distance between the substrate and the semiconductor chip, the molded element is arranged in a suitable way such that it protrudes into the filler.",
"The molded element is in this case preferably designed as a cylinder.",
"In the interests of high thermal conductivity, it is favorable for a metal layer to be applied to the substrate in the form of large interconnected metal areas.",
"Similarly, the thermal conductivity and its distribution are enhanced by applying a plurality of relatively small cylinders to the metal layer.",
"Since metal has good thermal conductivity, the molded elements are preferably formed from metal.",
"The described structure of the metal layer and the molded elements can be produced, for example, by a mask etching process or by electrodepositing the molded elements.",
"The molded elements or cylinders are embedded into the filling material before mounting the semiconductor chip.",
"In the mounted state, the molded elements or cylinders protrude as far as the chip surface, so that an improved heat path is produced from the semiconductor chip through the molded elements into the metal layer.",
"From the metal layer, the heat is dissipated through solder joints or solder balls that are not electrically connected and that are referred to as no-connects, and by thermal vias in the printed circuit board into metal tracks of the printed circuit board.",
"These thermal vias are, for example, metal-filled holes in the printed circuit board (board), which can be produced by known processes, for example by electrodeposition.",
"If the molded elements are arranged on the substrate in an appropriate number and with appropriate area coverage, a significant improvement in the heat resistance is achieved, while at the same time retaining the elastic and isolating properties of the filling material.",
"A further refinement of the invention provides a molded element applied to the metal layer and arranged on a side of the substrate facing the semiconductor chip and to the side of the semiconductor chip.",
"This opens a further heat path to the side of the semiconductor chip, which altogether increases the thermal conductivity of the FBGA package arrangement.",
"The mechanical stability of the described package arrangement can be increased by what is known as a support ring, i.e.",
"a frame which surrounds the semiconductor chip.",
"An application of this type is expedient, for example, for package arrangements in which the array of solder joints or the interposer extends out over the area of the chip (“fan out”).",
"The arrangement of this frame as specified by the invention consequently provides it with the function of a heat sink in addition to the mechanically stabilizing function.",
"If, in addition, the frame is electrically conductive and connected to ground potential, additional electrical improvements are obtained.",
"The frame, which has, with respect to the housing, the form of a ring antenna, produces a general shielding of all of the electrical paths of the metal layer.",
"In addition, the inductance of the ground terminals connected to the frame is decreased, which reduces, in particular, the noise caused by rapid changes in current (“delta I noise”).",
"At the same time, the capacitance of the ground terminals is increased, which leads to better radio frequency decoupling of the voltage supply system.",
"The inductance of further electrical connections, for example, of data lines or address lines, is likewise lowered by the additional ground reference that the frame represents.",
"In a development of the invention, the frame is applied directly to the metal layer and is fastened by a heat-conducting adhesive.",
"If the metal layer is located on a side of the substrate lying opposite from the frame, the frame is applied to the metal layer and connected to it in a heat-conducting manner through a clearance in the substrate by using a conducting adhesive layer.",
"This connection may also be performed by bonding.",
"To improve the heat dissipation via the frame, in a development of the invention, an additional, electrically non-conducting, thermally conductive connection is arranged between the semiconductor chip and the molded element.",
"This thermally conductive connection may be established, for example, by a heat-conducting paste.",
"Moreover, further improved heat dissipation from the semiconductor chip can be achieved by connecting wiring terminals of the semiconductor chip, which have no electrical function, to the metal layer of the interposer.",
"The invention is explained in more detail below on the basis of the figures represented in the drawing.",
"FIGS.",
"1 and 2 are cross sectional views of an embodiment of the invention; FIG.",
"3 is a plan view of an embodiment of the invention; FIG.",
"4 is a three-dimensional representation of an embodiment of the invention; FIG.",
"5 is a sectional view of an embodiment of a package arrangement; FIGS.",
"6 and 7 show details from FIG.",
"5; and FIG.",
"8 is a plan view of the embodiment of the invention shown in FIG.",
"5.FIG.",
"1 shows a cross section of an embodiment of the invention constructed as an FBGA package arrangement, before mounting the semiconductor chip.",
"It shows the substrate 3 (interposer), to which a metal layer 5 is applied.",
"The metal layer 5 has a molded element 7 of heat-dissipating material, which is designed in FIG.",
"1 as a cylinder.",
"Shown alongside the cylinder are further identical cylinders that are, for example, formed from metal, and that are connected in a heat-conducting manner to the metal layer.",
"Applied above the metal layer 5 is the filler 4 (cushioning material).",
"FIG.",
"2 shows a cross section of the structure shown in FIG.",
"1 after mounting the chip and solder balls and after being soldered onto the printed circuit board 8.The substrate 3 is arranged between the semiconductor chip 2 and the printed circuit board 8 and serves for routing wiring terminals (known as bonding pads) of the semiconductor chip 2 to the printed circuit board 8 by using the interconnect 13.The substrate 3 is connected to the printed circuit board 8 by solder joints 6.The filler 4 is arranged between the semiconductor chip 2 and the substrate 3 and serves for mechanically isolating the semiconductor chip 2 and the solder joints 6.The printed circuit board 8 is made up of the PCB 81 itself and a copper layer 82 of a larger surface area (power plane of the PCB).",
"After the semiconductor chip 2 has been laminated on, a comparatively small distance 10 is obtained between the semiconductor chip 2 and the molded elements 7.This distance is determined essentially by the technical aspects of the process and is, for example, 10-20 μm.",
"The filler 4 usually consists of a polyimide-based material.",
"The substrate 3 consists, for example, of a supporting material FR-4.The lost power generated in the semiconductor chip 2 is dissipated via the metal layer 5 and the solder joints 6 by way of the represented thermal via 9 into the printed circuit board 8, represented by the heat-conducting path 20.Since, over the molded element 7, the distance between the metal layer 5 and the semiconductor chip 2 is reduced, the thermal conductivity along the heat-conducting path 20, and consequently of the semiconductor component 1 as a whole, is increased.",
"FIGS.",
"3 and 4 respectively show a plan view and a three-dimensional representation of the embodiment of the invention shown in FIGS.",
"1 and 2.The wiring terminals 11 of the semiconductor chip 2 are connected to the solder joints or solder balls 6 by the terminals 61 for the solder joints.",
"The terminals 62 designate terminals for“thermal”, i.e.",
"electrically inactive, solder joints 6.The metal layer 5 is arranged in the form of large interconnected metal areas.",
"The molded elements 7 are applied to the metal layer 5 in a correspondingly dense coverage of the surface.",
"FIG.",
"5 shows a sectional representation of further package arrangement.",
"According to a further embodiment of the invention, the molded element 7 is arranged on the side of the substrate 5 facing the semiconductor chip 2 and to the side of the semiconductor chip 2.The molded element 7 is designed in the form of a frame which surrounds the semiconductor chip 2.The frame also contributes to the mechanical stabilization of the package.",
"The sectional representation shown in FIG.",
"5 is respectively represented as a detail in FIGS.",
"6 and 7.The molded element 7 is applied to the metal layer 5 for example by a heat-conducting adhesive.",
"In this exemplary embodiment, a plurality of nubbins 14 are arranged between the semiconductor chip 2 and the substrate 3.The nubbins 14 consist, for example, of an organic material containing silicone rubber.",
"The molded element 7 or the frame is expediently formed from metal.",
"To improve the thermal conductivity between the semiconductor chip 2 and the molded element 7, an additional thermally conductive connection 15 is arranged between the semiconductor chip 2 and the molded element 7, for example, in the form of a heat-conducting paste, as represented in FIG.",
"7.The adhesive layer, not represented in FIG.",
"6 or FIG.",
"7, may also be replaced by a bonding.",
"The metal layer 5 is formed here, as in the exemplary embodiments previously described, for example, from copper.",
"FIG.",
"8 shows a plan view of the embodiment of the invention shown in FIG.",
"5 with interconnects 13 and the wiring terminals 11.The metal layer 5 is arranged in the form of interconnected metal areas.",
"For the connection of the molded element or the frame to ground potential, the metal layer 5 is correspondingly connected by solder balls (not represented in the Fig.)",
"to ground terminals (known as ground pins)."
]
] | Patent_10149892 |
[
[
"Termination device e.g. for a multiconnector",
"A termination device e.g.",
"for a multiconnector comprises a cover (2) which is rotatably hinged to a housing (1), and which has a hole for receiving a cable containing a plurality of wires (5a-e).",
"When the individual wires are arranged in respective slits in the cover, the housing and the cover may be rorated mutually to a closed position, so that the wires are gradually inserted into respective terminals for providing an electrical connection.",
"This reduces the required closing force, and the cover may therefore be closed by hand without the use of a special tool."
],
[
"1.A termination device, e.g.",
"for a multiconnector and comprising a plurality of protruding terminals and a cover which, upon closing of the device, is adapted to guide and force a wire associated with each terminal down into the terminal to provide an electrical connection between the wire and the terminal, and wherein the cover is hinged to the device such that the wires may be positioned correctly in an open position, and such that the wires are in place in the respective terminals in a closed position, characterized in that a first number of terminals distributed in directions crosswise of the hinge axis is greater than a second number of terminals distributed in direction parallel to said axis and that the distance from the hinge axis to the terminals varies substantially evenly distributed between a smallest distance and a greatest distance.",
"2.A device according to claim 1, characterized in that all terminals are mutually differently spaced from the hinge axis.",
"3.A device according to claims 1-2, characterized in that the terminals are elastically movable transversely to the hinge axis.",
"4.A device according to claims 1-3, characterized in that the slit between the terminals for receiving a wire is arc-shaped and substantially concentric with the hinge axis.",
"5.A device according to claims 1-4, characterized in that the cover has grooves for receiving an associated wire and for guiding the wire when the cover is closed.",
"6.A device according to claim 5, characterized in that the grooves have a width corresponding to the thickness of the wires.",
"7.A device according to claim 5, characterized in that the grooves close to the hinge axis are wider than the grooves further away from the hinge axis, measured transversely to the hinge axis.",
"8.A device according to claims 5-7, characterized in that the grooves are open out toward a pair of opposed sides of the cover.",
"9.A device according to claims 5 8, characterized in that the grooves are positioned at the bottom of the cover, and that the cover has a through hole from top to bottom for receiving a cable which contains said wires.",
"10.A device according to claims 5-9, characterized in that at least one of the sides of the cover has a hole for receiving a cable which contains said wires."
],
[
"The invention relates to a termination device e.g.",
"for a multiconnector and comprising a plurality of protruding terminals and a cover which, upon closing of the device, is adapted to guide and force a wire associated with each terminal down into the terminal to provide an electrical connection between the wire and the terminal.",
"A multiconnector having a closure device of the above-mentioned type is known e.g.",
"from European Patent Application No.",
"907 226.With this and other known techniques, the wires must first be arranged in a cover which is a loose part relative to the part containing the terminals, so that it is difficult to guide the cover and the terminals relative to each other during closing.",
"The operation is additionally impeded in that it is normally necessary to use a special tool, because the known covers are adapted such that all terminals are affected simultaneously for insertion of a wire.",
"The object of the invention is to provide a termination device which may be closed more easily and without the use of tools relative to the prior art.",
"The invention is not restricted to multiconnectors, but will be explained below in relation to multiconnectors.",
"This object is achieved in that the cover is hinged to the multiconnector such that the wires may be positioned correctly in an open position, and such that the wires are in place in the respective terminals in a closed position, and that the distance from the hinge axis to the terminals varies substantially evenly distributed between a smallest distance and a greatest distance.",
"When the terminals are differently spaced from the hinge axis, the various terminals will gradually cooperate with the respective wires as the cover is closed around the hinge axis.",
"Thus, it is not so that all terminals have to be expanded at the same time, and it is therefore easier to force the wires down into the terminals in turn.",
"The hinge also means that the parts are better positioned relative to each other during the closing movement.",
"An optimum distribution of the applied forces during the closing movement is achieved in that all terminals are differently spaced from the hinge axis.",
"Since terminals and wires will describe a circular arc movement around the hinge axis when the cover is closed, it is expedient that the terminals are elastic transversely to the hinge axis.",
"Another embodiment may be that the slit between the terminals for receiving a wire is arc-shaped and positioned concentrically around the hinge axis.",
"Before the multiconnector is closed, the wires are arranged in position in the cover, which has grooves for receiving an associated wire.",
"The grooves may have a width which corresponds to the thickness of the wire so that the wires are fixed in the groove, but compensation for the above-mentioned arc-shaped movement may also be achieved in that the grooves close to the hinge axis are wider than the grooves which are positioned further away from the hinge axis.",
"Preferably, the grooves are open toward a pair of opposed sides of the cover so that the wire ends can protrude freely substantially in parallel with the hinge axis, which makes it very easy to cut off the wires so that they are flush with said sides before the cover is closed.",
"In a preferred embodiment, a through hole is provided from the top of the cover to the bottom of the cover, and as the grooves are preferably positioned at the bottom of the cover, a cable may be inserted from the top, following which the wires of the cable may be arranged in the grooves at the bottom of the cover.",
"Alternatively, another of the sides of the cover may be provided with a hole for receiving a cable.",
"The invention will be explained more fully by the following description of some embodiments with reference to the drawing, in which FIGS.",
"1 and 2 show a first embodiment of the termination device according to the invention, FIGS.",
"3 and 4 show the termination device shown in FIGS.",
"1 and 2, but with an inserted cable containing a plurality of wires, while FIGS.",
"5-7 show a second embodiment of the termination device according to the invention.",
"FIG.",
"1 shows a housing 1 and a cover 2 which is rotatably mounted on the housing 1 about an axis of rotation 3.The housing and the cover may form a multiconnector, so that the bottom of the housing might be adapted to receive a counterpart of a multiconnector, but the housing 1 might also have downwardly extending solder pins for mounting on a printed circuit board.",
"It is also conceivable that the bottom of the housing 1 is provided with a cover corresponding to the cover 2 with associated terminals.",
"The embodiments will be described below under the designation multiconnector.",
"FIG.",
"2 shows the parts shown in FIG.",
"1 in a closed position, while FIGS.",
"3 and 4 show the same as FIGS.",
"1 and 2, but with an inserted cable 4.In the embodiment shown, the cable 4 may contain up to eight wires which are shown at 5a-h.",
"The cable is inserted into an opening 6 in the cover, as will be seen best in FIG.",
"2, and the outer jacket is removed so that the individual wires protrude freely.",
"Then, the wires may be arranged in respective slits 7a-h.",
"The open end of the slits facing upwards in FIG.",
"1 is bevelled so that it is easy to introduce the wires 5a-h into the terminals.",
"The terminals are also positioned such that it is easy to shorten the wires with cutting pliers, as is indicated in FIG.",
"4.FIG.",
"5 shows another housing 11, and the associated cover 12 can be seen in FIGS.",
"6 and 7.In FIG.",
"5, the terminals are designated 8a-e, and the distances a-e drawn in the figure show the distances of the respective terminals from the axis of rotation 3.According to the invention, the terminals are differently spaced from the axis of rotation 3, and in a preferred embodiment all distances are mutually different.",
"This means that the closing force may be reduced relative to the prior art where all wires are forced down into the respective slits at the same time.",
"The invention also covers the embodiment that the terminals are just arranged at two mutually different distances from the axis of rotation 3.It will be appreciated that the terminals and/or the individual wires 5a-e will describe a circular arc movement around the axis of rotation 3 when the cover is closed, and one would therefore believe that it might cause problems to prevent in particular the innermost wires from getting squeezed during the closing movement.",
"This is prevented in several ways.",
"For one thing, it will be seen that the uppermost opening of the terminals is V-shaped so that the respective wire is moved down into the terminal during the closing movement, but according to the invention the terminals 8a-e may also be resilient transversely to the axis of rotation 3.Even though it is preferred that the slits 7a-e are so narrow that they can fix the wires temporarily during the closing movement, it is also conceivable that in particular the innermost slits 7a and 7e are wider than the outermost ones 7d and 7h.",
"Another solution is that the slits of the terminals might be arc-shaped and positioned substantially concentrically relative to the axis of rotation 3.A further option is indicated in FIG.",
"5.The protective rails, which are shown at 13 in FIG.",
"1, are removed in FIG.",
"5 to show that, at the top, the terminals have a wire-receiving part and below this a wide slit, which means that the terminals are elastic in a direction transversely to the axis of rotation 3.However, it has been found in practice that there is no appreciable need for compensation for the circular arc movement of the wires.",
"When the device according to the invention is intended for the mounting of copper wires in tinned terminals, the invention also utilizes the circumstance that a certain flow of said metals takes place to ensure a good electrical connection.",
"The cover 12 shown in FIGS.",
"6 and 7 also has a through hole 6 for a cable, but, as will appear from FIG.",
"7, a side of the cover may also comprise a hole 9 for receiving a cable.",
"In the embodiment shown in FIG.",
"7, the lowermost part of the wall, in which the hole 9 is provided, serves as a locking boss 10 which locks the cover 2 to the housing 1 in the position shown in FIG.",
"2.It will be appreciated that the cover may be hinged and formed in other ways so that the cable may be inserted and relieved in any direction."
]
] | Patent_10168605 |
[
[
"Electrical circuit board and a multiconnector",
"An electrical circuit board and multiconnector having protruding contact pins, which together form contact areas for providing contact with a complementary connector, are arranged such that the contact pins (6-13) extend out from one of the sides of the circuit board (1) substantially in a direction K which corresponds to the direction of coupling of a multiconnector and a complementary connector."
],
[
"1.An electrical circuit board having protruding contact pins which together form the contact area in a male connector or in a corresponding female connector, so that an electrical multiconnector connection may be established for data transmission by moving the male and female connectors together in respective directions of coupling, and wherein the contact pins are arranged such that cross-talk between the signals being transferred via the multiconnector connection is reduced, characterized in that the contact pins extend out from the circuit board substantially transversely to it and to the contact area in directions which are substantially indicated by the respective direction of coupling.",
"2.A circuit board according to claim 1, characterized in that the contact pins extend from the surface of the circuit board in two or more planes transversely to said surface from which the contact pins extend toward the contact area.",
"3.A circuit board according to claim 1 or 2, characterized in that the contact pins extend a distance past the contact area where they form an acute angle with the respective direction of coupling.",
"4.A multiconnector comprising a housing and contact pins, whose one end extends through the bottom of the housing for mounting on an electrical circuit board, and wherein the contact pins are arranged such that cross-talk between the signals being transferred via the multiconnector is reduced, and wherein the housing has an opening for receiving a complementary multiconnector to provide electrical connections via coupling areas on the contact pins, characterized in that the contact pins of the multiconnector extend from the bottom of the housing via said contact areas toward the opening of the housing.",
"5.A multiconnector according to claim 4, characterized in that the housing is box-shaped, and that the bottom and the opening of the housing are positioned on opposite sides of the housing.",
"6.A multiconnector according to claim 4, wherein the housing is box-shaped, characterized in that the bottom and the opening of the housing are positioned on adjacent sides of the housing."
],
[
"The invention relates to an electrical circuit board having protruding contact pins which together form the contact area in a male connector or in a corresponding female connector, so that an electrical multiconnector connection may be established for data transmission by moving the male and female connectors together in respective directions of coupling, and wherein the contact pins are arranged such that cross-talk between the signals being transferred via the multiconnector connection is reduced.",
"The invention also relates to a multiconnector comprising a housing and contact pins whose one end extends through the bottom of the housing for mounting on an electrical circuit board, and wherein the contact pins are arranged such that cross-talk between the signals being transferred via the multiconnector is reduced, and wherein the housing has an opening for receiving a complementary multiconnector to provide electrical connections via coupling areas on the contact pins.",
"It is a common feature of such circuit boards or multiconnectors that they comprise a plurality of protruding contact pins whose location is of great importance to the cross-talk that may occur between the electrical signals which are to be transferred via the contact pins.",
"Reference may be made e.g.",
"to the prior art disclosed in British Patent Application 2 329 530.It is thus also known to provide a form of electrical compensation of the cross-talk by means of various techniques, but nevertheless the aim must be that the interfering cross-talk is as small as possible.",
"The object of the invention is to provide a solution where the cross-talk is smaller than has been achieved so far by the prior art.",
"This object is achieved in that the contact pins extend out from the circuit board substantially transversely to it and to the contact area in directions which are substantially indicated by the respective direction of coupling.",
"Hereby, the distance which the signals have to travel along the contact pins is as short as possible in contrast e.g.",
"to the technique where the contact pins first extend a distance in the direction of coupling and then turn and extend rearwards from the turning point.",
"Also in connection with a multiconnector according to the invention, the shortest possible signal distance is achieved in that the contact pins of the multiconnector extend from the bottom of the multiconnector via said contact areas toward the opening of the multiconnector.",
"In the preferred embodiment, the contact pins extend from the surface of the circuit board in two or more planes transversely to said surface from which the contact pins extend toward the contact area.",
"This ensures not only the shortest signal distance, but also that the effective mutual spacing of the individual contact pins is as great as possible for further reduction of the cross-talk.",
"When the invention is performed in connection with a multiconnector, it may comprise a housing which is box-shaped, and it will be appreciated that the contact pins may extend out from any one of the sides of the housing, and that any other of the sides of the housing may have an opening for receiving a complementary multiconnector.",
"The invention will be explained more fully by the following description of some embodiments with reference to the drawing, in which FIG.",
"1 shows an embodiment of a circuit board according to the invention, FIG.",
"2 shows the same as FIG.",
"1, but in a scale which corresponds to FIGS.",
"3-5, where FIG.",
"3 shows the circuit board of FIG.",
"2 arranged in a holder for support of the contact pins, FIG.",
"4 shows a housing for the circuit board of FIGS.",
"2 and 3, FIG.",
"5 shows the housing of FIG.",
"4 and the circuit board of FIG.",
"3 in an assembled position, while FIG.",
"6 shows an embodiment of a multiconnector according to the invention.",
"FIG.",
"1 shows a circuit board 1 having eight terminals, of which only the four terminals 2-5 can be seen.",
"The terminals are connected to respective contact pins 6-13 preferably via circuit components which are adapted to counteract the cross-talk that occurs between the signals when they propagate between a contact area 0 and the respective terminals.",
"FIG.",
"3 shows the circuit board of FIGS.",
"1 and 2 inserted into a support 14 for the contact pins 6-13.The last-mentioned parts are configured to be pushed into a housing 15, which is shown in FIG.",
"4, whereby the multiconnector will look as shown in FIG.",
"5.Then, wires may be passed down into the terminals by means of a cover or in another manner, and the bottom of the housing 15 may have an opening for receiving a complementary connector which may provide an electrical connection along the contact area 0, which is shown in FIG.",
"1.FIG.",
"5 moreover shows an arrow K which indicates the direction in which the housing 5 is to be moved toward a complementary connector to provide the above-mentioned electrical connection.",
"The direction K is also drawn in FIG.",
"1, and it will thus be seen that the contact pins 6-13 extend out from one of the sides of the circuit 1 in directions which are substantially indicated by K. According to the invention, the contact pins extend substantially in this direction from the circuit board to the contact area 0.This provides the shortest possible distance which the signals travel along the contact pins.",
"It moreover appears from FIG.",
"1 that the contact pins 6, 8, 10, 12 extend from a level on the circuit board 1 which is higher than the level from which the contact pins 7, 9, 11 and 13 extend.",
"Moreover, the contact pins 6-13 are bent so that they extend substantially directly from the respective level to the contact area 0.The cross-talk between the contact pins is hereby reduced additionally.",
"Finally, it will be seen in FIG.",
"1 that the outer ends of the contact pins 6-13 form an acute angle with the direction of coupling K, which is expedient for the joining of the circuit board according to the invention and a complementary connector.",
"FIG.",
"6 shows an embodiment of a multiconnector 15 according to the invention and a complementary connector 16.The multiconnector 15 comprises a housing 17 with an opening 18 for receiving the complementary connector 16, which has a locking hook 19 for securing the connector 16 in the housing 7.The connector 16 has a plurality of contact areas located at 20, which are adapted to establish an electrical connection with respective contact pins in the housing 7, the last-mentioned contact pins being configured according to the invention, as will appear from the explanation above.",
"The multiconnector 15 is adapted to be secured to an electrical circuit board 23, as the contact pins, of which the pins 21 and 22 can be seen in FIG.",
"6, extend out through one of the sides of the housing 7 so that they may be secured to a circuit board.",
"It will be seen in FIG.",
"6 that the contact pins 21 and 22 extend out from the solder area or the circuit board on which the housing 7 is secured, in the same manner as is explained in connection with FIG.",
"1.It will thus be seen clearly how the contact pin 22 extends from a higher level than the contact pin 21, when the contact pins extend obliquely upwards toward the contact area which is shown by the arrow 0.The configuration according to the invention means that the signals are to propagate the shortest possible distance along the contact pins, i.e.",
"the shortest possible distance from the contact area 20 to the solder ends 21 and 22 is achieved when the connectors 15 and 16 are interconnected.",
"It will be appreciated that the opening 18 must not necessarily be the opposite end of the housing 7 relative to the side where the contact pins 21 and 22 extend outside the housing."
]
] | Patent_10168606 |
[
[
"System for reproducing three-dimensional images",
"The present invention describes a system that is able to reproduce images in three dimensions, that is, stereoscopic, three-dimensional or integral, without the need for glasses or any other device in front of observer's eyes.",
"The number of images necessary is small because the system is able to direct the images to the eyes of each observer.",
"In the case of stereoscopic reproductions, this number is only two and in the case of three-dimensional or integral reproductions it is ten or several tens.",
"Its fundamental characteristics are: it has a great focus field depth of the images which allows these to be directed to the observers' eyes wherever the latter are situated, it uses a single discriminating element of these images for each observer, it is able to reproduce bi-dimensional images both by transparency and by diffusion and it does not use movable elements."
],
[
"1-31.",
"(canceled) 32.A system for reproducing images in three dimensions, capable of reproducing stereoscopic, three-dimensional or integral images for at least one observer comprising: a retro-reflective screen for reflecting in a first direction and an opposite, second direction a beam of light rays; a bi-dimensional image-reproducer; a discriminating element for discriminating a plurality of bi-dimensional images from among each other; a first electronic device for multiplexing said plurality of bi-dimensional images, said first electronic device for emitting a synchronising signal, said synchronising signal for identifying said plurality of bi-dimensional images being reproduced at each moment; a monitor for sensing a position of an observer's head and providing a head position signal representative of said observer's head; and a second electronic device for receiving said synchronising signal and said head position signal and for controlling said discriminating element in response to said received signals.",
"33.A system for reproducing images in three dimensions in accordance with claim 32, wherein said retro-reflective screen comprises a plurality of reflecting elements, each of said plurality of reflecting elements having at least three flat mirrors, at least two of said at least three flat mirrors being disposed perpendicular with respect to one another.",
"34.A system for reproducing images in three dimensions in accordance with claim 33, wherein said discriminating element is a semi-transparent sheet.",
"35.A system for reproducing images in three dimensions in accordance with claim 33, wherein said discriminating element is at least one sheet of bi-refringent material and a de-polarising sheet, said at least one sheet of bi-refringent material and said de-polarising sheet both being disposed in parallel relation with respect to each other, and a plurality of retro-reflective screens.",
"36.A system for reproducing images in three dimensions in accordance with claim 32, wherein said retro-reflective screen and said discriminating element comprises two equal sections, each of said two equal sections being formed on a flat translucent sheet, said flat transparent sheet having a first side and a second side, said flat translucent sheet having a plurality of convergent micro-lenses on said first side, said second side being flat and being connected to said first side, wherein said flat translucent sheet is situated in a focal plane.",
"37.A system for reproducing images in three dimensions in accordance with claim 36, in which at least one section of convergent micro-lenses comprises: two lens sections having convex sides and respective axes being perpendicular to each other, said convex sides facing each other and contacting each other, said two lens sections having an focal distance that is equal to said spherical micro-lens.",
"38.A system for reproducing images in three dimensions, without vertical parallax, in accordance with claim 32, wherein said retro-reflective screen and said discriminating element are a plurality of reflecting elements, said plurality of reflecting elements comprising: a flat mirror; and a specular lens section having a plurality of horizontal cylinders, wherein said plurality of reflecting elements and said specular lens section are disposed perpendicular with respect to each other, wherein the least one observer is situated side by side and not situated one behind another.",
"39.A system for reproducing images in three dimensions, without vertical parallax, in accordance with claim 32, wherein said retro-reflective screen and said discriminating element comprise: a plurality of reflecting elements having a first surface, a second surface and a third surface, said first and said second surfaces being two flat mirrors, said first and said second surface being disposed perpendicular to each other and said third surface being a cylindrical mirror, wherein the at least one observer is situated side by side and not one behind another.",
"40.A system for reproducing images in three dimensions, without vertical parallax, in accordance with claim 32, wherein said retro-reflective screen and said discriminating element are two equal lens sections, said retro-reflective screen and said discriminating element comprising: a plurality of vertical cylindrical lenses having a flat translucent sheet disposed therebetween, said plurality of vertical cylindrical lenses and said translucent sheet being disposed at a focal distance, the at least one observer being situated side by side and not situated one behind another.",
"41.A system for reproducing images in three dimensions in accordance with claims 34, further comprising a single image-reproducing element for modulating a light source by transparency, wherein said bi-dimensional image reproducer is a liquid crystal.",
"42.A system for reproducing images in three dimensions in accordance with claim 34, wherein said bi-dimensional image reproducer is situated between said discriminating element and said retro-reflective element.",
"43.A system for reproducing images in three dimensions in accordance with claim 34, wherein said bi-dimensional image reproducer is situated between the observer and said discriminating element.",
"44.A system for reproducing images in three dimensions in accordance with claim 34, wherein said images are reproduced by one of the group consisting of a diffuser, a cathode ray tube, a plasma screen, an light emitting diode unit, a back-lit liquid crystal with a flat diffusing screen, a liquid crystal display, a cinema projector, a television projector and any combinations thereof.",
"45.A system for reproducing images in three dimensions in accordance with claims 43, further comprising a second retro-reflective element disposed perpendicular to said retro-reflective screen.",
"46.A system for reproducing images in three dimensions in accordance with claim 41, wherein said discriminating element is a light source and an electronic shutter.",
"47.A system for reproducing images in three dimensions in accordance with claim 41, wherein said discriminating element is a plurality of light sources being disposed adjacent to one another.",
"48.A system for reproducing images in three dimensions in accordance with claim 44, wherein said discriminating element is a flat mirror and an electronic shutter.",
"49.A system for reproducing images in three dimensions in accordance with claim 44, wherein said discriminating element is at least two polarised filters, each of said at least two polarised filters having a first and a second polarisation planes, said planes being disposed perpendicular to each other.",
"50.A system for reproducing images in three dimensions in accordance with claim 35, wherein said discriminating element comprises a plurality of light sources, said plurality of light sources being disposed adjacent and in spaced relation above an observer's head.",
"51.A system for reproducing images in three dimensions in accordance with claim 35, wherein said discriminating element comprises a light source and an electronic shutter, said discriminating element being disposed in spaced relation above the observer.",
"52.A system for reproducing images in three dimensions in accordance with claim 32, wherein said bi-dimensional image-reproducer reproduces at least two images.",
"53.A system for reproducing images in three dimensions in accordance with claim 52, wherein said first electronic device multiplexes at least two images, said first electronic device emitting said synchronising signal having information about said at least two images.",
"54.A system for reproducing images in three dimensions in accordance with claim 53, wherein said second electronic device processes said head position signal from said monitor, said monitor providing said head position signal in response to a first position of the observer's head, said second electronic device receiving said head position signal, and said second electronic device, in response to said head position signal, controlling said discriminating element, said discriminating element corresponding to the at least one observer.",
"55.A system for reproducing images in three dimensions in accordance with claim 32, wherein said bi-dimensional image-reproducer reproduces at least ten images.",
"56.A system for reproducing images in three dimensions in accordance with claim 55, wherein said first electronic device multiplexes a plurality of images, said first electronic device emitting said synchronising signal having information about said plurality of images, said second electronic device controlling said discriminating element in response thereto.",
"57.A system for reproducing images in three dimensions in accordance claim 56, wherein said bi-dimensional image reproducer reproduces images, said images being reproduced with an angle of vision, said angle of vision being insufficient to cover a first width equal to a width which the observer's eyes may occupy, wherein said second electronic device processes said head position signal from said monitor, said monitor sensing said position of said observer's head, said second electronic device controlling said discriminating element in response to said head position signal.",
"58.A system for reproducing images in three dimensions in accordance with claim 34, further comprising a plurality of semi-transparent sheets corresponding in number to each of the at least one observer.",
"59.A system for reproducing images in three dimensions in accordance with claim 34, wherein said semi-transparent sheet is disposed in front of said retro-reflective screen.",
"60.A system for reproducing images in three dimensions in accordance with claim 34, wherein said single semi-transparent sheet is a false ceiling.",
"61.A system for reproducing images in three dimensions in accordance with claim 34, for stereoscopic reproduction with only two images, comprising: at least two image-reproducing elements by transparency; at least two overhead-projecting elements being disposed perpendicular to each other; a semi-transparent screen being disposed at an angle of about 45° with respect to said at least two overhead-projecting elements; and two light sources corresponding to each eye of the observers.",
"62.A system for reproducing images in three dimensions in accordance with claim 34, for stereoscopic reproductions with at least two images, comprising: at least two bi-dimensional image reproducers; at least two overhead-projecting elements being disposed perpendicular to one another, said semi-transparent screen being disposed at an angle of about 45° with respect to said at least two overhead-projecting elements; and a plurality of light sources disposed in a row."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Independently of systems developed from 1947, based on the production of images through the coherent interference of light beams called holographic systems, the other systems, including that described in the present invention, are to be classified in one of the following groups: stereoscopic, three-dimensional and integral.",
"The term “stereoscopic” is used here to designate systems which use only two different images in the reproduction, one for each eye.",
"The term “three-dimensional” is used to designate systems which use more than two images in the reproduction and which allow observation with parallax within a wide range of horizontal vision, without observers having to be bothered by placing any device in front of their eyes.",
"The term “integral” is used to designate systems which use a large number of images in the reproduction, thus allowing observation with horizontal and vertical parallax within a wide range of vision.",
"Most of the systems marketed up to the present time in film projection belong to the “stereoscopic” group.",
"In these systems two single images are captured from the objectives whose optical centres are separated horizontally between each other.",
"Many procedure have been used to make each image reach a different eye.",
"The first solution was proposed by D'Almeida in 1858.His solution consisted of placing a rotating shutter in front of the observer, in such a way that it interrupted the passage of light to one eye or the other alternately.",
"The shutter had to be synchronised with the projector which successively projected the images of the left and right eye.",
"This procedure was abandoned due to the noise, mechanical complication and electrical risk.",
"An up-dated version of this procedure consists of placing a liquid crystal in front of each eye which prevents light from passing to one eye while allowing it to pass to the other, the images being projected in synchrony with this alternation.",
"In 1891 Ducos du Hauron suggested, the anaglyphic method of image separation.",
"Images are printed or projected in complementary colours and observed with the filters of the same colours inverted.",
"If the image of the left eye is blue, green and the right is red, the observer's left eye will see the latter through a red filter and vice versa.",
"The most important drawbacks of this procedure are that it is only possible to project images in black and white and that because of the fact that each eye sees a different coloured image, the so-called phenomenon of retina confusion occurs, which causes headaches in many people and nausea in others.",
"Projection with polarised light eliminates these drawbacks.",
"The method was patented by Anderson in 1891 and was not made commercially practicable until 45 years later when E. H. Land invented the Polaroid in the U.S.A.",
"The Polaroid is a relatively cheap sheet of polarised plastic material.",
"The process consists of projecting the image of one eye through a filter that is linearly polarised in a perpendicular direction to the filter used for the other eye.",
"The images projected on a metallized screen, which diffuses the light without de-polarising it, are observed by each viewer with a filter, in each eye, polarised in parallel directions to the filters of the projectors.",
"The biggest drawbacks of this method are that the unwanted image is not completely eliminated; and, if the observer tilts his head, the polarisation planes of the filters turn at the same time and the system loses its effectiveness.",
"The latter drawback has been resolved in recent times using circularly polarised filters, with left-handed polarisation being used for one eye and right-handed polarisation for the other.",
"An attempt to free viewers from having to wear glasses with polarised, coloured or shutter filters was started by Ives in the U.S.A., continued by Gabor in Great Britain; much research and many experiments being completed in the Soviet Union.",
"All these attempts mainly involve a special screen which receives the two images and channels them separately to each of the observer's eyes.",
"Essentially, the screen is made up of a series of opaque plates separated at a distance equal to their width and mounted in front of a diffusing surface.",
"This device is called a trace.",
"The images corresponding to the right and left eyes are projected onto the screen from projectors separated by an appropriate distance and the trace cuts the images into vertical bands.",
"Viewers must sit in a position such that the trace hides one image from one eye and allows the other image to be observed.",
"This system has several drawbacks among which we may mention its low luminous output and the fact that observers have to keep their heads absolutely still.",
"Several variants of this system have been proposed, but it seems that none of them are capable of commercial success.",
"Regardless of which procedure is used to make a different image reach each eye, there is an additional drawback, common to all stereoscopic systems.",
"It arises from the fact that all observers perceive the same parallax, regardless of the observation point they occupy.",
"In the view of a real scene parallax is less for distant observers than for nearby ones.",
"As it is possible to give only a single parallax value for all observers in the stereoscopic system, corresponding to a determinate observation distance, the most distant observers will see objects with disproportionate depth and the nearest observers will see them in the opposite way.",
"To summarise the stereoscopic systems, it may be said that the two drawbacks common to all of them are: The need to trouble observers either by placing filters or some other device in front of their eyes or by immobilising their head.",
"The impossibility of reproducing with a parallax appropriate for each observation distance.",
"This causes a distortion in the third dimension or depth of the reproduced image which is a function of said distance.",
"The three-dimensional systems arose later.",
"They partly avoid these drawbacks.",
"Most three-dimensional systems capture images by means of a series of conventional objectives situated in different spatial positions arranged according to a horizontal line or curve.",
"The reproduction system is different according to the different authors.",
"In the process described in U.S. Pat.",
"No.",
"1,918,705, Ives uses one or two sections of vertical, convergent cylindrical lenses, depending on whether it is frontal or rear projection and a diffusing surface parallel to said sections.",
"In this system, the maximum angle of orthoscopic vision is limited by the angle of opening of the convergent cylindrical elements which make up the section.",
"In reproduction rooms which need greater angles of vision than the above, these systems are not satisfactory.",
"The amount of information managed by these three-dimensional systems depends on the number of images reproduced.",
"As the maximum angle of vision is limited, the maximum number of images depends on the minimum angle occupied by each of them.",
"This number is limited by the optical quality of the cylindrical elements making up the section and, in practice, is insufficient for quality reproductions with distant observers.",
"In the three-dimensional system described by the author of this invention in U.S. Pat.",
"Nos.",
"5,004,335; 5,013,147 and 5,357,368, two sections of convergent or divergent cylindrical lenses are used.",
"In this system the orthoscopic angle of vision is not limited, and can reach 180°, nor is the number of images that can be used.",
"For these reasons the system is appropriate for reproduction rooms with any angle and any distance of observation.",
"Nonetheless, when the angles and distances of observation required by normal reproduction rooms are used, the number of images needed and therefore the amount of information to be captured and processed are enormously high.",
"The author of this invention describes, in PCT/ES96/00092, a device for reproducing three-dimensional images without using sections of lenses.",
"The different images are reproduced sequentially by transparency and appear supported in a liquid crystal that is observed by means of a special illumination.",
"This device seems to be adequate for reproductions on screen sizes similar to domestic televisions.",
"Its drawback is the large number of images necessary in the reproductions of acceptable angles of vision and therefore the large amount of information that it is necessary to process and represent sequentially in the liquid crystal.",
"The liquid crystals currently on the market are scarcely able to respond to the required frame rate.",
"In order to overcome the difficulties arising from the large amount of information that has to be processed and sequentially reproduced by transparency, devices have been designed which are analogous to the foregoing, that is, which reproduce the different two-dimensional images by transparency, illuminate it with a special system and focus the luminous beam with the help of a convergent optical system on the eyes of the observer.",
"There are systems, mentioned below, which do not need mechanical movements for monitoring the observer's head, and others which by incorporating mechanical movements, as well as adding other drawbacks arising from such movements, distance themselves from the device described in this invention, without solving any of the problems described here and will therefore not be mentioned here.",
"British patent 2,272,579, by David Ezra, describes a device configured analogously to those mentioned above, whose purpose is stereoscopic reproduction, as it uses only two or three different images for a single observer.",
"Therefore, the element that reproduces images by transparency needs only an output that is sufficient for the sequential reproduction of two or three images.",
"In PCT/US93/08412, Eichenlaub describes a stroboscopic system of illumination which may also be used for stereoscopic reproductions, that is, with only two images and for a single observer.",
"Other work, such as that mentioned below, deals with a larger number of observers.",
"European patent 0,576,106 A1, by Eichenlaub, describes a device with a configuration analogous to that of David Ezra.",
"Although his system of illumination and focussing may be used for a large number of observers, due to its lack of field depth, the observers' eyes have to be situated in a flat surface which, although it may be parallel to the floor, means a significant restriction.",
"It also uses a very small number of two-dimensional images, which is possible because the focussing system directs the image corresponding to the left eye to all the left eyes of the observers and analogously in the case of the image corresponding to the right eye.",
"In European patent 0,656,555 A1, Woodgate et al.",
"describe several reproduction systems all of which are based, like the foregoing, on one or two devices which reproduce images two-dimensionally by transparency, a special system of illumination and a device which focusses the luminous beam onto the observers' eyes.",
"The lack of focus field depth of the illumination system makes it necessary for observers to be situated in a plane that is parallel to the reproduction system, that is, no observer can be situated behind another.",
"The illumination devices used in the foregoing patents are reasonably simple because they are used for one or very few observers who are specially situated.",
"However, when the illumination device has to cover a wide field of observation with randomly situated observers, it is especially complex and expensive, as we explain below.",
"The condition to be fulfilled by illumination devices is to make a different image reach each eye of each observer.",
"In order to fulfill this condition when the observers are not situated in the same plane, the observation angle of each observer must be equal to or greater than the angle of illumination of each lens of the illumination device, assuming moreover that these lenses have no other type of optical aberration.",
"If one assumes the same focussing plane for all the lenses or objectives of illumination making up the illumination device, the angle of observation is the angle under which the optical centres of the observer's eyes are seen most distant from said focussing plane, from the geometric centre of said plane.",
"The angle of illumination of each illuminating objective is the angle under which the optical centres of two contiguous objectives of illumination are seen from the geometric centre of the above-mentioned focussing plane.",
"The distance between two contiguous optical centres of two lenses or objectives of the illumination device coincides with the width and height these should have, as there cannot be dark areas between two contiguous elements.",
"It can be demonstrated that the angle of observation must be greater than that of illumination so that the panel of objectives of illumination is able to direct a different image to each eye of any observer.",
"The angle of observation is determined by the distance to the focussing plane of the most distant observer and by the distance between his eyes.",
"This value is the maximum the angle of illumination can have.",
"In general, this maximum cannot be chosen as an angle of illumination because monitoring of the observer's head requires the detection of much smaller distances than that between the eyes of any observer.",
"With this value of the angle of illumination and the distance from the focussing plane to the panel of objectives of illumination, their maximum width is determined.",
"The fact that the angle of illumination must be small and the distance to the focussing plane must be great means that the size of the lenses or the objectives making up the system must be very small.",
"The surface occupied by the illumination sources of each lens or projecting objective must be sufficient to contain at least a number of horizontal and vertical sources that is equal to the number of possible eyes.",
"Given the necessarily limited size of this surface, the surface occupied by each source and the distance between them has to be extremely small, which means that its position must be controlled very accurately, as it can be affected even by distortions due to temperature changes.",
"There must be as many sources as the product of lenses and number of eyes, whose situation is controlled by a single electronic processor.",
"The area of illumination covered by the illumination device depends on the quotient between the diameter of the objective or lens of illumination and its focal distance.",
"If it is intended to cover a large area of illumination, the focal distance of the objectives of illumination must be very small in comparison to their diameter.",
"This complicates the design of these devices.",
"The foregoing explanation makes it clear that when the illumination systems are designed to cover large areas of illumination and for many randomly situated observers, they need a very complex design and are very expensive to manufacture.",
"Moreover, in the devices described above all the images are reproduced sequentially by transparency and in their entirety on a single electronic reproduction element, or on two of the same size and characteristics, therefore image reproducers by diffusion, such as cinema or TV projectors, cathode ray tubes, plasma screens, led units etc., cannot be used.",
"The German patent 4,123,895, by D. Dieter and the European patent 0,114,406, by Meacham and G. B. Kirby, describe another device for reproducing three-dimensional images without using lens sections, which achieve three-dimensional reproduction for a large number of observers and a small number of images.",
"The different images are projected sequentially upon a conventional diffusing surface and are observed by means of a shutter panel made, preferably, of liquid crystal placed in front of the observer.",
"The difficulties of this system arise from the inconvenience of placing a panel of liquid crystal in front of and close to each observer.",
"Reproduction systems which, in addition to horizontal parallax, as used by stereoscopic and three-dimensional systems, also reproduce vertical parallax, are called integral systems.",
"The invention of integral photography is due to M. G. Lippman in 1908.The device he conceived consists of a sheet made up of an enormous number of convergent lenses.",
"Behind each of these lenses a different image is captured.",
"Viewing these images through the same lenses reproduces the scene with both horizontal and vertical parallax in pseudoscopic form.",
"A second capturing of this pseudoscopic scene would allow the orthoscopic viewing of the first scene.",
"It is advisable that the lenses and the film in this device make up a single unit so that they remain rigidly joined throughout the process, thus avoiding difficult adjustments.",
"This system suffers from logical drawbacks arising from the fact that the lenses used are very small and therefore so is the size of the images captured by them.",
"It is also necessary to control the aperture of each lens so as not to invade the field of capture or reproduction of neighbouring lenses.",
"In view of the foregoing, the designs have been complex and practically impossible to market, even in the case of static images.",
"In U.S. Pat.",
"No.",
"5,013,147, PCT 90/00014 and patent application PCT/ES96/00092, the author of this work proposed an integral reproduction system based, firstly, on capturing the scene by means of a series of conventional objectives situated in the vertices of a rectangular reticle; and then on the projection from the same number of projecting objectives of these two-dimensional images onto two lens sections made up of convergent or divergent cylindrical optical elements that are perpendicular to each other.",
"In Spanish patent application No.",
"9700076, by the same author, an integral reproduction system is described which is based, firstly, on capturing the scene by means of a series of conventional objectives situated in the vertices of a rectangular reticle; and then on the projection from the same number of projecting objectives of these two-dimensional images directly, without any optical support, onto where the different images are focussed.",
"To do this, the input pupils of the projection objectives must have a rectangular shape, the sides of the contiguous rectangles being in contact with each other and without any gaps between them.",
"In the latter cases, even more than in the first ones, the number of images needed and therefore the amount of information to be processed is enormously high.",
"Although examination of the drawbacks in the foregoing systems has been limited to those arising in reproduction, capturing is logically much more complex the greater the number of images used.",
"In three-dimensional and integral systems that are appropriate for reproductions with a large number of observers, the capture of such a large number of images is required that an enormously complex and voluminous capturing device must be used, it being impossible in most cases to respond to the needs of filming.",
"To summarise, it is required that systems of reproduction of images in three-dimensions capture and reproduce a small number of images without troubling observers with any device in front of their eyes.",
"It has been explained that in systems that have been designed up to now, the difficulties become greater as the number of observers increases.",
"The device described in this invention resolves these and other drawbacks because it uses a reflexive or refractive optical device, made up of a large number of elements that are “sufficiently small” to provide it with the necessary field depth required by the random situation of observers.",
"It has the additional possibility that for all these elements a single source of illumination can be used, or another type of discriminator for each observation eye, these sources being situated inside a “sufficiently large volume” so as not to need precise positioning.",
"Also, any type of image reproducer can be used, whether by transparency or by diffusion and without using movable elements."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 shows a reflector element for light rays contained in a horizontal plane.",
"FIG.",
"2 shows a reflector element of light rays in any direction and a retro-reflective screen made up of a large number of these elements.",
"FIG.",
"3 shows a separating element made up of a semi-transparent sheet with the retro-reflective screen.",
"FIG.",
"4 shows a separating element made up of a bi-refringent material and a de-polarising sheet.",
"FIG.",
"5 shows a retro-reflective screen which works by refraction.",
"FIG.",
"6 shows the retro-reflective screen of FIG.",
"5 together with a separating element and another symmetrical optical section.",
"FIG.",
"7 shows the screen of FIG.",
"6 working by refraction.",
"FIG.",
"8 shows a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane made up of two specular elements.",
"FIG.",
"9 shows a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane made up of three specular elements.",
"FIG.",
"10 shows a lens section acting as a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane.",
"FIG.",
"11 shows two lens sections working by refraction and acting as a retro-reflective screen in the horizontal plane and a diffusor in the vertical plane.",
"FIG.",
"12 show the situation of an image-reproducing element by transparency and a semi-transparent sheet as a separating element.",
"FIG.",
"13 shows the situation of an image-reproducing element on a diffusing screen and a semi-transparent sheet as a separating element.",
"FIG.",
"14 shows the situation of an image-reproducing element by transparency and a bi-refringent sheet as a separating element together with a de-polarising sheet.",
"FIG.",
"15 shows the situation of an image-reproducing element by transparency and a screen working by refraction.",
"FIG.",
"16 shows different types of discriminating elements.",
"FIG.",
"17 shows the discriminating elements responding to the head movements of observers.",
"FIG.",
"18 shows a real arrangement of the model with a separating element for each observer and close to the latter.",
"FIG.",
"19 shows a real arrangement of the model with a single separating element next to the retro-reflective screen and reproduction by transparency.",
"FIG.",
"20 shows a real arrangement of the model with a single separating element as a false ceiling and reproduction by diffusion.",
"FIG.",
"21 shows a real arrangement of the model with a single separating element as a false ceiling.",
"FIG.",
"22 shows a real arrangement of the model which uses a screen that works by refraction.",
"FIG.",
"23 shows a real arrangement of the model using a bi-refringent sheet as a separating element and a de-polarising element.",
"FIG.",
"24 shows a diagrammatic arrangement of the model with two image-reproduction systems by transparency and two rows of light sources.",
"FIG.",
"25 shows a diagrammatic arrangement of the model with two image-reproduction systems by transparency and a single row of light sources.",
"FIG.",
"26 shows a diagrammatic arrangement of the model analogous to FIG.",
"24 , but working by refraction.",
"FIG.",
"27 shows a diagrammatic arrangement of the model the same as FIG.",
"26 with a single row of light sources.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"OBJECT OF THE INVENTION The present invention describes a system that is capable of reproducing images in three dimensions, that is, stereoscopic, three-dimensional or integral, without the need for glasses, nor any other device in front of the observer's eyes.",
"BACKGROUND OF THE INVENTION Independently of systems developed from 1947, based on the production of images through the coherent interference of light beams called holographic systems, the other systems, including that described in the present invention, are to be classified in one of the following groups: stereoscopic, three-dimensional and integral.",
"The term “stereoscopic” is used here to designate systems which use only two different images in the reproduction, one for each eye.",
"The term “three-dimensional” is used to designate systems which use more than two images in the reproduction and which allow observation with parallax within a wide range of horizontal vision, without observers having to be bothered by placing any device in front of their eyes.",
"The term “integral” is used to designate systems which use a large number of images in the reproduction, thus allowing observation with horizontal and vertical parallax within a wide range of vision.",
"Most of the systems marketed up to the present time in film projection belong to the “stereoscopic” group.",
"In these systems two single images are captured from the objectives whose optical centres are separated horizontally between each other.",
"Many procedure have been used to make each image reach a different eye.",
"The first solution was proposed by D'Almeida in 1858.His solution consisted of placing a rotating shutter in front of the observer, in such a way that it interrupted the passage of light to one eye or the other alternately.",
"The shutter had to be synchronised with the projector which successively projected the images of the left and right eye.",
"This procedure was abandoned due to the noise, mechanical complication and electrical risk.",
"An up-dated version of this procedure consists of placing a liquid crystal in front of each eye which prevents light from passing to one eye while allowing it to pass to the other, the images being projected in synchrony with this alternation.",
"In 1891 Ducos du Hauron suggested, the anaglyphic method of image separation.",
"Images are printed or projected in complementary colours and observed with the filters of the same colours inverted.",
"If the image of the left eye is blue, green and the right is red, the observer's left eye will see the latter through a red filter and vice versa.",
"The most important drawbacks of this procedure are that it is only possible to project images in black and white and that because of the fact that each eye sees a different coloured image, the so-called phenomenon of retina confusion occurs, which causes headaches in many people and nausea in others.",
"Projection with polarised light eliminates these drawbacks.",
"The method was patented by Anderson in 1891 and was not made commercially practicable until 45 years later when E. H. Land invented the Polaroid in the U.S.A.",
"The Polaroid is a relatively cheap sheet of polarised plastic material.",
"The process consists of projecting the image of one eye through a filter that is linearly polarised in a perpendicular direction to the filter used for the other eye.",
"The images projected on a metallized screen, which diffuses the light without de-polarising it, are observed by each viewer with a filter, in each eye, polarised in parallel directions to the filters of the projectors.",
"The biggest drawbacks of this method are that the unwanted image is not completely eliminated; and, if the observer tilts his head, the polarisation planes of the filters turn at the same time and the system loses its effectiveness.",
"The latter drawback has been resolved in recent times using circularly polarised filters, with left-handed polarisation being used for one eye and right-handed polarisation for the other.",
"An attempt to free viewers from having to wear glasses with polarised, coloured or shutter filters was started by Ives in the U.S.A., continued by Gabor in Great Britain; much research and many experiments being completed in the Soviet Union.",
"All these attempts mainly involve a special screen which receives the two images and channels them separately to each of the observer's eyes.",
"Essentially, the screen is made up of a series of opaque plates separated at a distance equal to their width and mounted in front of a diffusing surface.",
"This device is called a trace.",
"The images corresponding to the right and left eyes are projected onto the screen from projectors separated by an appropriate distance and the trace cuts the images into vertical bands.",
"Viewers must sit in a position such that the trace hides one image from one eye and allows the other image to be observed.",
"This system has several drawbacks among which we may mention its low luminous output and the fact that observers have to keep their heads absolutely still.",
"Several variants of this system have been proposed, but it seems that none of them are capable of commercial success.",
"Regardless of which procedure is used to make a different image reach each eye, there is an additional drawback, common to all stereoscopic systems.",
"It arises from the fact that all observers perceive the same parallax, regardless of the observation point they occupy.",
"In the view of a real scene parallax is less for distant observers than for nearby ones.",
"As it is possible to give only a single parallax value for all observers in the stereoscopic system, corresponding to a determinate observation distance, the most distant observers will see objects with disproportionate depth and the nearest observers will see them in the opposite way.",
"To summarise the stereoscopic systems, it may be said that the two drawbacks common to all of them are: The need to trouble observers either by placing filters or some other device in front of their eyes or by immobilising their head.",
"The impossibility of reproducing with a parallax appropriate for each observation distance.",
"This causes a distortion in the third dimension or depth of the reproduced image which is a function of said distance.",
"The three-dimensional systems arose later.",
"They partly avoid these drawbacks.",
"Most three-dimensional systems capture images by means of a series of conventional objectives situated in different spatial positions arranged according to a horizontal line or curve.",
"The reproduction system is different according to the different authors.",
"In the process described in U.S. Pat.",
"No.",
"1,918,705, Ives uses one or two sections of vertical, convergent cylindrical lenses, depending on whether it is frontal or rear projection and a diffusing surface parallel to said sections.",
"In this system, the maximum angle of orthoscopic vision is limited by the angle of opening of the convergent cylindrical elements which make up the section.",
"In reproduction rooms which need greater angles of vision than the above, these systems are not satisfactory.",
"The amount of information managed by these three-dimensional systems depends on the number of images reproduced.",
"As the maximum angle of vision is limited, the maximum number of images depends on the minimum angle occupied by each of them.",
"This number is limited by the optical quality of the cylindrical elements making up the section and, in practice, is insufficient for quality reproductions with distant observers.",
"In the three-dimensional system described by the author of this invention in U.S. Pat.",
"Nos.",
"5,004,335; 5,013,147 and 5,357,368, two sections of convergent or divergent cylindrical lenses are used.",
"In this system the orthoscopic angle of vision is not limited, and can reach 180°, nor is the number of images that can be used.",
"For these reasons the system is appropriate for reproduction rooms with any angle and any distance of observation.",
"Nonetheless, when the angles and distances of observation required by normal reproduction rooms are used, the number of images needed and therefore the amount of information to be captured and processed are enormously high.",
"The author of this invention describes, in PCT/ES96/00092, a device for reproducing three-dimensional images without using sections of lenses.",
"The different images are reproduced sequentially by transparency and appear supported in a liquid crystal that is observed by means of a special illumination.",
"This device seems to be adequate for reproductions on screen sizes similar to domestic televisions.",
"Its drawback is the large number of images necessary in the reproductions of acceptable angles of vision and therefore the large amount of information that it is necessary to process and represent sequentially in the liquid crystal.",
"The liquid crystals currently on the market are scarcely able to respond to the required frame rate.",
"In order to overcome the difficulties arising from the large amount of information that has to be processed and sequentially reproduced by transparency, devices have been designed which are analogous to the foregoing, that is, which reproduce the different two-dimensional images by transparency, illuminate it with a special system and focus the luminous beam with the help of a convergent optical system on the eyes of the observer.",
"There are systems, mentioned below, which do not need mechanical movements for monitoring the observer's head, and others which by incorporating mechanical movements, as well as adding other drawbacks arising from such movements, distance themselves from the device described in this invention, without solving any of the problems described here and will therefore not be mentioned here.",
"British patent 2,272,579, by David Ezra, describes a device configured analogously to those mentioned above, whose purpose is stereoscopic reproduction, as it uses only two or three different images for a single observer.",
"Therefore, the element that reproduces images by transparency needs only an output that is sufficient for the sequential reproduction of two or three images.",
"In PCT/US93/08412, Eichenlaub describes a stroboscopic system of illumination which may also be used for stereoscopic reproductions, that is, with only two images and for a single observer.",
"Other work, such as that mentioned below, deals with a larger number of observers.",
"European patent 0,576,106 A1, by Eichenlaub, describes a device with a configuration analogous to that of David Ezra.",
"Although his system of illumination and focussing may be used for a large number of observers, due to its lack of field depth, the observers' eyes have to be situated in a flat surface which, although it may be parallel to the floor, means a significant restriction.",
"It also uses a very small number of two-dimensional images, which is possible because the focussing system directs the image corresponding to the left eye to all the left eyes of the observers and analogously in the case of the image corresponding to the right eye.",
"In European patent 0,656,555 A1, Woodgate et al.",
"describe several reproduction systems all of which are based, like the foregoing, on one or two devices which reproduce images two-dimensionally by transparency, a special system of illumination and a device which focusses the luminous beam onto the observers' eyes.",
"The lack of focus field depth of the illumination system makes it necessary for observers to be situated in a plane that is parallel to the reproduction system, that is, no observer can be situated behind another.",
"The illumination devices used in the foregoing patents are reasonably simple because they are used for one or very few observers who are specially situated.",
"However, when the illumination device has to cover a wide field of observation with randomly situated observers, it is especially complex and expensive, as we explain below.",
"The condition to be fulfilled by illumination devices is to make a different image reach each eye of each observer.",
"In order to fulfill this condition when the observers are not situated in the same plane, the observation angle of each observer must be equal to or greater than the angle of illumination of each lens of the illumination device, assuming moreover that these lenses have no other type of optical aberration.",
"If one assumes the same focussing plane for all the lenses or objectives of illumination making up the illumination device, the angle of observation is the angle under which the optical centres of the observer's eyes are seen most distant from said focussing plane, from the geometric centre of said plane.",
"The angle of illumination of each illuminating objective is the angle under which the optical centres of two contiguous objectives of illumination are seen from the geometric centre of the above-mentioned focussing plane.",
"The distance between two contiguous optical centres of two lenses or objectives of the illumination device coincides with the width and height these should have, as there cannot be dark areas between two contiguous elements.",
"It can be demonstrated that the angle of observation must be greater than that of illumination so that the panel of objectives of illumination is able to direct a different image to each eye of any observer.",
"The angle of observation is determined by the distance to the focussing plane of the most distant observer and by the distance between his eyes.",
"This value is the maximum the angle of illumination can have.",
"In general, this maximum cannot be chosen as an angle of illumination because monitoring of the observer's head requires the detection of much smaller distances than that between the eyes of any observer.",
"With this value of the angle of illumination and the distance from the focussing plane to the panel of objectives of illumination, their maximum width is determined.",
"The fact that the angle of illumination must be small and the distance to the focussing plane must be great means that the size of the lenses or the objectives making up the system must be very small.",
"The surface occupied by the illumination sources of each lens or projecting objective must be sufficient to contain at least a number of horizontal and vertical sources that is equal to the number of possible eyes.",
"Given the necessarily limited size of this surface, the surface occupied by each source and the distance between them has to be extremely small, which means that its position must be controlled very accurately, as it can be affected even by distortions due to temperature changes.",
"There must be as many sources as the product of lenses and number of eyes, whose situation is controlled by a single electronic processor.",
"The area of illumination covered by the illumination device depends on the quotient between the diameter of the objective or lens of illumination and its focal distance.",
"If it is intended to cover a large area of illumination, the focal distance of the objectives of illumination must be very small in comparison to their diameter.",
"This complicates the design of these devices.",
"The foregoing explanation makes it clear that when the illumination systems are designed to cover large areas of illumination and for many randomly situated observers, they need a very complex design and are very expensive to manufacture.",
"Moreover, in the devices described above all the images are reproduced sequentially by transparency and in their entirety on a single electronic reproduction element, or on two of the same size and characteristics, therefore image reproducers by diffusion, such as cinema or TV projectors, cathode ray tubes, plasma screens, led units etc., cannot be used.",
"The German patent 4,123,895, by D. Dieter and the European patent 0,114,406, by Meacham and G. B. Kirby, describe another device for reproducing three-dimensional images without using lens sections, which achieve three-dimensional reproduction for a large number of observers and a small number of images.",
"The different images are projected sequentially upon a conventional diffusing surface and are observed by means of a shutter panel made, preferably, of liquid crystal placed in front of the observer.",
"The difficulties of this system arise from the inconvenience of placing a panel of liquid crystal in front of and close to each observer.",
"Reproduction systems which, in addition to horizontal parallax, as used by stereoscopic and three-dimensional systems, also reproduce vertical parallax, are called integral systems.",
"The invention of integral photography is due to M. G. Lippman in 1908.The device he conceived consists of a sheet made up of an enormous number of convergent lenses.",
"Behind each of these lenses a different image is captured.",
"Viewing these images through the same lenses reproduces the scene with both horizontal and vertical parallax in pseudoscopic form.",
"A second capturing of this pseudoscopic scene would allow the orthoscopic viewing of the first scene.",
"It is advisable that the lenses and the film in this device make up a single unit so that they remain rigidly joined throughout the process, thus avoiding difficult adjustments.",
"This system suffers from logical drawbacks arising from the fact that the lenses used are very small and therefore so is the size of the images captured by them.",
"It is also necessary to control the aperture of each lens so as not to invade the field of capture or reproduction of neighbouring lenses.",
"In view of the foregoing, the designs have been complex and practically impossible to market, even in the case of static images.",
"In U.S. Pat.",
"No.",
"5,013,147, PCT 90/00014 and patent application PCT/ES96/00092, the author of this work proposed an integral reproduction system based, firstly, on capturing the scene by means of a series of conventional objectives situated in the vertices of a rectangular reticle; and then on the projection from the same number of projecting objectives of these two-dimensional images onto two lens sections made up of convergent or divergent cylindrical optical elements that are perpendicular to each other.",
"In Spanish patent application No.",
"9700076, by the same author, an integral reproduction system is described which is based, firstly, on capturing the scene by means of a series of conventional objectives situated in the vertices of a rectangular reticle; and then on the projection from the same number of projecting objectives of these two-dimensional images directly, without any optical support, onto where the different images are focussed.",
"To do this, the input pupils of the projection objectives must have a rectangular shape, the sides of the contiguous rectangles being in contact with each other and without any gaps between them.",
"In the latter cases, even more than in the first ones, the number of images needed and therefore the amount of information to be processed is enormously high.",
"Although examination of the drawbacks in the foregoing systems has been limited to those arising in reproduction, capturing is logically much more complex the greater the number of images used.",
"In three-dimensional and integral systems that are appropriate for reproductions with a large number of observers, the capture of such a large number of images is required that an enormously complex and voluminous capturing device must be used, it being impossible in most cases to respond to the needs of filming.",
"To summarise, it is required that systems of reproduction of images in three-dimensions capture and reproduce a small number of images without troubling observers with any device in front of their eyes.",
"It has been explained that in systems that have been designed up to now, the difficulties become greater as the number of observers increases.",
"The device described in this invention resolves these and other drawbacks because it uses a reflexive or refractive optical device, made up of a large number of elements that are “sufficiently small” to provide it with the necessary field depth required by the random situation of observers.",
"It has the additional possibility that for all these elements a single source of illumination can be used, or another type of discriminator for each observation eye, these sources being situated inside a “sufficiently large volume” so as not to need precise positioning.",
"Also, any type of image reproducer can be used, whether by transparency or by diffusion and without using movable elements.",
"DESCRIPTION OF THE INVENTION In order to explain the operation of the system that is the object of this invention, each of the six basic parts which comprise it will be described.",
"As the first component of the system, a refractive or reflexive element may be used.",
"However, the system will be described with the reflexive element and the refractive element will be considered as a variant.",
"Thus the first component of the system that is the object of this invention is a retro-reflective screen.",
"This retro-reflective screen is made up of a large number of reflecting elements.",
"A reflector is understood to be the optical part that ensures reflection in the reverse direction to a beam of parallel rays.",
"The simplest reflector element is made up of two mirrors joined together at an angle of 90° and situated in such a way that the reflecting surfaces are perpendicular to the plane that contains the incident beam.",
"This limitation is overcome if a specular regular tetrahedron is used as a reflecting element, that is, an assembly of three flat mirrors in which every two of them are perpendicular to each other.",
"The first component of the system that is the object of this invention is a surface with back-reflecting or self-collimating properties, such as that described above, which ensures reflection in the reverse direction and in which the triple mirrors are so small that the direction of the reflected ray, parallel to that of the incident ray, has a small distance so that these directions are to be considered coincident.",
"Any beam of rays, homocentric at any point, which strikes the retro-reflective surface will, after reflection, become a beam of rays that are practically homocentric at the same point, travelling in the reverse direction.",
"The second component of the system is an optical element that is able to separate the incident and reflected light beams.",
"The most simple optical part with this property is a semi-transparent mirror forming a non-zero angle with the retro-reflective screen.",
"Incorporating this second element allows the point from which the beam of divergent rays departs to occupy a physical position different from the point at which the rays converge after back-reflection in the above-described screen.",
"Thus, corresponding to all divergent beams of rays that are homocentric at any point in space there is another beam of convergent rays at another point distinct from the former.",
"With the two foregoing components, it is possible to make a bi-univocal correspondence between any light-emitting point and another one, distinct and single, where all the rays emitted by the former, after back-reflecting, converge.",
"In this way, for each eye, considered as a light convergence point, there will be another unique light divergence point situated elsewhere.",
"The totality of observers' eyes will occupy a determinate volume.",
"The points from which the light rays diverge will occupy another volume of the same size situated elsewhere.",
"In another variant of the same invention a bi-refringent sheet together with a de-polarising sheet may be used as the second component, both being parallel to the retro-reflective screen, since these optical elements can also separate the divergent incident rays from the convergent reflected ones, as will be explained hereinbelow.",
"In general, at the point where the rays converge an observer's eye will be situated and at the point from where they diverge there will be a light source or simply a point from which a ray of light diverges.",
"The light captured by a determinate eye is that which is emitted by a single light source after back-reflecting in the screen described in the first place.",
"This eye will see the overhead projection screen uniformly illuminated by that single light source.",
"The same thing will happen for the other eyes of the observers.",
"As a variant of the foregoing reflexive components, the use of refractive elements may be considered.",
"A retro-reflective element made of refractive material is the screen made up of a large number of convergent lenses, analogous to that used by Lippman for his integral photography, whose rear surface is a diffusing plane.",
"The main drawback of this retro-reflective screen are the unwanted reflections on the convex surface of the convergent lenses.",
"However, if another identical screen is added to this one in such a way that the diffusing plane, in this case translucent, is sandwiched between these two screens like the former, said reflections are avoided.",
"So that each eye sees a two-dimensional image on the retro-reflective screen it will be necessary to modulate in intensity and colour the light that passes through each element making up the screen described above.",
"The third component of the system is used as a light intensity and colour modulating element.",
"The third component of the system is a screen for reproducing two-dimensional images.",
"This screen can generate the images by transparency, as in the case of liquid crystals, by diffusion, as in the case of cinema and video projectors, or by generation of diffuse light, as in the case of cathode ray tubes, plasma screens, led screens or liquid crystal screens illuminated by diffuse light from behind.",
"In the first case, that is, when the screen reproduces the image by transparency, all light rays are modulated in intensity and colour when they pass through any point of said screen.",
"Therefore this screen, which reproduces by transparency, will be situated parallel to the overhead projection screen mentioned as the first component of the system.",
"The light from the light sources can be modulated in intensity and colour once or twice, according to whether the screen for reproducing images by transparency is situated between the observer and the separating element or between the latter and the reproducing element.",
"In the second case, that is, when the screen reproduces the image by projection or generates it directly on its surface, the arrangement of the elements is the same, although it is different from the case of reproduction by transparency.",
"The diffusing screen is to have the same size as the overhead projector screen and will be situated in front of the elements that emit divergent light rays which, in this case, will have been converted into simple flat mirrors.",
"The images reproduced, whether by transparency or diffusion, are two-dimensional images.",
"To obtain stereoscopic images, the number of these images must be two; for three-dimensional or integral reproductions, it must be ten or several tens.",
"If the reproduction is done in a single element by transparency, reproduction of the different images will always be done by multiplexing in time.",
"If reproduction is done on diffuse screen or by transparency on two different reproducing elements, it will not always be necessary to do it by multiplexing in time.",
"In stereoscopic reproductions, the two images may be projected onto a metallized diffusing screen and each image polarised in a polarisation plane perpendicular to that of the other image, as in the case of ordinary cinema with polarised glasses, or each image may be reproduced on a different reproducing element by transparency, as explained hereinbelow.",
"The device that is the object of this invention must make a different image reach each eye of each observer without troubling said observer by placing glasses or other devices in front of his eyes.",
"To achieve this, the fourth component of the system is used.",
"The fourth element that makes up the system is responsible for making a different image reach each eye.",
"In the most general case in which images are reproduced by multiplexing in time, the fourth element is a simple shutter situated in front of each geometrical place where the generating points of divergent light beams are located, synchronised with the rhythm with which the different images are reproduced.",
"The optical effect is the same as if each of the discriminating elements were situated in front of each eye of each observer and however the observers are not troubled by any device being placed in front of their eyes.",
"If the screen which modulates the light intensity and the colour carries out this function by transparency, a variant included in this same invention consists of replacing the shutter and the light source by several sources, one next to another, flashing in synchrony with the generating sequence of the different images.",
"In the specific case of stereoscopic reproductions, that is, with two single images projected with polarised light in polarisation planes that are perpendicular to each other, the discriminating element may be made up of a pair of polarised filters in the same polarisation planes with which the images are projected.",
"Except in this case for synchronising the shutter with the image sequence, electronic means are needed which, in addition to multiplexing the images, generate and emit a synchronising signal to the different discriminating elements.",
"These electronic means constitute the fifth element making up the system.",
"What has been described up to here would allow an appropriate model to be made for stereoscopic projections with only two images, if the observer kept his head still; and for three-dimensional or integral projections if the angle of vision in these covered the entire area in which each observer could situate his eyes.",
"If, optionally, one wishes to make a stereoscopic or three-dimensional or integral system with a small angle of vision, but which allows observers to move their head freely, it is necessary to incorporate a sixth component in the system.",
"The sixth component of the system is made up of electronic means that are able to process the information from a detector of the head position of each observer and, according to this information, to control the electronic position of the image-discriminating element to which we referred as the fourth element making up the system.",
"The system described allows many variants.",
"For example, if one chooses the semi-transparent sheet as the second component element or element that is able to separate convergent light beams from divergent ones, depending on the situation of this sheet, there are different solutions.",
"A separating element may be situated in front of and close to each observer, a single separating element may be situated for all observers between them and the retro-reflective screen and close to the latter, or this separating element may be situated by way of a false ceiling in the projection room, as explained hereinbelow with its corresponding drawings.",
"A bi-refringent material may also be used as a separating element, thus giving rise to another variant of the same invention described here.",
"In cases in which the observers are never situated one behind another, a screen may be used as the first component element, made of reflexive material, which reflects only in the horizontal plane and diffuses in the vertical plane.",
"This diffusion may be used as a separating element, in which case it is not necessary to make additional use of an element to separate the convergent beams from the divergent ones.",
"As the first component element a retro-reflective screen may also be used, made of refractive material, such as a lens section which has a translucent rear surface to act as a separating element and a second lens section symmetrical to the first with respect to this translucent surface which prevents anomalous reflections.",
"Using the same model described in this invention, one may make as many variants as different types of retro-reflective screens in the horizontal and vertical planes, as explained hereinbelow with its corresponding drawing.",
"In short, the device described in this invention is a system for reproducing images in three dimensions that is able to reproduce stereoscopic, three-dimensional or integral images for any number of observers and wherever they are situated without the need for them to use glasses or any other device in front of their eyes, characterised in that it has a retro-reflective screen that is able to ensure the reflection in the same direction and in the reverse path of all rays belonging to any beam of light rays; it also has an element that can separate the light beams according to their direction of travel, a reproducer of two-dimensional images, an element that is able to discriminate among the different two-dimensional images, a first electronic device that can multiplex the different images and emit a synchronising signal identifying the image which is being reproduced at all times and a second electronic device that is able to receive the synchronising signal and control, in accordance with the latter, the discriminating element and, optionally, the latter may also process the electronic signals which indicate the position of the observer's eyes, from a monitor of the head of each observer, and electronically command the image discriminator in accordance with said signals.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a reflector element for light rays contained in a horizontal plane.",
"FIG.",
"2 shows a reflector element of light rays in any direction and a retro-reflective screen made up of a large number of these elements.",
"FIG.",
"3 shows a separating element made up of a semi-transparent sheet with the retro-reflective screen.",
"FIG.",
"4 shows a separating element made up of a bi-refringent material and a de-polarising sheet.",
"FIG.",
"5 shows a retro-reflective screen which works by refraction.",
"FIG.",
"6 shows the retro-reflective screen of FIG.",
"5 together with a separating element and another symmetrical optical section.",
"FIG.",
"7 shows the screen of FIG.",
"6 working by refraction.",
"FIG.",
"8 shows a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane made up of two specular elements.",
"FIG.",
"9 shows a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane made up of three specular elements.",
"FIG.",
"10 shows a lens section acting as a retro-reflective screen in the horizontal plane and a diffuser in the vertical plane.",
"FIG.",
"11 shows two lens sections working by refraction and acting as a retro-reflective screen in the horizontal plane and a diffusor in the vertical plane.",
"FIG.",
"12 show the situation of an image-reproducing element by transparency and a semi-transparent sheet as a separating element.",
"FIG.",
"13 shows the situation of an image-reproducing element on a diffusing screen and a semi-transparent sheet as a separating element.",
"FIG.",
"14 shows the situation of an image-reproducing element by transparency and a bi-refringent sheet as a separating element together with a de-polarising sheet.",
"FIG.",
"15 shows the situation of an image-reproducing element by transparency and a screen working by refraction.",
"FIG.",
"16 shows different types of discriminating elements.",
"FIG.",
"17 shows the discriminating elements responding to the head movements of observers.",
"FIG.",
"18 shows a real arrangement of the model with a separating element for each observer and close to the latter.",
"FIG.",
"19 shows a real arrangement of the model with a single separating element next to the retro-reflective screen and reproduction by transparency.",
"FIG.",
"20 shows a real arrangement of the model with a single separating element as a false ceiling and reproduction by diffusion.",
"FIG.",
"21 shows a real arrangement of the model with a single separating element as a false ceiling.",
"FIG.",
"22 shows a real arrangement of the model which uses a screen that works by refraction.",
"FIG.",
"23 shows a real arrangement of the model using a bi-refringent sheet as a separating element and a de-polarising element.",
"FIG.",
"24 shows a diagrammatic arrangement of the model with two image-reproduction systems by transparency and two rows of light sources.",
"FIG.",
"25 shows a diagrammatic arrangement of the model with two image-reproduction systems by transparency and a single row of light sources.",
"FIG.",
"26 shows a diagrammatic arrangement of the model analogous to FIG.",
"24, but working by refraction.",
"FIG.",
"27 shows a diagrammatic arrangement of the model the same as FIG.",
"26 with a single row of light sources.",
"DETAILED DESCRIPTION OF THE FIGURES FIG.",
"1 shows the simplest reflector element.",
"This element is made up of two mirrors 81 and 82 that are perpendicular to each other.",
"In the drawing, the intersecting straight line of the reflecting planes is perpendicular to the plane of the paper.",
"Any incident ray contained in the plane of the paper, for example 71 or 73, will be reflected in a parallel direction and in the opposite path 72 or 74.FIG.",
"2 shows a reflector element in the form of a regular tetrahedron in which each of its sides 83, 84, 85 is a flat mirror.",
"Any incident ray, no matter what its direction, for example 75, is returned after three reflections, one in each side of the tetrahedron, in a parallel and opposite direction 76.Also shown in this figure is the screen made up of a large number of small triple mirrors, like those described above, arranged upon a flat rectangular surface 11.This screen constitutes a surface with back reflecting or self-collimating properties and is the first component of the system that is the object of this invention.",
"FIG.",
"3 shows the retro-reflective screen 11, detailed in FIG.",
"2.The semi-transparent sheet 21 has been placed in front of said screen and said semi-transparent sheet is to be considered as the second component of the system that is the object of this invention.",
"This sheet has the property that it allows approximately half the light energy to pass through it and reflects approximately the other half.",
"The light rays 77, 78, 79 emitted by any light source 41 will strike the retro-reflective screen 11 after being reflected in the semi-transparent sheet 21.This divergent light beam will be reflected by the retro-reflective screen 11, forming a beam of rays 710, 711, 712 which converges at the point 91 after passing through the semi-transparent sheet 21.The optical effect produced by this sheet is to spatially separate the point 41, from which the light rays emerge, from the point 91 where they converge.",
"The rays emerging from any other different light source, for example 42, converge at another point also situated elsewhere, such as at point 92.FIG.",
"4 shows the retro-reflective screen 11, detailed in FIG.",
"2.A bi-refringent sheet 221 and a de-polarising sheet 222 have been placed in front of it.",
"All divergent beams of rays 713, 714, 715 generated by the light source 43 after being reflected undergo a double refraction.",
"A beam of rays reflected after passing through both sheets will follow the direction of the incident beam, but in the opposite path and will converge on the light source 43.The other convergent beam of rays 716, 717, 718 will converge on the point 94, separated vertically from point 43.The optical effect produced by the group of bi-refringent and de-polarising sheets is to spatially separate the point 43, from which the light rays emerge, from the point 94 where they converge.",
"This effect is analogous to that produced by the semi-transparent sheet shown in FIG.",
"3 and, therefore, this device is considered as a variant of the second component of the system that is the object of this invention.",
"FIG.",
"5 shows another type of retro-reflective screen which works by refraction.",
"This screen is made up of multiple convergent spherical micro-lenses, such as 1211, 1212, 1213, 1214 in whose focal plane an opaque diffusing surface 23 is situated.",
"All beams of rays 716, 717, 718 striking any micro-lens, for example 1213, is reflected in the same direction but in the reverse path 718, 717 and 716.Likewise for the other convergent beams and any other lens.",
"The group of micro-lenses forms a screen 121-23 such as that shown in said figure, which has back-reflecting properties analogous to that shown in FIG.",
"2.However, it has the serious drawback of generating unwanted reflections on the convex surface of each micro-lens, which makes it practically useless as a first component of the system.",
"FIG.",
"6 shows the retro-reflective screen shown in FIG.",
"5, in which the opaque diffusing surface 23 has been replaced by a translucent diffusing surface 24 and another identical section of convergent spherical micro-lenses, symmetrical with respect to the diffusing plane, has been added.",
"Any beam of rays 719, 720, 721 striking any micro-lens is focussed on the translucent diffusing surface 24.This focussed beam is dispersed by the surface 24, forming a beam of rays 722, 723, 724 which, after passing through the second section, continues in a direction symmetrical to the incident rays 719, 720, 721.As may be observed, the convergent beam is perfectly separated from the divergent beam and said property may be used to illuminate on one side and observe on the other, thus preventing the unwanted reflections mentioned in the description of FIG.",
"5.This group of a double section of micro-lenses 121-24-122, which hereinafter we shall call 12, is considered as a variant of the first and second components of the system that is the object of this invention.",
"The optical effect is the same as that obtained by a convergent micro-lens with two convergent cylindrical lenses whose axes are perpendicular to each other.",
"In this way, one or both of the above-mentioned spherical sections may be replaced by a pair of convergent cylindrical sections with the same focal distances and equal to that of the spherical micro-lens, with their convex sides facing each other and one next to another.",
"FIG.",
"7 shows the compound system 12 carrying out the same function as that effected by the first and second component of the system described in this invention.",
"The light rays 725, 726, 727, 728, 729 emitted by any light source 44 affect the optical system 12.This divergent light beam is refracted by this optical system 95, forming a beam of rays 730, 731, 732, 733, 734 that converge at the point 95.The optical effect is to spatially separate the point 44, from which the light rays emerge, from the point 95 where they converge.",
"The rays emerging from any other light sources converge at other points situated elsewhere.",
"FIG.",
"8 shows a screen 13 designed by Ives, which is back-reflecting in the horizontal plane and diffusing in the vertical plane.",
"Each reflecting element of which it is made up has two specular surfaces perpendicular to each other 131, 25 in an arrangement analogous to that shown in FIG.",
"1.One of said specular surfaces is flat 131 and the other 25 is a lens section of specular and divergent horizontal cylinders.",
"This screen 13 may be used as a retro-reflective element and as a separating element, the first and second components of the system that is the object of this invention, for applications in which observers are never situated one behind another and for reproductions with only horizontal parallax.",
"Separation of the divergent incident beam 730 from the convergent reflected beam is due to the fact that the screen diffuses the light vertically 731, 732, 733.Therefore it is easy to situate the points from which the light diverges at a place that is vertically separated from the points at which it converges.",
"FIG.",
"9 shows a retro-reflective screen in the horizontal plane and a diffusing screen in the vertical plane.",
"Each reflecting element of which it is made up has three specular surfaces 86, 87, 26 in an arrangement analogous to that shown in FIG.",
"2.Two of said surfaces 86 and 87 are flat and the third 26 is divergent and cylindrical.",
"This screen 14 can be used as a retro-reflective element and as a separating element, the first and second components of the system that is the object of this invention, for applications in which observers are always situated side by side and never one behind another and for reproductions with only horizontal parallax.",
"Separation of the divergent incident beam 734, 735 from the reflected beam is due to the fact that the screen diffuses the light vertically 736, 737.Therefore it is easy to situate the points from which the light diverges at a place that is vertically separated from the points at which it converges.",
"FIG.",
"10 shows a lens section working by refraction made up of vertical cylinders 151, 152, 153, 154 in whose focal plane 27 an opaque diffusing surface is situated.",
"Any incident beam of rays 738, 739, 740 is reflected in the same direction but in the reverse path 740, 739 and 738.The group of vertical cylinders and the diffusing plane form a retro-reflective screen with properties analogous to those described above in FIGS.",
"8 and 9.However, it has the serious drawback of generating unwanted reflections on the convex surface of each cylinder, which are difficult to prevent by tilting the screen and which make it practically useless as a first component of the system.",
"FIG.",
"11 shows the retro-reflective screen shown in FIG.",
"10, in which the opaque diffusing surface 27 has been replaced by a translucent diffusing surface 28 and another section of lens section identical to the first and symmetrical with respect to the diffusing plane 28, has been added.",
"Any incident diverging beam of rays 741, 742, 743 is focussed on the translucent diffusing surface 28.This surface disperses the beam, forming a new beam of rays 744, 745, 746 which, after passing through the second section, continues in a direction symmetrical to the incident ray with respect to the translucent surface.",
"The convergent beam is perfectly separated from the divergent beam and said property is used to illuminate on one side and observe on the other, thus avoiding the unwanted reflections mentioned in the description of FIG.",
"10.This screen 161-28-162, which hereinafter we shall call 15, may be used as a retro-reflective element and as a separating element, the first and second components of the system that is the object of this invention, for applications in which observers are never situated one behind another and for reproductions with only horizontal parallax.",
"Although the right-hand part of this figure is equal to the right-hand part of FIG.",
"6, they represent two different optical devices.",
"FIG.",
"6 represents spherical lenses, whereas FIG.",
"11 represents cylindrical lenses, although logically their profile is the same.",
"FIG.",
"12 represents the same optical diagram as FIG.",
"3, to which has been added the image-reproducing element by transparency 31 parallel to the retro-reflective screen 11 and between the latter and the separating element 21.This reproducing element is considered as the third component of the system that is the object of this invention.",
"All incident beams of light 77, 78, 79 are modulated in intensity and colour by the sheet 31 twice.",
"The first time before being reflected in the screen and the second time after being reflected.",
"The convergent beam with the information from a two-dimensional image, after passing through the sheet 21, converges at the point 91 where an eye may be situated that will see the image reproduced in 31 when the light source 41 is active.",
"Analogously for the source 42 and the eye 92.The eyes 91 and 92 could correspond to those of a single observer.",
"This observer would see a different image with each eye if, when the source 41 is activated, the image corresponding to that eye were reproduced in 31.Moments later 41 will be deactivated and 42 activated at the instant in which the screen 31 reproduces the image corresponding to the other eye.",
"If the image-reproducing element 31 is situated between the observer 91, 92 and the separating element 21, for example at E31, the modulation of light in intensity and colour is carried out only once.",
"In this case a second retro-reflective screen E11 may also be situated, which would duplicate the light energy reaching the observers.",
"FIG.",
"13 represents the same optical diagram as FIG.",
"3, to which has been added the image-reproducing element by diffusion 32 parallel to the retro-reflective screen 11 and of the same size as the latter.",
"The light source 41 of FIG.",
"12 has been replaced by a flat mirror situated at the same place 43.The image-producing screen by diffusion 32 is considered as a variant of the third component of the system that is the object of this invention.",
"All beams of rays 77, 78, 79 coming from the diffusing screen 32 with the information from the reproduced image will strike the flat mirror 43 where they will be reflected, forming a beam of divergent rays which will be reflected firstly in the semi-transparent sheet 21 and then in the retro-reflective surface 11 to form the beam of convergent rays 710, 711, 712 which will meet at the point 91 where an eye may be situated.",
"It must be pointed out that this FIG.",
"13 represents only an optical diagram which serves to explain the operation of the system that is the object of this invention with reproductions of images upon a diffusing screen, such as images reproduced with the help of cathode ray tubes, plasma screens, cinema or video projectors, led screens or diffusely back-lit liquid crystal screens.",
"The system will be represented hereinbelow closer to its real-life operation.",
"It should be remarked that reproductions of images on diffusing surfaces require that the element 11 should be a specular screen made up of trios of flat mirrors perpendicular to each other, as we explained in FIG.",
"2.To double the light flow reaching the observer, another retro-reflective element E11 may be situated.",
"FIG.",
"14 represents the same optical diagram as FIG.",
"4, to which has been added, as a third component of the system, the image-reproducing screen by transparency 31 parallel to the retro-reflective screen 11.All incident beams of rays 713, 714, 715 are modulated in intensity and colour by the sheet 31 twice.",
"The first time before being reflected in the screen 11 and the second time after being reflected.",
"The convergent beam with the information from the two-dimensional image reproduced in 31 converges at the point 94, where an eye may be situated that will see the image when the light source 44 is active.",
"FIG.",
"15 represents the same optical diagram as FIG.",
"7, to which has been added the image-reproducing element by transparency 31 parallel to the refracting optical system 13.All divergent incident beams 725, 726, 727, 728, 729 emerging from the source 44, after passing through the system 13, are modulated in intensity and colour by the sheet 31.The convergent beam 730, 731, 732, 733, 734 with the information from the two-dimensional image reproduced at 31, converges at the point 95, where an eye may be situated.",
"If the eyes 95 and 96 correspond to those of an observer, the latter would see a different image with each eye.",
"When the source 44 is activated, the image corresponding to that eye 95 is reproduced at 31.After the source 44 is de-activated, 45 is activated and the image corresponding to eye 96 is reproduced at 31.FIG.",
"16 shows different types of image-discriminating elements.",
"These discriminating elements constitute the fourth component of the system that is the object of this invention.",
"In the optical diagrams shown in FIGS.",
"12, 13 and 14, each eye 91, 92, 94 may be considered as a convergence point of light beams which originated at another point 41, 42, 43, 44, spatially separated from the first ones.",
"In FIG.",
"12 the light sources 41 and 42 can be activated and de-activated alternately, and each eye 91, 92 of an observer would see the image reproduced on the reproduction screen 31 according to that alternation.",
"When the source 41 was active, the source 42 would be de-activated and the eye situated at 91 would see the image reproduced at 31.Analogously, when the source 42 was active, the source 41 would be de-activated and only the eye 92 would see the image.",
"In this way, alternating the sources 41 and 42 functions as a discriminating element and therefore these sources 46, 47 have been included as a discriminating element in the upper part of FIG.",
"16.This alternation may also be achieved with a single light source 472 in front of which a shutter 471 has been placed.",
"This shutter will be able to block the right half, leaving the left half transparent and vice versa.",
"In the diagram shown in FIG.",
"13 a shutter may be used such as the foregoing 481, acting as a discriminating element by being situated in front of the mirror 482 which replaced 43 and achieving the same effect.",
"However, as in FIG.",
"13 the reproduction is made upon a diffusing surface, the possibility exists of reproducing upon said surface a pair of stereoscopic images projected with polarised light, each one in a polarisation plane perpendicular to that of the other.",
"In this case, the discriminating element could be two filters 491 polarised in the same perpendicular planes as above, and a flat mirror 492 to replace 43.In the case of three-dimensional reproductions in which one needs to reproduce ten or several tens of two-dimensional images, again, as many light sources 410 as images for each observer or an electronic shutter 4111 could be used as a discriminating element and a single light source 4112 in which the transparent window moves from left to right or from right to left in synchrony with the projection of the different images.",
"The width of the light source, or of the transparent window in the case of shuttering, may be different sizes according to the observation distance of each observer; in this way, parallax can be accommodated to the observation distance.",
"Finally, in the case of integral reproductions, the discriminating element could be a series of light sources 412 whose lighting up cadence would be from left to right and from up to down or an electronic shutter 4131 in front of a single source 4132 whose transparent observation window is a square which moves from left to right and from up to down.",
"In both cases the lighting up of the source or the movement of the transparent window will be carried out in synchrony with the reproduced image.",
"The size of the source and of the transparent window may be made different for each observer according to his observation distance, and parallax can be accommodated to that distance.",
"Although the luminous area of the sources has been represented in all the figures with a circular shape, in reality it may have any other shape, the most logical one being rectangular.",
"In order to synchronise the lighting up of the sources or the movement of the transparent window of the shutter, it will be necessary to use electronic means, which constitute the fifth component of the system.",
"This fifth component will carry out the following functions: generate the images for the reproduction screen, emit a synchronising signal for the lighting up of the source or the opening of the shutter's transparent window and, finally, command the opening of said window.",
"The transmission of the synchronising signal may be done electro-magnetically, by radio or light waves, electrically, through a connection cable or electro-acoustically.",
"The discriminators described above are effective in stereoscopic reproductions with only two reproduced images, if the observer keeps his head still and in three-dimensional or integral reproductions, if the observer moves within the area of vision covered by the sources or by the shutters.",
"If it is desired that the observer moves freely, it will be necessary to detect the head movement and make the discriminating element to respond appropriately to this movement.",
"This operation will be compulsory in stereoscopic reproductions and optional in three-dimensional or integral reproductions.",
"To carry out said operation electronic means are needed, constituting the sixth component of the system which, using the input data provided by a detector of the observer's head position, are able to generate an output signal to control the discriminator.",
"FIG.",
"17 shows the response of the discriminators to the observer's head movement.",
"In the case of stereoscopic images and if different illumination sources are used as a discriminating element, the latter are to be arranged in a row 410, as indicated in said FIG.",
"17.The width of the row must be sufficient to cover the whole area in which the observer may move.",
"Depending on the position of the observer's nose, line 01, the lighting up of the sources situated to the left of said line will synchronise with the reproduction cycles of the image of that eye, and analogously with the sources situated to the right of 01 and the right eye.",
"The movement of the observer to left or right is translated into the movement of the line 01 to left or right, as shown in this figure.",
"In the case of stereoscopic images and if a shutter is used, such as 471, in correspondence with the movement to left or right of the observer's nose, the vertical line 02 separating the transparent window from the opaque one or the vertical line 03 separating the polarisation planes that are perpendicular to each other in the shutter 491 will move at the same speed and the same distance.",
"In the case of three-dimensional or integral reproductions, it will be necessary to act upon the discriminating elements only if the area of possible position for each eye is greater than the area covered by the discriminator, in which case the space covered by the shutter 4111 in the three-dimensional reproduction will move horizontally as far as necessary to cover both the observer's eyes when he moves; and in the case of integral reproduction, the area covered by the shutter 4131 will move horizontally and/or vertically over the distance necessary to cover both the observer's eyes.",
"FIGS.",
"18, 19, 20, 21 and 22, which are commented on below, show some examples of the system that is the object of this invention in reproduction rooms for many observers.",
"The possibilities of placing the different components of the system are very great and a special design must be made according to each room and its shape.",
"The remaining figures show simple guiding diagrams.",
"When several discriminating elements may be used in the figures, they are designated by 4**, where each asterisk represents several different digits.",
"FIG.",
"18 shows an arrangement of the different elements of the model for multiple observers.",
"The observers have been situated in tiers so that all of them can see without disturbing each other.",
"A specular overhead projecting screen 11 is used as the first element of the system, such as that shown in FIG.",
"2.A semi-transparent sheet 21, different for each viewer, is used as a separating element.",
"The reproduction is carried out by transparency on the screen 31 whose signal with the images is received from the electronic means 51.This device 51 also generates a signal with information about which image is being reproduced at all times, which will be received by other electronic means 52.The electronic device 52 receives the synchronising signal from 51 and regulates the discriminating element.",
"Optionally, the electronic device 52 can also receive the signal from a monitor of the observer's head 61 and control the discriminating element with this signal.",
"The discriminator can be any of those shown in FIG.",
"16, except for elements 481, 491 and the mirrors 482, 492.In the lower part, a single observer is shown, such as the player on a video game.",
"The elements are the same as in the rest of the drawing, the only difference lying in the situation of the separating element 21, which in this case have been situated close to the reproducing screen 31 and distant from the observer.",
"Modulation of the image in intensity and colour is carried out by the reproducing element 31 twice, once when the light beams are directed towards the screen 11 and again after being reflected in the screen.",
"In the drawing in the lower part, where a single observer is represented, a single modulation can be achieved by situating the reproducing screen 31 at position E31.In this case a second retro-reflective screen may also be situated at E11, so that the light flow reaching the observers will be double that of the first case.",
"FIG.",
"19 shows an arrangement of the different elements of the model for multiple observers.",
"The observers are situated in tiers so that they do not obstruct each others' vision.",
"A specular overhead projecting screen 11 is used as the first element of the system, such as that shown in FIG.",
"2.A single semi-transparent sheet 21 is used as a separating element for all viewers.",
"Reproduction is carried out by transparency on the screen 31 whose image signal is received from the electronic means 51.The electronic device 52 receives the synchronising signal from 51 and regulates the discriminating element.",
"Optionally, the electronic device 52 can also receive the signal from a monitor of the observer's head 61 and control the discriminating element in accordance with this signal.",
"The discriminator can be any of those shown in FIG.",
"16, except for elements 481, 491 and the mirrors 482, 492.The light emerging from this discriminating element undergoes two reflections before reaching the reproducing screen 31.The first occurs in the flat mirror 88 and the second in the separating element, in this case a semi-transparent sheet 21.The object of the mirror 88 is to situate the discriminating elements along and upon the ceiling of the projection room.",
"However, in another arrangement not shown in the drawing, said mirror can be suppressed and the discriminating elements situated symmetrically with the eyes with respect to the sheet 21.Modulation of the image is carried out by the reproducing element 31, situated between the separating element 21 and the back-reflector 11, twice, once when the light beams are directed towards the screen 11 and again after being reflected in the screen.",
"A single modulation can also be achieved if the reproducing screen is situated at E31.In this case a second retro-reflective screen may also be situated at E11, so that the light flow reaching the observers will be double that of the foregoing case.",
"FIG.",
"20 shows an arrangement of the different elements of the model for multiple observers.",
"The observers are situated in tiers so that they do not obstruct each others' vision.",
"This drawing shows the profile of a reproduction room and each observer represents a row of observers.",
"A specular retro-reflective screen 11 is used as the first element of the system, such as that shown in FIG.",
"2.A single semi-transparent sheet 21 is used as a separating element, in the form of a false ceiling, for all viewers.",
"The reproduction is carried out by diffusion on the screen 32 whose image signal is received from the electronic means 51 or from a cinema projector.",
"This device 51 also generates a signal with information about which image is being reproduced on the screen 32 at all times, which will be received by the other electronic means 52.The electronic device 52 receives the synchronising signal from 51 and regulates the discriminator.",
"Optionally, the electronic device 52 can also receive the signal from a monitor of the observer's head 61 and control the discriminator with this signal.",
"The discriminator can be 481-482, 491-492 as shown in FIG.",
"16.FIG.",
"21 is another arrangement with the same elements as those in FIG.",
"18 and with the same features.",
"The difference lies in the situation of the separating element or semi-transparent sheet 21, is now situated as a false ceiling.",
"FIG.",
"22 shows an arrangement of the different elements of the model for multiple observers.",
"The observers are situated in tiers so that they do not obstruct each others' vision.",
"A double section of convergent spherical micro-lenses 12 is used as the first and second elements of the system, joined in the form of a sandwich to a translucent sheet situated in the focal plane of both sections, as described in FIG.",
"7.Reproduction is carried out by transparency on the screen 31 whose image signal is received from the electronic means 51.The electronic device 52 receives the synchronising signal from 51 and regulates the discriminating element.",
"Optionally, the electronic device 52 can also receive the signal from a monitor of the observer's head 61 and control the discriminating element in accordance with this signal.",
"The discriminator can be any of those shown in FIG.",
"16, except for elements 481 and 491 and the mirrors 482 and 492.The light emerging from this discriminating element undergoes two reflections before reaching the optical system, after being reflected in the latter it reaches the reproducing screen 31.These two reflections in the mirrors 88 and 89 serve to situate the space occupied by the discriminating elements in the ceiling of the reproduction room.",
"Other arrangements may be chosen, such as for example, situating the discriminating elements symmetrically with the eyes of the observers with respect to the optical system.",
"In the lower part of this drawing a single observer is shown, such as a player on a video game.",
"The elements are the same as in the rest of the drawing, except for the situation of the discriminating element, which in this case emits its light onto the optical system by means of a single flat mirror 810.FIG.",
"23 shows another arrangement of the different elements of the model for multiple observers.",
"The first element of the system is a specular retro-reflective screen 11, such as that shown in FIG.",
"2.A bi-refringent sheet together with a de-polarising sheet 22 are used as a separating element, both being parallel to the retro-reflective screen 11.Reproduction is carried out by transparency on a reproduction screen 31 whose image signal is received from the electronic means 51.The electronic device 52 receives the synchronising signal from 51 and regulates the discriminating element.",
"Optionally, the electronic device 52 can also receive the signal from a monitor of the observer's head 61 and control the discriminating element in accordance with this signal.",
"The discriminator can be any of those shown in FIG.",
"16, except for elements 481 and 491 and the mirrors 482 and 492.The discriminating elements are situated above the heads of the observers.",
"The light emerging from these discriminating elements before and after its back-reflection in the screen 11 undergoes a double refraction which allows the divergent incident beam to be separated from the convergent reflected beam.",
"These light beams are modulated by the reproducing screen by transparency 31.For the sake of simplicity, in the case of reproducing images by transparency, we have preferred throughout the foregoing description to do so upon a single element by multiplexing in time.",
"In the case of stereoscopic reproductions, that is, reproductions of only two bi-dimensional images, one may, as mentioned above, reproduce each image in a different reproducing element by transparency, that is, by using two reproducing elements.",
"FIG.",
"24 shows a diagrammatic arrangement of said solution analogous to that of FIG.",
"12, using the following: four specular elements E11, E′11, two image-reproducers by transparency E31, E′31, three semi-transparent sheets 21 and two rows of light sources 41, 42, one for each eye.",
"FIG.",
"25 shows the same diagrammatic arrangement as that shown in FIG.",
"24, in which a single light source 41 is used together with an additional semi-transparent sheet 21.This sheet and the one which receives light by reflection from it are turned so that the single row of light sources 42 directs its light to two different places, one for each eye.",
"FIG.",
"26 shows a diagrammatic arrangement analogous to that of FIG.",
"15, but with two refractive elements 12, two image-reproducers by transparency E31, E′31, a semi-transparent sheet 21 and two rows of light sources 41, 42, one for each eye.",
"FIG.",
"27 shows the same diagrammatic arrangement as that shown in FIG.",
"26, in which a single light source 43 is used together with an additional semi-transparent sheet 21 and two flat mirrors 88.This sheet and the one which receives light by reflection from it are turned so that the single row of light sources 42 directs its light to two different places, one for each eye.",
"FIGS.",
"8 and 9 represent two types of retro-reflective screens 13, 14 that are specular in the horizontal plane and diffusing in the vertical plane, which may be used as the first and second components of the model.",
"In this case the reproductions will be solely in horizontal parallax.",
"The separating element is the specular surface of vertical diffusion 25 and 26.The discriminating elements which can be used are those shown in FIG.",
"16, except for 482, 491, 492, 412, 4131, 4132 and they must be situated as indicated in FIG.",
"18 or 23, with the proviso that observers can never be one behind another.",
"FIG.",
"11 shows a type of screen 15 which works by selective refraction in the horizontal plane and diffusion in the vertical plane, which may be used as the first and second components of the model.",
"Only horizontal parallax can be reproduced.",
"The separating element is the vertical diffusion screen itself 28.The discriminating elements which can be used are those shown in FIG.",
"16, except for 482, 491, 492, 412, 4131, 4132.FIG.",
"22 shows a possible physical positioning of the elements with the proviso that observers can never be one behind another."
]
] | Patent_10168652 |
[
[
"Compounds and methods for the treatment of pain",
"2-substituted 7-chloro-4-hydroxy-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione compounds, pharmaceutical compositions and methods for the treatment of pain are disclosed."
],
[
"1.A compound selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"2.A method for treating a subject suffering from pain comprising administering a pain-ameliorating effective amount of a compound selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)ethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"3.A pharmaceutical composition comprising a pain-ameliorating effective amount of a compound selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)ethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; together with a pharmaceutically-acceptable excipient or diluent;"
],
[
"<SOH> FIELD OF THE INVENTION <EOH>This invention relates to the treatment or prevention of pain or nociception."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It has now been discovered that certain compounds which exhibit the property of binding to the NMDA receptor glycine site have utility for the amelioration of pain and particularly for the amelioration of neuropathic pain.",
"Therefore, in a first aspect the present invention provides compounds, or pharmaceutically-acceptable salts thereof, selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"In another aspect the invention comprises a pharmaceutical composition comprising a pain-ameliorating effective amount of a compound, or a pharmaceutically-acceptable salt thereof, selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)eth-1-yl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(−1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; together with a pharmaceutically-acceptable excipient or diluent; In a further aspect, the invention provides a method for the treatment of pain comprising administering a pain-ameliorating effective amount of a compound selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,0-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)ethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"Other aspects of the invention are methods for making the compounds disclosed herein.",
"Yet other aspects of the invention are pharmaceutical compositions which contain a compound disclosed herein; the use of such compounds for the preparation of medicaments and pharmaceutical compositions, and a method comprising binding a compound of the invention to the NMDA receptor glycine site of a warm-blooded animal, such as a human being, so as to beneficially inhibit the activity of the NMDA receptor.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION This invention relates to the treatment or prevention of pain or nociception.",
"RELATED ART Pain is a sensory experience distinct from sensations of touch, pressure, heat and cold.",
"It is often described by sufferers by such terms as bright, dull, aching, pricking, cutting or burning and is generally considered to include both the original sensation and the reaction to that sensation.",
"This range of sensations, as well as the variation in perception of pain by different individuals, renders a precise definition of pain difficult, however, many individuals suffer with severe and continuous pain.",
"Pain that is caused by damage to neural structures is often manifest as a neural supersensitivity or hyperalgesia and is termed “neuropathic” pain.",
"Pain can also be “caused” by the stimulation of nociceptive receptors and transmitted over intact neural pathways, such pain is termed “nociceptive” pain.",
"The level of stimulation at which pain becomes noted is referred to as the “pain threshold.” Analgesics are pharmaceutical agents which relieve pain by raising the pain threshold without a loss of consciousness.",
"After administration of an analgesic drug a stimulus of greater intensity or longer duration is required before pain is experienced.",
"In an individual suffering from hyperalgesia an analgesic drug may have an anti-hyperalgesic effect.",
"In contrast to analgesics, agents such as local anaesthetics block transmission in peripheral nerve fibers thereby blocking awareness of pain.",
"General anaesthetics, on the other hand, reduce the awareness of pain by producing a loss of consciousness.",
"Tachykinin antagonists have been reported to induce antinociception in animals, which is believed to be analogous to analgesia in man (Maggi et al, J. Auton.",
"Pharmacol.",
"(1993) 13, 23-93).",
"In particular, non-peptide NK-1 receptor antagonists have been shown to produce such analgesia.",
"For example, the NK-1 receptor antagonist RP 67,580 produced analgesia with potency comparable to that of morphine (Garret et al, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA (1993) 88, 10208-10212).",
"The opioid analgesics are a well-established class of analgesic agents with morphine-like actions.",
"Synthetic and semi-synthetic opioid analgesics are derivatives of five chemical classes of compound: phenanthrenes; phenylheptylamines; phenylpiperidines; morphinans; and benzomorphans.",
"Pharmacologically these compounds have diverse activities, thus some are strong agonists at the opioid receptors (e.g.",
"morphine); others are moderate to mild agonists (e.g.",
"codeine); still others exhibit mixed agonist-antagonist activity (e.g.",
"nalbuphine); and yet others are partial agonists (e.g.",
"nalorphine).",
"Whilst an opioid partial agonist such as nalorphine, (the N-alkyl analogue of morphine) will antagonize the analgesic effects of morphine, when given alone it can be a potent analgesic in its own right.",
"Of all of the opioid analgesics, morphine remains the most widely used, but, in addition to its therapeutic properties, it has a number of drawbacks including respiratory depression, decreased gastrointestinal motility (resulting in constipation), nausea and vomiting.",
"Tolerance and physical dependence also limit the clinical uses of opioid compounds.",
"Aspirin and other salicylate compounds are frequently used in treatment to interrupt amplification of the inflammatory process in rheumatoid diseases and arthritis and temporarily relieve the pain.",
"Other drug compounds used for these purposes include phenylpropionic acid derivatives such as Ibuprofen and Naproxen, Sulindac, phenyl butazone, corticosteroids, antimalarials such as chloroquine and hydroxychloroquine sulfate, and fenemates (J. Hosp.",
"Pharm., 36:622 (May 1979)).",
"These compounds, however, are ineffective for neuropathic pain.",
"Available therapies for pain also have drawbacks.",
"Some therapeutic agents require prolonged use before an effect is experienced by the patient.",
"Other existing drugs have serious side effects in certain patients, and subjects must be carefully monitored to ensure that any side effects are not unduly threatening.",
"Most existing drugs provide only temporary relief from pain and must be taken consistently on a daily or weekly basis.",
"With disease progression the amount of medication needed to alleviate the pain often increases, thus increasing the potential for adverse side effects.",
"NMDA receptors are defined by the binding of N-methyl-D-aspartate (NMDA) comprise a receptor/ion channel complex with several different identified binding domains.",
"NMDA itself is a molecule structurally similar to glutamate (Glu) which binds at the glutamate binding suite and is highly selective and potent in activating the NMDA receptor (Watkins (1987); Olney (1989)).",
"Many compounds are known that bind at the NMDA/Glu binding site (for example CPP, DCPP-ene, CGP 40116, CGP 37849, CGS 19755, NPC 12626, NPC 17742, D-AP5, D-AP7, CGP 39551, CGP43487, MDL-100,452, LY-274614, LY-233536, and LY233053).",
"Other compounds, referred to as non-competitive NMDA antagonists, bind at other sites in the NMDA receptor complex (examples are phencyclidine, dizocilpine, ketamine, tiletamine, CNS 1102, dextromethorphan, memantine, kynurenic acid, CNQX, DNQX, 6,7-DCQX, 6,7-DCHQC, R(+)—HA-966, 7-chloro-kynurenic acid, 5,7-DCKA, 5-iodo-7-chloro-kynurenic acid, MDL-28,469, MDL-100,748, MDL-29,951, L-689,560, L-687,414, ACPC, ACPCM, ACPCE, arcaine, diethylenetriamine, 1,10-diaminodecane, 1,12-diaminododecane, ifenprodil, and SL-82.0715).",
"These compounds have been extensively reviewed by Rogawski (1992) and Massieu et.",
"al., (1993), and articles cited therein.",
"In addition to its physiological function, glutamate (Glu) can be neurotoxic.",
"Glu neurotoxicity is referred to as “excitotoxicity” because the neurotoxic action of Glu, like its beneficial actions, is mediated by an excitatory process (Olney (1990); Choi (1992)).",
"Normally, when Glu is released at a synaptic receptor, it binds only transiently and is then rapidly removed from the receptor by a process that transports it back into the cell.",
"Under certain abnormal conditions, including stroke, epilepsy and CNS trauma, Glu uptake fails and Glu accumulates at the receptor resulting in a persistent excitation of electrochemical activity that leads to the death of neurons that have Glu receptors.",
"Many neurons in the CNS have Glu receptors, so excitotoxicity can cause an enormous amount of CNS damage.",
"Acute excitotoxicity injury can occur as a result of ischemic events, hypoxic events, trauma to the brain or spinal cord, certain types of food poisoning which involve an excitotoxic poison such as domoic acid, and seizure-mediated neuronal degeneration, which can result from persistent epileptic seizure activity (status epilepticus).",
"A large body of evidence has implicated the NMDA receptor as one receptor subtype through which Glu mediates a substantial amount of CNS injury, and it is well established that NMDA antagonists are effective in protecting CNS neurons against excitotoxic degeneration in these acute CNS injury syndromes (Choi (1988); Olney (1990)).",
"In addition to neuronal damage caused by acute insults, excessive activation of Glu receptors may also contribute to more gradual neurodegenerative processes leading to cell death in various chronic neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, AIDS dementia, Parkinson's disease and Huntington's disease (Olney (1990)).",
"It is generally considered that NMDA antagonists may prove useful in the therapeutic management of such chronic diseases.",
"In the 1980's it was discovered that PCP (also known as “angel dust”) acts at a “PCP recognition site” within the ion channel of the NMDA Glu receptor.",
"PCP acts as a non-competitive antagonist that blocks the flow of ions through the NMDA ion channel.",
"More recently it has become evident that drugs which act at the PCP site as non-competitive NMDA antagonists are likely to have psychotomimetic side effects.",
"Further, it is now recognized that certain competitive and non-competitive NMDA antagonists can cause similar pathomorphological effects in rat brain (Olney et.",
"al., (1991); Hargreaves et.",
"al., (1993)).",
"Such compounds also have psychotomimetic effects in humans (Kristensen et.",
"al., (1992); Herrling (1994); Grotta (1994)).",
"The glycine binding site of the NMDA receptor complex is distinguishable from the Glu and PCP binding sites.",
"Also, it has recently been discovered that NMDA receptors occur as several subtypes which are characterized by differential properties of the glycine binding site of the receptor.",
"Many compounds that bind at the NMDA receptor glycine site, useful for the treatment of stroke and neurodegenerative conditions, have been described in U.S. Pat.",
"Nos.",
"5,604,227; 5,733,910; 5,599,814; 5,593,133; 5,744,471; 5,837,705 and 6,103,721.SUMMARY OF THE INVENTION It has now been discovered that certain compounds which exhibit the property of binding to the NMDA receptor glycine site have utility for the amelioration of pain and particularly for the amelioration of neuropathic pain.",
"Therefore, in a first aspect the present invention provides compounds, or pharmaceutically-acceptable salts thereof, selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"In another aspect the invention comprises a pharmaceutical composition comprising a pain-ameliorating effective amount of a compound, or a pharmaceutically-acceptable salt thereof, selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)eth-1-yl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-[2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(−1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; together with a pharmaceutically-acceptable excipient or diluent; In a further aspect, the invention provides a method for the treatment of pain comprising administering a pain-ameliorating effective amount of a compound selected from: 7-chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,0-dione; 7-chloro-4-hydroxy-2-(tetrahydrofuran-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[5-(4-methoxyphenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 7-chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)ethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione; 4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde; N-(1-aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide; 2-({4-2-aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione; N-(-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide, and 2-{[4-(2-aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione.",
"Other aspects of the invention are methods for making the compounds disclosed herein.",
"Yet other aspects of the invention are pharmaceutical compositions which contain a compound disclosed herein; the use of such compounds for the preparation of medicaments and pharmaceutical compositions, and a method comprising binding a compound of the invention to the NMDA receptor glycine site of a warm-blooded animal, such as a human being, so as to beneficially inhibit the activity of the NMDA receptor.",
"DETAILED DESCRIPTION OF THE INVENTION Compounds of the invention are those exemplified hereafter.",
"Suitable pharmaceutically-acceptable salts of compounds of the invention include acid addition salts such as methanesulphonate, fumarate, hydrochloride, hydrobroncide, citrate, tris(hydroxymethyl)aminomethane, maleate and salts formed with phosphoric and sulphuric acid.",
"In other embodiments, suitable salts are base salts such as an alkali metal salts for example sodium, alkaline earth metal salts for example calcium or magnesium, organic amine salts for example triethylamine, morpholine, N-methylpiperidine, N-ethylpiperidine, procaine, dibenzylamine, choline, N,N-dibenzylethylamine or amino acids such as lysine.",
"To use a compound of the invention or a pharmaceutically-acceptable salt thereof for the therapeutic treatment, which may include prophylactic treatment, of pain in mammals, which may be humans, the compound can be formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.",
"Suitable pharmaceutical compositions that contain a compound of the invention may be administered in conventional ways, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal administration or by inhalation.",
"For these purposes a compound of the invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.",
"A preferred route of administration is orally by tablet or capsule.",
"In addition to a compound of the present invention a pharmaceutical composition of this invention may also contain one or more other pharmacologically-active agents, or such pharmaceutical composition may be simultaneously or sequentially co-administered with one or more other pharmacologically-active agents.",
"Pharmaceutical compositions of this invention will normally be administered so that a pain-ameliorating effective daily dose is received by the subject.",
"The daily dose may be given in divided doses as necessary, the precise amount of the compound received and the route of administration depending on the weight, age and sex of the patient being treated and on the particular disease condition being treated according to principles known in the art.",
"A preferred dosage regime is once daily.",
"A further embodiment of the invention provides a pharmaceutical composition which contains a compound of the invention as defined herein or a pharmaceutically-acceptable salt thereof, in association with a pharmaceutically-acceptable additive such as an excipient or carrier.",
"A yet further embodiment of the invention provide the use of a compound of the invention, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament useful for binding to the NMDA receptor glycine site in a warm-blooded animal such as a human being.",
"Still another embodiment of the invention provides a method of binding a compound of the invention to the NMDA receptor glycine site of a warm-blooded animal, such as a human being, in need of treatment for pain, which method comprises administering to said animal an effective amount of a compound of structural diagram I or a pharmaceutically-acceptable salt thereof.",
"Definitions: Generally in the methods, processes and examples described herein: concentrations were carried out by rotary evaporation in vacuo; operations were carried out at ambient temperature, that is in the range 18-26° C. and under a nitrogen atmosphere; column chromatography (by the flash procedure) was performed on Merck Kieselgel silica (Art.",
"9385) unless otherwise stated; yields are given for illustration only and are not necessarily the maximum attainable; the structure of the end-products of the formula I were generally confirmed by NMR and mass spectral techniques, proton magnetic resonance spectra were determined in DMSO-d6 unless otherwise stated using a Varian Gemini 2000 spectrometer operating at a field strength of 300 MHz; chemical shifts are reported in parts per million downfield from tetramethylsilane as an internal standard (δ scale) and peak multiplicities are shown thus: s, singlet; bs, broad singlet; d, doublet; AB or dd, doublet of doublets; t, triplet, dt, double of triplets, m, multiplet; bm, broad multiplet; fast-atom bombardment (FAB) mass spectral data were obtained using a Platform spectrometer (supplied by Micromass) run in electrospray and, where appropriate, either positive ion data or negative ion data were collected, in this application, (M+H)+ is quoted; IR data was obtained with a Nicolet Avatar 360 FT-IR; intermediates were not generally fully characterized and purity was in general assessed mass spectral (MS) or NMR analysis.",
"The following abbreviations and definitions when used, have the meanings, as follows: CDCl3 is deuterated chloroform; CMC is 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate; DCM is dichloromethane; DCU is dicyclohexyl urea; DHC is 1,3-dicyclohexylcarbodiimide; DMAP is 4-(dimethylamino)pyridine; DMF is N,N-dimethylformamide; DMSO is dimethylsulphoxide; m/s is mass spectroscopy; NMP is N-methylpyrrolidinone; NMR is nuclear magnetic resonance; p.o.",
"is per os; THF is tetrahydrofuran, and t.i.d.",
"is three times daily.",
"The examples and tests described herein are intended to illustrate but not limit the invention.",
"EXAMPLES Example 1 7-Chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione 2-p-Tolyl-[1,3,4]oxadiazole Triethyl orthoformate (5 mL) was added to p-toluic hydrazide (2.0 g, 13.3 mmol) and the solution was heated to reflux for 6 hours.",
"The volatiles were removed under reduced pressure, and the organic materials dissolved in ethyl acetate (100 mL).",
"The organic layer was extracted with 10% aqueous potassium carbonate, washed with aqueous sodium chloride (saturated, 1×10 mL), and dried over Na2SO4.The organic layer was filtered and removed under reduced pressure to give the title compound as a tan solid (2.1 g, 99%).",
"1H NMR (300 MHz, DMSO-d6): δ 2.40 (s, 3H); 7.42 (d, 2H, J=8.1 Hz); 7.88 (d, 2H, J=8.1 Hz); 9.30 (s, 1H).",
"2-(4-Bromomethyl-phenyl)-[1,3,4]oxadiazole 2-p-Tolyl-[1,3,4]oxadiazole (1.44 g, 9.03 mmol) was slurried in carbon tetrachloride (40 mL) and to this was added N-bromosuccinimide (1.68 g, 9.45 mmol), and benzoyl peroxide (0.21 g, 0.90 mmol).",
"The mixture was heated to reflux for 6 hours, and then cooled to room temperature.",
"The solids were filtered off and discarded.",
"The remaining material was dissolved into DCM (100 mL) and extracted with aqueous sodium bicarbonate (saturated, 2×50 mL).",
"The organic layer was washed with aqueous sodium chloride (saturated), and dried over Na2SO4.The organic layer was filtered and removed under reduced pressure to give the title compound as a tan solid (1.70 g, 79%).",
"(Contains about 5-10% of the α,α,-dibrominated material).",
"1H NMR (300 MHz, DMSO-d6): δ 4.79 (s, 2H), 7.67 (d, 2H, J=8.4 Hz); 8.02 (d, 2H, J=8.4 Hz); 9.32 (s, 1H).",
"N′-(4-[1,3,4]Oxadiazol-2-yl-benzyl)-hydrazinecarboxylic Acid tert-butyl Ester 2-(4-Bromomethyl-phenyl)-[1,3,4]oxadiazole (0.56 g, 2.35 mmol) was dissolved in DMF (8 mL) and to this was added tert-butyl carbazate (0.28 g, 2.11 mmol) followed by N,N-diisopropylethylamine (0.33 g, 0.45 mL, 2.62 mmol).",
"The solution was heated to 90° C. for 12 hours and then cooled to room temperature.",
"The material was dissolved into ethyl acetate (150 mL) and extracted with water (3×40 mL) and aqueous sodium chloride (saturated, 1×15 mL).",
"The organic layer was dried over Na2SO4, and evaporated to a thick oil.",
"This residue was chromatographed (SiO2, 1:1 hexanes:ethyl acetate) to give the title compound as a white solid (0.12 g, 20%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.37 (s, 9H); 3.96 (d, 2H, J=3.9 Hz); 4.93 (m, 1H); 7.55 (d, 2H, J=8.4 Hz); 7.93 (d, 2H, J=8.4 Hz); 9.31 (s, 1H).",
"Dimethyl 7-chloro-4-hydroxyquinoline-2,3-dicarboxylate A stirred mixture of methyl 2-amino-4-chlorobenzoate (2.50 g, 13.5 mmol) and dimethyl acetylenedicarboxylate (2.05 g, 14.4 mmol) in tert-butanol (22 ml) was refluxed for 7 hours under a nitrogen atmosphere.",
"After adding additional dimethyl acetylenedicarboxylate (1.16 g, 8.13 mmol) and refluxing another 2.5 hours, the reaction mixture was allowed to cool to room temperature and potassium tert-butoxide (1.56 g, 13.9 mmol) was added in one portion.",
"A precipitate formed and the resulting mixture was refluxed for 1.5 hours.",
"The mixture was cooled to room temperature and filtered to separate the solids, which were washed with tert-butanol and diethyl ether.",
"The solids were dissolved in water and acidified with 1 N sulfuric acid to form a precipitate.",
"The resulting mixture was extracted with DCM and the combined extracts were washed with brine and water, dried over MgSO4, filtered and concentrated to give a green solid.",
"Recrystallization of this material from methanol provided the title compound (1.15 g, 47%) as an off-white solid, mp 232-233° C.; MS (CI):296 (M+H).",
"Analysis for C13H10ClNO5: Calc'd: C, 52.81; H, 3.41; N, 4.74; Found: C, 52.75; H, 3.47; N, 4.69.3-Carbomethoxy-7-chloro-4-hydroxyquinoline-2-carboxylic Acid To a stirred suspension of dimethyl 7-chloro-4-hydroxyquinoline-2,3-dicarboxylate (1.0 g, 3.38 mmol) in water (20 mL) was added an aqueous solution of sodium hydroxide (0.27 g, 6.75 mmol).",
"Upon addition, the suspension dissolved.",
"The reaction mixture was warmed to 60° C. for 1 hour.",
"After this time the reaction was cooled to room temperature and acidified with concentrated hydrochloric acid.",
"The product was then extracted into diethyl ether and ethyl acetate.",
"The organic extracts were dried over MgSO4, filtered and concentrated in vacuo to provide the title compound as a solid (900 mg).",
"This material was purified by recrystallization employing an ethyl acetate/hexane co-solvent system to provide the title compound (571 mg, 60%) as a white solid mp 296° C. (dec); MS (CI)=238 (M+H).",
"Analysis for C12H8NO5Cl.0.45 CH3CO2CH2CH3.0.10 H2O: Calc'd: C, 51.30; H, 3.68; N 4.34, Found: C, 51.28; H, 3.62; N 3.97 1H NMR δ 8.22 (d, J=8.7 Hz, 1H), 7.92 (d, J=1.8 Hz, 1H), 7.28 (dd, J=8.7, 1.8 Hz, 1H), 3.90 (s, 3H).",
"3-Carbomethoxy-2-pyrrolidinocarbamide-7-chloro-4-hydroxyquinoline To a suspension of 3-carbomethoxy-7-chloro-4-hydroxyquinoline-2-carboxylic acid (2.25 g, 8.0 mmol) in THF (20 mL) at ambient temperature under a N2 atmosphere was added DHC (1.65 g, 8.0 mmol) and pyrrolidine (0.596 g, 8.4 mmol).",
"The reaction was stirred room temperature for 15 hours after which time the by-product urea was removed via filtration.",
"The desired product was purified via flash column chromatography employing 5% methanol in chloroform to provide the title compound (2.52 g, 94.3%) as a tan solid, mp=215° C.; MS (CI): 335 (M+H).",
"300 MHz 1H NMR (DMSO-d6): δ 8.12 (d, J=8.7 Hz, 1H), 7.60 (d, 1H, J=1.8 Hz), 7.47 (dd, 1H, J=8.8, 2.0 Hz), 3.69 (s, 3H), 3.40-3.49 (m, 2H), 3.27-3.33 (m, 2H), 1.80-1.96 (m, 4H).",
"7-Chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic Acid To a suspension of 3-carbomethoxy-2-pyrrolidinocarbamide-7-chloro-4-hydroxy quinoline (2.52 g, 7.5 mmol) in de-ionized water (40 mL) was added dropwise a solution (20 mL) of an aqueous potassium hydroxide (882 mg, 15.75 mmol).",
"Upon complete addition, the reaction was warmed to 60° C. After 3 hours, the reaction was filtered to remove a small amount of insoluble material.",
"The filtrate was then acidified to pH=1 which yield a white precipitate.",
"The solid was isolated by vacuum filtration, washed with water, and dried at 30° C. in vacuo for 16 hours.",
"This provided the title compound (1.5 g, 64%) as a white solid, mp=225-8° C.; MS (CI): 321 (M+H).",
"300 MHz 1H NMR (DMSO-d6): δ 8.28 (d, J=8.8 Hz, 1H), 7.77 (s, 1H), 7.64 (d, 1H, J=8.7), 3.52-3.57 (m, 2H), 3.17-3.19 (m, 2H), 1.83-1.98 (m, 4H).",
"N′-[7-Chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroquinoline-3-carbonyl-N-(4-1,3,4]oxadiazol-2-yl-benzyl)-hydrazinecarboxylic Acid tert-butyl Ester To a stirred slurry of 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid (0.12 g, 0.39 mmol,) in THF (8 mL) was added CMC (0.19 g, 0.45 mmol) and the reaction was stirred for five minutes.",
"To this mixture was added, via dropwise addition, a solution of N′-(4-[1,3,4]oxadiazol-2-yl-benzyl)-hydrazinecarboxylic acid tert-butyl ester (0.11 g, 0.45 mmol) and DMAP (ca.",
"0.003 g) in THF (2 mL).",
"The mixture was then refluxed for 16 hours.",
"The solution was filtered, washed with DCM (5 mL) and the insolubles collected.",
"This insoluble material was then washed with water (50 mL) and then diethyl ether (20 mL).",
"The remaining insoluble material was dried in vacuo to give the title compound as a white solid.",
"(0.20 g, 89%).",
"The material was used in the following reaction without further purification.",
"7-Chloro-4-hydroxy-2-(4-[1,3,4]oxadiazol-2-yl-benzyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione To a stirred solution of N′-[7-chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroquinoline-3-carbonyl]-N′-(4-[1,3,4]oxadiazol-2-yl-benzyl)-hydrazinecarboxylic acid tert-butyl ester (0.20 g, 0.33 mmol) in THF (5 mL) was added methanesulfonic acid (0.80 mL) and the reaction was heated to 70° C. for 1 hour.",
"The mixture was cooled to room temperature and stirred for 16 hours.",
"The resultant orange solid was, collected by filtration and washed with THF.",
"The material was sonicated for 20 minutes in diethyl ether/methyl alcohol (1:1, 5 mL) and then filtered.",
"The material was dried to give the title compound as an off-white solid (0.56 g, 17%).",
"1H NMR (300 MHz, DMSO-d6): δ 2.31 (s, 3H); 5.18 (s, 2H); 7.44 (m, 3H); 7.83 (d, 2H, J=8.1 Hz); 8.04 (s, 1H); 8.14 (d, 1H, J=8.7 Hz); 10.38 (br s, 1H); 11.30 (s, 1H); 11.95 (br s, 1H).",
"Example 2 7-Chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4.5-b]quinoline-1,10-dione.",
"3-(1H-Tetrazol-5-yl)-benzaldehyde 3-Cyanobenzaldehyde (2.0 g, 15.2 mmol) was dissolved in 2-methoxyethanol (8 mL) and to this was added sodium azide (0.91 g, 15.2 mmol) and lithium chloride (0.96 g, 22.8 mmol).",
"The mixture was stirred and heated to reflux for 16 hours and then poured into a mixture of concentrated hydrochloric acid (3 mL) and crushed ice (˜30 g).",
"The thick suspension was stirred until a fine pink solid had formed.",
"The solids were filtered, washed with water and diethyl ether to give the title compound (1.21 g, 46%).",
"1H NMR (300 MHz, DMSO-d6): δ 7.86 (t, 1H, J=7.8 Hz); 8.13 (dd, 1H, J=1.5, 7.8 Hz); 8.37 (ddd, 1H, J=1.5, 1.5, 7.8 Hz); 8.56 (d, 1H, J=1.5 Hz); 10.13 (s, 1H).",
"N′-[3-(1H-Tetrazol-5-yl)-benzylidene]-hydrazinecarboxylic Acid tert-butyl Ester 3-(1H-Tetrazol-5-yl)-benzaldehyde (1.21 g, 6.95 mmol) was dissolved in THF (40 mL) and to this was added tert-butyl carbazate (0.96 g, 7.30 mmol).",
"The reaction was stirred at room temperature for 16 hours and the THF removed under reduced pressure.",
"The residual solid was triturated with hexanes to give the title compound as an off-white solid (2.0 g, 100%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.48 (s, 9H); 7.69 (t, 1H, J=7.8 Hz); 7.78 (d, 1H, J=7.3 Hz); 8.03 (d, 1H, J=7.8 Hz); 8.09 (s, 1H); 8.31 (s, 1H); 11.08 (br s, 1H).",
"N′-[3-(1H-Tetrazol-5-yl)-benzyl]-hydrazinecarboxylic Acid tert-butyl Ester N′-[3-(1H-Tetrazol-5-yl)-benzylidene]-hydrazinecarboxylic acid tert-butyl ester (0.50 g, 1.7 mmol) was added to a Parr shaker bottle and slurried in methyl alcohol (20 mL).",
"To this was added 10% palladium-on-carbon (−100 mg) and the reaction was hydrogenated at 42 psi for 5 hours.",
"The catalyst was filtered on diatomaceous earth, washed with methyl alcohol (2×100 mL), and the solvents were removed in vacuo to give a oil.",
"The oil was dissolved in DCM (20 mL) and this was removed under reduced pressure to azeotrope away any residual methyl alcohol.",
"The title compound was isolated as an oil (0.40 g, 60%).",
"1H NMR (300 MHz, DMSO-d6): δ 3.96 (br s, 2H); 4.94 (br s, 1H); 7.50 (m, 2H); 7.89 (m, 1H); 8.01 (s, 1H); 8.27 (br s, 1H).",
"MS (−CI) m/z 289, 290.N′-[7-Chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroguinoline-3-carbonyl]-N′-[3-(1H-tetrazol-5-yl)-benzyl]-hydrazinecarboxylic Acid tert-butyl Ester To a stirred slurry of 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid, Example 1, (0.43 g, 1.34 mmol) in THF (20 mL) was added CMC (0.68 g, 1.60 mmol) and the reaction was stirred for five minutes.",
"To this mixture was added, via dropwise addition, a solution of N′-[3-(1H-tetrazol-5-yl)-benzyl]-hydrazinecarboxylic acid tert-butyl ester (0.42 g, 1.47 mmol) and DMAP (0.05 g, 0.41 mmol) in THF (5 mL), and the mixture was then refluxed for 16 hours.",
"The solution was filtered and the insolubles washed with THF.",
"The THF was removed under reduced pressure to give a sticky yellow oil.",
"This material was chromatographed (SiO2, 90/10) to give the title compound as a yellow solid (0.48 g, 60%).",
"The material was used without further purification in the following reaction.",
"7-Chloro-4-hydroxy-2-[3-(1H-tetrazol-5-yl)-benzyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione To a stirred solution of N′-[7-chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroquinoline-3-carbonyl]-N′-[3-(1H-tetrazol-5-yl)-benzyl]-hydrazinecarboxylic acid tert-butyl ester (0.48 g, 0.81 mmol) in THF (15 mL) was added methanesulfonic acid (1.9 mL) and the reaction was stirred at room temperature for 16 hours.",
"The THF was removed under reduced pressure to yield an orange oil.",
"To this was added diethyl ether (50 mL) and the reaction stirred for 10 minutes.",
"The ether was carefully decanted to leave a thick orange oil.",
"To this was added water (5 mL) and a fluffy yellow precipitate formed.",
"The mixture was stirred for 10 minutes and then filtered.",
"The solids were washed with water (10 mL) followed by diethyl ether (20 mL).",
"The solids were collected and sonicated in methyl alcohol/diethyl ether (1:5, 5 mL) for 10 minutes.",
"The solids were collected and dried under reduced pressure to give the title compound as a cream-colored solid (0.14 g, 41%,m.p.",
">275° C.).",
"1H NMR (300 MHz, DMSO d6): δ 5.22 (s, 2H); 7.44 (d, 1H, J=8.1 Hz); 7.56 (m, 2H); 7.95 (m, 2H); 8.03 (s, 1H); 8.15 (d, 1H, J=8.7 Hz), 11.97 (br s, 1H); 12.69 (br s, 1H).",
"Example 3 7-Chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione Methanesulfonate N-{1-Aza-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]vinyl}(tert-butoxy)-carboxamide Hydrochloride To a stirred solution of tert-butylcarbazate (1.22 g, 9.82 mmol) in THF (40 mL) was added pyridoxal hydrochloride (2.00 g, 9.82 mmol).",
"After 2 h at room temperature, the reaction was refluxed for 4 h, and the solvent was removed in vacuo.",
"The resultant solid was triturated with hexanes and filtered to give the title compound as a white solid (3.00 g, 96%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.50 (s, 9H); 2.59 (s, 3H); 4.70 (s, 2H); 8.19 (s, 1H); 8.51 (s, 1H); 12.06 (s, 1H); 12.72 (s, 1H).",
"(tert-Butoxy)-N-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridylmethyl)]carboxamide N-{1-Aza-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]vinyl}(tert-butoxy)-carboxamide hydrochloride (3.0 g, 9.46 mmol) was dissolved in methyl alcohol (90 mL) and placed in a Parr shaker bottle.",
"To this was added 10% palladium-on-carbon (300 mg) and the reaction was hydrogenated at 40 psi for 24 h. The mixture was filtered through diatomaceous earth, which was then washed with methyl alcohol (3×100 mL).",
"The combine filtrate and washes were concentrated in vacuo.",
"The resulting solid residue was dissolved in DCM (400 mL) and the resulting solution washed with saturated sodium bicarbonate.",
"The organic layer was washed with brine, dried over MgSO4 and concentrated to give the title compound as a white solid (1.4 g, 52%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.39 (s, 9H); 2.53 (s, 3H); 3.16 (s, 1H); 4.16 (s, 2H); 4.61 (s, 2H); 8.11 (s, 1H); 8.76 (br s, 1H).",
"N-[(tert-Butoxy)carbonylamino][7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)(3-hydroquinolyl]-N-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridylmethyl)]carboxamide To a stirred slurry of 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid, Example 1, (1.5 g, 4.94 mmol) in THF (100 mL) was added CMC (2.4 g, 5.68 mmol) and the reaction was stirred for five minutes.",
"To this mixture was added via dropwise addition a solution of (tert-butoxy)-N-[3-hydroxy-5-hydroxymethyl-2-methyl-(4-pyridylmethyl)]carboxamide (1.4 g, 4.94 mmol) and DMAP (0.250 g, 2.04 mmol) in THF (20 mL), and the mixture was stirred at room temperature for 1 hour.",
"The reaction mixture was filtered and the collected solids washed with DCM (2×150 mL).",
"The combined filtrate and washes were washed with water (100 mL) and brine and then dried over MgSO4 and filtered.",
"The filtrate was concentrated to dryness to give the title compound as a yellow foam (1.7 g, 58%).",
"7-Chloro-4-hydroxy-2-[3-hydroxy-5(hydroxymethyl)-2-methyl(4-pyridyl)]methyl-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione Methanesulfonate To a stirred solution of N-[(tert-butoxy)carbonylamino][7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)(3-hydroquinolyl]-N-[3-hydroxy-5-hydroxymethyl-2-methyl-(4-pyridylmethyl)] carboxamide (1.72 g, 1.5 mmol) in THF (40 mL) was added methanesulfonic acid (5 mL) and the reaction was stirred overnight.",
"The volatiles were removed in vacuo and the resultant oil was poured on to crushed ice.",
"To this mixture was carefully added 10 N sodium hydroxide until a solid precipitate formed.",
"The solution was filtered to give an orange solid.",
"The resultant aqueous layer formed a fine precipitate which was collected by filtration, washed with diethyl ether and dried to give the title compound as an off-white powder (m.p.",
"dec at 275° C.).",
"1H NMR (300 MHz, DMSO-d6): δ 2.30 (s, CH3SO3H); 2.39 (s, 3H); 4.84 (s, 2H); 5.21 (s, 2H); 7.45 (dd, 1H, J=2.1, 9.0 Hz); 8.03 (m, 2H); 8.13 (d, 1H, J=7.2 Hz).",
"Calc'd.",
"for C18H13ClN4O3.0.5 CH3SO3H.H2O: C, 49.63; H, 3.87; N, 11.84.Found: C, 49.74; H, 3.84; N, 11.74.Example 4 7-Chloro-4-hydroxy-2-(tetrahydro-furan-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione 4-(2-Hydroxy-ethyl)-piperazine-1-carboxylic Acid tert-butyl Ester 1-(2-Hydroxy-ethyl)-piperazine (2.00 g, 1.88 mL, 15.3 mmol) was dissolved in DCM (80 mL) and to this was added di-tert-butyl dicarbonate (3.35 g, 15.3 mmol).",
"The reaction was stirred at room temperature for 72 hours, diluted with DCM (100 mL) and extracted with water (1×50 mL).",
"The organic layer was then washed with aqueous sodium chloride (saturated, 1×50 mL) and dried over Na2SO4.The organic layer was collected and removed under reduced pressure to give the title compound as an oil (3.5 g, 99%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.38 (s, 9H); 2.39 (m, 6H); 3.28 (t, 4H, J=5.1 Hz); 3.46 (dd, 2H J=6.0 Hz); 4.39 (s, 1H, J=6.0 Hz).",
"4-(2-Hydroxy-ethyl)-piperazine, Polystyrene Bound 4-(2-Hydroxy-ethyl)-piperazine-1-carboxylic acid tert-butyl ester (3.3 g, 14.3 mmol) was dissolved in THF (80 mL) and to this was added solid potassium tert-butoxide (1.57 g, 13.9 mmol).",
"The mixture was stirred for 30 minutes at 0° C., and then at room temperature for 2 hours.",
"The reaction mixture was then transferred to an Erlenmeyer flask which was charged with chloromethylated styrene/divinylbenzene copolymer resin (1% cross linked, 1 meq./gram, 2.8 g).",
"The reaction flask was shaken for 48 hours, and the resin collected by filtration.",
"The resin was washed with THF (2×50 mL), water (2×50 mL), water/DMF (1/1, 2×50 mL), DCM (2×50 mL) and ethyl acetate (2×50 mL).",
"The resin was dried under reduced pressure to give polystyrene resin bound 4-(2-hydroxy-ethyl)-piperazine-1-carboxylic acid tert-butyl ester (3.1 g).",
"IR (on resin) 1696 (s); 1492 (w); 1452 (w); 1242 (s); 1172 (s) cm−1.Polystyrene bound 4-(2-hydroxy-ethyl)-piperazine-1-carboxylic acid tert-butyl ester (3.1 g) was slurried in DCM (50 mL) and trifluoracetic acid (10 mL) at room temperature for 2 hours.",
"The resin was collected by filtration and washed with 2 N ammoniated methanol (2×30 mL), DMF (1×40 mL), and DCM (3×30 mL).",
"The resin was dried under reduced pressure to give the title compound (2.45 g).",
"IR (on resin) 1601 (w); 1493 (w); 1452 (w).",
"7-Chloro-2-chlorocarbonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid Methyl Ester 3-Carbomethoxy-7-chloro-4-hydroxyquinoline-2-carboxylic acid (1.00 g, 3.55 mmol) was slurried in chloroform (25 mL, ethanol free) and to this was added thionyl chloride (˜2 mL).",
"The mixture was heated to reflux for 6 hours, and then cooled to room temperature.",
"The volatiles were removed under reduced pressure to give the title compound as a solid.",
"This material was used in the following reaction without further purification.",
"1H NMR (300 MHz, CDCl3): δ 4.06 (s, 3H); 7.58 (dd, 1H, J=8.1 Hz); 8.06 (dd, 1H, J=1.8 Hz); 8.29 (d, 1H, J=8.1 Hz); 12.59 (s, 1H).",
"7-Chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid, Polystyrene Bound Polystyrene resin-bound 4-(2-hydroxy-ethyl)-piperazine (2.45 g) was slurried in THF (50 mL) and to this was added 7-chloro-2-chlorocarbonyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid methyl ester (1.0 g, 3.55 mmol).",
"The mixture was shaken for 16 hours, and the resin was collected by filtration.",
"The resin was washed with THF (50 mL), DMF/water (1/1, 50 mL), DMF (50 mL), methyl alcohol (50 mL), and methanolic ammonia (0.5 N, 2×50 mL).",
"The resin was dried under reduced pressure to give polystyrene resin bound 7-chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]4-oxo-1,4-dihydroquinoline-3-carboxylic acid methyl ester (2.10 g).",
"IR (on resin) 1656 (w); 1601 (w); 1492 (w); 1467 (w); 1452 (w).",
"Polystyrene resin-bound 7-chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid methyl ester (2.10 g) was added to a mixture of THF (10 mL) and water (4 mL).",
"To this was added potassium hydroxide (0.35 g, 6.3 mmol) and the reaction stirred at reflux for 6 hours.",
"The reaction was quenched by the addition of aqueous hydrochloric acid (3.10 mL, 2N) and the resin collected by filtration.",
"The resin was washed with water (30 mL), DMF/water (1/1, 50 mL), DMF (50 mL), methyl alcohol (50 mL), and DCM (50 mL).",
"The resin was then dried under reduced pressure to give the title compound (0.75 g).",
"IR (on resin) 1666 (w); 1602 (w); 1582 (w); 1492 (w); 1452 (w).",
"N-(1-Aza-2-(2-furanyl)vinyl)(tert-butoxy)carboxamide To a stirred slurry of tert-butylcarbazate (131.5 g, 0.99 mol) in hexane (1000 mL) was added 2-furaldehyde (91.9 g, 0.95 mol).",
"The slurry was refluxed for 2.5 h, and then cooled to room temperature.",
"The resultant tan solid was filtered, dried and used as is in the next reaction (200 g, 99%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.52 (s, 9H); 6.45 (dd, 1H, J=3.3, 1.2 Hz); 6.71 (d, 1H, J=3.3 Hz); 7.47 (d, 1H, J=1.2 Hz); 7.91 (s, 1H).",
"(+/−)—N′-(Tetrahydro-furan-2-ylmethyl)-hydrazinecarboxylic Acid tert-butyl Ester A solution of N′-(1-azz-2-(2-furanyl)vinyl(tert-butoxy)carboxamide (2.05 g, 9.80 mmol) was dissolved in 50 methyl alcohol (50 mL) and charged into a Parr shaker bottle.",
"To this was added palladium on carbon (10%, 0.310 g) and the mixture shaken at 40 psi for 4 hours.",
"The mixture was filtered on diatomaceous earth and solvent removed under reduced pressure to give the title compound as an oil.",
"The material was used in the following reaction without further purification.",
"(+/−)-N′-{7-Chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]-4-oxo-1,4-dihydroquinoline-3-carbonyl}-N′-(tetrahydro-furan-2-ylmethyl)-hydrazinecarboxylic Acid tert-butyl Ester, Polystyrene Bound Polystyrene resin bound 7-chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]4 oxo-1,4-dihydroquinoline-3-carboxylic acid (0.50 g) was added to THF (30 mL) and to this was added CMC (1.0 g, 2.3 mmol).",
"The reaction was allowed to shake for 15 minutes at which point the resin turned bright yellow.",
"To this was added N′-(tetrahydro-furan-2-ylmethyl)-hydrazinecarboxylic acid tert-butyl ester (0.44 g, 2.05 mmol) and N,N-dimethylaminopyridine (0.10 g, 0.08 mmol) and the mixture was shaken for 16 hours.",
"The resin was collected by filtration and washed with water (30 mL), DMF/water (1/1, 50 mL), DMF (30 mL), methyl alcohol (2×30 mL) and DCM (2×30 mL).",
"The resin was dried under reduced pressure to give the title compound (0.40 g).",
"IR (on resin) 1666 (w); 1602 (w); 1582 (w); 1492 (w); 1451 (w).",
"(+/−)-7-Chloro-4-hydroxy-2-(tetrahydro-furan-2-ylmethyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione Polystyrene resin bound N′-{7-chloro-2-[4-(2-hydroxy-ethyl)-piperazine-1-carbonyl]4-oxo-1,4-dihydroquinoline-3-carbonyl}-N′-(tetrahydro-furan-2-ylmethyl)-hydrazinecarboxylic acid tert-butyl ester (0.40 g) was added to THF (15 mL) and to this was added methanesulfonic acid (1 mL).",
"The mixture was shaken at room temperature for 24 hours.",
"The resin was filtered and washed with THF.",
"The solvent was evaporated and the residual oil diluted with of THF (3 mL).",
"This mixture was poured into mixture of ice/water (80 mL) and stirred for 10 minutes.",
"The resultant solid that formed was filtered and washed with diethyl ether (5 mL).",
"The material was dried under reduced pressure to give the title compound as an off-white solid (0.01 g).",
"1H NMR (300 MHz, DMSO-d6): δ 1.65 (m, 1H); 1.85 (m, 3H); 3.61 (m, 1H); 3.75 (m, 1H); 3.87 (m, 1H); 3.95 (m, 1H); 4.20 (m, 1H); 7.42 (d, 1H, J=8.1 Hz); 8.02 (s, 1H); 8.14 (d, 1H, J=8.1 Hz); 12.35 (br, s).",
"MS (+CI) m/z 348/349.Example 5 7-Chloro-4-hydroxy-2-[5-(4-methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione 2-Chloromethyl-5-(4-methoxy-phenyl)-[1,3,4]oxadiazole 4-Methoxybenzoic hydrazide (0.70 g, 4.21 mmol) was dissolved in ethyl alcohol (10 mL) and to this was added 2-chloromethyl acetimidate hydrochloride (Schindler H., Helv.",
"Chim.",
"Acta 37 472-82 1954) (1.0 g, 6.36 mmol).",
"The solution was stirred and heated to reflux for 2 hours.",
"The mixture was dissolved in ethyl acetate (100 mL) and extracted with aqueous sodium bicarbonate (saturated, 1×20 mL), water (1×20 mL) and aqueous sodium chloride (saturated, 1×20 mL).",
"The organic layer was dried over Na2SO4 and solvent removed under reduced pressure to give the title compound as a white solid.",
"(0.83 g, 94%).",
"1H NMR (300 MHz, DMSO-d6): δ 3.86 (s, 3H); 5.12 (s, 2H); 7.16 (d, 2H, J=8.4 Hz); 7.92 (d, 2H, J=8.4 Hz).",
"N′-[5-(4-Methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-hydrazinecarboxylic Acid tert-butyl Ester 2-Chloromethyl-5-(4-methoxy-phenyl)-[1,3,4]oxadiazole (0.83 g, 3.70 mmol) was dissolved in DMF (5 mL) and to this was added tert-butyl carbazate (1.22 g, 9.26 mmol) and N,N-di-iso-propylethylamine (0.71 g, 0.96 mL, 5.55 mmol).",
"The solution was stirred and heated to 90° C. for 5 hours.",
"The mixture was dissolved in ethyl acetate (150 mL) and extracted with water (3×15 mL), and brine (1×20 mL).",
"The material was dried over Na2SO4 and the organic solvent removed under reduced pressure.",
"The residual oil was triturated with hexanes/ethyl acetate (7:3, 10 mL) giving rise to a white precipitate.",
"The precipitate was filtered and washed with small portions of hexanes/ethyl acetate (3×5 mL) giving the title compound as a white solid (0.75 g, 64%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.34 (s, 9H); 3.85 (s, 3H); 4.08 (d, 2H, J=4.2 Hz); 5.27 (m, 1H); 7.15 (d, 2H, J=8.4 Hz); 7.93 (d, 2H, J=8.4 Hz); 8.39 (br s, 1H).",
"N′-[7-Chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroguinoline-3-carbonyl]-N′-[5-(4-methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-hydrazinecarboxylic Acid tert-butyl Ester To a stirred slurry of 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid, Example 1, (0.54 g, 1.66 mmol) in THF (10 mL) was added CMC (0.87 g, 2.07 mmol) and the reaction was stirred for five minutes.",
"To this mixture was added, via dropwise addition, a solution of N′-[5-(4-methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-hydrazinecarboxylic acid tert-butyl ester (0.56 g, 1.75 mmol) and DMAP (0.01 g, 0.08 mmol) in THF (2 mL), and the mixture was then refluxed for 6 hours.",
"The solution was filtered and the insolubles washed with THF.",
"The THF was removed under reduced pressure to give a sticky yellow oil.",
"The material was used without further purification in the following reaction (˜1 gram material isolated).",
"7-Chloro-4-hydroxy-2-[5-(4-methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione To a stirred solution of N′-[7-chloro-4-oxo-2-(pyrrolidine-1-carbonyl)-1,4-dihydroquinoline-3-carbonyl]-N′-[5-(4-methoxy-phenyl)-[1,3,4]oxadiazol-2-ylmethyl]-hydrazinecarboxylic acid tert-butyl ester (0.30 g, 0.48 mmol) in THF (15 mL) was added methanesulfonic acid (1.0 mL) and the reaction was stirred at room temperature for 2 hours.",
"The THF was removed under reduced pressure to yield an orange oil.",
"To this was added diethyl ether (50 mL) and the reaction stirred for 10 minutes.",
"The ether was carefully decanted to leave a thick orange oil.",
"To this was added water (20 mL) and a fluffy yellow precipitate formed.",
"The mixture was stirred for 10 minutes and then filtered.",
"The solids were washed with water (10 mL) followed by diethyl ether (20 mL).",
"The solids were collected and sonicated in methyl alcohol/diethyl ether (1:9, 5 mL) for 10 minutes.",
"The solids were collected and dried under reduced pressure to give the title compound as a cream-colored solid (0.53 g, 25%).",
"1H NMR (300 MHz, DMSO-d6): δ 3.83 (s, 3H); 5.42 (s, 2H); 7.13 (d, 2H, J=8.7 Hz); 7.45 (d, 1H, J=8.7 Hz); 7.89 (d, 2H, J=8.7 Hz); 8.04 (s, 1H); 8.15 (d, 1H, J=8.7 Hz); 12.03 (br s, 1H); 12.89 (br s, 1H).",
"MS (+CI) m/z 452/454.Example 6 (+/−)-7-Chloro-4-hydroxy-2-[1-(4-methoxycarbonylphenyl)ethyl]-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione 4-[1-(tert-Butoxycarbonyl-hydrazino)-ethyl]-benzoic Acid Methyl Ester To a solution of methyl 4-acetylbenzoate (5.20 g, 29.2 mmol) and tert-butylcarbazate (3.85 g, 29.2 mmol) in THF (70 mL) was added concentrated HCl (3 drops).",
"Upon stirring 2 hours a solid formed.",
"At that time the mixture was evaporated to dryness by rotary evaporation.",
"The resulting solid was triturated with hexanes (3×50 mL) and dried in vacuo (500 mTorr, 30° C.) for 30 minutes to give the title compound as an off-white solid (8.36 g, 98%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.49 (s, 9H); 2.22 (s, 3H); 3.34 (s, 1H); 3.86 (s, 3H); 7.87 (d, 2H, Jo=8.4 Hz); 7.97 (d, 2H, Jo=8.4 Hz); 9.97 (s, 1H).",
"4-[1-(tert-Butoxycarbonyl-hydrazino)-ethyl]-benzoic Acid Methyl Ester To a suspension of 4-[1-(tert-butoxycarbonyl-hydrazino)-ethyl]benzoic acid methyl ester (8.35 g, 28.6 mmol) in ethanol (200 mL) was added 10% palladium on carbon (ca.",
"0.5 g) and the mixture was shaken under hydrogen at 50 psi on a Parr shaker for 3.5 hours.",
"All the organic material dissolved during this time.",
"The catalyst was removed by vacuum filtration through diatomaceous earth.",
"The filtrate was evaporated by rotary evaporation.",
"The residual solid was subjected to chromatography (silica gel, gradient of ethyl acetate/DCM beginning from 10/90 and ending at 20/80, respectively) to give the title compound as a white solid (6.41 g, 76%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.18 (d, 3H, J=6.6 Hz); 1.35 (s, 9H); 3.84 (s, 3H); 4.16 (m, 1H); 4.71 (m, 1H); 7.48 (d, 2H, Jo=8.3 Hz); 7.89 (d, 2H, Jo=8.3 Hz); 8.15 (s, 1H).",
"4-(1-{N′-tert-Butoxycarbonyl-N-[7-chloro-4-hydroxy-2-(pyrrolidine-1-carbonyl)-quinoline-3-carboxyl]-hydrazino}-ethyl)-benzoic Acid Methyl Ester To a stirred slurry of 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid, Example 1, (2.15 g, 6.7 mmol) in THF (50 mL) was added CMC (5.72 g, 13.5 mmol) and the reaction was stirred for five minutes.",
"To this mixture was added a solution of 4-[1-(tert-butoxycarbonyl-hydrazino)-ethyl]-benzoic acid methyl ester (1.97 g, 6.7 mmol) and DMAP (0.10 g, 0.84 mmol) in THF (10 mL), and the mixture was heated to reflux for 3 hours.",
"The reaction mixture was allowed to cool, then the solids were filtered off and washed with DCM (2×50 mL).",
"The combined filtrates were evaporated to dryness by rotary evaporation.",
"The residual solid was subjected to chromatography (silica gel, 5/95 methanol/DCM) to give the title compound as an off-white solid (3.53 g, 71%).",
"MS (CI) m/z 597/599.",
"(+/−)-7-Chloro-4-hydroxy-2-[ethyl-1-(4-carbomethoxyphenyl)-1,2,5,10-tetrahydropyridazino[4,5-b]quinoline-1,10-dione To a solution of 4-(1-{N′-tert-butoxycarbonyl-N-[7-chloro-4-hydroxy-2-(pyrrolidine-1-carbonyl)-quinoline-3-carboxyl]-hydrazino}-ethyl)-benzoic acid methyl ester (3.45 g, 5.78 mmol) in THF (50-mL) was added methanesulfonic acid (5 mL).",
"After 18 hours the solvent was removed by rotary evaporation and the product was precipitated by addition of water (100 mL).",
"The solid was collected by vacuum filtration and washed with water (2×50 mL) then diethyl ether (50 mL), and dried in vacuo (500 mTorr, 30° C.) for 1.5 hours.",
"The solid was suspended in diethyl ether (175 mL) and methanol (25 mL) and sonicated for 20 minutes.",
"The solid was collected by vacuum filtration and dried in vacuo (500 mTorr, 30° C.) for 18 hours.",
"The resulting yellow solid (2.05 g, 83%) was found to be of insufficient purity by 1H NMR.",
"A portion of this material (1.73 g) was suspended in acetonitrile (10 mL) and water (10 mL) to which was added 20% choline hydroxide in water dropwise until all the solids dissolved, then purified by preparative HPLC (41.4 mm×25 cm C-18 Dynamax 60A column using a gradient 10-35% of acetonitrile in water with 0.1% TFA over 50 minutes with a flow rate of 20 ml/min, monitoring, 210 nM).",
"Product was precipitated from the fractions containing title material by addition of 6M HCl to pH 3.The solid was collected by vacuum filtration, washed with water (2×50 ml) then diethyl ether (2×50 ml) and dried in vacuo (500 mTorr, 30° C.) for 18 hours to give the title compound as a yellow solid (1.23 g, 59%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.69 (d, 3H, J=6.9 Hz); 3.84 (s, 3H); 6.26 (q, 1H, J=7.2 Hz); 7.38-7.50 (m, 3H); 7.92 (d, 2H, Jo=8.4 Hz); 8.03 (d, 1H, Jm=1.8 Hz); 8.15 (d, 1H, Jo=8.7 Hz); 11.94 (s, 1H); 12.57 (s, 1H).",
"MS (CI) m/z 426/428.Calc'd.",
"for C21H16ClN3O5.0.2 H2O: C 58.74H 3.85 N 9.79.Found C 58.71-58.85H 3.89-3.90 N 9.93-9.95.Example 7 4-[(7-Chloro-4-hydroxy-1,10-dioxo-2.5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde N-{1-Aza-2-[4-(diethoxymethyl)phenyl]vinyl}-(tert-butoxy)carboxamide 4-(Diethoxymethyl)phenyl carboxaldehyde (2.0 g, 10.0 mmol) was dissolved in methyl alcohol and to this was added tert-butyl carbazate (1.33 g, 10 mmol).",
"The reaction was stirred overnight and used without further purification in the next reaction.",
"Analysis: 1H NMR (300 MHz, DMSO-d6): δ 1.15 (t, 6H, J=6.9 Hz); 1.47 (s, 9H); 3.55 (m, 4H); 7.43 (d, 2H, J=8.1 Hz); 7.60 (d, 2H, J=8.1 Hz); 8.01 (br s, 1H); 10.93 (s, 1H).",
"(tert-Butoxy)-N-{[4-((diethoxymethyl)phenyl)methyl]amino}Carboxamide The methanolic solution from the previous step was added to a Parr shaker apparatus and charged with 10% palladium on carbon (0.300 g) and N-{1-aza-2-[4-(diethoxymethyl)phenyl]vinyl}-(tert-butoxy)carboxamide (2.50 g, 7.75 mmol) in methanol (40 mL) was hydrogenated (40 psi) at room temperature for 2 hours.",
"The reaction was filtered through diatomaceous earth and the filtrate evaporated under reduced pressure to give the title compound as a gold oil (2.26 g, 70% for two steps).",
"1H NMR (300 MHz, DMSO-d6): δ 1.13 (t, 6H, J=6.9 Hz); 1.37 (s, 9H); 3.45 (m, 4H); 3.85 (s, 2H); 4.73 (br s, 1H); 7.32 (app s, 4H); 8.22 (br s, 1H).",
"N-{[4-diethoxymethyl)phenyl]methyl}N-[(tert-butoxy)carbonylamino][7-chloro-4-oxo-2-(pyrrolidynlcarbonyl)(3-hydroquinolyl)]carboxamide 7-chloro-4-oxo-2-(pyrrolidinylcarbonyl)hydroquinoline-3-carboxylic acid, Example 1, (2.13 g, 6.66 mmol), was slurried in DCM (60 mL) and to this was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.20 g, 17.0 mmol).",
"The reaction was stirred for five minutes at which point (tert-butoxy)-N-{[4-((diethoxymethyl)phenyl)methyl]amino}carboxamide (2.26 g, 6.99 mmol) and DMAP (0.19 g, 1.55 mmol) were added rapidly as a solution in DCM.",
"The reaction was refluxed for three hours and then stirred at room temperature overnight.",
"The reaction was further diluted with DCM (60 mL) and then extracted with water (30 mL), sodium bicarbonate (1×30 mL) and sodium chloride (20 mL).",
"The organic layer was dried over Na2SO4 and concentrated to give the title compound as a yellow foam.",
"The material was used without further purification.",
"4-[(7-chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde A solution of N-{[4-diethoxymethyl)phenyl]methyl}N-[(tert-butoxy)carbonylamino] [7-chloro-4-oxo-2-(pyrrolidynlcarbonyl)(3-hydroquinolyl)]carboxamide (1.69 g, 2.71 mmol) was dissolved in THF (100 mL) and to this was added methanesulfonic acid (7 mL).",
"The reaction was refluxed for one hour and then the volatiles removed in vacuo.",
"To the residual oil was added diethyl ether (50 mL) and the mixture stirred for five minutes.",
"The ether layer was decanted to leave a brown oil.",
"To this oil was added water (50 mL) and the reaction stirred until a precipitate formed.",
"The precipitate was collected by vacuum filtration, washed with diethyl ether, and dried in vacuo to give the title compound as a yellow solid (0.82 g, 80%).",
"1H NMR (300 MHz, DMSO-d6): δ 5.21 (s, 2H); 7.42 (d, 1H, J=8.7 Hz); 7.49 (d, 2H, J=8.1 Hz); 7.87 (d, 2H, J=8.1 Hz); 8.03 (s, 1H); 8.15 (d, 1H, J=8.7 Hz); 9.98 (s, 1H); 11.96 (s, 1H); 12.71 (s, 1H).",
"MS (+CI) m/z 382/384.Examples 8-11 The compounds of Examples 8-11 were made by the following general procedure: 4-[(7-Chloro-4-hydroxy-1,10-dioxo-2,5-dihydropyridazino[4,5-b]quinolin-2-yl)methyl]benzaldehyde (0.1 g, 0.2 mmol) was slurried in methyl alcohol and to this was added the indicated hydrazine (0.2 mmol).",
"The reaction mixture was refluxed for two hours, and cooled to room temperature.",
"In most cases, a precipitate had formed which was filtered off, washed with diethyl ether and dried in vacuo to give the title compound.",
"If a precipitate did not form, the methyl alcohol was removed in vacuo and the residual solid was sonicated in methyl alcohol/diethyl ether (1:1, 5 mL).",
"This solid was collected by vacuum filtration, washed with diethyl ether and dried in vacuo to give the title compound.",
"Example 8 N-(1-Aza-2-{[4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)(tert-butoxy)carboxamide The title compound was prepared from tert-butyl carbazate as the starting hydrazine (8%).",
"1H NMR (300 MHz, DMSO-d6): δ 1.45 (s, 9H); 5.12 (s, 2H); 7.32 (d, 2H, J=8.1 Hz); 7.43 (d, 1H, J=8.7 Hz); 7.55 (d, 2H, J=8.1 Hz); 7.96 (s, 1H); 8.02 (s, 1H); 8.15 (d, 1H, J=8.7 Hz); 10.86 (br s, 1H); 11.92 (s, 1H); 12.66 (br s, 1H).",
"MS (+CI) m/z 496/498.Example 9 2-{[4-[2-Aza-2-(2-pyridylamino)vinyl]phenyl}methyl)-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione The title compound was prepared from 2-hydrazinopyridine as the starting hydrazine (48%).",
"1H NMR (300 MHz, DMSO-d6): δ 5.12 (s, 2H); 6.75 (t, 1H, J=6.3 Hz); 7.20 (d, 1H, J=8.1 Hz); 7.32 (d, 2H, J=8.1 Hz); 7.42 (d, 1H, J=8.7 Hz); 7.60 (d, 2H, J=8.7 Hz); 7.99 (s, 1H); 8.02 (s, 1H); 8.09 (d, 1H, J=4.8 Hz); 8.14 (d, 1H, J=8.7 Hz); 10.86 (br s, 1H); 11.94 (br s, 1H); 12.67 (br s, 1H).",
"MS (+CI) m/z 473/475.Example 10 N-((-1-aza-2-{4-[(7-chloro-4-hydroxy-1,10-dioxo(2,5-dihydropyridazino[4,5-b]quinolin-2-yl))methyl]phenyl}vinyl)benzamide The title compound was prepared from benzhydrazide as the starting material (41%).",
"1H NMR (300 MHz, DMSO-d6): δ 5.15 (s, 2H); 7.38 (d, 2H, J=8.1 Hz); 7.42 (d, 1H, J=8.7 Hz); 7.55 (m, 3H); 7.68 (d, 2H, J=8.1 Hz); 7.90 (d, 2H, J=8.1 Hz); 8.03 (s, 1H); 8.15 (d, 1H, J=8.7 Hz); 8.43 (s, 1H); 11.83 (s, 1H); 11.95 (br s, 1H); 12.65 (br s, 1H).",
"MS (+CI) m/z 500/502.Example 11 2-{[4-(2-Aza-2-{[(2,4,6-trimethylphenyl)sulfonyl]amino}vinyl)phenyl]methyl}-7-chloro-4-hydroxy-2,5-dihydropyridazino[4,5-b]quinoline-1,10-dione The title compound was prepared from 2,3,6-trimethylsulfonyl hydrazide as the starting material (46%).",
"1H NMR (300 MHz, DMSO-D6): δ 2.22 (s, 3H); 2.62 (s, 6H); 5.10 (s, 2H); 7.02 (s, 2H); 7.28 (d, 2H, J=8.1 Hz); 7.43 (m, 3H); 7.87 (s, 1H); 8.02 (s, 1H); 8.14 (d, 1H, J=8.7 Hz); 11.55 (br s, 1H); 11.96 (br s, 1H); 12.64 (br s, 1H).",
"Tests for Biological Function: Test A: Inhibition of Binding of [3H]-MDL105,519: Binding of compounds to the NMDA receptor glycine site may be assessed by measuring the ability of test compounds to inhibit the binding of tritiated MDL105,519 to brain membranes bearing the receptor.",
"Rat Brain Membranes: The rat brain membranes used in the experiments were obtained from Analytical Biological Services Inc., and were prepared substantially in accordance with the method of B. M. Baron et al., J. Pharmacol.",
"Exp.",
"Ther.",
"250, 162 (1989).",
"Briefly, fresh brain tissue including cerebral cortex and hippocampus from male Sprague Dawley rats was homogenized in 0.32 M sucrose and centrifuged at low speed to separate cellular membranes from other cellular components.",
"The membranes were then washed 3 times using deionized water, followed by treatment with 0.04% Triton X-100.Finally, membranes were washed six times in 50 mM Tris citrate buffer, pH 7.4, and frozen at −80° C. until use.",
"[3H]MDL105,519 (72 Ci/mmol) was purchased from Amersham.",
"Cold MDL105,519 was purchased from Sigma/RBI.",
"Binding assays were performed substantially in accordance with the protocol of B. M. Baron et al., J. Pharmacol.",
"Exp.",
"Ther.",
"279, 62 (1996), as follows.",
"On the day of the experiment, brain membranes were thawed at room temperature and suspended in 50 mM tris acetate buffer, pH 7.4 (“TAB”).",
"Seventy-five micro grams per milliliter protein (by using the BioRad dye) were used for competition binding.",
"The experiments were carried out using 96-well plates.",
"Membranes were incubated with 20 μL of compounds of various concentrations and 1.2 nM [3H]MDL105,519 for 30 minutes at room temperature in a total volume of 250 μL.",
"Non specific binding was determined by using 100 μM of unlabeled MDL105,519.The unlabeled MDL105,519 and compounds were dissolved as 12.5 mM stock solutions in DMSO.",
"Final DMSO concentration in each well was kept below 1%, which concentration was found not to alter the binding results.",
"After incubation, unbound [3H]MDL105,519 was removed by filtration onto GF/B Unifilter plates using a Packard harvester.",
"Filters were washed four times with ice cold TAB (total of 1.2 mL buffer).",
"The plates were dried overnight at room temperature and bound radioactivity was measured on a Packard TopCount after the addition of 45 μL per well of the MICROSCINT O.",
"Human Brain Membranes: Human brain membranes were obtained from Analytical Biological Services Inc., and assays were performed as described for rat membranes.",
"Data analysis: Data was analyzed using a Microsoft Excel spreadsheet and GraphPad Prizm software and potency of compounds is expressed as the Ki (nM).",
"Test B: Formalin test: The Formalin test is an assay that assesses the capacity of a compound to inhibit formalin-induced nociceptive behaviors in rats (D. Dubuisson, et al., Pain 4, 161-174 (1977); H. Wheeler-Aceto et al., Psychopharmacology 104, 3544 (1991); T. J. Coderre, et al., Pain 54, 43-50 (1993)).",
"In the test, two distinctive phases of formalin-induced behaviors are observed.",
"A first phase response, caused by acute nociception to the noxious chemical (formalin) injected into the paw, occurs between zero and five minutes.",
"A quiescent period of 5 to 15 min post injection follows.",
"After the quiescent period a second phase response, caused by sensitization of the central neurons in the dorsal horn, occurs after 15 minutes and lasts up to 60 minutes.",
"Sensitization of the central neurons in the spine augments a noxious afferent input and causes a stronger pain barrage to be transmitted to the brain.",
"Therefore, inhibition of the second phase response indicates a central mechanism of drug action.",
"The procedure for the formalin test may be performed as follows: male rats are placed in a plexiglass chamber and observed for 30-45 min.",
"to observe their baseline activity.",
"Animals would either be pretreated with vehicle or with different doses of a test compound and are dosed with vehicle or test compound three hours prior to injection of 0.05 mL of sterile 1% formalin under the dorsal skin of a hind paw.",
"The number of paw flinches (responses) during the first phase (0-5 min.)",
"and the second phase (20-35 min.)",
"are scored and recorded.",
"Flinch response can be compared with the mean score of a saline control group and calculated as percentage inhibition.",
"The ED50 is the dose of compound which produced 50% inhibition of nociceptive response in the first or second phase response.",
"% inhibition of nociceptive response can be calculated as: 100 × ( number of responses in vehicle group - number of responses in compound group ) ( number of responses in vehicle group ) Student's t-test can be used for statistical analysis to determine the significance of compound effects.",
"Test C: Neuropathic Pain Model (Chronic Constriction Injury): The anti-hyperalgesic properties of a compound may be tested with the Chronic Constriction Injury (“CCI”) model.",
"The test is a model for neuropathic pain associated with nerve injuries that can arise directly from trauma and compression, or indirectly from a wide range of diseases such as infection, cancer, metabolic conditions, toxins, nutritional deficiencies, immunological dysfunction, and musculoskeletal changes.",
"In the model a unilateral peripheral hyperalgesia is produced in rats by nerve ligation (G. J. Bennett, et al., Pain 33, 87-107 (1988)).",
"Procedurally, Sprague-Dawley rats (250-350 g) are anesthetized with sodium pentobarbital and the common sciatic nerve exposed at the level of the mid thigh by blunt dissection through the biceps femoris.",
"A section of nerve (about 7 mm), proximal to the sciatic trifucation, is freed of tissue and ligated at four positions with chromic gut suture, with the suture tied with about 1 mm spacing between ligatures.",
"The incision is closed in layers and the animals allowed to recuperate.",
"Thermal hyperalgesia is measured using a paw-withdrawal test (K. Hargreaves, et al., Pain 32, 77-88 (1988)).",
"To perform the test, animals are habituated on an elevated glass floor and a radiant heat source aimed at the mid-plantar hindpaw (sciatic nerve territory) through the glass floor with a 20 second cut-off to prevent injury to the skin.",
"The latencies for the withdrawal reflex in both hind paws are recorded.",
"In this test, paws with ligated nerves show shorter paw withdrawal latencies compared to the unoperated or sham operated paws.",
"Responses to test compounds are evaluated at different times after oral administration to determine the onset and duration of compound effect.",
"When performing the test, groups of CCI rats would receive either vehicle or the test compound orally three times daily for 5 days.",
"Paw withdrawal latencies can be measured each day 10 min.",
"before and two or three hr.",
"after the first daily dose.",
"Compound efficacy is calculated as mean percentage decrease of hyperalgesia compared to a vehicle-treated group.",
"Compound potencies may be expressed as the minimum effective dose (MED) in mg/Kg/day that yields a % decrease in hyperalgesia that is statistically significant, where the % anti-hyperalgesic effect may be calculated as follows: ( Mean of vehicle group - Mean of compound group ) ( Mean of vehicle group ) × 100 Data analysis can be performed by the multiple means comparison (Dunnett's test).",
"Table 1 shows the results from Test A for the compounds of the invention.",
"TABLE 1 Test A Ki (nM) Ex.",
"1 95.8 Ex.",
"2 42.7 Ex.",
"3 1150 Ex.",
"4 449 Ex.",
"5 146 Ex.",
"6 268 Ex.",
"7 173 Ex.",
"8 124 Ex.",
"9 159 Ex.",
"10 91 Ex.",
"11 227"
]
] | Patent_10168755 |
[
[
"Repressible sterility of animals",
"A construct which allows animals to be bred in captivity but renders them infertile in the wild by allowing reversible control over fertility and reproduction.",
"The construct comprises: a first promoter that is activated in a defined spatial (tissue specific) or temporal manner linked to DNA encoding a transactivating protein; and a second promoter, which is activated by the transacting protein, linked to DNA encoding a blocker molecule which disrupts gametogenesis or embryogenesis.",
"Feeding an animal a molecule that prevents the transactivating protein binding the second promoter controls fertility."
],
[
"1.A method of controlling fertility in an animal comprising the steps of: 1) stably transforming an animal cell or single celled embryo with a construct comprising: a) a first nucleic acid molecule, which is activated in a defined spatio-temporal pattern, and which is operably linked to b) a second nucleic acid molecule, which encodes a transactivating protein; and c) a third nucleic acid molecule, which is operably linked to a fourth nucleic acid molecule, wherein activation of said first nucleic acid molecule controls the expression of the second nucleic acid molecule, which in turn activates the third nucleic acid molecule, which effects the expression of the fourth nucleic acid molecule which encodes a blocker molecule which disrupts gametogenesis or embryogenesis in the animal; and 2) and growing a whole animal directly from that cell or implanting the cell into a host animal, whereby a whole animal develops from the implanted cell.",
"2.The method of claim 1, wherein said first or said fourth nucleic acid molecule is transiently activated or transiently affects development in a defined spatio-temporal pattern.",
"3.The method of claim 1, wherein each of the first, second, third and fourth nucleic acids is genomic DNA, cDNA, RNA, or a hybrid molecule thereof.",
"4.The method of claim 3, wherein the nucleic acid molecule is a full-length molecule, or a biologically active fragment thereof.",
"5.The method of claim 1, wherein the first nucleic acid molecule is a DNA molecule encoding a promoter region.",
"6.The method of claim 5, wherein the promoter is activated only during embryonic development and/or gametogenesis, and is crucial for completion of embryogenic development and/or gametogenesis.",
"7.The method of claim 5, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:1, SEQ.",
"ID NO:8, or SEQ ID NO:60.8.The method of claim 1, wherein the second nucleic acid molecule is a cDNA molecule encoding a tetracycline-responsive transcriptional activator protein.",
"9.The method of claim 8, wherein said tetracycline-responsive transcriptional activator protein comprises the nucleotide sequence of SEQ ID NO:2.10.The method of claim 1, wherein the third nucleic acid molecule encodes a repressible promoter.",
"11.The method of claim 10, wherein the promoter consists of a tet-responsive element (TRE) which is coupled to and tightly regulates a minimal promoter region.",
"12.The method of claim 11, wherein said minimal promoter region is a PminCMV promoter region that comprises the sequence of SEQ ID NO:3.13.The method of claim 1, wherein the fourth nucleic acid molecule encodes a blocker molecule selected from the group consisting of an antisense RNA, a double-stranded RNA (dsRNA), a sense RNA and a ribozyme.",
"14.The method of claim 13, wherein the molecule is dsRNA or sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"15.The method of claim 13, wherein the RNA is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID:61.16.The method of claim 1, wherein said animal cell or said single celled embryo is transformed with said construct by microinjection, transfection or infection, wherein said construct stably integrates into the genome of said cell or said single celled embryo by homologous recombination.",
"17.A nucleic acid molecule, which encodes a promoter and is transiently activated in a defined spatio-temporal pattern, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:8, or SEQ ID NO:60.18.A nucleic acid molecule, which encodes a promoter having: a) a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60; b) a biologically active fragment of the sequence in a); c) a nucleic acid molecule which has at least 85% sequence homology to the sequence in a) or b); or d) a nucleic acid molecule which is capable of hybridizing to the sequence in a) or b) under stringent conditions.",
"19.A nucleic acid molecule that encodes the coding region of a gene including: a) a nucleotide sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO 61; b) a biologically active fragment of any one of the sequences in a); c) a nucleic acid molecule that has at least 85% sequence homology with any one of the sequences disclosed in a) or b); or d) a nucleic acid molecule that specifically hybridizes to any one of the sequences disclosed in a) or b) under stringent conditions.",
"20.A nucleic acid molecule that encodes a blocker molecule that disrupts gametogenesis or embryogenesis in an animal, wherein the blocker molecule is encoded, or partially encoded, by a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23 and SEQ ID NO:61.21.The nucleic acid molecule of claim 20, wherein the blocker molecule is selected from the group consisting of an antisense RNA, a dsRNA, a sense RNA and a ribozyme.",
"22.The nucleic acid molecule of claim 21, wherein the molecule is a dsRNA or a sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"23.A transgenic non-human animal stably transformed with the nucleic acid molecule of claim 17.24.The transgenic non-human animal of claim 23, wherein the animal is selected from the group consisting of fish, mammals, amphibians, and molluscs."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Feral animals are one of the world's major environmental problems.",
"Goats, cats, rabbits and carp are only the more prominent of hundreds of species traded internationally for recreation or agriculture that have escaped into the wild and formed destructive populations.",
"Terrestrial, freshwater and marine ecosystems are all conspicuously degraded by these species, to the extent that public concern over feral animals has become a major issue for industries seeking to introduce new species in order to compete on world markets.",
"A good recent example is the Pacific oyster.",
"Despite the promise of new jobs in coastal communities and an industry that is worth $50-75 million annually, recent applications to expand the geographic area for Pacific oyster mariculture facilities in Australia and the United States have been rejected indefinitely until the problem of feral oysters can be overcome.",
"Even plans to expand the size of the industry in areas where farming already occurs are being blocked for the same reason, following very public and often acrimonious debate between industry and conservation-minded elements of the community.",
"Attempts to solve the problem using current techniques such as triploidy and sterile hybrids have not been successful.",
"Neither technique can guarantee a zero risk of producing feral populations, and both also suffer major technical difficulties.",
"In the case of oysters, for example, animals sterilised via chemical or genetic manipulation of ploidy do not produce significant amounts of roe, which substantially reduces their market value.",
"Moreover, these animals still produce a small number of viable gametes.",
"So the debate continues to focus on whether degraded beaches are an acceptable price for new industries and jobs.",
"Hundreds of species of exotic animals are shipped internationally each day, mainly for recreational purposes.",
"Inevitably, either accidentally and/or through intentional release, some animals will escape, and establish feral populations.",
"Sterilisation prior to importation of such exotics would prevent the establishment of feral populations and remove the risk of forming new problem pest species.",
"A generic means of sterilisation that prevents development of these feral populations would have huge economic and environmental benefits.",
"More recently, the containment of genetically modified animals has caused concern.",
"For example, Salmon containing genes for enhanced production of growth hormones have been produced in Europe, New Zealand and North America.",
"Concern has been expressed about the impact of these fish as “super-competitors”, should they escape and form feral populations.",
"Similar concerns have been expressed about other genetic improvements that deliberately or accidentally enhance competitiveness.",
"This concern has now grown to a point where there is pressure to ban such modified organisms in toto.",
"However, given their economic significance, it may be preferable to have effective biological controls in place which enable these organisms to be contained within a specific locality.",
"A sterile feral construct inserted into the genetically enhanced stock would prevent development of viable feral populations, as well as preventing integration of enhanced genes into populations of wild con-specifics.",
"Accordingly, some of the major benefits that a sterile feral construct would offer include: 1.Provision of a fail-safe system for preventing the establishment of feral populations of exotic species.",
"This could fundamentally change the risk of importing these species, and would reduce public antagonism to farming of those that have the potential to be environmentally destructive.",
"2.Protection of investments in breeding stocks, for example those developed by extensive selective breeding programs.",
"Currently, the commercial advantages from improved stock can be lost when live, reproductively capable animals are marketed (eg oysters, prawns, and sheep).",
"Repressible sterility can be used as a “lock and key” process whereby improved stock could only breed when provided the correct combination of repressers (and optionally inducers) in exactly the right sequence.",
"3.Production of animals for intentional release that are guaranteed to be sterile.",
"Release of such sterile animals has been used as a control mechanism for certain highly fecund pest species, eg.",
"insects.",
"Repressible sterility technology makes it possible to apply similar approaches to other, existing pest species, for which there are currently no “sterile male” equivalents.",
"4.Provision of an effective containment mechanism for genetically modified organisms.",
"Repressible sterility provides just such a security system for future applications of molecular engineering in animal production, yet enables safe propagation of these individuals using conventional rearing facilities.",
"Linking a genetically engineered process (faster growth, longer spawning seasons, etc.)",
"to a repressible sterility construct ensures that genetic enhancements of exotic or native species do not enter wild populations.",
"One method of containing genetically-modified organisms, namely, plants, is the so-called “terminator gene” or Technology Protection System (TPS).",
"This approach was developed by Delta and Pine Land Company (D&PL), who jointly owns the rights for this invention with USDA-ARS, as disclosed in U.S. Pat.",
"No.",
"5,723,765, which is incorporated herein by reference.",
"Essentially, the method stops the seeds of certain plants from germinating, and utilizes: 1.A transiently-active promoter operably linked to a first (toxic, hence lethal) gene, but separated by a blocking sequence which prevents the lethal gene expression; 2.A second gene, encoding a recombinase which, upon expression, excises the blocker sequence; and 3.A third gene, encoding a tetracycline-controllable blocker of the recombinase.",
"Unless the seeds of the plants are transformed with all three genes, and receive the tetracycline at a precise point, the recombinase is expressed, resulting in the blocker sequence being excised, and the toxic gene being expressed.",
"While this method may function well in plants, it would not function in many animal species.",
"Few recombinases have been identified that will function in animals (and vertebrates in particular) and those that have been identified (eg., Cre and Flp recombinase) function in only a limited number of species.",
"Moreover, the use of a toxic substance in animals may be unacceptable, particularly for those likely to be consumed.",
"Further, the system requires a number of complex steps, which are not readily achieved, and once the blocker sequence has been excised it is virtually impossible to reverse the control process.",
"Accordingly, there is still a need to provide methods of preventing the escape of exotic and/or genetically modified animals.",
"We have now developed such a method.",
"We have designed certain genetic constructs that allow animals to be bred in captivity, but render them reproductively non-viable or infertile in the wild.",
"Moreover, these constructs provide reversible control over fertility and reproduction, and are applicable to a wide variety of animal species."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>In its most general aspect, the invention disclosed herein provides a nucleic acid construct which may be inserted into the genome of any target organism.",
"The construct can use any promoter/gene combinations, provided that they satisfy the criteria of being activated only during embryonic development and/or gametogenesis, and being crucial for completion of embryogenic development and/or gametogenesis.",
"One type of construct, which is designed to function in a variety of target species, comprises: a) a native promoter of a crucial gene; b) a blocking DNA sequence (blocker) contoured for and designed to abrogate the crucial gene's function or to cause its mis-expression; and c) a genetic switch to regulate controlled expression/repression of the blocker/gene knockout.",
"In captivity, expression of the blocker can be repressed in the presence of a trigger molecule, supplied via the diet or in soluble form, so that fertilisation occurs and embryos complete development.",
"In the wild, where the trigger molecule is unavailable, the blocker remains active and the critical gene is disrupted, leading to early death of invasive progeny.",
"Accordingly, in a first aspect, the present invention provides a construct for disrupting gametogenesis or embryogenesis in animals, comprising: a) a first nucleic acid molecule, which is activated in a defined spatio-temporal pattern, and which is operably linked to b) a second nucleic acid molecule, which encodes a transactivating protein; and c) a third nucleic acid molecule, which is operably linked to a fourth nucleic acid molecule, wherein activation of said first nucleic acid molecule controls the expression of the second nucleic acid molecule, which in turn activates the third nucleic acid molecule, which effects the expression of the fourth nucleic acid molecule which encodes a blocker molecule which disrupts gametogenesis or embryogenesis in the animal.",
"Either or both the first and fourth nucleic acid molecules are transiently activated or transiently affect development in a defined spatio-temporal pattern.",
"Each of the first, second, third and fourth nucleic acids may be genomic DNA, cDNA, RNA, or a hybrid molecule thereof.",
"It will be clearly understood that the term nucleic acid molecule encompasses a full-length molecule, or a biologically active fragment thereof.",
"Preferably the first nucleic acid molecule is a DNA molecule encoding a promoter region.",
"More preferably the promoter is activated only during embryonic development and/or gametogenesis, and is crucial for completion of embryogenic development and/or gametogenesis.",
"Most preferably this DNA molecule has the nucleotide sequence shown in SEQ ID NO:1, SEQ.",
"ID NO:8 SEQ ID NO:60.A sample of SEQ ID NO.1 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09098.A sample of SEQ ID NO.8 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.60 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09106.Preferably the second nucleic acid molecule is a cDNA molecule encoding the tetracycline-responsive transcriptional activator protein (tTA), as defined herein, having a nucleotide sequence of SEQ ID NO:2.A sample of SEQ ID NO.2 cDNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09099.Preferably the third nucleic acid molecule is DNA molecule encoding a repressible promoter.",
"More preferably the promoter consists of the tet responsive element (TRE) which is coupled to and tightly regulates a minimal promoter region.",
"Most preferably this comprises the tet responsive element (TRE) and the P minCMV as shown in SEQ ID NO:3.A sample of SEQ ID NO.3 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.Preferably the fourth nucleic acid molecule encodes a blocker molecule selected from the group consisting of antisense RNA, double-stranded RNA (dsRNA), sense RNA and ribozyme.",
"More preferably the molecule is dsRNA or sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"Most preferably this RNA molecule is encoded by the nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID:61.A sample of SEQ ID NO.13 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ ID NO:62 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.23 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09101.A sample of SEQ ID NO.24 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09102.A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In a second aspect, the present invention provides a nucleic acid molecule, which encodes a promoter and is transiently activated in a defined spatio-temporal pattern.",
"More preferably, the promoter is active only during a narrow window during embryogenesis or larval development.",
"Most preferably the nucleic acid is a promoter having a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60.In a third aspect, the present invention provides a nucleic acid molecule, which encodes a promoter having: a) a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60; or b) a biologically active fragment of the sequence in a); or c) a nucleic acid molecule which has at least 75% sequence homology to the sequence in a) or b); or d) a nucleic acid molecule which is capable of hybridizing to the sequence in a) or b) under stringent conditions.",
"In a fourth aspect, the present invention provides a nucleic acid molecule that encodes the coding region of a gene including: a) a nucleotide sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO 61 or b) a biologically active fragment of any one of the sequences in a); or c) a nucleic acid molecule which has at least 75% sequence homology with any one of the sequences disclosed in a) or b); or d) a nucleic acid molecule that is capable of binding to any one of the sequences disclosed in a) or b) under stringent conditions.",
"A sample of SEQ ID NO.63 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ ID NO.23 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09101.A sample of SEQ ID NO.24 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09102.A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In a fifth aspect, the present invention provides a nucleic acid molecule which encodes a blocker molecule wherein the blocker molecule is capable of disrupting gametogenesis or embryogenesis in an animal.",
"Preferably the blocker molecule is selected from the group consisting of antisense RNA, dsRNA, sense RNA and ribozyme.",
"More preferably the molecule is dsRNA or sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"Most preferably the blocker molecule is encoded, or partially encoded, by a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23 and SEQ ID NO:61.A sample of SEQ ID NO.13 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ.",
"ID NO.62 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In an sixth aspect, the present invention provides a construct for disrupting gametogenesis or embryogenesis in animals, comprising: a) a first nucleic acid molecule, which is transiently activated in a defined spatio-temporal pattern, and which is operably linked to b) a second nucleic acid molecule, which encodes a blocker molecule.",
"wherein activation of said first nucleic acid molecule controls the expression of the second nucleic acid which disrupts gametogenesis or embryogenesis in the animal.",
"In a seventh aspect, the present invention provides a method of preventing embryogenesis in animals comprising the steps of: 1) stably transforming an animal cell with a construct according to the invention; and 2) implanting the cell into a host organism, whereby a whole animal develops from the implanted cell.",
"Preferably, the stable transformation is effected by microinjection, transfection or infection, wherein the construct stably integrates into the genome by homologous recombination.",
"In an eighth aspect, the present invention provides a transgenic animal stably transformed with a construct according to the invention.",
"Preferably the host organism is of the same genus as the transformed cell.",
"More preferably the host organism is any animal, including vertebrates and invertebrates.",
"Most preferably the host organism is selected from the group consisting of fish, mammals, amphibians, and mollusc.",
"Fish include; but are not limited to, zebrafish, European carp, salmon, tilapia and trout.",
"Mammals include; but are, not limited to, cats, dogs, donkeys, camels, rabbits, rats, and mice.",
"Molluscs include; but are not limited to, Pacific oysters, zebra mussels, striped mussels, abalone, pearl oysters, and scallops.",
"Modified and variant forms of the constructs may be produced in vitro, by means of chemical or enzymatic treatment, or in vivo by means of recombinant DNA technology.",
"Such constructs may differ from those disclosed, for example, by virtue of one or more nucleotide substitutions, deletions or insertions, but substantially retain a biological activity of the construct or nucleic acid molecule of this invention."
],
[
"FIELD OF THE INVENTION This application is concerned with the control of animal reproduction, and especially with preventing the spread of feral and/or genetically modified animals.",
"In particular, the present invention relates to constructs and methods that allow animals to be bred in captivity, but renders them infertile in the wild, by allowing reversible control over fertility and reproduction.",
"BACKGROUND OF THE INVENTION Feral animals are one of the world's major environmental problems.",
"Goats, cats, rabbits and carp are only the more prominent of hundreds of species traded internationally for recreation or agriculture that have escaped into the wild and formed destructive populations.",
"Terrestrial, freshwater and marine ecosystems are all conspicuously degraded by these species, to the extent that public concern over feral animals has become a major issue for industries seeking to introduce new species in order to compete on world markets.",
"A good recent example is the Pacific oyster.",
"Despite the promise of new jobs in coastal communities and an industry that is worth $50-75 million annually, recent applications to expand the geographic area for Pacific oyster mariculture facilities in Australia and the United States have been rejected indefinitely until the problem of feral oysters can be overcome.",
"Even plans to expand the size of the industry in areas where farming already occurs are being blocked for the same reason, following very public and often acrimonious debate between industry and conservation-minded elements of the community.",
"Attempts to solve the problem using current techniques such as triploidy and sterile hybrids have not been successful.",
"Neither technique can guarantee a zero risk of producing feral populations, and both also suffer major technical difficulties.",
"In the case of oysters, for example, animals sterilised via chemical or genetic manipulation of ploidy do not produce significant amounts of roe, which substantially reduces their market value.",
"Moreover, these animals still produce a small number of viable gametes.",
"So the debate continues to focus on whether degraded beaches are an acceptable price for new industries and jobs.",
"Hundreds of species of exotic animals are shipped internationally each day, mainly for recreational purposes.",
"Inevitably, either accidentally and/or through intentional release, some animals will escape, and establish feral populations.",
"Sterilisation prior to importation of such exotics would prevent the establishment of feral populations and remove the risk of forming new problem pest species.",
"A generic means of sterilisation that prevents development of these feral populations would have huge economic and environmental benefits.",
"More recently, the containment of genetically modified animals has caused concern.",
"For example, Salmon containing genes for enhanced production of growth hormones have been produced in Europe, New Zealand and North America.",
"Concern has been expressed about the impact of these fish as “super-competitors”, should they escape and form feral populations.",
"Similar concerns have been expressed about other genetic improvements that deliberately or accidentally enhance competitiveness.",
"This concern has now grown to a point where there is pressure to ban such modified organisms in toto.",
"However, given their economic significance, it may be preferable to have effective biological controls in place which enable these organisms to be contained within a specific locality.",
"A sterile feral construct inserted into the genetically enhanced stock would prevent development of viable feral populations, as well as preventing integration of enhanced genes into populations of wild con-specifics.",
"Accordingly, some of the major benefits that a sterile feral construct would offer include: 1.Provision of a fail-safe system for preventing the establishment of feral populations of exotic species.",
"This could fundamentally change the risk of importing these species, and would reduce public antagonism to farming of those that have the potential to be environmentally destructive.",
"2.Protection of investments in breeding stocks, for example those developed by extensive selective breeding programs.",
"Currently, the commercial advantages from improved stock can be lost when live, reproductively capable animals are marketed (eg oysters, prawns, and sheep).",
"Repressible sterility can be used as a “lock and key” process whereby improved stock could only breed when provided the correct combination of repressers (and optionally inducers) in exactly the right sequence.",
"3.Production of animals for intentional release that are guaranteed to be sterile.",
"Release of such sterile animals has been used as a control mechanism for certain highly fecund pest species, eg.",
"insects.",
"Repressible sterility technology makes it possible to apply similar approaches to other, existing pest species, for which there are currently no “sterile male” equivalents.",
"4.Provision of an effective containment mechanism for genetically modified organisms.",
"Repressible sterility provides just such a security system for future applications of molecular engineering in animal production, yet enables safe propagation of these individuals using conventional rearing facilities.",
"Linking a genetically engineered process (faster growth, longer spawning seasons, etc.)",
"to a repressible sterility construct ensures that genetic enhancements of exotic or native species do not enter wild populations.",
"One method of containing genetically-modified organisms, namely, plants, is the so-called “terminator gene” or Technology Protection System (TPS).",
"This approach was developed by Delta and Pine Land Company (D&PL), who jointly owns the rights for this invention with USDA-ARS, as disclosed in U.S. Pat.",
"No.",
"5,723,765, which is incorporated herein by reference.",
"Essentially, the method stops the seeds of certain plants from germinating, and utilizes: 1.A transiently-active promoter operably linked to a first (toxic, hence lethal) gene, but separated by a blocking sequence which prevents the lethal gene expression; 2.A second gene, encoding a recombinase which, upon expression, excises the blocker sequence; and 3.A third gene, encoding a tetracycline-controllable blocker of the recombinase.",
"Unless the seeds of the plants are transformed with all three genes, and receive the tetracycline at a precise point, the recombinase is expressed, resulting in the blocker sequence being excised, and the toxic gene being expressed.",
"While this method may function well in plants, it would not function in many animal species.",
"Few recombinases have been identified that will function in animals (and vertebrates in particular) and those that have been identified (eg., Cre and Flp recombinase) function in only a limited number of species.",
"Moreover, the use of a toxic substance in animals may be unacceptable, particularly for those likely to be consumed.",
"Further, the system requires a number of complex steps, which are not readily achieved, and once the blocker sequence has been excised it is virtually impossible to reverse the control process.",
"Accordingly, there is still a need to provide methods of preventing the escape of exotic and/or genetically modified animals.",
"We have now developed such a method.",
"We have designed certain genetic constructs that allow animals to be bred in captivity, but render them reproductively non-viable or infertile in the wild.",
"Moreover, these constructs provide reversible control over fertility and reproduction, and are applicable to a wide variety of animal species.",
"SUMMARY OF THE INVENTION In its most general aspect, the invention disclosed herein provides a nucleic acid construct which may be inserted into the genome of any target organism.",
"The construct can use any promoter/gene combinations, provided that they satisfy the criteria of being activated only during embryonic development and/or gametogenesis, and being crucial for completion of embryogenic development and/or gametogenesis.",
"One type of construct, which is designed to function in a variety of target species, comprises: a) a native promoter of a crucial gene; b) a blocking DNA sequence (blocker) contoured for and designed to abrogate the crucial gene's function or to cause its mis-expression; and c) a genetic switch to regulate controlled expression/repression of the blocker/gene knockout.",
"In captivity, expression of the blocker can be repressed in the presence of a trigger molecule, supplied via the diet or in soluble form, so that fertilisation occurs and embryos complete development.",
"In the wild, where the trigger molecule is unavailable, the blocker remains active and the critical gene is disrupted, leading to early death of invasive progeny.",
"Accordingly, in a first aspect, the present invention provides a construct for disrupting gametogenesis or embryogenesis in animals, comprising: a) a first nucleic acid molecule, which is activated in a defined spatio-temporal pattern, and which is operably linked to b) a second nucleic acid molecule, which encodes a transactivating protein; and c) a third nucleic acid molecule, which is operably linked to a fourth nucleic acid molecule, wherein activation of said first nucleic acid molecule controls the expression of the second nucleic acid molecule, which in turn activates the third nucleic acid molecule, which effects the expression of the fourth nucleic acid molecule which encodes a blocker molecule which disrupts gametogenesis or embryogenesis in the animal.",
"Either or both the first and fourth nucleic acid molecules are transiently activated or transiently affect development in a defined spatio-temporal pattern.",
"Each of the first, second, third and fourth nucleic acids may be genomic DNA, cDNA, RNA, or a hybrid molecule thereof.",
"It will be clearly understood that the term nucleic acid molecule encompasses a full-length molecule, or a biologically active fragment thereof.",
"Preferably the first nucleic acid molecule is a DNA molecule encoding a promoter region.",
"More preferably the promoter is activated only during embryonic development and/or gametogenesis, and is crucial for completion of embryogenic development and/or gametogenesis.",
"Most preferably this DNA molecule has the nucleotide sequence shown in SEQ ID NO:1, SEQ.",
"ID NO:8 SEQ ID NO:60.A sample of SEQ ID NO.1 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09098.A sample of SEQ ID NO.8 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.60 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09106.Preferably the second nucleic acid molecule is a cDNA molecule encoding the tetracycline-responsive transcriptional activator protein (tTA), as defined herein, having a nucleotide sequence of SEQ ID NO:2.A sample of SEQ ID NO.2 cDNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09099.Preferably the third nucleic acid molecule is DNA molecule encoding a repressible promoter.",
"More preferably the promoter consists of the tet responsive element (TRE) which is coupled to and tightly regulates a minimal promoter region.",
"Most preferably this comprises the tet responsive element (TRE) and the PminCMV as shown in SEQ ID NO:3.A sample of SEQ ID NO.3 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.Preferably the fourth nucleic acid molecule encodes a blocker molecule selected from the group consisting of antisense RNA, double-stranded RNA (dsRNA), sense RNA and ribozyme.",
"More preferably the molecule is dsRNA or sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"Most preferably this RNA molecule is encoded by the nucleotide sequence shown in SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID:61.A sample of SEQ ID NO.13 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ ID NO:62 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.23 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09101.A sample of SEQ ID NO.24 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09102.A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In a second aspect, the present invention provides a nucleic acid molecule, which encodes a promoter and is transiently activated in a defined spatio-temporal pattern.",
"More preferably, the promoter is active only during a narrow window during embryogenesis or larval development.",
"Most preferably the nucleic acid is a promoter having a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60.In a third aspect, the present invention provides a nucleic acid molecule, which encodes a promoter having: a) a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:60; or b) a biologically active fragment of the sequence in a); or c) a nucleic acid molecule which has at least 75% sequence homology to the sequence in a) or b); or d) a nucleic acid molecule which is capable of hybridizing to the sequence in a) or b) under stringent conditions.",
"In a fourth aspect, the present invention provides a nucleic acid molecule that encodes the coding region of a gene including: a) a nucleotide sequence selected from the group consisting of SEQ ID NO:63, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO 61 or b) a biologically active fragment of any one of the sequences in a); or c) a nucleic acid molecule which has at least 75% sequence homology with any one of the sequences disclosed in a) or b); or d) a nucleic acid molecule that is capable of binding to any one of the sequences disclosed in a) or b) under stringent conditions.",
"A sample of SEQ ID NO.63 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ ID NO.23 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09101.A sample of SEQ ID NO.24 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number NM99/09102.A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In a fifth aspect, the present invention provides a nucleic acid molecule which encodes a blocker molecule wherein the blocker molecule is capable of disrupting gametogenesis or embryogenesis in an animal.",
"Preferably the blocker molecule is selected from the group consisting of antisense RNA, dsRNA, sense RNA and ribozyme.",
"More preferably the molecule is dsRNA or sense RNA that when mis-expressed disrupts development in a defined spatio-temporal pattern.",
"Most preferably the blocker molecule is encoded, or partially encoded, by a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:62, SEQ ID NO:23 and SEQ ID NO:61.A sample of SEQ ID NO.13 DNA was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09100.A sample of SEQ.",
"ID NO.62 DNA was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"A sample of SEQ ID NO.61 DNA was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999, and accorded the accession number NM99/09107.In an sixth aspect, the present invention provides a construct for disrupting gametogenesis or embryogenesis in animals, comprising: a) a first nucleic acid molecule, which is transiently activated in a defined spatio-temporal pattern, and which is operably linked to b) a second nucleic acid molecule, which encodes a blocker molecule.",
"wherein activation of said first nucleic acid molecule controls the expression of the second nucleic acid which disrupts gametogenesis or embryogenesis in the animal.",
"In a seventh aspect, the present invention provides a method of preventing embryogenesis in animals comprising the steps of: 1) stably transforming an animal cell with a construct according to the invention; and 2) implanting the cell into a host organism, whereby a whole animal develops from the implanted cell.",
"Preferably, the stable transformation is effected by microinjection, transfection or infection, wherein the construct stably integrates into the genome by homologous recombination.",
"In an eighth aspect, the present invention provides a transgenic animal stably transformed with a construct according to the invention.",
"Preferably the host organism is of the same genus as the transformed cell.",
"More preferably the host organism is any animal, including vertebrates and invertebrates.",
"Most preferably the host organism is selected from the group consisting of fish, mammals, amphibians, and mollusc.",
"Fish include; but are not limited to, zebrafish, European carp, salmon, tilapia and trout.",
"Mammals include; but are, not limited to, cats, dogs, donkeys, camels, rabbits, rats, and mice.",
"Molluscs include; but are not limited to, Pacific oysters, zebra mussels, striped mussels, abalone, pearl oysters, and scallops.",
"Modified and variant forms of the constructs may be produced in vitro, by means of chemical or enzymatic treatment, or in vivo by means of recombinant DNA technology.",
"Such constructs may differ from those disclosed, for example, by virtue of one or more nucleotide substitutions, deletions or insertions, but substantially retain a biological activity of the construct or nucleic acid molecule of this invention.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 shows the plasmid map of pBAC5/H11.FIG.",
"2 shows the plasmid map of pZBMP2(1.4)-EGFP.",
"The transcriptional unit consists of the modified EGFP coding sequences (Cormac et al., 1996), under the regulation of a 1,414 bp zBMP2 promoter.",
"FIG.",
"3 shows zBMP2 promoter-driven EGFP expression in zebrafish embryo at 9.5 h pi.",
"Right, latero-ventral view, anterior to right.",
"Panel A shows a typical zebrafish embryo showing EGFP expression predominantly in the anterio-ventral region.",
"Panel B shows a light micrograph of the embryo on left.",
"PO, polster.",
"FIG.",
"4 shows EGFP expression in 9.5 hpi old zebrafish embryo.",
"Lateral views, with dorsal to top and anterior to left.",
"Panel A shows EGFP expression driven by zBMP2 promoter.",
"Panel B shows a light micrograph of the embryo on left.",
"PO, polster; TB, tail bud.",
"FIG.",
"5 shows anterior region of a zebrafish embryo, showing EGFP expression driven by zpBMP2 at 24-h pi.",
"Panel A shows the left, dorso-lateral view.",
"EGFP expression is seen in domains of native zBMP2 expression.",
"Panel B shows light micrograph of the embryo on left.",
"Left, lateral view.",
"PE, posterior margin of eye; OV, otic vesicle; FB, pectoral fin bud.",
"FIG.",
"6 shows the plasmid map of pSMAD5-EGFP.",
"A sample of pSMAD5-EGFP was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"The zebrafish smad5 promoter drives expression of the EGFP.",
"FIG.",
"7 shows a shield stage zebrafish embryo, showing ubiquitous expression of EGFP (panel A) driven by zebrafish smad5 promoter Panel B represents the light micrograph of the embryo on left.",
"FIG.",
"8 shows middle section of a typical 24 hpi zebrafish embryo injected with pSMAd5-EGFP.",
"The EGFP expression is predominantly restricted to ventral tissues.",
"D, dorsal; V, ventral.",
"FIG.",
"9 shows dorsalized phenotypes of zebrafish, resulting from zBMP2 antisense (A) and dsRNA (B) injections.",
"Developments of ventral structures are perturbed in both instances.",
"FIG.",
"10 shows the ventralized chordino phenotypes of zebrafish resulting from zBMP2 sense transcript injections.",
"Enlarged blood island (A and B, arrow) and multiplicated ventral margin of tail fin (C, arrow).",
"FIG.",
"11 shows the plasmid map of the antisense EGFP fusion construct, pzBMP2-As-EGFP.",
"A sample of pzBMP2-As-EGFP was deposited at the Australian Government Analytical Laboratories on 22 Dec. 1999, and accorded the accession number MM99/09102.FIG.",
"12 shows the plasmid map of pzBMP2-dsRNA.",
"The zBMP2 promoter drives the expression of about 800 bp of zBMP2 cDNA, designed to fold back on itself as a dsRNA.",
"FIG.",
"13 shows the plasmid map of pzBMP2-Tet-Off.",
"This construct was engineered to drive expression of tTA under the regulation of zBMP2 promoter.",
"FIG.",
"14 shows the plasmid map of the complete sterile feral construct, pSF1.The zBMP2 promoter drives the expression of tTA, which in turn activates the expression of EGFP and the zBMP2 double stranded RNA blocker, in the absence of doxycycline.",
"FIG.",
"15 shows a plasmid map of zebrafish Sterile feral Construct pSF2.This construct is identical to pSF1, except that CMV promoter drive's the tTA.",
"A sample of pSF2 was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"FIG.",
"16 shows a plasmid map of zebrafish Sterile feral Construct pSF3.This construct is identical to pSF2, except that the zebrafish smad5 promoter drives the tTA.",
"A sample of pSF3 was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"FIG.",
"17 shows a plasmid map of zebrafish Sterile feral Construct pSF4.This construct is identical to pSF3, except that the zBMP2 double stranded RNA blocker is replaced by zBMP2 sense cDNA.",
"A sample of pSF4 was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number FIG.",
"18 (A-C) show 24-hpi zebrafish embryos following the injection of pSF4.Panel A, two-zebrafish embryos with enlarged blood islands (arrow), typical of ventralized mutations.",
"Panel B, close up view of 24 hpi zebrafish embryo tail, with enlarged blood island (arrow).",
"Panel C, EGFP micrograph of embryo in panel B, showing close association of EGFP expression and ventralization (arrow).",
"FIG.",
"19 shows the amino acid alignments of closely related HOXCG1 and HOXCG3 genes in various animals.",
"FIG.",
"20 shows (a) typical control D-hinge larvae with a single velum and (b) a larvae exhibiting the multiple velum phenotype as a consequence of blocking expression of Hox CG1 with double stranded HOXG1 RNA.",
"FIG.",
"21 shows the plasmid map of the double stranded blocking construct for oyster Hox gene, pBiT(dHSP)-RFP-oHoxDS/BH.",
"A sample of pBiT(dHSP)-RFP-oHoxDS/BH was deposited at the Australian Government Analytical Laboratories on ______, and accorded the accession number ______.",
"FIG.",
"22 shows the amino acid alignments of closely related goosecoid genes in various animals.",
"FIG.",
"23 shows the mechanisms of action of regulatory elements of the mouse goosecoid gene promoter region.",
"FIG.",
"24 shows the plasmid map of the mouse goosecoid promoter driving expression of the enhanced green fluorescent protein reporter (pSFM 1).",
"FIG.",
"25 shows the plasmid map of the tetracycline transactivated TRE driving expression of the mouse goosecoid cDNA (pSFM 2).",
"FIG.",
"26 shows the mouse goosecoid promoter driving expression of mouse goosecoid cDNA fused to the red fluorescent protein reporter (pSFM 6).",
"FIG.",
"27 shows the plasmid map of the mouse goosecoid promoter driving expression of the tetracycline transactivator tTA protein (pSFM 7).",
"FIG.",
"28 shows the plasmid map of the mouse goosecoid promoter driving expression of the luciferase+ protein reporter (pSFM 20).",
"FIG.",
"29 shows the plasmid map of the promoter-less luciferase+ protein reporter (pSFM 21).",
"FIG.",
"30 shows the plasmid map of the CMV promoter driving expression of the luciferase+ protein reporter (pSFM 23).",
"FIG.",
"31 shows the plasmid map of the tetracycline transactivated TRE driving expression of the enhanced green fluorescent protein reporter (pSFM 24).",
"FIG.",
"32 shows the plasmid map of the tetracycline transactivated TRE driving expression of the luciferase+ protein reporter (pSFM 25).",
"FIG.",
"33 shows an agarose gel demonstrating the presence of mouse goosecoid mRNA expression in P19 cells as detected by RT-PCR amplification of mRNA using goosecoid-specific primers.",
"Lane 1: PCR product from P19 cells using goosecoid primers; Lane 2: PCR product from 1 fg of pSFM 2 as a positive goosecoid control; Lane 3: PCR product from P19 cells with GAPDH primers; Lane 4: DNA MW marker.",
"FIG.",
"34 shows the plasmid map of the tetracycline transactivated TRE driving expression of the mouse goosecoid dsRNA blocker construct (pSFM 5).",
"FIG.",
"35 shows the plasmid map of the CMV promoter driving expression of the mouse goosecoid antisense RNA blocker construct (pSFM 8).",
"FIG.",
"36 shows the plasmid map of the tetracycline transactivated TRE driving expression of the mouse goosecoid antisense blocker construct (pSFM 9).",
"A sample of pSFM 9 was deposited at the Australian Government Analytical Laboratories on 23 Dec. 1999 and accorded the accession number NM99/09107.FIG.",
"37 shows the cellular locations of CMV promoter-driven expression of red fluorescent protein in P19-SFM 7 cells (A,B), CMV promoter-driven expression of red fluorescent protein fused to the mouse goosecoid protein (C) and TRE tetracycline responsive enhanced green fluorescent protein expression in cells co-transfected with CMV promoter-driven expression of red fluorescent protein fused to the mouse goosecoid protein (D).",
"DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention employs, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art.",
"Such techniques are well known to the skilled worker, and are explained fully in the literature.",
"See, e.g., “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” 12th edition (1989).",
"Definitions The description that follows makes use of a number of terms used in recombinant DNA technology.",
"In order to provide a clear and consistent understanding of the specification and claims, including the scope given such terms, the following definitions are provided.",
"A “nucleic acid molecule” or “polynucleic acid molecule” refers herein to deoxyribonucleic acid and ribonucleic acid in all their forms, i.e., single and double-stranded DNA, cDNA, mRNA, and the like.",
"A “double-stranded DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its normal, double-stranded helix.",
"This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.",
"Thus this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.",
"In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).",
"A DNA sequence “corresponds” to an amino acid sequence if translation of the DNA sequence in accordance with the genetic code yields the amino acid sequence (i.e., the DNA sequence “encodes” the amino acid sequence).",
"One DNA sequence “corresponds” to another DNA sequence if the two sequences encode the same amino acid sequence.",
"Two DNA sequences are “substantially similar” when at least about 85%, preferably at least about 90%, and most preferably at least about 95%, of the nucleotides match over the defined length of the DNA sequences.",
"Sequences that are substantially similar can be identified in a Southern hybridization experiment, for example under stringent conditions as defined for that particular system.",
"Defining appropriate hybridization conditions is within the skill of the art.",
"See e.g., Sambrook et al., “Molecular Cloning: a Laboratory Manual” 12th edition (1989), vols.",
"I, II and III.",
"Nucleic Acid Hybridization.",
"However, ordinarily, “stringent conditions” for hybridization or annealing of nucleic acid molecules are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C., or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× “Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.",
"A “heterologous” region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.",
"Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.",
"Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene).",
"Allelic variations or naturally occurring mutational events do not give rise to a heterologous region of DNA as defined herein.",
"A “gene” includes all the DNA sequences associated with the promoter and coding region and non-coding region such as introns and 5′ and 3′ non-coding sequences and enhancer elements.",
"A “coding region” is an in-frame sequence of codons from the start codon, normally ATG, to the stop codon TAA, and which may or may not include introns.",
"A “coding sequence” is an in-frame sequence of codons that correspond to or encode a protein or peptide sequence.",
"Two coding sequences correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences.",
"A coding sequence in association with appropriate regulatory sequences may be transcribed and translated into a polypeptide in vivo.",
"A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.",
"A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence.",
"A coding sequence is “under the control” of the promoter sequence in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA, which is then in turn translated into the protein encoded by the coding sequence.",
"For the purposes of the present invention, the promoter sequence is bounded at its 3′ terminus by the translation start codon of a coding sequence, and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.",
"Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.",
"Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes, prokaryotic promoters contain Shine-Delgarno sequences in addition to the −10 and −35 consensus sequences.",
"A cell has been “transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell wall.",
"Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell.",
"In prokaryotes and yeast, for example, the exogenous DNA may be maintained on an episomal element such as a plasmid.",
"With respect to eukaryotic cells, a stably transformed cell is one in which the exogenous DNA is inherited by daughter cells through chromosome replication.",
"This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.",
"“Integration” of the DNA may be effected using non-homologous recombination following mass transfer of DNA into the cells using microinjection, biolistics, electroporation or lipofection.",
"Alternative methods such as homologous recombination, and or restriction enzyme mediated integration (REMI) or transposons are also encompassed, and may be considered to be improved integration methods.",
"A “clone” is a population of cells derived from a single cell or common ancestor by mitosis.",
"“Cell,” “host cell,” “cell line,” and “cell culture” are used interchangeably herewith and all such terms should be understood to include progeny.",
"A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.",
"Thus the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom, without regard for the number of times the cultures have been passaged.",
"It should also be understood that all progeny might not be precisely identical in DNA content, due to deliberate or inadvertent mutations.",
"Vectors are used to introduce a foreign substance, such as DNA, RNA or protein, into an organism.",
"Typical vectors include recombinant viruses (for DNA) and liposomes (for protein).",
"A “DNA cloning vector” is an autonomously replicating DNA molecule,” such as plasmid, phage or cosmid.",
"Typically the DNA cloning vector comprises one or a small number of restriction endonuclease recognition sites, at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning.",
"The cloning vector may also comprise a marker suitable for use in the identification of cells transformed with the cloning vector.",
"An “expression vector” is similar to a DNA cloning vector, but contains regulatory sequences which are able to direct protein synthesis by an appropriate host cell.",
"This usually means a promoter to bind RNA polymerase and initiate transcription of mRNA, as well as ribosome binding sites and initiation signals to direct translation of the mRNA into a polypeptide.",
"Incorporation of a DNA sequence into an expression vector at the proper site and in correct reading frame, followed by transformation of an appropriate host cell by the vector, enables the production of mRNA corresponding to the DNA sequence, and usually of a protein encoded by the DNA sequence.",
"“Plasmids” are DNA molecules that are capable of replicating within a host cell, either extrachromosomally or as part of the host cell chromosome(s), and are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.",
"The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids by methods disclosed herein and/or in accordance with published procedures.",
"In certain instances, as will be apparent to the ordinarily skilled worker, other plasmids known in the art may be used interchangeably with plasmids described herein.",
"“Control sequences” refers to DNA sequences necessary for the expression of an operably linked nucleotide coding sequence in a particular host cell.",
"The control sequences suitable for expression in prokaryotes, for example, include origins of replication, promoters, ribosome binding sites, and transcription termination sites.",
"The control sequences that are suitable for expression in eukaryotes, for example, include origins of replication, promoters, ribosome binding sites, polyadenylation signals, and enhancers.",
"An “exogenous” element is one that is foreign to the host cell, or is homologous to the host cell but in a position within the host cell in which the element is ordinarily not found.",
"“Digestion” of DNA refers to the catalytic cleavage of DNA with an enzyme that acts only at certain locations in the DNA.",
"Such enzymes are called restriction enzymes or restriction endonucleases, and the sites within DNA where such enzymes cleave are called restriction sites.",
"If there are multiple restriction sites within the DNA, digestion will produce two or more linearized DNA fragments (restriction fragments).",
"The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements as established by the enzyme manufacturers are used.",
"Restriction enzymes are commonly designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restriction enzyme originally was obtained and then a number designating the particular enzyme.",
"In general, about 1 μg of DNA is digested with about 1-2 units of enzyme in about 20 μl of buffer solution.",
"Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer, and/or are well known in the art.",
"“Recovery” or “isolation” of a given fragment of DNA from a restriction digest typically is accomplished by separating the digestion products, which are referred to as “restriction fragments,” on a polyacrylamide or agarose gel by electrophoresis, identifying the fragment of interest on the basis of its mobility relative to that of marker DNA fragments of known molecular weight, excising the portion of the gel that contains the desired fragment, and separating the DNA from the gel, for example by electroelution.",
"“Ligation” refers to the process of forming phosphodiester bonds between two double-stranded DNA fragments.",
"Unless otherwise specified, ligation is accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.",
"“Oligonucleotides” are short-length, single- or double-stranded polydeoxynucleotides that are chemically synthesized by known methods (involving, for example, triester, phosphoramidite, or phosphonate chemistry), such as described by Engels et al., Agnew.",
"Chem.",
"Int.",
"Ed.",
"Engl.",
"28:716-734 (1989).",
"They are then purified, for example, by polyacrylamide gel electrophoresis.",
"“Polymerase chain reaction,” or “PCR,” as used herein generally refers to a method for amplification of a desired nucleotide sequence in vitro, as described in U.S. Pat.",
"No.",
"4,683,195.In general, the PCR method involves repeated cycles of primer extension synthesis, using two oligonucleotide primers capable of hybridizing preferentially to a template nucleic acid.",
"Typically, the primers used in the PCR method will be complementary to nucleotide sequences within the template at both ends of or flanking the nucleotide sequence to be amplified, although primers complementary to the nucleotide sequence to be amplified also may be used.",
"See Wang et al., in PCR Protocols, pp.70-75 (Academic Press, 1990); Ochman et al., in PCR Protocols, pp.",
"219-227; Triglia, et al., Nuc.",
"Acids Res.",
"16:8186 (1988).",
"“PCR cloning” refers to the use of the PCR method to amplify a specific desired nucleotide sequence that is present amongst the nucleic acids from a suitable cell or tissue source, including total genomic DNA and cDNA transcribed from total cellular RNA.",
"See Frohman et al., Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 85:8998-9002 (1988); Saiki et al., Science.",
"239:487-492 (1988); Mullis et al., Meth.",
"Enzymol.",
"155:335-350 (1987).",
"“zBMP2 promoter” refers to a promoter encoded by the nucleotide sequence set forth in SEQ ID NO.",
":1.“zSMAD promoter” refers to a promoter encoded by the nucleotide sequence set forth in SEQ ID NO.",
":8.“goosecoid promoter” refers to a promoter encoded by the nucleotide sequence set forth in SEQ ID NO:60.“Blocker molecule” refers to either antisense RNA, dsRNA, sense RNA or DNA that preferably encodes BMP2, GSC, HoxCG1 or HoxCG3 and includes the sequences shown in SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:61.However, it will be appreciated by those skilled in the art that any nucleic acid molecule capable of disrupting gametogenesis or embryogenesis is encompassed.",
"Accordingly, the terms “blocker molecule RNA” and “blocker molecule DNA” as used herein are interchangeable depending upon whether it is a species of RNA or DNA, that is being addressed.",
"“HoxCG” refers to genes HoxCG1 and HoxCG3 isolated from Pacific oyster encoded by the nucleotide sequences set forth in SEQ ID NO.",
":23 and SEQ ID NO:24, respectively.",
"Sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG blocker molecules may be made synthetically, for example, by site-directed or PCR mutagenesis, or may exist naturally, as in the case of allelic forms and other naturally occurring variants of the nucleotide sequences set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24, respectively, that may occur in fish and other animal species.",
"zBMP2 promoter, SMAD promoter, goosecoid promoter HoxCG, and blocker molecule nucleotide sequence variants are included within the scope of the invention, provided that they are functionally active.",
"As used herein, “functionally active” and “functional activity” with reference to zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG means that the zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG variants are able to function in a similar way to naturally occurring zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG.",
"With reference to the blocker molecule “functionally active” and “functional activity” means that the blocker molecule variants are capable of disrupting gametogenesis or embyrogenesis in an animal.",
"Therefore, zBMP2 promoter, SMAD promoter, goosecoid promoter HoxCG and blocker molecule nucleotide sequence variants generally will share at least about 75%, preferably greater than 80% and more preferably greater than 90%, sequence identity with the nucleotide sequences set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24 respectively, after aligning the sequences to provide for maximum homology, as determined, for example, by the Fitch et al., Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 80:1382-1386 (1983), version of the algorithm described by Needleman et al., J. Mol.",
"Biol.",
"48:443-453 (1970).",
"Nucleotide sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter HoxCG and blocker molecule are prepared by introducing appropriate nucleotide changes into zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA, or by in vitro synthesis.",
"Such variants include deletions from, or insertions or substitutions of, nucleotides within the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule nucleotide sequences set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO: 60, SEQ ID NO:23, and SEQ ID NO:24.Any combination of deletion, insertion, and substitution may be made to arrive at a nucleotide sequence variant of zBMP2 promoter, SMAD promoter, goosecoid promoter HoxCG or blocker molecule provided that such variants possess the desired characteristics described herein.",
"Changes that are made in the nucleotide sequence set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24, respectively, to arrive at nucleotide sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter and HoxCG blocker molecules also may result in further modifications of the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule upon their activation in host cells.",
"There are two principal variables in the construction of nucleotide sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule nucleic acid: the location of the mutation site and the nature of the mutation.",
"These are variants from the nucleotide sequences set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO 60, SEQ ID NO:23, and SEQ ID NO:24 and may represent naturally occurring allelic forms of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule or predetermined mutant forms of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule made by mutating zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA, either to arrive at an allele or a variant not found in nature.",
"In general, the location and nature of the mutation chosen will depend upon the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule characteristic to be modified.",
"Nucleotide sequence deletions generally range from about 1 to 30 nucleotides, more preferably about 1 to 10 nucleotides, and are typically contiguous.",
"Nucleotide sequence insertions include fusions ranging in length from one nucleotide to hundreds of nucleotides, as well as intrasequence insertions of single or multiple nucleotides.",
"Intrasequence insertions (i.e., insertions made within the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO:23, and SEQ ID NO:24) may range generally from about 1 to 10 nucleotides, more preferably 1 to 5, most preferably 1 to 3.The third group of variants are those in which nucleotides in the nucleotide sequences set forth in SEQ ID NO.",
":1, SEQ ID NO:8, SEQ ID NO:60, SEQ ID NO.23, and SEQ ID NO:24 have been substituted with other nucleotides.",
"Preferably one to four, more preferably one to three, even more preferably one to two, and most preferably only one nucleotide has been removed and a different nucleotide inserted in its place.",
"The sites of greatest interest for making such substitutions are those sites that are likely to be important to the functional activity of the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule.",
"zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA is obtained from cDNA or genomic DNA libraries, or by in vitro synthesis.",
"Identification of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA within a cDNA or a genomic DNA library, or in some other mixture of various DNAs, is conveniently accomplished by the use of an oligonucleotide hybridization probe labelled with a detectable moiety, such as a radioisotope.",
"See Keller et al., DNA Probes, pp.149-213 (Stockton Press, 1989).",
"To identify DNA encoding zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA, the nucleotide sequence of the hybridization probe is preferably selected so that the hybridization probe is capable of hybridizing preferentially to DNA encoding homologues of the equivalent zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA in other species, or variants or derivatives thereof as described herein, under the hybridization conditions chosen.",
"Another method for obtaining zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule is chemical synthesis using one of the methods described, for example, by Engels et al., Agnew.",
"Chem.",
"Int.",
"Ed.",
"Engl.",
"28:716-734 (1989).",
"If the entire nucleotide coding sequence for zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule is not obtained in a single cDNA, genomic DNA, or other DNA, as determined, for example, by DNA sequencing or restriction endonuclease analysis, then appropriate DNA fragments (e.g., restriction fragments or PCR amplification products) may be recovered from several DNA's, and covalently joined to one another to construct the entire coding sequence.",
"The preferred means of covalently joining DNA fragments is by ligation using a DNA ligase enzyme, such as T4 DNA ligase.",
"“Isolated” zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule nucleic acid is zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule nucleic acid that is identified and separated from (or otherwise substantially free from), contaminant nucleic acid encoding other polypeptides.",
"The isolated zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule can be incorporated into a plasmid or expression vector, or can be labeled for probe purposes, using a label as described further herein in the discussion of assays and nucleic acid hybridization methods.",
"It will be appreciated that if the desired result of the present invention is sterilized adult feral animals then the blocker molecules may be expressed in vitro, isolated, purified, and then delivered to specific organisms.",
"The mode of delivery may be any known procedure including injection and ingestion.",
"Moreover, constructs of the present invention which are capable of expressing blocker molecules may also be delivered to adult feral animals by viral vectors like adenovirus.",
"Isolated zBMP2 promoter, SMAD promoter and goosecoid promoter nucleic acid is also used to control the expression of other desired genes or blocker molecules in vivo.",
"Indeed, the zBMP2 promoter, SMAD promoter and goosecoid promoter may be used in any vector, or construct where the expression of a gene, cDNA, or coding sequence is desirably controlled to be at a particular spatio-temporal point during embyrogenesis.",
"It will be appreciated that while the zBMP2 promoter and SMAD promoter are particularly useful in controlling the expression of nucleic acids in fish, they are equally useful in other organisms.",
"In various embodiments of the invention, host cells are transformed or transfected with recombinant DNA molecules comprising an isolated zBMP2 promoter or SMAD promoter DNA or goosecoid promoter operably linked to a desired nucleic acid molecule, wherein the expression of the desired molecule is directly or indirectly under the control of the zBMP2 promoter or SMAD promoter or goosecoid promoter.",
"Isolated HoxCG nucleic acid is also used to produce HoxCG by recombinant DNA and recombinant cell culture methods.",
"In various embodiments of the invention, host cells are transformed or transfected with recombinant DNA molecules comprising an isolated HoxCG DNA, to obtain expression of the HoxCG DNA and thus the production of HoxCG in large quantities.",
"DNA encoding amino acid sequence variants of HoxCG is prepared by a variety of methods known in the art.",
"These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants of HoxCG), or preparation by site-directed or oligonucleotide-mediated mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA encoding a variant or a non-variant form of HoxCG.",
"Site-directed mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of HoxCG DNA, or other DNA such as the zBMP2 promoter, SMAD promoter, and blocker molecule DNA.",
"This technique is well known in the art; see Zoller et al., Meth.",
"Enz.",
"100:4668-500 (1983); Zoller, et al., Meth.",
"Enz.",
"154:329-350 (1987); Carter, Meth.",
"Enz.",
"154:382-403 (1987); Horwitz et al., Meth.",
"Enz.",
"185:599-611 (1990), and has been used to produce amino acid sequence variants of trypsin and T4 lysozyme, which variants have certain desired functional properties.",
"Perry et al., Science 226:555-557 (1984); Craik et al., Science 228:291-297 (1985).",
"Briefly, in carrying out site-directed mutagenesis of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA, the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA.",
"After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA as a template.",
"Thus the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA.",
"Oligonucleotides for use as hybridization probes or primers may be prepared by any suitable method, such as purification of a naturally occurring DNA or in vitro synthesis.",
"For example, oligonucleotides are readily synthesized using various techniques in such as those described by Narang et al., Meth.",
"Enzymol.",
"68:90-98 (1979); Brown et al., Meth.",
"Enzymol.",
"68:109-151 (1979); Caruther et al., Meth.",
"Enzymol.",
"154:287-313 (1985).",
"The general approach to selecting a suitable hybridization probe or primer is well known.",
"Keller et al., DNA Probes, pp.11-18 (Stockton Press, 1989).",
"Typically, the hybridization probe or primer will contain 10-25 or more nucleotides, and will include at least 5 nucleotides on either side of the sequence encoding the desired mutation so as to ensure that the oligonucleotide will hybridize preferentially to the single-stranded DNA template molecule.",
"Multiple mutations are introduced into HoxCG DNA to produce amino acid sequence variants of HoxCG comprising several or a combination of insertions, deletions, or substitutions of amino acid residues as compared to the amino acid sequences set forth in FIG.",
"20.If the sites to be mutated are located close together, the mutations may be introduced simultaneously using a single oligonucleotide that encodes all of the desired mutations.",
"If, however, the sites to be mutated are located some distance from each other (separated by more than about ten nucleotides), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes.",
"Instead, one of two alternative methods may be employed.",
"In the first method, a separate oligonucleotide is generated for each desired mutation.",
"The oligonucleotides are then simultaneously annealed to the single-stranded template DNA, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.",
"The alternative method involves two or more rounds of mutagenesis to produce the desired mutant.",
"The first round is as described for introducing a single mutation: a single strand of a previously prepared HoxCG DNA is used as a template, an oligonucleotide encoding the first desired mutation is annealed to this template, and a heteroduplex DNA molecule is then generated.",
"The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.",
"Thus this template already contains one or more mutations.",
"The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.",
"This resultant DNA can be used as a template in a third round of mutagenesis, and so on.",
"PCR mutagenesis is also suitable for making nucleotide sequence variants of zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule.",
"Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990); Vallette et al., Nuc.",
"Acids Res.",
"17:723-733 (1989).",
"Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.",
"For introduction of a mutation into a plasmid DNA, for example, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a nucleotide sequence within the opposite strand of the plasmid DNA, but this sequence can be located anywhere along the plasmid DNA.",
"It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced.",
"PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.",
"See Wagner et al., in PCR Topics, pp.69-71 (Springer-Verlag, 1991).",
"If the ratio of template to product amplified DNA is extremely low, the majority of product DNA fragments incorporate the desired mutation(s).",
"This product DNA is used to replace the corresponding region in the plasmid that served as PCR template using standard recombinant DNA methods.",
"Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the plasmid fragment in a three (or more)-part ligation.",
"Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).",
"The starting material is the plasmid (or other vector) comprising the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA to be mutated.",
"The codon(s) in the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG or blocker molecule DNA to be mutated are identified.",
"There must be a unique restriction endonuclease site on each side of the identified mutation site(s).",
"If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG and blocker molecule DNA.",
"The plasmid DNA is cut at these sites to linearize it.",
"A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques.",
"This double-stranded oligonucleotide is referred to as the cassette.",
"This cassette is designed to have 5′ and 3′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.",
"This plasmid now contains the mutated zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG, or blocker molecule DNA sequence.",
"zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG, and blocker molecule DNA, whether cDNA or genomic DNA or a product of in vitro synthesis, is ligated into a replicable vector for further cloning or for expression.",
"“Vectors” are plasmids and other DNA's that are capable of replicating autonomously within a host cell, and as such, are useful for performing two functions in conjunction with compatible host cells (a vector-host system).",
"One function is to facilitate the cloning of the nucleic acid that encodes the zBMP2 promoter, SMAD promoter, goosecoid promoter, HoxCG, and blocker molecule, i.e., to produce usable quantities of the nucleic acid.",
"The other function is to direct the expression of HoxCG.",
"One or both of these functions are performed by the vector-host system.",
"The vectors will contain different components depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression.",
"To produce HoxCG, an expression vector will contain nucleic acid that encodes HoxCG as described above.",
"The HoxCG of this invention may be expressed directly in recombinant cell culture, or as a fusion with a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the junction between the heterologous polypeptide and the HoxCG.",
"In one example of recombinant host cell expression, cells are transfected with an expression vector comprising HoxCG DNA and the HoxCG encoded thereby is recovered from the culture medium in which the recombinant host cells are grown.",
"But the expression vectors and methods disclosed herein are suitable for use over a wide range of prokaryotic and eukaryotic organisms.",
"Prokaryotes may be used for the initial cloning of DNA's and the construction of the vectors useful in the invention.",
"However, prokaryotes may also be used for expression of mRNA or protein encoded by HoxCG.",
"Polypeptides that are produced inprokaryotic host cells typically will be non-glycosylated.",
"Plasmid or viral vectors containing replication origins and other control sequences that are derived from species compatible with the host cell are used in connection with prokaryotic host cells, for cloning or expression of an isolated DNA.",
"For example, E. coli typically is transformed using pBR322 a plasmid derived from an E. coli species.",
"Bolivar et al., Gene 2:95-113 (1987).",
"PBR322 contains genes for ampicillin and tetracycline resistance so that cells transformed by the plasmid can easily be identified or selected.",
"For it to serve as an expression vector, the pBR322 plasmid, or other plasmid or viral vector, must also contain, or be modified to contain, a promoter that functions in the host cell to provide messenger RNA (mRNA) transcripts of a DNA inserted downstream of the promoter.",
"Rangagwala et al., Bio/Technology 9:477-479 (1991).",
"In addition to prokaryotes, eukaryotic microbes, such as yeast, may also be used as hosts for the cloning or expression of DNA's useful in the invention.",
"Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism.",
"Plasmids useful for cloning or expression in yeast cells of a desired DNA are well known, as are various promoters that function in yeast cells to produce mRNA transcripts.",
"Furthermore, cells derived from multicellular organisms also may be used as hosts for the cloning or expression of DNA's useful in the invention.",
"Mammalian cells are most commonly used, and the procedures for maintaining or propagating such cells in vitro, which procedures are commonly referred to as tissue culture, are well known.",
"Kruse & Patterson, eds., Tissue Culture (Academic Press, 1977).",
"Examples of useful mammalian cells are human cell lines such as 293, HeLa, and WI-38, monkey cell lines such as COS-7 and VERO, and hamster cell lines such as BHK-21 and CHO, all of which are publicly available from the American Type Culture Collection (ATCC), Rockville, Md.",
"20852 USA.",
"Expression vectors, unlike cloning vectors, should contain a promoter that is recognized by the host organism and is operably linked to the HoxCG nucleic acid.",
"Promoters are untranslated sequences that are located upstream from the start codon of a gene and that control transcription of the gene (that is, the synthesis of mRNA).",
"Promoters typically fall into two classes, inducible and constitutive.",
"Inducible promoters are promoters that initiate high level transcription of the DNA under their control in response to some change in culture conditions, for example, the presence or absence of a nutrient or a change in temperature.",
"A large number of promoters are known, that may be operably linked to HoxCG DNA to achieve expression of HoxCG in a host cell.",
"This is not to say that the promoter associated with naturally occurring HoxCG DNA is not usable.",
"However, heterologous promoters generally will result in greater transcription and higher yields of expressed HoxCG.",
"Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoters, Goeddel et al., Nature 281:544-548 (1979), tryptophan (trp) promoter, Goeddel et al., Nuc.",
"Acids Res.",
"8:4057-4074 (1980), and hybrid promoters such as the tac promoter, deBoer et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 80:21-25 (1983).",
"However, other known bacterial promoters are suitable.",
"Their nucleotide sequences have been published, Siebenlist et al., Cell 20:269-281 (1980), thereby enabling a skilled worker operably to ligate them to DNA encoding HoxCG using linkers or adaptors to supply any required restriction sites.",
"See Wu et al., Meth.",
"Enz.",
"152:343-349 (1987).",
"Suitable promoters for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, Hitzeman et al., J. Biol.",
"Chem.",
"255:12073-12080 (1980); Kingsman et al., Meth.",
"Enz.",
"185:329-341 (1990), or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.",
"Dodson et al., Nuc.",
"Acids res.",
"10:2625-2637 (1982); Emr, Meth.",
"Enz.",
"185:231-279 (1990).",
"Expression vectors useful in mammalian cells typically include a promoter derived from a virus.",
"For example, promoters derived from polyoma virus, adenovirus, cytomegalovirus (CMV), and simian virus 40 (SV40) are commonly used.",
"Further, it is also possible, and often desirable, to utilize promoter or other control sequences associated with a naturally occurring DNA that encodes HoxCG, provided that such control sequences are functional in the particular host cell used for recombinant DNA expression.",
"In particular, in the present invention it may be desirable to utilize the zBMP2 promoter or SMAD promoter or goosecoid promoter such that a spatio-temporal expression of the HoxCG occurs.",
"Other control sequences that are desirable in an expression vector in addition to a promoter are a ribosome-binding site, and in the case of an expression vector used with eukaryotic host cells, an enhancer.",
"Enhancers are cis-acting elements of DNA, usually about from 10-300 bp, that act on a promoter to increase the level of transcription.",
"Many enhancer sequences are now known from mammalian genes (for example, the genes for globin, elastase, albumin, α-fetoprotein and insulin).",
"Typically, however, the enhancer used will be one from a eukaryotic cell virus.",
"Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.",
"See Kriegler, Meth.",
"Enz.",
"185:512-527 (1990).",
"Expression vectors may also contain sequences necessary for the termination of transcription and for stabilizing the messenger RNA (mRNA).",
"Balbas et al., Meth.",
"Enz.",
"185:14-37 (1990); Levinson, Meth.",
"Enz.",
"185:485-511 (1990).",
"In the case of expression vectors used with eukaryotic host cells, such transcription termination sequences may be obtained from the untranslated regions of eukaryotic or viral DNA's or cDNAs.",
"These regions contain polyadenylation sites as well as transcription termination sites.",
"Birnsteil et al., Cell 41:349-359 (1985).",
"In general, control sequences are DNA sequences necessary for the expression of an operably linked coding sequence in a particular host cell.",
"“Expression” refers to transcription and/or translation.",
"“Operably linked” refers to the covalent joining of two or more DNA sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed.",
"For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.",
"Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame.",
"Linking is accomplished by ligation at convenient restriction sites.",
"If such sites do not exist, then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods.",
"Expression and cloning vectors also will contain a sequence that enables the vector to replicate in one or more selected host cells.",
"Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosome(s), and includes origins of replication or autonomously replicating sequences.",
"Such sequences are well known for a variety of bacteria, yeast, and viruses.",
"The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2 μ plasmid origin is suitable for yeast, and various viral origins (for example, from SV40, polyoma, or adenovirus) are useful for cloning vectors in mammalian cells.",
"Most expression vectors are “shuttle” vectors, i.e.",
"they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.",
"For example, a vector may be cloned in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.",
"The expression vector may also include an amplifiable gene, such as that comprising the coding sequence for dihydrofolate reductase (DHFR).",
"Cells containing an expression vector that includes a DHFR gene may be cultured in the presence of methotrexate, a competitive antagonist of DHFR.",
"This leads to the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of other DNA sequences comprising the expression vector, Ringold et al., J. Mol.",
"Apl.",
"Genet.",
"1:165-175 (1981), such as a DNA sequence encoding HoxCG.",
"In that manner, the level of HoxCG produced by the cells may be increased.",
"DHFR protein encoded by the expression vector also may be used as a selectable marker of successful transfection.",
"For example, if the host cell prior to transformation is lacking in DHFR activity, successful transformation by an expression vector comprising DNA sequences encoding HoxCG and DHFR protein can be determined by cell growth in medium containing methotrexate.",
"Also, mammalian cells transformed by an expression vector comprising DNA sequences encoding HoxCG, DHFR protein, and aminoglycoside 3′ phosphotransferase (APH) can be determined by cell growth in medium containing an aminoglycoside antibiotic such as kanamycin or neomycin.",
"Because eukaryotic cells do not normally express an endogenous APH activity, genes encoding APH protein, commonly referred to as neor genes, may be used as dominant selectable markers in a wide range of eukaryotic host cells, by which cells transfected by the vector can easily be identified or selected.",
"Jiminez et al., Nature, 287:869-871 (1980); Colbere-Garapin et al., J. Mol.",
"Biol.",
"150:1-14 (1981); Okayama & Berg, Mol.",
"Cell.",
"Biol., 3:280-289 (1983).",
"Many other selectable markers are known that may be used for identifying and isolating recombinant host cells that express HoxCG.",
"For example, a suitable selection marker for use in yeast is the trp1 gene present in the yeast plasmid YRp7.Stinchcomb et al., Nature 282:39-43 (1979); Kingsman et al., Gene 7:141-152 (1979); Tschemper et al., Gene 10:157-166 (1980).",
"The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No.",
"44076 or PEP4-1 (available from the American Type Culture Collection, Rockville, Md.",
"20852 USA).",
"Jones, Genetics 85:12 (1977).",
"The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.",
"Similarly, Leu2-deficient yeast strains (ATCC Nos.",
"20622 or 38626) are complemented by known plasmids bearing the Leu2 gene.",
"Particularly useful in the invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding HoxCG.",
"In general, transient expression involves the use of an expression vector that is able to efficiently replicate in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.",
"Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNA's, as well as for the rapid screening of such polypeptides for desired biological or physiological properties.",
"Yang et al., Cell 47:3-10 (1986); Wong et al., Science 228:810-815 (1985); Lee et al., Proc.",
"Nat Acad.",
"Sci.",
"USA 82:4360-4364 (1985).",
"Thus, transient expression systems are particularly useful in the invention for expressing DNA's encoding amino acid sequence variants of HoxCG, to identify those variants which are functionally active.",
"Since it is often difficult to predict in advance the characteristics of an amino acid sequence variant of HoxCG, it will be appreciated that some screening of such variants will be needed to identify those that are functionally active.",
"Such screening may be performed in vitro, using routine assays for receptor binding, or assays for cell proliferation, cell differentiation or cell viability, or using immunoassays with monoclonal antibodies that selectively bind to HoxCG that effect the functionally active HoxCG, such as a monoclonal antibody that selectively binds to the active site or receptor binding site of HoxCG.",
"As used herein, the terms “transformation” and “transfection” refer to the process of introducing a desired nucleic acid, such a plasmid or an expression vector, into a host cell.",
"Various methods of transformation and transfection are available, depending on the nature of the host cell.",
"In the case of E. coli cells, the most common methods involve treating the cells with aqueous solutions of calcium chloride and other salts.",
"In the case of mammalian cells, the most common methods are transfection mediated by either calcium phosphate or DEAE-dextran, or electroporation.",
"Sambrook et al., eds., Molecular Cloning, pp.",
"1.74-1.84 and 16.30-16.55 (Cold Spring Harbor Laboratory Press, 1989).",
"Following transformation or transfection, the desired nucleic acid may integrate into the host cell genome, or may exist as an extrachromosomal element.",
"Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype.",
"The culture conditions, such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent to those skilled in the art.",
"Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, oysters, lower vertebrate, and mammalian host cells.",
"Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli, Bacillus species such as B. subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans.",
"In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for zBMP2, HoxCG and blocker molecule-encoding vectors.",
"Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.",
"However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Beach and Nurse, Nature 290:140-142 (1981), Pichia pastoris, Cregg et al., Bio/Technology 5:479-485 (1987); Sreekrishna, et al., Biochemistry 28:4117-4125 (1989), Neurospora crassa, Case, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 76:5259-5263 (1979), and Aspergillus hosts such as A. nidulans, Ballance et al., Biochem.",
"Biophys.",
"Res.",
"Commun.",
"112:284-289 (1983); Tilburn et al., Gene 26:205-221 (1983); Yelton et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 81:1470-1474 (1984), and A. niger, Kelly et al., EMBO J.",
"4:475-479 (1985).",
"Suitable host cells for the expression of HoxCG also are derived from multicellular organisms.",
"Such host cells are capable of complex processing and glycosylation activities.",
"In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture.",
"It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher et al., Ann.",
"Rev.",
"Biochem.",
"57:785-838 (1988), the extent or pattern of glycosylation of HoxCG in a foreign host cell typically will differ from that of HoxCG obtained from a cell in which it is naturally expressed.",
"Examples of invertebrate cells include insect and plant cells.",
"Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori host cells have been identified.",
"Luckow et al., Bio/Technology 6:47-55 (1988); Miller et al., in Genetic Engineering, vol.",
"8, pp.277-279 (Plenum Publishing, 0.1986); Maeda et al., Nature 315:592-594 (1985).",
"Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.",
"Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens.",
"During incubation of the plant cells with A. tumefaciens, the DNA is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the introduced DNA.",
"In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter.",
"Depicker et al., J. Mol.",
"Appl.",
"Gen. 1:561-573 (1982).",
"Herrera-Estrella et al., Nature 310:115-120 (1984).",
"In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue.",
"European Pat.",
"Pub .",
".",
".",
".",
"No.",
"EP 321,196 (published Jun.",
"21, 1989).",
"However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years.",
"Kruse & Patterson, eds., Tissue Culture (Academic Press, 1973).",
"Examples of useful mammalian host cells are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293 (or 293 cells subcloned for growth in suspension culture), Graham et al., J. Gen Virol.",
"36:59-72 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (including DHFR-deficient CHO cells, Urlaub et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 77:421.6-4220 (1980); mouse sertoli cells (TM4, Mather, Biol.",
"Reprod.",
"23:243-251 (1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.",
"Sci.",
"383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).",
"Construction of suitable vectors containing the nucleotide sequence encoding HoxCG and appropriate control sequences employs standard recombinant DNA methods.",
"DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required.",
"For analysis to confirm correct sequences in the vectors constructed, the vectors are analyzed by restriction digestion (to confirm the presence in the vector of predicted restriction endonuclease) and/or by sequencing by the dideoxy chain termination method of Sanger et al., Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 72:3918-3921 (1979).",
"The mammalian host cells used to produce the HoxCG of this invention may be cultured in a variety of media.",
"Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells.",
"In addition, any of the media described in Ham, et al., Meth.",
"Enz.",
"58:44-93 (1979); Barnes et al., Anal.",
"Biochem.",
"102:255-270 (1980); Bottenstein et al., Meth.",
"Enz.",
"58:94-109 (1979); U.S. Pat.",
"Nos.",
"4,560,655; 4,657,866; 4,767,704; or 4,927,762; or in PCT Pat.",
"Pub.",
"Nos.",
"WO 90/03430 (published Apr.",
"5, 1990), may be used as culture media for the host cells.",
"Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source.",
"Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.",
"The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.",
"The host cells referred to in this disclosure encompass cells in culture in vitro as well as cells that are within a host animal, for example, as a result of transplantation or implantation.",
"It is further contemplated that the HoxCG of this invention may be produced by homologous recombination, for example, as described in PCT Pat.",
"Pub.",
"No.",
"WO 91/06667 (published May 16, 1991).",
"Briefly, this method involves transforming cells containing an endogenous gene encoding HoxCG with a homologous DNA, which homologous DNA comprises (1) an amplifiable gene, such as DHFR, and (2) at least one flanking sequence, having a length of at least about 150 base pairs, which is homologous with a nucleotide sequence in the cell genome that is within or in proximity to the gene encoding HoxCG.",
"The transformation is carried out under conditions such that the homologous DNA integrates into the cell genome by recombination.",
"Cells having integrated the homologous DNA then are subjected to conditions which select for amplification of the amplifiable gene, whereby the HoxCG gene amplified concomitantly.",
"The resulting cells then are screened for production of desired amounts of HoxCG.",
"Flanking sequences that are in proximity to a gene encoding HoxCG are readily identified, for example, by the method of genomic walking, using as a starting point the HoxCG nucleotide sequence set forth in SEQ ID NO.",
":23 and SEQ ID NO.",
":24.See Spoerel et al., Meth.",
"Enz.",
"152:598-603 (1987).",
"Gene amplification and/or gene expression may be measured in a sample directly, for example, by conventional Southern blotting to quantitate DNA, or Northern blotting to quantitate mRNA, using an appropriately labeled oligonucleotide hybridization probe, based on the sequences provided herein.",
"Various labels may be employed, most commonly radioisotopes, particularly 32P.",
"However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide.",
"The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radioisotopes, fluorophores, chromophores, or the like.",
"Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.",
"The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.",
"Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of the gene product, HoxCG.",
"With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like.",
"A particularly sensitive staining technique suitable for use in the present invention is described by Hsu et al., Am.",
"J. Clin.",
"Path., 75:734-738 (1980).",
"Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal.",
"Conveniently, the antibodies may be prepared against a synthetic peptide based on the DNA sequences provided herein.",
"Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, means “including but not limited to” and is not intended to exclude other additives, components, integers or steps.",
"The invention will now be further described by way of reference only to the following non-limiting examples.",
"It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above.",
"Amino acid sequences referred to herein are given in standard single letter code.",
"EXAMPLE 1 Isolation of Stage-Specific Promoters for a Sterile Feral Construct In order to identify a good candidate promoter and/or gene for the proposed construct, the applicant examined a number of animals, both vertebrate and invertebrate.",
"The applicant finally decided on the well-studied model for fish, the zebrafish (Brachydanio rerio).",
"This fish model was chosen as it is reasonably well characterized, and the fish are small and relatively easily breed and reared.",
"Moreover, the zebrafish has a high degree of nucleotide and amino acid sequence homology to most other fish species studied, and as will be shown later, a reasonably high degree of sequence homology with other non-fish species.",
"This degree of similarity can permit the identification of genes in other species by comparison with those of zebrafish.",
"Accordingly, it was considered, that this model was most appropriate for locating and testing a promoter which may function across all species.",
"At least it was a useful model for testing the broad “sterile feral construct” concept.",
"The applicant examined mutant screens in zebrafish for a target gene that was essential for a short period in larval development, but which had no adult functions.",
"The applicant focused on 6 mutations that cause dorso-ventral patterning defects (Mullins et al 1996), and in particular on the swirl mutant, which exhibits severe dorsalization and the complete lack of ventral structures such as blood and pronephros.",
"Swirl encodes the zebrafish homologue of BMP2 and was named zBMP2 (Kishimoto et al., 1997).",
"In zebrafish the dorsalised swirl mutant phenotype is rescued by injection of zBMP2 mRNA at the single cell stage (Kishimoto et al., 1997), which indicates that the gene is essential only during early larval development and plays no maternal role.",
"BMPs (Bone Morphogenetic Proteins) are a subfamily of the larger transforming growth factor beta (TGF-β) superfamily of signalling molecules that play a central role in establishing the early animal body plan and in organogenesis (Hogan, 1996).",
"The cDNA for the zBMP2 gene was obtained from M. Hammerschmidt (Max Plank Institute, Frieburg) as a 1,732 bp fragment subcloned into a plasmid designated pzBMP2b.",
"This plasmid was transformed into XL-1 blue strain of E. coli according to the instructions of the supplier (Stratagene).",
"A resulting positive clone carrying the plasmid was grown according to standard protocols, and the cDNA from the bacterial culture was isolated by standard procedures.",
"After digestion with EcoRI, a 422 bp fragment spanning the 5′ untranslated region was isolated and labelled with 32P.",
"This was then used as a probe for a zebrafish genomic library.",
"The zebrafish genomic BAC library was purchased in the form of arrayed filter sets, from Genome Systems Inc (GSI), and screened using the labelled probe by standard hybridization techniques as described previously.",
"Five positive clones (BMP-BAC5, BMP-BAC10, BMP-BAC15, BMP-BAC17, and BMP-BAC21) were then purchased from Genome Systems Inc (GSI).",
"Preliminary sequencing of all five positive BAC clones using primers specific of the 5′-untranslated region of the cDNA revealed that the clones were identical to each other and to the region of the BMP2 cDNA.",
"Two of the BAC clones (BMP-BAC5 and BMP-BAC10) were subcloned as HindIII fragments into pGEM-7ZF(+) by standard procedures.",
"We obtained 6,915 bp of sequence from these clones which represented from −3879 to +3035 bp relative to the translation start site.",
"The coding sequence obtained was identical to the zebrafish zBMP2 cDNA sequence previously described by Nikido et al.",
"(1997) and Lee et al.",
"(1998).",
"This suggested that BAC 5 and 10, and perhaps the remaining three BAC clones, contained authentic zebrafish BMP2 genomic DNA.",
"However, based on the genomic sequences we obtained, the previously designated start site, at 376 bp in the cDNA (Lee et al., 1998), lies in the second exon and the first exon is untranslated.",
"Further definition and isolation of the zBMP2 promoter was accomplished by sequencing these HindIII subclones to isolate candidate fragments which resided 5′ of the sequence homologous to the cDNA coding for zBMP2 gene.",
"One of these subclones had a 5,901 bp insert that was positive for zBMP2 gene.",
"FIG.",
"1 shows the resultant plasmid pBAC5/H11.The insert was also found to include a 1,414 bp region that was 5′ of the presumptive start codon of zBMP2, and which was considered to be a possible location of the zBMP2 promoter.",
"A 1,414 bp fragment was excised from pBAC5/H11 with SmaI/EcoRI and subcloned into the multiple cloning site of pBluescript-II-SK.",
"This fragment contained the putative zBMP2 promoter from about 60 bp 5′ of the first splice site.",
"A SacI-KpnI fragment was then excised from this plasmid and directionally cloned into pGEM-EGFP containing the modified GFP reporter gene (GM2, see Cormack et al., 1996) resulting in the construct pzBMP2(1.4)-EGFP as shown in FIG.",
"2.We considered that the control of expression of zBMP2 gene likely resided in this SacI-KpnI fragment, and would be useful in controlling the “Sterile-Feral” construct.",
"However, we are sure that any promoter with an appropriate spatial-temporal pattern could be used in the final “Sterile-Feral” construct.",
"The construct pzBMP2(1.4)-EGFP was inserted into zebrafish embryos to test whether it followed a similar spatial-temporal expression pattern as reported for the zBMP2 promoter.",
"This construct and all subsequent constructs were prepared using the following procedures and introduced into the developing embryos by microinjection.",
"All the DNA preparations were appropriately linearized and gel purified (Qiaquick Gel Extraction Kit) before injection.",
"Needles were made from borosilicate glass capillaries with filaments (GC100TF-15, Clark Electromedical instruments) using a P-80PC micropipette puller (Sutter Instrument Co.).",
"The needle was back-filled with purified DNA diluted to 100 ng/μl in 1× injection buffer (10×; 50 mM Tris; 5 mM EDTA; 1M KCl, pH7.2) using a hand pulled pipette.",
"Injections were carried out on a dissection microscope fitted with two, 3-dimensional Narshige MN-151 micromanipulators.",
"Embryos were held in place during injection by a hydraulically (mineral oil) driven holding pipette.",
"Injection of DNA solution was facilitated pneumatically using a 3-way foot operated plunge valve (Festo Engineering), connected between the injection needle holder and nitrogen tank.",
"Injection was performed on one-cell stage embryos, unless specifically indicated otherwise.",
"Injected embryos were incubated and reared as described above.",
"Post-injection, early-stage embryos were examined under UV illumination in a Zeiss microscope equipped with standard fluorescent isothiocynate (FITC) filter set, while later-stage embryos were anaesthetized in embryo medium containing 0.125%, 2-phenoxyethanol (Sigma P-1126), before examination.",
"Photomicrographs of embryos expressing EGFP were obtained for analysis.",
"Table 1 summarises the injection trials.",
"The percentage of embryos expressing EGFP at 10 h post injection (pi), varied from batch to batch, ranging from 0% to 42.7%.",
"Expression was detectable as early as dorsal shield stage (6 h pi) in most of the expressing embryos.",
"At 9.5 h pi, the majority of the expressing embryos had expression that was limited to anterior ventral regions (FIG.",
"3a); however, 3 embryos expressed EGFP all along the ventral margin (FIG.",
"4a).",
"The patchiness is typical of the mosaic expression expected in founder transgenic animals.",
"Nonetheless, expression domains extended from polster region (FIG.",
"3a;PO) anteriorly to the region of future tail bud, posteriorly (FIG.",
"3a;TB).",
"TABLE 1 Results of EGFP expression in embryos injected with pzBMP2(1.4)-EGFP at about 9.5-10 h Post Injection Number with No.",
"with Total No.",
"Anterio- entire Number with ventral ventral Batch Observed Expression expression domain 1 28 0 0 0 2 21 3 1 2 3 20 2 0 2 4 28 12 2 10 At about 24 h pi, expression was predominantly in the ventral domains (FIG.",
"5a), mimicking the native zBMP2 expression—in the region of the developing eye, otic vesicle, and pectoral fin bud.",
"Abolition of tail bud expression at 24 h pi suggests that the cloned promoter may lack regulatory elements responsible for maintenance of BMP2 expression at this stage.",
"No EGFP expression was detected by 48 h pi, suggesting that the zBMP2 gene is not required this late in development.",
"The zBMP2 promoter sequence is shown in SEQ ID NO:1.EXAMPLE 2 Isolation of Second Promoter for Sterile Feral Construct As the applicant was concerned about the potential shortcomings/delays of the BMP2 promoter in combination with a tet-responsive (tetOff) element to effectively block its own native transcripts, an early acting, but temporally restricted promoter sharing spatial domains with that of BMP2 was considered preferable.",
"One such candidate was the zebrafish SMAD5.Similar to BMP2, mutation in the zebrafish SMAD5 results in a dorsalized mutation designated somitabun (sbn) and the dorsalised mutant phenotype has been shown to be rescued by injection of SMAD5 mRNA at the single cell stage (Hild et al., 1999).",
"This indicated that the gene is essential only during early larval development.",
"It has also been implied that the SMAD5 acts as a transducer of BMP2 signalling with potential upstream and downstream functions.",
"The functional association between the BMP2 and SMAD5 suggested that the two genes share the same spatial expression domains.",
"Further the maternal expression of SMAD5 and also the relative early onset of zygotic SMAD5 expression ensure that the cells are competent to process BMP2 signalling (Hild et al., 1999; Dick et al., 1999).",
"Therefore, we considered that by employing a SMAD5 promoter to drive the expression of a BMP2 blocker would alleviate some of the potential temporal delays associated with employing the BMP2 promoter.",
"The cDNA for the SMAD5 gene was amplified from zebrafish shield stage cDNA using following primers SMADu1: 5′-TGCAGGTGGACTTTGGATCCG-3′ SEQ.",
"ID.",
"NO.",
": 4 SMADL1: 5′-GCCTAAAGGCAACAGATGCTA-3′ SEQ.",
"ID.",
"NO.",
": 5 The primers were designed based on the published zebrafish SMAD5 cDNA sequences (Hild et al., 1999).",
"The amplified 2285 bp product was cloned into pGem-T-Easy vector as per the cloning instructions of the manufacturer (Promega, Madison USA) and confirmed by sequencing.",
"A resulting positive clone carrying the plasmid was grown according to standard protocols, and the cDNA from the bacterial culture was isolated by standard procedures.",
"A 366 bp fragment spanning the 5′ untranslated region was isolated and labelled with 32P.",
"This was then used as a probe for a zebrafish genomic library.",
"Four positive clones (SMAD-BAC1, SMAD-BAC8, SMAD-BAC13, and SMAD-BAC 17) were then purchased from GSI.",
"Preliminary sequencing of all four positive BAC clones using primers specific of the 5′-untranslated region of the cDNA revealed that the clones were identical to each other and to the region of the BMP2 cDNA.",
"One of the BAC clones (SMAD-BAC51) was subcloned as HindIII fragments into pGEM-7ZF(+) by standard procedures.",
"We obtained a positive subclone of about 8 KB (psBAC1/H12), that contained 1,005 bp of putative promoter sequence 5′ of the start codon.",
"The coding sequence obtained was identical to the zebrafish SMAD5 cDNA sequence previously described by Hild et al.",
"(1999).",
"A 1,005 bp putative promoter fragment was then amplified from psBAC1/H12 with the following primers M13 forward: 5′-GTAAAACGACGGCCAGT SEQ ID NO: 6 SMAD L2: 5′-TAGTGCTGGGCTGCACCAG SEQ ID NO: 7 The amplified fragment was ligated into pGEM-Teasy vector and the orientation and sequence confirmed (pSMAD5′).",
"The promoter was again excised as SmaI/EcoRI fragment, blunt ended and ligated into the SmaI linearized pGEM-EGFP.",
"A positive clone, pSMAD5-EGFP (FIG.",
"6) in the correct orientation was selected and tested in vivo in zebrafish embryos.",
"Injection trials of pSMAD5-EGFP into the zebrafish embryo resulted in expression of the EGFP as early as 4 hp.",
"The expression pattern was ubiquitous initially as late as shield stage (FIG.",
"7), then predominantly restricting to ventral tissues at about 24 hpi (FIG.",
"8).",
"The experimental evidence suggested that the zygotic expression of SMAD5 was activated marginally ahead of zBMP2.Although preliminary, our promoter analysis experiments suggested that the SMAD5 promoter was indeed activated slightly ahead of bmp2 promoter (data not shown).",
"No EGFP expression was detected by 48 hpi, suggesting that the SMAD5 gene was not required this late in development.",
"The zebrafish SMAD5 promoter sequence is shown in SEQ ID NO; 8.EXAMPLE 3 Zebrafish Model Breeding and rearing protocols for zebrafish generally follow Westerfield (1995).",
"Stock was obtained from a local pet store; however, it would be appreciated by those skilled in the art that zebrafish could equally be obtained from laboratories around the world (e.g., Institute of Neuroscience, eugene, Oreg., USA) and maintained at 27-28° C. in an in-house re-circulatory flow-through system.",
"Embryos were obtained by natural matings, transferred into Embryo Medium (Westerfield, 1995), and incubated in a bench top incubator at 26-27° C. until 3-4 days old.",
"They were then transferred into nursery tanks maintained at 27-28° C., and reared on finely ground commercial fish flakes (Tetramin), and live Artemia.",
"After approximately 3 months, the fish were transferred into standard fish tanks alongside the adult fish.",
"The adult fish were fed daily with flakes and occasionally supplemented with either freshly hatched or frozen Artemia.",
"EXAMPLE 4 Blocking Expression of zBMP2 The applicant tested three options for blocking expression of the candidate genes: mis/over-expression of sense (see below), antisense (Izant and Weintraub 1984) and double stranded RNA (dsRNA).",
"(Guo and Kemphues, 1995).",
"The latter appears to be more potent than antisense at inducing interference in C. elegans (Fire et al., 1998) and has been employed to silence native and reporter genes in plants (Waterhouse et al., 1998).",
"To develop and optimise the blocking component of the “sterile feral” construct, the applicant assayed sense, antisense, and dsRNA of zBMP2 by injection in zebrafish embryos.",
"Results indicated that both antisense and dsRNA block gene expression, whereas sense strand injection resulted in over-expression.",
"Capped full-length sense and antisense zBMP2 RNA transcripts were generated by linearizing the plasmid pzBMP2b, whereas the truncated versions of just the 5′- or the 3′-regions were generated by appropriately linearised pzBMP2-ApaI or pzBMP2-BstXI, respectively.",
"All in vitro transcriptions were carried out using T3/T7 mMESSAGE mMACHINE™ (Ambion), as appropriate.",
"dsRNA was prepared by annealing sense and antisense RNA in RNAase free injection buffer at 37° C. for 5 minutes for the truncated and 10 minutes for the full-length transcripts.",
"Annealing of respective sense and antisense strands as dsRNA was confirmed by running a sample on a non-denaturing agarose gel.",
"About 3-5 picolitres of RNA solutions, ranging between 100-250 ng/μl, were injected into 1-2 cell stage embryos as described above in Example 1.In the case of 2-cell stage injections, both the cells were injected.",
"In embryos injected with full-length antisense or dsRNA of zBMP2, the proportion of normal embryos was significantly reduced and some weakly dorsalised embryos resembling zebrafish swirl mutant were seen (FIG.",
"9a&b).",
"Sense injections resulted in mild ventralization of the embryos, which in some cases resembled the zebrafish chordino mutant phenotype (FIG.",
"10).",
"Chordino is the dorsally expressing zebrafish homologue of chordin, known to interact antagonistically with BMPs (in this case swirl) in a dose dependent manner (Kishimoto et al., 1997).",
"To obtain molecular data to support hypothesised interference of the dsRNA on expression of zBMP2, the applicant injected truncated forms of zBMP2 ds RNA, so as to use the uninjected portion as probe to detect and quantify the native transcript levels in the injected embryos.",
"The percentage of deformed embryos in groups injected with 3′-zBMP2 and 5′-zBMP2 dsRNA was 43.4% and 40.2%, as compared to 9.2% and 2.4% in the corresponding controls (Table 2).",
"TABLE 2 Results of Truncated zBMP2 dsRNA Injection Into One-Cell Stage Embryos Transcript Number Number Number Injected Conc.",
"ng/μl injected Survivors* deformed* 3′-zBMP2 150 123 83 36 (67.5) (43.4) Control 0 66 54 5 (81.8) (9.2) 5′-zBMP 250 88 67 27 (76.1) (40.2) Control 0 53 42 1 (79.2) (2.4) *Results in parenthesis indicate percentages EXAMPLE 5 Combined Promoter and Blocker DNA Construct On confirming the ability of in vitro transcribed BMP2 antisense and double stranded transcripts to disrupt larval development, DNA constructs capable of expressing the antisense and double stranded transcripts in vivo were developed and tested.",
"A 711 bp ApaI fragment of the zBMP2 cDNA was excised from the plasmid pzBMP2b and inserted into the ApaI linearized pzBMP2(1.4)-EGFP resulting in the pzBMP2As-EGFP (FIG.",
"11).",
"Antisense orientation of zBMP2 fragment in pzBMP2AS-EGFP was confirmed both by restriction analysis and sequencing.",
"The pzBMP2As-EGFP was a fusion construct capable of co-expressing BMP2 antisense and EGFP.",
"Co-expression of EGFP with the BMP2 antisense provided an easily detectable marker to distinguish the mutant embryos emanating from antisense interference and those potentially resulting from spontaneous or background mutations.",
"pzBMP2As-EGFP was linearized with NotI for injection into the embryos.",
"For the double stranded knockout, four segments of the zBMP2 gene were arranged to express double stranded mRNA in vivo (FIG.",
"12).",
"The first-section comprised the 1,414 bp “HindIII-EcoRI” promoter region retained in the pGEM 7zf(+) vector backbone, obtained by excising the EcoRI-SacI coding region of the zBMP2 from pBAC5/H11 subclone.",
"The second segment was a 510 bp fragment of the zBMP2 cDNA from sequence 301-810 in the published cDNA sequence (Lee et al., 1998).",
"This fragment was amplified using the following primers: zfEx1-3.EcoF Forward Primer 5′-ACCCCGAATTCATGAGGAACTTAGGA-3′ SEQ ID NO: 9 zfEx1-3.SalR Reverse Primer 5′-ATCAGCTCGTCGACAGGAATGGAGGTAAG-3′ SEQ ID NO: 10 The amplified product generated had an EcoRI site on the 5′-end and a SalI site on the 3′-end for ease of cloning.",
"The third section was a 286 bp fragment of cDNA (bases 307-592) which was amplified using the following primers: Bex1i.PstF 2 Forward Primer 5′-ACACCTGCAGATGAGGAACTTAGGAGACGAC-3′ SEQ ID NO: 11 Bex1i.SalR Reverse Primer 5′-TACTGAGGGTCGACTGCCGATTTGCT-3′ SEQ ID NO: 12 These primers generated a PstI site on the 5′ end and SalI site on the 3′ end for cloning.",
"When ligated to the second fragment, the third segment formed an inverted repeat of the 5′ end of the cDNA (bases 307 through 592).",
"The final segment was a PstI-SacI fragment containing a poly A tail section, excised from the pGT2-ns-GM2f construct that was kindly donated by Dr. Shou Lin, Institute of Molecular Medicine and Genetics, Medical College of Georgia.",
"The DNA sequence for the double stranded BMP2 construct is given as SEQ ID NO:13.Results of the BMP2 antisense-EGFP fusion construct injection are presented in the Table 3.TABLE 3 Results of NotI linearized pzBMP2-As-EGFP Injection into the One-Cell Zebrafish Embryos Number Conc.",
"Number Number Number with EGFP Batch μg/ml injected Survivors* deformed* expression 1 100 48 36 1 0 (75) (2.7) 0 40 29 0 0 (72.5) 2 100 36 16 6 5 (44.4) (37.5) (31.3) 0 16 9 0 0 (56.2) 3 100 20 12 4 3 (60) (33.3) (75) 0 23 15 0 0 (65.2) *Figures in parenthesis indicate percentages.",
"The number of deformed individuals in the injected groups ranged from 0% to 37.5%.",
"The majority of the deformed individuals (83.3% and 75% in batches 1 and 2, respectively) expressed EGFP, indicating that the antisense was effective in disrupting the larval development.",
"None of the individuals in the control group and non-deformed individuals in the injected group had EGFP expression.",
"Results of the zBMP2-double stranded construct are given in Table 4.TABLE 4 Results of pzBMP2-ds Injection into 1-4 Cell Stage Zebrafish Embryos Treatment Number Number of Number Batch Conc.",
"(μg/ml Treated mortality Deformed Injected 0 37 4 (10.8) 0 Control Uninjected — 123 24 (20.5) 1 (0.8)* control dsRNA injected 100 143 20 (14.3) 21 (14.7) Uninjected — 51 11 (17.1) 0 control dsRNA injected 100 47 7 (16.5) 22 (45.7) Figures in parenthesis indicate percentages.",
"*denotes a deformed control fish that had deformities that did not resemble the swirl mutants.",
"Of 211 control embryos (mock-injected with buffer only or permitted to develop normally), only one embryo was deformed.",
"The deformity did not resemble the swirl mutant.",
"In the two dsDNA treatment groups, 14.7% and 45.7% of the embryos expressed the swirl mutation.",
"EXAMPLE 6 The Repressible Element The proof-of-concept used a commercially available repressible element as the externally keyed genetic switch or Tet-responsive PhCMV*-1 promoter.",
"PhCMV*-1 contains the Tet-responsive element (TRE) which consists of seven copies of the 42 bp tet operator sequence (tetO).",
"This element is just upstream of the minimal CMV promoter (PminCMV), which lacks the enhancer that is part of the complete CMV promoter.",
"Therefore, PhCMV*-1 is silent in the absence of binding of transactivator protein (tTA) to the tetO.",
"The tetracycline-sensitive element is described by Gossen and Bujard (1992; tet-off), Gossen et al.",
"(1995; Tet-on), and Kistner et al.",
"(1996).",
"In the tetracycline-regulated system (Tet-Off system) developed by Hermann Bujard, addition of tetracycline (Tc) or doxycycline Dox; a Tc derivative) prevents the binding of a tTA, to the Tet-responsive element.",
"This then blocks gene expression from the TRE until the drug is removed.",
"A complementary system has also been developed (Tet-On system).",
"In the Tet-On system, addition of doxycycline allows the binding of a reverse transactivater, rtTA, to the tetO promoter, leading to gene expression from the TRE.",
"Gene expression continues from the TRE until removal of the drug.",
"A tetracycline responsive element has the advantage of ease of administering.",
"Tetracycline is a routinely used antibiotic in fish and shellfish culture (see Stoffregan et al., 1996), readily traverses cutaneous membranes while retaining its biological activity, and can be administered by whole organism immersion.",
"Use of the Tet-On/Off controllable expression systems is covered by U.S. Pat.",
"No.",
"5,464,758, assigned to BASF Aktiengesellschaft.",
"The applicant first tested the functionality of the Tet-off system in zebrafish cell cultures.",
"The cell culture was established using ZF4 cells as previously described (Driever and Rangini, 1993).",
"Cells were transfected with the DNAs using Effectene liposomes (Qiagen) according to the manufacturer's instructions.",
"Cells were initially transfected with pTet-Off and placed under neomycin selection for 1 month.",
"Neomycin-resistant cells were then transfected with pTRE-EGFP, and the selection plasmid pTK-Hyg, and placed under hygromycin selection for two weeks.",
"EGFP expression was determined by examining and counting cells with obvious fluorescence and by examination of cell lysates using a fluorometer.",
"Cells were grown in medium with or without doxycycline (0.2 μg/ml) for 72 h prior to assessment of gene expression, or were rinsed of doxycycline and assessed for reporter gene expression 72 h after removal of doxycycline.",
"In the absence of doxycycline, EGFP fluorescence was detected in a small percentage (approximately 6%) of cells (Table 5).",
"TABLE 5 Transfection % cells expressing EGFP expression in Treatment EGFP cell lysates None 0 0 ± 15 pTet-Off 0 0 ± 12 pTRE-EGFP 0 0 ± 9 pTet-Off + pTRE- 5.9 ± 1.2 86 ± 11 EGFP pTet-Off + pTRE- 0.2 ± 0.1 5 ± 3 EGFP + Dox (72 h) pTet-Off + pTRE- 2.6 ± 0.9 49 ± 6 EGFP + removal of Dox (72 h) Values represent the average and standard errors for 3 separate transfection experiments, each containing 4 replicates.",
"The low percentage of cells expressing the reporter gene presumably reflects the efficiency of simultaneously transfecting the cells with two plasmids (pTRE-EGFP and pTK-Hyg).",
"When doxycycline was added, EGFP gene expression dropped substantially, to approximately 3% of expression levels seen in cells not exposed to doxycycline.",
"Interestingly, washing the cells and removing as much of the doxycycline as possible could reverse the repression of reporter gene expression.",
"Fluorometric assays of cell lysates performed using a BMG FluoStar showed similar results to cell counts, with repression of the EGFP fluorescence being repressed in the presence of doxycycline.",
"The reversal of the repression following removal of doxycycline appeared greater in these assays, most likely because the fluorometer could detect low levels of fluorescence not detected by microscopic examination.",
"Next the applicant tested the tet-off system in whole zebrafish embryos.",
"The Tet-On™ and Tet-off™ gene expression system and the Tet responsive bidirectional vectors pBI and pBI-EGFP were purchased from a commercial source (Clontech).",
"The pzBMP2-Tet-Off construct (FIG.",
"13) was engineered by excising PminCMV promoter as SpeI and EcoRI fragment from pTet-Off and replacing it with the 1,414 bp zBMP2 promoter as XbaI/EcoRI, from pzBMP2-(1.4), by directional cloning.",
"The pzBMP2-Tet-Off and pBI constructs were linearised with SacI and PuvII, respectively and column purified using a PCR purification column (Qiagen).",
"Eluted DNA were quantified and mixed in equimolar ratio to yield a final concentration of about 150 ng/μl in injection buffer.",
"Injections were carried out using one-cell stage embryos as described in Example 1.Of the 84 embryos co-injected, EGFP expression was detectable in 7 (8.3%) individuals at about 24 h pi.",
"A low percentage of transformed embryos is typical of co-injection experiments.",
"The spatial pattern of EGFP expression (along the anterio-ventral regions) is similar to that we previously observed when EGFP was directly under the regulation of zBMP2 promoter.",
"EXAMPLE 7 Complete Zebrafish Sterile Feral Construct A single tet responsive double stranded RNA blocker construct under the regulation of zBMP2 promoter, pBIT(Bmp2)-Bmp2ds (FIG.",
"14), was built using pBI-EGFP as the backbone.",
"The bidirectional tet responsive construct with EGFP as a marker was chosen to provide a visible marker.",
"First, the SV40 PolyA was excised from the vector pBI-EGFP (Clontech, PT3146-5) following digestion with AatII and SalI.",
"The resulting fragment was blunt ended with T4 DNA polymerase and religated to form pBi(−SV), an intermediate plasmid.",
"This was then cut with HindIII and used in a subsequent ligation with a HindIII fragment containing the BMP2 promoter, which was obtained from BMP-tetOff plasmid (SEQ ID NO:2, NM99/09099).",
"The resulting plasmid, called pBi.tTA was then cut with with PvuII, dephosphorylated, and added to a ligation reaction containing a second fragment (blunt ended with T4 DNA polymerase), which contained the a 510 bp fragment of the zBMP2 cDNA from sequence 301-810 in the published cDNA sequence (Lee et al., 1998) and was obtained by digesting dsRNA (BMP2) (SEQ ID NO:13, NM99/09100) with EcoRI and HindIII followed by gel purification.",
"This ligation reaction produced the construct pSF1.The pBIT(Bmp2)-bmp2ds construct is shown in FIG.",
"14 and SEQ ID NO: 14 and here through refereed to as pSF1.Similarly pBIT(Cmv)-BMP2ds (pSF2), a zbmp2 double stranded RNA blocker construct in which the tet-Off (tTA) is under the regulation of CMV promoter, was built as follows.",
"Commercially purchased pTet-Off construct was digested with HindIII, XhoI and SapI.",
"A 2250 bp XhoI/HindIII fragment containing CMV promoter, tTA and SV40 PolyA and a 2000 bp SapI/XhoI fragment containing vector backbone were gel purified.",
"Meanwhile the pBIT(bmp)-bmp2ds was digested with HindIII/SapI and a 3,459 bp fragment containing EGFP and double stranded bmp2 RNA, with β-globin poly A was gel purified.",
"Finally the three fragments were ligated directionally to yield the pBIT(CMV)-bmp2ds (pSF2, FIG.",
"15, SEQ ID NO:15) construct.",
"The applicant constructed two more candidate sterile feral constructs, with tTA driven by the zebrafish SMAD5 promoter: one used BMP2 double stranded RNA as developmental blocker [pBIT(smad)-BMP2ds] and another used zBMP2 sense, to be mis-expressed, as a blocker [pBIT(smad)-BMP2sense).",
"An intermediate construct, pSmadTet-Off, was built by excising the CMVmin1 promoter as XbaI and SpeI fragmet from pTet-Off and replacing it with a 965 bp zebrafish SMAD5 promoter.",
"Subsequently, pBIT(smad)-BMP2ds (pSF3, FIG.",
"16, SEQ ID NO:16) was made by excising CMV promoter as a XhoI/SphI fragment from pBIT(CMV)-bmp2ds and replacing it with XhoI/SphI SMAD5 promoter fragment from pSmadTet-Off.",
"The construct was confirmed by restriction analysis and sequencing.",
"The construct was renamed pSF3.The pBIT(smad)-BMP2sense(pSF4, FIG.",
"17; SEQ ID NO:17) was constructed as follows.",
"Firstly a 1,440 bp zebrafish BMP2 cDNA was excised as EcoRI and XhoI fragment from pzBMP2b, blunt ended and ligated into PvuII linearized pBI-EGFP.",
"The sense orientation of the bmp2 cDNA in the bi-directional vector was confirmed by restriction analysis and sequencing.",
"A resulting clone (pBI-bmp2-Sense) in the correct orientation was prepared for further use.",
"The double stranded RNA blocker in the pBIT(smad)-bmp2ds (pSF3) was excised as EagI/MluI fragment and replaced with EagI/MluI fragment from pBI-bmp2-Sense construct.",
"The resulting pBIT(smad)-bmp2-Sense construct (pSF4, FIG.",
"17 and SEQ ID NO:17) was confirmed by restriction analysis and sequencing.",
"Table 6 summarises the pooled results of three different batches of pSF1 construct injections into zebrafish embryos.",
"TABLE 6 Results of pSF1 (100 ng/μl) injections into the zebrafish embryo.",
"No No.",
"dead dead No.",
"No.",
"Glow No.",
"Non Glow Treatment Total 5 hpi 24 hpi Live Deformed Normal Deformed Normal SF1 166 65 11 90 2 34 0 52 Injected (54.2) (2.2) (37.7) (57.7) Buffer 143 56 17 70 0 0 0 0 Control (48.9) Although about 40% of the embryos had EGFP expression, only 2.2% had the associated deformity resembling the dorsalized swirl mutation.",
"This is in stark contrast to 14-40% swirl like deformities the applicant observed by injection of a double stranded RNA construct (pzBMP2-ds) that was driven directly by the BMP2 promoter.",
"The lack of correlation between the deformity and EGFP expression may be attributed to several reasons, including the delay associated with the indirect expression of the blocker by the BMP2 promoter mediated via the expression of tTA.",
"Table 7 summarises the results of pSF2 injected into the embryos of zebrafish.",
"TABLE 7 Results of injecting pSF2 (100 ng/μl) into the embryos of zebrafish.",
"No No.",
"dead dead No.",
"No.",
"Glow No.",
"Non Glow Treatment Total 5 hpi 24 hpi Live Deformed Normal Deformed Normal SF2 Dox 175 44 30 101 3 8 6 84 (57.7) (2.9) (7.9) (5.9) (83.1) No Dox 183 28 53 102 11 49 2 40 (55.7) (10.7) (48.0) (1.9) (39.2) Control 118 23 14 81 0 0 0 0 Dox (68.6) No Dox 107 13 18 76 0 0 0 0 (71.0) CMV, a ubiquitously active promoter, drives the pSF2.In all these sets of experiments, about half the injected and control fish were immersed in a solution of 150 ppm doxycycline (dox) to evaluate the efficiency of repression.",
"The data were pooled from 3 separate sets of injections.",
"Following pSF2 injection and repression, the proportion of embryos expressing EGFP in the dox treated group was much lower from that of untreated group (11% vs 59%).",
"These results confirm Example 6 that the applicant has achieved temporal control of genes under the regulation of tet responsive promoter in zebrafish.",
"However, as in case of pSF1, there was no correlation between the embryos expressing EGFP and those with a dorsalized deformity.",
"Although the CMV is a ubiquitously expressing promoter, the applicant hypothesized that the mosaic distribution of injected construct may have precluded consistent expression in the BMP2 expression domains.",
"The results from injection and repression of pSF3, in which the tTA is driven by zebrafish SMAD5 promoter are presented in Table 8.TABLE 8 Results of the repression experiment following injection of pSF3 and pSF2 constructs (100 ng/μl) in the zebrafish embryos.",
"Numbers in parenthesis are percentages.",
"Dead Dead Glow No Glow Treatment Total 5 HPI 24 HPI # Live Deformed Normal Total Deformed Normal Total pSF3 No Dox 60 9 13 38 15 11 26 0 12 12 (63.3) (39.4) (28.9) (68.4) (31.5) (31.5) Dox 52 11 14 27 5 5 10 1 16 17 125 ppm (51.9) (18.5) (18.5) (37.0) (3) (59.25) (62.9) Control No Dox 85 29 0 56 — — — 5 51 56 (65.8) (8.9) (91.0) (100) Dox 86 24 20 42 — — — 0 42 42 150 ppm (48.8) (100) (100) pSF2 No Dox 58 11 6 41 8 16 24 2 15 17 (70.6) (19.5) (39.0) (58.5) (4.8) (36.5) (41.4) Dox 58 18 14 26 1 2 3 9 14 23 150 ppm (44.8) (3.8) (7.6) (11.5) (34.6) (53.8) (88.4) The applicant included pSF2 injections in this set of experiments as positive controls for repression.",
"Repression of embryos injected with pSF3 were carried out in rearing medium containing 125 ppm dox, unlike the 150 ppm employed for pSF2 injected groups.",
"This was because in preliminary experiments the applicant encountered higher mortality associated with 150 ppm dox and pSF3 injected embryos (data not shown).",
"As for pSF2, treatment with dox reduced substantially the percentage of surviving embryos exhibiting EGFP expression and swirl-like deformies, confirming repression.",
"Unlike the pSF2 construct, there was a clear association between.",
"EGFP expression and a dorsalizied mutation, the two co-expressing in close to 40% of the embryos surviving past 24 hpi.",
"This confirms that the SMAD5 promoter effectively expressed the BMP2 double-stranded blocker, causing developmental arrest in un-repressed embryos.",
"The applicant hypothesize that the increased efficiency of SMAD5 promoter in the complete Sterile Feral Construct over that of BMP2 promoter results from its potential early zygotic activation, ensuring the transcription of blocker molecules much before expression of the native BMP2 transcripts.",
"Since the smad5 is known to be expressed maternally (Hild et al., 1999), it is likely to function even more effectively in permanently transformed lines.",
"The applicant also built and tested a Sterile Feral Construct for zebrafish using mis-expression of the BMP2 gene as the blocker sequence (pSF4).",
"As predicted, injection of pSF4 resulted in overexpression of BMP2, resulting in fish with ventralizied mutations (FIG.",
"18A-C, arrow.",
"Majority of the deformed fish co-expressed EGFP and in some instances the EGFP expression was closely associated with the ventralized tissue (FIG.",
"18C).",
"As summarized in Table 9, the large majority of the EGFP expressing embryos also had ventralized phenotypes as shown in FIG.",
"18A-C. TABLE 9 Results of pSF4 injection (100 ng/μl) into zebrafish embryos Total No.",
"Dead No No Glowing No.",
"non-Glowing Treatment No.",
"5 HPI 24 HPI Live Deformed Normal Deformed Normal PSF4 234 104 37 93 33 31 7 22 Injected (44.4) (15.8) (39.7) (35.4) (33.3) (7.5) (23.6) Control 118 46 10 68 — — 3 65 (4.4) (95.5) EXAMPLE 8 Transfection of Pacific Oysters Mature oysters (Crassostrea gigas) were obtained from local hatcheries in Tasmania and New South Wales, and held in artificial seawater at 10° C. until required.",
"Eggs were collected from 2-3 females by stripping the gonads and were pooled, rinsed on a 20 μm mesh, and left to condition in artificial sea water for 2 h. Sperm were stripped from male gonads, diluted to approximately 10,000 gametes/μl, and used immediately for electroporation-mediated nucleic acid delivery.",
"Plasmid DNA (50 μg/ml) or double-stranded RNA (dsRNA; 1 μg/ml) was delivered into 1×106 sperm using a BioRad Gene Pulser II electroporator in 0.2 cm gap electroporation cuvettes.",
"Sperm were subjected to a single electroporation pulse (50 V, 100% modulation, 10 kHz, 12.5 msec) and immediately mixed with 5000 oocytes.",
"Fertilized embryos and developing larvae were reared at 20° C. in artificial seawater containing 0.1 μg/ml chloramphenicol.",
"Surviving larvae were counted after 24 h development.",
"For experiments in which the Drosophila melanogaster heat shock promoter was used to drive expression of the delivered genes, a 1 h heat shock at 37° C. was provided either at 2 h or 18 h post fertilization, and development was then permitted to proceed at 20° C. The applicant developed and tested transfection techniques for Pacific oyster eggs and larvae using genes encoding enhanced green fluorescent protein (EGFP, Clontech), glucuronidase (GUS), and red fluorescent protein (RFP, Clontech).",
"Efficacy of electroporation as a transfection method of oyster sperm, using EGFP as a reporter gene was tested.",
"Two different constructs, containing the EGFP gene under the control of either the CMV or Drosophila heat shock (Hsp) promoter were delivered into sperm using electroporation, and EGFP fluorescence was monitored using microscopy and fluorometric assays.",
"Oyster embryos and larvae displayed a moderate level of autofluorescence that obscured detection of low levels of EGFP.",
"Consequently, it was seldom possible to visually distinguish transfectants from non-transfectants when the EGFP gene was under the control of the CMV promoter using the construct pBiT(CMV)-EGFP (SEQ ID NO:18) as compared to EGFP expression levels observed using pBiT(dHSP)-EGFP (SEQ ID NO:19) following heat shock.",
"However, EGFP and RFP were easily detected when expressed under the control of the D. melanogaster heat shock promoter, using constructs pBiT(dHSP)-EGFP (SEQ ID NO:19) and pBiT(dHSP)-RFP-oHoxDS/BH (SEQ ID NO:20) respectively.",
"By visual inspection, it was estimated that approximately 60% of the surviving trochophore larvae were transfected (Table 10).",
"TABLE 10 Genetic Electro- construct % larvae with EGFP fluorescence poration Promoter/ Heat EGFP relative applied Reporter shock fluorescence1 to controls − − − 0 ± 0.2 1 ± 0.2 + − − 0 ± 0.2 1 ± 0.2 − CMV/EGFP − 1 ± 0.5 1 ± 0.3 + CMV/EGFP − 5 ± 3 1.5 ± 0.2 − Hsp/EGFP + 4 ± 1 1.2 ± 0.3 + Hsp/EGFP − 24 ± 10 2.4 ± 0.7 + Hsp/EGFP + 61 ± 15 14.3 ± 1.1 1Larvae with EGFP fluorescence visibly greater than that seen in non-transfected controls The values represent the means and standard errors for three separate experiments.",
"To quantitatively assess EGFP and RFP fluorescence, larvae were homogenized in homogenization buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 10 mM mercaptoethanol), and protein extracts were measured for fluorescence using a BMG FLUOstar fluorometer.",
"Transfection efficiencies were also assessed in a second set of experiments that examined delivery of pHSP-GUS construct (SEQ ID NO:21).",
"The pHSP-GUS construct was made in a two step fashion.",
"First, the D. melanogaster heat shock promoter and terminator were isolated from the pCaSpeR-hs plasmid (Thummel and Pirrotta, 1992, Drosophila Information Service 71: 150) by PCR using the two primers: Dmhsp Forward Primer 5′-GAATTCCTAGAATCCCAAAACAAACTGG-3′ SEQ ID NO: 31 Dmhst Reverse Primer 5′- GGATCCTGACCGTCCATCGCAATAAAATGAGCC-3′ SEQ ID NO: 32 The resulting amplicon was cloned into the T-tailed vector pGEM-T-Easy (Promega) according to the manufacturer's directions to produce the pGEMhsp70 plasmid.",
"The second step involved excision of the gene encoding the β-glucuronidase gene (gus) from the plasmid pBacPak8-GUS (Clontech) using the restriction endonucleases NcoI and EcoRI.",
"The ends of the 1.8 kb gus fragment were then converted to blunt ends using the Klenow fragment of E. coli DNA polymerase.",
"The pGEMhsp70 plasmid was then linearized at the polylinker site between the promoter and terminator sequences using BglII and the ends were converted to blunt ends using the Klenow fragment.",
"The 1.8 kb gus gene fragment was finally ligated into the blunt-end BglII site to produce the pHSP-GUS plasmid (SEQ ID NO:21).",
"The pHSP-GUS construct expresses GUS under the control of the D. melanogaster heat shock promoter (Table 11).",
"TABLE 11 Efficacy of electroporation as a transfection method of oyster sperm, using GUS as a reporter gene.",
"Genetic GUS construct activity Electro- Promoter/ Heat % GUS relative to poration reporter gene shock survival activity1 controls − — none 100 ± 4 4.2 ± 0.3 1.0 + — none 95 ± 5 4.3 ± 0.3 1.0 − CMV/GUS none 93 ± 5 4.4 ± 0.4 1.0 + CMV/GUS none 92 ± 6 6.7 ± 0.8 1.6 − Hsp/GUS yes 92 ± 5 4.5 ± 0.5 1.1 + Hsp/GUS none 91 ± 6 10.5 ± 0.5 2.5 + Hsp/GUS yes 90 ± 4 83.2 ± 5.4 19.8 1GUS activity expressed as fluorescence units/μg protein.",
"Values represent the mean and standard error for three separate spawning experiments, each with three replicates.",
"GUS activity in these experiments was measured using a fluorometric assay as previously described (Jefferson, R A 1987).",
"Fluorometric assays of larval extracts confirmed that electroporation of sperm could deliver foreign DNA into oyster embryos (Tables 10 and 11).",
"In the absence of electroporation, little or no reporter gene expression was detected in transfected larvae.",
"With electroporation, clear differences were observed in the relative strengths of the two different gene promoters tested.",
"Expression of the reporter genes was approximately 1.6 times higher using the heat shock promoter, even in the absence of heat shock, compared to expression levels observed using the CMV promoter.",
"With heat shock, reporter gene expression increased another 6-8 fold.",
"EXAMPLE 9 The Repressible Element in Oysters Tet-Off™ control of EGFP expression was first assessed in oyster heart primary cell culture, using culturing methods previously described (Mol.",
"Marine Biol.",
"Biotech.",
"5: 167-174).",
"Oyster cells were first transfected with the pTet-Off plasmid (Clontech, Genbank ACC# U89929)., using Effectene liposomes (Qiagen), and placed under neomycin selection for 2 weeks.",
"The cells were then co-transfected with the pBI-EGFP reporter gene plasmid (Clontech #PT3146-5) and the selection plasmid pTK-Hyg (Clontech, GenBank Accession #: U40398).",
"Dually transfected cells were then treated with 1 μg/ml doxycycline and EGFP expression was assessed 72 h later.",
"Doxycycline was then removed from the medium, cells were washed in PBS, and incubated for a further 96 h to determine if EGFP expression had changed.",
"It can be seen from Table 12 that a small percentage of cells were observed to express EGFP in the absence of doxycycline.",
"TABLE 12 Tet-Off ™ Control of EGFP Expression in Oyster Cell Culture Transfection and doxycycline (Dox) % cells expressing Treatment EGFP None 0 pTet-Off 0 pBI-EGFP 0 pTet-Off + pBI-EGFP (no Dox) 2.2 ± 0.4 pTet-Off + pBI-EGFP (+ Dox for 72 h) 0 pTet-Off + pBI-EGFP (+ Dox for 72 h, 0.5 ± 0.2 followed by removal of Dox for 96 h) The low double transfection rates are presumably due to most cells acquiring the pTK-Hyg plasmid without acquiring the pBI-EGFP plasmid.",
"Addition of doxycycline to the medium resulted in complete repression of the EGFP reporter gene expression.",
"When the doxycycline was removed, the level of reporter gene expression increased after 96 h, indicating that the repression is reversible.",
"The results in Table 12 indicated that gene expression in oyster cells can be regulated using the Tet-Off™ system, and hence similar experiments were conducted in oyster larvae.",
"Oyster embryos were transfected with the pBiT(HSP)-EGFP plasmid (SEQ ID NO:19), which encodes the tetracycline (or doxycycline)-controlled transactivator (tTA=Tet-Off™) under control of a heat shock promoter, and contains the EGFP reporter gene under the control of the tetracycline (doxycycline) response element (TRE).",
"The construct pBiT(HSP)-EGFP (SEQ ID NO:19) was prepared as follows.",
"Four fragments were prepared and ligated together to create the construct.",
"The first, was obtained by digesting pHSP70-1MCS (SEQ ID NO:22) with XhoI and XbaI followed excision and gel purification of the appropriate XhoI/XbaI fragment containing the Drosophila HSP70 promoter.",
"The second was obtained by digesting pTet-Off (Genbank ACC# U89929) with XbaI and HindIII and gel purifying the appropriate fragment containing the tet-responsive transcriptional activator (tTA) and SV40 poly adenylation signal.",
"The third fragment was obtained by digesting pBI-EGFP (Clontech, PT3146-5) with HindIII and SapI and gel purifying the appropriate fragment containing the TRE and CMVmin bidrectional promoter and multiple cloning site.",
"The fourth fragment was obtained by digesting pTet-Off (Genbank ACC# U89929) with XhoI and SapI and gel purifying the appropriate fragment containing the vector backbone and ampicilin resistance gene.",
"The construct expresses the tet-responsive transcriptional activator (tTA) from the Drosophila HSP70 promoter (PHSP70) which in turn activates expression of EGFP under control of the tetracycline-response element, or TRE.",
"Oyster sperm were transfected with the construct using electroporation, and oocytes were fertilized and allowed to develop for 24 hours in the presence or absence of 5 μg/μl doxycycline.",
"In the absence of doxycycline, EGFP was expressed in transfected oyster larvae, and when doxycycline was added, the EGFP expression levels dropped to levels equal to that of non-transfected embryos (Table 13).",
"The results from the tissue culture and embryo transfections indicate that transgene expression in oysters can be effectively controlled using the Tet-Off™ system.",
"TABLE 13 Regulation of EGFP Expression Using Doxycycline in Oyster Larvae Transfected with pBiT(HSP)-EGFP (SEQ ID NO.19) Fluorescence (FU/μg protein) Total fluorescence (incl.",
"Corrected for Treatment Regime autofluorescence) autofluorescence Non-transformed 320 (±21) 0 (±21) control −Dox, − heat shock 426 (±24) 106 (±24) −Dox, + heat shock 1025 (±78) 705 (±78) +Dox, + heat shock 215 (±27) 0 (±27) Values represent the mean and standard deviation for two separate spawning experiments, each with three replicates.",
"EXAMPLE 10 Blocking Expression of a Developmental Gene in Oysters The applicant has identified conserved gene functions which are crucial to larval development in oysters and characterised two suitable candidate sequences as targets for antisense or dsRNA knockout.",
"Disrupting this gene function is then lethal to the animal (larvae) because transcription factors are prevented from binding and initiating cascades of gene activity required for morphogenesis (body construction).",
"The applicant chose to target the DNA binding ability of a class of transcription factors known as “Helix-loop-Helix” factors that bind DNA during the development of animal body plans (reviewed by Stein et al., 1996; and also see de Rosa, 1999).",
"The applicant isolated two partial gene sequences comprising this crucial and highly conserved DNA binding sequence from a Pacific oyster cDNA library (HoxCg1 and HoxCg3; SEQ ID NOS.",
": 23 and 24, respectively).",
"Alignments of the sequence of this evolutionary conserved class of genes and phylogenetic analysis have revealed that this sequence is indeed a HOX gene and is previously undescribed in oysters (FIG.",
"19).",
"The applicant identified two oligonucleotide sequences that are candidates for antisense larval pesticides.",
"An oyster specific antisense: 5′-GAGATCGTTCAGTCAGCG-3′ SEQ ID NO:25.and a broader spectrum antisense 5′-CATGSGSSGGTTTTGGA 3′ SEQ ID NO:26.wherein “S” represents the base guanine or cytosine.",
"These sequences are potentially capable of truncating vital gene products, and hence preventing their function in vivo.",
"The applicant synthesized and tested antisense and double stranded blockers for the gus gene from Escherichia coli, Hox CG1 (SEQ ID NO:23), and Hox CG3 (SEQ ID NO:24).",
"RNA was prepared by in vitro synthesis for these three different genes or gene fragments: the 1.8 kb open reading frame of the gus gene from E. coli; the 129 bp fragment of oyster gene Hox Cg1 (SEQ ID NO:23, AGAL ref# NM99/09101); and the 129 bp fragment of the oyster gene HoxCG3 (SEQ ID NO:24, AGAL Ref#NM99/09102).",
"The DNA fragments were each cloned into pBluescript SK(+), the vectors were linearized with either HindIII or PstI, and T3 or T7 RNA polymerase (Promega) was used to generate sense or antisense RNAs, respectively using a commercially available in vitro transcription kit (Promega, Madison Wis.).",
"The resulting samples were then digested with DNase I for 15 minutes at 37° C. To produce double stranded RNA (dsRNA), equimolar amounts of the sense and antisense RNAs were mixed and heated to 80° C. and allowed to cool slowly to room temperature thus forming dsRNA.",
"The RNA was extracted with phenol/chloroform and then chloroform, precipitated with ethanol, and resuspended in 10 mM Tris-HCl, pH 9.Formation of dsRNA was confirmed by resolving the annealed and non-annealed RNAs on a 1% agarose gel in TBE (90 mM Tris-borate, 2 mM EDTA, pH 8.0).",
"The in vitro transcribed dsRNAs, plus sense, and antisense RNAs for the GUS, HoxCG1 and HoxCG3 genes were delivered into oyster sperm by electroporation using a set of conditions previously found to be optimal for delivery of a reporter gene construct (dHSP70-GUS).",
"Transfections for the control treatments were carried out in RNA free sea water.",
"Delivery of sense and antisense RNAs had no or only a small effect on the number of individuals that developed, relative to the non-treated controls (Table 14).",
"TABLE 14 Effect on Early Larval Development of Oyster Transfected with In Vitro Transcribed Sense (S), antisense (AS), and double-stranded (DS) RNAs of three different gene sequences, GUS, HoxCG1, HoxCG3 RNA delivered % survivors at 24 h % arrested into sperm development1 development2 control 100 ± 3 5 ± 1 GUS - (DS) 94 ± 5 7 ± 3 HoxCG1 - (S) 91 ± 5 9 ± 4 HoxCG1 - (AS) 85 ± 9 17 ± 5 HoxCG1 - (DS) 71 ± 7 79 ± 10 HoxCG3 - (S) 92 ± 4 8 ± 4 HoxCG3 - (AS) 87 ± 6 15 ± 3 HoxCG3 - (DS) 79 ± 7 23 ± 5 1Percentage of embryos that developed into trochophores, relative to non-treated controls 2Includes all individuals (embryos and larvae) that failed to develop to the D-hinge larval stage Transfection with dsRNA for the GUS gene had no obvious effect on development, but transfection with dsRNAs specific to the HoxCG genes resulted in increased numbers of individuals showing arrested early larval development.",
"The dsRNA specific to the HoxCG1 gene was the most effective dsRNA, with almost 80% of individuals failing to develop beyond the trochophore stage of larval development (Table 14).",
"Screening for mutant phenotypes in the resulting larvae revealed severe developmental mutants especially in the treatments containing dsRNA for both gene constructs, but not the RNA-free controls (FIG.",
"20, Table 14).",
"Fatal embryonic distortions due to the double stranded blocker of HoxCG1 can be broadly classified as defects in the anterior/posterior axis formation including associated structures (such as the velum) and for HoxCG3 as defects in velum and body—perhaps premature velum release.",
"To test whether dsRNA could reduce expression of a gene in oyster cells, primary cell cultures were first transfected with the pHSP-GUS plasmid (SEQ ID NO:21).",
"After two days of growth, the dsRNA specific to the gus gene was delivered into these cells by transfection using Effectene liposomes (Qiagen).",
"After 72 h, the level of GUS activity was measured.",
"The cells transfected with the dsRNA showed a 76% reduction in the reporter gene activity compared to similarly aged gus-transfected cells (Table 15).",
"TABLE 15 Reduced GUS Transgene Expression in Oyster Cells Transfected with In Vitro Transcribed dsRNA GUS Gene Expression (ρmol MU produced/min) % decrease in No dsRNA added dsRNA added gene expression 42 ± 13 10 ± 4 76 In vivo expression of dsRNA was achieved by transfecting oyster larvae with the pBiT(dHSP)-RFP-oHoxDS/BH plasmid (FIG.",
"21; SEQ ID NO:20), which contains the heat inducible promoter (PHSP70) from D. melanogaster driving the expression of a hairpin RNA molecule specific to the HoxCG1 gene.",
"The construct was prepared as follows.",
"SEQ ID NO:23 (AGAL ref #: NM99/09101) was used as a template to generate a PCR fragment using the following primers: CG1.1.Sal.for Forward primer: 5′-ATGGATGTCGACTCAGACGCTGGAG-3′ SEQ ID.",
"NO.",
": 27 And CG1.1.Pst.rev Reverse primer: 5′-GATTCACTAGTCAATTCCTGCAGTT-3′ SEQ ID NO: 28 This fragment was then cloned into the pCR®2.1-TOPO (Invitrogen) cloning vector.",
"Two separate fragments, an EcoRI/EcoRI and a SalI/PstI, both containing the HoxCG1.1 (SEQ ID NO:23), were digested out of this construct for use in further ligations.",
"The latter fragment (SalI/PstI) was inserted into the dsRNA(BMP2) construct (AGAL ref# NM99/09100) which had been digested with SalI and PstI to remove the inverted BMP2 sequence.",
"This intermediate construct was then digested with EcoRI and SpeI to produce a fragment containing both the a 510 bp fragment of the zBMP2 cDNA from sequence 301-810 in the published cDNA sequence (Lee et al., 1998) and the Hox CG1.1 (SEQ ID NO:23) fragment.",
"This EcoRI/SpeI fragment and the EcoRI/EcoRI fragment containing HoxCG1.1 were then combined into a ligation reaction with pHSP70-1MCS (SEQ ID NO:22, containing the Drosophila heat shock promoter dHSP70 and its poly adenylation signal) digested with EcoRI and XbaI, to produce pHSP-oHoxDS/BH (SEQ ID NO:29).",
"This latter construct uses the Drosophila heat shock promoter to drive expression of an mRNA consisting of an inverted section of the HoxCG1.1 followed by a section of BMP2 cDNA in sense orientation followed by a segment of the HoxCG1.1 fragment in sense orientation followed by the poly adenalation signal of the Drosophila heat shock promoter.",
"Oyster sperm were transfected with the DNA using electroporation, and oocytes were fertilized and larvae allowed to develop for 96 hours.",
"Embryos were heat shocked for one hour at 3 hours post fertilization to induce transcription of the dsRNAs.",
"Even without heat shock, approximately a third of the larvae failed to develop beyond the trochophore larval stage, and died within a few days (Table 16).",
"TABLE 16 Arrested Development of Oyster Embryos Transfected with pHSP-oHoxDS/BH plasmid (SEQ ID NO: 29) % arrested development no heat shock with heat shock non-transfected 5 ± 1 4 ± 1 phsp-GUS 6 ± 2 8 ± 3 pHSP-oHoxDS/BH 33 ± 9 67 ± 16 With heat shock, over 65% of the larvae failed to develop.",
"Since all larvae are not transfected by the electroporation procedure, it is likely that those individuals that developed normally were not transfected with the genetic construct.",
"Non-transfected oyster embryos and embryos transfected with a plasmid expressing dsRNA for the GUS gene showed no obvious reduction in survivorship (Table 16).",
"EXAMPLE 10 Complete Sterile Feral Construct for Oysters Two different plasmids were prepared that used Tet-Off™ to control the in vivo expression of dsRNAs specific to developmental genes.",
"The first, pBiT(CMV)-EGFP-zfBMP(DS), (SEQ ID NO:30), was designed to express the reporter gene EGFP as well as dsRNA specific to the zebrafish BMP2 gene in the absence of tetracycline or doxycycline.",
"The construct was prepared as follows: An intermediate constuct was first engineered using three separate fragments.",
"The first was an XhoI/HindIII fragment that was obtained by digesting pTet-Off (Genbank ACC# U89929) with XhoI and HindIII and gel purifying the appropriate fragment containing the CMV promoter, tet-responsive transcriptional activator (tTA), and SV40 poly adenylation signal.",
"The second fragment was obtained by digesting pBI-EGFP (CLONTECH) with HindIII and SapI and gel purifying the appropriate fragment containing the TRE and CMVmin bidrectional promoter and multiple cloning site (MCS).",
"The third fragment was obtained by digesting pTet-Off (Genbank ACC# U89929) with XhoI and SapI and gel purifying the appropriate fragment containing the vector backbone and ampicilin resistance gene.",
"These three fragments were ligated together to form the intermediate construct pBiT(CMV)-EGFP (SEQ ID NO:18).",
"A fourth fragment, obtained by digesting Seq.ID#4 (dsRNA(BMP2), AGAL Ref# NM99/09100) with EcoRI and HindIII and gel purifying the appropriate fragment containing a 510 bp segment of the zBMP2 cDNA from sequence 301-810 and the inverted 286 bp segment of the cDNA (Bases 307-592) of the published zebrafish BMP2 cDNA sequence (Lee et al., 1998).",
"This EcoRI/HindIII fragment was then blunt ended with T4 DNA polymerase and ligated into the unique PvuII site of the MCS of pBiT(CMV)-EGFP to form the construct pBiT(CMV)-EGFP-zfBMP(DS) (SEQ ID NO:30).",
"This construct expresses the tet-responsive transcriptional activator (tTA) from the strong immediate early promoter of cytomegalovirus (PCMV).",
"The tTA functions to drive gene expression via the tetracycline-response element, or TRE.",
"In the absence of tetracyline or doxycyline both EGFP and the blocker gene (double stranded BMP2 mRNA, cloned into the MCS) are expressed.",
"Sperm were transfected with either pBiT(dHSP)-EGFP (SEQ ID NO:19) or pBiT(CMV)-EGFP-zfBMP(DS) DNA, (SEQ ID NO:30), oocytes were fertilized, and allowed to develop for 24 hours in the presence or absence of 5 μg/μl doxycycline.",
"Embryos transfected with the pBiT(dHSP)-EGFP DNA were not heat shocked so that EGFP expression would be similar in both transfections.",
"When oyster embryos were transfected with this construct, lower hatch rates and poorer larval survival rates than those of non-transfected controls were observed (Table 17).",
"TABLE 17 Tet-Off ™ Control of EGFP and dsRNA-zfBMP Expression in Oyster Embryos % survival (relative to EGFP (FU/μg Construct control) protein) injected −Dox +Dox −Dox +Dox Non-transfected 100 + 5 100 + 3 0 + 10 0 + 11 pBiT(dHSP)-EGFP 77 + 6 95 + 3 31 + 8 0 + 8 pBiT(CMV)-EGFP- 71 + 8 92 + 4 20 + 11 0 + 9 zfBMP(DS) When doxycycline was added to the water, this trend was reversed.",
"Most of this arrested development however, may be caused by expression of EGFP, as similar levels of arrested development were observed when embryos were transfected with the pBiT(dHSP)-EGFP plasmid (without exposure to heat shock), and normal developmental rates were restored when doxycycline was added to the water.",
"It cannot be excluded however, that the zebrafish dsRNA has caused some small degree of developmental arrest in the oysters, as the BMP2 may have an as yet unidentified orthologue with enough sequence identity to zfBMP2 to be affected by this dsRNA molecule.",
"The second Sterile Feral Construct tested for oysters, expresses the tTA under the Drosophila HSP.",
"The tTA then drives expression of red fluorescent protein and double stranded oyster Hox via the TRE.",
"Three separate fragments were ligated together to form this construct.",
"The first fragment was obtained by digestion of pBiT(dHSP)-EGFP, (Seq ID NO:19), with HindIII and NheI followed by gel purification of the appropriate fragment containing the Drosophila HSP promoter.",
"The second fragment was obtained by digesting pBiT(dHSP)-EGFP with NotI and MluI followed by gel purification of the appropriate fragment containing the TRE.",
"The third fragment was obtained by digesting pHSP-oHoxDS/BH with MluI and.",
"SpeI and gel purifying the appropriate fragment containing the 510 bp fragment of the zBMP2 cDNA from sequence 301-810 in the published cDNA sequence (Lee et al., 1998).",
"The fourth fragment was obtained by firstly subcloning into pGEM3zf a KpnI/XbaI fragment containing the coding region of red fluorescent protein (RFP) that was excised from pDsRed1-N1 (Clontech, PT3405-5) vector.",
"The resulting plasmid was then subjected to digestion with HindIII and PspOMI and the appropriate fragment containing the coding region of RFP was then gel purified from this reaction.",
"This HindIII/PspOMI fragment was combined with the NheI/HindIII, NotI/MluI, and MluI/SpeI fragments to form the second sterile feral oyster construct pBiT(dHSP)-RFP-oHoxDS/BH (SEQ ID NO:20; FIG.",
"21).",
"Sperm were transfected with the plasmid, oocytes were fertilized, and allowed to develop for 72 hours in the presence or absence of 5 μg/μl doxycycline.",
"When oyster embryos were transfected with the second repressible sterile feral construct, a considerable percentage (67%) failed to develop beyond the trochophore stage of larval development and subsequently died before reaching the D-hinge stage (Table 18).",
"TABLE 18 Reversible Arrested Oyster Larval Development Following Transfection with the Tetracycline-Responsive Plasmid phsp-BiT-RFP/dsRNA-HoxCG1 Construct used for % arrested development transfection No doxycycline With doxycycline Non transfected 0 ± 5 0 ± 3 phsp-GUS 5 ± 3 4 ± 3 pCMV-RFP 5 ± 2 4 ± 3 phsp-BiT-dsRNA- 67 ± 8 9 ± 4 HoxCG1/RFP Addition of doxycycline to the water virtually prevented the developmental arrest, and most embryos developed properly to the D-hinge larval stage, relative to the non-treated controls.",
"RFP expression was not easily detected by microscopy in embryos transfected with the RFP gene under the control of either a heat shock or a CMV promoter.",
"A small amount of RFP was detected using fluorometric measurements of larvae transfected with the pCMV-RFP construct, but little RFP could be detected in larvae transfected with the repressible anti-development construct (results not shown).",
"As many of the embryos transfected with this latter construct fail to develop, the lack of RFP expression is not surprising.",
"Attempts to detect RFP in early and late staged embryos were unsuccessful, using either RFP-expressing construct.",
"EXAMPLE 11 Development of a Repressibly Sterile Mouse Development of the sterile feral construct for mice parallels that detailed above for zebrafish, and involves identification of a suitable target gene and associated promoter, engineering these into a construct with the Tet On/Off repressible system, and then-testing, in this case in cell lines, prior to production of a transgenic mouse model for the sterile-feral concept.",
"There are many genes known to have adverse effects on fertilisation, development or reproduction in mice.",
"These genes can be readily identified through literature and database searches (Medline, mouse knock out database, Genbank etc.).",
"These candidate genes fall mainly into the category of genes that are required for specific developmental processes during embryogenesis.",
"Furthermore, genes that are involved in stages of fertilisation and implantation are also potential candidate genes for this fertility control technology.",
"Developmental stages identified as potential sterile feral construct targets are classified under one of the following general areas: fertilisation, preimplantation, post implantation (until neurulation) and organogenesis stages.",
"The latter stages include factors such as those associated with the specification of male and female reproductive organs (Cunha et al., 1976).",
"Proteins involved in these stages may have different roles such as morphogens, master genes, growth factors or receptors.",
"Genes associated with fertilisation include such factors as protein receptors or ligands required for successful fertilisation.",
"Preimplantation genes that can be manipulated to control their gene expression and so achieve controllable fertility are also covered by this patent and include genes encoding proteins such as growth factors, signaling molecules and their receptors.",
"The homeobox gene goosecoid is one of the first genes to be transcribed in the organizer region of the mouse at the onset of gastrulation and RNA transcripts first appear in the dorsal mesoderm of the late blastula (Blumberg et al., 1991).",
"The goosecoid gene is also highly conserved among different species (FIG.",
"22).",
"During mouse embryogenesis, expression of the goosecoid gene takes place in two different phases.",
"In the first phase of expression, goosecoid gene expression can be detected in the organizer between 6.4 to 6.7 days (Blum et al., 1992) and in the second phase it is detected during organogenesis from 10.5 day onwards (Gaunt et al., 1993) and expressed in some parts of head, the limbs and the ventrolateral body wall.",
"The homozygous knockout mutation of goosecoid in the mouse leads to defects late in development of the embryos.",
"In particular, null homozygous goosecoid embryos are born with numerous developmental defects and die within 24 hours of birth (Rivera-Perez et al., 1995).",
"The observed phenotype is in accordance with late expression of goosecoid in normal embryos, and it has been proposed that the lack of an earlier phenotype is due to functional compensation by other orthologous genes such as gsc2.At the promoter level, molecular studies have demonstrated that expression of goosecoid in Xenopus is mediated by the combined effects of two regions of the promoter, the distal element (DE) and the proximal element (PE).",
"The DE responds directly to dorsal mesoderm inducing signals such as activin and Vg1 (members of the TGF-β super family), whereas the PE responds indirectly to wnt signaling (McKendry et al., 1998).",
"Sequence comparison among different species shows that these proximal and distal elements are conserved among different species and there may be a common mechanism for its activation (Blum et al., 1992).",
"It was proposed that the DE responds directly to mesoderm inducing signals such as activin, whereas the PE responds indirectly to Wnt signaling (Laurent and Cho, 1999) (FIG.",
"23).",
"Studies involving the goosecoid promoter in mouse and other species have shown that the promoter region carrying these two elements are adequate for reporter gene activity studies.",
"These two elements are generally located within 500 bp from the transcriptional start site.",
"The goosecoid gene, in the form of sterile feral constructs, can be used to demonstrate how a developmentally active gene can be manipulated to maintain its temporal and spatial gene specification under repressible promoter elements.",
"EXAMPLE 12 Cloning the Goosecoid Gene Promoter The goosecoid promoter was amplified by PCR using BALB/c genomic DNA.",
"Primers were designed from Mus musculus goosecoid homeobox gene, promoter sequence, of the Genbank accession number Y13151.The primers were as follows: Forward Primer 5′-GGAGACAGGCAGTCCCGGTAGATC-3′ SEQ ID NO: 33 Reverse Primer 5′-TGGGAATTGTCCCACTCTCTGCTC-3′ SEQ ID NO: 34 The PCR conditions were as follows: 95° C.×3 min, 72° C.×1 min (hotstart), 58° C.×1 min, 72° C.×1 min for 1 cycle.",
"Then 95° C.×45 sec, 58° C.×1 min, 72° C.×1 min for 28 cycles.",
"The reaction was completed by incubating the reaction at 72° C.×10 min and 25° C.×5 min).",
"The PCR product for the goosecoid promoter was ligated into pGEM-T-Easy cloning vector (Promega Cat # A1360).",
"EXAMPLE 13 Selection and Construction of Reporter Plasmids for Testing Promoter Function Reporter genes for promoter expression in mammals are available in two forms.",
"Firstly reporter genes can be used to determine location of expression of a gene product.",
"Examples of such commercially available reporters include the Enhanced Green Fluorescent Protein (EGFP) and Red Fluorescent Protein (RFP).",
"Alternatively, other reporter genes can be used to quantitate relative levels of expression and include firefly luciferase (LUC+) modified for optimal expression in mammalian systems.",
"The reporter genes EGFP and LUC+ were selected for use in testing sterile feral constructs based on the goosecoid promoter in the mouse.",
"pSFM 1: goosecoid promoter expressing enhanced green fluorescent protein (FIG.",
"24; SEQ ID 35).",
"The goosecoid promoter produced by PCR and cloned into pGEM-T-Easy (see above) was subcloned into the pEGFP-1 vector (Clontech Cat.",
"# 6086-1) by digestion with EcoR1 and cloned into the EcoR1 site of the MCS of pEGFP-1.The orientation of the goosecoid promoter was confirmed by both restriction enzyme mapping and sequencing.",
"pSFM 2: goosecoid cDNA in pTRE (FIG.",
"25; SEQ ID 36).",
"A goosecoid cDNA equivalent was prepared from a goosecoid genomic DNA clone.",
"The goosecoid DNA clone was prepared by PCR using BALB/c mouse genomic DNA.",
"Primers were designed from the published sequence of goosecoid (Genbank Accession # M85271).",
"The goosecoid gene coding region is comprised of 3 exons.",
"PCR primers were designed to produce each of the exons individually and were cloned into bacterial plasmid vectors using standard molecular biology techniques.",
"The cDNA for goosecoid was then reconstructed by tandemly ligating the individual exons together to form a new clone.",
"The exons can also be joined in other orientations to encode for various combinations of dsRNA or antisense of the goosecoid RNA.",
"The Primers used were designed from the entire coding region of the genomic DNA (Sequence locations referred to goosecoid Genbank Accession Number=M85271) and were: Design of PCR primers to amplify goosecoid exons 1, 2, 3.exon 1 (bp 296-650); exon 2 (bp 1159-1418); exon 3 (bp 1765-1920): SEQ ID NO: 37 Exon 1 forward (bp 296-316) 5′-GGTTAAGCTTATGCCCGCCAGCATGTTCAGC-3′ SEQ ID NO: 38 Exon 1 reverse (bp 631-650) 5′-GCGGGGCCCTCGTAGCCTGGGGGCGTCGGGACGCAG-3′ SEQ ID NO: 39 Exon 2 forward (bp 1165-1183) 5′-CGAGGGCCCCGGTTCTGTACT-3′ SEQ ID NO: 40 Exon 2 reverse (bp 1398-1418) 5′-TTTGAGCTCCACCTTCTCCTCCCGAAG-3′ SEQ ID NO: 41 Exon 3 forward (bp 1765-1785) 5′-GTCTGGTTTAAGAACCGCCGA-3′ SEQ ID NO: 42 Exon 3 reverse (bp 1900-1920 5′-GGAATTCTCAGCTGTCCGAGTCCAAATC-3′ Three exons were amplified by PCR using the above primers and the following conditions; 95° C.×2 min, 40° C.×30 sec, 72° C.×45 sec for 1 cycle.",
"Then 95° C.×30 sec, 40° C.×30 sec, 72° C.×45 sec for 30 cycles.",
"The reaction was stopped by incubation at 72° C.×10 min and 25° C.×5 min.",
"Goosecoid exon 1-3 PCR products were cloned into Promega (Cat # A1360) pGEM-T-Easy cloning vectors.",
"These clones were named pME 1, pME 2 and pME 3 for exon 1-3 in pGem-T-Easy respectively.",
"The strategy for producing the equivalent clone for the complete goosecoid cDNA coding region was as follows: pME 2 was cut with ApaI and religated, to remove the EcoR1 site.",
"Pfu polymerase PCR of clone pME 3 was undertaken using the primers and conditions for exon 3 as described above.",
"This generated a blunt-ended fragment which was then digested with EcoRI.",
"Following religation of pME2 (see step 1 above) with EcoIcR1.Ligated together pME2 from (3) and digested PCR product from (2) to produce pME 4.Cut pME 1 with HindIII and then partial digest with ApaI (band size 370 bp, external ApaI site).",
"Cut pME 4 with ApaI, followed by EcoR1.Cut pBluescript SK− with HindIII followed by EcoRI Ligated (7) above with pME 4 product and pME 1 product to produce the complete goosecoid cDNA coding region.",
"This clone was confirmed by sequencing and designated pCMH142 (SEQ ID 43).",
"pSFM 6: Goosecoid promoter expressing goosecoid cDNA fused to red fluorescent protein (FIG.",
"26).",
"A 0.9 kb PCR fragment containing the full coding sequence of mouse goosecoid was amplified from pCMH142 using two PCR primers: gsc F4 - 5′-TTAAGCTTGCCACCATGCCCGCCAGCATGT-3′ SEQ ID 44 gsc R4 - 5′-TTGGATCCGCGCTGTCCGAGTCCAAATC-3′ SEQ ID 45 These primers produced a goosecoid-containing fragment where the TGA stop codon was replaced with an alanine codon.",
"The PCR primers were also used to introduce a HindIII site upstream of the ATG start codon and a BamHI site downstream of the alanine codon.",
"This fragment was restricted with HindIII and BamHI and then inserted into the plasmid pDSRed1-N1 (Clontech 6921-1) cut with HindIII and BamHI in order to generate pSFM 6 (SEQ ID 46).",
"pSFM 7: Mouse goosecoid promoter expressing the tetracycline transactivator protein tTA (FIG.",
"27).",
"SEQ ID 47.The goosecoid tetracycline dependent transactivator plasmid was constructed by replacing EGFP of pSFM 1 with the 1008 bp coding region region (Genbank accession # U89930 bp 774-1781) of the tet-responsive transcriptional activator (tTA) from the pTET-OFF plasmid (Clontech, Cat # K1620-A).",
"The tTA coding region was amplified by PCR using Pfu polymerase, restricted by Age1 and EcoR1 and cloned into pSFM 1 to produce pSFM 7.pSFM 20: goosecoid promoter expressing luciferase+ protein (FIG.",
"28).",
"SEQ ID 48.A 0.7 kb (NotI end-filled with Klenow+BamHI) fragment coding for green fluorescent protein region from pSFM1 was replaced with 1.6 kb (XbaI end filled with Klenow enzyme+BamHI) luciferase+ coding fragment derived from pXP1-G (Promega E1751).",
"pSFM 21: Promoterless luciferase+ (FIG.",
"29).",
"SEQ ID 49.A 1.6 kb luciferase coding EcoRI fragment was deleted from pSFM 20.pSFM 23: pCMV promoter expressing luciferase+ (FIG.",
"30).",
"SEQ ID 50.A 1.6 kb (SacI+StuI) luciferase+ coding fragment of pSFM 20 was cloned into pEGFP-N1 (Clontech 6085-1) cut with SacI+StuI.",
"pSFM 24: Equivalent to the tet-responsive enhanced green fluorescent protein expression vector pTRE-EGFP (Clontech 6241-1)(FIG.",
"31) SEQ ID 51.pSFM 25: Tet-responsive expression vector pTRE-luciferase+ (FIG.",
"32).",
"SEQ ID 52.A 0.77 kb SalI+XbaI EGFP containing fragment of pSFM 24 was replaced by a 1.7 kb SalI+XbaI luciferase+ containing fragment derived from pXP1-G (Promega).",
"EXAMPLE 14 Selection of Mammalian Cell Lines Mouse goosecoid was selected to demonstrate whether a developmental gene can be tightly regulated in the form of sterile feral constructs in mammalian cell lines.",
"Most of the mainpulations using sterile feral constructs based on goosecoid were therefore carried out in the mouse embryo cell lines P19 teratocarcinoma since it has been shown previously that the mouse goosecoid gene product is constitutively expressed in P19 teratocarcinoma cell lines.",
"NIH/3T3 cells (in which goosecoid gene expression is absent) were used as controls.",
"In addition goosecoid reporter constructs were tested in non-transformed mouse primary embryonic fibroblasts.",
"These cells display monolayered, anchorage dependent and contact inhibited growth in tissue culture.",
"Using transient transfection with reporter and other plasmid constructs (reporters and blockers) the observed effects on these plasmids is expected to reflect the anticipated effect in the whole organism.",
"Chromatin structure surrounding the inserted gene is also likely affect the pattern of regulation of gene expression and so the choice of stable cell lines for gene expression is essential.",
"For example, it is known that transfected DNA does not display the same accessibility to transcriptional factors as chromosomal DNA (Archer et al., 1992).",
"Another important factor to consider is that the goosecoid promoter contains only 1.1 kb upstream to the transcription start site leading to potential restriction of access by nuclear and other transcriptional factors by surrounding DNA sequences and chromatin structure.",
"All cell lines were obtained from American Type Culture Collection unless otherwise stated.",
"These are P19 teratocarcinoma cells (ATCC number CRL-1825) and NIH/3T3 cells (ATCC number CRL-1658).",
"For transient transfection assays, P19 cells were cultured on gelatinized dishes in DMEM supplemented with 10% fetal bovine serum.",
"Cells (0.3 million per well in 6-well cluster plates) were transfected with 5 μg reporter plasmid using transfection reagent ‘Geneporter’ from Gene Therapy Systems according manufacturer's recommendation.",
"Stably integrated P19 clones were obtained by using BioRad Gene Pulser II electroporation system.",
"30 μg DNA electroporated into 10 million cells under following conditions 960 μF and 0.16 kV in a 0.4 cm cuvette (0.4 kV/cm).",
"The next day normal media were replaced with appropriate selection media (300 μg/ml G418).",
"Reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm that the goosecoid gene is actively expressed in P19 cell lines with the goosecoid specific primers exon 2 forward (SEQ.",
"ID 39) and exon 3 reverse (SEQ ID 42): RT-PCR cDNA was synthesized in a 50 μl reaction using 100 ng of poly(A) RNA extracted from various tissues and cell lines.",
"The RNA was heated with a mixture of random 6 base pair and oligo(dT) primers for 5 min at 65° C. and cooled to room temperature for 10 min.",
"Reverse transcription was performed at 37° C. for 1 h after adding 5 μl.",
"10×RT buffer (Promega), 20 U RNase inhibitor (Promega), 2 μl of 0.1 mM dNTPs and 50 U MMLV reverse transcriptase.",
"The cDNA mixture was then heated for 5 min at 90° C. and stored at −20° C. until needed.",
"RT-PCR was conducted using 2 μl of cDNA in a 50 μl final reaction using goosecoid specific primers (FIG.",
"33).",
"By comparison, RT-PCR amplification on NIH/3T3 cells gave negative results for goosecoid.",
"In both cells, RT-PCR of a general housekeeping gene GADPH gave positive bands.",
"In addition GFP expression from P19 cells containing the reporter plasmid pSFM 1 stably integrated was unaffected by repeated passaging or freezing and thawing.",
"In order to measure the activity of the goosecoid gene, a cell culture system was developed that responds to tetracycline repression and permits the measurement of gene activity using both fluorescence reporters.",
"Fluorescent and transmitted light images were acquired using a CCD camera with a microscope.",
"Fluorescence filter sets had an excitation wavelength of 480 nm, dichroic cut-on filter at 505 nM and an emission filters at 535 nM and 605 nM.",
"The luminescence assays were conducted by using a dark 96 well plate was done by Victor2 from Wallac or by Topcount NXT from Can berra Packard.",
"P19 cells were transiently transfected in 6 well plates with pSFM 20 (goosecoid promoter-luciferase), pSFM 21 (promoterless luciferase) and pSFM 23 (CMV promoter-luciferase) using Gene Porter.",
"Cells were harvested at various times post-tranfection and assayed for luciferase activity using a Promega kit (Cat.",
"# E1501) in a Top Count NXT luminometer.",
"Table 19 shows the luciferase activities of promoter reporter constructs shown in counts per second (cps) of transiently transfected in P19 cells.",
"TABLE 19 Hours pSFM 21 pSFM 23 pSFM20 24 254 78778 1263 48 604 145403 3707 72 252 49936 1692 Maximum luciferase activity was observed 48 hours post-transfection for all plasmids.",
"Luciferase activity from the goosecoid promoter construct (pSFM 20) was 6 fold higher compared to the promoterless construct (pSFM 21).",
"CMV driven luciferase activity (pSFM 23) was 200-300 fold higher than for the promoterless luciferase (pSFM 21).",
"Therefore 48 hours post transformation was selected for optimal detection of luciferase expression.",
"Selection of a P19 cell line stably integrated with a goosecoid-dependent TET-OFF transactivator P19 cells were electroporated with pSFM 7 (Goosecoid promoter-TET/OFF) linearised with ApaLI and selected for stable integration.",
"Table 20 showes the luciferase activities of pSFM 25 (TRE luciferase+) shown in counts per second (cps) of transiently transfected in P19-pSFM 7 cells.",
"TABLE 20 pSFM 25 pSFM 25 Clone number without doxycycline with doxycycline 9 582529 54858 12 417268 4396 29 616604 48260 32 272888 19548 46 703470 8013 From 100 clones, one clone (46) was selected which demonstrated the highest luciferase activity when transiently transfected with the reporter plasmid pSFM 25 (TRE-luciferase+).",
"Addition of doxycycline at 1 μg/ml reduced luciferase activity from pSFM 25 in this clone by 90 fold.",
"This clone, containing stably integrated pSFM 7 was therefore designated P19-pSFM 7 and used for further testing.",
"Reporter plasmids pSFM 20 (goosecoid promoter luciferase+), pSFM 21 (promoterless luciferase+), pSFM 23 (CMV promoter luciferase+) and pSFM 25 (TRE luciferase+) were transiently transfected into either P19 or P19-pSFM 7 (Goosecoid TET/OFF) cells to test the effectiveness of the TET-OFF genetic switch driven by goosecoid promoter.",
"Table 21 shows the luciferase activities of transient transfection of reporter plasmids in P19 and P19-pSFM 7 cell lines.",
"TABLE 21 Pl9-pSFM 7 cells P19 cells Plasmids Average Fold Average Fold pSFM 20 365 6 610 5 pSFM 21 60 1 121 1 pSFM 23 16031 267 44491 367 pSFM 25 368 6 183 1.5 P19-pSFM 7 but not the P19 cells show a 6 fold increase in luciferase+ reporter activity when transfected with pSFM 25 compared to the promoterless plasmid pSFM 21.This increase is comparable to the increase seen when the cells are transfected with plasmids containing the luciferase driven by the goosecoid promoter (pSFM 20).",
"Therefore the P19-pSFM 7 cell line can be used to drive expression through pTRE plasmids to the same level as plasmids driving expression from the goosecoid promoter directly.",
"EXAMPLE 15 Construction and Testing of Blocker Plasmids Antisense and double stranded blockers specific for goosecoid were constructed.",
"pSFM 5: Tet-responsive expression vector pTRE-goosecoid double strand RNA (FIG.",
"34).",
"SEQ ID 57.pSFM5 was derived from pSFM 2 and pSFM 9.A 0.48 kb PstI+BamHI fragment of pSFM 9 was inserted into a 3.9 kb PstI partial+BamHI fragment of pSFM 2 to produce pSFM 5.pSFM 8: pCMV promoter expressing goosecoid antisense RNA (FIG.",
"35).",
"SEQ ID 58.A 0.8 kb EcoRI+KpnI fragment of pSFM 9 containing the goosecoid cDNA was inserted into pdsRED-N1 (Clontech 6921-1) cut with KpnI+EcoR1.This clone was then cut with SmaI+HpaI to remove the RFP and religated to produce pSFM 8.pSFM 9: Tet-responsive expression vector pTRE-goosecoid antisense RNA (FIG.",
"36).",
"SEQ ID 59.A 0.78 kb HindIII Klenow end-filled+EcoRI fragment of pCMH142 was cloned into pTRE cut with BamHI end-filled with Klenow+EcoRI.",
"The first stage for testing blocker constructs is to set up an appropriate cell system to detect expression of reporter constructs.",
"Initially, either pdsRED-N1 (CMV promoter RFP), pSFM 6 (CMV promoter goosecoid cDNA fused to RFP) or pSFM 24 (TRE EGFP) were transfected into P19-pSFM 7 cells to test the expression patterns of the EGFP, RFP and goosecoid-fused to RFP proteins (FIG.",
"37).",
"These tests show that RFP is expressed in the cytoplasm when driven from a CMV promoter (FIG.",
"37,B).",
"When goosecoid is fused to RFP and driven from a CMV promoter however, the RFP signal is now detected in the nucleus (FIG.",
"37C,D), whereas the EGFP is expressed in the cytoplasm of the same cells when expressed through the TRE promoter (FIG.",
"37D).",
"This shows therefore, that goosecoid is efficiently transferred to the nucleus when fused to the reporter gene RFP and that this system can be used to test co-transfected blocker plasmids against goosecoid.",
"In these cases, RFP expression fused to goosecid in the nucleus is expected to be inhibited in the presence of an appropriate blocker.",
"In order to assess various antisense and dsRNA blockers, pSFM 6 (CMV promoter goosecoid fused to RFP) was transiently cotransfected into the P19-pSFM 7 (Goosecoid promoter TET/OFF) cells along with either pSFM 5 (TRE promoter dsRNA goosecoid), pSFM 8, (CMV promoter antisense goosecoid), pSFM 9 (TRE promoter antisense goosecoid) or pSFM 24 (TRE promoter EGFP).",
"In these cases, significant difference could not be detected between the various treatments in either the intensity or number of cells expressing RFP in the nucleus.",
"There are several potential reasons for the absence of RNA blocker effects.",
"First, antisense and dsRNA blockers may not be expressed at levels high enough to effectively interfere with the target mRNA molecules.",
"Secondly, there may be cellular mechanisms in mammals that recognize and interfere with such constructs.",
"Thirdly, the RNA inhibitory molecules may not be able to access and block the RNA target.",
"The goosecoid gene, in the form of sterile feral constructs, was tested in mammalian cells to demonstrate whether plasmids DNA coding for SF blockers have effect any effect on blocking goosecoid expression.",
"We have demonstrated the methods for producing stably integrated cell lines and the testing of blocker constructs based on goosecoid dsRNA and goosecoid anti-sense.",
"Our results suggest that post-transcriptional silencing through double strand RNA is unlikely to be very effective in mice.",
"We therefore conclude that either the system described here is insufficiently sensitive to detect RNA interference using the current blockers or that these inhibitors are relatively ineffective in the P19 mammalian cell line.",
"Nevertheless, small effects in cell culture can translate into severe phenotypic abnormality when introduced into mice.",
"By contrast, over-expression and mis-expression of genes leading to developmental abnormalities has been demonstrated in mice (Zwijsen et al., 1999; Goodrich et al., 1999).",
"It can be reasonably expected therefore that sterile feral blockers that cause over-expression or mis-expression of developmental genes through at tetracycline repressible system will succeed.",
"However, sense constructs cannot be easily tested using reporter systems.",
"It is necessary to stably introduce such constructs into embryonic stem (ES) cells and produce transgenic mice to evaluate the extent to which development can be disrupted.",
"EXAMPLE 16 Production of Transgenic Mice Using the Goosecoid Promoter By using the goosecoid gene promoter (or similar) to drive expression of known proteins critical to early embryogenesis a transgenic mouse can be made.",
"Candidate sense blockers for expression from the goosecoid promoter are gene products that are critical for development in the mouse and are also normally expressed in the embryo during gastrulation at the same time as the goosecoid gene product.",
"Two other proteins, Chordin and Noggin, are known to expressed within the same embryonic region at times and locations similar to that of goosecoid (Bachiller et al., 2000).",
"In particular, Chordin is expressed in the same region as goosecoid at embryonic stage TS 11 in the primitive streak and node.",
"Double knock-out mice for Chordin and Noggin have been produced and these show severe phenotypic defects in the prosenchephalon.",
"Both of these proteins are therefore essential for successful development in the mouse.",
"These two genes are antagonisers of another gene product, BMP-4, which is expressed in the region adjacent to the primitive streak.",
"Together, these three gene products contribute to the anterior/posterior structural features of the developing mice.",
"Therefore, misexpression of BMP-4 using the goosecoid promoter, within the primitive streak, where Noggin and Chordal are expressed, will interefere with the balance between these gene products and be expected to produce a phenotype that will match the double knock-out for Chordin and Noggin.",
"Many other developmental genes, particularly those involved with early embryogenesis could be misexpressed in a similar manner.",
"The following process can be used to generate a transgenic mouse line expressing repressible developmentally regulated blockers.",
"Gene targeting in mice is regularly achieved using two different methods.",
"One is by oocyte injection and the other is through gene insertion into embryonic stem cells.",
"The embryonic stem cell method is the most preferred for manipulations using the goosecoid gene since this gene is usually activated following removal of leukemic inhibitory factor (a factor used to maintain the cells in undifferentiated state) from the culture medium (Savatier et al., 1996).",
"Testing for effectiveness of reversible blockers on goosecoid expression in cell cuture can therefore be tested in embryonic stem cells before being transferred into mouse but not in system using directly injected oocytes.",
"The manipulations and production of repressibly steile transgenic mice is readily achievable to those practiced in the art (Hogan et al., 1994).",
"This involves the following steps: Transfection, stable integration and selection of embryonic stem cells with a sterile feral construct consisting of the goosecoid promoter driving expression of tTA (Tet-Off) such as pSFM 7 (SEQ ID NO:48).",
"Transfection, stable integration and selection of the teracycine dependent effector construct consisting of the TRE (Tet-reponsive promoter) driving expression of one of the following: goosecoid antisense or dsRNA in constructs such as pSFM 9 (SEQ ID NO:59) and pSFM 5 (SEQ ID NO:57) or the cDNA for genes essential for development in the embryo around the time of primitive streak formation (such as BMP-4).",
"Conclusions One type of “sterile feral” construct encompassed by the present invention consists of three components, a developmental or constitutive promoter, a gene blocker sequence, and a repressible promoter from Clontech™'s commercially available Tet-Off system.",
"The developmental or constitutive promoter functions to drive expression of Tet-Off represser protein (tTA, Clontech™) which binds to the tet responsive element (TRE-CMVmin, Clontech™) that in turn drives expression of the gene blocker sequence.",
"Expression of the blocker DNA sequence results in production of either antisense or double stranded mRNA to ultimately knock-out function of the target gene or mis-expression of a sense sequence, that causes distorted development and embryo death.",
"Correct function of the sterile feral construct requires that functions of both the developmental promoter and the target gene are confined to either oogenesis or embryogenesis.",
"This can be achieved optimally by using a stage-specific promoter, though it can also be achieved through use of a developmental blocker who's effects are also spatio-temporally confined to early embryogenesis.",
"Repression of the blocker sequence function is accomplished through exposure to tetracyline which prevents the binding of the tTA to the TRE-CMVmin.",
"REFERENCES Archer, T. K., P. Lefebvre, et al.",
"(1992).",
"Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation [published erratum appears in Science 1992 Apr.",
"10;256(5054):161].",
"Science 255(5051): 1573-6.Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sridman, J. G., Smith J.",
"A. and Struhl, K.",
"(Eds.",
"; 1996).",
"Current protocols in molecular biology.",
"John Wiley and Sons Inc, USA.",
"Vol 1-3.Bachiller, D., J. Klingensmith, et al.",
"(2000).",
"The organizer factors Chordin and Noggin are required for mouse forebrain development.",
"Nature 403 (6770): 658-61.Blum, M., S. J. Gaunt, et al.",
"(1992).",
"Gastrulation in the mouse: the role of the homeobox gene goosecoid.",
"Cell 69(7): 1097-106.Blumberg, B., C. V. Wright, et al.",
"(1991).",
"Organizer-specific homeobox genes in Xenopus laevis embryos.",
"Science 253(5016): 194-6.Cormack B P, Valdivia R H, Falkow S (1996) FACS-optimised mutants of the green fluorescent protein (GFP).",
"Gene 173:33-38.Cunha, G. R. (1976).",
"Stromal induction and specification of morphogenesis and cytodifferentiation of the epithelia of the Mullerian ducts and urogenital sinus during development of the uterus and vagina in mice.",
"J Exp Zool 196(3): 361-70.Dale, L., Howes, G., Price, B. M. J. and Smith J. C. (1992) Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development.",
"Development 115:573-585 DeRobertis, E. M. and Sasai, Y (1996).",
"A common plan for dorsoventral patterning in bilateria.",
"Nature 380:37-40.Dick et al (1999).",
"Smad1 and Smad5 have distinct roles during dorsoventral patterning of the zebrafish embryo.",
"Developmental Dynamics 216:285-298.Driever et al (1996).",
"A genetic screen for mutations affecting embryogenesis in zebrafish.",
"Development.",
"123:37-46 Driever, W. and Rangini, Z.",
"(1993).",
"Characterization of a cell line derived from zebrafish (Bracydanio rerio) embryos.",
"In Vitro (Cell.",
"Dev.",
"Biol.",
"29A: 749-754.factor encoded by the short gastrulation gene.",
"Genes Dev 8:2602-2616.Ferguson, E. L. and Anderson, K. V. (1992).",
"Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo.",
"Cell 71:451-461.Fire, A., Xu, S., Montogomery M. K., Kostas, S. A. Driver, S. E. and Mello C. C. (1998).",
"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.",
"Nature.391:806-811.Francois, V., Solloway, M., O'Neill, J. W., Emery, J. and Bier, E. (1994) Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth Gaunt, S. J., M. Blum, et al.",
"(1993).",
"Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall.",
"Development 117(2): 769-78.Goodrich, L. V., D. Jung, et al.",
"(1999).",
"Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube.",
"Dev Biol 211(2): 323-34.Gossen, M. & Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline responsive promoters.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 89:5547-5551.Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. & Bujard, H. (1995) Transcriptional activation by tetracycline in mammalian cells.",
"Science 268:1766-1769.Hafter et al (1996).",
"The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.",
"Development.",
"123:1-36.Hammerschmidt, M., Serbedzija, N. and McMahon, A. P. (1996) genetic analysis of dorsoventral pattern formation in the zebrafish: requirement of a BMP-like ventralizing activity and its dorsal represser.",
"Genes Dev.",
"10:2452-2461.Hild et al (1999).",
"The snad5 mutation somitabun blocks Bmp2b signalling during early dorsoventral patterning of the zebrafish embryo.",
"Development 126: 2149-2159.Hogan, B., R. Beddington, et al., Eds.",
"(1994).",
"Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press.",
"Holley, S. A., Jackson, P. D., Sasai, Y., Lu, B., De Robertis, E. M., Hoffmann, F. M., and Ferguson, E. L. (1995) A conserved system for dorsal-ventral patterning in insects and Animals involving sog and chordin.",
"Nature 376: 249-253 Izant J G, Weintraub H (1984).",
"Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis.",
"Cell 36:1007-1015.Jefferson, R. A.",
"(1987).",
"Assaying chimeric genes in plants: The GUS gene fusion system.",
"Plant Mol.",
"Biol.",
"Rep. 5: 387-405.Jones, C. M., Dale, L., Hogan, B. L. M., Wright C. V. E. and Smith, J. C. (1996) Bone morphogenetic protein-4 (BMP4) acts during gastrula stages to cause ventralization of Xenopus embryos.",
"Development 122:1545-1554.Kishimoto, Y., Lee, K. H., Zon, L., Hammerschmidt, M. and Schulte-Merker, S. (1997).",
"The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning.",
"Development.",
"124:4457-4466.Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, and Bujard H. (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.",
"Proc Natl Acad Sci USA 93:10933-10938.Laurent, M. N. and K. W. Cho (1999).",
"Bone morphogenetic protein antagonism of Spemann's organizer is independent of Wnt signaling.",
"Dev Biol 206(2): 157-62.Lemaire, P. and Yasuo H. (1998).",
"Developmental signalling: a careful balancing act.",
"Curr Biol.8:228-231 McKendry, R., R. M. Harland, et al.",
"(1998).",
"Activin-induced factors maintain goosecoid transcription through a paired homeodomain binding site.",
"Dev Biol 204(1): 172-86.mechanisms of dorsal-ventral patterning.",
"Development 121:4319-4328 Mishihina, Y., Susuki, A., Ueno.",
"N. and Behringer, R. R. (1995).",
"BMPr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis.",
"Genes and Dev.",
"9:3027-3037.Miya, T., Morita, K., Suzuki, A., Ueno, N. and Satoh, N. (1997).",
"Functional analysis of an ascidian homologue of Animal BMP-2/BMP-4 suggests its role in Piccolo, S., Sasai, Y., Lu, B. and DeRobertis, E. M. (1996).",
"A possible molecular mechanism for Spemann organizer function: Inhibition of ventral signals by direct binding of Chordin to BMP-4.Cell 85:589-598.Rivera-Perez, J.",
"A., M. Mallo, et al.",
"(1995).",
"Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development.",
"Development 121(9): 3005-12.Sambrook, J., Fritsch, E. F. and Maniatis, T.",
"(eds.",
"; 1989).",
"Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press.",
"Vol 1-3.Savatier, P., H. Lapillonne, et al.",
"(1996).",
"Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells.",
"Oncogene 12(2): 309-22.Schmidt, J., Francois, V., Bier, E. and Kimelman, D. (1995).",
"Drosophila short gastrulation induces an ectopic axis in Xenopus: evidence for conserved Schulte-Merker, S., Lee, K., McMahon A. P. and Hammerschmidt, M. (1997).",
"The zebrafish organizer requires chordino.",
"Nature 387:862-863.Stoffregen, D. A., Bowser, P. R. and Babish J. G. (1996).",
"Antibacterial chaemotherapeutants for finfish aquaculture: A synopsis of laboratory and field efficacy and saftey studies.",
"J. aquatic animal health.",
"8:181-207.Suzuki Y, Yandell M D, Roy P J, Krishna S, Savage-Dunn C, Ross R M, Padgett R W, Wood W B (1999).",
"A BMP homolog acts as a dose-dependent regulator of body size and male tail patterning in Caenorhabditis elegans.",
"Development 126(2): 241-250 the inhibition of neural fate specification.",
"Development.124:5149-5159 Tjian R, Maniatis T (1994).",
"Transcriptional activation: a complex puzzle with few easy pieces.",
"Cell.",
"8:77:5-8.Waterhouse, P., Graham, M. W. and Wang, M-B.",
"(1998).",
"Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"95:13959-13964.Westerfield M. (1995).",
"The zebrafish book: University of Oregon Press.",
"Zwijsen, A., M. J. Goumans, et al.",
"(1999).",
"Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.",
"Dev Dyn 214(2): 141-51."
]
] | Patent_10169050 |
[
[
"Compositions and methods for gene silencing",
"DNA constructs are provided for disrupting gene expression in targeted organisms."
],
[
"1.An IR gene construct encoding an inverted repeat gene, said construct comprising: a) a promoter element operably linked in a 5′ to 3′ direction to a first coding sequence and a second sequence, said first coding sequence being in a sense orientation, said second sequence being the first coding sequence in an antisense orientation; and b) a transcription termination element operably linked 3′ to said first and second coding sequences.",
"2.The IR gene construct of claim 1 inserted into an expression vector.",
"3.A IR gene construct as claimed in claim 1, said promoter being inducible.",
"4.A IR gene construct as claimed in claim 2, said promoter being selected from the group consisting of a heat shock promoter, a metallotheinine promoter, a glucocorticoid promoter, CMV promoter, SV40 promoter, nervous system specific promoters, unc-119, mec-4, odr-4, muscle promoters unc-54, myo-2, act-l and ben-1, and a CaMV promoter.",
"5.The DNA construct of claim 1, wherein a spacer sequence is inserted between said first coding and second sequences.",
"6.A host cell containing the DNA construct of claim 1.7.A method for production of a phenocopy knock out mutant by introducing an inverted repeat gene into an organism, said inverted repeat gene comprising: a) a promoter element operably linked in a 5′ to 3′ direction to a first coding sequence and a second sequence, said first coding sequence being in a sense orientation, said second sequence being the first coding sequence in an antisense orientation; and b) a transcription termination element operably linked 3′ to said first and second coding sequences.",
"8.A method as claimed in claim 7, wherein said inverted repeat gene is introduced into C. elegans via a process selected from the group consisting of microinjection, soaking, and DNA coated particle bombardment.",
"9.A method as claimed in claim 7, wherein said inverted repeat gene contains the coding sequence of a gene selected from the group consisting of green fluorescent protein gene, C3782.5, F26F12.7, T14G8.1, efk-1, mec-4, unc-8, unc-119, degenerinis ZB770.1, T28B8.5, T28F24.2, C.24G7.2 and T28D9.7.10.A method as claimed in claim 7, wherein said inverted repeat gene is passed onto progeny thereby generating phenocopy mutants upon induction of expression of said inverted repeat gene.",
"11.A method as claimed in claim 7, wherein said organism is selected from the group consisting of plants, mice, humans, insects and nematodes.",
"12.An IR gene construct expression vector as claimed in claim 2 for the treatment of Alzheimers disease as shown in FIG.",
"4.13.An IR gene construct expression vector as claimed in claim 2 for the treatment of Parkinson's disease as shown in FIG.",
"5.14.An IR gene construct expression vector as claimed in claim 2 for the treatment of tomato leaf curl geminivirus as shown in FIG.",
"6."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains.",
"Complete citations may be found at the end of the specification.",
"The disclosure of each of these publications is incorporated by reference herein.",
"RNA interference was discovered by Guo and Kemhues in the course of attempts to use antisense RNA to block gene expression in the maternal germ line.",
"To their surprise, they found that both antisense and sense RNA preparations induced remarkably precise phenocopies of the targeted gene.",
"Since then, both the efficacy and apparent lack of strand specificity associated with this interference process have been borne out in many subsequent studies.",
"The mystery surrounding the mechanism of interference was recently deepened with the discovery that double-stranded RNA (dsRNA) is at least an order of magnitude more potent at inducing interferences than are preparations of either single strand.",
"The surprising properties of this interference mechanism prompted users to abandon the term “antisense” and to begin referring to the process merely as “RNA interference”.",
"The robust nature of the interference effect and the high degree of specificity have allowed RNAi to gain wide acceptance as a reverse genetic tool.",
"To date, most of these studies entail the microinjection of dsRNA duplexes corresponding to particular segments of targeted genes.",
"While these methods are effective most of the time, the phenotype is not inherited.",
"Accordingly, subsequent generations of targeted organisms must be continuously microinjected in order to attain the desired gene silencing effects.",
"Likewise the technique is limited to organisms which are amenable to injection or application of dsRNA.",
"It is an object of the present invention to overcome these limitations of the prior art."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to materials and methods which facilitate gene silencing in targeted organisms.",
"In one embodiment of the invention, a DNA construct encoding an inverted repeat gene is provided.",
"The construct comprises i) a promoter element operably linked in a 5′ to 3′ direction to a first coding sequence and a second sequence, the first coding sequence being in a sense orientation, the second sequence being the first coding sequence in an antisense orientation linked to the 3′ end of the first coding sequence; and ii) a transcription termination element operably linked 3′ to said first and second coding sequences.",
"In an alternative embodiment, the first coding sequence of the IR construct is in an antisense orientation and the second coding sequence is in a sense orientation.",
"In a preferred aspect of the invention, the inverted repeat gene of the invention is inserted into an expression vector.",
"In a preferred embodiment, the DNA constructs of the invention contain an inducible promoter to maximize expression of the inverted repeat genes of the invention.",
"It should be apparent to those of skill in the art however that any promoter which acts to drive expression of the inverted repeat genes are also within the scope of the invention.",
"Promoters contemplated for use in the DNA construct described above include, without limitation, heat shock promoters, metallothioneine promoter, glucocorticoid promoter, CMV promoter, SV40 promoter, nervous system specific C. elegans promoters, such as unc-119, mec-4, odr-4, and muscle promoters such as unc-54, myo-2, act-1 and ben-1.Optionally, the DNA constructs of the invention may include a spacer sequence between the first coding and second sequences.",
"Such spacer sequences can be about 300, 500, 700, 1000 or 1500 nucleotides in length.",
"In yet another aspect of the invention, host cells containing the DNA constructs encoding the inverted repeat genes of the invention are provided.",
"Such cells include, by way of illustration, C. elegans , yeast, Dictostelium, drosophila, mice, plants, insects, human cells and other nematodes Methods for production of phenocopy knock out mutants via introduction of an inverted repeat gene into a target organism are also provided.",
"The inverted repeat genes of the invention may be introduced into target organisms, such as C. elegans , via a process selected from the group consisting of microinjection, soaking, and DNA coated particle bombardment.",
"Suitable targets for gene silencing include the following: green fluorescent protein gene, C3782.5, F26F12.7, T14G8.1, efk-1, mec-4, unc-8, unc-119, degenerinis ZB770.1, T28B8.5, T28F24.2, C24G7.2 and T28D9.7.Several exemplary IR gene construct expression vectors are provided herein.",
"IR gene constructs for reducing or inhibiting the expression of the beta amyloid protein are shown in FIG.",
"4 .",
"IR gene construct expression vectors for inhibiting or reducing the expression of alpha-synuclein are provided in FIG.",
"5 .",
"An exemplary vector for inhibiting geminivirus infection in tomato is provided in FIG.",
"6 .",
"According to one aspect of the present invention, a method is provided for inhibiting or preventing the production of a pre-determined protein in a living organism.",
"The method comprises providing a vector encoding IR ds RNA molecules which are capable of binding specifically to an mRNA sequence of interest.",
"The vectors encoding the IR gene constructs of the invention are administered to the living organism under conditions whereby the vector enters cells, is expressed and thereafter specifically binds to the nucleic acid encoding the protein of interest, in an amount sufficient to reduce or inhibit production of the protein of interest.",
"According to another aspect of the present invention, a method is provided for treating a pathological condition related to an abnormal accumulation of disease-associated proteins.",
"Examples of such abnormal pathological conditions include, without limitation, Alzheimer's disease, Parkinson's and Huntington's Disease.",
"The method comprises administering to a patient having such a pathological condition a pharmaceutical preparation comprising vector having an IR gene construct contained therein capable of entering a cell expressing the protein of interest.",
"Expression of the IR gene construct results in the generation of a nucleic acid molecule which specifically binds to a nucleic acid encoding the protein of interest, in an amount sufficient to affect the level of production of the protein of interest, thereby alleviating the pathological condition.",
"According to another aspect of this invention, a pharmaceutical preparation is provided for treating a pathological condition related to the abnormal accumulation of disease-related proteins.",
"This pharmaceutical preparation comprises, in a biologically compatible medium, a vector having an IR gene construct contained therein capable of entering a cell and causing targeted gene silencing, in an amount sufficient to inhibit or reduce the level of production of the disease-associated protein.",
"The biologically compatible medium is preferably formulated to enhance the lipophilicity and membrane-permeability of the IR gene construct expression vector.",
"The use of an inverted repeat RNAi expression construct as a delivery vehicle exploits the ability of such a vector to continue to generate multiple dsRNA copies, thereby prolonging the expression of the inhibitory RNA molecules indefinitely in vivo.",
"This feature presents a distinct advantage over that of conventional modes for introduction of dsRNA, which provide a nonrenewable source of dsRNA in a one time delivery system.",
"The methods and IR RNAi expression vectors of the present invention provide notable advantages over currently available compounds and methods for treating diseases associated with the abnormal expression, or accumulation of proteins in cells observed in many viral and neurodegenerative disorders."
],
[
"FIELD OF THE INVENTION This invention relates the fields of molecular biology and gene silencing.",
"More specifically, the invention provides compositions and methods for heritable and inducible gene silencing in target organisms.",
"BACKGROUND OF THE INVENTION Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains.",
"Complete citations may be found at the end of the specification.",
"The disclosure of each of these publications is incorporated by reference herein.",
"RNA interference was discovered by Guo and Kemhues in the course of attempts to use antisense RNA to block gene expression in the maternal germ line.",
"To their surprise, they found that both antisense and sense RNA preparations induced remarkably precise phenocopies of the targeted gene.",
"Since then, both the efficacy and apparent lack of strand specificity associated with this interference process have been borne out in many subsequent studies.",
"The mystery surrounding the mechanism of interference was recently deepened with the discovery that double-stranded RNA (dsRNA) is at least an order of magnitude more potent at inducing interferences than are preparations of either single strand.",
"The surprising properties of this interference mechanism prompted users to abandon the term “antisense” and to begin referring to the process merely as “RNA interference”.",
"The robust nature of the interference effect and the high degree of specificity have allowed RNAi to gain wide acceptance as a reverse genetic tool.",
"To date, most of these studies entail the microinjection of dsRNA duplexes corresponding to particular segments of targeted genes.",
"While these methods are effective most of the time, the phenotype is not inherited.",
"Accordingly, subsequent generations of targeted organisms must be continuously microinjected in order to attain the desired gene silencing effects.",
"Likewise the technique is limited to organisms which are amenable to injection or application of dsRNA.",
"It is an object of the present invention to overcome these limitations of the prior art.",
"SUMMARY OF THE INVENTION The present invention is directed to materials and methods which facilitate gene silencing in targeted organisms.",
"In one embodiment of the invention, a DNA construct encoding an inverted repeat gene is provided.",
"The construct comprises i) a promoter element operably linked in a 5′ to 3′ direction to a first coding sequence and a second sequence, the first coding sequence being in a sense orientation, the second sequence being the first coding sequence in an antisense orientation linked to the 3′ end of the first coding sequence; and ii) a transcription termination element operably linked 3′ to said first and second coding sequences.",
"In an alternative embodiment, the first coding sequence of the IR construct is in an antisense orientation and the second coding sequence is in a sense orientation.",
"In a preferred aspect of the invention, the inverted repeat gene of the invention is inserted into an expression vector.",
"In a preferred embodiment, the DNA constructs of the invention contain an inducible promoter to maximize expression of the inverted repeat genes of the invention.",
"It should be apparent to those of skill in the art however that any promoter which acts to drive expression of the inverted repeat genes are also within the scope of the invention.",
"Promoters contemplated for use in the DNA construct described above include, without limitation, heat shock promoters, metallothioneine promoter, glucocorticoid promoter, CMV promoter, SV40 promoter, nervous system specific C. elegans promoters, such as unc-119, mec-4, odr-4, and muscle promoters such as unc-54, myo-2, act-1 and ben-1.Optionally, the DNA constructs of the invention may include a spacer sequence between the first coding and second sequences.",
"Such spacer sequences can be about 300, 500, 700, 1000 or 1500 nucleotides in length.",
"In yet another aspect of the invention, host cells containing the DNA constructs encoding the inverted repeat genes of the invention are provided.",
"Such cells include, by way of illustration, C. elegans, yeast, Dictostelium, drosophila, mice, plants, insects, human cells and other nematodes Methods for production of phenocopy knock out mutants via introduction of an inverted repeat gene into a target organism are also provided.",
"The inverted repeat genes of the invention may be introduced into target organisms, such as C. elegans, via a process selected from the group consisting of microinjection, soaking, and DNA coated particle bombardment.",
"Suitable targets for gene silencing include the following: green fluorescent protein gene, C3782.5, F26F12.7, T14G8.1, efk-1, mec-4, unc-8, unc-119, degenerinis ZB770.1, T28B8.5, T28F24.2, C24G7.2 and T28D9.7.Several exemplary IR gene construct expression vectors are provided herein.",
"IR gene constructs for reducing or inhibiting the expression of the beta amyloid protein are shown in FIG.",
"4.IR gene construct expression vectors for inhibiting or reducing the expression of alpha-synuclein are provided in FIG.",
"5.An exemplary vector for inhibiting geminivirus infection in tomato is provided in FIG.",
"6.According to one aspect of the present invention, a method is provided for inhibiting or preventing the production of a pre-determined protein in a living organism.",
"The method comprises providing a vector encoding IR ds RNA molecules which are capable of binding specifically to an mRNA sequence of interest.",
"The vectors encoding the IR gene constructs of the invention are administered to the living organism under conditions whereby the vector enters cells, is expressed and thereafter specifically binds to the nucleic acid encoding the protein of interest, in an amount sufficient to reduce or inhibit production of the protein of interest.",
"According to another aspect of the present invention, a method is provided for treating a pathological condition related to an abnormal accumulation of disease-associated proteins.",
"Examples of such abnormal pathological conditions include, without limitation, Alzheimer's disease, Parkinson's and Huntington's Disease.",
"The method comprises administering to a patient having such a pathological condition a pharmaceutical preparation comprising vector having an IR gene construct contained therein capable of entering a cell expressing the protein of interest.",
"Expression of the IR gene construct results in the generation of a nucleic acid molecule which specifically binds to a nucleic acid encoding the protein of interest, in an amount sufficient to affect the level of production of the protein of interest, thereby alleviating the pathological condition.",
"According to another aspect of this invention, a pharmaceutical preparation is provided for treating a pathological condition related to the abnormal accumulation of disease-related proteins.",
"This pharmaceutical preparation comprises, in a biologically compatible medium, a vector having an IR gene construct contained therein capable of entering a cell and causing targeted gene silencing, in an amount sufficient to inhibit or reduce the level of production of the disease-associated protein.",
"The biologically compatible medium is preferably formulated to enhance the lipophilicity and membrane-permeability of the IR gene construct expression vector.",
"The use of an inverted repeat RNAi expression construct as a delivery vehicle exploits the ability of such a vector to continue to generate multiple dsRNA copies, thereby prolonging the expression of the inhibitory RNA molecules indefinitely in vivo.",
"This feature presents a distinct advantage over that of conventional modes for introduction of dsRNA, which provide a nonrenewable source of dsRNA in a one time delivery system.",
"The methods and IR RNAi expression vectors of the present invention provide notable advantages over currently available compounds and methods for treating diseases associated with the abnormal expression, or accumulation of proteins in cells observed in many viral and neurodegenerative disorders.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 provides a schematic drawing of an IR gene construct expression vector of the invention.",
"FIGS.",
"2A and 2B depict the strategy for generation of heritable and inducible RNAi in the nematode.",
"A strong inducible promoter is fused to a direct inverted repeat gene.",
"Upon induction of expression in transgenic animals harboring this gene, transcripts are generated which fold back in a uni-molecular reaction to generate double stranded RNA within all cells that express the heat shock gene.",
"The size of the single stranded loop that occurs after foldback is not known (FIG.",
"2A).",
"Construction of inducible inverted repeat genes is shown in FIG.",
"2B.",
"Exon-rich genomic DNA (or CDNA) is amplified using two primers that introduce unique restriction sites at the fragment ends.",
"One restriction site is used to generate the inverted repeat and is ultimately situated at the inversion point (IP).",
"The other restriction site (designated as end) is ultimately used to join the inverted repeat to the vector.",
"Amplified fragments are digested with the enzyme situated at the IP restriction site (IPRS) and ligated together.",
"Digestion at the end restriction site (ERS) enables the fragment to be cloned into a similarly digested, CIAP-treated C. elegans expression vector.",
"In our work, vector pPD49.7822, which includes the hsp16-2 promoter and the 3′ untranslated region of muscle myosin unc-54, was utilized (FIG.",
"2B).",
"FIGS.",
"3A and 3B show that double stranded RNA synthesized in vivo can disrupt C. elegans gene expression.",
"FIG.",
"3A: Enzymatic assay for in vivo RNAi-induced disruption of the eEf2 kinase gene.",
"CeEFK-1 activity was assayed as described11 in reactions in which 0.5 μg rabbit reticulocyte eEF-2 was added to worm protein extracts.",
"Arrow indicates the eEF-2 protein position.",
"Lane 1, Wild type; lane 2, line harboring extrachromosomal hsp16-2pCefk-1 (IR), non-heat shocked; lane 3, a transgenic line harboring extrachromosomal parental vector pPD49.78, heat shocked; lane 4, line harboring extrachromosomal hsp16-2pCefk-1(IR), heat shocked; lane 5, Tc1 insertion Cefk-1 mutant.",
"FIG.",
"3B: Use of in vivo RNAi to disrupt GFP expression in neurons and pharyngeal muscle.",
"Progeny of transgenic lines harboring extrachromosomal unc-119pGFP (panels 1, 4; unc-119 is expressed in all neurons11), integrated mec-4pGFP (panels 2,5; mec-4 is expressed in six touch sensory neurons14) or myo-2pGFP (panels 3, 6; myo-2 is expressed in pharyngeal muscle16) and hsp16-2pGFP(IR) were compared at 20° C. or consequent to parental heat shock at the L4 stage (35° C., 4 h).",
"Progeny of similarly heat-shocked unc119pGFP, mec-4pGFP or myo-2pGFP lines exhibited no apparent reduction in intensity of neuronal fluorescence (data not shown).",
"In parallel conventional RNAi experiments, 6 of 210 progeny of an unc-119pGFP parent, 11 of 270 progeny of a mec-4pGFP parent, and 57 of 240 progeny of a myo-2pGFP parent exhibited detectable reduction in GFP signal.",
"FIG.",
"4 is a schematic diagram of an IR gene construct expression vector suitable for inhibiting or reducing expression of the beta-amyloid protein.",
"FIG.",
"5 is a schematic diagram of an IR gene construct expression vector suitable for inhbiting or reducing expression of the a-synuclein protein.",
"FIG.",
"6 is an schematic diagram of an IR gene construct expression vector suitable for expressing the NP protein of a tomato geminivirus thereby inhibiting viral replication in the targeted tomato plant.",
"The detailed description set forth below describes preferred methods for practicing the present invention.",
"Methods for selecting and preparing the IR gene constructs of the invention and expression vectors containing the same are described, as well as methods for administering the IR gene construct containing compositions in vivo.",
"Specific in vitro and in vivo diagnostic and therapeutic applications of the IR gene construct compositions are also set forth.",
"DETAILED DESCRIPTION OF THE INVENTION Double-stranded RNA interference (RNAi) is an effective method for disrupting specific gene expression in C. elegans and other organisms1-5.However, this powerful reverse genetics tool is most often employed in nematodes and plants because introduced dsRNA is not stably inherited1.Another difficulty is that late-acting genes are not as efficiently knocked-out by RNAi as embryonically expressed genes.",
"This may be due to a lowering of the concentration of dsRNA as cellular division proceeds during organismal development1.In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference.",
"It is an object of the invention to extend the applicability of RNAi by the controlled in vivo expression of heritable inverted-repeat (IR) genes.",
"The efficacy of in vivo-driven RNAi has been assessed in three situations for which heritable, inducible RNAi would be advantageous: 1) production of large numbers of animals deficient for gene activities required for viability or reproduction, 2) generation of large populations of phenocopy mutants for biochemical analysis, and 3) effective gene inactivation in the nervous system.",
"It is demonstrated herein that heritable inverted-repeat genes confer potent and specific gene inactivation for each of these applications, significantly broadening the already remarkable utility of RNAi for C. elegans reverse genetics.",
"Many neurodegenerative disorders result from the aberrant expression and/or accumulation of proteins to toxic levels.",
"The IR gene constructs of the invention may be utilized to inhibit the expression of such proteins thereby alleviating the pathological symptoms of the disorder.",
"In a similar fashion, the IR gene construct expression vectors of the invention may be engineered to inhibit the expression of viral proteins in infected cells.",
"Such viruses include both plant and animal viruses.",
"The IR gene constructs also have utility for the treatment of neoplastic diseases.",
"For example, the aberrant expression of oncogenes in certain cancers may be targeted for gene silencing using the compositions and methods of the invention.",
"Such oncogenes include, without limitation, ras, myc, myb, bcl-1, bcl-2, bcl-6, erb-a, erb-b, fgr, fos, src, lck, and lyn, The IR gene construct expression vectors of the invention are also suitable for the generation of transgenic knock-out mice.",
"Such mice provide ideal in vivo models for studying the contribution of particular genes to embryonic development, growth and disease.",
"Finally, the IR gene construct expression vectors of the invention may be used to advantage to generate disease resistant plants.",
"For example, geminiviruses are plant pathogens that infect a wide range of vegetable crops in tropical and subtropical regions with devastating consequences (Brown et al.",
"(1992) Plant Disease, 76:220-225).",
"Traditional breeding methods have failed to generate cultivars that are resistant to geminiviruses.",
"Transformation of target crops with the IR gene constructs of the invention encoding viral genes required for replication should effectively result in viral disease resistance.",
"I. Definitions The following definitions are provided to aid in understanding the subject matter regarded as the invention.",
"“Nucleic acid” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.",
"In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5′ to 3′ direction.",
"With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used.",
"This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.",
"For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.",
"When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above.",
"Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues).",
"An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.",
"The terms “percent similarity”, “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.",
"The phrase “inverted repeat gene” as used herein refers to an expression construct containing a promoter element operably linked in the 5′ to 3′ direction to a first coding sequence in a sense orientation which is in turn operably linked to a second sequence consisting of the first coding sequence in an anti-sense orientation.",
"Alternatively, the first coding sequence may be in an antisense orientation and the second coding sequence may be in a sense orientation.",
"The first and second coding sequences range between 20 and 2500 nucleotides in length.",
"Preferably the coding sequences may be between 100-300 nucleotides in length, 300-500 nucleotides in length, 500-800 nucleotides in length or 800-1500 nucleotides in length.",
"Most preferably the first and second coding sequences are about 1000 nucleotides in length.",
"The expression construct also contains 3′ regulatory regions which facilitate transcription of the inverted repeat gene in the targeted organism and the processing, expression, and translocation of its transcript.",
"The IR gene constructs of the invention may optionally comprise a spacer sequence between the first and second coding sequences.",
"Such spacer regions may be between 300-1000 nucleotides in length.",
"In certain embodiments, the spacer comprises an intronic region.",
"The term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.",
"The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:.",
"For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.",
"A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control.",
"A replicon may be either RNA or DNA and may be single or double stranded.",
"A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.",
"An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.",
"Promoters may be inducible or constitutive.",
"Inducible promoters include without limitation, heat shock promoter, metallothienine promoter, glucocorticoid promoter.",
"Constitutive promoters suitable for the practice of the present invention in worms include, without limitation, nervous system promoters, such as unc-119, mec-4, odr-4, muscle promoters, such as unc-54 and myo-2 and general promoters, such as act-1 and ben-1.In higher organisms, promoters such as CMV are suitable.",
"Other mammalian promoters are known to those of skill in the art and include for example, SV40, Mt promoter, glucocorticoid promoters.",
"When plants are targeted for gene silencing, the term “DNA construct” refers to genetic sequence used to transform plants and generate progeny transgenic plants.",
"These IR gene constructs may be administered to plants in a viral or plasmid expression vector.",
"The biolistic process of transformation is preferred for practice of the present invention.",
"Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using electroporation are also contemplated to be within the scope of the present invention.",
"In a preferred embodiment of the present invention, the associated plant promoter is a strong and non tissue- or developmental-specific plant promoter (e.g.",
"a promoter that strongly expresses in many or all tissue types).",
"Examples of such strong, “constitutive” promoters include, but are not limited to, the CaMV 35S promoter, the T-DNA mannopine synthetase promoter, and their various derivatives.",
"In another embodiment of the present invention, it may be advantageous to engineer a plant with a gene construct operably associating a tissue- or developmental-specific promoter with a sequence encoding the desired enzyme.",
"For example, where expression in photosynthetic tissues and organs are desired, promoters such as those of the ribulose bisphosphate carboxylase (RUBISCO) genes or chlorophyll a/b binding protein (CAB) genes may be used; where expression in seed is desired, promoters such as those of the various seed storage protein genes may be used; where expression in nitrogen fixing nodules is desired, promoters such those of the legehemoglobin or nodulin genes may be used; where root specific expression is desired, promoters such as those encoding for root-specific glutamine synthetase genes may be used (see Tingey et al., 1987, EMBO J.6:1-9; Edwards et al., 1990, Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 87:3459-3463).",
"In an additional embodiment of the present invention, it may be advantageous to transform a plant with an IR gene construct expression vector operably associating an inducible promoter with a sequence encoding the IR gene construct.",
"Examples of such promoters are many and varied.",
"They include, but are not limited to, those of the heat shock genes, the defense responsive gene (e.g., phenylalanine ammonia lyase genes), wound induced genes (e.g., hydroxyproline rich cell wall protein genes), chemically-inducible genes (e.g., nitrate reductase genes, gluconase genes, chitinase genes, etc.",
"), dark-inducible genes (e.g., asparagine synthetase gene (Coruzzi and Tsai, U.S. Pat.",
"No.",
"5,256,558, Oct. 26, 1993, Gene Encoding Plant Asparagine Synthetase) to name just a few.",
"The plant IR gene construct expression vectors of the invention may also comprise 3′ terminator sequences to stabilize the mRNA encoded by the construct.",
"Such sequences include, without limitation, poly A sequences, and the os or nos 3′ terminator sequence.",
"The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.",
"A probe may be either single-stranded or double-stranded.",
"The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.",
"For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.",
"The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence.",
"This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions.",
"Therefore, the probe sequence need not reflect the exact complementary sequence of the target.",
"For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand.",
"Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specfically.",
"The term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).",
"In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.",
"The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.",
"When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product.",
"The primer may vary in length depending on the particular conditions and requirement of the application.",
"For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length.",
"The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme.",
"It is not required that the primer sequence represent an exact complement of the desired template.",
"For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer.",
"Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.",
"All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.",
"The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.).",
"More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound.",
"Purity is measured by methods appropriate for the given compound (e.g.",
"chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).",
"The terms “transform”, “transfect”, “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning.",
"Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.",
"The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.",
"In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.",
"Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.",
"In other manners, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.",
"A “clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.",
"A “cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.",
"A “disease related protein may be a viral, bacterial or aberrant endogenously produced protein associated with a particular disease phenotype.",
"Such human proteins include, without limitation, those set forth in Table I below: TABLE I Increased dosage/Gain-of-function disease genes in humans 1.>gi|6681203|ref|NP_031894.1|| dystrophin, muscular dystrophy [Mus musculus] 2.>gi|6995996|ref|NP_000483.2|| cystic fibrosis TM conductance regulator 3.>gi|4753163|ref|NP_002102.2|| huntingtin [Homo sapiens] 4.>gi|4502167|ref|NP_000475.1|| amyloid beta (A4) precursor protein 5.>gi|2135246|pir||A56993 presenilin 2 - human 6.>gi|4506435|ref|NP_000312.1|| retinoblastoma 1 (osteosarcoma) [Homo sapiens] 7.>gi|1082578|pir||S50830 Machado-Joseph disease MJD1a protein - human 8.>gi|1709040|sp|P54252|MJD1_HUMAN MACHADO-JOSEPH DISEASE PROTEIN 1 9.>gi|3063388|dbj|BAA25751.1| Parkin [Homo sapiens] 10.>gi|4506113|ref|NP_000302.1|| prion protein (p27-30)[Homo sapiens] 11.>gi|2498924|sp|Q16637|SMN1_HUMAN SURVIVAL MOTOR NEURON PROTEIN 1 12.>gi|4507891|ref|NP_000542.1|| von Hippel-Lindau syndrome tumor suppressor 13.>gi|4507091|ref|NP_000335.1|| survival of motor neuron 1, telomeric 14.>gi|6166210|sp|Q92902|HPS_HUMAN HERMANSKY-PUDLAK SYNDROME PROTEIN 15.>gi|2228793|gb|AAC51731.1| Jagged1 [Homo sapiens] 16.>gi|904119|gb|AAB46416.1| S182 gene product, Alzheimer Disease Chr.",
"14 17.>gi|950348|gb|AAC42012.1|E5-1 protein Alzheimer Disease Chr.",
"1 18.>gi|4557365|ref|NP_000048.1|| Bloom syndrome protein [Homo sapiens] 19.>gi|4502839|ref|NP_000072.1|| Chediak-Higashi syndrome 1 [Homo sapiens] 20.>gi|6166193|sp|Q16595|FRDA_HUMAN FRATAXIN (FRIEDREICH'S ATAXIA PROTEIN) 21.>gi|6166210|sp|Q92902|HPS_HUMAN HERMANSKY-PUDLAK SYNDROME PROTEIN 22.>gi|4504455|ref|NP_000183.1|| Holt-Oram syndrome [Homo sapiens] 23.>gi|4557683|ref|NP_000207.1|| Kallmann syndrome 1 protein [Homo sapiens] 24.>gi|7531135|sp|Q12809|HERG_HUMAN VOLTAGE-GATED POTASSIUM CHANNEL 25.>gi|400664|sp|Q01968|OCRL_HUMAN LOWE'S OCULOCEREBRORENAL SYNDROME PROTEIN 26.>gi|1709040|sp|P54252|MJD1_HUMAN MACHADO-JOSEPH DISEASE PROTEIN 1 27.>gi|2135606|pir||I39294 McLeod syndrome-associated protein XK - human 28.>gi|4502321|ref|NP_000043.1|| ATPase, Cu++ transporting, (Menkes syndrome) [Homo sapiens] 29.>gi|5729770|ref|NP_000382.3|| ceroid-lipofuscinosis, neuronal 2, [Homo sapiens] 30.>gi|4557803|ref|NP_000262.1|| Niemann-Pick disease, type C1 [Homo sapiens] 31.>gi|4505339|ref|NP_002476.1|| Nijmegen breakage syndrome 1; nibrin [Homo 32.>gi|4505833|ref|NP_000287.1|| polycystic kidney disease 1 [Homo sapiens] 33.>gi|3126905|gb|AAC16004.1|polycystic kidney disease type II protein 34.>gi|4507091|ref|NP_000335.1|| survival of motor neuron 1, [Homo sapiens] 35.>gi|4506793|ref|NP_000323.1|| ataxin 1; spinocerebellar ataxia 1 36.>gi|4506795|ref|NP_002964.1|| ataxin 2; spinocerebellar ataxia 2 37.>gi|4506797|ref|NP_000324.1|| ataxin 7; spinocerebellar ataxia 7 [Homo sapiens] 38.>gi|4507891|ref|NP_000542.1|| von Hippel-Lindau syndrome tumor suppressor 39.>gi|482301|pir|A38080 Wilms tumor susceptibility protein WT1 - human 40.>gi|7513430|pir||A55197 Wiskott-Aldrich syndrome protein WASP - human 41.>gi|5174749|ref|NP_005996.1|| Wolfram syndrome [Homo sapiens] 42.>gi|6094278|sp|O60880|SH2A_DOMAIN PROTEIN 1A(DUNCAN'S DISEASE SH2-PROTEIN) 43.>gi|4502889|ref|NP_000077.1|| ceroid-lipofuscinosis, Spielmeyer-Vogt disease 44.>gi|189356|gb|AAA59964.1|Lowe Syndrome 45.>gi|515873 Membrane transport protein, McLeod Syndrome 46.>gi|34705|emb|CAA49145.1| Menkes Disease 47.>gi|307177|gb|AAA36206.1| protein kinase, Myotonic Dystrophy 48.>gi|292292|gb|AAA36212.1| Neurofibromatosis, Type 2 49.>gi|1163234|gb|AAA91041.1| Dpc4, Pancreatic Carcinoma 50.>gi|1314871|gb|AAC50481.1| retinitis pigmentosa GTPase regulator 51.>gi|1237181|gb|AAA98132.1| glypican, Simpson-Golabi-Behmel syndrome 52.>gi|624186|gb|AAA66242.1| survival motor neuron 53.>gi|1737213|gb|AAC52047.1| neuronal apoptosis inhibitory protein [Homo sapiens] 54.>gi|529662|emb|CAA55793.1| ataxin-1 [Homo sapiens] Spinocerebral Ataxia 55.>gi|731120|sp|P40337|VHL_HUMAN VON HIPPEL-LINDAU DISEASE TUMOR SUPPRESSOR (G7) 56.>gi|435421|gb|AAA03628.1| PAX-3 Waardenburg Syndrome II.",
"Preparation of Nucleic Acid Molecules Encoding the Inverted Repeat Genes of the Invention Nucleic acid molecules comprising the inverted repeat genes of the invention may be prepared by two general methods: (1) synthesis from appropriate nucleotide triphosphates, or (2) isolation from biological sources.",
"Both methods utilize protocols well known in the art.",
"The availability of nucleotide sequence information for genes targeted for knock-out enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.",
"Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices.",
"The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC).",
"Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods.",
"Thus, for example, a 1.9 kb double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity.",
"Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment.",
"Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 1.9 kb double-stranded molecule.",
"A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.",
"Nucleic acid sequences encoding the inverted repeat genes of the invention may be isolated from appropriate biological sources using methods known in the art.",
"In one embodiment, a clone is amplified from a DNA expression library of from the desired species of origin.",
"Suitable primers for this purpose are derived from sequences within the gene targeted for silencing.",
"Such primers may be between 15 and 40 nucleotides in length.",
"Alternative approaches for obtaining DNA for the inverted repeat genes of the invention, include cloning the inverted repeat fragments directly or chemically synthesizing the entire inverted repeat gene.",
"In accordance with the present invention, nucleic acids having the appropriate level of sequence homology with the protein coding region of genes targeted for silencing may be identified by using hybridization and washing conditions of appropriate stringency.",
"For example, hybridizations may be performed, according to the method of Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), using a hybridization solution comprising: 5× SSC, 5× Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.",
"Hybridization is carried out at 37-42 C. for at least six hours.",
"Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2× SSC and 1% SDS; (2) 15 minutes at room temperature in 2× SSC and 0.1% SDS; (3) 30 minutes-1 hour at 3-7 C. in 1× SSC and 1% SDS; (4) 2 hours at 42-65 C. in 1× SSC and 1% SDS, changing the solution every 30 minutes.",
"One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989) is as follows: Tm=81.5° C.+16.6Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp in duplex.",
"As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology.",
"Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C. Rather than direct cloning of genes targeted for silencing, an alternative approach entails identification of target genes by homology searches in available the available nucleic acid databases such as Genbank.",
"The inverted repeat genes of the present invention may be maintained as DNA in any convenient cloning vector.",
"In a preferred embodiment, clones are maintained in a plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, Calif.), which is propagated in a suitable E. coli host cell.",
"Other vectors suitable for the practice of the present invention, include, without limitation, pBlue/TOPO (Invitrogen), PCR-Blunt-TOPO (Invitrogen) and the pCDNA series from Invitrogen.",
"II.",
"Selection and Preparation of Expression Vectors Containing the IR Gene Constructs of the Invention Selection of a suitable sequence targeted for knock-out depends on knowledge of the nucleotide sequence of the target mRNA, or gene from which the mRNA is transcribed.",
"Although targeting to mRNA is preferred and exemplified in the description below, it will be appreciated by those skilled in the art that other forms of nucleic acid, such as pre-mRNA or genomic DNA, may also be targeted.",
"Double-stranded IR transcripts should correspond to regions present in the transcript encoding the targeted protein.",
"Such sequences include, but are not limited to 5′ untranslated regions, coding regions and the 3′ untranslated regions.",
"Various genetic regulatory control elements may be incorporated into the expression vectors containing the IR gene constructs of the invention to facilitate propagation in both eucaryotic and procaryotic cells.",
"Different promoters may be utilized to drive expression of the IR gene construct sequences, the cytomegalovirus immediate early promoter being preferred for use in humans as it promotes a high level of expression of downstream sequences.",
"Polyadenylation signal sequences are also utilized to promote mRNA stability.",
"Sequences preferred for use in human cells include, but are not limited to, bovine growth hormone polyadenylation signal sequences or thymidine kinase polyadenylation signal sequences.",
"Antibiotic resistance markers are also included in these vectors to enable selection of transformed cells.",
"These may include, for example, genes that confer hygromycin, neomycin or ampicillin resistance.",
"A variety of different vectors are available for use in the methods of the invention.",
"These include without limitation, those set forth in Table II.",
"The listed vector have been utilized to express the indicated proteins.",
"Conventional molecular biological techniques may be utilized to replace the protein coding sequence with the IR gene constructs of the invention.",
"TABLE II VECTORS FOR USE IN THE METHODS OF THE INVENTION Name Description pACCMVpLmP1(−)loxP-SSP Adenoviral shuttle plasmid with unique restriction tag, SwaI, SfiI, Pmel, CMV promoter, pUC19 polylinker, mP1 splicing signal/poly A, loxP pACCMVpLpA(−)loxP Adenoviral shuttle plasmid, CMV promoter, pUC19 polylinker, SV40 splice/polyA, loxP PACCMVpLPA(−)loxP-SSP Adenoviral shuttle plasmid with unique restriction tag, Swal, Sfil, PmeI, CMV promoter, pUC19 polylinker, SV40 splice/polyA, loxP pACpL + loxP Adenoviral shuttle plasmid, no promoter pACpL + loxP-SSP Adenoviral shuttle plasmid, no promoter, SwaI, Sfil, Pmel unique site cluster pAd-HSV tkHSV tk, thymidine kinase, pAD BglII pAdBgl II Ampicillin Resistance, Recombinant Adenovirus pAdEF1 alpha loxP Adenovirus, EF 1 alpha promoter, loxP pAdMCSlacZ multiple cloning sites, lacZ gene pAdMCSloxP Adenoviral shuttle vector, polylinker, loxP pAdMCSpA multiple cloning sites, SV40 PolyA signal pAdMCSpA/lacZ multiple cloning sites, lacZ gene, SV40 poly(A) signal pAdRSV4 RSV promoter, multiple cloning sites, SV40 poly(A) pNGVL1 PstI, SalI, HindIII, EcoRV, BglII polylinker, kanamycin resistance pNGVL1-CAT chloramphenicol transferase pNGVL1-hGM-CSF human granulocyte-monocyte colony stimulating factor pNGVL1-hpAP human placental alkaline phospbatase pNGVL1-mGM-CSF mouse granulocyte-monocyte colony stimulating factor pNGVL1-ntbeta-gal nuclear targeted beta- galactosidase pNGVL1-tk thymidine kinase pNGVL2 polylinker deleted, kanamycin resistance pNGVL3 12 site polylinker, kanamycin resistance pNGVL3-4070a-env Amphotropic 4070A virus env.",
"pNGVL3-gag-pol retrovirus gag-pol helper plasmid, kanamycin resistant pNGVL3-hFL human full length Flt3 ligand cDNA pNGVL3-hFLex human Flt3 ligand, secreted pNGVL3-hIL10 human interleukin 10 pNGVL3-hIL12 human interleukin-12, internal ribosome entry site pNGVL3-hIL15 human interleukin-15 pNGVL3-hIL2 human interleukin-2 pNGVL3-hIL2/IL15 human IL2-IL15 fusion pNGVL3-hIL4 human interleukin 4 pNGVL3-hIL7 human interleukin 7 pNGVL3-mFL mouse FLT3 ligand pNGVL3-mFLex Extracellular domain of mouse FLT3 ligand pNGVL3-mIL10 mouse interleukin 10 pNGVL3-mIL12 mouse interleukin 12, internal ribosome entry site pNGVL3-mIL15 mouse interleukin-15 pNGVL3-mIL2 Mouse interleukin-2 pNGVL3-mIL4 mouse interleukin 4 pNGVL3-mIL7 mouse interleukin 7 pNGVL3-shIL15R soluble human interleukin 15 receptor pNGVL3-smIL15R soluble mouse interleukin-15 receptor pNGVL4a immunostimulatory sequence pNGVL4b immunostimulatory sequence pNGVL5 IL2 secretory signal peptide pNGVL6a IL2 secretion signal peptide pNGVL7 pNGVL7, CMV, tpa RVNL3(+) CMV promoter, ATG(−), non- episomal, retrovirus vector IV.",
"Uses of Nucleic Acids Encoding Inverted Repeat Genes Gene silencing provides a powerful technique for the elucidation of molecular and biochemical mechanisms associated with homeostasis, growth and development.",
"The inverted repeat genes of the invention may be used to advantage to “knock out” the activity of any target gene, provided the sequence of the target gene is known.",
"The inverted repeat gene constructs have been designed such that they are both inducible and heritable.",
"Expression of the inverted repeat genes of the invention driven from strong inducible promoters facilitates the formation of a double stranded “snap back” RNA endogenously, thereby abrogating functional expression of the target gene.",
"Gene expression manipulation is extremely important to the pharmaceutical industry.",
"Beneficial uses of this invention include specific gene inactivation for the investigation of gene function and reverse genetic studies.",
"When C. elegans is used as the target organism, the compositions and methods of the invention facilitate the production of phenocopy mutants which can be induced also in offspring.",
"Thus the invention provides for the production of large populations of nematodes that can be subjected to gene inactivation at any time during growth and development which in turn may be used to advantage for drug screening, large scale genetic mutant assessment, and biochemical studies.",
"The invention also provides for disruption of gene activity, markedly improving current protocols which appear to be ineffective in neuronal “knock out”.",
"The invention also provides for large scale preparations for analysis of the RNAi mechanism itself.",
"Additionally, this protocols described herein may be used to inactivate or disrupt gene activity in any organism.",
"Any organism which may be transformed with exogenous DNA corresponding to a target gene having a defined promoter may be subjected to gene silencing using the compositions and methods of the present invention.",
"Such organisms include, without limitation, yeast, Dictostelium, drosophila, mice, insects, plants, human cells and other nematodes.",
"Such methods facilitate an analysis of specific gene function by phenocopy knockout.",
"Additionally, other harmful or potentially harmful gene expression may be inhibited or prevented in accordance with the invention.",
"Such genes include by way of illustration, genes required for oncogenisis, productive HIV infection and genes required for successful infection by a variety of pathogenic organisms.",
"The availability of sequence information encoding nucleic acids targeted for gene silencing enables the production of strains of laboratory mice carrying the IR gene constructs of the invention.",
"Such mice provide an in vivo model for assessing growth development and disease.",
"The compositions and methods provided herein enables the production of knockout mice in which the endogenous gene corresponding to the IR gene construct has been specifically inactivated.",
"Methods of introducing IR gene construct expression vectors in laboratory mice are known to those of skill in the art.",
"Three common methods include: 1.integration of retroviral vectors encoding the foreign gene of interest into an early embryo; 2.injection of DNA into the pronucleus of a newly fertilized egg; and 3.the incorporation of genetically manipulated embryonic stem cells into an early embryo.",
"Production of the transgenic mice described above will faciliate the molecular elucidation of the role predetermined target genes play in embryonic development and disease.",
"A “transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus.",
"The term “transgenic animal” is not meant to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule.",
"This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA.",
"The term “germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring.",
"If such offspring, in fact, possess some or all of that alteration or genetic information, then they, too, are transgenic animals.",
"The DNA used for altering expression of a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated MRNA templates, direct synthesis, or a combination thereof.",
"A type of target cell for transgene introduction is the embryonal stem cell (ES).",
"ES cells may be obtained from pre-implantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc.",
"Natl.",
"Acad.",
"Sci.",
"83:9065-9069).",
"Transgenes can be efficiently introduced into the ES cells by standard techniques such as DNA transfection or by retrovirus-mediated transduction.",
"The resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.",
"The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.",
"One approach to the problem of determining the contributions of individual genes and their expression products is to use IR gene constructs to selectively inactivate the wild-type gene in totipotent ES cells (such as those described above) and then generate transgenic mice.",
"The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described, and is reviewed elsewhere (Frohman et al., (1989) Cell 56:145-147; Bradley et al., (1992) Bio/Technology 10:534-539).",
"Techniques are available to inactivate or alter any genetic region to a mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles.",
"However, in comparison with homologous extrachromosomal recombination, which occurs at a frequency approaching 100%, homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10−6 and 10−3.Nonhomologous plasmid-chromosome interactions are more frequent occurring at levels 105-fold to 102-fold greater than comparable homologous insertion.",
"To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants.",
"One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening of individual clones.",
"Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly.",
"One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists.",
"The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter.",
"Non-homologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with effective herpes drugs such as gancyclovir (GANC) or (1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodouracil, (FIAU).",
"By this counter selection, the number of homologous recombinants in the surviving transformants can be increased.",
"Methods of use for the transgenic mice of the invention are also provided herein.",
"Therapeutic agents for the treatment or prevention of disease may be screened in studies using transgenic mice harboring the IR gene constructs of the invention.",
"As mentioned previously, the IR gene construct expression vectors of the invention may be used for gene silencing in any organism which may be targeted with exogenous DNA.",
"Uses of the expression vectors for the treatment of human and plant diseases is also exemplified herein.",
"V. Administration of Plasmid Vectors Producing the IR Genes of the Invention The IR gene construct containing expression vectors as described herein are generally administered to a patient as a pharmaceutical preparation.",
"The term “patient” as used herein refers to human or animal subjects.",
"The pharmaceutical preparation comprising the IR gene construct expression vector of the invention is conveniently formulated for administration with a acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.",
"The concentration of expression vector in the chosen medium will depend on the hydrophobic or hydrophilic nature of the medium, as well as the length and other properties of the vector molecule.",
"Solubility limits may be easily determined by one skilled in the art.",
"As used herein, “biologically acceptable medium” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph.",
"The use of such media for pharmaceutically active substances is known in the art.",
"Except insofar as any conventional media or agent is incompatible with the IR gene construct expression vectors to be administered, its use in the pharmaceutical preparation is contemplated.",
"Selection of a suitable pharmaceutical preparation depends upon the method of administration chosen.",
"For example, IR gene construct expression vectors may be administered by direct injection into the region of the brain containing the targeted cell type.",
"In this instance, a pharmaceutical preparation comprises the IR gene construct expression vector dispersed in a medium that is compatible with cerebrospinal fluid.",
"In a preferred embodiment, artificial cerebrospinal fluid (148 mM NaCl, 2.9 mM KCl, 1.6 mM MgCl2 6H2O, 1.7 mM CaCl2, 2.2 mM dextrose) is utilized, and IR gene construct expression vectors are provided directly to neurons by intraventricular injection.",
"IR gene construct expression vectors for use in gene silencing may also be administered parenterally by intravenous injection into the blood stream, or by subcutaneous, intramuscular or intraperitoneal injection.",
"Pharmaceutical preparations for parenteral injection are commonly known in the art.",
"If parenteral injection is selected as a method for administering the IR gene construct expression vector, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.",
"The lipophilicity of the IR gene construct expression vectors, or the pharmaceutical preparation in which they are delivered may have to be increased so that the molecules can cross the blood-brain barrier to arrive at their target locations.",
"Furthermore, the IR gene construct expression vectors may have to be delivered in a cell-targeted carrier so that sufficient numbers of molecules will reach the target cells.",
"Methods for increasing the lipophilicity of a molecule are known in the art, and include the addition of lipophilic groups to the IR gene construct expression vector.",
"Several techniques have been used to increase the stability, cellular uptake and biodistribution of DNA expression vectors.",
"The expression vector of the present invention may be encapsulated in a lipophilic, targeted carrier, such as a liposome.",
"One technique is to use as a carrier for the expression vector a liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (D OT MA; lipofectin).",
"The vectors of the present invention may be complexed to liposomes.",
"To further facilitate targeting of the IR gene construct expression vector, liposomes may be “studded” with antibodies specific for certain regions of the brain (Leserman et al., (1980) Nature 288:604).",
"In a preferred embodiment, cationic liposomes are complexed with (1) the IR gene construct expression vector; and (2) antibodies specific for the desired region of the brain.",
"Vector containing antibody-studded-liposome complexes are expected not only to be targeted and specifically expressed in the desired regions of the brain, but also to be expressed for indefinitely.",
"The pharmaceutical preparation is formulated in dosage unit form for ease of administration and uniformity of dosage.",
"Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment.",
"Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier.",
"Procedures for determining the appropriate dosage unit are well known to those skilled in the art.",
"The pharmaceutical preparation comprising the IR gene construct expression vector may be administered at appropriate intervals, for example, twice a day until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level.",
"The appropriate interval in a particular case would normally depend on the condition of the patient.",
"When performing gene silencing in plants, the IR gene construct expression vector is delivered to plant cells using biolistic or Agrobacterium mediated DNA transfer.",
"Selection and propagation of transformed plant cells is performed according to methods well known to those of ordinary skill in the art.",
"The following protocols are provided to facilitate the practice of the present invention.",
"Nematode strains.",
"C. elegans strains were reared and maintained as described21.We constructed transgenic lines by injection of plasmid DNAs each at 100 ng/μl using standard protocols22.In all experiments we used plasmid pRF423, which harbors a dominant rol-6 allele that causes a readily distinguished roller phenotype in transgenic animals, as a co-transformation marker.",
"Construction of inverted repeat genes.",
"We PCR amplified exon-rich genomic DNA (or cDNA) using two primers that introduce unique restriction sites at the fragment ends.",
"We digested the amplified fragment with one of the enzymes and ligated to generate an inverted repeat (outlined in FIG.",
"2b).",
"We then digested with the other enzyme, the restriction site for which is now positioned at the IR fragment ends, and ligated into CIAP-treated vector pPD49.7822, which includes the hsp16-2 promoter and the 3′ untranslated region of muscle myosin unc-54.The cDNA and genomic DNA we amplified for RNAi ranged from 0.58-1.45 kb in length.",
"Alternative cloning strategies include: 1) digestion at two naturally occurring restriction sites to excise the gene fragment of interest with subsequent two-step ligation as above, or 2) direct tri-molecular ligation of the doubly digested fragment into CIAP-treated vector previously linearized with one of the enzymes at the fragment end.",
"In an alternative embodiment spacer loops are included between the inverted repeat gene segments.",
"We found the efficiency of cloning inverted repeats to be acceptable in the E. coli DH5 strain (in general, a few per hundred candidates screened) and relatively high in the E. coli SURE strain (Stratagene), a bacterial host tolerant of inverted repeats (about 1/20 candidate constructs correct).",
"The hsp16-2punc-8 (IR) construct, however, proved highly difficult to generate (1000 candidates screened, 0.58 kb of cDNA sequence in the repeat) for reasons that are not clear.",
"Slower growing bacterial transformant colonies appear to have an enhanced chance of harboring the IR gene.",
"The yield of plasmid DNA from IR genes harbored in E. coli DH5 strain is low (about 3-5 μg per 50 ml culture); when the SURE strain is the host, yields are improved (80-100 μg per 50 ml culture).",
"While this method relates to PCR amplification of the target genes, it will be appreciated by those of skill in the art that other methods for obtaining the DNA for the inverted repeat genes of the invention are available.",
"These include direct synthesis of the target gene on a DNA synthesizer and direct cloning using conventional hybridization and DNA isolation procedures.",
"RNA interference assays.",
"For standard RNAi, we prepared dsRNA from cDNAs or coding sequence-rich genomic DNAs, 0.58-1.2 kb in length are injected into N2 adults/group as described1.We scored progeny born to injected adults (10 adults per group) 12 hours or more after injection (older progeny exhibit a much lower phenocopy rate).",
"For genetically directed RNAi mediated by expression of inverted repeat genes, we selected 50 transgenic roller L4s from lines harboring various hsp-16p(IR) constructs plus co-transformation plasmid pRF423 (array transmission frequency >60%) and reared continuously at 20° C. (non-heat shock) or heat shocked for 4 hours at 35° C., before returning to 20° C. Progeny of these animals were scored for phenotypes of interest at embryonic or larval stages as appropriate; behavioral assays and phenotypic analysis as indicated in Table 3 and Figure legends.",
"On average at least half of lines for a given gene assayed conferred potent interference upon heat-activation.",
"The following examples are provided to illustrate various embodiments of the invention.",
"They are not intended to limit the invention in any way.",
"EXAMPLE I To test the feasibility of specific gene disruption via in vivo expression of double stranded RNA, we constructed transgenic nematodes that synthesize hairpin ds RNA3 from IR genes under the control of the strong heat shock-inducible promoter, hsp16-2 6-8 (FIG.",
"2).",
"We first compared effects of conventional RNAi via injection of dsRNA, expression of sense and antisense genes, and in vivo production of dsRNA using C. elegans predicted gene C37A2.5, an essential gene required for progression past the L2 larval stage (N. Tavernarakis, S. Wang, M., Driscoll, unpublished observations).",
"Conventional RNAi via injection of C37A2.5 dsRNA1 produces a high yield of L2 stage-arrested F1 progeny (Table 3).",
"Expression of the antisense strand, which can be an effective method for specific gene inactivation9, confers a modest percentage of phenocopy progeny, whereas expression of the sense stand is ineffective.",
"To test in vivo RNAi, we heat-shocked young adults of transgenic lines harboring extrachromosomal hsp16-2pC37A2.5(IR).",
"In vivo promoter-driven RNAi proved effective in reproducing the C37A2.5 null phenotype, with efficiencies approaching that of direct injection of dsRNA (Table 3).",
"Likewise, promoter-driven RNAi efficiently disrupted the Mi-2 chromatin remodeling homolog F2612.710 to phenocopy the sterile phenotype of a deletion of this gene (Table 1).",
"We conclude that in vivo driven RNAi can be effective and that this technique should enable convenient generation of large populations of phenocopy mutants, even when development or reproduction is blocked.",
"TABLE 3 In vivo dsRNA interference Gene disruption approach Trial/Line 1 Trial/Line 2 Trial/Line 3 Trial/Line 4 dsRNA C37A2.5 94 ± 8 89 ± 4 97 ± 5 89 ± 7 injected pPD49.78 (hsp16-2p alone) + heat 0 0 0 0 shock hsp16-2pC37A2.5 sense + heat 0 0 0 0 shock hsp16-2pC37A2.5 antisense + heat 9 ± 4 9 ± 4 11 ± 6 — shock hsp16-2pC37A2.5(IR) − heat 0 0 0 0 shock hsp16-2pC37A2.5(IR) + heat 67 ± 3 79 ± 6 84 ± 5 56 ± 7 shock hsp16-2pF26F12.7(IR) − heat 1 ± 0.9 2 ± 1 1 ± 0.9 3 ± 1.3 shock hsp16-2p F26F12.7(IR) + heat 58 ± 4 59 ± 5 75 ± 8 82 ± 6 shock ds mec-4 RNA 12 ± 7 19 ± 5 15 ± 6 — injected hsp16-2pmec-4(IR) − heat 0 0 0 — shock hsp16-2pmec-4(IR) + heat 58 ± 4 60 ± 7 61 ± 8 — shock ds unc-8 RNA 0 0.8 ± .01 0 0 injected hsp16-2punc-8(IR) − heat 0 0 0 0 shock hsp16-2punc-8(IR) + heat 17 ± 3 11 ± 5 14 ± 2 13 ± 3 shock Results for four injection trials using conventional RNAi or heat shock induced in vivo RNAi in 4 transgenic lines (unless otherwise noted) are indicated.",
"In all experiments, at least 100 animals were scored per experimental trial.",
"Gene C37A2.5 is required for developmental progression past the L2 stage.",
"Numbers indicate the percentage of F1 progeny arrested at the L2 stage ± SD.",
"Co-expression of sense and antisense genes, which can be effective24, # was not tested.",
"Deletion of chromatin remodeling gene homolog F26F12.7 causes sterility (S. Wang and M. Driscoll, unpublished).",
"Treated progeny of transgenic lines harboring hsp16-2pF26F12.7(IR) were scored for % that fail to develop into fertile adults.",
"A similar strategy for in vivo disruption of a second MI-2 homolog, T14G8.1, yielded 59% and 72% sterile in progeny of two lines scored after heat shock (data not shown).",
"mec-4 is expressed in six # mechanosensory neurons and is required for touch sensitivity.",
"ds mec-4 RNA or plasmid hsp16-2pmec-4(IR) was introduced into wild type animals and progeny were scored for touch insensitivity.",
"unc-8(n491) is a dominant gain-of-function mutation that causes coiling and backward paralysis; locomotion in a loss of function mutant is nearly normal15.ds unc-8 RNA or plasmid hsp16-2punc-8(IR) was introduced into the n491 background and progeny were assayed for # backing proficiency.",
"Note that to regain backing ability, gene expression must be knocked down in the majority of unc-8-expressing cells, approximately 60 neurons.",
"C. elegans translation elongation factor 2 kinase eEF-2 (efk-1)11 phosphorylates eEF-2, an activity abolished by a Tc1 insertion into the active site (A. Ryazanov, C. Mendola, L. Zhang and J. Culotti, unpublished observations) (FIG.",
"3a).",
"We find that kinase activity in the offspring of heat-shocked hsp16-2pefk-1 (IR) transgenic parents is reduced at least several fold in 4/6 lines we assayed.",
"An analogous assay could not be performed on a population of phenocopy mutants induced by conventional RNAi, since several hundred animals are required.",
"We conclude that inducible IR genes are effective in generating populations amenable to biochemical analysis.",
"Injected dsRNA is not uniformly effective in disrupting gene expression in the nervous system.",
"For example, we find that only 6/210 progeny from three lines harboring integrated unc-119pGFP (expressed in all neurons) injected with double-stranded GFP RNA exhibited a detectable reduction in fluorescence (FIG.",
"3b).",
"To examine more closely effects of endogenously expressed dsRNA species on gene inactivation in the differentiated nervous system, we first constructed a plasmid that directs in vivo expression of double-stranded GFP RNA upon heat shock and tested for extinction of fluorescent signals generated by cell-specific GFP reporter fusions (FIG.",
"3b).",
"We co-introduced the hsp16-2pGFP(IR) construct and unc-119pGFP (pIM17512; expressed at high levels throughout the nervous system13), selected lines exhibiting strong GFP fluorescence, heat shocked in the L4 stage, and examined fluorescence in their progeny.",
"Approximately 79% of roller progeny from 3 (of 5) lines harboring unc-119pGFP and hsp16-2pGFP(IR) exhibit easily distinguished knockdown effects, with fewer than 10 cells readily detectable in most (FIG.",
"3b).",
"We did not detect any consistent pattern of cells that appeared refractory to fluorescence inactivation, suggesting that all cells in the nervous system are susceptible to the effects of in vivo RNAi.",
"We also tested effects of heat shock induction of hsp16-2pGFP (IR) on expression of an integrated mec-4pGFP gene, which is specifically expressed in the six touch receptor neurons14.On average, 85% of roller progeny of heat shocked parents harboring the extragenic hsp-16pGFP(IR) transgene had GFP signals that were either eliminated or markedly attenuated (2 of 4 lines; FIG.",
"3b).",
"By contrast, we observed similar effects in only 11 of 270 progeny of a line harboring an integrated mec-4pGFP reporter injected with dsGFP RNA.",
"We also tested for dsRNA-mediated inactivation of C. elegans neuronal genes.",
"Conventional RNAi mediated by introduced mec-4 dsRNA induced touch-insensitivity in 46/300 (15%) offspring of injected wild type parents.",
"On average, 60% progeny of heat-shocked lines harboring hsp16-2pmec-4 (IR) were touch insensitive (Table 1).",
"As another example, we tested the effectiveness of in vivo-directed RNAi in the inactivation of unc-8, a neuronally expressed gene that in our hands has been resistant to the effects of conventional RNAi.",
"unc-8 gain of function allele n491 dominantly induces uncoordinated locomotion characterized by the inability to back up; the loss of function phenotype appears nearly wild type15.Injection of unc-8 dsRNA is not effective in knocking out the gf phenotype (2 phenocopy mutants generated among 1300 progeny of injected parents).",
"Progeny of heat shocked unc-8(n491) mutants harboring hsp16-2punc-8(IR) are effectively targeted about 13% of the time (Table 3).",
"Taken together, our results indicate that sequences expressed in terminally differentiated neurons can be targeted by in vivo induced RNAi and in some instances effects are more potent than those observed after injection of dsRNA.",
"For all nine cases we investigated, heat shock of control lines carrying the expression vector alone or low temperature growth of lines carrying the hsp16-2p(IR) genes did not produce any readily apparent phenotypes (we assayed for the anticipated knockout phenotype, morphological and locomotion defects, fertility, and developmental abnormalities; >100 animals examined per line).",
"Thus, effects of in vivo RNAi appear highly specific, consistent with reported tight regulation of the hsp16-2 promoter8 and the selective precision of RNAi knock out capacity1.Moreover, in vivo RNAi appears effective in many tissue types, including neurons (FIG.",
"2b, note that C37A2.5 and efk-1 are expressed early in development and later in a broad range of cells including body wall and pharyngeal muscles, neurons, hypodermis and intestine (N. Tavernarakis, A. Ryazanov and M. Driscoll, unpublished); Mi2 homolog F2612.7 is expressed in the hypodermis; Mi2 homolog T14G8.1 is expressed in the hypodermis and pharynx (S. Wang, N. Tavernarakis and M. Driscoll unpublished); myo-2 is expressed in pharyngeal tissue16).",
"Our analysis establishes that endogenous inverted repeat genes can be expressed to generate dsRNA species with biological effects similar to, and superior than that of directly injected dsRNA.",
"Advantages of expressing heritable inverted repeat genes include that: 1) stable lines harboring the potential for gene inactivation can be easily maintained, 2) assays requiring large numbers of mutant phenocopies are feasible, and 3) inhibition can be inducible, and thus may be used for stage-specific gene inactivation.",
"In some cases, the endogenous high level of dsRNA product produced upon heat shock appears to make for more potent inhibition than germline injected dsRNA.",
"Although we have focused our initial studies on the use of the inducible hsp16-2 promoter, our findings indicate that it us possible to inactivate specific genes for the duration of their expression period by integrating a transgene in which the promoter of the gene of interest drives transcription of an inverted repeat segment of the same gene.",
"In addition, since dsRNA can inactivate genes in flies, plants, trypanosomes and planaria17-20, in vivo directed RNAi could be effective in other organisms.",
"These observations indicate that a similar strategy for in vivo driven RNAi can be applied to inactivate specific genes in organisms that can be genetically engineered but are not readily amenable to direct injection of dsRNA.",
"EXAMPLE II IR Constructs for Use in the Prevention and Treatment of Neurodegenerative Disorders Neurodegenerative disorders, such as Alzheimer's and Parkinson's disease disproportionately affect the elderly, the most rapidly growing sector of the population.",
"Additionally, many neural viral infections, such as those caused by HIV and encephalitis viruses, cause irreversible destruction of brain tissue, thereby compromising the quality of life for the patient.",
"Accordingly prophylactic and therapeutic treatments are highly desirable for preventing and/or inhibiting the neurodegeneration associated with such diseases.",
"Neurodegenerative disorders are generally classified as heritable or spontaneous in origin.",
"Parkinsons disease (PD) and Alzheimer's disease (AD), appear to be both heritable and spontaneous diseases.",
"Disorders such as spinocerebellar ataxia 1 and 3 are heritable diseases.",
"Many of these disorders are characterized by proteinaceous cellular inclusions (either inside or outside cells) encoded by genes whose expression is implicated in disease.",
"In all cases, inappropriate accumulation of such proteinaceous cellular inclusions is associated with the progression of the disease and, in some cases, is causally linked to disease etiology.",
"During viral infection and replication in brain tissue, harmful viral proteins accumulate and cellular destruction occurs.",
"In HIV infection, CD4+ T cells are targeted for destruction.",
"During viral encephalitis, neuronal cells are destroyed.",
"Inhibition of viral replication can effectively prevent this type of neuronal cell damage.",
"Given that the aberrant, toxic accumulation of a cellular protein(s) to toxic levels is a theme common to diseases mentioned above and many other disorders, therapeutic treatments designed to reduce levels of such toxic proteins have general utility in the treatment of patients suffering from neurodegenerative disorders.",
"The present example provides compositions and methods suitable for reducing the levels of such toxic proteins utilizing RNAi generated from inheritable inverted repeat (IR) gene constructs.",
"Such compositions can be employed as a single agent or can be utilized in combination with other therapies.",
"Such therapeutic approaches should result in an amelioration of symptoms associated with the disease and potentially reverse the course of the disease by eliminating the causative agent.",
"Moreover, the compositions and methods of the present invention may be used to advantage prophylactically to delay or prevent the onset of a disorder.",
"This application has particular utility for preventing or delaying the onset of disease in patients with a known genetic predisposition for a specific heritable neurodegenerative disorder.",
"In the present example, the usefulness of the compositions and methods of the invention for the treatment of Alzheimer's disease (AD) and Parkinson's disease is demonstrated.",
"AD is a spontaneous neurodegenerative disorder which is caused by cell death in the brain and is characterized by the deposition of amyloid plaques.",
"A major component of these plaques is β-amyloid, which is a cleavage product of the amyloid precursor protein (APP).",
"Brain cell death is associated with an increase in a 42 amino acid fragment of β-amyloid, called Aβ, which is generated by an aberrant processing event of APP.",
"A critical goal in AD therapy is the development of therapeutic agents which effectively reduce the production of this fragment.",
"To achieve this goal, an IR construct containing a fragment of DNA encoding Aβ can be placed under the control of a brain specific promoter.",
"Following administration to a patient, which can be achieved by specific delivery to the brain or systemic introduction (see above), expression of the Aβ inverted repeat double stranded RNA molecule should dramatically reduce production of this neurotoxic Aβ fragment.",
"This, in turn, should result in a dramatic reduction in plaque formation and delay or prevent disease onset.",
"An AS-IR expression construct can be generated as follows.",
"A 1 kb fragment of Aβ nucleic acid is obtained.",
"The GenBank Accession number for the genomic sequence of Aβ is D87675.The Genbank accession number for the cDNA is Y00264.The sequence can be amplified using Aβ specific primers that incorporate unique restriction sites at the IR fragment 5′ and 3′ ends and another restriction site to generate the inverted repeat which is ultimately situated at the inversion point (IP).",
"See FIG.",
"4.The 5′ and 3′ terminal restriction sites (designated as end A and B) can be used to insert the Aβ inverted repeat into an expression vector.",
"Two Aβ products can then be generated in parallel by PCR amplification of human cDNA using (1)primers A and IP and (2) primers B and IP, respectively.",
"Amplified Aβ fragments A-IP and B-IP can be digested with the appropriate restriction enzyme located at the IP restriction site (IPRS) to generate compatible termini which could then be ligated to produce an Aβ-IR.",
"Digestion of the Aβ-IR at the 5′ and 3′ restriction sites A and B facilitates ligation into an expression vector that has been linearized with restriction enzymes to generate compatible sites.",
"The New England Biolabs catalog provides a wide variety of restriction enzymes and the corresponding restriction sites which can be utilized in the construction of the IR repeat constructs of the invention.",
"In an alternative cloning strategy, two naturally occurring restriction sites found within DNA encoding the Aβ fragment could be used to excise an Aβ fragment.",
"In a two-step ligation reaction, this Aβ DNA fragment could be ligated end-to-end to generate an inverted repeat, and consequently ligated into an appropriately linearized vector as described above.",
"In this cloning strategy it is necessary to treat the linearized expression vector with calf intestine alkaline phosphatase.",
"In yet another approach each of the desired sequence elements for the IR construct may be synthesized separately, blunt ended and then ligated using DNA ligase.",
"A variety of vectors are available for expressing exogenous nucleic acids in human cells.",
"The plasmid vector pCEP4 (Invitrogen), for example, is comprised of components that facilitate selection and expression in human cells.",
"pCEP4 contains the following elements: a CMV promoter, a TKpA—thymidine kinase polyadenylation signal, a Hygromycin resistance gene, a ColE1 origin, an Ampicillin resistance gene, an Epstein Barr Virus Nuclear Antigen (EBNA-1) and an EBV origin (OriP EBV) for episomal replication in EBV transformed cell.",
"In another example, the plasmid clone pCR3 (Invitrogen) could also be used to drive high level IR gene expression in mammalian cells.",
"This plasmid vector includes a CMV promoter—Cytomegalovirus immediate—early promoter for high-level expression of the cloned IR gene; BGHpA—Bovine growth hormone polyadenylation signal for mRNA stability; ColE1—origin for replication, maintenance, and high copy number in E. coli; TKPA—thymidine kinase polyadenylation signal; Neomycin—neomycin resistance gene for selection of stable mammalian cell lines; PSV40/ori—origin for episomal replication in cells containing the SV40 large T antigen; Ampicillin—resistance gene for selection and maintenance in E. coli; F1 ori-origin for rescue of sense strand for mutagenesis and single strand sequencing.",
"In another example, the CMV-Script-Ex vector (Stratagene) could also be used to drive high level IR gene expression in mammalian cells.",
"Several vectors suitable for use in the present invention are set forth in Table II.",
"Using Alzheimer's disease as an exemplary disease model, and the β-amyloid encoding nucleic acid as the target for inhibition, the selected nucleic acid sequence corresponds to approximately 1000 contiguous nucleotides from the sequences set forth in the Genbank Accession Nos.",
"provided above, operably linked in a sense and antisense orientation.",
"An exemplary expression construct for use in inhibiting the expression of beta-amyloid protein is shown in FIG.",
"4.As mentioned previously, the compositions and methods of the invention also have utility in the treatment and prevention of Parkinson's disease.",
"For example, alpha-synuclein has been implicated in the pathology of familial Parkinson's disease.",
"The nucleic acid sequence encoding the alpha-synuclein protein is known.",
"See Genbank Accession No.",
"D31839.Accordingly, an IR construct can be generated in accordance with the present invention to reduce alpha synuclein accumulation in the affected patient.",
"An appropriate expression vector for this purpose is shown in FIG.",
"5, which contains 1081 nucleotides from GenBank Accession No.",
"D31839 operably linked in a sense and antisense orientation.",
"Methods for delivering the alpha-synuclein targeted IR constructs of the invention to the dopaminergic neurons of the substania nigra (the neurons affected in the disease) are set forth hereinabove.",
"EXAMPLE III Use of IR Gene Construct Expression Vectors for Controlling Plant Disease Geminiviruses are plant pathogens that infect a wide range of vegetable crops in tropical and subtropical regions with devastating consequences (Brown et al.",
"(1992) Plant Disease, 76:220-225).",
"Major epidemics of geminivirus infections of beans and tomatoes have occurred recently on several continents, thereby threatening the livelihood of farmers producing these crops and causing shortages which adversely impact the consumer population.",
"The intransigent nature of the problem is underscored by a failure to generate cultivars that are resistant to geminiviruses by traditional breeding methods.",
"The compositions and methods of the invention are suitable for generating geminivirus resistant strains of a variety of vegetable crops that are susceptible to this family of viruses.",
"In brief, the heritable system of RNAi described herein facilitates the generation of transgenic plant strains that produce dsRNA molecules which inhibit the expression of viral genes critical for productive infection.",
"Transgenic plant strains can be engineered to express the inhibitory RNA molecule under the control of either a constitutive or an inducible promoter.",
"In one embodiment of the invention, an IR construct containing a fragment of DNA encoding an essential geminivirus protein may be placed under the control of a constitutive promoter that functions in plant cells.",
"Viral genes encoding suitable target proteins for such inhibition include, but are not limited to, those essential for viral replication and capsid assembly.",
"Plant cells transformed with such an IR gene construct expression vector should be resistant to viral infection.",
"Progeny of plant cells containing stably integrated IR gene construct expression vectors inherit resistance to geminivirus infection.",
"In a particularly preferred embodiment of the invention, the transformed plant cell is a seed from which resistant plant stock can be derived.",
"In the present example, an IR gene construct expression vector is described which can confer disease resistance to the tomato yellow leaf curl (TYLC) geminivirus.",
"The TYLC virus infects the cultivated tomato (Lycopersicon esculentum), with devastating consequences.",
"The virus which has a single monopartite genome, is a subgroup III type of geminivirus, transmitted by the whitefly.",
"Navot, N. et al., (1991) Virology, 151-161.Notably, a whitefly-transmitted TYLC-like geminivirus having a bipartite genome has also been cloned.",
"See Rochester, D. E., et al., (1990) Virology, 520-526.The TYLC viral genome comprises six open reading frames.",
"Two open reading frames, V1 and V2, are located on the virion or plus strand, whereas the four remaining open reading frames, C1, C2, C3, and C4 are located on the complementary or minus strand.",
"The C2 open reading frame displays partial overlap with the C1 and C3 open reading frames.",
"The C1 open reading frame, which is sometimes referred to as AC1, includes the C4 open reading frame.",
"Partial or complete sequence data are available for several TYLC viral isolates.",
"The entire genome of an Israeli isolate of TYLC virus, for example, has been cloned and sequenced.",
"Navot, N. et al., (1991) Virology, 151-161.Sequence data are also available for TYLC viral isolates from Sardinia, Australia, Thailand, Egypt, and Sicily.",
"Kheyr-Pour, A., et al., (1991) Nucl.",
"Acids Res., 19:6763-6769.Dry et al.",
"(1993) J Gen. Virol.74:147-151.Padidam et al.",
"(1995) J. Gen. Virol.",
"76:249-263.The entire genome sequence of the Tomato yellow leaf curl virus can be found at GenBank Accession No.",
"NC—001996.In an exemplary IR gene construct expression vector, the geminivirus Nia-protease (NP) gene, which is required for productive infection is targeted for gene silencing.",
"A fragment of DNA which encodes a portion of the NP can be generated from either PCR of available TYLC isolates or by RT-PCR of RNA derived from infected plant cells amplified using suitable forward and reverse primers.",
"Suitable primer sequences corresponding to NP (of approximately 20-25 bases) can be selected from sequence information available in a variety of data bases.",
"The NP specific primers are designed to incorporate unique restriction sites at the IR fragment 5′ and 3′ ends and another unique restriction site to generate the inverted repeat which is ultimately situated at the inversion point (IP) (See figure).",
"The 5′ and 3′ terminal restriction sites (designated as end A and B) can be used to insert the NP inverted repeat into a plant expression vector of choice.",
"Specifically, two NP products would be generated in parallel by PCR amplification of TYLC genomic DNA or cDNA using (1) primers A and IP and (2) primers B and IP, respectively.",
"Amplified NP fragments A-IP and B-IP would be digested with the appropriate restriction enzyme located at the IP restriction site (IPRS) to generate compatible termini which could then be ligated to produce an NP-IR.",
"Digestion of the NP-IR at the 5′ and 3′ restriction sites A and B facilitates ligation into a plant expression vector that has been linearized with restriction enzymes to generate compatible sites.",
"In an alternative cloning strategy, two naturally occurring restriction sites found within an NP gene fragment could be used to excise the fragment.",
"In a two-step ligation reaction, this NP fragment could be ligated end-to-end to generate an inverted repeat, and consequently ligated into an appropriately linearized expression vector as described above.",
"In this cloning strategy it is suggested to treat the linearized expression vector with calf intestine alkaline phosphatase to prevent the recircularization of vector without the incorporation of the desired NP-IR insert.",
"In yet another approach, each of the IR gene construct expression vector sequence elements may be cloned and isolated separately and then operably linked into an expression vector using DNA ligase.",
"As mentioned previously, the first and second coding regions of the IR gene construct may optionally include a spacer sequence between the first and second coding sequences to facilitate expression or stability of the clone.",
"A variety of plant expression vectors are available that would be of utility for the present invention.",
"In a preferred embodiment of the invention, the cauliflower mosaic virus derived expression vector CMV35S could be utilized to drive expression of the NP-IR in tomato cells (Gleave, 1992).",
"Once fully constructed, the NP-IR expression vector would then be transformed into a suitable bacterial strain such as, for example, E. coli strain DH5 or SURE.",
"Transformation into bacteria facilitates the generation of large quantities NP-IR expression vector, which can be used in the production of TYLC resistant tomato plant strains.",
"In one application of the present invention, tomato plants are transformed using Agrobacterium which requires a T-DNA producing binary vector (pBIN19 for example, Stanford et al., 1990).",
"In another application of the present invention, tomato plants can be transformed using particle bombardment, for which there are no requirements for specific DNA constructs.",
"Methods are available for the isolation of stable plant transformants.",
"In a preferred embodiment, the IR gene construct expression vector optionally includes a selectable marker gene.",
"Such markers include, but are not limited to, the NPTII gene which confers resistance to the antibiotic kanamycin or the BAR gene which confers resistance to the plant herbicide Bialophos.",
"Examples of tobacco vectors are: pART7 and pART 27 (Gleave, 1992 Plant Mol.",
"Biol.",
"20: 1203-1207).",
"A suitable rice vector is pVec4 (Wang et al., 1998 Acta Hortic.",
"461:401-405.).",
"References 1.Fire, A. et al.",
"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.",
"Nature 391, 806-811 (1998).",
"2.Montgomery, M. K., Xu, S., & Fire, A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 95, 15502-15507 (1998).",
"3.Timmons, L. & Fire, A.",
"Specific interference by ingested dsRNA.",
"Nature 395, 854 (1998).",
"4.Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression.",
"Trends Genet.",
"14, 255-258 (1998).",
"5.Tabara, H., Grishok, A.",
"& Mello, C. C. RNAi in C. elegans—Soaking in the genome sequence.",
"Science 282, 430-431 (1998).",
"6.Jones, D., Russnak, R. H., Kay, R. J.",
"& Candido, E. P. M. Structure, expression and evolution of a heat-shock gene locus in C. elegans that is flanked by repetitive elements.",
"J. Biol.",
"Chem.",
"261, 12006-12015 (1986).",
"7.Stringham, E. G., Dixon, D. K., Jones, D. & Candido, E. P. M. Temporal and spatial expression patterns of the small heat shock (hsp-16) genes in transgenic Caenorhabditis elegans.",
"Mol.",
"Biol.",
"Cell 3, 221-233 (1992).",
"8.Fire, A., Harrison, S. W., & Dixon, D. A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans.",
"Gene 93, 189-198 (1990).",
"9.Lu, X.",
"& Horvitz, H. R. lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48.Cell 95, 981-991 (1998).",
"10.Zhang.",
"Y., LeRoy, G., Seelig, H. P., Lane, W. S. & Reinberg, D. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.",
"Cell 95, 279-89 (1998).",
"11.Ryazanov, A. G. et al.",
"Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 94, 4884-4889 (1997).",
"12.Maduro, M. & Pilgrim, D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.",
"Genetics 141, 977-988 (1995).",
"13.Ren, X.",
"-C., Kim, S., Fox, E., Hedgecock, E. & Wadsworth, W. G. Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans.",
"J. Neurobiol.",
"39, 107-118 (1999).",
"14.Mitani S., Du, H., Hall, D., Driscoll, M. & Chalfie, M. Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans.",
"Development 119, 773-783 (1993).",
"15.Park, E. -C. & Horvitz, H. R. Mutations with dominant effects on the behavior and morphology of the nematode C. elegans.",
"Genetics 113, 821-852 (1986).",
"16.Dibb, N. J., Maruyama, I. N., Krause, M. & Karn, J. Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family.",
"J. Mol.",
"Biol.",
"205, 603-613 (1989).",
"17.Kennerdell, J. R. & Carthew, R. W. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.",
"Cell 95,1017-1026 (1998).",
"18.Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 95,14687-14692 (1998).",
"19.Voinnet, O., Vain, P., Angell, V. & Baulcombe, D. C. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA.",
"Cell 95, 177-187 (1998).",
"20.Sanchez Alvarado.",
"A.",
"& Newmark, P. A. Double-stranded RNA specifically disrupts gene expression during planarian regeneration.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 96, 5049-5054 (1999).",
"21.Brenner, S. The genetics of Caenorhabditis elegans.",
"Genetics 77, 71-94 (1974).",
"22.Mello, C. C. & Fire, A. DNA transformation.",
"in, Methods in Cell Biology.",
"Caenorhabditis elegans: Modern Biological Analysis of an Organism.",
"H. F. Epstein & D. C. Shakes (Eds).",
"(Academic Press, Inc. San Diego, 1995) pp.",
"451-482.23.Kramer, J. M., French, R. P., Park, E. -C. & Johnson, J. J.",
"The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.",
"Mol.",
"Cell.",
"Biol.",
"10, 2081-2089 (1990).",
"24.Tabara, H. et al.",
"The rde-1 gene, RNA interference, and transposon silencing in C. elegans.",
"Cell 99, 123-132 (1999).",
"While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments.",
"Various modifications may be made to the invention without departing from the scope and spirit thereof as set forth in the following claims."
]
] | Patent_10169287 |
[
[
"Image pickup device, and image pickup device assembling method",
"The invention integrally arranges a lens, an optical filter, a semiconductor imaging device, on-chip components, and a printed circuit board on a three-dimensional circuit board and contrives the lens attachment structure, bonding method, color of an adhesive, masking of the lens, etc.",
"in order to implement a structure that allows high performance, compact, lightweight and rigid design, and mass production, as well as a high-quality picture."
],
[
"1.Imaging apparatus comprising: a semiconductor imaging device on a surface of the imaging apparatus; and a three-dimensional circuit board above said semiconductor imaging device.",
"2.Imaging apparatus comprising: a three-dimensional circuit board having a leg and a cylindrical barrel provided on the leg; a semiconductor imaging device attached on back of said leg; and a lens, supported inside said barrel, to impinge a light onto said semiconductor imaging device.",
"3.Imaging apparatus according to claim 1 or 2, characterized in that said three-dimensional circuit board includes: a barrel of a bottomed cylinder shape supporting said lens; a leg connected to said barrel, inside of which is formed said wiring pattern; and an opening formed at the boundary between said barrel and the leg, that said optical filter is arranged above said opening of said three-dimensional circuit board, and that said semiconductor imaging device and said on-chip components are arranged below said opening.",
"4.Imaging apparatus according to claim 1 or 2, characterized in that said three-dimensional circuit board comprises a recessed shoulder where a wiring pattern is formed inside said leg and that on said recessed shoulder is arranged a printed circuit board with an LSI and on-chip components joined on one surface or both surfaces.",
"5.Imaging apparatus according to claim 3, characterized in that said three-dimensional circuit board has a wiring pattern for directly providing an electric connection between said three-dimensional circuit board and another printed circuit board on the bottom of said leg.",
"6.Imaging apparatus according to claim 5, characterized in that said three-dimensional circuit board has a plurality of adhesive injection grooves for injecting an adhesive on the upper area of the inner circumference of said barrel.",
"7.Imaging apparatus according to claim 6, characterized in that the inner circumference of said barrel of said three-dimensional circuit board is formed while tapering downward adjacent to said adhesive injection grooves.",
"8.Imaging apparatus according to claim 6 or 7, characterized in that said adhesive is black.",
"9.Imaging apparatus according to any one of the claims 6 through 8, characterized in that in the lower area of said barrel of said three-dimensional circuit board is provided an adhesive reservoir.",
"10.Imaging apparatus according to any one of the claims 1 through 9, characterized in that the sections other than the effective section of the lens on the front of the lens are masked.",
"11.Imaging apparatus according to any one of the claims 1 through 10, characterized in that the said optical filter is omitted.",
"12.A imaging apparatus assembling method characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to said barrel inside of which is formed a wiring pattern and an opening formed at the boundary between said barrel and the leg, said method comprises a step of joining a lens with the inner circumference of said barrel, a step of joining a semiconductor imaging device with said wiring pattern so as to block said opening from the back of said leg, and a step of joining on-chip components with said wiring pattern.",
"13.A imaging apparatus assembling method characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to said barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between said barrel and the leg, said method comprises a step of joining a lens with the inner circumference of said barrel, a step of joining a semiconductor imaging device with said wiring pattern so as to block said opening from the back of said leg, a step of joining on-chip components with said wiring pattern, and a step of joining with said recessed shoulder a printed circuit board with an LSI and on-chip components joined in advance on one surface or both surfaces.",
"14.A imaging apparatus assembling method characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to said barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between said barrel and the leg, said method comprises a step of joining an optical filter so as to block said opening from said barrel side, a step of joining a lens with the inner circumference of said barrel, a step of joining a semiconductor imaging device with said wiring pattern so as to block said opening from the back of said leg, and a step of joining on-chip components with said wiring pattern.",
"15.A imaging apparatus assembling method characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to said barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between said barrel and the leg, said method comprises a step of joining an optical filter so as to block said opening from said barrel side, a step of joining a lens with the inner circumference of said barrel, a step of joining a semiconductor imaging device with said wiring pattern so as to block said opening from the back of said leg, a step of joining on-chip components with said wiring pattern, and a step of joining with said recessed shoulder a printed circuit board with an LSI and on-chip components joined in advance on one surface or both surfaces.",
"16.A imaging apparatus assembling method according to any one of the claims 12 through 15 characterized in that said method comprises a step of joining an assembled three-dimensional circuit board to another printed circuit board via a wiring pattern formed on the bottom of its leg."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Conventionally, concerning imaging apparatus of this type, as the semiconductor imaging device to convert a picture shot by lenses to an electric signal has become smaller and high-performance, the camera has become compact and has been used in various applications thus enhancing the society's convenience.",
"Smaller cameras have widely used in the market of a camera as a picture input sensor.",
"The imaging apparatus that uses a conventional.",
"semiconductor device has combined components such as a lens, a semiconductor-imaging device and an LSI into case or a structure respectively.",
"The printed circuit board of the apparatus has a form of a plane on which components necessary for driving the semiconductor-imaging device are mounted.",
"However, in the method for assembling conventional cameras, reduction of the size of the camera is limited as long as the components are connected to each other even when the semiconductor-imaging device is downsized, that is, an expertise is required for assembling or an automatic machine cannot be used for the assembling procedure.",
"The present invention solves the above problems and provides imaging apparatus that allows further downsizing and assembling using an automatic machine and its assembling method."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a perspective view of imaging apparatus in Embodiment 1 of the invention.",
"FIG.",
"2 is a sectional view of imaging apparatus in Embodiment 1 of the invention.",
"FIG.",
"3 is a sectional view of imaging apparatus in Embodiment 2 of the invention.",
"FIG.",
"4 is a perspective view of the bottom of a three-dimensional circuit board in the embodiments of the invention.",
"FIG.",
"5 is a schematic view of an optical system in the embodiments of the invention.",
"FIG.",
"6 is a characteristic diagram showing the sensitivity characteristic of a semiconductor imaging device in the embodiments of the invention.",
"FIG.",
"7 is a schematic view of another optical system in the embodiments of the invention.",
"FIG.",
"8 is a perspective view of imaging apparatus showing the adhesive injecting method in the embodiments of the invention.",
"FIG.",
"9 is a sectional view of imaging apparatus showing the adhesive injecting method in the embodiments of the invention.",
"FIG.",
"10 is a sectional view of imaging apparatus showing the irregular reflection prevention method in the embodiments of the invention.",
"FIG.",
"11 is a partial sectional view of imaging apparatus showing the adhesive seeping prevention method in the embodiments of the invention.",
"FIG.",
"12 is a perspective view of imaging apparatus showing the lens mask in the embodiments of the invention.",
"FIG.",
"13 is front view of a lens equipped with a lens mask in the embodiments of the invention.",
"FIG.",
"14 is front view of a lens without a lens mask in the embodiments of the invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?",
"In the figures, a numeral 1 represents a three-dimensional circuit board, 1 A a leg, 1 B a barrel, 1 C an opening, 1 D a recessed shoulder, 2 a lens, 3 an optical filter, 4 a semiconductor imaging device, 5 bonding points (adhesive injection grooves), 6 a lens mask, 7 an adhesive reservoir, 8 on-chip components, 9 an adhesive, 10 a printed circuit board, 11 a an LSI, 11 b on-chip components, 12 a dropping pipet, 13 a printed circuit board, 14 solder, 15 a taper, 16 incident lights, 17 an optical axis, 18 a bottom of a three-dimensional circuit board, 19 a side of a three-dimensional circuit board, 22 a pattern, and 23 a video amplifier circuit."
],
[
"TECHNICAL FIELD The present invention relates to imaging apparatus such as a monitoring camera, a camera for medical use and a vehicle-mounted camera downsized using a semiconductor-imaging device.",
"BACKGROUND OF THE INVENTION Conventionally, concerning imaging apparatus of this type, as the semiconductor imaging device to convert a picture shot by lenses to an electric signal has become smaller and high-performance, the camera has become compact and has been used in various applications thus enhancing the society's convenience.",
"Smaller cameras have widely used in the market of a camera as a picture input sensor.",
"The imaging apparatus that uses a conventional.",
"semiconductor device has combined components such as a lens, a semiconductor-imaging device and an LSI into case or a structure respectively.",
"The printed circuit board of the apparatus has a form of a plane on which components necessary for driving the semiconductor-imaging device are mounted.",
"However, in the method for assembling conventional cameras, reduction of the size of the camera is limited as long as the components are connected to each other even when the semiconductor-imaging device is downsized, that is, an expertise is required for assembling or an automatic machine cannot be used for the assembling procedure.",
"The present invention solves the above problems and provides imaging apparatus that allows further downsizing and assembling using an automatic machine and its assembling method.",
"DISCLOSURE OF THE INVENTION Imaging apparatus of the invention has a semiconductor imaging device on the surface and comprises a three-dimensional printed circuit board above the semiconductor imaging device.",
"With this configuration, all the components necessary for the imaging apparatus can be integrated on a three-dimensional printed circuit board for downsizing.",
"Imaging apparatus of the invention comprises a three-dimensional circuit board having a leg and a cylindrical barrel provided on the leg, a semiconductor imaging device attached on the back of the leg and a lens, supported inside the barrel, to impinge a light onto the semiconductor imaging device.",
"With this configuration, dislocation on the optical axis between the semiconductor device and the lens is reduced.",
"Imaging apparatus of the invention is characterized in that the three-dimensional circuit board includes a barrel of a bottomed cylinder shape supporting the lens, a leg connected to the barrel, inside of which is formed the wiring pattern and an opening formed at the boundary between the barrel and the leg, that the optical filter is arranged above the opening of the three-dimensional circuit board, and that the semiconductor imaging device and the on-chip components are arranged below the opening.",
"With this configuration, it is possible to do without a supporting component or joint assembly dedicated to a lens, an optical filter, a semiconductor imaging device, an LSI, on-chip components, etc.",
"that constitute imaging apparatus and downsize the imaging apparatus by integrating all of such components on a three-dimensional circuit board.",
"It is also possible to reduce the length between the LSI that drives the semiconductor imaging device and peripheral components thereby preventing the distortion of a clock signal and unwanted radiation.",
"Imaging apparatus of the invention is characterized in that the three-dimensional circuit board comprises a recessed shoulder where a wiring pattern is formed inside of the leg and that on the recessed shoulder is arranged a printed circuit board with an LSI and on-chip components joined on one surface or both surfaces.",
"With this configuration, it is possible to add further features to the imaging apparatus.",
"Imaging apparatus of the invention is characterized in that the three-dimensional circuit board has a wiring pattern for directly providing an electric connection between the three-dimensional circuit board and another printed circuit board on the bottom of the leg.",
"With this configuration, it is possible to directly attach an assembled three-dimensional circuit board to the main printed circuit board of a portable telephone set, a PC or various sensors.",
"This implements a compact imaging system and a lightweight structure owing to elimination of a connector, etc.",
"Imaging apparatus of the invention is characterized in that the three-dimensional circuit board comprises a plurality of adhesive injection grooves for injecting an adhesive on the upper area of the inner circumference of the barrel.",
"With this configuration, by instilling an adhesive using a jug having the shape of a dropping pipet into the adhesive injection grooves, it is possible to uniformly permeate an adhesive onto the periphery of the lens in order to readily fix the lens on the three-dimensional circuit board.",
"Imaging apparatus of the invention is characterized in that the inner circumference of the barrel of the three-dimensional circuit board is formed while tapering downward adjacent to the adhesive injection grooves.",
"With this configuration, it is possible to uniformly permeate an adhesive onto the periphery of the lens on the inner circumference of the barrel to assure precise bonding.",
"It is also possible to provide a structure whereby a lens is easily inserted into a three-dimensional circuit board by using an automatic machine from above or when a three-dimensional circuit board is elevated from beneath to attach the three-dimensional circuit board to the lens in fixed position.",
"Imaging apparatus of the invention is characterized in that the adhesive is black.",
"With this configuration, it is possible to absorb with black color of the adhesive an irregular light inside the lens reaching the lens side thus preventing reflection of the light on the lens side.",
"It is thus possible to prevent a light in the direction of the optical axis caused by the poor surface accuracy of the lens or dust on the lens from generating optical interference inside the lens or from reaching the semiconductor imaging device thus degrading the quality of the picture obtained.",
"Imaging apparatus of the invention is characterized in that in the lower area of the barrel of the three-dimensional circuit board is provided an adhesive reservoir.",
"With this configuration, it is possible to prevent an adhesive from seeping through the bonding site of the lens to cause internal contamination when an adhesive is injected in excess or is less viscous.",
"Imaging apparatus of the invention is characterized in that the sections other than the effective section of the lens on the front of the lens are masked.",
"With this configuration, it is possible to prevent an incident light from a section other than the effective section of the lens thus preventing distortion of a picture or degrading of picture quality caused by irregular reflection.",
"Imaging apparatus of the invention is characterized in that the an optical filter is omitted from the foregoing configuration.",
"With this configuration, it is possible to provide imaging apparatus fit for a specific purpose of use, that is, imaging apparatus that is sensitive enough to detect in the nighttime the basic signal Y of a picture alone, or a sensor that can extract a video signal in the infrared region.",
"A imaging apparatus assembling method of the invention is characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to the barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between the barrel and the leg, the method comprises a step of joining a lens with the inner circumference of the barrel, a step of joining a semiconductor imaging device with the wiring pattern so as to block the opening from the back of the leg, and a step of joining on-chip components with the wiring pattern.",
"With this method, it is possible to separate the manufacturing process by performing bonding of components to be assembled from above the three-dimensional circuit board and by performing electric connection of components from beneath the three-dimensional circuit board by way of soldering, a conductive adhesive, or ultra sound joint, there by assuring efficient manufacturing that allows easy assembly using an automatic machine.",
"A imaging apparatus assembling method of the invention is characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to the barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between the barrel and the leg, the method comprises a step of joining a lens with the inner circumference of the barrel, a step of joining a semiconductor imaging device with the wiring pattern so as to block the opening from the back of the leg, a step of joining on-chip components with the wiring pattern, and a step of joining with the recessed shoulder a printed circuit board with an LSI and on-chip components joined in advance on one surface or both surfaces.",
"With this method, it is possible to separate the manufacturing process by performing bonding of components to be assembled from above the three-dimensional circuit board and by performing electric connection of components from beneath the three-dimensional circuit board by way of soldering, a conductive adhesive, or ultrasound joint, thereby assuring efficient manufacturing that allows easy assembly using an automatic machine.",
"A imaging apparatus assembling method of the invention is characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to the barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between the barrel and the leg, the method comprises a step of joining an optical filter so as to block the opening from the barrel side, a step of joining a lens with the inner circumference of the barrel, a step of joining a semiconductor imaging device with the wiring pattern so as to block the opening from the back of the leg, and a step of joining on-chip components with the wiring pattern.",
"With this method, it is possible to separate the manufacturing process by performing bonding of components to be assembled from above the three-dimensional circuit board and by performing electric connection of components from beneath the three-dimensional circuit board by way of soldering, a conductive adhesive, or ultrasound joint, thereby assuring efficient manufacturing that allows easy assembly using an automatic machine.",
"A imaging apparatus assembling method of the invention is characterized in that, with respect to a three-dimensional circuit board having a barrel of a bottomed cylinder shape, a leg connected to the barrel, inside of which is formed a wiring pattern and an opening formed at the boundary between the barrel and the leg, the method comprises a step of joining an optical filter so as to block the opening from the barrel side, a step of joining a lens with the inner circumference of the barrel, a step of joining a semiconductor imaging device with the wiring pattern so as to block the opening from the back of the leg, a step of joining on-chip components with the wiring pattern, and a step of joining with the recessed shoulder a printed circuit board with an LSI and on-chip components joined in advance on one surface or both surfaces.",
"With this method, it is possible to separate the manufacturing process by performing bonding of components to be assembled from above the three-dimensional circuit board and by performing electric connection of components from beneath the three-dimensional circuit board by way of soldering, a conductive adhesive, or ultrasound joint, thereby assuring efficient manufacturing that allows easy assembly using an automatic machine.",
"A imaging apparatus assembling method of the invention is characterized in that the method comprises a step of joining an assembled three-dimensional circuit board to another printed circuit board via a wiring pattern formed on the bottom of its leg.",
"With this method, it is possible to directly attach a three-dimensional circuit board mounting a lens, an optical filter, a semiconductor imaging device, an LSI, on-chip components, etc.",
"to the main printed circuit board of a portable telephone set, a PC or various sensors.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view of imaging apparatus in Embodiment 1 of the invention.",
"FIG.",
"2 is a sectional view of imaging apparatus in Embodiment 1 of the invention.",
"FIG.",
"3 is a sectional view of imaging apparatus in Embodiment 2 of the invention.",
"FIG.",
"4 is a perspective view of the bottom of a three-dimensional circuit board in the embodiments of the invention.",
"FIG.",
"5 is a schematic view of an optical system in the embodiments of the invention.",
"FIG.",
"6 is a characteristic diagram showing the sensitivity characteristic of a semiconductor imaging device in the embodiments of the invention.",
"FIG.",
"7 is a schematic view of another optical system in the embodiments of the invention.",
"FIG.",
"8 is a perspective view of imaging apparatus showing the adhesive injecting method in the embodiments of the invention.",
"FIG.",
"9 is a sectional view of imaging apparatus showing the adhesive injecting method in the embodiments of the invention.",
"FIG.",
"10 is a sectional view of imaging apparatus showing the irregular reflection prevention method in the embodiments of the invention.",
"FIG.",
"11 is a partial sectional view of imaging apparatus showing the adhesive seeping prevention method in the embodiments of the invention.",
"FIG.",
"12 is a perspective view of imaging apparatus showing the lens mask in the embodiments of the invention.",
"FIG.",
"13 is front view of a lens equipped with a lens mask in the embodiments of the invention.",
"FIG.",
"14 is front view of a lens without a lens mask in the embodiments of the invention.",
"In the figures, a numeral 1 represents a three-dimensional circuit board, 1A a leg, 1B a barrel, 1C an opening, 1D a recessed shoulder, 2 a lens, 3 an optical filter, 4 a semiconductor imaging device, 5 bonding points (adhesive injection grooves), 6 a lens mask, 7 an adhesive reservoir, 8 on-chip components, 9 an adhesive, 10 a printed circuit board, 11a an LSI, 11b on-chip components, 12 a dropping pipet, 13 a printed circuit board, 14 solder, 15 a taper, 16 incident lights, 17 an optical axis, 18 a bottom of a three-dimensional circuit board, 19 a side of a three-dimensional circuit board, 22 a pattern, and 23 a video amplifier circuit.",
"BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the invention will be described referring to the drawings.",
"(Embodiment 1) FIG.",
"1 is a perspective view showing the first embodiment of imaging apparatus according to the invention.",
"FIG.",
"2 is a sectional view thereof.",
"In FIGS.",
"1 and 2, a three-dimensional circuit board 1 is composed of a leg 1A having rectangular frustum shape and a barrel 1B thereon having the shape of a bottomed cylinder, and an opening 1C is formed at the boundary between the leg 1A and the barrel 1B and a wiring pattern 22 is formed on the back side of the leg 1A.",
"Onto the inner circumference of the barrel 1B of the three-dimensional circuit board 1 is fit a lens 2, about whose optical axis 17 is arranged an optical filter 3 above the opening 1C, below which are arranged a semiconductor imaging device 4 and on-chip components 8.All of these components are completed when attached to a three-dimensional circuit board 1.Beneath the three-dimensional circuit board is arranged a printed circuit board 13 as a main circuit board of a portable telephone set, a PC or various sensors to which the three-dimensional circuit board 1 is attached.",
"Next, the assembly sequence of imaging apparatus in this embodiment will be briefly described.",
"An optical filter 3 is fixed to the three-dimensional circuit board 1 from above, a lens 2 equipped with a lens mask 6 in advance on the surface is attached, then the lens 2 is fixed by injecting an adhesive from bonding points (adhesive injection grooves) 5 formed on the inner periphery of the barrel 1.An excess volume of the injected adhesive is stored in the adhesive reservoir 7 below the injection point 5.Next, from beneath the three-dimensional circuit board 1, a semiconductor imaging device 4, an LSI or on-chip components 8 such as a resistor and a capacitor are electrically connected to a pattern 22 by way of soldering 14, a conductive adhesive, or ultrasound joint, etc.",
"and a leg 1A is electrically connected to the pattern of a printed circuit board 13 via the pattern 22 by way of soldering, a conductive adhesive, or ultrasound joint, etc.",
"In this way, according to Embodiment 1 of the invention, it is possible to provide a compact, lightweight and rigid structure by arranging the lens 2 on the three-dimensional circuit board 1 and arranging the optical filter 3 and the semiconductor imaging device 4 on the optical axis 17 of the lens 2, and by integrally fixing the on-chip components 8 to the pattern 22 of the three-dimensional printed circuit board 1.This approach allows assembly by an automatic machine and is fit for mass production.",
"Further, by mounting the on-chip components 8 on the three-dimensional printed circuit board 1 to perform driving of the semiconductor imaging device 4 and de-coupling of circuits, it is possible to do without a lead wire of a length corresponding to wiring to the drive circuit for the semiconductor imaging device 4 and components mounted on the circuit board thus providing a simple structure, thereby reducing a round waveform of a clock signal flowing in the lead wire as well as reducing unwanted radiation byway of de-coupling of circuits.",
"(Embodiment 2) FIG.",
"3 is a sectional view of imaging apparatus in Embodiment 2 of the invention.",
"For simplicity, similar members to those in Embodiment 1 are assigned the similar signs.",
"In FIG.",
"3, a three-dimensional printed circuit board 1 is composed of a leg 1A having a rectangular frustum shape and a barrel 1B thereon having the shape of a bottomed cylinder, and an opening 1C is formed at the boundary between the leg 1A and the barrel 1B and a recessed shoulder 1D is formed inside the leg 1A.",
"On the back-side of the leg 1A including the recessed shoulder 1D is formed a wiring pattern 22.Onto the inner circumference of the barrel 1B of the three-dimensional circuit board 1 is fit a lens 2, about whose optical axis 17 is arranged an optical filter above the opening 1C, below which are arranged a semiconductor imaging device 4 and on-chip components 8.On the recessed shoulder 1D beneath is arranged a printed circuit board 10 mounting an LSI 11a and on-chip components 11b on one surface or both surfaces, and finally a printed circuit board of a portable telephone set, etc.",
"is arranged.",
"Next, the assembly sequence of imaging apparatus Embodiment 2 will be briefly described.",
"An optical filter 3 is fixed to the three-dimensional circuit board 1 from above, a lens 2 equipped with a lens mask 6 in advance on the surface is attached, then the lens 2 is fixed by injecting an adhesive from bonding points 5 formed on the inner periphery of the barrel 1B.",
"An excess volume of the injected adhesive is stored in the adhesive reservoir 7 below the injection point 5.Next, from beneath the three-dimensional circuit board 1, a semiconductor imaging device 4, an LSI or on-chip components 8 such as a resistor and a capacitor are electrically connected to a pattern 22 by way of soldering 14, a conductive adhesive, or ultrasound joint, etc., a printed circuit board 10 is electrically connected to the pattern 22 of the recessed shoulder 1D by way of soldering 14, and finally a leg 1A is electrically connected to the pattern of a printed circuit board 13 via the pattern 22 by way of soldering, a conductive adhesive, or ultrasound joint, etc.",
"In this way, according to Embodiment 2 of the invention, it is possible to add further features to the imaging apparatus on top of the advantages of Embodiment 1 by arranging the lens 2 on the three-dimensional circuit board 1 and arranging the optical filter 3 and the semiconductor imaging device 4 on the optical axis 17 of the lens 2, and by integrally fixing the printed circuit board 10 mounting the LSI 11a and on-chip components 8 to the pattern 22 of the three-dimensional circuit board 1.Next, description common to the aforementioned Embodiment 1 and Embodiment 2 will be given.",
"FIG.",
"4 shows the bottom of the three-dimensional circuit board 1.In order to electrically connect imaging apparatus where components are attached by way of joint and electrical connection from above and from beneath the three-dimensional circuit board 1 to the printed circuit board 13 for applications such as a portable telephone set, a PC, etc., the pattern 22 where the semiconductor imaging device 4, the printed circuit board 10 are electrically connected is formed by way of printing or bonding along a bottom 18 of the three-dimensional circuit board 1 and so as to reach a side 19 of the three-dimensional circuit board.",
"Via the pattern 22 the bottom 18 of the three-dimensional circuit board is arranged on the printed circuit board 13 for the application and electrically joined with the printed circuit board 13 by way of soldering 14, a conductive adhesive, or ultrasound joint, etc., and a video signal converted to an electric signal by the semiconductor imaging device is output to the main body of the application.",
"FIG.",
"5 shows an optical system in the aforementioned embodiments.",
"Incident lights 16 are condensed d by a lens 2, passes through an optical filter 3 that suppresses the sensitivity in the infrared spectrum, and impinges on the semiconductor imaging device 4.A CCD as a representative semiconductor imaging device 4 shows a flat sensitivity characteristic in the visible light region as the characteristic B obtained by passing through the optical filter 3, amplified by a video amplifier circuit 23 and corresponds to a color picture as a video signal of a flat characteristic.",
"In the CCD sensitivity characteristic shown in FIG.",
"6, the characteristic A without the optical filter 3 shows an extremely high sensitivity in the infrared region so that the imaging apparatus can be used for imaging pictures in a dark place or as an infrared sensor by utilizing the sensitivity in the infrared region when imaging an object in a dark place.",
"Thus, as shown in FIG.",
"7, By removing the filter 3 for suppressing the sensitivity in the infrared region and configuring the imaging apparatus by the lens 2 and the semiconductor imaging device 4, it is possible to use the imaging apparatus as high-sensitivity imaging apparatus or an infrared sensor thus enhancing the application range of the imaging apparatus.",
"FIG.",
"8 shows a method for fixing by bonding a lens 2 with a three-dimensional circuit board 1 in the embodiments.",
"The structure shown comprises four bonding points (adhesive injection grooves) 5 inside the location where the lens 2 of the three-dimensional circuit board 1 is inserted thus facilitating the bonding work.",
"The number of bonding points 5 is increased/decreased depending on the viscosity of the adhesive 9.When the adhesive 9 is less viscous, the number of bonding points 5 is decreased and the adhesive 9 is injected in the form of instillation 9a, which forms adhesive spread 9b to bond the lens 2 with the three-dimensional circuit board 1.When the adhesive 9 is more viscous, an appropriate volume of the adhesive 9 is injected in the form of instillation 9a from a plurality of bonding points 5.This structure is fit for effectively performing the instillation 9a from the dropping pipet 12 when imaging apparatus is successively produced using an automatic machine.",
"FIG.",
"9 shows a structure where the inner periphery of the three-dimensional circuit board 1 is a taper 15 so that the adhesive 9 may permeate into the boundary between the lens 2 and the three-dimensional circuit board 1 when the lens 2 is bonded with the three-dimensional circuit board 1.By doing this, the instillation 9a injected from bonding points 5 in successive production of imaging apparatus flows along the taper 15 to form the adhesive spread 9b and is applied uniformly in the peripheral of the lens 2.The taper 15 can be used to give a margin to the insertion of an automatic machine when the lens 2 is attached in place from above, in successive production of imaging apparatus.",
"In other words, it is possible to insert the lens 2 from above with good margin while following the taper.",
"FIG.",
"10 shows a structure where the adverse effects on a picture caused by irregular reflection are minimized.",
"When incident lights 16 are impinged and condensed by the lens 2 and focused on the semiconductor imaging device 4, an irregularly reflected light 16a caused by dust on the surface of the lens 2 or irregularity of lights inside the lens 2 impinges on a lens side 2a and reflects thereon, which may interfere with the incident lights 16 or reach the semiconductor imaging device 4 thus causing distortion or disturbances in a picture.",
"In order to prevent this, a dark (black) colored adhesive 9 is used for bonding on the outer surface of the lens 2 thus suppressing reflection of light on the lens side 2a.",
"This allows an optical signal to suppress reflection of irregularly reflected light 16a to be output from the lens side 2a to the semiconductor imaging device 4.FIG.",
"11 shows a structure that prevents seeping of an excess volume of the adhesive 9 through the inside of the three-dimensional circuit board.",
"In case the volume of the instillation 9a is excessive when the lens 2 is bonded with the three-dimensional circuit board 1 with an adhesive, the adhesive 9a drops inside the three-dimensional circuit board 1 thus forming seeping 9c from the lens contact surface la thus contaminating the inside.",
"In order to prevent this, an adhesive reservoir 7 is provided in the lower area of the inner periphery of the barrel of the three-dimensional circuit board 1.FIG.",
"12 is a structure where the adverse effects on a picture caused by lights impinging on the sections other than the effective lens section are minimized.",
"The lens 2 is more lightweight than a glass lens and is often manufactured by way of resin molding in order to adjust the refractive index as an aspheric lens.",
"In the case of a resin-molded lens, as shown in FIG.",
"13, when incident lights 21a, 21b impinge on a plane 2b separate from the effective lens section, the lights that impinged enter the lens due to irregular reflection on the lens surface, such as an irregularly reflected light 21c, thus interfering with the incident lights 21 or reaching the semiconductor imaging device 4, which may distort the picture or degrade the picture quality otherwise.",
"In order to prevent this, as shown in FIG.",
"14, the structure is equipped with a lens mask 6 by way of a black system coating or metal material onto the plane 2b, so that lights may not impinge from the plane 2b separate from the effective lens section 2a of the lens 2.In this way, it is possible to commercialize imaging apparatus at a high level by way of particular assembly of the lens 2, optical filter 3, semiconductor imaging device 4, printed circuit boards 10, 13 with the three-dimensional circuit board 1.It is also possible to provide a structure where electric connection with a printer circuit board for an application using direct connection without connectors, etc.",
"Another structure is possible where the printed wiring pattern 22 provided on the bottom and sides of the three-dimensional circuit board 1 is used to insert the three-dimensional circuit board into connectors provided on a printer circuit board for an application thus providing electric connection.",
"<Industrial Applicability> As described hereinbefore, the invention has a structure where a three-dimensional circuit board is provided having a wiring pattern for connecting a semiconductor imaging device and on-chip components, and a lens, as well as an optical filter and the semiconductor imaging device arranged on the optical axis of the lens are integrally incorporated on the three-dimensional circuit board.",
"It is possible to provide a compact, lightweight and rigid imaging apparatus where all the components necessary for imaging apparatus are integrated on the three-dimensional circuit board, as well as implement an assembling method that allows automatic mounting of components and excellent in terms of mass production."
]
] | Patent_10169359 |
[
[
"Vessel provided with a sealing ring",
"A vessel for containing a fluid is provided.",
"A composite wall encloses the fluid chamber and is connected, at at least one location, to a shaft-like body which traverses the fluid chamber and extends through the composite wall.",
"A sealing ring is further provided to which the composite wall is connected at the connection location(s) and is slidable around the shaft-like body, subject to being limited in at least one axial direction by a stop means."
],
[
"1.A vessel for containing fluid, comprising: a composite wall enclosing a fluid chamber; a shaft-like body which traverses the fluid chamber and extends through the composite wall, the composite wall being, at at least one connecting location, connected to the shaft-like body; the composite wall comprising a fluid-tight inner lining around which fibers are provided; a ring to which the composite wall is connected at the at least one connecting location to the shaft-like body, the ring being designed as a sealing ring and arranged in an axially slidable and sealing manner around the shaft-like body; and stop means for limiting in at least one axial direction the distance over which the sealing ring can be slid relative to the shaft-like body.",
"2.A vessel according to claim 1, wherein the stop means comprises: a first press-on surface being arranged on the sealing ring; and a second press-on surface being arranged on the shaft-like body; the first and second press-on surfaces being positioned such that through axial sliding of the sealing ring along the shaft-like body, the first and second press-on surfaces can be moved towards each other, thereby clamping the fibers of the composite wall therebetween.",
"3.A vessel according to claim 1, wherein: the fibers are wound around the inner lining as one or more tension-loadable cords; and the shaft-like body traversing the chamber comprises a tension body reaching through the composite wall at two locations situated opposite each other.",
"4.A vessel according to claim 2, wherein the second press-on surface extends at least partly along a radially outwardly extending flange part of the shaft-like body.",
"5.A vessel according to claim 1, wherein at least a part of the connecting locations the fibers and the inner lining of the composite wall are separately connected with the sealing ring.",
"6.A vessel according to claim 1, wherein the sealing ring comprises a curved, throat-shaped contact surface along which a correspondingly curved part of the inner lining slidably abuts.",
"7.A vessel according to claim 6, wherein the inner lining cooperates bondlessly with the contact surface.",
"8.A vessel according to claim 1, wherein: the sealing ring comprises a cylindrical channel in which a cylindrical part of the shaft-like body is slidably received; and the cylindrical channel comprises at least one groove in which an O-ring is received for sealing in a gas-tight manner the intermediate space between the channel and the cylindrical part of the shaft-like body.",
"9.A vessel according to claim 2, wherein the sealing ring comprises a curved, throat-shaped contact surface along which a correspondingly curved part of the inner lining slidably abuts.",
"10.A vessel according to claim 2, wherein: the sealing ring comprises a cylindrical channel in which a cylindrical part of the shaft-like body is slidably received; and the cylindrical channel comprises at least one groove in which an O-ring is received for sealing in a gas-tight manner the intermediate space between the channel and the cylindrical part of the shaft-like body.",
"11.A vessel according to claim 3, wherein at at least a part of the connecting locations the fibers and the inner lining of the composite wall are separately connected with the sealing ring.",
"12.A method of manufacturing a vessel for containing fluid, comprising the steps of: providing a composite wall enclosing a fluid chamber with a fluid-tight inner lining; providing fibers around the fluid-tight inner lining; providing a shaft-like body which traverses the fluid chamber and extends through the composite wall; arranging a sealing ring in an axially slidable and sealing manner around the shaft-like body; connecting the composite wall at at least one connecting location to the shaft-like body with the ring; and providing stop means for limiting in at least one axial direction the distance over which the sealing ring can be slid relative to the shaft-like body.",
"13.The method according to claim 12, wherein the step of providing the stop means comprises the steps of: arranging a first press-on surface on the sealing ring; arranging a second press-on surface on the shaft-like body; positioning the first and second press-on surfaces such that through axial sliding of the sealing ring along the shaft-like body, the first and second press-on surfaces can be moved towards each other, thereby clamping the fibers of the composite wall therebetween.",
"14.The method according to claim 12, wherein the step of providing fibers comprises the step of winding the fibers around the inner lining as one or more tension-loadable cords.",
"15.The method according to claim 12, wherein the step of arranging the second press-on surface comprises the step of extending the second press-on surface at least partly along a radially outwardly extending flange part of the shaft-like body.",
"16.The method according to claim 12, wherein the step of connecting comprises separately connecting the fibers and the inner lining of the composite wall with the sealing ring at at least a part of the connecting locations.",
"17.The method according to claim 12, further comprising the steps of: providing a cylindrical channel in which a cylindrical part of the shaft-like body is slidably received in the sealing ring; and receiving in at least one groove in the cylindrical channel an O-ring for sealing in a gas-tight manner the intermediate space between the channel and the cylindrical part of the shaft-like body."
],
[
"The invention relates to a vessel, comprising a composite wall enclosing a fluid chamber and being, at least at one connecting location, connected to a shaft-like body traversing the fluid chamber and extending through the composite wall, which composite wall comprises a fluid-tight inner lining around which fibers are provided and which composite wall, at the at least one connecting location, is connected via a ring to the shaft-like body.",
"Such a type of vessel is known from practice and is often used for storing a gas or liquid supply.",
"The composite wall is often built up of a relatively flexible plastic inner lining around which fibers are provided in a relatively stiff support layer.",
"The advantage of this is, that the wall of the vessel, compared to a conventional steel wall, can be of a relatively light and low cost design, while having a comparable strength.",
"In the known vessel, at the connecting location, the composite wall is rigidly connected, via the ring, to the shaft-like body.",
"A drawback of the known vessel is that the sealing between the composite wall and the shaft-like body at the connecting location is often insufficiently reliable.",
"In particular, the chance exists that, upon impact or shock loading of the vessel, the composite wall breaks off or becomes damaged at the location of the connection to the ring.",
"In practice, therefore, it has been found to be a problem to connect the fibers of the support layer and the inner lining of the composite wall, which is relatively flexible in comparison to the fibers, to the shaft-like body such that the sealing is guaranteed, while the chance of damage to the support layer and/or the inner lining is small.",
"The object of the invention is a vessel of the type mentioned in the preamble, in which the above mentioned problems are avoided.",
"To that end, a vessel according to the invention is characterized in that the ring is designed as a sealing ring which is provided in an axially slidable and sealing manner around the shaft-like body and that stop means are provided for limiting in at least one axial direction the distance over which the sealing ring can be slid relative to the shaft-like body.",
"What is thereby achieved is that, while maintaining the sealing, an axial displacement of the fibers and/or the inner lining in relation to the shaft-like body is possible, so that tensions between the fibers and/or the inner lining and the shaft-like body due to displacement can be reduced.",
"By using stop means it is achieved that damage to the composite wall by too large a displacement can be prevented.",
"By designing the stop means as cooperating press-on surfaces which are provided at the location of a connecting location on the sealing ring and the shaft-like body, respectively, it is achieved that fibers of the composite wall situated between the press-on surfaces can be clamped when the press-on surfaces are moved towards each other, for instance under pressure of fluid in the fluid chamber.",
"This has as an advantage that possible play between the fibers during the pressing-on can be removed, so that a maximum number of enclosed fibers can be used for transmitting forces between the composite wall and the shaft-like body.",
"In a further embodiment, the fibers of the vessel are designed as tension-loadable cords, which are wound around the inner lining, and the shaft-like body which traverses the chamber comprises a tension body which extends through the composite wall at two connecting locations.",
"The fibers are then preferably wound dry, i.e.",
"without matrix material, around the inner lining, while, optionally, for protecting the fibers on the outside, a preferably elastomeric sealing layer can be provided.",
"With such a vessel, a fluid, for example LPG, can be stored under pressure.",
"Via the inner lining the fluid pressure can then be transmitted to the sealing ring so that, subsequently, for instance with the help of the above described press-on surfaces, intermediately situated fibers can be clamped between the sealing ring and the shaft-like body.",
"Especially in such a pressure vessel the operational safety and the transmission of forces of the connection between the composite wall and the shaft-like body are of particular importance.",
"It is noted that by dry-winding the fibers, it can be prevented that the composite wall becomes damaged by the fibers breaking loose from intermediately situated matrix material, for instance as a result of an impact or shock load to the vessel.",
"Furthermore, by dry-winding the fibers, the manufacture of the vessel can be carried out quicker, since no time for hardening of the matrix material needs to be taken into account.",
"In a further advantageous embodiment, at at least a part of the connecting locations, the fibers and the inner lining of the composite wall are separately connected to the sealing ring.",
"Thus, it is achieved that both the connection between the fibers and the sealing ring, and the connection between the inner lining and the sealing ring can be optimized for the function to be fulfilled by the connection, and that, for both connections, the nature of the materials to be connected can be taken into account.",
"For instance, the fibers can be rigidly clamped into a position in which the clamped part of the fibers smoothly aligns with the non-clamped part of the fibers in order to reduce the risk of wear and breakage of the fibers, while the connection between the inner lining and the sealing ring can, for instance, be slidable, so that, while maintaining the sealing action, displacement of the inner lining relative to the sealing ring is possible.",
"This is particularly advantageous when the inner lining, for instance during manufacture, shrinks or when the composite wall undergoes an impact or shock load.",
"Further advantageous embodiments are described in the subclaims.",
"It is noted that in this context, fluid should be understood to mean not only liquid or liquid solid matter, but also gas or vapor.",
"The invention will be further elucidated on the basis of an exemplary embodiment which is represented in the drawing.",
"In the drawing: FIG.",
"1 shows a schematic cross section of the vessel; FIG.",
"1A shows a detailed view of the connecting location of the vessel of FIG.",
"1; and FIG.",
"1B shows a cross section of one side of the sealing ring of FIG.",
"1A.",
"It is noted that the Figures are only schematic representations of an advantageous embodiment.",
"In the Figures, identical or corresponding parts are designated with the same reference numerals.",
"FIG.",
"1 shows a vessel 1.The vessel 1 comprises a composite wall 2 which encloses a fluid chamber 3.At two connecting locations 4 opposite each another, the composite wall 2 is connected to a shaft-like body 5 which traverses the fluid chamber 3.In the exemplary embodiment, the shaft-like body 5 is provided with a tension body 18 which, at the connecting locations, reaches through the composite wall 2, which is represented in detail in FIG.",
"1A.",
"Near its end parts, the tension body 18 is provided with flange parts 20, extending radially outwards.",
"Referring to FIG.",
"1A, the composite wall 2 comprises a fluid-tight inner lining 6 around which fibers 7 are provided in a support layer.",
"In this exemplary embodiment, the fibers 7 of the composite wall 2 are designed as tension-loadable cords 19 which are wound around the flexible, fluid-tight inner lining 6.The inner lining 6 is designed as a flexible core which, in relation to the layer of fibers 7, is relatively flexible, for instance a core of polyethylene, which, at least under its own weight load, retains its shape.",
"The tension-loadable cords 19 are designed as strands of fibers, for instance glass, carbon and/or polyamide fibers which are bundled to a strand in the longitudinal direction.",
"Preferably, one tension-loadable cord is wound around the inner lining 6 several times.",
"A vessel, the fibers of whose composite wall and a central shaft are tension-loadable, is known per se.",
"For a detailed description of such a vessel and its manner of manufacture, reference is therefore made to the published European patent application 0 879 381.At the connecting location 4, the composite wall 2 is connected to the shaft-like body 5 via a sealing ring 8 mounted around the shaft-like body 5 so as to be axially and freely slidable along the longitudinal axis A.",
"In an advantageous manner, the sealing ring 8 is provided with a cylindrical channel in which a cylindrical part of the shaft-like body 5 is received.",
"The cylindrical channel can comprise one or more grooves 14 in which an O-ring 15 is received.",
"Thus, it is achieved that in a simple manner a reliable, gas-tight sealing between the sealing ring 8 and the shaft-like body 5 can be realized.",
"It will be clear that the sealing can also be realized in a different manner, for instance by a spring ring or an interference fit.",
"The vessel 1 is provided with stop means for limiting, in relation to the fluid chamber 3, the distance in axially outward direction along the tension body 18, over which the sealing rings 8 can be slid along the longitudinal axis of the tension body 18.The stop means comprise first press-on surfaces 21 which are provided on the sealing rings 8, and second press-on surfaces 22 provided on the flange parts 20.The first and second press-on surfaces 21, 22 are positioned such that, by axially and, in relation to the fluid chamber 3, outwardly displacing the sealing rings 8 along the longitudinal axis A, along the tension body 18, the press-on surfaces 21, 22 are moved towards each other while clamping the intermediately situated cords 19.The press-on surfaces 21, 22 are provided with a curvature such that the fibers can be clamped into a position in which the clamped part of the fibers substantially smoothly aligns with the adjacent, non-clamped part of the fibers.",
"This is represented in detailed view FIG.",
"1A.",
"The cords 19 and the inner lining 6 are separately connected to the sealing ring 5.When the fluid chamber 3 is provided with a fluid under pressure, the inner lining 6, while taking with it the sealing rings 8 attached thereto, will be pressed outward.",
"The cords 19 are now tension-loaded and limit the outward displacement of the inner lining 6.The displacement of the sealing ring 8 is limited by cooperation of the first press-on surfaces 21 with the second press-on surfaces 22.In this manner, the cords 19 are clamped, free of play, in a position in which each of the clamped fibers can transmit force to the tension body 18.The sealing ring 8 comprises a curved, throat-shaped contact surface 25 along which a correspondingly curved part 26 abuts in a sliding manner.",
"By having the curved part 26 of the inner lining cooperate in a sliding manner with the throat-shaped contact surface 25, it is achieved that a good force transmission between the sealing ring 8 and the inner lining 6 is possible, while the inner lining 6, while maintaining the sealing action, can slide to some extent along the contact surface.",
"This is particularly important when the vessel is put under pressure by filling the fluid chamber 3 with fluid.",
"Referring to FIG.",
"1B, the cross section of the sealing ring 8 is represented in detail therein.",
"In the Figure, it can be seen that the first press-on surface 21 is provided with a curvature such that the cords 19, from the area G, where they separate from the inner lining 6, can align smoothly with the press-on surface 21.Near the area G, the contact surface 21 is provided with a rounding II, such that the chance of damage to the cords 19 and/or the inner lining 6 can be reduced.",
"The curved contact surface 25 is provided with a throat-shaped, concave curvature III, such that a middle part M thereof is situated closer to the longitudinal axis A of the shaft-like body 5 than are the adjacent side parts IVa, IVb.",
"Thus, it is achieved that forces between the inner lining 6 and the sealing ring 8 can be transmitted better in the direction of the longitudinal axis A.",
"Further, it is achieved that, with an inward deformation of the inner lining 6, i.e., towards the fluid chamber 3, it is rendered increasingly difficult for the inner lining to become detached from the contact surface 25 of the sealing ring 8.In this manner, it is achieved that the chance of damage to the inner lining 6 upon an inward movement of the composite wall 2 is small, while a good sealing remains ensured.",
"It is noted that this manner of sliding cooperation of the throat-shaped curved contact surface and the correspondingly curved part of the inner lining can be applied as such in an advantageous manner in vessels whose inner lining of the composite wall has to be fixedly connected to a body.",
"It will be clear that the invention is not limited to the exemplary embodiments described here, but that many variations are possible.",
"For example, other connections between the composite wall and the sealing ring are also possible, for instance glue connections.",
"Also, the stop means can be designed differently, for instance such that they limit axial displacement in two directions.",
"Also, the fibers of the composite wall can be relatively short and these fibers can be received with mutually crossing orientations in a matrix material.",
"Additionally, it is possible for the cords to consist of only one fiber.",
"Also, the inner lining can be designed from different material than plastic, for instance from metal film.",
"Further, the vessel can comprise only one connecting location, for instance in an embodiment of the vessel in which the shaft-like body is designed as a carrier traversing the fluid chamber and which supports the inner lining at a side opposite the connecting location.",
"Also, the vessel can comprise more than two connecting locations and the vessel can be provided with several tension bodies.",
"Such variants will be clear to the skilled person and are understood to fall within the scope of the invention as set forth in the following claims."
]
] | Patent_10169529 |
[
[
"Method for preparing hydroxypicolinic scid derivatives",
"The invention concerns a method for preparing compounds of general formula (I) wherein: n, Q1, Q2, X1, X2, Y and Z are as defined in the description."
],
[
"1.A process for preparing compounds of general formula (I): in which: n represents 0 or 1, Q1 is chosen from an oxygen or sulfur atom, a group NR1 and a group N—NR4R5, Q2 is chosen from a group OR2 or SR3 and a group —NR4R5, or Q1 and Q2 may together form a ring of 5 to 7 atoms containing 2 to 3 oxygen and/or nitrogen atoms, optionally substituted with one or more radicals, which may be identical or different, chosen from halogens and alkyl and haloalkyl radicals, Z is chosen from a hydrogen atom, a cyano radical and an alkyl, allyl, aryl, arylalkyl, propargyl, cycloalkyl, halocycloalkyl, alkenyl, alkynyl, cyanoalkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, alkoxycarbonylaminoalkyl, aminocarbonylaminoalkyl, alkoxycarbonyl, N-alkylaminocarbonyl, N,N-dialkylaminocarbonyl, acyl, thioacyl, alkoxythiocarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, alkoxysulfonyl, aminosulfonyl, N-alkylaminosulfonyl, N,N-dialkylaminosulfonyl, arylsulfinyl, arylsulfonyl, aryloxysulfonyl, N-arylaminosulfonyl, N,N-diarylaminosulfonyl or N,N-arylalkylaminosulfonyl radical; Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, a cycloalkyl, halocycloalkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, alkenylthio or alkynylthio group, an amino, N-alkylamino, N,N-dialkylamino, —NHCOR10, —NHCSR10, N-alkylaminocarbonylamino, N,N-dialkylaminocarbonylamino, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, thioacylamino, alkoxythiocarbonylamino, N-alkylaminothiocarbonylamino, N,N-dialkylaminothiocarbonylamino, N,N-arylalkylaminocarbonylamino, N-alkylsulfinylamino, N-alkylsulfonylamino, N-arylsulfinylamino, N-arylsulfonylamino, N-alkoxysulfonylamino, N-alkoxysulfinylamino, N-haloalkoxysulfinylamino, N-haloalkoxysulfonylamino, N-arylamino, N,N-diarylamino, arylcarbonylamino, alkoxycarbonylamino, N-arylaminocarbonylamino, N,N-diarylaminocarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino, N-arylaminothiocarbonylamino, N,N-diarylaminothiocarbonylamino or N,N-arylalkylaminothiocarbonylamino group, an acyl, carboxyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, lower alkoxycarbonyl, N-arylcarbamoyl, N,N-diarylcarbamoyl, aryloxycarbonyl or N,N-arylalkylcarbamoyl group, and an imino group of formula: X1 and X2 are identical or different and chosen, independently of each other, from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, and an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, or X1 and X2 may be joined together, thus forming a saturated, partially unsaturated or totally unsaturated 4- to 8-membered ring optionally comprising one or more hetero atoms chosen from sulfur, oxygen, nitrogen and phosphorus, R2 and R3 or different and chosen, independently of each other, from an alkyl radical comprising from 1 to 12 carbon atoms, a haloalkyly, cycloalkyl, halocycloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl or acyl radical, a nitro, cyano, carboxyl, carbamoyl or 3-oxetanyloxycarbonyl radical, and an N-alkylcarbamoyl, N,N-dialkylcarbamoyl, alkoxycarbonyl, alkylthiocarbonyl, haloalkoxycarbonyl, alkoxythiocarbonyl, haloalkoxythiocarbonyl, alkylthiothiocarbonyl, alkenyl, alkynyl, N-alkylamino, N,N-dialkylamino, N-alkylaminoalkyl or N,N-dialkylaminoalkyl radical, or a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, or R1, R4, R5, R6 and R7 are identical or different and chosen, independently of each other, from a hydrogen atom, an optionally substituted alkyl radical comprising from 1 to 12 carbon atoms in a linear or branched chain, a haloalkyl, cycloalkyl, halocycloalkyl, alkoxy, aryloxy, arylalkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl or acyl radical, a nitro, cyano, carboxyl, carbamoyl or 3-oxetanyloxycarbonyl radical, and an N-alkylcarbamoyl, N,N-dialkylcarbamoyl, alkoxycarbonyl, alkylthiocarbonyl, haloalkoxycarbonyl, alkoxythiocarbonyl, haloalkoxythiocarbonyl, alkylthiothiocarbonyl, alkenyl, alkynyl, N-alkylamino, N,N-dialkylamino, N-alkylaminoalkyl or N,N-dialkylaminoalkyl radical, or a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, or R4 and R5, on the one hand, or R6 and R7, on the other hand, may be joined together, thus forming a saturated, partially unsaturated or totally unsaturated 4- to 8-membered ring optionally comprising one or more hetero atoms chosen from sulfur, oxygen, nitrogen and phosphorus, T represents a direct bond or a divalent radical chosen from a radical —(CH2)m—, m taking a value between 1 and 12, limits included, the said radical optionally being interrupted or ending with one or two hetero atoms chosen from nitrogen, oxygen and/or sulfur, and an oxyalkylene, alkoxyalkylene, carbonyl (—CO—), oxycarbonyl (—O—CO—), carbonyloxy (—CO—O—), sulfinyl (—SO—), sulfonyl (—SO2—), oxysulfonyl (—O—SO2—), sulfonyloxy (—SO2—O—), oxysulfinyl (—O—SO—), sulfinyloxy (—SO—O—), thio (—S—), oxy (—O—), vinyl (—C═C—), ethynyl (—C≡C—), —NR9—, —NR9O—, —ONR9—, —N═N—, —NR9—NR10—, —NR9—S—, —NR9—SO—, —NR9—SO2—, —S—NR9—, —SO—NR9—, —SO2—NR9—, —CO—NR9—O— or —O—NR9—CO— radical, R8 is chosen from a hydrogen atom and an aryl or heterocyclyl radical, R9 and R10, which may be identical or different, are chosen, independently of each other, from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, and an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, arylalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, their salts and metal and metalloid complexes, a process characterized in that a compound of formula (II) in which X1 and X2 are as previously defined, is reacted with a cyanide, alkali metal derivatives or alkaline-earth metal derivatives of hydrocyanic acid in the presence of an alkylating agent and a solvent, or with trimethylsilyl cyanide in the presence of dimethylcarbamoyl chloride and a solvent, to give the compounds of formula (III).",
"in which X1 and X2 are as previously defined, the compounds of formula (III) above can be converted into corresponding halo derivatives of formula (IVa): in which X1 and X2 are as previously defined and X represents a halogen atom chosen from fluorine, chlorine, bromine and iodine, by reaction with an acyl halide in the presence of a solvent, the halo derivatives of formula (IVa) then being hydrolyzed to compounds of formula (Ia): in which X, X1 and X2 are as previously defined, by the action of hot hydracid—or of a strong mineral base, optionally in the presence of aqueous hydrogen peroxide solution—and optionally of boron tribromide, the compounds of formula (III) or (IVa) then possibly being placed in contact with an alcohol or an alkoxide in the presence of a solvent such as, preferably, but not exclusively, a protic or aprotic polar solvent, to give the compounds of formula (IVb): in which X1 and X2 are as previously defined, and then used in a hydrolysis reaction under operating conditions similar to those used for the formation of the compounds of formula (Ia), to give the compounds of respective formulae (Ib) and (Ib′) in which X1 and X2 are as previously defined, the compounds of formula (IVa) also possibly being converted into picolinic acid derivatives of formula (Va): in which X1, X2 and R6 are as previously defined, by reacting a compound of formula R6SH, or a corresponding alkali metal salt or alkaline-earth metal salt, in an aprotic polar solvent, at a temperature of between 0° C. and the boiling point of the solvent, the nitrites of formula (Va) then being used in a hydrolysis reaction to give the corresponding acids of formula (Ic): in which X1, X2 and R6 are as previously defined, according to a reaction similar to that used to form the compounds of formula (Ia), the halides of formula (IVa) also possibly being treated with an azothydric acid salt, to give the compounds of formula (Vb): in which X1 and X2 are as previously defined, this reaction being carried out at a temperature of between 0° C. and the boiling point of the solvent, the compounds of formula (Vb) can then be hydrolyzed according to techniques similar to those presented for the preparation of the acids of formula (Ia) above, to give the acids of formula (Id): in which X1 and X2 are as previously defined, the azides of formula (Id) then being optionally reduced to amine derivatives of formula (Ie): in which X1 and X2 are as previously defined, by the action of a reducing agent, the acids of formulae (Ia) to (Ie) possibly being converted into thio acids, imino derivatives (—C(═NR1)) or amino imino derivatives (—C(═N—NR4R5)) according to conventional techniques, that are well known to those skilled in the art of organic synthesis, the acids (Ia) to (Ie), or the thio, imino and imino-amino derivatives thereof defined above, substituted in position 3 (relative to the pyridine nitrogen atom) with —OH or -methoxy then possibly being subjected to various reactions already known in the prior art in order to give the corresponding derivatives substituted in position 3 (relative to the pyridine nitrogen atom) with —O-Z, Z being as defined for the compounds of formula (I), the compounds of formula (I) previously defined for which Y represents an amino radical (—NH2) possibly being placed in contact with an acylating agent in the presence of a solvent and optionally of a base, to give the compounds of formulae (VIa) and (VIb): in which X1, X2, Q1, Q2, Z and R10 are as previously defined, the picolinic acid derivatives of formula (VII): in which X1, X2, Q1, n, Z and Y are as previously defined, capable of being placed in contact with a reagent of formula R2OH, R3SH or HNR4R5, R2, R3, R4 and R5 being as previously defined, at a temperature between −80° C. and 180° C. (preferably between 0° C. and 150° C.) or at the boiling point of the solvent, to give the respective compounds of formulae (VIIIa), (VIIIb) and (VIIIc), which form the set of compounds of formula (VIII): which are special cases of the compounds of formula (I) in which Q2 represents —OR2, —SR3 and —NR4R5, respectively, the compounds of general formula (IX): which are special cases of the compounds of formula (I) for which n is equal to 1, can be obtained by a process consisting in placing a compound of formula (X): which is a special case of the compounds of formula (I) for which n is equal to zero, in contact with an oxidizing agent, aqueous hydrogen peroxide solution or carboxylic, boronic or sulfuric peracids.",
"2.The preparation process as claimed in claim 1, characterized in that the solvent used for the halogenation reaction of the compounds of formula (III) to the compounds of formula (IV) is chosen from diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane.",
"3.The process as claimed in either of the preceding claims, characterized in that the hydrolysis reaction of the compounds of formula (IV) into compounds of formula (Ia) consists in treating the nitrile of formula (IVa) with an acid, in particular hydrochloric acid, hydriodic acid or hydrobromic acid, or alkyl sulfonic acids, this hydrolysis reaction being performed in the excess acid, in the absence or presence of a solvent, at reflux or at a temperature of between 20° C. and 200° C. 4.The process as claimed-in claim 3, characterized in that the acid used is chosen from hydrochloric acid, hydriodic acid, hydrobromic acid and alkylsulfonic acids.",
"5.The process as claimed in any one of the preceding claims, characterized in that the solvent used in the reaction for conversion of the compounds of formula (IVa) into compounds of formula (Va) is chosen from dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylprolyleneurea and dimethyl sulfoxide.",
"6.The process as claimed in any one of the preceding claims, characterized in that the solvent used in the reaction for conversion of the compounds of formula (IVa) into compounds of formula (Vb) is chosen from dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylprolyleneurea and dimethyl sulfoxide.",
"7.The process as claimed in any one of the preceding claims, characterized in that the reducing agent used for the reduction of the compounds of formula (Id) to compounds of formula (Ie) is chosen from lithium aluminium hydride, triphenylphosphine and hydrogen in the presence of a catalyst.",
"8.The process as claimed in any one of the preceding claims, characterized in that the acylating agent used in the step for preparing the compounds of formulae (VIa) and (VIb) is chosen from an acyl halide, an anhydride, an acid, an ester and a primary amide, and thio homologues thereof.",
"9.The process as claimed in any one of the preceding claims, characterized in that the activating agent used for the formation of the compounds of formula (VIII) is chosen from thionyl chloride, oxalyl chloride, dicyclocarbodiimide, 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole and phosphorus oxychloride.",
"10.The process as claimed in any one of the preceding claims, characterized in that the solvent used in the reaction for obtaining the compounds of formula (VIII) is chosen from pentane, hexane, heptane, octane, benzene, toluene, xylenes, halobenzenes, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, dichloromethane, chloroform, 1,2-dichloroethane, 1,1,1-trichloroethane, methyl acetate, ethyl acetate, acetonitrile, propionitrile, benzonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylpropyleneurea, dimethyl sulfoxide, pyridine and water, mixtures of these various solvents also being able to be used.",
"11.The process as claimed in any one of the preceding claims, characterized in that the reaction time for obtaining the compounds of formula (VIII) is between 0.1 and 48 hours.",
"12.The process as claimed in any one of the preceding claims, characterized in that the reaction for obtaining the compounds of formula (VIII) is performed in the presence of an organic or mineral base chosen from alkali metal hydroxides and alkaline-earth metal hydroxides, alkali metal alkoxides and alkaline-earth metal alkoxides, alkali metal hydrides and alkaline-earth metal hydrides, alkali metal carbonates and bicarbonates and alkaline-earth metal carbonates and bicarbonates, and organonitrogen bases.",
"13.The process as claimed in claim 12, characterized in that the organic or mineral base is chosen from sodium hydroxide, potassium hydroxide, cesium hydroxide or calcium hydroxide, potassium tert-butoxide, sodium hydride, potassium hydride or caesium hydride, sodium carbonate, potassium carbonate or calcium carbonate, sodium bicarbonate, potassium bicarbonate or calcium bicarbonate, pyridine, trimethylamine, triethylamine or diisopropylethylamine, 1,5-diazobicyclo[4.3.0]non-5-ene and 1,8-diazabicyclo-[5.4.0]undec-7-ene.",
"14.The process as claimed in claim 11, characterized in that an excess of liquid base chosen from pyridine and alkylpyridines is used, thus replacing the solvent.",
"15.The process as claimed in any one of the preceding claims, characterized in that 3-hydroxy-picolinic acid derivatives of general formula (I) are such that: X1 and X2 each represent a hydrogen atom, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"16.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) are such that: Q1 is chosen from an oxygen atom and a sulfur atom, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"17.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) are such that: Z is chosen from an alkyl radical, a hydrogen atom and an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkyl-aminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothio-carbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"18.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) are such that: Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, and an amino, N-alkylamino, N,N-dialkyl-amino, —NHCOR10, —NHCSR10, N-arylamino, N,N-diaryl-amino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothio-carbonylamino group, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"19.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) are such that: Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical containing from 1 to 12 carbon atoms in a linear or branched chain, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl and alkynyl and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or-aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"20.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) have the following characteristics, taken separately or in combination: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical, a hydrogen atom and an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkyl-aminoalkyl, acylaminoakyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothio-carbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano, or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, and an amino, N-alkylamino, N,N-dialkyl-amino, —NHCOR10, —NHCSR10, N-arylamino, N,N-diaryl-amino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothio-carbonylamino group, Q1 is chosen from an oxygen atom and a sulfur atom, Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical containing from 1 to 12 carbon atoms in a linear or branched chain, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl and alkynyl and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"21.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) have the following characteristics: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical, a hydrogen atom and an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkyl-aminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothio-carbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, and an amino, N-alkylamino, N,N-dialkyl-amino, —NHCOR10, —NHCSR10, N-arylamino, N,N-diaryl-amino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothio-carbonylamino group, Q1 is chosen from an oxygen atom and a sulfur atom, Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical containing from 1 to 12 carbon atoms in a linear or branched chain, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl and alkynyl and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"22.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) have the following characteristics: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical, a hydrogen atom and an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkyl-aminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothio-carbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, azido, alkoxy, alkylthio or alkylsulfonyl radical and an amino, —NHCOR10 and —NHCSR10 group, Q1 represents an oxygen atom, Q2 represents a group NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an aryl, arylalkyl, heterocyclyl and heterocyclylalkyl radical, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined for the general formula (I), and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes of the compounds of formula (I) as they have just been defined.",
"23.The process as claimed in any one of the preceding claims, characterized in that the 3-hydroxypicolinic acid derivatives of general formula (I) are: 3-hydroxy-N-{[3-(trifluoromethyl)benzyl]oxy}-2-pyridine carboxamide, 1-{3-hydroxy-2-[(4-phenoxyanilino)carbonyl]-4-pyridinyl}-1,2-triazadien-2-ium, 4-amino-3-hydroxy-N-{4-[4-(trifluoromethyl)-phenoxy]phenyl}-2-pyridine carboxamide, 4-amino-3-hydroxy-N-[4-(4-methylphenoxy)phenyl]-2-pyridine carboxamide, 4-(formylamino)-3-hydroxy-N-{4-[3-(trifluoromethyl)phenoxy]phenyl}-2-pyridine carboxamide N-[4-(4-chlorophenoxy)phenyl]-4-(formylamino)-3-hydroxy-2-pyridine carboxamide, 4-(formylamino)-3-hydroxy-N-{4-[4-(trifluoromethyl)phenoxy]phenyl}-2-pyridine carboxamide and N-[4-(benzyloxy)phenyl]-4-(formylamino)-3-hydroxy-2-pyridine carboxamide, and also the possible N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts, metallic and metalloid complexes thereof.",
"24.Use of the compounds obtained by the process according to one of the preceding claims, as fungicidal active materials in the agrochemical field, and also in human and animal therapy."
],
[
"The present invention relates to a new process for preparing derivatives of 3-hydroxypicolinic acid, and more particularly of picolinic acid derivatives substituted in position 3 with an oxygen atom and optionally substituted in position 4.Such 3-hydroxypicolinic acid derivatives are known in the literature and in particular in patent application WO-A-99/11127 and in the publication by Kuzo Shibata et al.",
"(The Journal of Antibiotics, 51 (12), (1998), 1113-1116), for their fungicidal properties against phytopathogenic fungi of plants.",
"However, the preparation processes presented do not, for example, allow access to 3-hydroxypicolinic acid derivatives substituted in position 4.Specifically, the compounds described in these publications are obtained from fermentation musks of natural compounds.",
"Other 3-hydroxypicolinamide derivatives are also known from patent application publications JP-11 228 542 and EP-A-0 690 061.There again the preparation-processes presented do not give access to all the derivatives in the present description.",
"Thus, the present invention concerns a new process for preparing 3-hydroxypicolinic acid derivatives of general formula (I): in which: n represents 0 or 1, Q1 is chosen from an oxygen or sulfur atom, a group NR1 and a group N—NR4R5, Q2 is chosen from a group OR2 or SR3 and a group —NR4R5, or Q1 and Q2 may together form a ring of 5 to 7 atoms containing 2 to 3 oxygen and/or nitrogen atoms, optionally substituted with one or more radicals, which may be identical or different, chosen from halogens and alkyl and haloalkyl radicals, Z is chosen from a hydrogen atom, a cyano radical and an alkyl, allyl, aryl, arylalkyl, propargyl, cycloalkyl, halocycloalkyl, alkenyl, alkynyl, cyanoalkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, alkoxycarbonylaminoalkyl, aminocarbonylaminoalkyl, alkoxycarbonyl, N-alkylaminocarbonyl, N,N-dialkylaminocarbonyl, acyl, thioacyl, alkoxythiocarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, alkoxysulfonyl, aminosulfonyl, N-alkylaminosulfonyl, N,N-dialkylaminosulfonyl, arylsulfinyl, arylsulfonyl, aryloxysulfonyl, N-arylaminosulfonyl, N,N-diarylaminosulfonyl or N,N-arylalkylaminosulfonyl radical; Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, a cycloalkyl, halocycloalkyl, alkenyl, alkynyl, alkenyloxy, alkynyloxy, alkenylthio or alkynylthio group, an amino, N-alkylamino, N,N-dialkylamino, —NHCOR10, —NHCSR10, N-alkylaminocarbonylamino, N,N-dialkylaminocarbonylamino, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, thioacylamino, alkoxythiocarbonylamino, N-alkylaminothiocarbonylamino, N,N-dialkylaminothiocarbonylamino, N,N-arylalkylaminocarbonylamino, N-alkylsulfinylamino, N-alkylsulfonylamino, N-alkyl(alkylsulfonyl)amino, N-arylsulfinylamino, N-arylsulfonylamino, N-alkoxysulfonylamino, N-alkoxysulfinylamino, N-haloalkoxysulfinylamino, N-haloalkoxysulfonylamino, N-arylamino, N,N-diarylamino, arylcarbonylamino, alkoxycarbonylamino, N-arylaminocarbonylamino, N,N-diarylaminocarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino, N-arylaminothiocarbonylamino, N,N-diarylaminothiocarbonylamino or N,N-arylalkylaminothiocarbonylamino group, an acyl, carboxyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, lower alkoxycarbonyl, N-arylcarbamoyl, N,N-diarylcarbamoyl, aryloxycarbonyl or N,N-arylalkylcarbamoyl radical, and an imino group of formula: X1 and X2 are identical or different and chosen, independently of each other, from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, and an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, or X1 and X2 may also be joined together, thus forming a saturated, partially unsaturated or totally unsaturated 4- to 8-membered ring optionally comprising one or more hetero atoms chosen from sulfur, oxygen, nitrogen and phosphorus, R2 and R3 are identical or different and chosen, independently of other, from an alkyl radical comprising from 1 to 12 carbon atoms, a haloalkyl, cycloalkyl, halocycloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl or acyl radical, a nitro, cyano, carboxyl, carbamoy or 3-oxetanyloxycarbonyl radical, and an N-alkylcarbamoyl, N,N-dialkylcarbamoyl, alkoxycarbonyl, alkylthiocarbonyl, haloalkoxycarbonyl, alkoxythiocarbonyl, haloalkokylthiothiocarbonyl, alkokythiothiocarbonyl, alkenyl, alkynyl, N-alkylamino, N,N-dialkylamino, N-alkylaminoalkyl or N,N-dialkylaminoalkyl radical, or a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, or R1, R4, R5, R6 and R7 are identical or different and chosen, independently of each other, from a hydrogen atom, an optionally substituted alkyl radical comprising from 1 to 12 carbon atoms in a linear or branched chain, a haloalkyl, cycloalkyl, halocycloalkyl, alkoxy, aryloxy, arylalkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, cyanoalkyl or acyl radical, a nitro, cyano, carboxyl, carbamoyl or 3-oxetanyloxycarbonyl radical, and an N-alkylcarbamoyl, N,N-dialkylcarbamoyl, alkoxycarbonyl, alkylthiocarbonyl, haloalkoxycarbonyl, alkoxythiocarbonyl, haloalkoxythiocarbonyl, alkylthiothiocarbonyl, alkenyl, alkynyl, N-alkylamino, N,N-dialkylamino, N-alkylaminoalkyl or N,N-dialkylaminoalkyl radical, or a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, or R4 and R5, on the one hand, or R6 and R7, on the other hand, may be joined together, thus forming a saturated, partially unsaturated or totally unsaturated 4- to 8-membered ring optionally comprising one or more hetero atoms chosen from sulfur, oxygen, nitrogen and phosphorus, T represents a direct bond or a divalent radical chosen from a radical —(CH2)m—, m taking a value between 1 and 12, limits included, the said radical optionally being interrupted or ending with one or two hetero atoms chosen from nitrogen, oxygen and/or sulfur, and an oxyalkylene, alkoxyalkylene, carbonyl (—CO—), oxycarbonyl (—O—CO—), carbonyloxy (—CO—O—), sulfinyl (—SO—), sulfonyl (—SO2—), oxysulfonyl (—O—SO2—), sulfonyloxy (—SO2O—), oxysulfinyl (—O—SO—), sulfinyloxy (—SO—O—), thio (—S—), oxy (—O—), vinyl (—C═C—), ethynyl (—C═C—), —NR9—, —NR9—, —ONR9, —N═N—, —NR9—NR10—, —NR9—S—, —NR9—SO—, —NR9—SO2—, —S—NR9—, —SO—NR9—, —SO2—NR9—, —CO—NR9—O— or —O—NR9—CO— radical, R8 is chosen from a hydrogen atom and an aryl or heterocyclyl radical, R9 and R10, which may be identical or different, are chosen, independently of each other, from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, and an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, arylalkyl, cyanoalkyl, cyanoalkoxy, cyanoalkylthio, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of the compounds of formula (I) as have just been defined.",
"The tautomeric forms of the compounds of formula (I) such as those defined above are also included in the invention.",
"By tautomeric forms there are to be understood all of the isomeric forms described in the work.",
"“The Tautomerism of Heterocycles, Advances in Heterocyclic Chemistry, Supplement 1, by J Elguero, C. Martin, A. R. Katritsky and P Linda, published by Academic Press, New York, 1976, pages 1-4.The following generic terms are used with the following meanings: halogen atom signifies the fluorine, chlorine, bromine or iodine atom the alkyl radicals as well as groups including these alkyl radicals, (alkoxy, alkylcarbonyl or acyl, etc.)",
"include, unless indicated to the contrary, from 1 to 6 carbons atoms in a linear or branched chain and are optionally substituted, the halogenated alkyl, alkoxy and halocycloalkyl radicals may comprise one or more identical or different halogen atoms, the cycloalkyl radicals comprise from 3 to 6 carbon atoms and are optionally substituted, the alkenyl and alkynyl radicals, as well as groups including such radicals, comprise, unless indicated to the contrary, from 2 to 6 carbon atoms in a straight or branched chain and are optionally substituted, the acyl radical signifies alkylcarbonyl or cycloalkylcarbonyl, the alkyl part containing from 1 to 6 carbon atoms and the cycloalkyl part containing 3 to 6 carbon atoms, unless indicated to the contrary and are optionally substituted, the alkylene radical designates the divalent —(CH2)m— radical where m represents an integer equal to 1, 2, 3, 4, 5 or 6, the term “aryl” in “aryl” and “arylalkyl” signifies phenyl or naphthyl, optionally substituted, the term “heterocyclyl” in “heterocyclyl” and “heterocyclylalkyl” signifies a ring of 4 to 10 members, saturated, partially unsaturated or unsaturated, optionally substituted, comprising one or more heteroatoms, identical or different, chosen from nitrogen, oxygen, sulfur, silicon and phosphorus, when the amino radical is disubstituted, the two substituents are identical or different or may together with the nitrogen atom which carries them form a saturated, partially unsaturated or unsaturated nitrogen-containing heterocycle, containing 5 or 6 atoms in total, when the carbamoyl radical is disubstituted, the two substituents are identical or different or may together with the nitrogen atom which carries them form a saturated, partially unsaturated or unsaturated nitrogen-containing heterocycle of 5 to 6 carbon atoms in total, unless indicated to the contrary, the expression “optionally substituted” qualifying an organic group applies to different radicals constituting the group and indicates that the different radicals are optionally substituted by one or more radicals R9 and/or aryl and/or arylalkyl, identical or different.",
"According to one variant of the present invention, the invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above and for which: X1 and X2 each represent a hydrogen atom, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as they have just been defined.",
"According to another variant of the present invention, this invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above and for which: Q1 is chosen from an oxygen atom and a sulfur atom, the other substituents being as previously defined, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as they have just been defined.",
"According to a third variant of the present invention, this invention relates to 3-hydroxypicolinic acid derivatives of general formula (I) as defined above and for which: Z is chosen from an alkyl radical and a hydrogen atom or a cleavable radical capable of donating hydrogen, for example an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula I as previously defined.",
"Another variant of the present invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above and for which: Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, an amino group, and an N-alkylamino, N,N-dialkylamino, —NHCOR10, —NHCSR10, N-arylamino, N,N-diarylamino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothiocarbonylamino group, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula I as previously defined.",
"According to yet another variant of the present invention, this invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above and for which: Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical comprising from 1 to 12 carbon atoms in a linear or branched chain, a haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl or alkynyl radical and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as previously defined.",
"More particularly, the present invention relates to a process for preparing picolinic acid derivatives of general formula (I) as defined above, which have the following characteristics, taken separately or in combination: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical and a hydrogen atom or a cleavable radical capable of donating hydrogen, for example an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, an amino group, and an N-alkylamino, N,N-dialkylamino, —NHCOR10, —NHCSR10, N-arylamino, N,N-diarylamino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothiocarbonylamino group, Q1 is chosen from an oxygen atom and a sulfur atom, Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical comprising from 1 to 12 carbon atoms in a linear or branched chain, a haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl and alkynyl radical and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as previously defined.",
"Even more particularly, the present invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above, which have the flowing characteristics: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical and a hydrogen atom or a cleavable radical capable of donating hydrogen, for example an alkoxyalkyl haloalkoxyalkyl, alkythioalkyl, haloalkylthioalkyl, N-alkylaminoal, N,N-dialkylaminoalkyl, acylaminoalkl,acyl, thioacyl, cyanoalkyl, alkoxythibcarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, mercapto, nitro, thiocyanato, azido, cyano or pentafluorosulfonyl radical, an alkyl, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl or alkoxysulfonyl radical, an amino group, and an N-alkylamino, N,N-dialkylamino, 13 NHCOR10, —NHCSR10, N-arylamino, N,N-diarylamino, arylcarbonylamino, arylthiocarbonylamino, aryloxythiocarbonylamino or N,N-arylalkylaminothiocarbonylamino group, Q1 is chosen from an oxygen atom and a sulfur atom, Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an optionally substituted alkyl radical comprising from 1 to 12 carbon atoms in a linear or branched chain, a haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, alkenyl and alkynyl radical and a radical chosen from aryl, arylalkyl, heterocyclyl and heterocyclylalkyl, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as previously defined.",
"Even more specifically, the present invention relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above, which have the following characteristics: X1 and X2 each represent a hydrogen atom, Z is chosen from an alkyl radical and a hydrogen atom or a cleavable radical capable of donating hydrogen, for example an alkoxyalkyl, haloalkoxyalkyl, alkylthioalkyl, haloalkylthioalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl, acylaminoalkyl, acyl, thioacyl, cyanoalkyl, alkoxythiocarbonyl, N-alkylaminothiocarbonyl, N,N-dialkylaminothiocarbonyl or alkylsulfinyl radical, Y is chosen from a hydrogen atom, a halogen atom, a hydroxyl, azido, alkoxy, alkylthio and alkylsulfonyl radical and an amino, —NHCOR10 and —NHCSR10 group, Q1 represents an oxygen atom, Q2 represents a group —NR4R5, in which R4 represents a hydrogen atom and R5 is chosen from an aryl, arylalkyl, heterocyclyl and heterocyclylalkyl radical, optionally substituted with one or more radicals R9 and/or aryl and/or arylalkyl, which may be identical or different, and/or a group -T-R8, the other substituents being as defined above, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes of compounds of formula (I) as previously defined.",
"In the context of the present invention, the term “aryl” means phenyl or naphthyl, the term “arylalkyl” means phenylalkyl or naphthylalkyl, more particularly benzyl, plenethyl, phenylpropyl, phenylbuty, naphhthylethyl, naphthylpropyl or naphthylbutyl.",
"It is understood that these various radicals may optionally be substituted with one or more radicals R9 and/or aryl and/or arylalkyl radicals, which may be identical or different.",
"The terms “heterocyclyl” and “heterocyclylal” are defined similarly, it being understood that “heterocycle” means saturated, partially unsaturated or unsaturated monocycle or bicycle containing from 4 to 10 ring units, comprising at least one hetero atom chosen from nitrogen, oxygen, sulfur, silicon and phosphorus.",
"More particularly, the term “heterocycle” is understood as being one of the rings (i) to (v) below: a 5-membered ring; described by formula (i): in which each of the groups of the list B1, B2, B3, B4 is chosen from carbon, nitrogen, oxygen and sulfur atoms such that the said list comprises from 0 to 3 carbon atoms, from 0 to 1 sulfur atom, from 0 to 1 oxygen atom and from 0 to 4 nitrogen atoms; a 6-membered ring described by formula (ii): in which each of the groups of the list D1, D2, D3, D4, D5 is chosen from carbon and nitrogen atoms such that the said list comprises from 1 to 4 carbon atoms and from 1 to 4 nitrogen atoms; two fused rings, each being 6-membered, described by formula (iii): in which each of the groups in the list E1, E2, E3, E4, E5, E6, E7, E8 is chosen from carbon and nitrogen atoms such that the said list comprises from 4 to 7 carbon atoms and from 1 to 4 nitrogen atoms; a 6-membered ring and a 5-membered ring fused together, described by formula (iv): in which: each of the groups in the list J1, J2, J3, J4, J5, J6 is chosen from carbon and nitrogen atoms such that the said list comprises from 3 to 6 carbon atoms and from 0 to 3 nitrogen atoms; and each of the groups in the list L1, L2, L3 is chosen from carbon, nitrogen, oxygen and sulfur atoms such that the said list comprises from 0 to 3 carbon atoms, from 0 to 1 sulfur atom, from 0 to 1 oxygen atom and from 0 to 3 nitrogen atoms; and each of the groups in the list J1, J2, J3, J4, J5, J6, L1, L2, L3 is chosen such that the said list comprises from 3 to 8 carbon atoms; two fused rings, each being 5-membered, described by formula (v): in which: each of the groups in the list M1, M2, M3 represents, carbon, nitrogen, oxygen or sulfur atoms such that the said list comprises from 0 to 3 carbon atoms, from 0 to 1 sulfur atom, from 0 to 1 oxygen atom and from 0 to 3 nitrogen atoms; each of the groups in the list T1, T2, T3 represents carbon, nitrogen, oxygen or sulfur atoms such that the said list comprises from 0 to 3 carbon atoms, from 0 to 1 sulfur atom, from 0 to 1 oxygen atom and from 0 to 3 nitrogen atoms; Z represents a carbon or nitrogen atom; each of the groups in the list M1, M2, M3, T1, T2, T3 is chosen such that the said list comprises from 0 to 6 carbon atoms.",
"In the present invention, the term “heterocycle” even more particularly means: furyl, pyrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3,4-tetrazinyl, 1,2,3,5-tetrazinyl, 1,2,4,5-tetrazinyl, benzimidazolyl, indazolyl, benzotriazolyl, benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, 1,2,3-benzoxadiazolyl, 2,5-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 1,2,5-benzothiadiazolyl, quinolyl, isoquinolyl, quinoxazolinyl, quinazolinyl, cinnolyl or phthalazyl, pteridinyl, benzotriazinyl, 1,5-naphthyridinyl, 1,6-naphthyridinyl, 1,7-naphthyridinyl, 1,8-naphthyridinyl, imidazo[2,1-b]thiazolyl, thieno[3,4-b]pyridyl, purine or pyrrolo[1,2-b]thiazolyl.",
"The present invention most particularly relates to a process for preparing 3-hydroxypicolinic acid derivatives of general formula (I) as defined above, which are: 3-hydroxy-N-{[3-(trifluoromethyl)benzyl]oxy}-2-pyridinecarboxamide, 1-{3-hydroxy-2-[(4-phenoxyanilino)carbonyl]-4-pyridinyl}-1,2-triazadien-2-ium, 4-amino-3-hydroxy-N-{4-[4-(trifluoromethyl)-phenoxy]-phenyl}-2-pyridinecarboxamide, 4-amino-3-hydroxy-N-[4-(4-methylphenoxy)phenyl]-2-pyridinecarboxamide, 4-(formylamino)-3-hydroxy-N-{4-[3-(trifluoromethyl)-phenoxy]phenyl}-2-pyridinecarboxamide, N-[4-(4-chlorophenoxy)phenyl]-4-(formylamino)-3-hydroxy-2-pyridinecarboxamide, 4-(formylamino)-3-hydroxy-N-{4-[4-(trifluoromethyl)-phenoxy]phenyl}-2-pyridinecarboxamide and N-[4-(benzyloxy)phenyl]-4-(formylamino)-3-hydroxy-2-pyridinecarboxamide, as well as the optional N-oxides, geometrical and/or optical isomers, enantiomers and/or diastereoisomers, tautomeric forms, salts and metal and metalloid complexes thereof.",
"The compounds of general formula (I) and the compounds which may be used as intermediates in the processes of preparation, and which will be defined in the description of these processes, can exist in one or more forms of geometrical isomers according to the number of double bonds in the compound.",
"The compounds of general formula (I) where Q1 is —NR1 or —N—NR4R5 can comprise 2 different geometrical isomers denoted (E) or (Z) depending on the configuration of the two double bonds.",
"The E and Z notation can be replaced, respectively, by the term syn and anti, or cis and trans.",
"Reference is made particularly to the work of E. Eliel and S. Wilen “Stereochemistry of Organic Compound”, published by Wiley (1994), for the description and use of these notations.",
"The compounds of general formula (I) and compounds which may be used as intermediates in the processes of preparation, and which will be defined in the description of the processes, can exist in one or more optical isomeric or chiral forms according to the number of asymmetric centres in the compound.",
"The invention thus also relates to all the optical isomers and their racemic or scalemic (scalemic designates a mixture of enantiomers in different proportions), as well as the mixtures of all possible stereoisomers in all proportions.",
"The separation of the diastereoisomers and/or optical isomers can be effected by known methods (E. Eliel ibid.).",
"The present invention thus relates to the process of preparation of the compounds of general formula (I) and compounds useful as intermediates in the processes of preparation, described in a general way below.",
"Although general, this method of preparation provides all of the operating conditions to be used for the synthesis of the compounds of formula (I) according to the present invention.",
"It will nevertheless be understood that the skilled worker will be able to adapt this method according to the specifics of each of the compounds which it is desired to synthesize.",
"The preparation of reagents used in one or other of the general methods of preparation is generally known and is generally described specifically in the prior art or in such a manner that the man skilled in the art can adapt it to the desired aim.",
"The prior art usable by the normally skilled worker in order to establish conditions for the preparation of reagents can be found in numerous general chemistry text books such “Advanced Organic Chemistry” by J.",
"March, published by Wiley (1992), “Methoden der organischen Chemie” (Houben-Weyl), published by Georg Thieme Verlag, or the “Chemical Abstracts” published by the American Chemical Society as well as in information data bases accessible to the public.",
"The compounds of general formula (I) may advantageously be prepared from a compound of formula (II) (described, for example, in patent U.S. Pat.",
"No.",
"5,652,363): in which X1 and X2 are as defined above, which compound is placed in contact with a cyanide, alkali metal derivatives or alkaline-earth metal derivatives of hydrocyanic acid in the presence of an alkylating agent and a solvent, or with trimethylsilyl cyanide in the presence of dimethylcarbamoyl chloride and a solvent, to give the compounds of formula (III) in which X1 and X2 are as defined above.",
"The compounds of formula (III) above can be converted into corresponding halo derivatives of formula (IVa) in which X1 and X2 are as defined above and X represents a halogen atom chosen from fluorine, chlorine, bromine and iodine, by reaction with an acyl halide in the presence of a solvent, such as, preferably, but not exclusively, an ethereal solvent such as diethyl ether, diisopropyl ether, tetrahyrofuran, dioxane or 1,2-dimethoxyethane.",
"The halo derivatives of formula (IVa) are then hydrolyzed into compounds of formula (Ia): in which X, X1 and X2 are as defined above, by the action of a hot hydracid—or a strong mineral base, optionally in the presence of aqueous hydrogen peroxide solution—and optionally boron tribromide as described in the abovementioned publications.",
"One variant which is possible for this hydrolysis reaction consists in treating the nitrile of formula (IVa) with an acid, in particular hydrochloric acid, hydroiodic acid or hydrobromic acid, or alkylsulfonic acids, this hydrolysis reaction being carried out in excess acid, in the absence or presence of a solvent, at reflux or at a temperature of between 20° C. and 200° C. The compounds of formula (III) or (IVa) may also be placed in contact with an alcohol or an alkoxide in the presence of a solvent such as, preferably, but not exclusively, a protic or polar aprotic solvent, to give the compounds of formula (IVb) in which X1 and X2 are defined above, and may then be used in a hydrolysis reaction under operating conditions similar to those used for the formation of the compounds of formula (Ia), to give the compounds of the 6respective formulae (Ib) and (Ib′): in which X1 and X2 are as defined above.",
"The compounds of formula (IVa) may also be converted into picolinic acid derivatives of formula (Va): in which X1, X2 and R6 are as defined above, by reacting a compound of formula R6SH, or a corresponding alkali metal or alkaline-earth metal salt, in an aprotic polar solvent, such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylprolyleneurea or dimethyl sulfoxide, at a temperature between 0° C. and the boiling point of the solvent.",
"The nitrites of formula (Va) may then be used in a hydrolysis reaction to give the corresponding acids of formula (Ic): in which X1, X2 and R6 are as defined above, according to a reaction similar to the one used for the formation of the compounds of formula (Ia).",
"The halides of formula (IVa) may also be treated with an azothydric acid salt, more particularly with sodium azide, to give the compounds of formula (Vb): in which X1 and X2 are as defined above, this reaction preferably being carried out in a polar aprotic solvent such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylprolyleneurea or dimethyl sulfoxide, at a temperature between 0° C. and the boiling point of the solvent.",
"The compounds of formula (Vb) may then be hydrolyzed according to techniques similar to those presented for the preparation of the acids of formula (Ia) above, to give the acids of formula (Id): in which X1 and X2 are as defined above.",
"The azides of formula (Id) are then optionally reduced to amine derivatives of formula (Ie): in which X1 and X2 are as defined above, by the action of reducing agents such as, for example, lithium aluminum hydride, triphenylphosphine or hydrogen in the presence of a catalyst, or alternatively any other reducing agent as described by J.",
"March, ibid, p. 1219-1220.The acids of formulae (Ia) to (Ie) can be converted into thio acids, imino derivatives (—C(═NR1)) or amino imino derivatives (—C(═N—NR4R5)) according to standard techniques that are well known to those skilled in the art specialized in organic synthesis.",
"Similarly, the acids (Ia) and (Ie), or the thio, imino and imino amino derivatives thereof defined above, substituted in position 3 (relative to the pyridine nitrogen atom) with —OH or -methoxy may be subjected to various reactions that are already known in the prior art, in order to give the corresponding derivatives substituted in position 3 (relative to the pyridine nitrogen atom) with —O-Z, Z being as defined for the compounds of formula (I).",
"The compounds of formula (I) defined above for which Y represents an amino (—NH2) radical may be placed in contact with an acylating agent in the presence of a solvent and optionally of a base.",
"The term acylating agent preferentially means, but in a non-limiting manner, an acyl halide, an anhydride, an acid, an ester or a primary amide, and the thio-homologs thereof, as described in J.",
"March, ibid, pages 417-424, to give the compounds of formulae (VIa) and (VIb): in which X1, X2, Q1, Q2, Z and R10 are as defined above.",
"The picolinic acid derivatives of formula (VII): in which X1, X2, Q1, n, Z and Y are as defined above, may be placed in contact with a reagent of formula R2OH, R3SH or HNR4R5, R2, R3, R4 and R5 being as defined above, to give the compounds of formulae (VIIIa), (VIIIb) and (VIIIc), respectively, forming the set of compounds of formulae (VIII): which are special cases of the compounds of formula (I) in which Q2 represents —OR2, —SR3 and —NR4R5, respectively.",
"The above reaction is carried out in the presence of an activating agent such as thionyl chloride, oxalyl chloride, dicyclocarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole or phosphorus oxichloride or the like, as described in the reference publications, in the presence of organic or inorganic base, in the absence or presence of a solvent.",
"These reagents may, where appropriate, be linked to a polymer resin.",
"The reaction is generally carried out at a temperature of between −80° C. and 180° C. (preferably between 0° C. and 150° C.) or at the boiling point of the solvent used.",
"The solvent which is suitable for this reaction may be an aliphatic hydrocarbon such as pentane, hexane, heptane or octane, an aromatic hydrocarbon such as benzene, toluene, xylene or halobenzenes, an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane or dimethoxyethane, a halogenated hydrocarbon such as dichloromethane, chloroform, 1,2-dichloroethane or 1,1,1-trichloroethane, an ester such as methyl acetate or ethyl acetate, a nitrile such as acetonitrile, propionitrile or benzonitrile, or a polar aprotic solvent such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylprolyleneurea, dimethyl sulfoxide, pyridine or water.",
"Mixtures of these various solvents may also be used.",
"The reaction time depends on the conditions used and is generally between 0.1 h and 48 h. As organic or inorganic bases that are suitable for this reaction, mention may be made of alkali metal hydroxides and alkali-earth metal hydroxides such as sodium hydroxide potassium hydroxide, cesium hydroxide or calcium hydroxide, alkali metal alkoxides and alkaline-earth metal alkoxides such as potassium tert-butoxide, alkali metal hydrides and alkaline-earth metal hydrides, such as sodium, hydride, potassium hydride or cesium hydride, alkali metal carbonates and bicarbonates or alkaline-earth metal carbonates and bicarbonates, such as sodium carbonate, potassium carbonate, calcium carbonate or sodium bicarbonate, potassium bicarbonate or calcium bicarbonate, organic bases, preferably organonitrogen bases, such as pyridine, alkylpyridines, alkylamines such as trimethylamine, triethylamine or diisopropylethylamine, and aza derivatives such as 1,5-diazabicylco[4.3.0]non-5-ene or 1,8-diazabicylco[5.4.0]undec-7-ene.",
"The reaction may be carried out using an excess of a base which is liquid at the reaction temperature, this base then also acting as solvent.",
"Mention may be made of organonitrogen bases such as pyridine or alkylpyridines.",
"There is no strict limitation on the relative proportions of the compounds of formula (VII) and of formula (VIII).",
"However, it is advantageous to select a (VIII)/(VII) molar ratio of between 0.1 and 10, preferably 0.5 to 2.The compounds of general formula (XI): which are special cases of the compounds of formula (I) for which n is equal to 1, may be obtained by a process which consists in placing a compound of formula (X): which is a special case of the compounds of formula (I) for which n is equal to zero, in contact with an oxidizing agent as described in J.",
"March, ibid., page 1200, in particular aqueous hydrogen peroxide solution or carboxylic, boronic or sulfuric peracids.",
"It should be understood that the reactions described in the preceding paragraphs may be carried out in any other order which is suitable to obtain the desired compounds of formula (I).",
"The order of the reactions will be determined most particularly by the compatibility requirements of the various substituents on the pyridine nucleus.",
"The compatibilities of the various radicals and reagents used are well known to the person skilled in the art, who may moreover refer to the examples for the preparation of the compounds of formula (I) described later in this description.",
"The 3-hydroxypicolinic acid derivatives of formula (I) described in the present invention are useful in the agrochemical field and in human and animal therapy.",
"Specifically, 3-hydroxypicolinic acid derivatives of formula (I) have advantageous antifungal properties enabling them to effectively combat fungal diseases of crops and also fungal diseases encountered in man and animals.",
"The large antifungal potential of the compounds of formula (I) also allows them to be used in any field for which it is required and/or necessary to combat microscopic fungi such as, for example, molds.",
"The following examples illustrate in a non-limiting manner several examples of fungicidal compounds according to the invention.",
"In the examples which follow, “MP” signifies “melting point” and is expressed ° Celsius (° C.).",
"EXAMPLE A Preparation of 2cyano-3-methoxy-4-nitropyridine A mixture of 12.5 g (12.5 moles) of the N-oxide of 3 methoxy-4-nitropyridine, 7.72 ml (1.1 eq.)",
"of methyl sulfate and 70 ml of 1,2-dichloroethane is heated at 70° C. for 2.5 hours.",
"It is allowed to cool and 70 ml of water are added.",
"It is cooled in a salt and ice bath and, in portions, 7.55 g (2.1 moles) of sodium cyanide are added, controlling the temperature so as not to exceed 10° C. After 4 hours stirring, the reaction mixture is extracted with ethyl ether, the organic phase is washed with water, concentrated and the residue chromatographed (ethyl acetate/dichloromethane).",
"There is obtained 7.06 g of a yellow oil (yield 53%).",
"EXAMPLE B Preparation of 4-bromo-2-cyano-3-methoxypyridine A mixture of 6 g (0.0335 moles) of 2-cyano-3-methoxy-4-nitropyridine obtained in Example a), 12.37 g (0.100 moles) of acetyl bromide and 36 ml of 1,2-dimethoxyethane is heated at 85° C. for 1.5 hours.",
"It is allowed to cool and the reaction mixture is poured onto 100 g of crushed ice.",
"30 ml of 1,2-dichloroethane are added and gently neutralized to pH=8 with a 28% aqueous solution of ammonia.",
"After extraction with 1,2-dichloroethane, washing with water, drying and concentration the residue is chromatographed (ethyl acetate/heptane, 3:7) to obtain 5.32 g (75% yield) of a white solid (MP=116° C.).",
"In a similar manner, replacing the acetyl bromide by acetyl chloride, there is obtained 4-chloro-2-cyano-3-methoxpyridine (83% yield) in the form of a white solid (MP=91° C.).",
"EXAMPLE C Preparation of 4-azido-2-cyano-3-methoxypyridine To 1 g (0.0155 moles) of sodium azide in 25 ml of dimethylformamide at 0° C., there is added gently 3 g (0.0141 moles) of 4-bromo-2-cyano-3-methoxypyridine from Example b), dissolved in 40 ml of dimethylformamide.",
"The mixture is stirred for 6 hours at ambient temperature.",
"The reaction mixture is diluted in 200 ml of iced water and extracted with dichloromethane.",
"The organic phase is washed twice with water, dried, concentrated and the residue chromatographed (ethyl acetate/heptane, 3:7).",
"There is obtained 0.87 g (35% yield) of a white solid (MP=102° C.).",
"EXAMPLE D Preparation of 4-chloro-3-hydroxypicolinic Acid A mixture of 2 g (0.012 moles) of 4-chloro-2-cyano-3-methoxypyridine obtained in Example b), and 7 ml of 37% hydrochloric acid is heated at 100° C. for 12 hours.",
"After cooling the solid formed is filtered, washed once with water and 3 times with acetone and dried under vacuum for 8 hours.",
"There is obtained 1.78 g (86% yield) of a yellow solid (MP=228° C.).",
"In the same manner, the following hydroxy acids are obtained: Y Hydracid Yield, MP (° C.) 4-bromo-3-hydroxy-picolinic acid HBr Yellow solid, 82%, 230° C. 4-azido-3-hydroxy-picolinic acid HCl Violet solid, 63% 3,4-dihydroxypicolinic acid HBr White solid, 74%, 264° C. EXAMPLE E Preparation of 2-cyano-3,4-dimethoxypyridine 3 g (0.017 moles) of 2-cyano-3-methoxy-4-nitropyridine obtained in Example a) and a sodium methoxide solution prepared from 0.77 g (0.033 moles) of sodium and 65 ml of methanol are stirred at ambient temperature for 4 hours.",
"There is added 100 ml of water, the methanol is eliminated and the aqueous phase is extracted with dichloromethane.",
"The organic phase is washed with water, dried, concentrated and the residue chromatographed (ethyl acetate/heptane, 1:1) to obtain 1.96 g (72% yield) of a white solid (MP=133° C.).",
"EXAMPLE F Preparation of 2-cyano-3-hydroxy-4-thiomethoxypyridine 2 g of 4-bromo-2-cyano-3-methoxypyridine obtained in Example b) and 2.16 g of sodium thiomethoxide in 40 ml of anhydrous dimethylformamide are heated at 85° C. for 5 hours.",
"After cooling and the addition of 20 ml of water, the reaction mixture is concentrated to dryness.",
"The residue is extracted three times with hot methanol.",
"The cooled methanolic phase is filtered and concentrated.",
"There is obtained 1.51 g (97% yield) of a brown syrup used crude.",
"EXAMPLE G Preparation of 3-hydroxy-4-thiomethoxypicolinic acid 2.5 g (0.015 moles) of 2-cyano-3-hydroxy-4-thiomethoxypyridine from Example f), 8.5 g of potassium hydroxide and 25 ml of water are heated at reflux for 2.5 hours.",
"After allowing to cool and in an ice bath the mixture is gently neutralized with 1 N hydrochloric acid to pH=2-3.The solid formed is filtered.",
"The solid is washed once with water and three times with acetone; it is dried under vacuum for 8 hours.",
"There is obtained 1.81 g (68% yield) of a white solid (MP=247° C.).",
"EXAMPLE H Preparation of 3,4-dimethoxypicolinic acid 1 g of 3,4-dimethoxy-2-cyanopyridine obtained in Example e) and 3.5 g of potassium hydroxide in 15 ml of water are heated to 85° C. for half an hour.",
"It is allowed to cool and in an ice bath hydrochloric acid is added gently to pH=2-3.After concentration to dryness, the residue is extracted three times with hot methanol, allowed to cool, filtered and concentrated.",
"There is obtained a solid used crude.",
"EXAMPLE I Preparation of the N-oxide of 3-hydroxypicolinic acid To a mixture of 20 ml acetic acid and 20 ml of hydrogen peroxide solution, are added 2 g of 3-hydroxypicolinic acid; the whole is heated at 80° C. for 5 hours.",
"After elimination of the solvents under vacuum, the solid obtained is washed with hot alcohol to obtain 2.02 g of compound in the form of a white solid (MP=182° C.).",
"EXAMPLE 1 3-Hydroxy-4-methoxy-N-para-phenoxyphenylpicolinamide 0.046 g of para-phenoxyaniline, 0.04 g of 3-hydroxy-4-methoxypicolinic acid (obtained in a manner similar to that described in Example g)), 0.034 g of 1-hydroxybenzotriazole and 0.060 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are heated in 2 ml of pyridine between 75 and 85° C. for 1 to 2 hours.",
"After cooling, the residue is taken up in a mixture of dichloromethane and 2 mL of IN hydrochloric acid.",
"After extraction with dichloromethane, concentration and chromatography on silica there is obtained 0.057 g of the title compound, a yellow solid (MP=186° C.).",
"EXAMPLE 2 4-amino-3-hydroxy-N-para-phenoxyphenylpicolinamide To 0.14 g of 4-azido-3-hydroxy-N-para-phenoxyphenylpicolinamide (obtained from the compound of Example 1 according to the methods described in Examples a) to g)) dissolved in a mixture of ethanol/ethyl acetate, 1:2, there is added a spatula tip of 10% palladium on carbon.",
"Hydrogenation is carried out at 20 bars pressure and ambient temperature for 4-5 hours.",
"After filtration, concentration and chromatography of the residue in ethyl acetate, there is obtained 0.099 g of a white solid (MP:197° C.).",
"EXAMPLE 3 4-formamido-3-hydroxy-N-para-phenoxyphenylpicolinamide There is heated at reflux 61.2 mg of acetic anhydride and 27.6 mg of formic acid for 4 hours and 46 mg of 4-amino-3-hydroxy-N-para-phenoxyphenylpicolin-amide of Example 2 is added, dissolved in 5 ml of tetrahydrofuran.",
"After 8 hours at reflux, the reaction mixture is concentrated and purified by chromatography to give 39 mg of a yellow solid MP 208° C. The compounds described in the following Tables 1 and 2 are prepared in a similar manner: TABLE 1 No Q2 O—Z Y MP 4 114 5 151 6 250 7 234 8 108 9 98 10 144 11 116 12 260 13 186 14 238 15 260 16 165 17 178 18 120 19 62 20 72 21 124 22 76 23 128 24 86 25 1.58* 26 138 27 186 28 29 30 31 32 122 33 162 34 248 35 36 174 37 161 38 144 39 170 40 182 41 162 42 210 43 158 44 45 140 46 159 47 116 48 134 49 138 50 51 52 168 53 155 54 145 55 118 56 86 57 165 58 250 59 255 60 235 61 162 62 292 63 168 64 135 65 165 66 161 67 160 68 122 69 256 70 198 71 162 72 139 73 150 74 208 75 210 76 242 77 243 78 212 79 80 185 81 118 82 172 83 214 84 172 85 122 86 248 87 168 88 186 89 120 90 146 91 148 92 147 93 110 94 66 95 150 96 246 97 260 98 226 99 140 100 101 166 102 124 103 174 104 166 105 164 106 120 107 279 108 76 109 156 110 284 111 265 112 138 113 114 271 115 274 116 252 117 272 118 294 119 296 120 121 108 122 106 123 thick oil 124 89 125 92 126 127 thick oil 128 132 129 254 130 212 131 121 132 thick oil 133 thick oil 134 102 135 114 136 137 136 138 197 139 199 140 141 148 142 143 277 144 288 145 278 146 147 276 148 187 149 227 150 260 151 152 152 205 153 161 154 148 155 137 156 165 157 248 158 154 159 178 160 148 161 135 162 169 163 130 164 185 165 187 166 162 167 144 168 154 169 192 170 121 171 113 172 105 173 106 174 135 175 138 176 113 177 131 178 171 179 165 180 175 181 182 183 184 170 185 142 186 187 188 189 190 191 192 193 194 117 195 196 214 197 252 198 232 199 246 200 227 201 208 202 208 203 185 204 198 205 173 206 170 207 143 208 230 209 128 210 232 211 AS90328 212 230 213 214 215 216 TABLE 2 N° Q2 O—Z Y MP 217 156 218 158 219 258 220 244 221 156 222 150 223 224 275 225 178 226 114 227 128 228 229 176 230 178 231 172 232 160 EXAMPLES OF BIOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION Example A in vivo Test on Alternaria Brassicae (Leaf Spot of Crucifers An aqueous suspension, with a concentration of 2 g/l , of the active material tested is obtained by grinding it finely in the following mixture: water Tween 80 surfactant (polyoxyethylenated derivative of sorbitan oleate) diluted to 10% in water: 5 ml/mg of active material clay: inert support q.s.",
"100%.",
"This aqueous suspension is then diluted with water so as to obtain the desired active material concentration.",
"Radish plants (Pernot variety) in starter cups, sown on a 50/50 peat soil-pozzolana substrate and grown at 18-20° C., are treated at the cotyledon stage by spraying with the aqueous suspension described above.",
"Plants, used as controls, are treated with an aqueous solution not containing the active material.",
"After 24 hours, the plants are contaminated by spraying them with an aqueous suspension of Alternaria brassicae spores (40,000 spores per cm3).",
"The spores are collected from a 12-13-day-old culture.",
"The contaminated radish plants are incubated for 6-7 days at about 18° C., under a humid atmosphere.",
"Grading is carried out 6 to 7 days after the contamination, in comparison with the control plants.",
"Under these conditions, good (at least 50%) or total protection is observed at a dose of 250 g/ha with the following compounds: 198, 200, 202, 206, 206.Example B in vivo Test on Septoria Nodorum (Septoria Disease of Wheat An aqueous suspension, with a concentration of 2 g/l, of the active material tested is obtained by grinding it finely in the following mixture: water Tween 80 surfactant (polyoxyethylenated derivative of sorbitan oleate) diluted to 10% in water: 5 ml/mg of active material clay: inert support q.s.",
"100%.",
"This aqueous suspension is then diluted with water so as to obtain the desired active material concentration.",
"Wheat plants (Scipion variety) in starter cups, sown on a 50/50 peat soil-pozzolana substrate and grown at 12° C., are treated at the 1-leaf stage (10 cm tall) by spraying them with the aqueous suspension described above.",
"Plants, used as controls, are treated with an aqueous solution not containing the active material.",
"After 24 hours, the plants are contaminated by spraying with an aqueous suspension of Septoria nodorum spores (500,000 spores per cm3).",
"The spores are collected from a seven-day-old culture.",
"The contaminated wheat plants are incubated for 72 hours at about 18° C., under a humid atmosphere, and then for 14 days at 90% relative humidity.",
"Grading is carried out 15 to 20 days after contamination, in comparison with the control plants.",
"Under these conditions, good (at least 50%) or total protection is observed, at a dose of 250 g/ha, with the following compounds: 198, 200, 202.Example C in vivo Test on Magnaporthe Grisea (Blast Disease of Rice An aqueous suspension, with a concentration of 50 mg/l, of the active material tested is obtained by grinding it finely in the following mixture: water, 2% acetone.",
"Rice plants (Koshihirakari variety), sown on Kureha soil and grown in 33 cm2 plastic pots up to the 3-4 leaf stage, are treated by spraying with the aqueous suspension described above.",
"Plants, used as controls, are treated with an aqueous solution not containing active material.",
"24 hours after treatment, the plants are contaminated by spraying with an aqueous suspension of Magnaporthe grisea spores (500,000 spores per cm3).",
"The contaminated rice plants are placed in an incubator for 24 hours at 25° C. under a humid atmosphere, and then for 5 to 7 days in an incubation chamber at 20-25° C. and 70-90% relative humidity.",
"Grading is carried out 5 to 7 days after the contamination, by counting the lesions on the first leaf of the plant.",
"Under these conditions, good (at least 50%) or total protection is observed, at a dose of 50 mg/l, with the following compounds: 24, 112, 204, 205."
]
] | Patent_10169855 |
[
[
"Ultrasonic diagnosing apparatus",
"An ultrasonic diagnostic apparatus transmits an ultrasonic beam into an object to be examined using a multi-ring arrangement formed with transducer elements arrayed two-dimensionally in concentric rings and receives an echo so as to create a tomogram or a three-dimensional image of the object.",
"To correct for focusing error due to the difference in length of ultrasound propagating paths, the ultrasonic diagnostic apparatus groups the transducer elements so as to form a multi-ring arrangement, transmits/receives ultrasonic beams with a delay to each ring of the multi-ring arrangement and scans the ultrasonic beam so as to create an ultrasonic image, measures delay error due to presence of a sound speed non-uniformity portion of the object and changes either the coupling of the multi-ring or the delay time based on the measurement error."
],
[
"1.An ultrasonic diagnostic apparatus comprising a probe having two-dimensional array of transducer elements for transmitting and receiving ultrasonic waves to an object to be examined, in which a circular pattern of transducer elements is formed for transmitting and receiving an ultrasound signal by bundling the transducer elements of said two-dimensional array electronically to compose a multi-ring arrangement of transducer elements in concentric rings, and an ultrasound beam is transmitted and received with application of a delay time between each ring of said multi-ring arrangement, wherein said ultrasonic diagnostic apparatus further comprises means for transmitting and receiving an ultrasonic beam that has been corrected using said multi-ring arrangement by measuring a focusing error due to the presence of a sound speed non-uniformity interior of said object and modifying at least one of the manner of bundling of said multi-ring arrangement or the delay time based on the measuring error, and means for imaging the object with an echo signal formed of the corrected ultrasonic beam.",
"2.An ultrasonic diagnostic apparatus according to claim 1, wherein the multi-ring arrangement of transducer elements formed by bundling transducer elements of the two-dimensional array in said probe in concentric rings is divided into a plural number of sections in a radial shape from the center to the outside of the circular pattern, and a focusing error due to the presence of a sound speed non-uniformity part in the object is measured between each section of the divided circular pattern or each section of the concentric rings, and the ultrasonic beam is corrected by feeding back a measured value to delay circuits in said transmitting and receiving means.",
"3.An ultrasonic diagnostic apparatus according to claim 1, wherein a bundled arrangement of transducer elements is provided that is different from that of said multi-ring arrangement composed by bundling said transducer elements of said two-dimensional array of said probe in concentric rings, and a focusing error due to the presence of a sound speed non-uniformity part in the object is measured between each circular pattern, and the ultrasonic beam is corrected by feeding back a measured value to delay circuits in said transmission and receiving means and returning the multi-ring arrangement to an initial setting.",
"4.An ultrasonic diagnostic apparatus comprising: a probe having a two-dimensional array of transducer elements; transducer selection means for electronically bundling transducer elements into a multi-ring arrangement composed of a plural number of rings, means for dividing said multi-ring arrangement into a plural number of sections; means for calculating a focusing error between sections in said multi-ring arrangement; means for feed-back correcting said calculated focusing error relative to a set delay time to produce a corrected focusing error signal; a beam-forming part comprised of delay circuits for applying a delay time to each ring of said multi-ring arrangement of transducer elements in response to a corrected focusing error signal and an adder circuit for adding the outputs of said each delay circuits; and means for imaging an output signal of said beam-forming part.",
"5.An ultrasonic diagnostic apparatus according to claim 5, wherein means for calculating the focusing error between sections in said multi-ring arrangement includes means for calculating the focusing error between rings in one section.",
"6.An ultrasonic diagnostic apparatus comprising: transducer selection means for initially forming a multi-ring arrangement of transducer elements composed of a plural number of rings from a two-dimensional array of transducer elements; means for specifying transducer elements having a focusing error in each ring of said multi-ring arrangement; ring correction means for changing a specified transducer element to a different ring from initial ring location to correct the focusing error; a beam-forming part comprised of delay circuits for applying a delay time to the transducer elements of each ring of said multi-ring arrangement and an adder circuit for adding the outputs of each of said delay circuits; and means for imaging an output signal of said beam-forming part.",
"7.Ultrasonic diagnosing apparatus according to claim 6, wherein means for specifying transducer elements having a focusing error includes means for bundling a group of adjacent transducer elements different from a ring and assigning a transmitter and beam-forming circuit to this bundled transducer group.",
"8.An ultrasonic diagnostic apparatus comprising: transducer selection means for initially forming a multi-ring arrangement of transducer elements composed of a plural number of rings from a two-dimensional of array transducer elements; means for setting a multi-ring arrangement to calculate a focusing error, separately from said formed multi-ring arrangement; a beam-forming part comprised of delay circuits for applying a delay time to a respective ring of said multi ring arrangement of transducer elements initially formed or the multi-ring arrangement for calculating said focusing error, and an adder circuit"
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>An apparatus having a plural number of transducer composed of a two-dimensional transducer array in which sector scanning is performed using an ultrasonic beam (in an arbitrary direction) generated by a transmitting/receiving circular pattern of transducers formed in the two-dimensional transducer array is known.",
"In this ultrasonic diagnostic apparatus, for example, if 64 64 transducers are disposed in the two-dimensional array, then the total number of transducers is 4,096.; and, ultrasonic scanning is performed with a separate delay control provided for each transducer element.",
"In this case, a beam-forming circuit with 4,096 channels is necessary.",
"Realizing a beam-forming circuit having such a large number of multi-channel delay circuits is difficult.",
"So, by thinning out the number of driven elements for forming one ultrasonic beam, the number of delay circuit channels in the beam-forming circuit is reduced.",
"But the S/N of signal acquired by thinning out the driven elements is deteriorated.",
"So, the apparatus is realized by comprising a beam-forming circuit having as many as delay channels possible.",
"For example, there is an apparatus that comprises a beam-forming circuit having a delay circuit of 256 channels for transmitting and 256 channels for receiving.",
"As shown in FIG.",
"4 , a linear scanning expanded field of view obtained by moving the transmitting/receiving circular pattern of transducer elements 1 , that are arrayed in a two-dimensional X, Y direction, and a scanning method in which a convex scanning is applied to two-dimensions are proposed.",
"In this case, the number of transducer elements 1 is more than in said sector scanning type apparatus.",
"So, for reducing the number of channels in the beam-forming circuit, an apparatus is known that produces an ultrasound transmitting/receiving circular pattern 2 by electronically bundling a plural number of transducer elements 1 in the two-dimensional array into a multi-ring arrangement with concentric rings, so as to give a consistent delay time to the transducer elements composing one ring of said multi-ring arrangement.",
"The apparatus transmits/receives an ultrasonic beam with a delay time between rings, and forms an ultrasonic image by moving said circular pattern 2 in X, Y directions.",
"As shown in FIG.",
"5 , in said multi-ring arrangement, each ring is formed by the bundling of transducer elements in concentric rings of which the distance L 1 , L 2 .",
".",
".",
", from the single focal spot F is almost the same, and the diameter of the most exterior ring is diameter 2 for ultrasound transmitting/receiving.",
"In this method, the bundling of transducer elements in concentric rings makes it possible to reduce greatly the number of channels in the beam forming circuit, which corresponds to the number of delay circuits, and the S/N of a signal acquired by using all elements in a circular pattern can be improved.",
"When considering the shape of an ultrasonic beam, a focusing calculation for an ultrasonic beam in the object to be examined is traditionally performed under the condition that the speed of sound in an ultrasonic propagation medium is uniform.",
"As shown in FIG.",
"6 , a traditional apparatus comprises delay circuits 4 , 4 , .",
".",
".",
"comprising one per transducer element of the probe 3 having a plural number of transducers and an adder 5 for adding received signals output from these delay circuits 4 , 4 , .",
".",
".",
"Although reflected signals from focus point 6 in the object propagate through medium 7 to reach each transducer element, the difference in the path length from the focus point to each transducer element causes a difference in the arrival time of each reflected signal.",
"In this case, the reflected signals reach the transducer elements located in the center of probe 3 early, and, on the other hand, they reach the transducer elements at the ends late.",
"So, the shape of the wave surface 8 in the received signals is not linear.",
"Thus, the signals output from transducer elements in the center part of the probe are delayed with a large amount of delay time in delay circuits 4 corresponding to each received signal, and the signals output from transducer elements at the ends of the probe are delayed with a small amount of delay time.",
"The signals are then output to adder 5 .",
"With these delay operations, the wave surface 9 of the signal output from delay circuits 4 is linear since the signals have the same phase.",
"The received signals having the same phase, such as shown by wave surface 9 in this condition, are added in adder 5 so as to form the combined signal 10 .",
"But actually, as shown in FIG.",
"7 , a sound speed non-uniformity part 11 typically exists on the path from the focus point 6 in the object to the probe 3 , so that the wave surface of the received signal is disturbed, as shown at 8 ′.",
"In this case, when performing the delay operation, while assuming that the speed of sound is uniform, the wave surface of the received signals output from delay circuits 4 is distorted.",
"as shown at 9 ′, so that they do not have the same phase.",
"Accordingly.",
"the output produced in adder 5 does not increase in intensity as the signals are added, so that its intensity is small, as shown by signal 10 ′.",
"On the Contrary, there is a technology referred to as an adaptive ultrasonic imaging method which operates to correct the delay amount produced in said delay circuits 4 in accordance with the speed of sound in the medium.",
"In the adaptive ultrasonic imaging technology, a mutual correlation method for correcting the delay amount by correction processing of respectively received signals between adjacent channels, and a maximum value brightness method for searching for a brightness maximum while changing the delay amount of the delay circuits are known.",
"FIG.",
"8 is a block diagram which illustrates the mutual correlation method.",
"In FIG.",
"8 , a signal received from each transducer element, which is not shown in the figure, is delayed by a predetermined amount by a respective delay circuit 4 , 4 , .",
".",
".",
"This delay is possible by use of analog delay circuits or digital delay circuits.",
"In this case, when outputs of adjacent channels in each transducer element, a mutual correlation processing is carried out with correlation device 12 , and the phase difference between the outputs of adjacent channels can be obtained.",
"By detecting the phase difference value, transforming it to focus data in correction processing part 13 , and feeding the transformed data back to focus controlling part 14 , the delay amount produced by the individual delay circuits 4 .",
"FIG.",
"9 is a block diagram illustrating the maximum value brightness method.",
"In FIG.",
"9 , a received signal from each transducer element, which is not shown in the figure, is delayed in a respective delay circuit 4 , 4 , .",
".",
".",
"by a predetermined amount.",
"The outputs delayed in the respective delay circuits 4 , 4 , .",
".",
".",
"are added in adder 5 ., and this output is input to maximum value detecting part 15 .",
"This maximum value detecting part 15 compares the input signal with the last input value, and, in case the input value is smaller than the last detected value, the focus data is slightly changed systematically in focus controlling part 14 .",
"Then, after the phasing of the received echo signal has been changed by this focus data, the output of adder 5 is inputted to the maximum value detecting part 15 , and judged again.",
"After repeating this operation, when the detected value is formed to converge at the maximum value, its data is used as focus data.",
"However, as shown in FIG.",
"4 and FIG.",
"5 , in an ultrasonic diagnostic apparatus in which a transmitting/receiving circular pattern 2 is formed by bundling transducer elements of a two-dimensional array into concentric rings to compose a multi-ring arrangement, in case there is a sound speed non-uniformity part 11 on the path from focus point 6 to the probe 3 in the object, since transducer elements 1 in the two-dimensional array are bundled in concentric rings as thus described, it is difficult to detect the phase difference due to said path difference for correcting for the influence of said sound speed non-uniformity part 11 .",
"Therefore, the phase difference of echo signals caused by said path difference, due to the existence of the sound speed non-uniformity part 11 , is not corrected.",
"As a result, the image quality is deteriorated because the ultrasonic beam becomes worse.",
"Thus, it is an object of the present invention to solve the above-mentioned problems by providing an ultrasonic diagnostic apparatus which is able to correct a focusing error by detecting the phase difference of echo signals which occur due to a difference in the ultrasonic propagating path, even when its multi-ring arrangement of transducers is composed by bundling transducers in a two-dimensional array for transducer elements in concentric rings."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>To achieve the foregoing object, an ultrasonic diagnostic apparatus is provided in accordance with the present invention, in which there is a probe comprising a plural number of transducer elements formed as a two-dimensional array for transmitting/receiving an ultrasound to an object to be examined.",
"A circular pattern of transducers for ultrasound transmitting/receiving is formed by bundling said two-dimensional array of transducer elements in concentric rings to form a multi-ring arrangement, and transmitting/receiving of an ultrasound is achieved with the application of a delay between each ring in said multi-ring arrangement, so that an ultrasonic image is formed by scanning said beam.",
"The ultrasonic diagnostic apparatus comprises means for measuring focusing error that is produced due to a sound speed non-uniformity in said object and for transmitting/receiving an ultrasonic beam that has been corrected based on the measured error by changing at least one of the bundling of said multi-ring arrangement of transducers or the delay time, and means for imaging the object by using an echo signal of the corrected ultrasonic beam.",
"In the apparatus of the present invention, the circular pattern formed by said multi-ring arrangement, in which transducer elements in the two-dimensional array are bundled in concentric rings in said probe is divided into a plural number of sections in a radial form from the center to the outside thereof.",
"And, the focusing error due to a sound speed non-uniformity in the object is measured between the ring sectors of each divided section and between each section, and the ultrasonic beam is corrected by feeding this measured value back to the delay circuits.",
"Furthermore, in the apparatus of the present invention, a bundled arrangement of transducer elements form a circular pattern that is different from that of said multi-ring arrangement composed by bundling said transducer elements of a two dimensional array in said probe in concentric rings.",
"And, the focusing error due to a sound speed non-uniformity in the object between each sector is measured, and the ultrasonic beam is corrected by feeding back this measured value to the delay circuits and returning the form of the multi-ring to an initial setting."
],
[
"FIELD OF THE INVENTION The present invention relates to an ultrasonic diagnostic apparatus of the type used for acquiring an ultrasonic image of a diagnostic part by scanning the interior of an object to be examined in real time with an ultrasonic beam formed with a two-dimensional transducer array; and, more particularly, the invention relates to an ultrasonic diagnostic apparatus which includes means for correcting a focusing error which occurs due to a difference in the length of propagating paths of ultrasound transmitted/received with a multi-ring type transducer in which a plural number of electronically transducers are bundled into concentric rings.",
"BACKGROUND OF THE INVENTION An apparatus having a plural number of transducer composed of a two-dimensional transducer array in which sector scanning is performed using an ultrasonic beam (in an arbitrary direction) generated by a transmitting/receiving circular pattern of transducers formed in the two-dimensional transducer array is known.",
"In this ultrasonic diagnostic apparatus, for example, if 64 64 transducers are disposed in the two-dimensional array, then the total number of transducers is 4,096.; and, ultrasonic scanning is performed with a separate delay control provided for each transducer element.",
"In this case, a beam-forming circuit with 4,096 channels is necessary.",
"Realizing a beam-forming circuit having such a large number of multi-channel delay circuits is difficult.",
"So, by thinning out the number of driven elements for forming one ultrasonic beam, the number of delay circuit channels in the beam-forming circuit is reduced.",
"But the S/N of signal acquired by thinning out the driven elements is deteriorated.",
"So, the apparatus is realized by comprising a beam-forming circuit having as many as delay channels possible.",
"For example, there is an apparatus that comprises a beam-forming circuit having a delay circuit of 256 channels for transmitting and 256 channels for receiving.",
"As shown in FIG.",
"4, a linear scanning expanded field of view obtained by moving the transmitting/receiving circular pattern of transducer elements 1, that are arrayed in a two-dimensional X, Y direction, and a scanning method in which a convex scanning is applied to two-dimensions are proposed.",
"In this case, the number of transducer elements 1 is more than in said sector scanning type apparatus.",
"So, for reducing the number of channels in the beam-forming circuit, an apparatus is known that produces an ultrasound transmitting/receiving circular pattern 2 by electronically bundling a plural number of transducer elements 1 in the two-dimensional array into a multi-ring arrangement with concentric rings, so as to give a consistent delay time to the transducer elements composing one ring of said multi-ring arrangement.",
"The apparatus transmits/receives an ultrasonic beam with a delay time between rings, and forms an ultrasonic image by moving said circular pattern 2 in X, Y directions.",
"As shown in FIG.",
"5, in said multi-ring arrangement, each ring is formed by the bundling of transducer elements in concentric rings of which the distance L1, L2 .",
".",
".",
", from the single focal spot F is almost the same, and the diameter of the most exterior ring is diameter 2 for ultrasound transmitting/receiving.",
"In this method, the bundling of transducer elements in concentric rings makes it possible to reduce greatly the number of channels in the beam forming circuit, which corresponds to the number of delay circuits, and the S/N of a signal acquired by using all elements in a circular pattern can be improved.",
"When considering the shape of an ultrasonic beam, a focusing calculation for an ultrasonic beam in the object to be examined is traditionally performed under the condition that the speed of sound in an ultrasonic propagation medium is uniform.",
"As shown in FIG.",
"6, a traditional apparatus comprises delay circuits 4, 4, .",
".",
".",
"comprising one per transducer element of the probe 3 having a plural number of transducers and an adder 5 for adding received signals output from these delay circuits 4, 4, .",
".",
".",
"Although reflected signals from focus point 6 in the object propagate through medium 7 to reach each transducer element, the difference in the path length from the focus point to each transducer element causes a difference in the arrival time of each reflected signal.",
"In this case, the reflected signals reach the transducer elements located in the center of probe 3 early, and, on the other hand, they reach the transducer elements at the ends late.",
"So, the shape of the wave surface 8 in the received signals is not linear.",
"Thus, the signals output from transducer elements in the center part of the probe are delayed with a large amount of delay time in delay circuits 4 corresponding to each received signal, and the signals output from transducer elements at the ends of the probe are delayed with a small amount of delay time.",
"The signals are then output to adder 5.With these delay operations, the wave surface 9 of the signal output from delay circuits 4 is linear since the signals have the same phase.",
"The received signals having the same phase, such as shown by wave surface 9 in this condition, are added in adder 5 so as to form the combined signal 10.But actually, as shown in FIG.",
"7, a sound speed non-uniformity part 11 typically exists on the path from the focus point 6 in the object to the probe 3, so that the wave surface of the received signal is disturbed, as shown at 8′.",
"In this case, when performing the delay operation, while assuming that the speed of sound is uniform, the wave surface of the received signals output from delay circuits 4 is distorted.",
"as shown at 9′, so that they do not have the same phase.",
"Accordingly.",
"the output produced in adder 5 does not increase in intensity as the signals are added, so that its intensity is small, as shown by signal 10′.",
"On the Contrary, there is a technology referred to as an adaptive ultrasonic imaging method which operates to correct the delay amount produced in said delay circuits 4 in accordance with the speed of sound in the medium.",
"In the adaptive ultrasonic imaging technology, a mutual correlation method for correcting the delay amount by correction processing of respectively received signals between adjacent channels, and a maximum value brightness method for searching for a brightness maximum while changing the delay amount of the delay circuits are known.",
"FIG.",
"8 is a block diagram which illustrates the mutual correlation method.",
"In FIG.",
"8, a signal received from each transducer element, which is not shown in the figure, is delayed by a predetermined amount by a respective delay circuit 4, 4, .",
".",
".",
"This delay is possible by use of analog delay circuits or digital delay circuits.",
"In this case, when outputs of adjacent channels in each transducer element, a mutual correlation processing is carried out with correlation device 12, and the phase difference between the outputs of adjacent channels can be obtained.",
"By detecting the phase difference value, transforming it to focus data in correction processing part 13, and feeding the transformed data back to focus controlling part 14, the delay amount produced by the individual delay circuits 4.FIG.",
"9 is a block diagram illustrating the maximum value brightness method.",
"In FIG.",
"9, a received signal from each transducer element, which is not shown in the figure, is delayed in a respective delay circuit 4, 4, .",
".",
".",
"by a predetermined amount.",
"The outputs delayed in the respective delay circuits 4, 4, .",
".",
".",
"are added in adder 5., and this output is input to maximum value detecting part 15.This maximum value detecting part 15 compares the input signal with the last input value, and, in case the input value is smaller than the last detected value, the focus data is slightly changed systematically in focus controlling part 14.Then, after the phasing of the received echo signal has been changed by this focus data, the output of adder 5 is inputted to the maximum value detecting part 15, and judged again.",
"After repeating this operation, when the detected value is formed to converge at the maximum value, its data is used as focus data.",
"However, as shown in FIG.",
"4 and FIG.",
"5, in an ultrasonic diagnostic apparatus in which a transmitting/receiving circular pattern 2 is formed by bundling transducer elements of a two-dimensional array into concentric rings to compose a multi-ring arrangement, in case there is a sound speed non-uniformity part 11 on the path from focus point 6 to the probe 3 in the object, since transducer elements 1 in the two-dimensional array are bundled in concentric rings as thus described, it is difficult to detect the phase difference due to said path difference for correcting for the influence of said sound speed non-uniformity part 11.Therefore, the phase difference of echo signals caused by said path difference, due to the existence of the sound speed non-uniformity part 11, is not corrected.",
"As a result, the image quality is deteriorated because the ultrasonic beam becomes worse.",
"Thus, it is an object of the present invention to solve the above-mentioned problems by providing an ultrasonic diagnostic apparatus which is able to correct a focusing error by detecting the phase difference of echo signals which occur due to a difference in the ultrasonic propagating path, even when its multi-ring arrangement of transducers is composed by bundling transducers in a two-dimensional array for transducer elements in concentric rings.",
"SUMMARY OF THE INVENTION To achieve the foregoing object, an ultrasonic diagnostic apparatus is provided in accordance with the present invention, in which there is a probe comprising a plural number of transducer elements formed as a two-dimensional array for transmitting/receiving an ultrasound to an object to be examined.",
"A circular pattern of transducers for ultrasound transmitting/receiving is formed by bundling said two-dimensional array of transducer elements in concentric rings to form a multi-ring arrangement, and transmitting/receiving of an ultrasound is achieved with the application of a delay between each ring in said multi-ring arrangement, so that an ultrasonic image is formed by scanning said beam.",
"The ultrasonic diagnostic apparatus comprises means for measuring focusing error that is produced due to a sound speed non-uniformity in said object and for transmitting/receiving an ultrasonic beam that has been corrected based on the measured error by changing at least one of the bundling of said multi-ring arrangement of transducers or the delay time, and means for imaging the object by using an echo signal of the corrected ultrasonic beam.",
"In the apparatus of the present invention, the circular pattern formed by said multi-ring arrangement, in which transducer elements in the two-dimensional array are bundled in concentric rings in said probe is divided into a plural number of sections in a radial form from the center to the outside thereof.",
"And, the focusing error due to a sound speed non-uniformity in the object is measured between the ring sectors of each divided section and between each section, and the ultrasonic beam is corrected by feeding this measured value back to the delay circuits.",
"Furthermore, in the apparatus of the present invention, a bundled arrangement of transducer elements form a circular pattern that is different from that of said multi-ring arrangement composed by bundling said transducer elements of a two dimensional array in said probe in concentric rings.",
"And, the focusing error due to a sound speed non-uniformity in the object between each sector is measured, and the ultrasonic beam is corrected by feeding back this measured value to the delay circuits and returning the form of the multi-ring to an initial setting.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a block diagram showing the overall arrangement of an embodiment of an ultrasonic diagnostic apparatus according to the present invention.",
"FIG.",
"2 is a diagram of a probe used in the first embodiment of the present invention.",
"FIG.",
"3 is a diagram of a probe used in the second embodiment of the present invention.",
"FIG.",
"4 is a diagrammatic plan view showing an example in which a multi-ring arrangement is set to probe in accordance with the present invention or in a traditional ultrasonic diagnostic apparatus, and in which transmitting and receiving of an ultrasonic beam and two-dimensional scanning are performed.",
"FIG.",
"5 is a diagram showing a principal of said multi-ring arrangement.",
"FIG.",
"6 is a diagram showing the formation of a traditional ultrasonic beam.",
"FIG.",
"7 is a diagram showing the beam formation in a case where a sound speed non-uniformity part is present in the medium when said ultrasonic beam is formed.",
"FIG.",
"8 is a block diagram showing a circuit arrangement for correcting the delay amount by using a mutual correlation method for producing adaptive image processing.",
"FIG.",
"9 is a block diagram showing a circuit arrangement for correcting the delay amount by using a maximum value brightness method for producing an adaptive image processing.",
"THE BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an embodiment of the present invention will be described in detail based on the accompanying drawings.",
"FIG.",
"1 is a block diagram showing an embodiment of an ultrasonic diagnostic apparatus according to the present invention.",
"This ultrasonic diagnostic apparatus forms an ultrasonic beam with a two-dimensional transducer array and acquires an ultrasonic image of a diagnostic part in the interior of an object to be examined by scanning an ultrasonic beam over the object in real time.",
"As shown in FIG.",
"1, it comprises a probe 20, an element selecting data part 21, a transmitting part 22, abeam-forming part 23, a transmitting/receiving separation circuit 24, a signal processing part 25, a scan converter 26, a monitor 27, and a controlling part 28.The probe 20 transmits an ultrasound to an object to be examined and receives its echo.",
"It is composed of a plural number of transducer elements 29, 29, .",
".",
".",
"arrayed two-dimensionally.",
"These transducer element 29, 29, .",
".",
".",
"are arrayed two-dimensionally such as 1˜m in the x direction and 1˜n in the y direction in planar view, as shown in FIG.",
"4.And, the transducer elements 29, that are arrayed two-dimensionally, are also bundled electronically to form a multi-ring arrangement in which there are concentric rings, and the multi-ring arrangement ahs the form of an ultrasonic transmitting/receiving circular pattern 30.A delay time is given between each ring of said multi-ring arrangement to transmit and receive the ultrasonic beam.",
"The ultrasonic beam scanning is performed by moving said circular pattern 30 at each cycle of the transmitting/receiving of the ultrasound, whereby an ultrasonic image is formed.",
"In addition, said multi-ring arrangement is made, for example, with fresnel-bundles.",
"In addition, connection switch groups 31 are connected for coupling an arbitrary delay channel in the beam-forming circuits to be described in later to selected transducer elements.",
"Furthermore, a switching control part 32 for controlling the switching operation is connected to the connection switch groups 31.Element selecting data part 21 memorizes element selecting data needed to form the transmitting/receiving circular pattern 30.In this regard, element selecting data read out from element selecting data part 21 is transferred to switching control part 32; and, by controlling said switching control part 32, the switching on and off of connection switch groups 31 is controlled to form the transmitting/receiving circular pattern 30.Transmitting part 22 supplies a transmitting signal for emitting an ultrasound to the respective ultrasonic transducer elements with the application of a delay time such that the ultrasound transmitted from each transducer element which forms the transmitting/receiving circular pattern 30 of said probe 20 is focused at a desired focus point set in the object.",
"The beam-forming part 23 performs a desired focus process on respective reflected echo signals received by the transducer elements 29 of said probe 20 and forms a receiving beam by phasing and adding these echo signals.",
"The transmitting/receiving separation circuit 24 changes the connection of said transmitting part 22 and beam-forming part 23 to selected transducer elements 29 to control whether an ultrasound is transmitted or received.",
"Signal processing part 25 inputs the signal output from said beam-forming part 23 and obtains the data of one scanning line by predetermined processing, such as detecting, compression, filtering processing, edge emphasizing etc.",
"The scan converter 26 inputs the data output from said signal processing part 25 and forms image data for display on the monitor 27, and it also performs scan conversion between ultrasonic scanning and scanning for display and interpolation processing of image data or the like.",
"Furthermore, monitor 27 displays the data received from said scan converter 26 as an ultrasonic image.",
"On the display screen, a three-dimensional image or an arbitrary slice image is displayed.",
"Controlling part 28 controls the operation of each element.",
"In accordance with the present invention, means is provided for measuring a delay error in receiving a signal due to the presence of a sound speed non-uniformity part in said object, and for correcting either the bundling of transducer elements in the multi-ring arrangement or the delay time or both.",
"In a first embodiment, the circular pattern 30, composed of the multi-ring arrangements formed by electronically bundling the two-dimensional array transducer elements 29, 29, .",
".",
".",
"of said probe 20 in concentric rings, is divided into a plural number of regions in radial form from the center to the outside; and, between the diameter of each divided region, the delay error (focusing error) due to the presence of a sound speed non-uniformity part (refer to 11 in FIG.",
"7) in the object is measured to the correct beam.",
"A probe according to this first embodiment has transducer elements arranged two-dimensionally.",
"Although this is omitted in FIG.",
"2, actually they are arrayed with a matrix arrangement in the directions X and Y as shown in FIG.",
"4.The transducer elements are electronically bundled into a ring shape to form a circular pattern of annular array (multi-ring).",
"This circular pattern is moved in the X direction or Y direction according to a cycle of transmitting/receiving for performing a linear scanning or a convex scanning with an ultrasonic beam, or sector scanning with the beam without moving the circular pattern.",
"With such linear scanning, convex scanning, or sector scanning, with this two-dimensional probe, image data of a three-dimensional volume is acquired from the object.",
"This image data is taken into scan converter 26 and transformed to the image data which is displayed on the screen of the monitor 27 as an ultrasonic slice image or a three-dimensional ultrasonic image.",
"To provide a simple illustration, a basic circular pattern for acquiring one image is shown in FIG.",
"2.In FIG.",
"2, the circular pattern composed of a multi-ring arrangement is effected by bundling said two-dimensional array transducer elements electronically in concentric rings so that the circular pattern is divided into a plural number of regions in radial form from the center to the outside.",
"For example, element selecting data is transferred from element selecting data part 21 shown in FIG.",
"1 to switching control part 32.With the controlling of said switching control part 32, the dividing lines L1, L2 crossing the center of said multi-ring arrangement are formed, and these dividing lines L1, L2 divide the multi-ring arrangement into four sectors Sa, Sb, Sc, Sd.",
"Each of the divided sectors Sa˜Sd is respectively connected to transmitting part 22 and beam-forming part 23, as shown in FIG.",
"1.And, to the multi-rings separated in each sector, delay data calculated on the condition that the speed of sound is uniform in medium 7 (refer to FIG.",
"6) is added.",
"That is to say, in FIG.",
"2, transmitting part 22 and beam-forming part 23 are connected respectively to ring sections a1, a2, a3, a4.b1,.b2, b3, b4, c1, c2, C3, C4, d1, d2, d3, d4 in each of the divided sectors Sa˜Sd.",
"Thus, delay control is performed individually for the respective divided rays.",
"Accordingly, the ultrasonic beam is focused at some point with the prescribed delay.",
"At this time, if the speed of sound in medium 7 is consistent with the speed of sound when the delay data is calculated, then the received signal increases in amplitude with the same phase adding because the phase surface in the received signal after receiving phasing is the same phase as the case shown in FIG.",
"6.On the other hand, if a sound speed non-uniformity part 11 exists in medium 7, then the received signal will be small because the phase surface in the received signal after receiving phasing is different, as shown in FIG.",
"7.In accordance with the present invention, as shown in FIG.",
"2, the multi-ring arrangement is divided into, for example, four sectors Sa˜Sd, so that the phase error (this corresponds to focusing error) is calculated with respect to the correlation of received signals after receiving phasing between the channel of transducer elements in each divided sector and is corrected in the same manner as shown in FIG.",
"8.That is to say, to each received signal after receiving phasing, the phase difference is calculated by mutual correlation between adjacent sectors in each ring divided in each sector Sa˜Sd, as shown in FIG.",
"2, and correlation processing is performed between each ring in each sector.",
"For example, in FIG.",
"2, in the multi-ring arrangement divided into four sectors Sa˜Sd, mutual correlation processing is performed between adjacent sectors, such as a1 and b1, b1 and c1, c1 and d1, d1 and a1 in the most interior ring.",
"In addition, mutual correlation processing is performed between adjacent sectors, such as a2 and b3, b2 and c2, c2 and d2, d2 and a2 in the second ring.",
"Furthermore, in the third and the fourth rings as well, mutual correlation processing is performed between adjacent sectors.",
"Also, to acquire a relationship between each ring in each sector, the correlation processing between adjacent rings a1 and a2, a2 and a3, a3 and a4 in sector Sa is performed.",
"In the sector Sb, the correlation processing is performed between adjacent rings b1 and b2, b2 and b3, b3 and b4.Furthermore, in sectors Sc, Sd as well, correlation processing is performed between adjacent rings.",
"In this regard, the method of correlation processing is not restricted to the above-described sequence.",
"Other methods can be used.",
"On the other hand, it is preferable to correct the phase error by calculating the delay amount that brings about maximum intensity by changing the delay amount of the beam-forming part between channels of transducer elements in each divided sector in the same way as shown in FIG.",
"9, under the condition that, for example, a multi-ring arrangement is divided into four sectors Sa˜Sd, as shown in FIG.",
"2.That is, it is preferable to correct the phase error by calculating the delay amount that brings about a maximum intensity by changing the delay amount consecutively to each ring, which is divided into each sector Sa˜Sd, a1,.a2, a3, a4, b1, b2, b3, b4, c1, c2, C3, C4, d1, d2, d3, d4.Next, a second embodiment of the present invention will be described.",
"In this second embodiment, it is preferable to form a bundled circular pattern which is different from the circular pattern formed by bundling transducer elements arrayed two-dimensionally in said probe 20 to form a multi-ring arrangement having concentric rings, and to measure delay error due to the presence of a sound speed non-uniformity part in the object between each circular pattern (refer to 11 in FIG.",
"7) and to correct the beam.",
"FIG.",
"3 is a diagram of a probe representing the second embodiment.",
"This probe has a two-dimensional array of transducer elements.",
"Each transducer element is connected to a respective delay circuit, and an ideal delay is given for some focus point to calculate delay error by correlating the outputs of the transducer elements.",
"In the same way as shown in FIG.",
"5, when a multi-ring arrangement is bundled, if the bundled transducer elements forming one ring are formed, for example, such that the difference in the travel time (distance) of the ultrasound to the focus point F is in the range of/10 (is wave-length of the ultrasonic beam), then the delay error is corrected by adding said calculated delay error to the travel time.",
"For example, it is assumed that a specified transducer element 33 has a delay error in the multi-ring arrangement n1, n2, n3, n4 shown in FIG.",
"3.Although transducer element 33 is bundled into ring n2 in the ideal state, when the delay error is added to it, a correction is performed such that it is bundled into ring n3, whereby the delay error is corrected.",
"And, after having calculated a correction value of delay error, the transducer element 33 is bundled so as to return to the ring in the original ideal state.",
"Thus, it is not the diameter for forming an ultrasonic beam actually (bundled ring n1˜n4), but the delay error that is detected with transducer element in another bundled ring.",
"And, the correction is performed so as to form the bundled ring in consideration of the delay error.",
"In the case mentioned above, if a transmitter and a beam-forming circuit are connected to all of the two-dimensional array transducer elements, as shown in FIG.",
"3, then the circuit scale is large.",
"Thus, for example, it is preferable to bundle adjacent transducer elements into rectangular blocks and connect the transmitter and the beam-forming circuits to bundled transducer elements as a unit.",
"In addition, also in the second embodiment, it is preferable to correct the phase error by calculating the delay amount in which the output signal is a maximum value by changing the delay amount of the beam-forming part, as shown in FIG.",
"9.As thus described, the present invention comprises means for measuring focusing error which occurs due to the presence of a sound speed non-uniformity part in the object and for correcting one or both of bundling or delay time of the multi-ring arrangement having two-dimensional array transducer elements in concentric rings.",
"Thus, the interior of the object can be imaged with a corrected beam, so that the image quality of the ultrasonic image is improved."
]
] | Patent_10169979 |
[
[
"Quad cable",
"The invention relates to a cable (10) having an elongate core (20) made of a first dielectric material and having a core outer surface (25); and a plurality of elongate conducting elements (30) arranged about the elongate core (20).",
"The elongate conducting elements (30) have an inner conductor (40) surrounded by an insulating layer (50) which is made of a second dielectric material.",
"The outer surface of the inner conductor (40) is divided into a first outer surface area (60) and a second outer surface are a (70) and the first outer surface area (60) is attached, for example by sintering, to the core outer surface (25).",
"The first and second dielectric materials are preferably expanded PTFE.",
"The cable (10) finds application in a streamer (100) for use in seismographic surveys of the ocean bottom sub surface."
],
[
"1.Cable (10) comprising: an elongate core (20) made of a first dielectric material and having a core outer surface (25); and a plurality of elongate conducting elements (30) disposed about the elongate core (20), the elongate conducting elements (30) having an inner conductor (40) surrounded by an insulating layer (50) made of a second dielectric material, and an outer surface divided into a first outer surface area (60) and a second outer surface area (70), wherein the first outer surface area (60) is attached to the core outer surface (25).",
"2.Cable (10) according to claim 1 wherein the second outer surface area (70) is exposed to the environment.",
"3.Cable (10) according to claim 1 wherein a jacket is disposed about the plurality of elongate elements (30).",
"4.Cable (10) according to claim 1 wherein said first dielectric material is selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"5.Cable (10) according to claim 4 wherein said first dielectric material is expanded polytetrafluoroethylene.",
"6.Cable (10) according to claim 1 wherein said second dielectric material is selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"7.Cable (10) according to claim 6 wherein said second dielectric material is expanded polytetrafluoroethylene.",
"8.Cable (10) according to claim 1 wherein the first outer surface area (50) is sintered to the core outer surface (25).",
"9.Cable (10) according to claim 1 wherein the insulating layer (50) comprises a plurality of insulating layers (50a, 50b, 50c).",
"10.Cable (10) according to claim 9 wherein an inner one of the plurality of insulating layers (50a, 50b, 50c) is made of expanded PTFE and an outer one of the plurality of insulating layers (50a, 50b, 50c) is made of full density PTFE.",
"11.Cable (10) according to claim 1 wherein said inner conductor (40) is made from the group of conducting materials selected from copper, nickel-plated copper, tin-plated copper, silver-plated copper, tin-plated alloys, silver-plated alloys or copper alloys.",
"12.Cable (10) according to claim 11 wherein said inner conductor (40) is made from silver-plated copper.",
"13.Cable (10) according to claim 1 having four elongate conducting elements (30).",
"14.Cable (10) according to claim 13 wherein said four elongate conducting elements (30) are equidistantly disposed about the elongate core (20).",
"15.Cable (10) according to claim 1 wherein said plurality of elongate conducting elements (30) are helically disposed about the elongate core (20).",
"16.Streamer (100) having a jacket (110) filled with fluid (120) and having a cable (10) disposed within said fluid (120), the cable (10) comprising: an elongate core (20) made of a first dielectric material and having a core outer surface (25); and a plurality of elongate conducting elements (30) disposed about the elongate core (20), the elongate conducting elements (30) having an inner conductor (40) surrounded by an insulating layer (50) made of a second dielectric material, and an outer surface divided into a first outer surface area (60) and a second outer surface area (70), wherein the first outer surface area (60) is attached to the core outer surface (25).",
"17.Streamer (100) according to claim 16 wherein said first dielectric material is selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"18.Streamer (100) according to claim 17 wherein said first dielectric material is expanded polytetrafluoroethylene.",
"19.Streamer (100) according to claim 16 wherein said second dielectric material is selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"20.Streamer (100) according to claim 19 wherein said second dielectric material is expanded polytetrafluoroethylene.",
"21.Streamer (100) according to claim 16 wherein the first outer surface area (50) is sintered to the core outer surface (25).",
"22.Streamer (100) according to claim 16 wherein the insulating layer (50) comprises a plurality of insulating layers (50a, 50b, 50c).",
"23.Streamer (100) according to claim 22 wherein an inner one of the plurality of insulating layers (50a, 50b, 50c) is made of expanded PTFE and an outer one of the plurality of insulating layers (50a, 50b, 50c) is made of full density PTFE.",
"24.Streamer (100) according to claim 16 wherein said inner conductor (40) is made from the group of conducting materials selected from copper, nickel-plated copper, tin-plated copper, silver-plated copper, tin-plated alloys, silver-plated alloys or copper alloys.",
"25.Streamer (100) according to claim 24 wherein said inner conductor (40) is made from silver-plated copper.",
"26.Streamer (100) according to claim 16 having four elongate conducting elements (30).",
"27.Streamer (100) according to claim 16 wherein said four elongate conducting elements (30) are equidistantly disposed about the elongate core (20).",
"28.Streamer (100) according to claim 16 wherein said plurality of elongate conducting elements (30) are helically disposed about the elongate core (20).",
"29.Method for the construction of a cable (10) comprising the following steps: providing an elongate core (20) made of a first dielectric material, disposing about the elongate core (20) a plurality of elongate conducting elements (30) having an insulating layer (50) made of a second dielectric material, attaching the plurality of elongate conducting elements (30) to the elongate core (20).",
"30.Method according to claim 29 wherein the step of attaching the plurality of elongate conducting elements (30) to the elongate core (20) is carried out by heat treatment.",
"31.Method according to claim 29 wherein the step of attaching the plurality of elongate conducting elements (30) to the elongate core (20) is carried out by placing an adhesive between a core outer surface (35) of the elongate core (20) and a first outer surface area (60) of the plurality of elongate conducting elements (30) and curing the adhesive.",
"32.Method according to claim 29 wherein the step of attaching the plurality of elongate conducting elements (30) to the elongate core (20) is carried out by sintering a core outer surface (25) of the elongate core (20) to a first outer surface area (60) of the plurality of elongate conducting elements (30).",
"33.Method according to claim 32 wherein the step of sintering is carried out in a salt bath.",
"34.Method according to claim 32 wherein the step of sintering is carried out in an oven.",
"35.Method according to claim 32 wherein the step of sintering is carried out at a temperature between 320° C. and 420° C. 36.Method according to claim 35 wherein the step of sintering is carried out at a temperature of approximately 390° C. 37.Cable (10) according to claim 1 wherein the average impedance of the plurality of elongate conducting elements (30) on wrapping a one metre length of the cable about a mandrel of 11 mm diameter varies by less than 5%."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Quad cables are known in the art, for example U.S. Pat.",
"No.",
"5,521,333 (Kobayawshi et al.)",
"assigned to Sumitomo Electric Industries, Ltd., teaches a transmission cable including four insulated electric wires with a low dielectric central interposition member or elongate core.",
"In the quad cable of this patent the elongate core is preferably made of polyethylene.",
"The insulation of the insulated electric wires are also preferably made from polyethylene.",
"A jacket of polyvinylchloride is disposed about the insulated wires.",
"The insulated electric wires of this patent are held in place about the elongate core by means of polyester tape wound about the wires.",
"Quad cables incorporating an elongate core or filler made from expanded polytetrafluoroethylene are known from U.S. Pat.",
"No.",
"5,574,250 (Hardie et al.)",
"assigned to W.L.Gore & Associates.",
"In this patent, the four insulated electric wires are held in position by a spacer layer.",
"A further quad cable is known from DE-A-31 04 429 (Fröscher) assigned to Siemens in which two signal conductors and two earth wires are disposed about a central core made of a dielectric material.",
"This application does not disclose the means by which the signal conductors or earth wires are held in place about the core.",
"The use of electrical cables in marine environments is known to be problematic since there is a risk that water may penetrate into the cable in the event of a cut or breach in the outer jacket.",
"The most common method of protecting the cable in the art involves the use of flooding materials to fill the interstices of the cable.",
"Such flooding materials include synthetic polymers and petroleum greases.",
"One known disadvantage of the use of these materials is that the dielectric constant of the cable is changed by the addition of these materials in the cable.",
"Furthermore, as the conductors move within the cable, the relative position of the interstices move which affects the electrical properties of the cable.",
"It has been particularly difficult in the past to provide water blocking quad cables since additional handling and processing steps are required which are both messy and inefficient.",
"U.S. Pat.",
"No.",
"5,949,018 (Esker) assigned to CommScope, Inc., teaches a water blocked cable construction which provides one way of solving this problem.",
"In this patent, a water swellable flooding material is taught which is placed between two metal braided shields.",
"The material used is a hydrogel polymer which is solid when dry but swells up and becomes gel-like on immersion in water.",
"U.S. Pat.",
"No.",
"5,734,126 (Siekierka et al.)",
"assigned to Belden Wire & Cable Company teaches a twisted pair cable which is suitable for high frequency signal transmission.",
"The cable has two solid, stranded or hollow conductor wires which are surrounded respectively by a cylindrical dielectric insulation layer.",
"Each of the conductors is disposed centrally within and thus substantially concentric with the corresponding insulation.",
"The insulated conductors are joined or bonded together along their entire length by an appropriate adhesive.",
"Alternatively, the adjacent dielectric insulation layers can be bonded together by causing material contact while the dielectrics are at elevated temperature and then cooling to provide a joined cable having no adhesive.",
"Similarly U.S. Pat.",
"No.",
"5,382,390 (Hubis et al.)",
"assigned to W.L.Gore & Associates, Inc., teaches a twisted pair in which the dielectric insulators of the insulated conductors are bonded together by heating.",
"Neither the Siekerka nor the Hubis patent make reference, however, to quad cables.",
"Indeed their teachings are not applicable since it is necessary in a quad cable to maintain a fixed spatial relationship between the individual conductors, e.g.",
"by use of a central spacing member or core, and this is not possible to implement using the teachings of these two patents."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It is the object of the invention to provide an improved quad cable.",
"It is furthermore an object of the invention to ensure that the individual conductors of a quad cable maintain a substantially fixed spatial relationship to each other throughout the length of the cable.",
"It is furthermore an object of the invention to improve the flexibility of the cable whilst maintaining its electrical properties.",
"It is yet a further object of the invention to provide a multi-conductor cable for use in a fluid environment such as a marine environment or in oil fields.",
"These and other objects of the invention are solved by providing a cable comprising an elongate core made of a first dielectric material with a core outer surface.",
"Four or more (a plurality of) elongate conducting elements are disposed about the elongate core.",
"Each of the elongate conducting elements has an inner conductor which is surrounded by an insulating layer made of a second dielectric material.",
"The outer surface of the insulating layer of the elongate conducting elements is divided into a first outer surface area and a second outer surface area.",
"The first outer surface area of the elongate conducting elements is attached to the core outer surface.",
"The use of the core in the cable provides a spacer in the centre of the cable about which the elongate conducting elements can be arranged.",
"In the case of the quad cable four elongate conducting elements are provided.",
"The outer surface of the insulators of the elongate conducting elements are attached to the outer surface of the core so that they elongate elements remain in a fixed position even when the cable is flexed.",
"Thus the electrical properties of the cable, e.g.",
"attenuation or characteristic impedance, remain substantially unchanged during flexing since the distance between the inner conductors of each of the elongate conducting elements remains substantially unchanged.",
"In one advantageous body of the invention, the second outer surface area of each of the elongate conducting elements is exposed to the environment, i.e.",
"the cable is not provided with an outer jacket about the elongate conducting elements.",
"Such a construction is particularly advantageous in environments in which a fluid, such as sea water or oil, could leak underneath the jacket and thus between the jacket and the elongate conducting elements, The fluid will locally alter the dielectric constant of the cable and thus affect the electrical properties of the cable.",
"Both the first and second dielectric materials are advantageously selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"Most preferably expanded polytetrafluoroethylene is used for both dielectric materials since this material has a low dielectric constant and can be bonded to each other by sintering.",
"In one embodiment of the cable the insulating layer comprises a plurality of insulating layers with different dielectric constants, for example an inner one of the plurality of insulating layers is made of expanded PTFE and an outer one of the plurality of insulating layers is made of full density PTFE.",
"This has the advantage that the effective dielectric constant of the elongate conducting elements and thus of the cable can be tailored to the required value.",
"The invention also provides for a method for the construction of a cable comprising the following steps: providing an elongate core made of a first dielectric material, disposing about the elongate core a plurality of elongate conducting elements having an insulating layer made of a second dielectric material, attaching the plurality of elongate conducting elements to the elongate core.",
"In this method the step of attaching the plurality of elongate conducting elements to the elongate core is carried out by heat treatment and preferably by sintering since this provides, with the correct choice of materials, an extremely strong bond between the plurality of elongate conducting elements and the elongate core.",
"The sintering can be carried out in a salt bath or in an oven at a temperature between 320° C. and 420° C. and preferably of approximately 390° C. The cable of the invention can be incorporated in a streamer for use in seismic surveys.",
"Such streamers need to be flushable, i.e.",
"the oil within the streamer can be flushed out of the interior of the streamer if required.",
"Since—as explained above—there are no interstices between the conducting elements in which the oil remains trapped and thus locally affects the dielectric constant, the electrical properties of the signal carrying cable are greatly improved."
],
[
"FIELD OF THE INVENTION The invention relates to a cable having a core to which are attached a plurality of elongate conducting elements.",
"In particular four conducting elements are used to form a quad cable.",
"BACKGROUND OF THE INVENTION Quad cables are known in the art, for example U.S. Pat.",
"No.",
"5,521,333 (Kobayawshi et al.)",
"assigned to Sumitomo Electric Industries, Ltd., teaches a transmission cable including four insulated electric wires with a low dielectric central interposition member or elongate core.",
"In the quad cable of this patent the elongate core is preferably made of polyethylene.",
"The insulation of the insulated electric wires are also preferably made from polyethylene.",
"A jacket of polyvinylchloride is disposed about the insulated wires.",
"The insulated electric wires of this patent are held in place about the elongate core by means of polyester tape wound about the wires.",
"Quad cables incorporating an elongate core or filler made from expanded polytetrafluoroethylene are known from U.S. Pat.",
"No.",
"5,574,250 (Hardie et al.)",
"assigned to W.L.Gore & Associates.",
"In this patent, the four insulated electric wires are held in position by a spacer layer.",
"A further quad cable is known from DE-A-31 04 429 (Fröscher) assigned to Siemens in which two signal conductors and two earth wires are disposed about a central core made of a dielectric material.",
"This application does not disclose the means by which the signal conductors or earth wires are held in place about the core.",
"The use of electrical cables in marine environments is known to be problematic since there is a risk that water may penetrate into the cable in the event of a cut or breach in the outer jacket.",
"The most common method of protecting the cable in the art involves the use of flooding materials to fill the interstices of the cable.",
"Such flooding materials include synthetic polymers and petroleum greases.",
"One known disadvantage of the use of these materials is that the dielectric constant of the cable is changed by the addition of these materials in the cable.",
"Furthermore, as the conductors move within the cable, the relative position of the interstices move which affects the electrical properties of the cable.",
"It has been particularly difficult in the past to provide water blocking quad cables since additional handling and processing steps are required which are both messy and inefficient.",
"U.S. Pat.",
"No.",
"5,949,018 (Esker) assigned to CommScope, Inc., teaches a water blocked cable construction which provides one way of solving this problem.",
"In this patent, a water swellable flooding material is taught which is placed between two metal braided shields.",
"The material used is a hydrogel polymer which is solid when dry but swells up and becomes gel-like on immersion in water.",
"U.S. Pat.",
"No.",
"5,734,126 (Siekierka et al.)",
"assigned to Belden Wire & Cable Company teaches a twisted pair cable which is suitable for high frequency signal transmission.",
"The cable has two solid, stranded or hollow conductor wires which are surrounded respectively by a cylindrical dielectric insulation layer.",
"Each of the conductors is disposed centrally within and thus substantially concentric with the corresponding insulation.",
"The insulated conductors are joined or bonded together along their entire length by an appropriate adhesive.",
"Alternatively, the adjacent dielectric insulation layers can be bonded together by causing material contact while the dielectrics are at elevated temperature and then cooling to provide a joined cable having no adhesive.",
"Similarly U.S. Pat.",
"No.",
"5,382,390 (Hubis et al.)",
"assigned to W.L.Gore & Associates, Inc., teaches a twisted pair in which the dielectric insulators of the insulated conductors are bonded together by heating.",
"Neither the Siekerka nor the Hubis patent make reference, however, to quad cables.",
"Indeed their teachings are not applicable since it is necessary in a quad cable to maintain a fixed spatial relationship between the individual conductors, e.g.",
"by use of a central spacing member or core, and this is not possible to implement using the teachings of these two patents.",
"SUMMARY OF THE INVENTION It is the object of the invention to provide an improved quad cable.",
"It is furthermore an object of the invention to ensure that the individual conductors of a quad cable maintain a substantially fixed spatial relationship to each other throughout the length of the cable.",
"It is furthermore an object of the invention to improve the flexibility of the cable whilst maintaining its electrical properties.",
"It is yet a further object of the invention to provide a multi-conductor cable for use in a fluid environment such as a marine environment or in oil fields.",
"These and other objects of the invention are solved by providing a cable comprising an elongate core made of a first dielectric material with a core outer surface.",
"Four or more (a plurality of) elongate conducting elements are disposed about the elongate core.",
"Each of the elongate conducting elements has an inner conductor which is surrounded by an insulating layer made of a second dielectric material.",
"The outer surface of the insulating layer of the elongate conducting elements is divided into a first outer surface area and a second outer surface area.",
"The first outer surface area of the elongate conducting elements is attached to the core outer surface.",
"The use of the core in the cable provides a spacer in the centre of the cable about which the elongate conducting elements can be arranged.",
"In the case of the quad cable four elongate conducting elements are provided.",
"The outer surface of the insulators of the elongate conducting elements are attached to the outer surface of the core so that they elongate elements remain in a fixed position even when the cable is flexed.",
"Thus the electrical properties of the cable, e.g.",
"attenuation or characteristic impedance, remain substantially unchanged during flexing since the distance between the inner conductors of each of the elongate conducting elements remains substantially unchanged.",
"In one advantageous body of the invention, the second outer surface area of each of the elongate conducting elements is exposed to the environment, i.e.",
"the cable is not provided with an outer jacket about the elongate conducting elements.",
"Such a construction is particularly advantageous in environments in which a fluid, such as sea water or oil, could leak underneath the jacket and thus between the jacket and the elongate conducting elements, The fluid will locally alter the dielectric constant of the cable and thus affect the electrical properties of the cable.",
"Both the first and second dielectric materials are advantageously selected from the group of polymers consisting of polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"Most preferably expanded polytetrafluoroethylene is used for both dielectric materials since this material has a low dielectric constant and can be bonded to each other by sintering.",
"In one embodiment of the cable the insulating layer comprises a plurality of insulating layers with different dielectric constants, for example an inner one of the plurality of insulating layers is made of expanded PTFE and an outer one of the plurality of insulating layers is made of full density PTFE.",
"This has the advantage that the effective dielectric constant of the elongate conducting elements and thus of the cable can be tailored to the required value.",
"The invention also provides for a method for the construction of a cable comprising the following steps: providing an elongate core made of a first dielectric material, disposing about the elongate core a plurality of elongate conducting elements having an insulating layer made of a second dielectric material, attaching the plurality of elongate conducting elements to the elongate core.",
"In this method the step of attaching the plurality of elongate conducting elements to the elongate core is carried out by heat treatment and preferably by sintering since this provides, with the correct choice of materials, an extremely strong bond between the plurality of elongate conducting elements and the elongate core.",
"The sintering can be carried out in a salt bath or in an oven at a temperature between 320° C. and 420° C. and preferably of approximately 390° C. The cable of the invention can be incorporated in a streamer for use in seismic surveys.",
"Such streamers need to be flushable, i.e.",
"the oil within the streamer can be flushed out of the interior of the streamer if required.",
"Since—as explained above—there are no interstices between the conducting elements in which the oil remains trapped and thus locally affects the dielectric constant, the electrical properties of the signal carrying cable are greatly improved.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a quad cable without jacket according to the invention.",
"FIG.",
"2 shows a quad cable with jacket according to the invention.",
"FIG.",
"3 shows a manufacturing apparatus for this invention.",
"FIG.",
"4 shows a streamer incorporating a quad cable of the invention.",
"FIG.",
"5 shows a further embodiment of a quad cable with a jacket.",
"DETAILED DESCRIPTION OF THE INVENTION A quad cable 10 comprising found elongate conducting elements 30 disposed about an elongate core 20 is shown in FIG.",
"1.The elongate core 20 is made of a dielectric material such as polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"Preferably expanded polytetrafluoroethylene such as that described in U.S. Pat.",
"No.",
"3,953,556, U.S. Pat.",
"No.",
"4,187,390 or U.S. Pat.",
"No.",
"4,443,657 is used for the elongate core.",
"The elongate core 20 has a core outer surface 25.The elongate elements 30 are made of an inner conductor 40 of AWG size, for example, 20 or 22 surrounded by an insulating layer 50.The inner conductor 40 can be made from any conducting material such as copper, nickel-plated copper, tin-plated copper, silver-plated copper, tin-plated alloys, silver-plated alloys or copper alloys.",
"Alternatively the inner conductor could be an optical fibre surrounded by a cladding layer.",
"The insulating layer 50 is made of a dielectric material such as polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"Preferably expanded polytetrafluoroethylene such as that described in U.S. Pat.",
"No.",
"3,953,556, U.S. Pat.",
"No.",
"4,187,390 or U.S. Pat.",
"No.",
"4,443,657 is used.",
"The elongate elements 30 have an outer surface area which is divided into a first outer surface area 60 and a second outer surface area 70.The materials from which the insulating layer 50 and the elongate core 20 are made are chosen such that the two elongate core 20 and the insulating layer 50 can be attached to each other by adhesive or direct bonding.",
"Preferably the attachment is made by direct bonding and most preferably by sintering as will be explained below.",
"The insulating layer 50 and the elongate core 20 are preferably made from the same material.",
"The attachment between the insulating layer 50 and the elongate core is made at the first outer surface area 60.The embodiment of the invention in FIG.",
"1 shows four elongate conducting elements 30 surrounding an elongate core 20.The four elongate conducting elements 30 are twisted about each other.",
"The lay length, i.e.",
"the number of twists per unit length, can vary between 19 mm and 45 mm.",
"The principles of the invention are also applicable to a different number of elongate conducting elements 30 surrounding an elongate core 20.The embodiment of the invention of FIG.",
"1 has no jacket.",
"This embodiment is particularly useful in applications in which the presence of voids or interstices in the cable is undesirable.",
"An example of such applications is fluid-filled environments such as the ocean or in oil wells in which fluids might potentially seep between the elongate conducting element's 30 and the jacket over some lengths of the cable to potentially create localised impedance differences which can affect the skew of the signals travelling down the cable.",
"A further embodiment of the invention is depicted in FIG.",
"2 in which the cable 10 of the embodiment of FIG.",
"1 is additionally surrounded by a jacket 80.The jacket 80 can be made from polyurethane, polyethylene, polyester, perfluoralkoxy, fluoroethylene-propylene, polypropylene, polymethylpentene, full density polytetrafluoroethylene or expanded polytetrafluoroethylene.",
"Preferably the jacket 80 is made from polyurethane extruded over the cable 10.Manufacture of the cable 10 for the preferred embodiment of the invention is carried out as shown diagrammatically in FIG.",
"3.Four elongate conducting elements 30 comprising an insulating layer 50 of ePTFE tape-wrapped about an inner conductor 40 are twisted about an elongate core 20 of ePTFE.",
"The elongate conducting elements 30 about the elongate core 20 are passed into a sintering oven 100 which is at a temperature between 320° C. and 420° C. To sinter ePTFE a temperature of around 390° C. is required.",
"The sintering oven 100 bonds the core outer surface 25 to the insulating layer 50.Instead of a sintering oven 100 a salt bath can be used.",
"The cable 10 is then passed through an extruder 110 if a jacket 80 is to be extruded onto the cable 10.The cable 10 can find one application in so-called streamers.",
"These are used by oil exploration companies to carry out seismographic surveys of the ocean bottom sub surface to search for hydrocarbons.",
"FIG.",
"4 shows one example of a streamer 100 including a cable 10 manufactured according to this invention.",
"Streamers are known in the art and are made by companies such as Teledyne or Geco-Prakla.",
"The streamer 100 comprises a tube 110 of approximately 7.5 cm diameter having walls made of polyurethane and being approximately 2 mm thick.",
"The streamer 100 is filled with an oil 120 such as Isopar M and further includes power cables 130.Other signal carrying cables such as twisted pairs may be found inside of the streamer 100 but are not shown on this Fig.",
"EXAMPLES Example 1 This cable 10 has the construction as shown in FIG.",
"1.The elongate core 20 is made of a 2.0 mm diameter ePTFE core.",
"Each of the four elongate elements 30 is made of an inner conductor 40 of silver plate copper of size AWG 2019 surrounded by an insulating layer 50 which comprises three layers 50a, 50b, 50c of tapes wrapped about the inner conductor 40.The first tape 50a is made of ePTFE and has a thickness of 101.6 μm.",
"The second tape 50b is wound over the first tape 50a and the third tape 50c is wound over the second tape 50b.",
"The second tape 50b and the third tape 50c are both made of full density PTFE and have a thickness of 101.6 μm and 76.2 μm respectively.",
"The elongate elements 30 have a lay length of 38 mm.",
"Prior to sintering the elongate elements 30 have a nominal outside diameter of 2.23 mm.",
"The elongate core 20 with the elongate elements 30 is passed at a maximum speed of 1.0 m per minute through the sintering oven 100 at 395° C. which results in a dwell time in the oven of around 3 minutes.",
"Subsequent to sintering the elongate elements 30 had a nominal outside diameter of 1.88 mm.",
"The cable 10 has a maximum diameter of 4.6 mm.",
"Example 2 This has the same construction and is made in the same way as the cable 10 of Example 1.The elongate elements 30 are made of an inner conductor 40 of size AWG 2219 about which is wrapped a single tape of ePTFE of 0.9 mm thickness.",
"The elongate core 30 is made of ePTFE and has a nominal outside diameter prior to sintering of 0.9 mm.",
"Example 3 The construction of Example 3 is shown in FIG.",
"5.The quad cable 10 of Example 1 was provided additionally with a jacket 80.In this example, the jacket 80 comprised an inner jacket 80a and an outer jacket 80b, both of which were made of polyurethane.",
"A braid 85 of a synthetic fiber, for example Technora, impregnated with PTFE was braided about the inner jacket 80a after extrusion of the inner jacket 80a and prior to extrusion of the outer jacket 80b.",
"The braided synthetic fibre acts as a strength member.",
"Example 4 This was made in the same manner as Example 1 except that the lay length was 19 mm.",
"The cable was passed through a 3 m oven at a rate of 1 m/min and thus had a dwell time of around 3 minutes in the oven.",
"Example 5 This was made in the same manner as Example 1 except that the lay length was 19 mm.",
"The cable was passed through a 3 m oven at a rate of 1.3 m/min and thus had a dwell time of around 2.3 minutes in the oven.",
"The four elongate elements 30 were poorly bonded to the elongate core 20.Example 6 This was made in the same manner as Example 1 except that the lay length was 19 mm.",
"The cable was passed through a 3 m oven at a rate of 1.5 m/min and thus had a dwell time of around 2 minutes in the oven.",
"The four elongate elements 30 were substantially not bonded to the elongate core 20.Results Measurements of the differential impedance, attenuation and weight were made of the cables manufactured in Examples 1-3 and are given in the table below.",
"Example 1 Example 2 Example 3 Impedance (Ω) 140 ± 7 142 ± 5 128 ± 5 Attenuation (dB/km) 45 <43.3 <35 Weight (kg/km) 35 ± 2 18.2 ± 0.9 88 The attenuation measurements were made in air at a frequency of 20 MHz for Examples 1 and 2 and at 10 MHz for Example 3.The impedance measurements were also made in air by a Tectronics network analyser CSA 803.Measurements on Example 2 were also made in 5% NaCl solution (equating to sea water).",
"In this case the impedance was 111±5Ω and the attenuation was less than 54 dB/km.",
"The same measurements were made using streamer fluid and the impedance was then 132±5Ω whilst the attenuation was less than 47 dB/km.",
"Approximately 1 m length of the cables 10 of Examples 4 to 6 were wrapped about an 11 mm outside diameter plastic mandrel and the impedance of a pair of the elongate elements 30 were measured before wrapping (as a straight cable) and after wrapping.",
"The results are given in the table below.",
"The impedance measurements in ohms along the 1 m length of the cable were made using the Tectronics network analyser CSA 803.Mean Impedance Mean Impedance Sample impedance - variation - impedance - variation - No straight cable straight cable wrapped cable wrapped cable 4 142.4 141-143 144.6 144-147 5 142.5 142-147 161.2 153-169 6 145.5 145-147 162.0 159-176 As can be seen from the table, the impedance variation along one of the pairs of the elongate elements 30 of Example 4 before and after wrapping is small as the elongate elements 30 remain bound to the elongate core 20.In Examples 5 and 6, however, there is poorer bonding or no bonding at all respectively.",
"Thus the elongate elements 30 on wrapping about the plastic mandrel separate at least partly from the elongate core 20 which leads to a higher impedance and a greater variability in impedance.",
"This experiment simulates the flexing of the cable 10 in real life which leads to bending of the cable and thus—if the elongate elements 30 are not sufficiently bonded—to separation from the elongate core 20."
]
] | Patent_10181667 |
[
[
"TORSION SENSOR",
"The invention relates to a device for providing an axial force subject to a torque, which device has a first shaft with a friction surface comprising at least a radial directional component; a second shaft which is placed parallel with at least an axial directional component to the first shaft; a friction member arranged with an axial screw thread on the second shaft for friction contact with the friction surface, which friction member displaces axially in the case of rotation relative to the second shaft; and pressing means for urging the friction member in an axial direction."
],
[
"1.Mechanical transmission, comprising: a frame; a first and second friction device, which device comprises: a first shaft with a friction surface comprising at least a radial directional component; a second shaft which is placed parallel with at least an axial directional component to the first shaft; a friction member arranged with an axial screw thread on the second shaft for friction contact with the friction surface, which friction member is displaced axially in the case of rotation relative to the second shaft; and pressing means for urging the friction member in an axial direction, wherein the first shaft of the first friction device forms an input shaft, which input shaft is arranged rotatably on the frame; wherein the second shaft of the second friction device forms an output shaft, which output shaft is arranged rotatably on the frame parallel to the input shaft and wherein the second shaft of the first friction device and the first shaft of the second friction device are mutually coupled to form a rotatable body, which rotatable body is arranged for at least radial displacement on the frame; a first push belt arranged between the friction surface and the friction member of the first friction device; a second push belt arranged between the friction surface and the friction member of the second friction device; wherein the friction surfaces are rotation-symmetrical, the friction surfaces comprise at least an axial component and comprise at least a radial directional component.",
"2.Transmission as claimed in claim 1, wherein the pressing means of the first device and of the second device are shared.",
"3.Transmission as claimed in claim 1, wherein at least one of the input shaft and the output shaft is axially displaceable and wherein bounding means are arranged in order to bound the axial displacement of the friction member associated with the axially displaceable shaft.",
"4.Transmission as claimed in claim 3, wherein the friction surface arranged on the displaceable shaft comprises a first engaging surface.",
"5.Transmission as claimed in claim 4, wherein a second engaging surface is arranged on the push belt which engages on the friction surface associated with the displaceable shaft, wherein the first engaging surface lies outside the plane of the friction surface such that only the first engaging surface can co-act with the second engaging surface."
],
[
"The invention relates to a device for providing an axial force subject to a torque.",
"In transmissions wherein a torque is transmitted via a friction surface, it is required that sufficient pressing force be applied to the friction surface, since otherwise slippage will occur, thereby limiting the torque to be transmitted.",
"If the torque varies, the same pressing force is then not constantly necessary.",
"If a fixed pressing force is chosen, it must always be so great that the maximum torque can be transmitted.",
"Due to this great pressing force the bearings of the transmission will be unnecessarily loaded in the case of partial load.",
"This problem occurs particularly in continuously variable transmissions.",
"In such transmissions a driving torque is transmitted by friction to an opposite surface serving as friction surface.",
"Depending on the demanded torque, a determined pressing force must be realized between the friction surfaces.",
"Devices are known for adjusting a certain pressing force which is subject to the torque, wherein the torque is measured and on the basis hereof a certain pressing force is then adjusted, for instance by means of hydraulics.",
"These devices consume energy and are complicated and therefore expensive to manufacture and maintain.",
"It is an object of the invention to provide a simple device according to the preamble which obviates the drawbacks of known devices.",
"This object is achieved according to the invention by a device according to the invention, comprising: a first shaft with a friction surface comprising at least a radial directional component; a first and second friction device, which device comprises: a first shaft with a friction surface comprising at least a radial directional component; a second shaft which is placed parallel with at least an axial directional component to the first shaft; a friction member arranged with an axial screw thread on the second shaft for friction contact with the friction surface, which friction member is displaced axially in the case of rotation relative to the second shaft; and pressing means for urging the friction member in an axial direction, wherein the first shaft of the first friction device forms an input shaft, which input shaft is arranged rotatably on the frame; wherein the second shaft of the second friction device forms an output shaft, which output shaft is arranged rotatably on the frame parallel to the input shaft and wherein the second shaft of the first friction device and the first shaft of the second friction device are mutually coupled to form a rotatable body, which rotatable body is arranged for at least radial displacement on the frame; a first push belt arranged between the friction surface and the friction member of the first friction device; a second push belt arranged between the friction surface and the friction member of the second friction device; wherein the friction surfaces are rotation-symmetrical, the friction surfaces comprise at least an axial component and comprise at least a radial directional component.",
"When the first shaft is rotated with a certain torque, the first shaft will rotate relative to the friction member due to the screw thread.",
"The friction member will hereby displace axially and press against the friction surface.",
"When the pressing force is large enough to transmit the torque onto the second shaft, the rotation of the first shaft relative to the friction member will stop and the torque is transmitted from the first shaft onto the second shaft.",
"The pressing means are arranged to ensure the first contact between the friction member and the friction surface.",
"The screw thread also provides a good radial centering of both shafts, whereby the bearing can remain relatively simple.",
"In a preferred embodiment roller bodies are arranged in the screw thread.",
"These roller bodies considerably reduce the friction in the screw thread, whereby the pressing force is adjusted more precisely.",
"In another embodiment according to the invention the pressing means comprise a cup spring.",
"In a preferred embodiment according to the invention, one of the two shafts is axially displaceable and bounding means are arranged which engage on the friction member in order to limit the axial displacement of the friction member.",
"The ability to displace one of the two shafts makes it possible to also provide a clutch function in the transmission.",
"It is particularly desirable in the case of such a mechanical transmission to have a sufficiently great pressing force.",
"On the other hand, this pressing force must not be too great, whereby unnecessary wear could occur.",
"In a preferred embodiment of the transmission according to the invention the pressing means of the first device and of the second device are shared.",
"In another embodiment of the transmission according to the invention at least one of the input shaft and the output shaft is axially displaceable and bounding means are arranged in order to bound the axial displacement of the friction member associated with the axially displaceable shaft.",
"A mechanical transmission with built-in clutch is formed using the above measures.",
"Because the friction member is bounded in its axial displacement, a larger axial displacement of one of the shafts enables one of the two friction surfaces to be disengaged from the friction member.",
"It is hereby possible to disengage the input shaft from the output shaft.",
"In a preferred embodiment according to the invention the friction surface arranged on the displaceable shaft comprises a first engaging surface.",
"When the mechanical transmission is also used as clutch, the friction surface arranged on the displaceable shaft will then wear excessively in that when the input shaft and the output shaft engage the push belt will slip over the friction surface.",
"The operation of the mechanical transmission will be affected because the friction surface wears excessively.",
"By now providing a special engaging surface only this surface will wear and the friction surface required for the mechanical transmission will remain intact.",
"In a further preferred embodiment according to the invention a second engaging surface is arranged on the push belt which engages on the friction surface associated with the displaceable shaft, wherein the first engaging surface lies outside the plane of the friction surface such that only the first engaging surface can co-act with the second engaging surface.",
"Owing to the slippage occurring between the push belt and the friction surface during engaging of the clutch, the push belt will also wear.",
"By now providing on the push belt a special second engaging surface which can only co-act with a first engaging surface, wear to either the push belt or the friction surface will not affect the operation of the mechanical transmission.",
"These and other features of the invention will be further elucidated with reference to the annexed drawings: FIG.",
"1 shows a cross-section of a device according to the invention; FIG.",
"2 shows a first embodiment of a mechanical transmission according to the invention; FIGS.",
"3a and 3b shows cross-sections of a second embodiment of a mechanical transmission according to the invention; and FIG.",
"4 shows a third embodiment of a mechanical transmission in which a device according to the invention is arranged.",
"FIG.",
"1 shows a device 20 according to the invention.",
"Such devices 20 are usually designated as torque sensor.",
"The device 20 has an input shaft 21 and an output shaft 22.Provided on input shaft 21 is a part 23 which has an internal screw thread 24.Arranged in this screw thread 24 are balls 25 which form a bearing for friction member 26.Friction member 26 is pressed against a friction surface 28 of output shaft 22 by means of cup springs 27.When input shaft 21 is driven, it will carry along the friction member 26.Since friction member 26 is pressed against friction surface 28 of the stationary output shaft by cup springs 27, friction member 26 is held back in the first instance, whereby it will displace axially as a result of the screw thread.",
"When the pressing force is sufficiently great, this axial displacement will stop and output shaft 22 will be carried along.",
"FIG.",
"2 shows a mechanical transmission 1 which comprises an input shaft 2 having thereon a friction surface 3, an output shaft 4 and a friction surface 5 arranged thereon.",
"Between friction surfaces 3 and 5 is arranged a displaceable friction member 6 with which the transmission ratio between input shaft 2 and output shaft 4 can be adjusted.",
"Friction member 6 comprises a frame 7 which is displaceable.",
"A bush 9 is mounted in this frame 7 via bearings 8.Bush 9 is provided on the inner side with a screw thread 10.Two bodies 12 and 13 are arranged in this screw thread by means of balls 11.Arranged between bodies 12 and 13 are cup springs 14 which urge the two bodies away from each other.",
"Bodies 12 and 13 are provided on the sides directed toward the respective friction surfaces 3 and 5 with a push belt 15 respectively 16.Cup springs 14 ensure that push belts 15, 16 are brought into contact with the respective friction surfaces 3 and 5.If a torque is now applied to shaft 2, the push belt 15, and therefore body 2, will be carried along by rotation of friction surface 3.Owing to the screw thread 10 the body 12 will now displace relative to bush 9 in the direction of friction surface 3.This will result in a certain pressing force of push belt 15 on friction surface 3.When the pressing force is sufficiently great, the bush 9 will be carried along by rotation of shaft 2.In this mechanical transmission two pressing devices according to FIG.",
"1 are in fact arranged, each ensuring the correct pressing force of one of the push belts.",
"Since in the first instance the output shaft 4 stands still, the body 13 will be held back due to friction between push belt 16 and friction surface 5.Because bush 9 rotates, the body 13 will now displace relative to this bush toward friction surface 5, so that the pressing force between push belt 16 and friction surface 5 increases.",
"As soon as the pressing force is sufficiently great, output shaft 4 will begin to rotate and a torque of input shaft 2 can thus be transmitted onto output shaft 4.FIGS.",
"3a and 3b show a variant of the device according to FIG.",
"2.Corresponding components are designated with the same reference numerals and for the sake of simplicity will not be further described here.",
"The difference from the embodiment of FIG.",
"2 is that output shaft 4 is displaceable in the axial direction A.",
"In bush 9 is arranged a securing member 17 which prevents the body 13 running out of the screw thread as a consequence of the cup springs 14 when output shaft 4 is moved away from friction member 6.FIG.",
"3a shows the disengaged position.",
"In FIG.",
"3b the output shaft 4 is once again placed against friction member 6, whereby body 13 is released from the securing member 17 and the mechanical transmission operates as shown in FIG.",
"2.FIG.",
"4 shows a mechanical transmission which in construction largely corresponds with the embodiment shown in FIG.",
"2.Corresponding components are therefore designated with the same reference numerals.",
"In this embodiment the push belt 15 is also arranged in a body 12.Push belt 16 is arranged in a body 30.Body 12 is mounted in body 30 via roller bodies 11 and screw thread 10.Placed coaxially on body 30 is a bush 31 which is axially displaceable relative to this body 30.This bush is mounted in frame 7 by means of bearings 8.The operation corresponds for the most part with the operation of the embodiment of FIG.",
"2.The rotation of body 12 relative to body 30 ensures that the distance between the two push belts 15, 16 changes.",
"In order to distribute the axial pressing force evenly over the two friction surfaces 3, 5, the body 30 will displace axially in bush 31.The advantage of this construction is that the bearings 8 are not axially loaded and the frame therefore also remains unloaded in axial direction."
]
] | Patent_10181796 |
[
[
"Purifying polyoxyethylated castor oils with activated charcoal and pharmaceutical formulations thereof",
"Disclosed are polyoxyethylated castor oils produced by preparing a suspension of activated charcoal and a polyoxyethylated castor oil; and separating the activated charcoal from the polyoxyethylated castor oil.",
"The process removes impurities such as colorants and alkali metal cations.",
"Also disclosed are compositions containing the castor oil and an active agent such as a pharmaceutical agent.",
"The formulations have prolonged storage stability."
],
[
"1.A polyoxyethylated castor oil produced by: preparing a suspension of activated charcoal and a polyoxyethylated castor oil wherein the activated charcoal has a median surface area of about 900 m2/g, and is present in the suspension in an amount of from 3 to about 20% (w/w); heating said suspension at a temperature of from about 30° C. to about 60° C. for a period of time of from about 1 to about 6 hours; and filtering the heated suspension to separate the activated charcoal from the castor oil.",
"2.The castor oil of claim 1 which is Cremophor EL®.",
"3.A formulation comprising the castor oil of claim 1 and an active agent.",
"4.The composition of claim 3 wherein said active agent is an anti-neoplastic agent.",
"5.The composition of claim 4 wherein said anti-neoplastic agent is a taxane.",
"6.The composition of claim 5 wherein said taxane is paclitaxel.",
"7.The composition of claim 5 further comprising an alcohol.",
"8.The composition of claim 7 wherein said alcohol comprises dehydrated ethanol.",
"9.The composition of claim 5 further comprising an acid.",
"10.The composition of claim 9 wherein said acid is citric acid.",
"11.A polyoxyethylated castor oil formulation produced by: preparing a suspension of activated charcoal and a polyoxyethylated castor oil wherein the activated charcoal has a median surface area of about 900 m2/g, and is present in the suspension in an amount of from 3 to about 20% (w/w); heating said suspension at a temperature of from about 30° C. to about 60° C. for a period of time of from about 1 to about 6 hours; filtering the heated suspension to separate the activated charcoal from the castor oil; and adding an active agent to the filtered castor oil.",
"12.The formulation of claim 11 wherein the active agent is a taxane 13.The formulation of claim 12 wherein the taxane is paclitaxel.",
"14.The formulation of claim 13 wherein said adding further comprises adding dehydrated ethanol.",
"15.The formulation of claim 14 wherein said adding further comprises adding citric acid.",
"16.The formulation of claim 15 wherein said castor oil is Cremophor EL®.",
"17.A polyoxyethylated castor oil produced by: preparing a suspension of activated charcoal and a polyoxyethylated castor oil; and separating the activated charcoal from the polyoxyethylated castor oil.",
"18.The castor oil of claim 17 wherein the charcoal is present in the suspension in an amount of from 3 to about 20% (w/w).",
"19.The castor oil of claim 17 wherein the activated charcoal is present in the suspension in an amount of from about 5 to about 10% (w/w).",
"20.The castor oil of claim 17 wherein the activated charcoal has a surface area ranging from about 500 m2/g to about 1300 m2/g.",
"21.The castor oil of claim 17 further comprising heating the suspension prior to said separating.",
"22.The castor oil of claim 21 wherein said heating is conducted at a temperature of from about 30° C. to about 60° C. 23.The castor oil of claim 22 wherein said heating is conducted for a period of time of from about 1 to about 6 hours.",
"24.The castor oil of claim 17 wherein said separating comprises filtering the suspension.",
"25.The castor oil of claim 24 wherein said filtering comprises at least two filtration steps using filters with successively smaller apertures."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Polyethoxylated castor oils are commonly used as solubilizing and/or dispersing agents for a variety of pharmaceutically active agents that are substantially insoluble in water.",
"Impurities tend to catalyze the decomposition of the pharmaceutically active agents, particularly anti-neoplastic agents such as paclitaxel.",
"In particular, it has been found that pharmaceutical compositions of paclitaxel in a co-solvent system containing dehydrated ethyl alcohol and commercial grade Cremophor EL® exhibit a loss of potency of greater than 60% after storage for 12 weeks at 50° C., the loss of which is attributable to the decomposition of paclitaxel during storage.",
"In addition to the impurities such as colorants and dopants that are contained in the castor oil solution that is used to prepare the pharmaceutical composition, other impurities are formed during storage of the ultimate pharmaceutical formulation.",
"Such impurities include fibrous precipitate of unknown composition, and which also cause loss of potency of the active agent.",
"U.S. Pat.",
"No.",
"5,504,101 to Agharkar, et al., teaches a method for treating a polyoxyethylated castor oil with an acid or contacted with alumina to reduce carboxylate anion content, thereby extending shelf life and lower amounts of degradation by-products.",
"U.S. Pat.",
"No.",
"5,925,776 to Nikolayev, et al., teaches methods of reducing cation contents of polyoxyethylated castor oils by pre-treating the castor oil with a strong cationic exchange resin such as styrene divinyl benzene.",
"International publication number WO 00/23070 to Ben Venue teaches a method for purifying polyoxyethylated castor oils by contacting a solution of castor oil and an alcohol with a column containing activated carbon, followed by contact with an ion exchange resin column to remove residual amounts of carbon."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Applicants have devised a method for removing impurities from polyethoxylated castor oils without requiring cumbersome chromatographic procedures.",
"Hence, one aspect of the present invention is directed to a polyoxyethylated castor oil produced by the steps of preparing a suspension of activated charcoal in a polyoxyethylated castor oil (e.g., a commercial grade castor oil), and separating the activated charcoal from the polyoxyethylated castor oil.",
"In preferred embodiments, the suspension is heated prior to separating and the separation of the activated charcoal from the castor oil is performed while the suspension is still heated.",
"In a more preferred embodiment, impurities are removed by prepared by preparing a suspension of activated charcoal and a polyoxyethylated castor oil wherein the activated charcoal has a median surface area of about 900 m 2 /g, and is present in the suspension in an amount of from 3 to about 20% (w/w); heating said suspension at a temperature of from about 30° C. to about 60° C. for a period of time of from about 1 to about 6 hours; and filtering the heated suspension to separate the activated charcoal from the castor oil.",
"The castor oils of the present invention have reduced impurities (e.g., colorants and alkali metal cations including K + and Na + ) relative to castor oils not having undergone the processes described herein.",
"Another aspect of the present invention is directed to the purification process per se, which entails preparing a suspension of activated charcoal in the polyoxyethylated castor oil under conditions of time and temperature so as to allow the impurities to be removed from the castor oil.",
"Another aspect of the present invention is directed to compositions and formulations containing a polyoxyethylated castor oil treated in accordance with the aforementioned process, and an active agent.",
"The agent is soluble or dispersible in the castor oil.",
"In preferred embodiments, the active agent is an anti-neoplastic agent, more preferably a taxane such as paclitaxel.",
"More preferred compositions also contain, in addition to the paclitaxel, dehydrated ethanol and citric acid.",
"Processes of making the compositions are also provided."
],
[
"TECHNICAL FIELD The present invention relates to the removal of impurities from polyethoxylated castor oils, and formulations containing the treated castor oils and active agents.",
"BACKGROUND OF THE INVENTION Polyethoxylated castor oils are commonly used as solubilizing and/or dispersing agents for a variety of pharmaceutically active agents that are substantially insoluble in water.",
"Impurities tend to catalyze the decomposition of the pharmaceutically active agents, particularly anti-neoplastic agents such as paclitaxel.",
"In particular, it has been found that pharmaceutical compositions of paclitaxel in a co-solvent system containing dehydrated ethyl alcohol and commercial grade Cremophor EL® exhibit a loss of potency of greater than 60% after storage for 12 weeks at 50° C., the loss of which is attributable to the decomposition of paclitaxel during storage.",
"In addition to the impurities such as colorants and dopants that are contained in the castor oil solution that is used to prepare the pharmaceutical composition, other impurities are formed during storage of the ultimate pharmaceutical formulation.",
"Such impurities include fibrous precipitate of unknown composition, and which also cause loss of potency of the active agent.",
"U.S. Pat.",
"No.",
"5,504,101 to Agharkar, et al., teaches a method for treating a polyoxyethylated castor oil with an acid or contacted with alumina to reduce carboxylate anion content, thereby extending shelf life and lower amounts of degradation by-products.",
"U.S. Pat.",
"No.",
"5,925,776 to Nikolayev, et al., teaches methods of reducing cation contents of polyoxyethylated castor oils by pre-treating the castor oil with a strong cationic exchange resin such as styrene divinyl benzene.",
"International publication number WO 00/23070 to Ben Venue teaches a method for purifying polyoxyethylated castor oils by contacting a solution of castor oil and an alcohol with a column containing activated carbon, followed by contact with an ion exchange resin column to remove residual amounts of carbon.",
"SUMMARY OF THE INVENTION Applicants have devised a method for removing impurities from polyethoxylated castor oils without requiring cumbersome chromatographic procedures.",
"Hence, one aspect of the present invention is directed to a polyoxyethylated castor oil produced by the steps of preparing a suspension of activated charcoal in a polyoxyethylated castor oil (e.g., a commercial grade castor oil), and separating the activated charcoal from the polyoxyethylated castor oil.",
"In preferred embodiments, the suspension is heated prior to separating and the separation of the activated charcoal from the castor oil is performed while the suspension is still heated.",
"In a more preferred embodiment, impurities are removed by prepared by preparing a suspension of activated charcoal and a polyoxyethylated castor oil wherein the activated charcoal has a median surface area of about 900 m2/g, and is present in the suspension in an amount of from 3 to about 20% (w/w); heating said suspension at a temperature of from about 30° C. to about 60° C. for a period of time of from about 1 to about 6 hours; and filtering the heated suspension to separate the activated charcoal from the castor oil.",
"The castor oils of the present invention have reduced impurities (e.g., colorants and alkali metal cations including K+ and Na+) relative to castor oils not having undergone the processes described herein.",
"Another aspect of the present invention is directed to the purification process per se, which entails preparing a suspension of activated charcoal in the polyoxyethylated castor oil under conditions of time and temperature so as to allow the impurities to be removed from the castor oil.",
"Another aspect of the present invention is directed to compositions and formulations containing a polyoxyethylated castor oil treated in accordance with the aforementioned process, and an active agent.",
"The agent is soluble or dispersible in the castor oil.",
"In preferred embodiments, the active agent is an anti-neoplastic agent, more preferably a taxane such as paclitaxel.",
"More preferred compositions also contain, in addition to the paclitaxel, dehydrated ethanol and citric acid.",
"Processes of making the compositions are also provided.",
"BEST MODE OF CARRYING OUT THE INVENTION Polyoxyethylated castor oils are produced by condensation of castor oil with ethylene oxide.",
"Preferred oils are commercially available under the trade names Cremophor EL® and Cremophor EL-P (BASF).",
"Other preferred castor oils include polyoxyl 35 castor oil and Cremphor RH60.Alternatively, Cremophor may be prepared in accordance with the methods disclosed in U.S. Pat.",
"No.",
"3,070,499.Activated charcoal is commercially available from several sources e.g., Calgon (Pittsburg, Pa.) and Spectrum Chemical Manufacturing Corp. (Gardena, Calif.).",
"Alternatively, charcoal may be activated in accordance with standard procedures, notably by chemical treatment, or steam or some other heat source, charcoal can be “activated”.",
"Bonhomme-Faivre, et al., Life Sci.",
"66(9):817-827 (2000), for example, discloses pinewood charcoal LSM (CECA-SA, 92 La Defense) and peat charcoal SX4 (Norit 93, Le Blanc Masnil), activated by steam and washed with phosphoric acid.",
"The surface absorption for pinewood charcoal is 1,000 m2/g and 650 m2/g for peat charcoal.",
"In general, the activated charcoal has a surface area ranging from about 500 to about 1300 m2/g (including sub-ranges thereof), and preferably a median surface area range of about 900 m2/g.",
"To reduce the impurities in a given polyoxyethylated castor oil, a predetermined amount of the oil is added together with activated charcoal to produce a suspension of the charcoal in the oil.",
"The product is thus a free suspension of the activated charcoal, in contrast to the Ben Venue patent publication which entails contacting the castor oil with a column containing the charcoal.",
"Persons skilled in the art will be able to determine the amounts of activated charcoal in order to remove impurities, in accordance with standard techniques.",
"In general, the charcoal is present in the suspension in an amount of from 3 to about 20% (w/w), preferably from about 5 to about 10% (w/w), including sub-ranges thereof.",
"The castor oil and activated charcoal are allowed to remain in contact under conditions (e.g., time and temperature) to allow removal or capture of impurities by the charcoal.",
"Although not intending to be bound by any particular theory of operation, it is believed that the activated charcoal adsorbs the impurities.",
"In preferred embodiments, the suspension is heated, generally at a temperature of from about 30° C. to about 60° C., and for a period of time from about 1 to about 6 hours, including sub-ranges thereof.",
"It is believed that the heating enhances absorption of impurities and thus facilitates their removal from the castor oil.",
"It is also preferred to stir the suspension, at least periodically.",
"The activated charcoal is then separated from the castor oil.",
"This separation is conveniently performed by at least one filtration step.",
"In the event that two or more filtration steps are used, filters having successively smaller apertures are used.",
"In preferred embodiments, the heated suspension is filtered while it is still heated.",
"It is believed that the lesser viscosity of the heated suspension facilitates filtration.",
"In less preferred embodiments, the heated suspension is allowed to cool to about room temperature, optionally with stirring.",
"Other techniques for separating or removing activated charcoal are known in the art.",
"The treatment or purification process removes impurities from the castor oil.",
"The main impurities removed include colorants (e.g., naturally occurring color-stuffs contained in castor oils, and dopants and other chemical additives that are included in the final commercial product) and alkali metal cations such as K+ and Na+.",
"In preferred embodiments, the thus-treated castor oil has a color content of no greater than about 0.045 absorbance units (AU), measured at 425 nm/1 cm cell.",
"In more preferred embodiments, the castor oil has a color content of no more than about 0.038 AU.",
"With respect to cations, the castor oils of the present invention have a K+ content is no more than about 80 ppm, more preferably no more than about 50 ppm, and a Na+ content no more than about 10 ppm.",
"In other preferred embodiments, the castor oil has a water content of no greater than about 2.5%.",
"The polyoxyethylated castor oil of the present invention disperse or solubilize a wide variety of active agents, including pharmaceutical agents e.g., anesthetics (e.g., benzocaine), immunosuppressive agents, anti-fungal agents (e.g., miconazole and clotrimazole), anti-bacterial agents (e.g., hexidine), non-steroidal anti-inflammatory agents (e.g., diclofenac), vitamins (e.g., A, D, E and K), cosmetics (e.g., deodorant and antiperspirant actives such as aluminum zirconium chloride, aluminum chloride and sodium bicarbonate) and feedstuff, and other agents such as excipients (e.g., solvents, thickeners, colors, dyes, flow aids, lubricants and non-volatile silicones such as cyclomethicone and clays e.g., bentonite.",
"In preferred embodiments, the active agent is an anti-neoplastic agent.",
"Suitable anti-neoplastic agents include teniposide, a semi-synthetic derivative of podophyllotoxin having the chemical name 4′-demethylepiodophyllotoxin 9-(4,6-O-2-thenylidene-β-D-glucopyranoside), camptothecin, a compound isolated from the stemwood of the Chinese tree, and taxanes (e.g., obtainable from the bark of Pacific yew trees).",
"In more preferred embodiments, the anti-neoplastic agent is a taxane.",
"Suitable taxanes include paclitaxel and its prodrugs (e.g., docetaxel), derivatives, pharmaceutically acceptable salts and metabolites thereof.",
"Docetaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl paclitaxel) is commercially available under the trade name TAXOTERE® (Rhone-Poulenc-Rohrer S.A.).",
"Taxol analogs and derivatives are disclosed in the literature, e.g., U.S. Pat.",
"Nos.",
"6,103,698 and 6,136,990; WO 94/03093; Kingston, DGI.",
"Taxol: the chemistry and structure-activity relationships of a novel anticancer agent.",
"Trends Biotechnology 12: 222-227 (1994); and Alder, J D et al.",
"Preclinical in vivo efficacy of two dihydrotaxane analogues against human and murine tumors.",
"Br.",
"J.",
"Cancer 73:560-564 (1996).",
"Other than docetaxel, other prodrugs include the 2′-onium salts of paclitaxel and docetaxel, particularly the 2′-methylpyridiniuin mesylate (2′-MPM) salts.",
"Preferred metabolites of paclitaxel, designated A, B and C, are represented by the following formula: In the formula, metabolite A: R1═H and R2═OH; metabolite B: R1═OH and R2═H; and metabolite C: R1═H and R2═H.",
"In the case of taxanes, it is particularly preferred that the castor oil is contained in admixture with an alcohol such as dehydrated ethanol.",
"A 50:50 mixture (w/w) is preferred.",
"Stabilizers may also be added.",
"Citric acid is a preferred stabilizer.",
"The products and methods of the present invention are further illustrated by the following example.",
"The presentation of this example is by no way intended to limit applicants' invention in any way.",
"Unless otherwise specified, all percentages are by weight.",
"EXAMPLE One hundred grams of charcoal were placed in an oven at 120° C. for about 16 hours.",
"Nine hundred grams Cremophor EL® were added to make a 10% charcoal (w/w) Cremophor EL® slurry solution.",
"The solution was heated to 45° C. with a hot plate for three hours with mechanical stirring, followed by cooling to room temperature, followed by stirring for two additional hours.",
"The slurry solution was then filtered twice using a 10 μm filter and a 0.45 μm filter.",
"The paclitaxel formulation was prepared by dissolving 6 mg/ml of paclitaxel in 50/50 (v/v) commercial or pre-treated Cremophor EL® and dehydrated ethanol.",
"Two ml of the formulation solution were placed in a 6 cc glass vial.",
"The vials were sealed with a Teflon-faced cap and stored for 48 hours at 80° C. They were analyzed by HPLC for the concentration of paclitaxel.",
"The results were as follows.",
"It was found that 98.2% of the paclitaxel remained in the sample treated with charcoal whereas 42.2% of the paclitaxel remained in the untreated sample.",
"Thus, the processes of the present invention and the formulations produced thereby possess greater storage stability, particularly compared to identical formulations made without the treatment or purification of the present invention.",
"The pH value of both samples was measured following 1:10 dilution with water.",
"The potassium concentration and the color of the samples were analyzed as well.",
"The results are shown in the Table below.",
"TABLE % Paclitaxel remaining Sample (80° C. for 48 hours) Charcoal Treated Cremophor EL ® 98.2 Untreated Cremophor EL ® 42.2 The pH value of the samples was measured following 1:10 dilution with water.",
"The potassium concentration of samples was analyzed.",
"The color of the samples also was measured as shown in following: Color (Vis.",
"Sample K (ppm) pH 425 nm, AU) Charcoal Treated Cremophor EL ® 137.5 4.27 0.0381 Untreated Cremophor EL ® 398.7 6.06 0.0516 As used herein, the term “about” is intended to convey that the numbers and ranges disclosed herein are flexible and that practice of the present invention using temperatures, concentrations, amounts, contents, surface areas, etc.",
"outside of the range or different from a single value will achieve the desired result, namely reducing impurities.",
"The term typically includes a deviation of ±10% of any value it modifies.",
"PUBLICATIONS 1.MacEachem-Keith, et al., Anal.",
"Chem.",
"69:72-77 (1997).",
"2.Sampedro, et al., J. Microencapsulation 11(3):309-318 (1993).",
"3.Balasubramanian, et al., Solvent- and Concentration-Dependent Molecular Interactions of Taxol (Paclitaxel), Journal of Pharmaceutical Sciences 83(10) (1994).",
"4.Merisko-Liversidge, et al., Formulation and Antitumor Activity Evaluation of Nanocrystalline Suspensions of Poorly Soluble Anticancer Drugs, Pharmaceutical Research 13(2)(1996).",
"5.Sharma, et al., Novel Taxol Formulations: Preparation and Characterization of Taxol-Containing Liposomes, Pharmaceutical Research 11(6) (1994).",
"6.Adams, et al., J. Natl.",
"Cancer Inst.",
"Monogr.",
"15,141 (1993).",
"7.Dordunoo, et al., International Journal of Pharmaceutics 133:191-201 (1996).",
"8.Tarr, et al., A New Parenteral Vehicle for the Administration of Some Poorly Water Soluble Anti-Cancer Drugs, Journal of Parenteral Science & Technology 41(1) (1987).",
"9.Kingston, Pharma.",
"Ther.",
"52:1-34 (1991).",
"10.Wenk, et al., Paclitaxel Partitioning into Lipid Bilayers, Journal of Pharmaceutical Sciences 85(2) (1996).",
"INDUSTRIAL APPLICABILITY The present invention is useful the preparation of compositions, particularly pharmaceutical compositions containing polyethoxylated castor oils.",
"All patent and non-patent publications cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.",
"All these publications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as being incorporated herein by reference."
]
] | Patent_10182041 |
[
[
"Christmas tree stand comprising securing wedges",
"The invention relates to a Christmas tree stand with a holding body which surrounds the lower end of the trunk of a Christmas tree and with securing wedges for the trunk which are connected to the holding body via blade wedges."
],
[
"1-8.",
"(canceled) 9.A Christmas tree stand comprising: a holding body that receives a lower end of a trunk of a Christmas tree; a securing wedge with a guide pin, and a blade wedge having a guide channel, wherein the securing wedge is movably coupled to blade wedge via the guide pin and the guide channel; wherein the blade wedge is disposed within the holding body, and wherein the securing wedge moves between a first position and a second position when the guide pin moves in the guide channel; and wherein the securing wedge contacts and secures the trunk of the Christmas tree in the first position and releases the trunk of the Christmas tree in the second position.",
"10.The Christmas tree stand of claim 9 wherein the securing wedge has an edge proximal to the trunk of the Christmas tree, and wherein the edge curves away from the trunk of the Christmas tree when the securing wedge is in the first position.",
"11.The Christmas tree stand of claim 9 wherein the holding body has a cylindrical horizontal cross section and a lower end, and wherein the lower end of the holding body is further coupled to a closing plate.",
"12.The Christmas tree stand of claim 9 wherein the holding body has a lower portion that includes a stabilizing plate for the blade wedge.",
"13.The Christmas tree stand of claim 9 wherein the holding body further includes a channel that receives the securing wedge.",
"14.The Christmas tree stand of claim 10 wherein the channel is vertical to the ground when the Christmas tree stand secures a Christmas tree.",
"15.The Christmas tree stand of claim 9 wherein the securing wedge has an impact surface that forms an angle of less than 90 degrees relative to a vertical axis of the Christmas tree stand.",
"16.The Christmas tree stand of claim 15 wherein the angle is between 80 degrees and 60 degrees.",
"17.The Christmas tree stand of claim 9 wherein the securing wedge is a triangular steel plate having a thickness of 3 to 10 millimeter.",
"18.The Christmas tree stand of claim 17 wherein the securing wedge is a triangular steel plate having a thickness of 3 to 5 millimeter."
],
[
"The invention relates to a Christmas tree stand with a holding body which surrounds the lower end of the trunk of a Christmas tree and with securing wedges for the trunk which are connected to the holding body via blade wedges.",
"In Christmas tree stands of the type referred to in the introduction, the holding body is in most cases designed as a tube which surrounds the lower end of the trunk of the Christmas tree.",
"In order for it to be possible to fix this lower end in the holding body, radially arranged threaded screws are provided in one version of the Christmas tree stand, which are in most cases designed as wing screws and are screwed against the trunk with their end which is frequently equipped with a disk on the inside.",
"In this way, the trunk can be not only secured but also positioned.",
"In another version, wooden wedges made from pieces of wood are driven between the trunk and the holding body in order to fix the trunk.",
"In the known Christmas tree stands, the lower end of the trunk usually has to be tapered.",
"In the previously known Christmas tree stands with a positioning device which has flat wooden wedges, it is disadvantageous that the wooden wedges can be used only once and, on each new use of the Christmas tree stand, wooden wedges first have to be provided.",
"Many of the previously known Christmas tree stands form an upwardly open, watertight trough which, like a vase, receives the lower end of the Christmas tree.",
"A Christmas tree stand is known from U.S. Pat.",
"No.",
"2,613,899, which, in addition to providing a support for the tree trunk via positioning means acting on the trunk, comprises a water reservoir in the stand foot, into which the lower end of the tree projects.",
"In the previously known Christmas tree stands, the disadvantage exists, however, that the contact surface between the trunk and the water is relatively small, because only the lower surface of the trunk, at which it was cut off, is available for this.",
"A considerable improvement of the Christmas tree stands known from the prior art, inter alia from U.S. Pat.",
"No.",
"2,613,899, is described in EP-A-763340.According to the solution of this specification, the Christmas tree stands known from the prior art are improved by virtue of the insertion of the Christmas tree into the stand being greatly simplified, the parts used being reusable, better centering being achieved and the tree trunk coming into contact with water in an improved way.",
"This is achieved by virtue of the fact that, below the positioning arrangement present in the Christmas tree stand described there, blade wedges protect from the holding body, which are formed by radially inwardly facing edges of triangular bodies which are connected to the holding body and are designed in such a manner that a lower end of the trunk, which is inserted into the holding body, meets the blade wedges and, as the pushing-in of the trunk continues, the blade wedges cut into the lower end of the trunk.",
"Although, in accordance with EP-A-763340, the use of the positioning arrangement in the form of several wedges and a ring holding these together makes possible simple centering and alignment of the tree, further simplified handling is desirable.",
"The object of the invention is therefore to provide a Christmas tree stand which is improved in relation to the last-mentioned specification and makes possible simpler handling and better guidance of the trunk in the positioning arrangement.",
"According to the invention, this is achieved by providing a Christmas tree stand having the features of the main claim.",
"The invention therefore relates to a Christmas tree stand with a holding body which surrounds the lower end of the trunk of a Christmas tree and with securing wedges, which each have a hole, for the trunk, blade wedges projecting from the holding body below the securing wedges, which blade wedges are formed by radially inwardly facing edges of triangular bodies which are connected to the holding body and are designed in such a manner that a lower end of the trunk, which is inserted into the holding body, meets the blade wedges and, as the pushing-in of the trunk continues, the blade wedges cut into the lower end of the trunk, a vertical channel being provided in each blade wedge, and in each case a pin being fixed by one end in the hole in the securing wedge, the other end of the pin being arranged vertically movably in the channel, so that each securing wedge can be displaced between an insertion position, in which the securing wedge is mounted rotatably, and a use position, in which each securing wedge is pressed against the lower end of the trunk.",
"By fixing the securing wedges via the axis of rotation in the channel, a guide rail for the securing wedge, and the vertical mobility thus ensured, optimum guidance and support of the trunk in the Christmas tree stand is made possible.",
"After insertion of the lower end of the trunk and continued pushing-in of the trunk, the blade wedges cut into the lower end of the trunk.",
"When the Christmas tree stand is filled with water, reliable access of the water to the lower end of the trunk is made possible and through the lateral cuts into the trunk is facilitated.",
"In order to secure the trunk in this position, the securing wedges (alternatively called positioning wedges) are raised out of the insertion position with radially outwardly facing securing wedges and pressed against the trunk.",
"By means of light hammer blows on the upwardly facing impact edge of the securing wedge, the latter is driven downwardly along the guide channels located in the lower blade wedges and at the same time cuts into the trunk via that corner of the triangular securing wedges facing the trunk.",
"Through uniform and, through opposite securing wedges, staggered driving-in of each securing wedge around the trunk, centering, fixing and securing of the trunk is thus achieved at in each case the highest point of a securing wedge.",
"When the securing wedge is driven forward in the direction of the channel in the blade wedge, at the same time a lever force is exerted on the securing wedge via the radially outer edge of the securing wedge when this edge meets the holding body, which force has, in addition to the force acting in the driving direction, a radially acting component which facilitates the cutting of the upper corners of the securing wedge into the trunk.",
"In a preferred embodiment, that edge of the securing wedge facing the trunk is designed not as a straight edge but as an arcuate edge curved away from the trunk.",
"In this embodiment, it is ensured that the distance between the upper pressure point at the upper end of the securing wedge (cutting-in point on the side facing the trunk) and the lower pressure point on the blade wedge is as great as possible, irrespective of whether thickenings, in the form of remains of branches which have been cut off, natural deformities etc., are present in the lower area of the trunk.",
"The great distance between the holding points for the trunk results in improved stable support, and Christmas trees of different size and of different trunk diameter can thus be secured.",
"Furthermore, the Christmas tree stand can correspondingly be manufactured in different sizes, so that it is also suitable for every size of Christmas tree.",
"The Christmas tree stand is just as suitable as a Christmas tree stand with a water trough as it is as one in which the trunk remains dry and stands in sand, earth or the like.",
"The removal of the securing wedges is easy; to this end, one or more projections, stops or holes can be provided on the securing wedges, which facilitate levering out, as long as the trunk is still located in the holding body, or which make it possible to knock the securing wedges out at a later stage.",
"A steel hook can be inserted into a hole of the securing wedge, and the securing wedge can then be pulled upward, so that the secure connection is freed.",
"According to the invention, the securing wedges are triangular and they can bear with the longitudinal edge against the upper edge of the holding body depending on the thickness of the trunk and the driving-in depth, and possibly, when the securing wedge is driven in completely, along an inner wall of the holding body.",
"A securing wedge angle of 10 to 25°, preferably 15°, has proved advantageous.",
"That corner of each securing wedge bearing against the trunk can preferably be designed as a cutting edge or have a sawtooth design and acts in the manner of a claw on the trunk, so that the trunk is, by means of the, preferably at least five, circumferentially spaced securing wedges, connected firmly “in a claw-like manner” and stably to the foot.",
"According to the invention, blade wedges are provided in the lower end area of the holding body, which are designed similarly to the contact surfaces of the securing wedges and can likewise cut into the trunk.",
"They are arranged on a circular ring and, seen from top to bottom, project to an increasing extent toward the center.",
"They are preferably provided in the same number as the securing wedges.",
"The blade wedges preferably also have cutting edges, so that they too open the trunk and make access of water possible in these places.",
"A vertical guide channel, which serves as a guide for the pin fixed to the securing wedge and by virtue of this allows vertical movement of the securing wedge, is provided in each blade wedge.",
"Further advantages and features of the invention emerge from the other claims and also from the description below of an illustrative embodiment of the invention which is to be understood as non-limiting and is explained in greater detail with reference to the drawing, in which: FIG.",
"1 shows a sectional view of a tubular holding body with channels for the securing wedges; FIG.",
"2 shows a plan view of the holding body according to FIG.",
"1 from above; FIG.",
"3 shows a plan view of a stabilizing plate for the blade wedges; FIG.",
"4 shows a plan view of a blade wedge; FIG.",
"5 shows a plan view of a preferred embodiment of a securing wedge; FIG.",
"6 shows a cross section through the holding body with stabilizing plate, blade wedge and securing wedge, and FIG.",
"7 shows a plan view from above of the connection between a blade wedge and a securing wedge.",
"As can be seen from FIGS.",
"1 and 2, the Christmas tree stand has a holding body (1) which, here, is in the form of a tubular portion.",
"Attached to this at the bottom is a plate which forms an adequate standing surface.",
"Five blade wedges (3) (not shown in FIGS.",
"1 and 2), which are arranged directly next to the guide channels (2), project from the inner wall of the tube of the holding body (1).",
"The blade wedges are formed by the radially inwardly facing edges of five triangular bodies which are connected to the tubular holding body (1), and if appropriate also, or only, to the stabilization plate (4), shown in FIG.",
"3, with the preferably horizontal slots (5) which stabilize the blade wedges.",
"As can be seen from FIG.",
"4, the blade wedges (3) run at an angle of roughly 15° to the vertical axis of the Christmas tree stand.",
"They are designed in such a manner that a lower end of a trunk, which is inserted into the tubular holding body (1), meets the blade wedges and, as the pushing-in of the Christmas tree continues, and also under its own weight, they cut into the trunk.",
"Preferably, the blade wedges can be ground on both sides and designed as cutting edges.",
"However, they can also have a sawtooth design.",
"In the latter embodiment, care must be taken that the sawteeth do not hinder too greatly the removal of the trunk when the Christmas tree is taken down.",
"In the illustrative embodiment shown, the diameter at which the blade wedges (3) are located at their uppermost position, where, for instance, they are arranged in the inner diameter of the holding body (1), is two and a half times the diameter of an inner circle at the foot of the five blade wedges, in other words immediately at the plate (4).",
"The blade wedge has a vertical guide channel (6) for the guidance of the securing wedge (8).",
"At the lower end facing the plate (4), the guide channel is enlarged to form a bore (7) which, when the wedge (8) is driven in completely, secures the latter in position on account of the lever force acting thereon.",
"As shown in FIG.",
"5, the securing wedge (8) is provided with the impact edge (9), which faces upward in the use position, the bore (10) for the guide pin (12) (not shown in FIG.",
"5), and a further bore (11) for a hook to be inserted for pulling the wedge out of the use position.",
"The securing wedge has a wedge angle of roughly 15° and two longitudinal sides, namely a curved edge (13) toward the trunk and a bearing edge (14) with which it bears against the holding body (1).",
"As shown in FIG.",
"5, the bearing edge is, at the end facing the guide pin (12), which can also be in the form of a rivet, preferably curved toward the curved edge (13) like the chord of a circle, so that both thicker and thinner trunks can be pushed into the support better and held securely.",
"The contact surface (13), which is preferably likewise curved like the chord of a circle facing away from the trunk, so that a type of web-like design of the wedge is produced at that end of the wedge facing the bore (10), is preferably ground at least in the area of the wedge corner (14); it can also have sawtoothing.",
"Owing to the configuration of the curves in the form of a wedge, preferably as shown in FIG.",
"5, no crushing of the trunk in the lower area is brought about, but the trunk is, owing to being guided in the blades fixed to the bottom, clamped (clutched) at the highest point of the wedges.",
"When a Christmas tree is inserted into the stand according to the invention, the trunk is inserted into the holding body (1) and pushed in from above.",
"In the process, the blade wedges (3), as has been explained above, cut into the wood of the trunk.",
"The trunk is centered in the bottom by means of the funnel-shaped arrangement of the blade wedges (3) and it is secured and centered in this way.",
"When the trunk has been driven sufficiently far into the holding body (1), the fixing and alignment takes place by means of the securing wedges.",
"First, the securing wedges (8), guided in the annular channel between the trunk and the upper edge of the holding body (1), are positioned in such a manner that they bear against the trunk with the wedge corner (14).",
"The individual securing wedges (8) are then driven into said gap, in the process of which, depending on the diameter of the trunk, they slide along the edge of the guide channel (2) or the inner wall of the holding body (1) and cut into the wood of the trunk.",
"An excellent secure connection is achieved by the penetration into the wood.",
"Depending on the extent to which a securing wedge is driven in, the tree is pressed more to one side, and a vertical alignment is achieved in this way.",
"It has proved advantageous to provide a number of guide channels (2) in the upper edge area of the holding body (1), in each of which a bearing side of a securing wedge can engage and which cause the securing wedges to be held essentially radially, in other words they cannot slide away laterally.",
"When the Christmas tree stand according to the invention is removed, the securing wedges (8) can first be pulled up by a suitable hook being hooked into the securing wedge bore provided and pulled upward.",
"The positioning arrangement has, for example, three to seven, preferably, five or more securing wedges.",
"The blade wedges (3) on the holding body (1) can in particular be designed as cutting edges or have a sawtooth-shaped design.",
"Preferably three to seven, in particular five, blade wedges (3) are provided.",
"The angle which the blade wedges (3) enclose with the axis of the Christmas tree stand lies in particular between 10 and 25° and is preferably 15°.",
"FIG.",
"6 shows a cross-sectional illustration through the Christmas tree stand according to the invention roughly in the center of a guide channel (2) in the holding body (1).",
"In this connection, the securing wedge (8) is shown in the rest position, while the securing wedge with reference number (8a) is illustrated, by a broken line, in the use position, which depends on the diameter of the Christmas tree trunk.",
"Via the pin (12), which, shown from above in cross section in FIG.",
"7, connects the blade wedge (3) and the securing wedge (8), the securing wedge (8) can be pivoted out of the rest position into the use position and displaced along the guide channel (6) to the catching position in the bore (7).",
"The blade wedges (3) are protected against lateral yielding via the locking plate (4)."
]
] | Patent_10182325 |
[
[
"Method and apparatus for signal transduction pathway profiling",
"An assay device for determining the presence of analytes in a cell lysate comprises a porous support member and a plurality of binding reagents arranged and immobilized at multiple reaction sites on the support member.",
"The binding reagents are selected and arranged to assess the status of a selected cellular signal transduction pathway/protein-protein interactive network.",
"In a further aspect, a method for assessing the status of a signal transduction pathway comprises generating a lysate of cells, the lysate retaining one or more pathway molecules present in one or more states and the pathway molecules reflecting signal transduction events taking place in the cells.",
"The method further includes applying the lysate to an immobilized series of binding reagents which can discriminate the pathway molecules and their states.",
"Binding events between the pathway molecules and the binding reagents are identified and the state of the selected signal pathway is determined."
],
[
"1.An assay device for use in determining the presence of a plurality of analytes in a lysate of a sample of cells, the device comprising: (a) a porous support member; and (b) a plurality of binding reagents arranged and immobilized at a plurality of reaction sites on the support member, the binding reagents selected and arranged to assess the status of a selected protein-protein interaction network when the lysate is applied thereto.",
"2.The device of claim 1, wherein the protein-protein interaction network is a signal transduction pathway.",
"3.The device of claim 2, wherein the binding reagents are arranged to quantitatively assess the status of the selected signal transduction pathway.",
"4.The device of claim 2, wherein each binding reagent selectively binds with one of a series of biological molecules, the signal transduction pathway comprising said series of biological molecules, the biological molecules selected from one or more proteins, a phosphorylated state of said one or more proteins, an activated state of said one or more proteins, and a binding partner of said one or more proteins.",
"5.The device of claim 1, wherein the protein-protein interaction network is relevant to a disease state.",
"6.The device of claim 5, wherein the disease state is selected from cancer, brain disease, cardiac disease, and an allergy.",
"7.The device of claim 2, wherein the binding reagents are ordered to at least partially recapitulate a temporal or spatial status of molecular complex formations or networks of protein interaction which characterize the selected protein-protein interactive network.",
"8.The device of claim 7, wherein the binding reagents are bound in an immobilized flow-through matrix such that a plurality of protein complexes comprising different combinations of the same molecules can be sorted to determine the relative proportion of at least one selected molecule within the protein complexes.",
"9.The device of claim 7, wherein the protein-protein interactive network is branched.",
"10.The device of claim 7, wherein the protein-protein interactive network is a signal transduction pathway.",
"11.The device of claim 10, wherein the binding reagents are ordered to at least partially recapitulate the selected cellular signal transduction pathway in reverse temporal order.",
"12.The device of claim 10, wherein the binding reagents are ordered to at least partially recapitulate the selected cellular signal transduction pathway in forward temporal order.",
"13.The device of claim 10, wherein the binding reagents are ordered to at least partially recapitulate one of a signal pathway branch point and a connection to another pathway.",
"14.The device of claim 1, which comprises a kit for providing information about the therapeutic, toxic, or diagnostic state of a cellular sample.",
"15.A method of elucidating complex protein-protein interactions within defined protein pathways and protein networks in cells, comprising: a) generating a lysate of cells containing one or more protein components of the protein-protein interactions; b) applying the lysate to an immobilized series of binding reagents which discriminate the one or more protein components; and c) identifying binding events between the protein components and the binding reagents.",
"16.A method for assessing the status of a selected signal transduction pathway in cells comprising: a) generating a lysate of cells containing components of signal pathway proteins, the lysate retaining, at least in part, one or more pathway molecules, the pathway molecules present in one or more states selected from inactive, activated, activity altered, phosphorylated, cleaved, modified, and bound, the one or more pathway molecules reflecting signal transduction events taking place in the cells; b) applying the lysate to an immobilized series of binding reagents which discriminate the one or more pathway molecules and the one or more states thereof; c) identifying binding events between the pathway molecules and the binding reagents; and d) determining the state of the selected signal pathway.",
"17.The method of claim 16, wherein the immobilized binding reagents are ordered to at least partially recapitulate a temporal and or spatial status of molecular complex formations or networks of protein interaction which characterize the selected cellular signal pathway.",
"18.The method of claim 16, wherein the immobilized binding reagents are arranged in a preselected arrangement and wherein the determining step comprises identifying a pattern of binding events based on said arrangement to form a pattern of binding events.",
"19.The method of claim 18, wherein the determining step further comprises using a heuristic to provide information about the temporal or spatial status of cellular pathways and networks.",
"20.The method of claim 18, wherein the heuristic is selected from one or both of a heuristic-based bioinformatic and a genetic algorithm.",
"21.The method of claim 18, wherein the determining step further comprises comparing the identified patterns of binding events with one or more previously identified patterns for cells of a known condition.",
"22.The method of claim 21, wherein the binding reagents are bound in an immobilized flow-through matrix such that protein complexes comprising different combinations of the same molecules can be sorted to determine the relative proportion of at least one chosen molecule within different complexes.",
"23.The method of claim 22, wherein a signal pathway is at least partially recapitulated in reverse temporal order.",
"24.The method of claim 22, wherein a signal pathway is at least partially recapitulated in forward temporal order.",
"25.A method of claim 22, wherein a signal pathway branch point or connection to another pathway is at least partially recapitulated.",
"26.The method of claim 16, wherein the signal pathway is a pathway selected from one of the following classes: cell growth, cell differentiation, cell death, cell movement, gene transcription regulation, hormonal autocrine or paracrine stimulation, and cell adhesion pathways.",
"27.A method for determining the state of at least a portion of a signal pathway or network in a cell, comprising: solubilizing the cellular contents; exposing the contents to a series of labeled binding reagents to form a pattern of binding events; and analyzing the pattern of binding events.",
"28.The method of claim 27, wherein the binding reagents comprise ligands selected from one or more of antibodies, nucleic acids, synthetic molecules, drugs, bacteriophage, engineered prokaryotic cells, and engineered eukaryotic cells.",
"29.The method of claim 27, in which the labeled binding reagents generate a signal via chemiluminescence, florescence, radiometric, electrochemical, photochemical, enzymatic, calorimetric, or optical readout.",
"30.The method of claim 28, wherein the binding reagents are immobilized on a planar surface.",
"31.The method of claim 30, wherein the binding reagents are arranged in one of a linear array and a two-dimensional grid.",
"32.The method of claim 28, wherein binding reagents are generally linearly arranged and the cell contents are passively or actively made to flow through multiple binding zones.",
"33.The method of claim 28, wherein the binding reagents are arranged in a three-dimensional matrix.",
"34.The method of claim 33, wherein the matrix comprises a circuit containing branch-points in a flow of molecular complexes within the said matrix.",
"35.The method of claim 33, wherein molecules bound in one zone are released during a flow state and the released molecules can bind to a separate zone downstream in the flow.",
"36.A method for identifying proteins involved in cellular signaling or networks comprising: a) exposing cells to one of a phosphatase inhibitor followed by a drug such that the phosphorylation state of one or more molecules is changed; b) analyzing patterns of groups of at least 2 phosphorylated molecules before and after exposure to the drug; c) exposing cells to a molecule which perturbs one or more pathways; and d) comparing the patterns before and after exposure of the cells to said molecule.",
"37.The method of claim 36, further including the step of: (e) identifying therapeutic targets by evaluating whether a candidate compound either binds to a phosphorylated protein or perturbs the pattern of groups of at least 2 phosphorylated proteins.",
"38.A method of claim 36, which is used to determine at least one of therapeutic efficacy and toxicity of a preselected molecule being evaluated for therapeutic potential.",
"39.The method of claim 15, further comprising identifying one or both of drug-induced toxicity and therapeutic efficacy by evaluating one or more patterns of protein-protein interactions.",
"40.The method of claim 39, wherein the patterns are evaluated through heuristically-based and/or genetic algorithm-based data mining.",
"41.The method of claim 16, further comprising identifying one or both of drug-induced toxicity and therapeutic efficacy by evaluating one or more patterns of protein-protein interactions.",
"42.The method of claim 41, wherein the patterns are evaluated through heuristically-based and/or genetic algorithm-based data mining.",
"43.A method of identifying a repertoire of proteins that serve as acceptors for phosphorylation, comprising: (a) treating a first biological sample with one or more compounds that inhibit one or both of protein tyrosine and serine/threonine phosphatase activity; (b) isolating and lysing cells of interest; (c) selecting and enriching for phosphorylated proteins using antibodies on an immobilized bait to produce an enriched faction; (d) separating the phosphoproteins in the enriched fraction; (e) identifying a primary amino acid sequence of the separated proteins; and (f) identifying binding molecules that specifically bind to the separated proteins.",
"44.The method of claim 43, wherein the biological sample is selected from cells, whole tissue specimens grown ex-vivo, and animal tissue treated in vivo.",
"45.The method of claim 43, wherein the compound that inhibits one or both of protein tyrosine and serine/threonine phosphatase activity is selected from one or more of sodium pervanadate, okadaic acid, calyculin A.",
"46.The method of claim 45, wherein the biological sample is treated for a period of time that is less than about 3 hours.",
"47.The method of claim 43, wherein the antibodies are selected from one or more of anti-phosphotyrosine, anti-phosphoserine, and anti-phosphothreonine.",
"48.The method of claim 43, wherein the phosphoproteins are separated by one or both of a chromatographic techniques and an electrophoretic technique.",
"49.The method of claim 43, wherein the sequence identifying step comprises: degrading the protein by one of enzymatic digestion and chemically-induced protein fragmentation; identifying the sequence using mass spectrometry.",
"50.The method of claim 43, wherein the binding molecules are antibodies.",
"51.The method of claim 43, wherein the step of separating the phosphoproteins in the enriched fraction is performed by applying the lysate to an assay device comprising a porous support member and a plurality of binding reagents arranged and immobilized at a plurality of reaction sites on the support member.",
"52.The method of claim 43, wherein the step of separating the phosphoproteins comprises: applying the lysate to an immobilized series of binding reagents which discriminate the one or more protein components; and identifying binding events between the protein components and the binding reagents.",
"53.The method of claim 43, further comprising: (g) forming an immobilized arraying said binding molecules; (h) treating a second biological sample with a drug; and (i) evaluating one or both of drug-induced toxicity and drug efficacy by identifying binding events between the immobilized array and proteins in the biological sample.",
"54.The method of claim 53, wherein the step of evaluating further comprises identifying a pattern of the binding events.",
"55.The method of claim 43, wherein the biological sample comprises a whole tissue sample, further comprising: fixing the tissue sample; selecting a portion of the tissue sample; and identifying a cellular localization of an identified phosphorylated protein."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to an apparatus and method for assessing the status of a cellular pathway.",
"Several biotechnology companies are developing DNA-chips which contain printed arrays of hundreds or thousands of genes, or more.",
"However, the DNA sequence of the genes or the level of gene expression do not reflect the actual functional state of the proteins encoded by the genes.",
"The functional state of each protein involves more than simply the amount of the proteins.",
"It includes, for example, post translational modifications (phosphorylation, glycosylation, etc.",
"), binding partners in cellular pathways, conformation, enzymatic function, and state of activation.",
"Moreover, each cell in the body has a pattern of functional proteins which reflects the current biologic working state of the cell (e.g., growing, differentiating, diseased, dying, senescent, etc.).",
"Therefore, there exists a need in the art for a device and method for determining the status of cellular pathways (e.g., the phosphorylation state or binding partners of involved proteins) in tissue samples, including human tissue samples, thus providing the diagnostician or clinician with an early warning of impending or occult toxicity, disease state, response to treatment, differentiated function, and so forth."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a molecular detection device having a plurality of binding or reaction sites comprising a series of immobilized recognition molecules or binding reagents selected and arranged to qualitatively or quantitatively assess the status of a series of signal transduction pathway proteins or other protein-protein interactive network, their phosphorylated or activated state, and their binding partners, in a cell sample to determine the status of a selected cellular signal transduction pathway.",
"Knowledge of the pathway status can then be used by the diagnostician or clinician to determine the health of the sampled cells, drug or other treatment efficacy or toxicity.",
"This knowledge can also be used to identify candidates for selected therapies, to aid in therapy selection for a given subject, and to determine drug toxicities.",
"Protein interactions are defined herein as the coupling of two or more proteins in a given space and time such that a binding reaction occurs, and/or one protein modifies the other protein.",
"The novel concept of the invention is that the protein networks and pathways, and their changing state in cells, can be recapitulated after the cells are dissolved and the proteins are solubilized.",
"The invention method recapitulates the proteins involved in networks because a) networks are built from proteins binding to other proteins to form larger complexes; and b) phosphorylation events cause specific proteins to interact with (or dissociate from) other proteins in a certain order.",
"Therefore, the upstream and downstream networks emanating from any given point can be elucidated by using the subject invention to find at least two proteins that are phosphorylated and forming complexes with other proteins.",
"This pattern changes with disease state or drug treatment or toxicity.",
"In a preferred embodiment, multiple nodes in a signal transduction pathway circuit within a cell are profiled to determine whether the entire pathway is functionally activated and in use by the cell or whether only a portion of the pathway is activated (e.g., upstream or downstream), thus indicating, for example, that the pathway may be regulated at intermediate points along the pathway circuit.",
"Also, the binding partners which complex with individual nodes of the pathway can be identified.",
"In still a further aspect, a method of identifying the full repertoire of proteins that could serve as acceptors for phosphorylation comprises treating cells, such as whole tissue specimens grown ex-vivo, or animal tissue treated in vivo with compounds that inhibit protein tyrosine and/or serine/threonine phosphatase activity such as sodium pervanadate, okadaic acid, calyculin A, for acute periods of time (e.g., less than 3 hours), and isolating the cells of interest.",
"The cells of interest are lysed and selected for the phosphorylated proteins using antibodies on an immobilized bait, such as anti-phosphotyrosine, anti-phosphoserine, and/or anti-phosphothreonine antibodies, and so forth.",
"The phosphoproteins in the enriched fraction are separated, e.g., by chromatographic and/or electrophoretic means and the primary amino acid sequence of the proteins is identified by enzymatic digestion or chemically-induced protein fragmentation followed by standard mass spectrometric techniques.",
"Finally, antibodies or binding molecules that specifically bind to the identified proteins are developed.",
"In further embodiments, protein pathways which reflect a disease state can be fingerprinted to pinpoint a therapeutic target.",
"In order to select a treatment or determine treatment efficacy, informatics or heuristics can be developed for various pathways, pathological states, therapies, and the like."
],
[
"RELATED APPLICATION This application claims the benefit under 35 U.S.C.",
"§ 119(e) of U.S. provisional application Ser.",
"No.",
"60/179,997, filed Feb. 3, 2000.U.S.",
"provisional application Ser.",
"No.",
"60/179,997 is incorporated herein by reference in its entirety.",
"BACKGROUND OF THE INVENTION The present invention relates to an apparatus and method for assessing the status of a cellular pathway.",
"Several biotechnology companies are developing DNA-chips which contain printed arrays of hundreds or thousands of genes, or more.",
"However, the DNA sequence of the genes or the level of gene expression do not reflect the actual functional state of the proteins encoded by the genes.",
"The functional state of each protein involves more than simply the amount of the proteins.",
"It includes, for example, post translational modifications (phosphorylation, glycosylation, etc.",
"), binding partners in cellular pathways, conformation, enzymatic function, and state of activation.",
"Moreover, each cell in the body has a pattern of functional proteins which reflects the current biologic working state of the cell (e.g., growing, differentiating, diseased, dying, senescent, etc.).",
"Therefore, there exists a need in the art for a device and method for determining the status of cellular pathways (e.g., the phosphorylation state or binding partners of involved proteins) in tissue samples, including human tissue samples, thus providing the diagnostician or clinician with an early warning of impending or occult toxicity, disease state, response to treatment, differentiated function, and so forth.",
"SUMMARY OF THE INVENTION The present invention provides a molecular detection device having a plurality of binding or reaction sites comprising a series of immobilized recognition molecules or binding reagents selected and arranged to qualitatively or quantitatively assess the status of a series of signal transduction pathway proteins or other protein-protein interactive network, their phosphorylated or activated state, and their binding partners, in a cell sample to determine the status of a selected cellular signal transduction pathway.",
"Knowledge of the pathway status can then be used by the diagnostician or clinician to determine the health of the sampled cells, drug or other treatment efficacy or toxicity.",
"This knowledge can also be used to identify candidates for selected therapies, to aid in therapy selection for a given subject, and to determine drug toxicities.",
"Protein interactions are defined herein as the coupling of two or more proteins in a given space and time such that a binding reaction occurs, and/or one protein modifies the other protein.",
"The novel concept of the invention is that the protein networks and pathways, and their changing state in cells, can be recapitulated after the cells are dissolved and the proteins are solubilized.",
"The invention method recapitulates the proteins involved in networks because a) networks are built from proteins binding to other proteins to form larger complexes; and b) phosphorylation events cause specific proteins to interact with (or dissociate from) other proteins in a certain order.",
"Therefore, the upstream and downstream networks emanating from any given point can be elucidated by using the subject invention to find at least two proteins that are phosphorylated and forming complexes with other proteins.",
"This pattern changes with disease state or drug treatment or toxicity.",
"In a preferred embodiment, multiple nodes in a signal transduction pathway circuit within a cell are profiled to determine whether the entire pathway is functionally activated and in use by the cell or whether only a portion of the pathway is activated (e.g., upstream or downstream), thus indicating, for example, that the pathway may be regulated at intermediate points along the pathway circuit.",
"Also, the binding partners which complex with individual nodes of the pathway can be identified.",
"In still a further aspect, a method of identifying the full repertoire of proteins that could serve as acceptors for phosphorylation comprises treating cells, such as whole tissue specimens grown ex-vivo, or animal tissue treated in vivo with compounds that inhibit protein tyrosine and/or serine/threonine phosphatase activity such as sodium pervanadate, okadaic acid, calyculin A, for acute periods of time (e.g., less than 3 hours), and isolating the cells of interest.",
"The cells of interest are lysed and selected for the phosphorylated proteins using antibodies on an immobilized bait, such as anti-phosphotyrosine, anti-phosphoserine, and/or anti-phosphothreonine antibodies, and so forth.",
"The phosphoproteins in the enriched fraction are separated, e.g., by chromatographic and/or electrophoretic means and the primary amino acid sequence of the proteins is identified by enzymatic digestion or chemically-induced protein fragmentation followed by standard mass spectrometric techniques.",
"Finally, antibodies or binding molecules that specifically bind to the identified proteins are developed.",
"In further embodiments, protein pathways which reflect a disease state can be fingerprinted to pinpoint a therapeutic target.",
"In order to select a treatment or determine treatment efficacy, informatics or heuristics can be developed for various pathways, pathological states, therapies, and the like.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention may take form in various components and arrangements of components, and in various steps and arrangements of steps.",
"The drawings are only for purposes of illustrating preferred embodiments and are not to be construed as limiting the invention.",
"FIG.",
"1 illustrates a cellular signal transduction pathway profiling device of the present invention.",
"FIGS.",
"2-6 illustrate exemplary pathway profiling methods in accordance with the present invention.",
"FIG.",
"7 illustrates a system and method for identifying therapeutic targets employing trapped lysates from cells of different pathological states.",
"FIG.",
"8 shows a multiplexed embodiment of the molecular detection device in accordance with the present invention.",
"FIG.",
"9 illustrates an exemplary pathway profiling device according to the present invention.",
"FIG.",
"10 illustrates autoradiography results for microdissected erb2+ and erb2−human breast cancer epithelium from tissue sample specimens probed for anti-ERK½ mouse monoclonal antibody using the pathway profiling device according to FIG.",
"9.FIG.",
"11 illustrates a method of the present invention for the detection of IFNA stimulation of the JAK-STAT signal pathway.",
"FIG.",
"12 illustrates the use of nitrocellulose engraved by a laser to manufacture raised porous columns that contain and channel the flow of cellular proteins through the zones.",
"In the depicted example, a high sensitivity and dose dependence of protein detection is noted for the intracellular human heart muscle protein troponin.",
"FIGS.",
"13A-13C, 14A, 14B, and 15-18 illustrate the use of activated glass beads as the flow-through matrix of the present invention.",
"FIG.",
"19 is a flow chart of a signal transduction pathway profiling method in accordance with the present invention.",
"FIGS.",
"20 and 21 illustrate the use of patterns for the identification of a pathway and/or disease state of a cell sample.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to FIG.",
"1, a cellular signal transduction pathway profiling device 100 includes a porous support member 110, such as a porous membrane or the like.",
"support member 110 may be fabricated from any material into which fluids may flow and readily pass through, such as nitrocellulose, cellulose, glass, nylon, or other fibrous material.",
"On the support 110, two or more, preferably three or more, distinct binding site regions or trapping zones 116 are formed by applying and immobilizing within each region a substance capable of reaction with an analyte contained within the test sample.",
"In a preferred embodiment, each region contains a single purified ligand, such as a protein, peptide, antibody, or drug capable of trapping a specific protein or modification thereof involved in a selected transduction pathway.",
"Alternately, the immobilized materials at the binding sites are complex mixtures (e.g., cellular lysates).",
"By “immobilized” is meant that the substance capable of holding an analyte is applied in a confined area on the surface of the membrane such that it is permanently bound or otherwise incapable of substantial movement to positions elsewhere on the support.",
"A lysate application region 114 and a chase application region 112 are provided at one end of the device 100.In this manner the lysate can be applied to the support 110, followed by a chase, such as a buffer or other physiological solution, to cause the lysate to flow through the trapping zones 116 to an outflow region 118.Preferably, the lysate is drawn through the reaction zones via wicking or capillary action, although other methods are contemplated as well, such as gravity, application of a pressure differential, electrical pumping, and so forth.",
"The trapping zones are applied in a manner such that subsequent capillary wicking of material for analysis will pass through the immobilized protein zone and not around it.",
"In one embodiment, the support material 110 forms a strip and the reaction zones 116 are applied in a line traversing the width of the strip.",
"When antibodies are used for trapping, they may be from any species, including but not limited to human, goat, rabbit, mouse, etc.",
"The antibodies can be either anti-protein specific (e.g., anti-ERK kinase) or anti-modified protein specific (e.g., anti-phosphorylated ERK kinase).",
"For signal transduction profiling, the sequence of the applied antibodies in the multiple reaction zones 116 are preferably ordered such that the protein components of a pathway that are activated last are captured first, although the ordering is otherwise not necessarily critical.",
"For example, the analysis of EGF signaling profiles from cells that are expressing differential amount of the erb receptor family can be analyzed by imprinting antibodies recognizing the phosphorylated forms of proteins in the EGF receptor signaling cascade.",
"The order of imprinting, from first capture zone to last capture zone, is shown below in TABLE 1.TABLE 1 Capture Zone Antibody 1 anti-phosphorylated c-myc 2 anti-phosphorylated c-jun 3 anti-phosphorylated p70RSK 4 anti-phosphorylated erk 5 anti-phosphorylated AKT 6 anti-phosphorylated c-RAF 7 anti-phosphorylated erb2 8 anti-phosphorylated erb1 Once the trapping zones 116 have been imprinted on the support 110, cellular lysates, DNA aptomer libraries, focussed or unfocussed drug libraries, and/or phage display libraries, can be applied to the top of the detection device 100 in the defined application zone 114 and allowed to actively wick through the support 110 by active capillary action using a buffer chase applied to region 112.Again, other methods of drawing the lysate through the zones 116 are also contemplated.",
"Analytes in the material for analysis, such as proteins, drugs, DNA, phages, will, depending on their abundance, specifically bind to a corresponding trapping zone region 116 having a substance capable of reaction therewith.",
"All other components will wick through the zone into the waste outlet region 118 at the bottom of the chip 100.Because these analyses are performed under native conditions, protein complexes, comprising activated proteins (e.g., phosphorylated proteins) and their binding partners will be trapped in each of the subsequent zones, depending on the degree of phosphorylation and the presence of the binding partners.",
"Subsequent analysis and/or identification of proteins trapped in the zones 116 is performed by countercurrent extraction, treatment with enzymes (e.g., trypsin), mimetics (e.g., phenylphosphate for the removal of phosphotyrosine-containing proteins), and so forth.",
"Analysis of extracted material can be performed by elution into other trap zones (three-dimensional separation) or by mass spectrometry (e.g., LCQ-MS, CE-ESI-MS) for identification and discovery purposes.",
"Additionally, quantitative analysis of trapped analyte composition can be performed by querying each trap zone with a tagged or detectably labeled antibody (e.g., biotinylated or alkaline phosphatase tagged) to generate a signal.",
"Exemplary methods for detecting the bound proteins are illustrated in FIGS.",
"2-6.An exemplary embodiment in which each trapping zone 116 is imprinted with lysates from cells of different types or different pathological states is illustrated in FIG.",
"7.Phage and/or DNA aptamer libraries 220, such as random phage and/or random aptamer libraries, can be screened and analyzed by successive rounds of trapping with a plurality of protein lysate zones 216a-216c.",
"For example, in a first binding site 216a, there is applied and immobilized a normal cell lysate.",
"In a second binding site 216b, there is applied and immobilized a lysate of cells in a predisease state.",
"In a third binding site 216c, there is applied and immobilized a lysate of diseased cells.",
"Each round of trapping is followed by amplification.",
"For example, each binding site can be cut out and amplified using PCR (for aptomer libraries) or amplification by infection (e.g., in E. coli for phage display peptide libraries).",
"Alternately, small molecule drugs can be identified by NMR or other like methodologies.",
"The amplified entities 222 can then be used for potential targeting therapies, e.g., as toxin-conjugated vehicles.",
"For example, an amplified entity which selectively binds to a protein representative of a disease state, such as cancer, can be conjugated or linked to a toxin for efficient delivery of the vehicle to the targeted cells.",
"Likewise, an amplified entity can be conjugated to an imaging reagent for diagnostic imaging of the targeted cells, such as x-ray contrast agents, MRI contrast agents, radiopharmaceuticals for nuclear medicine diagnostic imaging, and so forth.",
"These entities can be further screened for DNA or peptide sequences that bind to unique cell-specific or tissue-specific proteins by repanning against immobilized bait trap surfaces.",
"Referring now to FIG.",
"8, there is shown another embodiment of the present invention in which a molecular detection device 300 comprises a plurality of support members 310 arranged in a multiplexed format.",
"Each support member includes a plurality of binding sites 316 to which is applied and immobilized a series of proteins and modifications thereof involved in a selected signal transduction pathway.",
"Such a format is suitable for high-throughput drug screening using cell lines and/or microdissected tissue cell lysates.",
"A lysate application zone 314 is provided for each strip 310.In alternate embodiments, the zones 314 are cell growth zones for selected cell lines, in which case an optional lysing buffer application zone 322 is provided such that the cells are lysed prior to reaching the trapping zones 316.A chase application zone 312 is provided for application of a buffer or other physiological solution to carry the lysate through the trapping regions 316.Analytes in the lysate will specifically bind to a corresponding trapping zone region 316 having a substance capable of reaction therewith and all other components will wick or otherwise be drawn through the zones into the waste outlet region 118 at the bottom of the device 300.The reduction of this method to practice has been demostrated in the following tests.",
"A pure nitrocellulose membrane (0.45 micron pore size, Shleister and Schuell) was cut as a 6 cm×1 strip beginning and ending with wider 3 cm×3 ends.",
"The following commercially available rabbit polyclonal antibodies (New England Biolabs, Upstate Biotechnology) at 100 microgram/ml concentration were applied undiluted in total applied volume of 2 microliters as traping zone “stripes” perpendicular to the length of the menbrane as shown in FIG.",
"9.The antibodies were applied in bands approximately 2-3 mm wide, spaced at repeated 0.5 cm intevals, and ordered from top to bottom as shown in TABLE 2.TABLE 2 Capture Zone Antibody 1 anti-phosphorylated c-myc 2 anti-phosphorylated p70 SK kinase 3 anti-phosphorylated elk 4 anti-phosphorylated STAT5 5 anti-phosphorylated AKT 6 anti-phosphorylated c-erb2 7 anti-phosphorylated c-erb1 8 anti-phosphorylated erk kinase The antibodies were allowed to bind to the membrane overnight, after which the entire strip was immersed in a commercially available casein blocking solution (Superblock, Pierce Chemical ) for 2 hours.",
"The membrane was then washed 3 times for 10 minutes with 20 milliliters of a 50 mM TRIS, 100 mM NaCl, 0.5% Tween−20 solution (TBST).",
"The strip was then allowed to air-dry for 5 hours.",
"Lysates comprised of 1500 cells procured via Laser Capture microdissection (LCM) of two human breast cancer specimens, one known to be highly reactive for erb2 (erb2+), and the other known to be weakly reactive (erb2−), were applied to the top of the strip (lysate application region) in a volume of 5 microliters of commercially available lysing buffer (T-PER, Pierce Chemical) with a commercially available protease inhibitor cocktail (Complete Tablets, Boehringer Mannheim) and 1 μl sodium vanadate as a phosphotyrosine phosphatase inhibitor.",
"25 microliters of the T-PER solution was applied as a chase immediately after the application of the lysate, and the lysates were allowed to actively “wick” through the strip over a period of 60 minutes.",
"An additional volume of 25 microliters of T-PER was applied to the chase zone when the buffer front was approximately ½ of the way through the strip.",
"Five minutes after the buffer front had passed through the last antibody trap zone, the entire strip was immersed and washed 3 times for 10 minutes in TBST.",
"The strip was then immersed in TBST+ mouse monoclonal anti-ERK (Transduction Laboratories) at a 1:2000 dilution for 1 hour.",
"The strip was then washed 3 times for 10 minutes each time with TBST, and then incubated for 1 hour with a biotinylated goat anti-mouse IgG antibody (Vector Laboratories) at a 1:5000 dilution.",
"The strip was then washed 3 times for 10 minutes each time in TBST, and developed according to the package insert from Vector Laboratories using a commercially available kit (ABC reagent) which generates luminescent output.",
"The strips were then exposed to standard autoradiography for 1 second to 30 second exposures.",
"The results of the autoradiography are illustrated in FIG.",
"10.The following experimental example demonstrates the recapitulation of the state of a cellular signal pathway using proteins dissociated from lysed human cells treated with a drug known to activate the selected signal pathway.",
"The Janus kinase-signal transducer and activator of ranscription (JAK-STAT) signaling pathway is important in he Interferon Alpha (IFNA) cellular response.",
"Binding of FNA induces the following sequence of events: Fusion of Interferon Alpha Receptor(IFNAR)1 and IFNAR2; Jak1 and Tyk2 are phosphorylated; IFNAR1 is phosphorylated allowing docking of Stat2; Stat2 is phosphorylated allowing Stat1 to dock; Stat1 is phosphorylated; Stat1(Pi)-Stat2(Pi) heterodimer is released.",
"Interferon Alpha Treatment of Peripheral Blood Lymphocytes Peripheral Blood Lymphocytes (PBLs) were isolated using Histopaque-1077 (Sigma).",
"The PBLs were suspended in PBS (0.01 M phosphate buffered saline, 0.138 M NaCl, 0.0027 M KCl), pH 7.4, and allowed to acquiesce overnight at 4° C. The cells were pelleted by centrifugation at 5000×g for 5 minutes.",
"The supernatant was decanted off, and the cells resuspended in RPMI-1640 media (Bio-Whittaker) at approximately 20 million cells/mL.",
"The cells were treated with 13,500 units/mL of recombinant human interferon alpha (BioSource) for 0, 3, 10 or 30 minutes.",
"Once the incubation was complete, cells were pelleted by centrifugation at 5000×g for 5 minutes.",
"Cells were washed with ice-cold PBS and resuspended in lysis buffer ((T-PER, Pierce), 10 mM beta-glycerophosphate, 1 mM sodium molybdate, 2 mM sodium orthovanadate, 2.5 mM AEBSF and 5% glycerol).",
"The cells were vortexed and placed on ice for 5 minutes, repeated.",
"Lysates were clarified by centrifugation at 10,000×g for 5 minutes.",
"The supernatant was snap frozen and stored at −80° C. Pervanadate Treatment of Peripheral Blood Lymphocytes Peripheral Blood Lymphocytes (PBLs) were isolated using Histopaque-1077.The PBLs were suspended in PBS and allowed to acquiesce overnight at 4° C. The cells were pelleted by centrifugation at 5000×g for 5 minutes.",
"The supernatant was decanted off, and the cells resuspended in RPMI-1640 media at approximately 20 million cells/mL.",
"A pervanadate solution was made as follows.",
"882 μL of 0.10 M sodium orthovanadate was added to a mixture containing 832 μL RMPI-1640 media and 50 μL 30% H2O2, mixed at room temperature, and let stand for 15 minutes.",
"The cells were treated with pervanadate 1 μL to 500 μL of cells for 30 minutes at 37° C. Cells were pelleted at 5700×g for 5 minutes.",
"Pellets were washed with ice-cold PBS and snap frozen.",
"T-PER (salt concentration was adjusted from 0.150 M to 0.20 M) was added to the pellet and the resulting lysate was vortexed rapidly for 1 minute.",
"The lysate was clarified by centrifugation at 15,000×g for 10 minutes.",
"The supernatant was snap frozen and stored at −80° C. Preparation of Flow-Through Trapping Zones.",
"FF85 (Schleicher and Schuell) nitrocellulose membranes were cut into approximately 8 cm×1 cm strips.",
"Stat2 antibody (C-20, Santa Cruz Biotechnology) was spotted down at 0.5 μL increments/2.5 μL total across the 1 cm width of the strip at approximately 6.0 cm from the top.",
"Stat1αp91 antibody (C-24, Santa Cruz Biotechnology) was strided down at 0.5 μL increments/2.5 μL total across the 1 cm width of the strip at approximately 7.0 cm from the top.",
"The treated membranes were dried for 45 minutes at room temperature and 6% relative humidity.",
"The strips were blocked in Superblock (Pierce Chemical Company) for 3 hours at room temperature with shaking.",
"The strips were washed 3 times with TBS-T (0.20 M Tris, 0.50 M NaCl, 0.1% Tween20), pH7.5, then dried at 37° C./6% relative humidity.",
"p-Stat1 Association Strip Assay The IFNA treated PBL lysates (25μL) were placed approximately 1 cm from the top of the strip.",
"The lysate was moved through the strip upon addition of TBS-T to the top of the strip.",
"Every 20 minutes over a period of 3 hours, 50 μL of TBS-T was placed at the top of the strip.",
"Upon completion the entire strip was washed 1 time with TBS-T.",
"The strips were incubated overnight at 4° C. with p-Stat1 (A-2, Santa Cruz Biotechnology) diluted at an appropriate concentration in Superblock.",
"The strips were washed 3 times with TBS-T.",
"The strips were then incubated at room temperature for 3 hours with Goat Anti-Mouse:Alkaline Phosphatase (Fortran, In House Conjugation to AP) diluted in Superblock.",
"The strips were washed 3 times with TBS-T.",
"The strips were incubated with CDP-Star (Tropix) for 5 minutes.",
"Light output/binding of p-Stat1 antibody was recorded using a CCD imager (NightOwl, Berthold Technologies).",
"As shown in FIG.",
"11, the invention method was able to detect IFNA stimulation of the JAK-STAT signal pathway in the expected time-dependent manner.",
"CTNI Example The following Example demonstrates how parallel arrays of flow-through binding zones useful for practicing the subject invention can be manufactured by using a laser engraver to build up porous matrix columns.",
"The raised columns on a plastic backing contain and direct the flow of solubilized proteins to be analyzed.",
"The example indicates the quantitative dose-dependent detection of troponin, an intracellular myofibril component of human cardiac muscle cells.",
"Nitrocellulose, purchased from Schleicher and Schuell, comprises an inert plastic (Mylar) backing support onto which a layer of directly cast nitrocellulose is deposited.",
"During our investigation of this matrix for flow-through diagnostic applications, it was discovered that it is possible to create channels in the material.",
"This was achieved by using the cutting power of a CO2 laser.",
"The laser is programmable through a computer software application, which enables the creation of highly complex patterns on the surface of the nitrocellulose.",
"Briefly, the laser vaporizes the cellulose beneath the beam, exposing the underlying mylar surface.",
"One of the simplest applications of this process is to take a 4 cm×4 cm square of nitrocellulose (or any other desired size) and use the laser to cut a series of vertical slots in the material.",
"In this manner, one creates in the nitrocellulose a number of “columns”, each column separated by an inert mylar barrier.",
"Interpreted another way, a series of “usable” ridges are manufactured on the matrix, each ridge usable for a similar or dissimilar diagnostic application.",
"The number of ridges created on the material varies with the width of the ridge and the length of the nitrocellulose piece.",
"The laser action also scores the upper surface of the mylar, allowing for easy detachment of a single column or multiple columns from the sheet.",
"A series of columns (3 mm wide) were cut into a 4 cm×4 cm length of nitrocellulose.",
"Seven columns were detached and treated as follows.",
"1 μl of a 1 mg/ml solution of neutravidin (Pierce Chemical Co., Rockford, Ill.) was applied to each ridge.",
"The protein was heat (37*) cured on the matrix for 2 hrs.",
"After washing, a 1 μl aliquot of biotinylated anti-cardiac troponin I antibody was overlayed onto the original neutravidin spot.",
"Thirty minutes later, excess antibody was removed and the whole surface of the matrix blocked in a PEG/PVP solution.",
"A series of troponin I calibrators (purified from human heart muscle), ranging in concentration from 0 to 160 ng/ml were reacted individually with an anti-troponin I antibody conjugate.",
"The latter recognizes a site on troponin I, which differs from the site recognized by the neutravidin bound antibody.",
"A 1 μl aliquot of each antigen/conjugate reaction was placed onto individual columns.",
"The placement of the liquid was immediately below the neutravidin complex.",
"The lower edge of the seven column composite was placed into a buffer allowing the latter to move through the material via capillary action (conventional TLC).",
"The length of time required for the buffer to reach the top of the device was 45 seconds.",
"The device was removed and coated in CDP* substrate (Tropix Corporation, Bedford, Massachusetts), light emission was recorded on a NightOwl CCD scanner of light emission.",
"The flow-through trapping zones created in nitrocellulose porous matrix columns efficiently retained antibody antigen complexes such that the troponin analyte could be measured in a dose-dependent manner as shown in FIG.",
"12 and TABLE 3.TABLE 3 Card Style Troponin Membrane; Dose Curve 1 μL(Sample + Conjugate) Troponin (ng/mL) Light output (cts/sec) 0 0 2.6 4955 5.2 7264 10.4 8477 20.8 13116 41.65 18199 83.5 21415 The porous matrix material through which the cellular protein flows has been successfully reduced to practice with porous or fibrous materials, such as nylon, cellulose or silica, and can be configured as raised porous interconnecting columns or suitably packed particles or beads.",
"The following example demonstrated the use of activated glass beads to construct a flow through matrix of the present invention.",
"Controlled pore glass (CPG) is a matrix whose surface is readily modified by reaction with a wide variety of bifunctional silanes.",
"The material exhibits a significant surface area to weight ratio.",
"The rigid nature of the glass, high porosity and non-compressibility lends itself to rapid passage of liquids or biological fluids through the matrix.",
"The present invention employing a series of capture zones having differing specificity for removal and quantitation of phosphorylated and non-phosphorylated protein complexes from stimulated cells can be achieved through the use of CPG.",
"By confining specific capture zones of CPG in a micro-capillary tube, each zone separated by either inert glass or an inert cellulose plug, biological fluids can be drawn through each zone (e.g., using a vacuum or positive displacement pump, or the like), to capture the desired entities in the zones.",
"The microenvironment of the capillary minimizes diffusion limitations thereby enhancing acceleration of the rate of binding of the ligand to the capturing agent.",
"Accordingly, CPG (Sigma Corporation, St. Louis, Mo.)",
"was reacted with 3-aminopropyl trimethoxysilane (Sigma Corporation, St. Louis, Mo.)",
"using standard published procedures.",
"Subsequently, the amino modified glass was reacted with iminothiolane (Traut's reagent, Pierce Chemical Co., Rockford, Ill.).",
"The latter procedure provides a sulphydryl group (thiol) for further reaction with maleimido modified proteins, drugs, nucleotides and other entities so modified.",
"Maleimido horseradish peroxidase was reacted with 20 mg of iminothiolane CPG.",
"After washing, small portions (2 mg×3) of the beads were loaded into a micro-capillary tube.",
"Each zone was separated by an inert glass or plug.",
"A chemiluminescent substrate (Duolux, Lumigen, Southfield, Mich.) was pulled rapidly into the capillary using vacuum.",
"The tube was transferred to a light measuring device (NightOwl CCD light scanner) and the light output read for 1 sec.",
"The results are shown in FIG.",
"13A.",
"In either case, three illumination zones are indicated, thereby demonstrating the feasibility of the system.",
"In a further iteration, an epitope of cardiac troponin I (P14 cTNI epitope, Research Genetics, Huntsville, Ala.), conjugated to thyroglobulin (porcine thyroglobulin, Sigma Corporation, St. Louis, Mo.)",
"was reacted with CPG.",
"An antibody (P3 cTNI antibody, Fortron Bioscience Inc., Morrisville, N.C.), conjugated to peroxidase or alkaline phosphatase (AP), was introduced into the requisite capillary, excess conjugate was removed through rapid washing and the bound antibody enzyme visualized with an appropriate chemiluminescent substrate.",
"The results are recorded in FIGS.",
"13B and 13C.",
"A single peroxidase bead, 100-200 microns in diameter, was exposed to Duolux and the light output recorded, compared with substrate background.",
"The calculated signal to noise ratio was 3.5, indicating that the system has the potential of being reasonably sensitive.",
"The results are shown in FIG.",
"14A.",
"In order to provide a generic CPG system, maleimido-neutravidin (neutravidin and maleimido peroxidase were purchased from Pierce Chemical Co., Rockford, Ill.) was reacted with iminothiolane glass.",
"Neutravidin binds biotinylated species aggressively.",
"Such biotinylated species include peptides, proteins, drugs, nucleotides and other entities so modified.",
"In this example, biotin containing Stat 1 and phosphorylated Stat 1 antibodies (Stat1{pY701} peptide, BioSource International, Camarillo, Calif.) were attached to CPG.",
"Bovine serum albumin (Sigma Corporation, St. Louis, Mo.)",
"containing phosphorylated Stat 1 peptide was drawn into a capillary, in which a zone of each was antibody was deposited.",
"After washing, the zones were probed with RC20-AP (RC20-AP antibody conjugate purchased from Transduction Laboratories, Lexington, Ky.).",
"Following a further wash, substrate was introduced and the light output recorded.",
"In this experiment it was anticipated that the luminescence from the p-Stat 1 zone would be greater than that from the Stat 1 zone.",
"This turned out to be the case.",
"The light output from the Stat1 zone is related to the poor quality of the RC-20 AP conjugate and probably represents non-specific binding to the bound matrix antibody.",
"To demonstrate that binding to a zone is reversible in the presence of a appropriate ligand, p-Stat 1 antibody beads were exposed to p-Stat 1 peptide peroxidase, both in the absence and presence of p-Stat1 peptide albumin (blocking agent).",
"As anticipated, the presence of the peptide reduced the binding of the conjugate, resulting in less light output.",
"The results are shown in FIG.",
"14B.",
"To further demonstrate the utility of our CPG capillary approach, neutravidin beads were reacted with a biotinylated antibody which recognizes a specific epitope on cardiac troponin I (cTNI).",
"A negative serum sample was drawn through the capillary (vacuum).",
"After washing, a secondary AP labeled antibody, with a different specificity for cTNI was introduced and excess conjugate removed immediately by washing.",
"Exposure of the beads to substrate produced the light output shown in FIG.",
"15 (-⋄-).",
"Using the same capillary and procedure, a positive sample of cTNI (490 pg/ml) was examined for light yield (-□-).",
"The calculated signal to noise ratio of 5 to 1 indicates that the system is capable of extreme sensitivity.",
"An overlay of the light output from the capillary is included in FIG.",
"15.Peripheral blood lymphocytes (PBLs) were exposed to alpha-interferon.",
"The cells were lysed and cellular debris removed by centrifugation.",
"In a similar fashion, non-treated cells were and lysed and the cell contents collected.",
"Fifty microliters of each lysate were drawn through separate capillaries, each containing Stat 1 antibody CPG beads.",
"The capillaries were probed with RC20-AP.",
"After washing, substrate (CDP*) was drawn into the tubes and the light emission recorded in a tube luminometer (Junior Luminometer, Berthold Technologies) or the Nightowl CCD scanner.",
"The data indicates that stimulation of PBLs with interferon, induces an increase of phosphorylated tyrosine species, which bind to Stat 1 protein.",
"This is indicative of binding of tyrosine phosphorylated Stat 2 to Stat 1, thus forming the recognized heterodimer, known to be part of the alpha-interferon/lymphocyte signaling cascade.",
"The results are illustrated in FIG.",
"16 An additional approach to confirm the observations from FIG.",
"16 is described here and shown in FIG.",
"17.Instead of probing with RC20-AP, a non-labeled mouse antibody specific for phosphorylated tyrosine in conjunction with a goat anti-mouse AP conjugate were employed sequentially.",
"Negatively and positively, alpha-interferon stimulated PBL lysates were drawn through individually packed capillaries containing Stat 1 antibody CPG.",
"After washing, etc., and exposure to the primary and secondary antibodies, the beads were exposed to CDP* substrate.",
"The increase in positive light emission supports our contention that stimulation of PBLs with alpha-interferon leads to an increase in phosphorylated Stat2, which in turn binds to Stat 1 protein.",
"The phosphatase activity of PBLs is significantly reduced in the presence of pervanadate.",
"Inhibition of phosphatase activity enhances the overall level of phosphorylated proteins within the lymphocyte.",
"The experiment shown and described with reference FIG.",
"18 illustrates this phenomenon quite nicely.",
"Briefly, CPG Stat 1 antibody beads (10 mg), were placed in a Z-spin well (Z-spin columns were obtained from Fisher Scientific, Pittsburgh, Pa.).",
"As a control, plain neutravidin CPG was used.",
"To each well, 50 μl of pervanadate treated PBL lysate was added.",
"The wells were centrifuged, washed and then exposed sequentially to mouse anti-phosphorylated tyrosine antibody and goat anti-touse AP conjugate.",
"Each addition was followed by a wash/centrifuge cycle.",
"The addition of CDP* substrate induced light emission which was recorded in the NightOwl.",
"The data indicates that inhibition of phosphatase activity enables measurement of the basal level of phosphotyrosine proteins in non stimulated PBLs.",
"In this instance, the data suggest that this type of methodology is capable of discriminating phosphorylated Stat2 from a whole range of known and as yet unknown phosphorylated signaling intermediates.",
"The following are experimental examples of more than 20 specific different trapping ligands that were successfully used in the subject invention for protein network profiling.",
"The invention methods led to identification of phosphorylated proteins and assignment of their functional participation in signal pathways.",
"The information derived is relevant to the following: a) identifying specific protein-protein interactions; b) assignment of the specific interactions into larger functional circuits and networks; c) downstream ordering of protein components in a given pathway; d) specific disruptions caused by drug treatments, disease, or toxicity; and e) identifying patterns of protein interactions or phosphorylations that are unique to disease state, drug treatment or toxicity.",
"1.100,000 primary human lymphocytes and 100,000 primary human monocytes obtained by tangential centrifugal elutriation and differential ficoll-percoll gradient selection were resuspended in 5 ml (each cell type) RPMI-1640 media.",
"2.1 ml aliquots were placed in eppendorf tubes 3.A 100× cocktail (herein referred to as PI or phosphatase inhibitor cocktail) consisting of the following: 2500 nM Okadaic acid (cell-permeable inhibitor of protein phosphatase); 500 micromolar sodium pervandate (membrane soluble form of sodium orthovanadate and an inhibitor of tyrosine phosphatases), 500 nM Calyculin A (cell-permeable inhibitor of protein phosphatase 2A and protein phosphatase); and 2500 micromolar phenylarsine oxide (cell-permeable phosphotyrosine phosphatase inhibitor).",
"4.The following experimental treatment protocol was then set up and performed with each 1 ml cell suspension (20,000 cells/ml): i. primary human lymphocytes/monocytes+ vehicle alone (10 microliters DMSO) 1 hour; ii.",
"primary human lymphocytes/monocytes+1× PI ½ hour; and iii.",
"primary human lymphocytes/monocytes+1× PI ½ hour+10 micromolar SB 203589 (p38 kinase inhibitor) ½ hour.",
"5.Cells were pulse centrifuged at 5000×g for 2.minutes, washed twice in ice cold PBS and the resultant cell pellet lysed in 100 microliters lysing solution (TPER (Pierce Chemical)+2 mM sodium orthovanadate (tyrosine phosphatase inhibitor), 10 mM B-glycerol phosphate (serine phosphatase inhibitor), 300 mM NaCl, 4 NM AEBSF (protease inhibitor)), vortexed rapidly for.",
"1 minute and clarified by centrifugation for 5 minutes.",
"At this point the lysates are analyzed by 4 main methods.",
"i.",
"Open faced array of antibodies immobilized on nitrocellulose (see No.",
"6 list below).",
"ii.",
"Flow-through devices striped with trapping zones containing protein binding ligands.",
"The analysis was complemented by the following additional standard methods: iii.",
"Two-dimensional or one-dimensional gel-based separation technologies.",
"iv.",
"Mass-spectrophotometric-based baiting technologies.",
"6.The resultant whole cell lysate was then incubated overnight at 4 degrees C. on an Oncyte multiwell (Grace Biolabs, Inc) nitrocellulose slide that was spotted with rabbit polyclonal phospho-specific antibodies recognizing the following 20 proteins: EGFR (Tyr1173); Tyk2 (Tyr1054/105); ATF-2 (Thr71); eIF2a (Ser51); eIF4E (Ser209); Cdc2 (Tyr15); p38 (Thr180/Tyr182); Cdk1 (Thr14/Tyr15); CREB (Ser133); Akt (Ser473); eNOS (Ser1177); CREB (Ser133).",
"; c-Jun(Ser63); Elk-1 (Ser383); ErbB2 (Tyr1248); Bad (Ser112); Jak1 (Tyr1022/1023); p70 S6 (Thr421/Ser424); ERK½ (Thr202/Tyr204); and Cdc25 (Ser216).",
"7.The slide was washed 3× with TBC-tween for 5 minutes, then incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody for 1 hour at 4 degrees C. 8.Chemiluminescent detection was performed using the ECL-plus kit from Amersham following recommended manufacturer procedures.",
"9.Proteins whose phosphorylation is dependent upon p38 kinase activity and lie downstream of the p38 kinase are subsequently identified by their inhibition of phosphorylation due to the pre-incubation of the specific p38 kinase inhibitor.",
"10.For 2D-PAGE applications, the lysates are 35 enriched for phosphorylated proteins via: i. Affinity chromatography-based or immuno-precipitation-based methods (i.e., using a anti-phosphotyrosine antibody, anti-phosphoserine antibody, etc.",
"); and ii.",
"Eluted by a competitive mimetic such as phenylphosphate or sodium pyrophosphate or a specific peptide used as the antigen for the development of the antibody.",
"iii.",
"The resultant eluate is then run on the gels directly, and phosphorylated proteins detected by traditional staining procedures compatible with mass spectroscopy-based protein identification (e.g., colloidal coomassie, ponceau S, SYPRO Ruby-red (Molecular Probes, Eugene, OR), and so forth) iv.",
"The visualized proteins are identified by mass spectroscopy and proteins whose activity lie downstream and are substrates for the p38 kinase are identified by the absence of the signal on the gel which separated lysates that were pre-treated with the specific kinase inhibitor.",
"11.For flow-through applications see the previous example for IFNA and ERB2.12.For mass-spectrophotometric applications a Ciphergen Inc. mass spec.",
"instrument was employed.",
"The resultant phosphoprotein-enriched eluates or whole cell extracts are incubated on bait traps that specifically bind phosphorylated proteins.",
"Such baits may be protein-based (e.g., anti-phosphotyrosine antibody) or chemical (e.g., iron, gallium ion, etc.).",
"The surfaces are then washed and phosphorylated proteins detected and identified by time-of-flight or collision-induced daughter ion spectral analysis.",
"Novel circuit profiles and maps were successfully obtained using ligands that recognized the phosphorylated versions of the 20 proteins listed in above.",
"1.The following proteins were identified to lie downstream of p38 kinase: CREB, CD25, cJUN, Cdk1, and e1f2A.",
"2.The following proteins can be activated in primary human monocytes: EGFR, Tyk2, E1F2a, E1F4E, p38, CDK1, CREB, c-Jun, Elk-1, BAD, Jak1, Erk ½, CD25.The following were specific to monocytes and not found in lymphocytes from the same patient: EGFR, Tyk2, E1F2a.",
"3.The following proteins are specifically activated in primary human lymphocytes but not monocytes from the same patient: ERB2.4.The following new unknown proteins changed their phosphorylation state in response to treatment with SB203589: mw.",
"100 kDa; mw.",
"85 kDa; mw.",
"35 kDa; mw.",
"30 kDa; mw.25 kDa; mw.",
"20 kDa.",
"5.ERB2 positive primary human breast cancer cells show that ERK is interacting and binding with the following proteins to a greater level than ERB2 negative human breast cancer cells: pERB2, pERB1, pELK, p70S6K.",
"6.Fresh human brain and prostate tissue cells can be treated ex vivo with phosphatase inhibitors under the subject invention resulting in the identification of proteins that are phosphorylated in a tissue and disease specific manner.",
"FIG.",
"19 is a flow chart of a molecular detection method in accordance with the present invention.",
"As indicated in step 402, the method includes arranging a series of recognition molecules or binding reagents which selectively bind to the various proteins, their phosphorylated or activated state, and their binding partners, of a signal transduction pathway to form a signal transduction pathway profiling chip 100 as described above by way of reference to FIG.",
"1.The method further includes applying (step 404) a cell lysate from a tissue of interest to the profiling chip.",
"Thereafter, the sample is allowed to bind or hybridize with the recognition molecules at the binding sites 116 (FIG.",
"1) as indicated by step 406 At step 408, binding events at the plurality of binding sites 116 are detected.",
"The detection can be performed by a number of labeling or other techniques to produce a detectable signal, such as spectrophotometric, fluorometric, colorimetric, chemiluminescent, radiometric, electrochemical, photochemical, enzymatic, or optical readout techniques, and the like.",
"In one embodiment, the step of detecting the binding events provides a qualitative indication of binding at each binding site.",
"More preferably, however, a quantitative determination of binding at each binding site is made at step 408, for example, by determining an intensity or magnitude of the detectable signal at each binding site.",
"At step 410, the detected binding events are used to determine a characteristic of the analyzed sample.",
"By identifying at which sites binding events occurred, the status of the selected pathway can be determined.",
"The determination can be made by visual detection of the binding sites.",
"Alternately, the characteristics can be determined automatically, for example, using a binding event sensor and pattern recognition software so that the pathway status can be determined under preprogrammed control.",
"Furthermore, heuristic algorithms are used to correlate binding patterns and/or pathway status with cellular conditions, such as normal, pre-disease, or disease states.",
"The use of patterns for the identification of a pathway and/or disease state of a cell sample is illustrated in FIGS.",
"20 and 21.Appendix A of 17 pages is incorporated herein by reference.",
"Appendix A presents further exemplary embodiments related to the protein interaction profiling system and method of the present invention.",
"The description above should not be construed as limiting the scope of the invention, but as merely providing illustrations to some of the presently preferred embodiments of this invention.",
"In light of the above description and examples, various other modifications and variations will now become apparent to those skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.",
"Accordingly, the scope of the invention should be determined solely by the appended claims and their legal equivalents."
]
] | Patent_10182354 |
[
[
"Defoaming agent for liquid hydrocarbons II",
"A process for inhibiting a liquid hydrocarbon from foaming involving: (a) providing a liquid hydrocarbon; (b) providing a defoamer additive containing: (i) an ester corresponding to formula (I) R1—COO—R2 (I) wherein R1 and R2 are, independent of each other, saturated or unsaturated, linear or branched alkyl radicals having from about 6 to 16 carbon atoms, as a defoamer; and (ii) optionally, a co-defoamer selected from the group consisting of a non-alkoxylated ester of a polyol having from about 2 to 6 carbon atoms, and from about 2 to 4 OH groups, a saturated or unsaturated, linear or branched fatty acid having from about 8 to 24 carbon atoms, and mixtures thereof; and (c) adding a foam-inhibiting effective amount of (b) to (a)."
],
[
"1-11.",
"(canceled) 12.A process for inhibiting a liquid hydrocarbon from foaming comprising: (a) providing a liquid hydrocarbon; (b) providing a defoamer additive containing: (i) an ester corresponding to formula (I) R1—COO—R2 (I) wherein R1 and R2 are, independent of each other, saturated or unsaturated, linear or branched alkyl radicals having from about 6 to 16 carbon atoms, as a defoamer; and (ii) optionally, a co-defoamer selected from the group consisting of a non-alkoxylated ester of a polyol having from about 2 to 6 carbon atoms, and from about 2 to 4 OH groups, a saturated or unsaturated, linear or branched fatty acid having from about 8 to 24 carbon atoms, and mixtures thereof; and (c) adding a foam-inhibiting effective amount of (b) to (a).",
"13.The process of claim 12 wherein in formula (I) R1 and R2, independently of each other, are alkyl radicals having from about 8 to 12 carbon atoms.",
"14.The process of claim 12 wherein R1 and R2 are selected from the group consisting of a mono-unsaturated alkyl radical, a poly-unsaturated alkyl radical, and mixtures thereof.",
"15.The process of claim 12 wherein both R1 and R2 have an identical number of carbon atoms.",
"16.The process of claim 12 wherein (i) is octyl octanoate.",
"17.The process of claim 12 wherein (b) is added to (a) in an amount of from about 1 to 5000 ppm.",
"18.The process of claim 12 wherein (b) is added to (a) in an amount of from about 1 to 3000 ppm.",
"19.The process of claim 12 wherein (b) is added to (a) in an amount of from about 1 to 2500 ppm.",
"20.The process of claim 12 wherein the liquid hydrocarbon is a lubricant.",
"21.The process of claim 12 wherein the co-defoamer is present in (b) in an amount of from about 1 to 10% by weight, based on the weight of the defoamer additive.",
"22.The process of claim 12 wherein the co-defoamer is a triglyceride of natural origin.",
"23.A low-foaming liquid hydrocarbon composition comprising: (a) a liquid hydrocarbon; and (b) a defoaming additive containing: (i) an ester corresponding to formula (I): R1—COO—R2 (1) wherein R1 and R2 are, independent of each other, saturated or unsaturated, linear or branched alkyl radicals having from about 6 to 16 carbon atoms; and (ii) optionally, a co-defoamer selected from the group consisting of a non-alkoxylated ester of a polyol having from about 2 to 6 carbon atoms, and from about 2 to 4 OH groups, a saturated or unsaturated, linear or branched fatty acid having from about 8 to 24 carbon atoms, and mixtures thereof.",
"24.The composition of claim 23 wherein in formula (I) R1 and R2 independently of each other, are alkyl radicals having from about 8 to 12 carbon atoms.",
"25.The composition of claim 23 wherein R1 and R2 are selected from the group consisting of a mono-unsaturated alkyl radical, a poly-unsaturated alkyl radical, and mixtures thereof.",
"26.The composition of claim 23 wherein both R1 and R2 have an identical number of carbon atoms.",
"27.The composition of claim 23 wherein the ester is octyl octanoate.",
"28.The composition of claim 23 wherein the ester is present in the composition in an amount of from about 1 to 5000 ppm, based on the weight of the composition.",
"29.The composition of claim 23 wherein the ester is present in the composition in an amount of from about 1 to 3000 ppm, based on the weight of the composition.",
"30.The composition of claim 23 wherein the ester is present in the composition in an amount of from about 1 to 2500 ppm, based on the weight of the composition.",
"31.The composition of claim 23 wherein the liquid hydrocarbon is a lubricant.",
"32.The composition of claim 23 wherein the co-defoamer is present in the composition in an amount of from about 1 to 10% by weight, based on the weight of the defoamer additive.",
"33.The composition of claim 23 wherein the co-defoamer is a triglyceride of natural origin."
],
[
"The present invention relates to the use of liquid esters for defoaming liquid hydrocarbons, a process for defoaming liquid hydrocarbons and also additives for defoaming liquid hydrocarbons.",
"In the field of lubricant technology, the high demands placed on lubricants today dictate not only that suitable base oils be used but also that their performance be supplemented and improved by various additives.",
"Examples of such additives include oxidation inhibitors, viscosity index improvers, pour point depressants, detergents and dispersants, high pressure (HP) additives, friction modifiers and defoamers.",
"The latter are required because many lubricant oils tend to foam which can sometimes drastically reduce the performance of the lubricant, in particular in closed lubricant circuits.",
"Foaming of lubricants accordingly has to be avoided at all costs.",
"The problem of foaming of lubricants is normally solved by incorporating foam inhibitors in the lubricants, for example, low molecular weight silicone oils or alkyl polyacrylates.",
"Preference is given to using low molecular weight silicone oils such as polydimethylsiloxanes, fluorosilicones or silicone glycols for this purpose.",
"However, silicones have the disadvantage that, in organic liquids which are then subjected to combustion, the reaction of the organosilicone polymer with oxygen leads to the formation of silicon oxides, which in finely divided form firstly cause environmental pollution and secondly lead to problems with filters and catalysts in the system to be lubricated.",
"This problem occurs in particular in the automobile sector or more precisely in internal combustion engines.",
"U.S. Pat.",
"No.",
"3,166,508 discloses defoamers for hydrocarbons based on alkyl acrylate polymers having a molecular weight of less than 10 000.However, such defoamers show a lower foam-inhibiting effect than the prior art silicone oils.",
"WO 94/06894 discloses reaction products of polyamines with carboxylic acids as defoamers or foam inhibitors for organic liquids.",
"In this case, disadvantages occur in that insufficient long-term stabilities were detected and the products were in the form of fine particles, which complicates their use in lubricant oils in motor vehicles.",
"Also, defoamers for lubricants, for example in gearboxes, have to maintain their effectiveness over a wide temperature range, frequently up to 80° C. or 100° C. The object of the present invention, then, was to provide suitable defoamers for liquid hydrocarbons which do not have the abovementioned disadvantages.",
"These shall in particular achieve defoaming performance on the order of magnitude of the known silicone defoamers, without the danger of formation of solid particles during combustion being observed.",
"It was also required that the defoamer effect should be retained over a wide temperature range.",
"Surprisingly, it was found that certain liquid ester compounds fulfill the abovementioned requirements.",
"A first part of the subject-matter of the present invention relates to the use of esters which are liquid at room temperature of the general formula (I) R′—COO—R″ (I) where R′ and R″ are independently saturated or unsaturated, linear or branched alkyl radicals having from 6 to 16 carbon atoms, as defoamers for liquid hydrocarbons.",
"These esters are compounds known per se which can be synthesized by known organic chemistry processes.",
"To this end, saturated or unsaturated monoalcohols of preferred chain length from C6 to C16 are esterified with monohydric, saturated or unsaturated carboxylic acids of chain length from C6 to C16, preferably from C8 to C12, for example in the presence of acid catalysts.",
"Examples of useful alcohols include caproic alcohol, caprylic alcohol, pelargonyl alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol and stearyl alcohol.",
"Examples of useful unsaturated alcohols include oleyl alcohol, elaidyl alcohol, ricinoleyl alcohol, linoeyl alcohol or linolenyl alcohol.",
"Branched alcohols, preferably 2-alkylalkanols, which may be obtained, for example, by the Guerbet synthesis, may also be used.",
"Examples of useful monocarboxylic acids include caproic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, palmitic acid and stearic acid.",
"Useful unsaturated acids include lauroleic acid, myristoleic acid, palmitoleic acid, petroselic acid, petroselaidic acid, oleic acid, elaidic acid, ricinoleic acid and linoleic acid or linolenic acid and linoaidic acid.",
"It has been shown that esters of the formula (I) where at least one alkyl radical R or R′ is olefinically mono- or polyunsaturated are preferred.",
"Particular preference is given to esters of the formula (I) where both alkyl radicals R′ and R″ are mono- and/or polyunsaturated.",
"Preference may also be given to both radicals R′ and R″ having the same chain length, i.e.",
"to obtaining symmetrical compounds.",
"In this context, particular preference is given to octyl octanoate as defoamer.",
"In the context of the present application, the term defoamer is used synonymously with the expression foam inhibitor or foam-preventing reagent.",
"The effect of the present compound of the formula (I) can be regarded as the suppression of foam formation or the faster degradation of foam which is already formed.",
"The compound according to formula (I) are added to liquid hydrocarbon quantities in quantities of from 1 to 5000 ppm, preferably from 1 to 3000 ppm and in particular from 1 to 2500 ppm (based in each case on the active substance of the formula (I)).",
"Particular preference is given to the range from 500 to 2500 ppm and 1000 to 2500 ppm.",
"The esters used according to the invention show a defoaming effectiveness comparable to the known silicone compounds without their disadvantages, in particular the formation of solid particles.",
"The esters used according to the invention are suitable for defoaming hydrocarbons which are liquid at room temperature (21° C.).",
"In the present application, the term hydrocarbons is used in a wide sense.",
"It does not only include crude oil raffinates, such as gasoline or diesel oil, but also base oils for lubricants in general which encompass not only polymers of olefins, condensation products of olefins or chloroparaffins with aromatics, and dechlorinated condensates of chloroparaffins, but also polyethers, carboxylic esters, phosphoric esters, phosphonic esters and also fluorinated compounds which are known to those skilled in the art as lubricants.",
"Preference is given to using the compounds of the formula (I) for defoaming synthetic lubricants which comprise ester oils.",
"The ester oils are compounds which are formed firstly from branched-chain primary alcohols and straight-chain dicarboxylic acids, from branched-chain monocarboxylic acids and straight-chain diols or polyalkylene glycols, from straight-chain primary alcohols and branched dicarboxylic acids or, in particular, from esters of neopentyl polyols with monocarboxylic acids.",
"The alcohols required for preparing such ester oils are obtained from the oxo process or aldol condensation.",
"In principle, all olefins are suitable for the oxo process, but for later use of the alcohols as ester oil components, preference is given to using tri- or tetrapropylene, diisobutene, mixed dimers of propylene and n-butene and also butenes or pentenes.",
"The oxo process alcohols are esterified as isomer mixtures, whereas the alcohols obtained by aldol condensation, for example, the 2-ethylhexan-1-ol obtained from n-butanal, are esterified as a substantially unitary compound.",
"The most important dicarboxylic acids are sebacic acid, adipic acid and azelaic acid.",
"Perlagonic acid which, as well as azelaic acid, results from the oxidation of oleic acid, is available as a monocarboxylic acid.",
"Sebacic acid is obtained by alkaline cleavage of ricinoleic acid.",
"The esters are prepared from acid and alcohol in the presence of acid catalysts and with distillative removal of the water formed, using benzene or toluene.",
"Particular importance attaches to what are known as the complex esters which are prepared using dicarboxylic acids, glycols (or polyglycols) and monocarboxylic acids of monoalcohols.",
"Depending on the desired product, glycol and dicarboxylic acid are first esterified and the end groups of this intermediate, depending on the molar ratio of the two components, are either reacted with a monocarboxylic acid or a monoalcohol.",
"The complex esters have higher molecular masses than the simple esters and accordingly substantially higher intrinsic viscosities.",
"Further details of such compounds can be found, for example, in Ullmanns Encyklopädie der technischen Chemie, 4th edition, 1981, pages 514 ff.",
"Further suitable lubricants include perfluoropolyalkyl ethers, tetrahydrofuran polymer oils, polythioether oils, polyphenyl ethers, ethylene and propylene polymers, polybutenes and polymers of higher olefins.",
"The present compounds of the formula (I) are also suitable for defoaming mixtures of these different base oils.",
"According to the invention, the compounds of the general formula (I) are added directly to the lubricant or hydrocarbon to be defoamed.",
"The hydrocarbons according to the present invention are generally water-free, i.e.",
"they contain water in quantities of less than 1% by weight, preferably in the ppm range, in particular of less than 500 ppm.",
"Where diesel and gasolines are concerned, preference is given to those hydrocarbons whose sulfur content is reduced.",
"The sulfur content of such hydrocarbons is preferably below 50 ppm, in particular in the range of less than 10 ppm.",
"A further part of the subject matter of the invention relates to the combination of defoamers of the formula (I) with nonalkoxylated esters of polyols having from 2 to 6 carbon atoms and from 2 to 4 OH groups and saturated or unsaturated, linear or branched fatty acids having from 8 to 24 carbon atoms.",
"It has been observed that the additional use of such compounds can have a synergistic effect on the defoaming performance.",
"Preference is given to room temperature liquid triglycerides which are of natural, in particular plant, origin.",
"Examples thereof include rapeseed oil, sunflower oil, soya oil, coconut oil and castor oil.",
"Particular preference is given to combining defoamers of the formula (I) with solvents and the triglycerides, and the additional use of alkoxylated alcohols may also be preferable.",
"A further part of the subject-matter of the present invention relates to additives for defoaming lubricants, and the additives preferably contain a) from 1 to 99% by weight of a compound of the formula (I), b) from 1 to 10% by weight of the above-described polyol esters or preferably of triglycerides and optionally further additives in quantities of up to a maximum of 10% by weight.",
"It is also possible and preferred to supplement the additives according to the invention by adding further additives known in the lubricant sector, for example, VI improvers, corrosion inhibitors, antioxidants, friction modifiers, HP additives, polymers, preferably vinyl polymers, etc., and to adapt their performance to the requirements of each practical use.",
"The present invention also relates to a process for defoaming liquid hydrocarbons, wherein compounds of the formula (I) are added to the liquid hydrocarbons in quantities of from 1 to 5000 ppm (of active substance).",
"EXAMPLES In order to demonstrate the effectiveness of the technical teaching according to the invention, defoamer tests were carried out to ASTM D892.To this end, 200 ml of the hydrocarbon were prepared with the additives to be tested in the desired concentrations and heated to different temperatures.",
"190 ml of this solution are transferred to a 1000 ml upright cylinder.",
"A gas distributor (sinter brick) is then saturated for five minutes and air is blown through the solution for two minutes (400 l per hour volume).",
"The foam volume was read off (in ml) and noted.",
"In the present case, two additives according to the invention in two transmission oils were investigated at 20, 60 and 90° C. As a comparison, the oils were measured without additives or with addition of a prior art silicone defoamer.",
"The composition of the additives according to the invention is reported in table 1.TABLE 1 Octyl Coconut ocanoate oil % by weight % by weight 1 99 1 2 100 — TABLE 2 a: Transmission oil I b: Transmission oil II 20° C. 60° C. 90° C. 20° C. 60° C. 90° C. h (ml) h (ml) h (ml) h (ml) h (ml) h (ml) 1 120* 250* 500* — 100* 240* 2 90** 110** 190** 50* 90* — C 90*** 140*** 350*** 141*** 270*** 122**** B 530 900 1000 550 680 >600 *500 ppm dose **1000 ppm dose ***25 ppm dose ****100 ppm dose The oils additivized according to the invention show distinctly better foaming behavior at different temperatures in comparison to nonadditivized oil.",
"It becomes clear that the additives according to the invention even have comparable performance to the known, expensive silicone reference defoamers."
]
] | Patent_10203859 |
[
[
"Stretch film and method for producing a stretch film",
"A stretch film for the packing of goods, particularly for the packing of goods stacked on a pallet, comprises a prestretched main film (10).",
"The main film (10) is reinforced by reinforcement strips (12) extending in the longitudinal direction.",
"The reinforcement strips (12) are made from film strips of prestretched film which have been folded a plurality of times in the longitudinal direction.",
"Between two adjacent reinforcement strips (12), the main film (10) can comprise two mutually staggered rows of holes (16,18).",
"This stretch film with perforations (14) is particularly useful for the packing of food items."
],
[
"1.A method for producing a stretch film, comprising the following steps: supply of film strips (34) from a first prestretched film (22), generating reinforcement strips (12) by folding these film strips (34) at least once in longitudinal direction, supply of a further prestretched main film (10), and connecting the reinforcement strips (12) to the main film (10) in such a manner that the reinforcement strips (12) extend in parallel to each other in the longitudinal direction of the main film (10).",
"2.The method according to claim 1 wherein the film strips (34) are generated by cutting a prestretched film (22).",
"3.The method according to claim 1 or 2 wherein the main film (10) is perforated.",
"4.The method according to claim 1 or 3 wherein the main film (10) is perforated in such a manner that perforations are formed only in the regions of the main film (10) between the reinforcement strips (12).",
"5.The method according to claim 3 or 4 wherein, between two reinforcement strips (12), a plurality of mutually staggered rows of holes (16,18) are formed to extend in the longitudinal direction of the main film (10).",
"6.The method according to any one of claims 1-5 wherein at least one of the films (10,22) includes additives for intensifying the connection between the films.",
"7.The method according to any one of claims 1-6 wherein, for connecting the main film (10) and the reinforcement strips (12), the main film (10) and the reinforcement strips (12) have different temperatures.",
"8.The method according to a any one of claims 1-7 wherein the main film (10) and the reinforcement strips (12) are connected by application of heat.",
"9.The method according to claim 1 wherein the film strips (34) are folded twice and preferably four times.",
"10.The method according to a any one of claims 1-9 wherein at least three reinforcement strips (12) are provided in parallel to each other.",
"11.A stretch film, comprising reinforcement strips (12) made from a first prestretched film (22) and extending in the longitudinal direction of a second prestretched main film (10), wherein the reinforcement strips (12) are film strips (34) made from the first prestretched film (22) which have been folded in the longitudinal direction.",
"12.The stretch film according to claim 11, characterized in that the main film (10) is perforated.",
"13.The stretch film according to claim 12, characterized in that the main film (10) is perforated only in the regions between the reinforcement strips (12).",
"14.The stretch film according to claim 12 or 13, characterized in that, between two reinforcement strips (12), the main film (10) is provided with a plurality of mutually staggered rows of holes (16,18) extending in the longitudinal direction.",
"15.The stretch film according to any one of claim 11-14, characterized in that the reinforcement strips (12) comprise film strips (34) which have been folded twice and preferably four times in the longitudinal direction.",
"16.The stretch film according to any one of claim 11-15, characterized in that at least three mutually parallel reinforcement strips (12) are provided.",
"17.Use of a stretch film according to any one of claims 11-16 for the packing of food items.",
"18.Use of a stretch film according to any one of claims 11-16 for wrapping a pallet load."
],
[
"The invention relates to a stretch film and method for producing a stretch film.",
"Stretch films are produced by prestretching a film.",
"Prestretching the film will increase the strength of the film.",
"Further, the prestretching generates a memory effect so that the film upon further stretching will remember its previous condition, i.e.",
"the film will contract.",
"Stretch films are used for packing goods for shipment.",
"Particularly, stretch films are utilized for tying together goods on a pallet or the like.",
"For this purpose, the goods are stacked on a pallet, and a stretch film is wrapped completely around them to thus hold the goods safely on the pallet.",
"Due to different tensions in the film while being wrapped around the goods and due to sharp edges of the goods, such stretch films can easily tear at the edges.",
"Because of the inherent tension in the film, this will cause the film sheet to tear off, so that the goods have to be wrapped again.",
"For reducing the danger that the outer edges of the stretch film might tear, it is known from EP 0 728 102 to fold the two outer edges of the longitudinal film inwards.",
"Also in such a stretch film, sharp edges or projecting edges of a carton may tear holes in the film.",
"Because of the tension in the longitudinal direction of the film, these holes will enlarge along the whole width of the film so that a safe hold of the goods on a pallet will not be guaranteed anymore.",
"It is an object of the invention to provide a stretch film and a method for producing a stretch film wherein the danger of tearing is reduced.",
"According to the invention, this object is achieved by claims 1 and 11, respectively.",
"The stretch film according to the invention comprises a prestretched main film and reinforcement strips extending in the longitudinal direction of the main film.",
"As provided by the invention, the reinforcement strips comprise film strips of prestretched film which are folded in the longitudinal direction.",
"The main film has a plurality of reinforcement strips connected thereto in the longitudinal direction of the main film.",
"The distances between the reinforcement strips can be selected in dependence from the number of the reinforcement strips and the width of the film.",
"Even a tearing of the film between two reinforcement strips will thus cause merely a small hole which cannot grow until extending along the whole width of the film.",
"Since the reinforcement strips provided as folded film strips, the reinforcement of the main film can be determined by the number of the folds.",
"The film strips are folded preferably twice, and particularly four times, in the longitudinal direction.",
"With usual film widths of 400 to 500 mm, preferably at least three mutually parallel reinforcement strips are connected to the main film.",
"By providing at least three reinforcement strips made from film strips that have been folded at least four times in the longitudinal direction, the holding force of the stretch film can be increased at least five times.",
"This advantageous particularly when packing heavy goods.",
"In a particularly preferred embodiment of the stretch film, the main film comprises perforations.",
"The main film can be perforated before or after being connected to the reinforcement strip.",
"Preferably, the perforations are formed after the application of the reinforcement strips because this will reduce the danger of a tearing of the stretch film during perforation.",
"Such perforated stretch films with reinforcement strips will reliably provide for an interchange of air and thus are suited particularly for the packing of food items.",
"To increase the strength of the film and to guarantee a safe connection of the reinforcement strips with the main film, the main film is perforated preferably only in the regions between the reinforcement strips.",
"To decrease the danger that even the perforated main film might tear, it can be provided, in addition to the reinforcement strips, that the main film comprises a plurality of longitudinal rows of holes which are staggered relative to each other.",
"Thus, a large strength of the stretch film is provided along with a good venting of the packed goods.",
"In the inventive method for producing the stretch film provided with the reinforcement strips, film strips are supplied which are made from a first prestretched film.",
"Thereafter, reinforcement strips are produced by folding these film strips at least once in the longitudinal direction.",
"Then, a second prestretched main film is supplied, and the reinforcement strips are connected to the main film in such a manner that the reinforcement strips will extend in parallel to each other in the longitudinal direction of the main film.",
"Producing the reinforcement strips through the folding of film strips offers the particular advantage that the main film and the film strips can consist of the same film and that a considerable increase of the strength is achieved by the folding.",
"Using the same film is further advantageous in that the connection between the reinforcement strips and the main film can be performed in a simple manner.",
"Preferably, the film strips are produced by cutting a prestretched film.",
"Thus, two identical film rolls can be used for producing the stiffened stretch film, one of the films serving as a main film while the reinforcement strips are produced from the other film.",
"In a preferably preferred variant of the method, the main film will be perforated.",
"The perforation of the main film can be performed before or after connecting the main film to the reinforcement strip.",
"Preferably, the perforation is carried out after the connection with the reinforcement strips.",
"Because of the perforations in the main film, the stretch film of the invention is particularly useful for the packing of food items.",
"Preferably, only the regions of the main film between the reinforcement strips are perforated.",
"Thus, the main film will not be weakened in the regions where the reinforcement strips are connected to the main film.",
"On the other hand, especially in case of very small holes, it is possible and advantageous under the aspect of production technology to perforate the main film prior to the connection with the reinforcement strips.",
"Preferably, the perforation of the main film is performed to the effect that two respective reinforcement strips have a plurality of rows of holes arranged therebetween which extend in the longitudinal direction of the main film.",
"These rows of holes are positioned in a mutually staggered arrangement to thus increase the strength of the stretch film.",
"Such a perforation will improve the venting of the packed goods, particularly in case of food items.",
"If particularly strong and tear-resistant stretch films are required, only one row of holes is provided between the reinforcement strips.",
"A further increase of the strength of the stretch film with continued reliable venting can be achieved by providing holes only between each second pair of reinforcement strips so that the regions between the reinforcement strips are alternately perforated and non-perforated.",
"The perforating of the film can be performed e.g.",
"by punching the holes.",
"Particularly in thin films, it is possible to carry out the perforation by exposure to heat.",
"For this purpose, use can be made e.g.",
"of heated protrusions which have a temperature above the melting point of the film, e.g.",
"preferably above 200° C. and particularly above 250° C. Such heated protrusions, which preferably comprise aluminum, can be arranged on a roller so that the perforating of the film is carried out during the transport of the film over that roller.",
"Preferably, immediately after perforating the film by application of heat, a cooling by air is performed.",
"The formation of the holes by melting the film is further of advantage in that the melting of the film will generate reinforced edges on the holes.",
"Thus, tearing of the film on the holes is prevented.",
"To connect the reinforcement strips to the main film, at least one of the films can include additives.",
"By use of such additives, the connection between the two films, i.e.",
"the main film and the reinforcement strips, is reinforced.",
"Preferably, the main film and the reinforcement strips have different temperatures so that the films will adhere to each other already due to the differences in temperature and will enter a sufficiently strong connection.",
"Of particular advantage is the connecting of the reinforcement strips and the main film by application of heat.",
"In this case, heat is supplied to at least one roller among a plurality of pressure rollers provided to press the reinforcement strips and the main film together.",
"To avoid an impairment of the characteristics of the stretch film, the supplied heat must not be so high that the two films melt to each other or become welded to each other.",
"The films used are preferably films produced by the sheet blowing method or flat-molded films.",
"The film material can be selected from a group comprising polyethylene, polyvinylchloride, ethylene-vinyl-acetate, ethylene-methylacetate and polyethylene.",
"To allow for a processing of film lengths as large as possible, the device for producing a reinforced, preferably perforated stretch film can be combined with a device for stretching the film.",
"The device for reinforcing and perforating the film is then arranged directly downstream of the device for stretching the film.",
"Winding up the stretched film prior to reinforcing and perforating the same is not required in this case.",
"The invention will be explained in greater detail hereunder in connection with a preferred embodiment with reference to the accompanying drawings.",
"FIG.",
"1 is a schematic plan view of a stretch film according to the invention, FIGS.",
"2a and 2b show a schematic view of a device for producing the stretch film of the invention, FIG.",
"3a is a schematic sectional view of a folded film strip, taken along the line I in FIG.",
"2a, FIG.",
"3b is a schematic sectional view of a folded film strip, taken along the line II in FIG.",
"2a, FIG.",
"3c is a schematic sectional view of a folded film strip, taken along the line III in FIG.",
"2a, and FIG.",
"3d is a schematic sectional view of a folded film strip, taken along the line IV in FIG.",
"2a.",
"The stretch film comprises a main film 10 which is provided as a prestretched film.",
"A plurality of reinforcement strips 12 are connected to the main film 10 in the longitudinal direction thereof.",
"The reinforcement strips 12 comprise film strips 34 (FIG.",
"2a) folded in the longitudinal direction and likewise made from prestretched film.",
"Provided between respectively two adjacent reinforcement strips 12 are two rows of holes 16,18 comprising a plurality of holes 14.The holes 14 of the row of holes 16 are staggered relative to the holes 14 of the row of holes 18 by respectively half the distance between adjacent holes 14 of a row of holes 16,18.The holes 14 of the row of holes 16 are thus positioned to fill the gaps relative to the holes 14 of the row of holes 18.The reinforcement strips 12 comprise the same material as the main film 10.To achieve a stronger reinforcement, the film for generating the reinforcement strips 12 is folded a plurality of times so that the reinforcement strips 12 comprise a plurality of layers.",
"Preferably, prior to being cut into individual strips to be folded, the film from which the reinforcement strips 12 are produced has the same width as the main film 10.Thus, use can be made of identical films both for the main film 10 and for the production of the reinforcement strips 12.For producing the reinforcement strips 12 (FIG.",
"2a), a first prestretched film sheet 22 is wound off a supply roll 20.The film sheet is supplied via a guide roller 24 to a cutting unit 26.The cutting unit comprises cutting blades 30 arranged on a holding support 28.By the transport of the film sheet 22 in the direction of the arrow 32, the film sheet 22 is cut into individual film strips 34 by the cutting blades 30.The film strips 34 are supplied, via a guide roller 36, to a curve roller 38.Curve roller 38 comprises curve elements 42 arranged for rotation on a shaft 40.By the curve elements 42 and the tension in the film strips 34, the outer longitudinal edges of the film strips 34 are folded upwards in the Figure so that each film sheet 34 is substantially U-shaped in cross section (FIG.",
"3a).",
"Thereafter, the film sheets 34 being U-shaped in cross section are supplied to a folding unit 44.The folding unit 44 comprises shaping rods 46.The shaping rods 46 are oriented transverse to the moving direction of the film strips and arranged at an inclination.",
"For setting the width of the folded regions, the shaping rods can be adjusted transversely the moving direction 32 of the film strips.",
"During the transport of the film strips 34 in the moving direction 32, the outer legs 48 (FIG.",
"3a) of the U-shaped film strips 34 are folded inwards.",
"Thus, the film sheet 34 will assume the cross section shown in FIG.",
"3b.",
"In the next step, the film strips 34 are guided around a guide roller in such a manner that the outer legs 48 of the film strips 34 point in the direction of the guide roller 50.Thereby, the outer legs 48 are pressed onto the region of film strip 34 arranged between the legs 48 (FIG.",
"3c).",
"The subsequent folding unit 52, also provided with shaping rods 54, will again fold the film sheets 34 in the longitudinal direction and thus lend them the shape illustrated in FIG.",
"3d.",
"Thereafter, film strip 34 is pressed together in a pressing unit, thus generating reinforcement strips 12 folded four times.",
"The pressing unit 56 comprises two pressure rollers 58 arranged to press against each other with the required force.",
"In this manner, trapped air will be pressed out from the reinforcement strips 12 and the individual film layers will be connected to each other.",
"From pressing unit 56, the film strips 12 are guided via guide rollers 60 into that region of the device where the reinforcement strips 12 are to be connected to the main film 10 (FIG.",
"2b).",
"As a main film 10, a second prestretched film is wound off a supply roll 62 and guided via a guide roller 64 to a pressing unit 66.The reinforcement strips 12 are also supplied to the pressing unit 66 while moving in parallel to the longitudinal direction of main film 10 and respectively in parallel to each other.",
"Similar to pressing unit 56, the pressing unit 66 comprises two rollers 68 arranged to press against each other with a predetermined force.",
"The connection of the reinforcement strips 12 to the main film 10 is performed in that either the reinforcement strips 12 and/or the main film 10 includes an additive.",
"Further, the pressing rollers 68 of the pressing units 66 can be heated so that the reinforcement strips 12 and the main film 10 will be connected to each other under the influence of heat.",
"In this regard, care must be taken that the temperature of the pressing rollers 68 does not cause a melting of the reinforcement strips 12 or the main film 10.In the next step, perforating rollers 70,72 will be operated to form holes into the main film 10 between the reinforcement strips 12.For this purpose, perforating roller 72 is provided with cylindrical protrusions 74 distributed about its periphery.",
"Since the illustrated embodiment is shown as a stretch film comprising three reinforcement strips 12 and consequently two rows of holes, perforating roller 72 is provided with two rows of cylindrical protrusions 74.The corresponding perforating roller 70 is formed with cylindrical recesses 76 corresponding to the cylindrical protrusions 74.The cylindrical recesses 76 are likewise arranged in two rows.",
"One or both of the two perforating rollers 70,72 are heated.",
"By the heating, the holes 14 are molded into main film 10.The holes are thus not generated by punching so that no film waste is generated.",
"The perforating roller 70 can also be omitted.",
"In this case, the perforating is performed solely by means of the perforating roller 72 with the protrusions 74.Because of the tension in the film, the film only slightly deviates from the perforating roller 72, thus adhering sufficiently close to the perforating roller 72 or the protrusions 74 for having the perforations formed therein.",
"Apart from circular holes 14, any desired other shape can be selected for the holes.",
"Particularly advantageous is an oval or elliptic hole shape having its longer semiaxis extending transversely to the longitudinal direction of the film sheet so that the shorter semiaxis is oriented in parallel to the tension direction prevailing in the stretch films.",
"After forming the holes 14 in the main film 10, the film sheet is deflected a plurality of times via guide rollers 78.In this manner, there is provided a cooling path in which the film sheet will be cooled again sufficiently to keep it from becoming bonded to itself when being wound up afterwards.",
"After the cooling path, the film sheet wound up to form a roll 80.Roll 80 moves vertically to the transport direction of the film sheet in the direction of the arrow 82, i.e.",
"in a horizontal direction.",
"This will safeguard that the reinforcement strips 12 when being wound onto roll 80 will not be arranged completely above each other.",
"By the reciprocating movement of the wind-up roll 80, the roll has a substantially identical thickness throughout its width.",
"Further, to guarantee a uniform winding onto roll 80, a press-on roller 84 is provided."
]
] | Patent_10203952 |
[
[
"Alloy for use in bonded magnet, isotropic magnet powder and anisotropic magnet powder and method for production thereof, and bonded magnet",
"An alloy for bonded magnet alloy of the present invention includes at least Fe as a main component, 11-15 at % rare-earth element (R) that includes yttrium (Y) and does not include lanthanum (La), 5.5-10.8 at % B and 0.01-1.0 at % La, and has superior corrosion resistance.",
"Using the obtained magnet powder by applying the d-HDDR process etc.",
"to this bonded magnet, bonded magnet with not only magnetic properties but also reliability such as corrosion resistance and heat resistance etc., can be achieved."
],
[
"1.An alloy for bonded magnet which includes at least Fe as a main component, 11-15 at % of rare earth element (hereafter referred to as [R]) that includes yttrium (Y) and does not include lanthanum (La), 5.5-10.8 at % of B and 0.01-1.0 at % of La, and has superior corrosion resistance.",
"2.An alloy for bonded magnet according to claim 1 which includes 0.05-1 at % of gallium (Ga) and 0.05-0.8 at % of niobium (Nb).",
"3.An alloy for bonded magnet according to claim 1 in which R is composed at least one of Nd, Pr and Dy.",
"4.Additionally, an alloy for bonded magnet according to claim 1 which includes 0.1-10 at % of cobalt (Co).",
"5.An isotropic magnet powder obtained by the HDDR process which includes putting an ingot, with alloy composition of at least the main component Fe, R including Y without La of 11-15 at %, B of 5.5-10.8 at % and La of 0.01-1.0 at %, through the hydrogenation step while maintaining the ingot between 1023-1173 K in a hydrogen atmosphere, and then after the hydrogenation step, carrying out the desorption step where hydrogen is removed, is used for bonded magnet with superior corrosion resistance.",
"6.An isotropic magnet powder obtained from the production method which includes the HDDR process which includes putting a magnet alloy, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the hydrogenation step while maintaining the alloy between 1023-1173 K in a hydrogen atmosphere, and then after the hydrogenation step, carrying out the desorption step where hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the hydrogenation process or the dehydrogenation process with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and their hydride (a simple substance of La, a La alloy as well as a hydride of La compound, hereafter referred as to La hydride), is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder, and when regarding the whole as 100 at %, it includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"7.An anisotropic magnet powder obtained by the d-HDDR process which includes putting an ingot, with alloy composition of at least the main component Fe, R including Y without La of 11-15 at %, B of 5.5-10.8 at %, and La of 0.01-1.0 at %, through the low temperature hydrogenation step while maintaining the ingot at less than 873 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the high-temperature hydrogenation step while maintaining 20-100 kPa and 1023-1173 K in a hydrogen atmosphere, and then the high-temperature hydrogenation step, carrying out the first evacuation step while maintaining 0.1-20 kPa and 1023-1173 K in a hydrogen atmosphere, and after the first evacuation step, carrying out the second evacuation step where the hydrogen is removed, is used for bonded magnet with superior corrosion resistance.",
"8.An anisotropic magnet powder obtained from the production method which includes the d-HDDR process which includes putting a magnet alloy for bonded magnet, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the low temperature hydrogenation step while maintaining the ingot at less than 873 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the high-temperature hydrogenation step while maintaining 20-100 kPa and 1023-1173 K in a hydrogen atmosphere, and then the high-temperature hydrogenation step, carrying out the first evacuation step while maintaining 0.1-20 kPa and 1023-1173 K in a hydrogen atmosphere, and after the first evacuation step, carrying out the second evacuation step where the hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the high-temperature, the first evacuation process or the second evacuation process with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and a La hydride, is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder, and when regarding the whole as 100 at %, it includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"9.An isotropic magnet powder obtained from the production method which includes the HDDR process which includes putting a magnet alloy for bonded magnet, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the low temperature hydrogenation step while maintaining the ingot at less than 1023-1173 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the desorption step where hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the hydrogenation step and the desorptin step with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and a La hydride, is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder, and when regarding the whole as 100 at %, it includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"10.Production method of an anisotropic magnet powder obtained from the production method which includes the d-HDDR process which includes putting a magnet alloy for bonded magnet, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the low temperature hydrogenation step while maintaining the ingot at less than 873 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the high-temperature hydrogenation step while maintaining 20-100 kPa and 1023-1173 K in a hydrogen atmosphere, and then the high-temperature hydrogenation step, carrying out the first evacuation step while maintaining 0.1-20 kPa and 1023-1173 K in a hydrogen atmosphere, and after the first evacuation step, carrying out the second evacuation step where the hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the high-temperature, the first evacuation process or the second evacuation process with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and a La hydride, is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder, and when regarding the whole as 100 at %, it includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"11.A bonded magnet having superior corrosion resistance which can be achieved by mixing binder with the obtained isotropic magnet powder through the HDDR process that is composed of at least the main component Fe, 11-15 at % of R including Y without La, 5.5-10.8 at % of B as well as 0.01-1.0 at % of La, and then by compression molding.",
"12.A bonded magnet having its superior corrosion resistance which can be achieved by mixing binder with the obtained anisotropic magnet powder through the d-HDDR process that is composed of at least the main component Fe, 11-15 at % of R including Y without La, 5.5-10.8 at % of B as well as 0.01-1.0 at % of La, and then by compression molding."
],
[
"<SOH> BACKGROUND ART <EOH>Hard magnets (permanent magnet) are used in motors and various other equipment and they are demanded to possess superior magnetic characteristics to achieve the miniaturization and high performance.",
"From this point of view, the development of a RFeB-type magnet (rare-earth magnet), made from a rare-earth element (R), Boron (B) and Iron (Fe), has up until now been popular.",
"However, to further increase the demand for rare-earth magnet, a stable exhibition of the excellent magnetic characteristics becomes important in order to secure the reliability of the product made from the magnet.",
"Because the main reason that rare-earth magnet degrade its magnetic characteristics lies on an oxidization of the main components such as Fe and R, rare-earth magnet is demanded to possess excellent corrosion resistance against the oxidization.",
"Especially, in the case of rare-earth magnet that gets exposed to high-temperature condition where the oxidization can easily proceed, the magnet is demanded to possess excellent corrosion resistance under high-temperature condition.",
"For example, in the case of use for each kinds of motors equipped in engine rooms of automobile cars, or in the case of use for driving motors in hybrid cars or electric cars, the rare-earth magnet has to maintain high magnetic characteristics at high-temperature range exceeding 100° C. However, a NdFeB-type magnet, for example, is poor in heat resistance with its generally large temperature-dependency (temperature coefficient), resulting in considerable decrease in coercivity at high-temperature range.",
"So far, to improve the temperature dependency itself has been difficult.",
"Therefore, up to this date, it has been dealt by initially enlarging coercivity (iHC) of rare-earth magnet, taking into account the degree of degradation in magnetic characteristics.",
"By the way, many of applications concerned with rare-earth magnets with enhanced initial magnetic characteristics have been made, for example, the following open patents can be listed.",
"{circle over (1)}U.S. Pat.",
"No.",
"4,802,931, U.S. Pat.",
"No.",
"4,851,058 In the former open patent, an alloy is disclosed which are composed of less than 40 atomic % (at %) R or R′ that are rare-earth elements, 0.5-10% at % B and a remainder of Fe, and whose main phase is (R, R′) 2 (Fe, TM) 14 B 1 (TM: transition element).",
"Indeed, what is disclosed as a magnetic powder composed of the alloy is only an isotropic magnetic powder produced by quenching solidification.",
"Moreover, concerning with time-variation properties or its controlling method of the isotropic magnetic powder or the applied hard magnets, nothing has been disclosed in this open patent.",
"In the latter open patent, although similar alloys where the above-mentioned R is neodymium (Nd) or praseodymium (Pr) have, in this case again, nothing is disclosed about the above-mentioned time-variation properties.",
"{circle over (2)} U.S. Pat.",
"No.",
"4,402,770 In this open patent, alloys composed of (M w X x B 1-x ) 1-y (R z La 1-z ) y have been disclosed.",
"M represent Fe or Co and so on, and X represents Si or Al and so on.",
"By making the alloy into amorphous through quenching solidification method and then crystallizing it through heat processing, an isotropic magnetic powder can be obtained.",
"Here La must be an essential component for formation of the above-mentioned amorphous structure.",
"Again, in this open patent, nothing is reported about --- or its controlling method of the isotropic magnetic powder.",
"{circle over (3)} Japanese examined Patent Publication No.",
"6-82575 (Japanese Patent No.",
"1947332), Japanese examined Patent Publication No.",
"7-68561 (Japanese Patent No.",
"2041426), Japanese Patent No.",
"2576671 and Japanese Patent No.",
"Alternative production methods, other than the above-mentioned quenching solidification method (melt spinning method), include the HDDR (hydrogenation-disproportionation-desorption-recombination) processing method and the d-HDDR processing method.",
"The HDDR processing method is used to produce RFeB-type isotropic magnet powder and RFeB-type anisotropic magnet powder, and it generally has two production steps.",
"That is to say, the first step of 3-phase decomposition (disproportionation) reaction (the hydrogenation step) is carried out while maintained at 773-1273 K in a hydrogen gas environment on the order of 100 kPa (1 atm), and after that is the dehydrogenation step (the second step) where dehydrogenation occurs under vacuum.",
"On the other hand, d-HDDR is used predominantly in as a production method for RFeB-type anisotropic magnet powder.",
"As reported in detail in commonly-known literature (Mishima, et.",
"al.",
": Journal of the Japan Applied Magnetics Society, 24 (2000), p. 407), it is defined as the control of the reaction rate between the RFeB-type alloy and hydrogen when going from room temperature to a high temperature.",
"In detail, the four principal production steps are the low-temperature hydrogenation step (step 1) where hydrogen is sufficiently absorbed into the RFeB-type alloy at room temperature, the high-temperature hydrogenation step (step 2) where the 3-phase decomposition (disproportionation) reaction occurs under low hydrogen pressure, the evacuation step (step 3) where hydrogen is decomposed under as high a hydrogen pressure as possible, and the desorption step (step 4) where the hydrogen is removed from the material.",
"The point, which differs from the HDDR process is that through the preparation of multiple production steps with different temperatures and hydrogen pressures, the rection rate of the RFeB-type alloy and hydrogen can be maintained relatively slow, thus homogeneously anisotropic magne powder.",
"In these four open patents above-mentioned, a magnetic powder produced by those processes is reported.",
"In Japanese Examined Patent Publication No.",
"6-82575 a RFeB-type isotropic magnet powder which possesses recrystallized tetragonal structure is disclosed, and in Japanese Examined Patent Publication No.",
"7-68561 the production method of RFeB-type isotropic magnet powder using the HDDR process is disclosed.",
"Indeed, even in these open Patent, nothing about time-variation properties etc.",
"of the isotropic magnet powder is disclosed.",
"In Japanese Patent No.",
"2576671 as well as Japanese Patent No.",
"2586198, RFeB-type anisotropic magnet powder with superior corrosion resistance as well as bonded magnet are disclosed.",
"The prior magnet powder produced through thermo-plastic process is poor in corrosion resistance due to an introduction of a plastic process strain.",
"On the other hand, as those open patents report, an corrosion resistance of magnet powder produced using the HDDR process improves with no introduction of a plastic process strain.",
"That is, in the case of magnet powder produced using the HDDR process, the corrosion resistance improves because there is no grain boundary in its recrystallized structure, nor is stress strain caused by plastic process.",
"However, the disclosed method in this open patent was far from sufficient in terms of improvement in corrosion resistance or magnetic characteristics of the magnet powder.",
"{circle over (4)} Japanese Unexamined Patent Publication No.",
"2002-93610 In this open patent, a method to diffuse (or coating) Nd or Dy on the surface or inside of anisotropic magnet powder by means of diffusion heat process, is disclosed.",
"The heat processed Nd or Dy acts as an oxygen getter, control as well as prevent a direct oxidization of R or Fe that compose main phase of magnet powder.",
"As a result, the diffusion heat processed magnet powder achieves an excellent corrosion resistance.",
"However, the corrosion resistance was not surely a sufficient level.",
"As mentioned above, even if the prior rare-earth magnet powder and the magnet have a superior initial magnetic characteristics, the corrosion resistance was insufficient.",
"In addition, even ones with improved corrosion resistance were not surely sufficient level, with no coexistence of magnetic characteristics and corrosion resistance on a high level.",
"However, to achieve a coexistence of high functionalization and high reliability of the product using rare-earth magnet, to improve corrosion resistance of RFeB-type magnet powder with superior magnetic characteristics and the applied rare-earth magnets is very important.",
"In addition, although many of the above-mentioned open patents are illustrating La as R that compose a main phase of RFeB-type magnet powder, none of them reports an example in which La is R. Also none of them utilizes La as to improve corrosion resistance of magnet powder."
],
[
"TECHNICAL FIELD This invention comprises the alloy for bonded magnet, the isotropic magnet powder as well as anisotropic magnet powder form which a bonded magnet with superior time-variation properties such as magnetic characteristics and corrosion resistance, and their production method, as well as the bonded magnet with superior magnetic characteristics and time-variation properties.",
"BACKGROUND ART Hard magnets (permanent magnet) are used in motors and various other equipment and they are demanded to possess superior magnetic characteristics to achieve the miniaturization and high performance.",
"From this point of view, the development of a RFeB-type magnet (rare-earth magnet), made from a rare-earth element (R), Boron (B) and Iron (Fe), has up until now been popular.",
"However, to further increase the demand for rare-earth magnet, a stable exhibition of the excellent magnetic characteristics becomes important in order to secure the reliability of the product made from the magnet.",
"Because the main reason that rare-earth magnet degrade its magnetic characteristics lies on an oxidization of the main components such as Fe and R, rare-earth magnet is demanded to possess excellent corrosion resistance against the oxidization.",
"Especially, in the case of rare-earth magnet that gets exposed to high-temperature condition where the oxidization can easily proceed, the magnet is demanded to possess excellent corrosion resistance under high-temperature condition.",
"For example, in the case of use for each kinds of motors equipped in engine rooms of automobile cars, or in the case of use for driving motors in hybrid cars or electric cars, the rare-earth magnet has to maintain high magnetic characteristics at high-temperature range exceeding 100° C. However, a NdFeB-type magnet, for example, is poor in heat resistance with its generally large temperature-dependency (temperature coefficient), resulting in considerable decrease in coercivity at high-temperature range.",
"So far, to improve the temperature dependency itself has been difficult.",
"Therefore, up to this date, it has been dealt by initially enlarging coercivity (iHC) of rare-earth magnet, taking into account the degree of degradation in magnetic characteristics.",
"By the way, many of applications concerned with rare-earth magnets with enhanced initial magnetic characteristics have been made, for example, the following open patents can be listed.",
"{circle over (1)}U.S. Pat.",
"No.",
"4,802,931, U.S. Pat.",
"No.",
"4,851,058 In the former open patent, an alloy is disclosed which are composed of less than 40 atomic % (at %) R or R′ that are rare-earth elements, 0.5-10% at % B and a remainder of Fe, and whose main phase is (R, R′)2(Fe, TM)14B1 (TM: transition element).",
"Indeed, what is disclosed as a magnetic powder composed of the alloy is only an isotropic magnetic powder produced by quenching solidification.",
"Moreover, concerning with time-variation properties or its controlling method of the isotropic magnetic powder or the applied hard magnets, nothing has been disclosed in this open patent.",
"In the latter open patent, although similar alloys where the above-mentioned R is neodymium (Nd) or praseodymium (Pr) have, in this case again, nothing is disclosed about the above-mentioned time-variation properties.",
"{circle over (2)} U.S. Pat.",
"No.",
"4,402,770 In this open patent, alloys composed of (MwXxB1-x)1-y(RzLa1-z)y have been disclosed.",
"M represent Fe or Co and so on, and X represents Si or Al and so on.",
"By making the alloy into amorphous through quenching solidification method and then crystallizing it through heat processing, an isotropic magnetic powder can be obtained.",
"Here La must be an essential component for formation of the above-mentioned amorphous structure.",
"Again, in this open patent, nothing is reported about --- or its controlling method of the isotropic magnetic powder.",
"{circle over (3)} Japanese examined Patent Publication No.",
"6-82575 (Japanese Patent No.",
"1947332), Japanese examined Patent Publication No.",
"7-68561 (Japanese Patent No.",
"2041426), Japanese Patent No.",
"2576671 and Japanese Patent No.",
"Alternative production methods, other than the above-mentioned quenching solidification method (melt spinning method), include the HDDR (hydrogenation-disproportionation-desorption-recombination) processing method and the d-HDDR processing method.",
"The HDDR processing method is used to produce RFeB-type isotropic magnet powder and RFeB-type anisotropic magnet powder, and it generally has two production steps.",
"That is to say, the first step of 3-phase decomposition (disproportionation) reaction (the hydrogenation step) is carried out while maintained at 773-1273 K in a hydrogen gas environment on the order of 100 kPa (1 atm), and after that is the dehydrogenation step (the second step) where dehydrogenation occurs under vacuum.",
"On the other hand, d-HDDR is used predominantly in as a production method for RFeB-type anisotropic magnet powder.",
"As reported in detail in commonly-known literature (Mishima, et.",
"al.",
": Journal of the Japan Applied Magnetics Society, 24 (2000), p. 407), it is defined as the control of the reaction rate between the RFeB-type alloy and hydrogen when going from room temperature to a high temperature.",
"In detail, the four principal production steps are the low-temperature hydrogenation step (step 1) where hydrogen is sufficiently absorbed into the RFeB-type alloy at room temperature, the high-temperature hydrogenation step (step 2) where the 3-phase decomposition (disproportionation) reaction occurs under low hydrogen pressure, the evacuation step (step 3) where hydrogen is decomposed under as high a hydrogen pressure as possible, and the desorption step (step 4) where the hydrogen is removed from the material.",
"The point, which differs from the HDDR process is that through the preparation of multiple production steps with different temperatures and hydrogen pressures, the rection rate of the RFeB-type alloy and hydrogen can be maintained relatively slow, thus homogeneously anisotropic magne powder.",
"In these four open patents above-mentioned, a magnetic powder produced by those processes is reported.",
"In Japanese Examined Patent Publication No.",
"6-82575 a RFeB-type isotropic magnet powder which possesses recrystallized tetragonal structure is disclosed, and in Japanese Examined Patent Publication No.",
"7-68561 the production method of RFeB-type isotropic magnet powder using the HDDR process is disclosed.",
"Indeed, even in these open Patent, nothing about time-variation properties etc.",
"of the isotropic magnet powder is disclosed.",
"In Japanese Patent No.",
"2576671 as well as Japanese Patent No.",
"2586198, RFeB-type anisotropic magnet powder with superior corrosion resistance as well as bonded magnet are disclosed.",
"The prior magnet powder produced through thermo-plastic process is poor in corrosion resistance due to an introduction of a plastic process strain.",
"On the other hand, as those open patents report, an corrosion resistance of magnet powder produced using the HDDR process improves with no introduction of a plastic process strain.",
"That is, in the case of magnet powder produced using the HDDR process, the corrosion resistance improves because there is no grain boundary in its recrystallized structure, nor is stress strain caused by plastic process.",
"However, the disclosed method in this open patent was far from sufficient in terms of improvement in corrosion resistance or magnetic characteristics of the magnet powder.",
"{circle over (4)} Japanese Unexamined Patent Publication No.",
"2002-93610 In this open patent, a method to diffuse (or coating) Nd or Dy on the surface or inside of anisotropic magnet powder by means of diffusion heat process, is disclosed.",
"The heat processed Nd or Dy acts as an oxygen getter, control as well as prevent a direct oxidization of R or Fe that compose main phase of magnet powder.",
"As a result, the diffusion heat processed magnet powder achieves an excellent corrosion resistance.",
"However, the corrosion resistance was not surely a sufficient level.",
"As mentioned above, even if the prior rare-earth magnet powder and the magnet have a superior initial magnetic characteristics, the corrosion resistance was insufficient.",
"In addition, even ones with improved corrosion resistance were not surely sufficient level, with no coexistence of magnetic characteristics and corrosion resistance on a high level.",
"However, to achieve a coexistence of high functionalization and high reliability of the product using rare-earth magnet, to improve corrosion resistance of RFeB-type magnet powder with superior magnetic characteristics and the applied rare-earth magnets is very important.",
"In addition, although many of the above-mentioned open patents are illustrating La as R that compose a main phase of RFeB-type magnet powder, none of them reports an example in which La is R. Also none of them utilizes La as to improve corrosion resistance of magnet powder.",
"DISCLOSURE OF THE INVENTION The present invention was developed in order to solve problems as those mentioned above.",
"That is to say, the purpose is to offer a bonded magnet with superior magnetic characteristics and small time-dependent deterioration, as well as RFeB-type magnet alloy and RFeB-type magnet powder which make such bonded magnet, and their production methods.",
"As a result of many types of systematic experiments and diligent research to solve these challenges, the present inventors found out that, by means of introducing proper amount of La into bonded magnet and then diffusing or coating, an excellent bonded magnet with superior time-variation properties such as corrosion resistance and with little decline in magnetic characteristics was achievable, and accomplished inventions as follows.",
"(Bonded Magnet Alloy) First, the component of the bonded magnet alloy of the present invention (hereafter simply referred to as magnet alloy) are at least Fe as a main component, 11-15 at % R that includes yttrium (Y) and does not include lanthanum (La), 5.5-10.8 at % B and 0.01-1.0 at % La, and has superior corrosion resistance.",
"La is a kind of R that composes RFeB-type magnet or RFeB-type magnet powder because La is also an rare-earth element.",
"However, the RFeB-type magnet containing La as R is inferior in the magnetic characteristics to the RFeB-type magnet containing neodymium (Nd), praseodymium (Pr), dysprosium (Dy) or terbium (Th) as R. For this reason, La is indeed rarely chosen as R. Furthermore, the actual condition is that in the RFeB-type magnet of which the magnetic characteristics is intended to be improved as good as possible, containing of La has been intended to be avoided.",
"However, on the contrary to such customary understanding, the present inventor paid attention to the La, and succeeded to improve the corrosion resistance (specifically, deterioration resistance against oxidization or oxidization resistance) of RFeB-type magnet powder, scarcely deteriorating the magnetic characteristics.",
"The reasons for this are considered as follows.",
"La has the largest oxidation potential of all rare-earth elements.",
"Because of this, in the case of the RFeB-type alloy containing La, La is used as a so-called oxygen getter and is selectively (at a priority) oxidized as compared with the above-mentioned R (Nd, Dy, etc.).",
"Consequently, a magnet powder containing La exhibits higher corrosion resistance with superior time-variation properties because the oxidization of the main phase RFeB-type crystal is markedly suppressed.",
"As mentioned above, Dy, Th, Nd, Pr, etc.",
"can be used in place of La.",
"However, compared with these elements, use of La brings about more sufficient oxidization suppression effect of magnet powder or bonded magnet, and with respect to cost, to use La is cheaper over these other elements.",
"Here, to harmonize corrosion resistance and magnetic characteristics on high level, a composition of La becomes important.",
"An avoidable impurity level of La is approximately 0.001 at %.",
"When La is added on a very small quantity exceeding the avoidable impurity level, corrosion resistance of bonded magnet improves.",
"And, in terms of achieving a sufficient improvement of corrosion resistance, La was added with the lower limit content of 0.01 at %.",
"On the other hand, more than 1.0 at % content yields the unfavorable result of lowering the iHc.",
"Thus, in terms of improvement of corrosion resistance and suppression of decline in iHc, 0.01-0.1 at % of La content is more favorable.",
"As stated above, a magnet powder and a hard magnet (bonded magnet) obtained from the raw material of a magnet alloy containing proper amount of La shows an superior time variation characteristics, scarcely deteriorating the superior magnetic characteristics.",
"In addition, the cost for this is lower compared to the use of Nd or Dy.",
"Because a bonded magnet produced from the raw material of this magnet alloy exhibits superior corrosion resistance, the use not only for equipment used under room temperature environment, but also the use for equipment used under high temperature environment where deterioration due to oxidization easily occur (for example, driving motors for hybrid cars or electric cars) is favorable.",
"By the way, the magnet alloy of the present invention can be an ingot melt and then cast by various melting method (high-frequency melting method, nuclear melting method), or a coarse powder achieved via hydrogen crushing or mechanical crushing.",
"Furthermore, it can be a magnet powder itself of the undermentioned isotropic magnet powder or anisotropic magnet powder.",
"Consequently, the magnet powder of the present invention does not regard the forms such as shape or grain diameter.",
"Additionally, it is sufficient if the alloy of the present invention is one used for production of bonded magnet or magnetic powder with superior corrosion resistance, and does not regard their production process.",
"For example, it can be what is supplied as the raw material for the undermentioned hydrogenation processing method (HDDR processing or d-HDDR processing).",
"Moreover, the alloy of the present invention is not limited to a single type of magnet alloy which has the above-mentioned composition.",
"That is to say, it is also favorable, mixing the plural kinds of alloys, and if the mixture as the whole forms an alloy which has at least the above-mentioned composition.",
"For example, the case of a mixed alloy in which a RFeB-type alloy containg 11-15 at % R and 5.5-10.8 at % B, and a La-type material (for example, a simple substance of La, a La alloy such as LaCo or its hydride) are mixed together, is also defined as a bonded magnet in the present invention.",
"And these mixed alloys also become the raw material to which HDDR processing or d-HDDR processing are applied.",
"The composition of magnet alloy in the present invention is what is described above, and the reason why R and B were restricted as above-mentioned is as follows.",
"With less than 11 at % of R, the primary crystal of α-Fe becomes liable to precipitation causing a decrease in iHc, with more than 15 at % of R, R2Fe14B phase decreases, decreasing the maximum energy product (BH) max, thus neither of them is unfavorable.",
"Here this R is more than one kind of scandium (Sc), yttrium (Y) and lanthanide (La).",
"To get superior magnetic properties, it is ideal to usee more than one of Y, cerium (Ce), Pr, Nd, samarium (Sm), gadolinium (Gd), Th, Dy, holmium (Ho), erbium (Er), thulium (Tm) as well as lutetium (Lu) for R. Out of these, it is especially desirable to use at least one of Pr, Nd or Dy for R in terms of cost as well as magnetic properties.",
"With less than 5.5 at % of B, a soft magnetic R2Fe17 phase precipitates, causing a decrease of magnetic properties, with more than 10.8 at %, R2Fe17B phase decreases, also decreasing magnetic properties, thus neither of them is favorable.",
"Additionally, in the magnet alloy of the present invention, it is favorable to include at least one of gallium (Ga) or aluminum (Al) (hereafter referred as to Group 1 Elements) with a total amount of 0.05-1.0 at %.",
"These elements improve the coercivity iHc of the magnet.",
"In the magnet alloy of the present invention, it is favorable to include niobium (Nb) (hereafter referred as to 2 Element) with a amount of 0.05-1.0 at %.",
"This element improve the residual remanence (Br) of the magnet.",
"The maximum energy product (BH)max can be improved with the addition of both elements from the Group 1 Elements and 2 Element.",
"In any case if the total is less than 0.05 at % there is no actual effest and if more than 1.0 at % the iHc, Br or (BH)max will decrease bringing on an unfavorable result.",
"If you think of cost and magnetic properties, it is desirable to have a content of 0.05-1.0 at % or better yet 0.2-0.4 at % (on the order of 0.3 at %) Ga and 0.05-0.8 at %, or better yet 0.1-0.4 at % (on the order of 0.2 at %) Nb.",
"In particular, it is more desirable to have both of a content of 0.05-1 at % Ga and a content of 0.05-0.8 at % Nb, improving both of iHc and Br.",
"Furthermore, in addition to the above-mentioned elements, it is desirable to have a content of 0.1-10 at %, or better yet 1-10 at % of cobalt (Co).",
"This is because Co is an element that will increase the Curie temperature, as well as increasing the heat durability.",
"With less than 0.1 at % of Co content, there is not an actual effect.",
"On the other hand, because Co is expensive, less than 10 at % of the content is desirable in terms of industrial cost.",
"Upon addition of La, if an alloy or a compound of La and Co is used as the row material, both of them can be contained in the magnet powder for low cost.",
"Needless to say, unavoidable impurities may exist to a certain extent in the magnet alloy of the present invention and the over all composition accounts for the difference in the Fe balance.",
"In addition, each indicated composition in the present detailed statement is given when defining the whole alloy or magnet powder as 100 at %.",
"What is mentioned above concerning a composition or shapes falls under the undermentioned magnet powder of the present invention, its production method, as well as bonded magnet.",
"(Magnet Powder and its Production Method) (1) An example of shapes or used forms of the above-mentioned alloy is a magnet powder.",
"For example, an isotropic magnetic powder given by employing the HDDR process to an ingot etc.",
"composed of the above-mentioned magnet alloy, or the d-HDDR processed anisotropic magnet powder.",
"That is to say, an isotropic magnet powder obtained by the HDDR process which includes putting an ingot, with alloy composition of at least the main component Fe, R including Y without La of 11-15 at %, B of 5.5-10.8 at % and La of 0.01-1.0 at %, through the hydrogenation step while maintaining the ingot between 1023-1173 K in a hydrogen atmosphere, and then after the hydrogenation step, carrying out the desorption step where hydrogen is removed, is used for bonded magnet with superior corrosion resistance.",
"On the other hand, anisotropic magnet powder obtained by the d-HDDR process which includes putting an ingot, with alloy composition of at least the main component Fe, R including Y without La of 11-15 at %, B of 5.5-10.8 at %, and La of 0.01-1.0 at %, through the low temperature hydrogenation step while maintaining the ingot at less than 873 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the high-temperature hydrogenation step while maintaining 20-100 kPa and 1023-1173 K in a hydrogen atmosphere, and then the high-temperature hydrogenation step, carrying out the first evacuation step while maintaining 0.1-20 kPa and 1023-1173 K in a hydrogen atmosphere, and after the first evacuation step, carrying out the second evacuation step where the hydrogen is removed, is used for bonded magnet with superior corrosion resistance.",
"(2) Additionally, not limited to these, magnet powder obtained by the following production method of the present invention is also a magnet powder involved in the present invention.",
"The production method of isotropic magnet powder of the present invention is a production which includes the HDDR process which includes putting a magnet alloy, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the hydrogenation step while maintaining the alloy between 1023-1173 K in a hydrogen atmosphere, and then after the hydrogenation step, carrying out the desorption step where hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the hydrogenation process or the dehydrogenation process with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and their hydride (a simple substance of La, a La alloy as well as a hydride of La compound, hereafter referred as to La hydride), is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder.",
"In addition, thus achieved isotropic magnet powder, when regarding the whole as 100 at %, includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"The production method of anisotropic magnet powder of the present invention is a production which includes the d-HDDR process which includes putting a magnet alloy, with alloy composition of at least the main component Fe, R including Y without La as well as B, through the low temperature hydrogenation step while maintaining the ingot at less than 873 K in a hydrogen atmosphere, and then after the low-temperature hydrogenation step, carrying out the high-temperature hydrogenation step while maintaining 20-100 kPa and 1023-1173 K in a hydrogen atmosphere, and then the high-temperature hydrogenation step, carrying out the first evacuation step while maintaining 0.1-20 kPa and 1023-1173 K in a hydrogen atmosphere, and after the first evacuation step, carrying out the second evacuation step where the hydrogen is removed, to which diffusion heat process is annexed or unified where a La blended powder that can be obtained by blending the obtained RFeB-type powder after the high-temperature, the first evacuation process or the second evacuation process with La-type powder composed of more than one kind that includes a simple substance of La, a La alloy, a La compound and a La hydride, is heated at 673-1123 K and La is diffused on the surface and the inside of the RFeB-type powder.",
"In addition, thus achieved anisotropic magnet powder, when regarding the whole as 100 at %, includes at least 11-15 at % of the above-mentioned R, 5.5-10.8 at % of the above-mentioned B as well as 0.01-1 at % of the above-mentioned La, and is used for bonded magnet with superior corrosion resistance.",
"In these magnet powder, the addition form of La is modified from the previously explained magnet powder.",
"That is to say, the previously stated magnet powder is produced form use of the raw material that includes La.",
"On the other hand, to the latter explained magnet powder, La is added on the way of its production, or La is added after production of the RFeB-type powder.",
"It goes without saying that, no matter which way is used, as long as La exists, corrosion resistance of magnet powder or bonded magnet improves and thus there are no problems within the scope of the present invention regarding the form that La addition takes.",
"It must be also said, for even more effective control of the oxidation of magnet powder which has La, which has an oxygen-getter function, that is even more favorable if the La exists on the surface of, or in the vicinity of, the magnet powder structural grains.",
"Consequently, rather than having La included initially in the magnet alloy, it is more advantageous to have La diffuse into, or onto the surface of the magnet powder by mixing La-type material with the RFeB-type powder during or after the production of the powder, for an achievement of magnet powder with superior corrosion resistance.",
"In a case La is added after the production of magnet powder, the diffusion heat process is conducted after the desorption process in the above-mentioned HDDR process or after the second evacuation process in the above-mentioned d-HDDR process.",
"In a case La is added during the production of magnet powder, the diffusion heat process is conducted after the hydrogenation process in the above-mentioned HDDR process or after the high-temperature hydrogenation process or the second evacuation process in the above-mentioned d-HDDR process.",
"Here, though each process of the HDDR process and d-HDDR process as well as the diffusion heat process can be conducted individually, it is more efficient if the both are conducted in the unified manner.",
"For example, a case in which the diffusion heat process and the second evacuation process are conducted simultaneously after the D-HDDR process.",
"The case in which each process is conducted individually corresponds to what is expressed as [annexation] in the present invention, and the case in which each process is conducted in the unified manner corresponds to what is expressed as [unification] in the present invention.",
"In addition, in a case La is added during the production of magnet powder, the partner material the RFeB-type powder exists, more or less, as a hydride state (hereafter referred as to the RFeBHx powder).",
"Because La is added after the hydrogenation process and before the hydrogenation process, or after the high-temperature hydrogenation process and before the second evacuation process.",
"In this RFeBHx powder etc., R or Fe are in a state where they can hardly be oxidized compared to the case where hydrogen is not included.",
"For this reason, diffusion or coating of La can be conducted in a state of controlled oxidation, and magnet powder with superior corrosion resistance can be produced at stable quality.",
"Also for the same reason, it is favorable if the La-type powder is on a hydride state.",
"For example, LaCoHx etc.",
"is favorable.",
"Additionally, to achieve a magnet powder with superior both magnetic characteristics and corrosion resistance, it is favorable if the RFeB-type powder is a recrystallized polycrystal (RFeBHx) due to that hydrogen is removed from the three-phase decomposed RH2 phase after (high-temperature) hydrogenation process and then crystal direction of Fe2B phase is transcribed.",
"This polycrystal can be obtained, for example, after the first evacuation step in the d-HDDR process.",
"Consequently, it is favorable if the production method of anisotropic magnet powder of the present invention is the one where the above-mentioned diffusion heat process step is conducted after the above-mentioned first evacuation step.",
"Moreover, it is efficient if the above-mentioned diffusion heat process step and the above-mentioned second evacuation step are unified and conducted in the unified manner.",
"By the way, the diffusion heat process step is the step where surface diffusion (coating) or center diffusion of La into the surface or center of each structural grain of the RFeB-type powder or the RFeBHx powder occurs.",
"This diffusion heat process step can be carried out after mixing with La-type material, or simultaneously with that mixing.",
"If the processing temperature is less than 673 K, sufficient diffusion process becomes difficult, as it is difficult for the La-type material to become a liquid phase.",
"On the other hand, if above 1123 K, with the crystal grain growth of the RFeB-type magnet powder, etc., sufficient improvement of the corrosion resistance (decrease of the permanent demagnetizaiton ratio) cannot be achieved with the decrease in iHc.",
"A duration of 0.5-5 hours for this processing time is ideal.",
"If less than 0.5 hours, the La diffusion will be in sufficient, and the corrosion resistance of the magnet powder will not be much improved.",
"On the other hand, if greater than 5 hours, a decrease in iHc will be induced.",
"As we all know, it is preferable that this diffusion heat process step be carried out in an atmosphere where oxidation is prevented (such as in a vacuum atmosphere).",
"In addition, in a case this diffusion heat process step is conducted in the unified manner with the dehydrogenation process in the HDDR process, or with the first evacuation process or the second evacuation process in the d-HDDR process, their processing temperature and processing time as well as processing atmosphere are adjusted to the common range to both.",
"Although there is no specific requirement for the forms such as grain size of the above-mentioned RFeB-type powder or the La-type powder, from the viewpoint of conducting the diffusion heat process step efficiently, not more than 1 mm average particle size of the RFeB-type powder and not more than 25 μm average grain size of the La-type powder is favorable.",
"The RFeB-type powder can be, according to degree of the step progress, a hydride, magnet powder, or the one whose structure has been three phase decomposed as well as their recrystal.",
"The La-type powder is composed of more than one kinds that include a simple substance, a La alloy, a compound of La or a hydride of La, in consideration of effects etc.",
"on magnetic properties, it becomes more favorable if it is composed of an alloy between transition metallic element (TM) and La, a compound (including metallic compound) or a hydride.",
"For, example, LaCO (Hx), LaNdCo (Hx), LaDyCo (Hx) and so on.",
"Additionally, in the case the La-type powder is composed of an alloy or a compound (including hydride), it is more favorable if the amount of the La contained in the alloy etc.",
"is not less than 20 at %, not less desirably 70 at %.",
"(Bonded Magnet) By using these magnet, bonded magnet with superior magnetic characteristics and corrosion resistance can be achieved.",
"For, example, in the case using isotropic magnet powder, the bonded magnet of the present invention is characterized by that it can be achieved by mixing binder with the obtained isotropic magnet powder through the HDDR process that is composed of at least the main component Fe, 11-15 at % of R including Y without La, 5.5-10.8 at % of B as well as 0.01-1.0 at % of La, and then by compression molding, as well as by its superior corrosion resistance.",
"Or, in the case using anisotropic magnet powder, the bonded magnet of the present invention is characterized by that it can be achieved by mixing binder with the obtained anisotropic magnet powder through the d-HDDR process that is composed of at least the main component Fe, 11-15 at % of R including Y without La, 5.5-10.8 at % of B as well as 0.01-1.0 at % of La, and then by compression molding, as well as by its superior corrosion resistance.",
"Additionally, the presented isotropic magnet powder and anisotropic magnet powder are not limited to ones produced by the above-mentioned production method.",
"The Best Conditions for Implementation of the Present Invention A.",
"Condition for implementation All of the following conditions for implementation are described detail in the present invention (1)HDDR Processing Method The HDDR process related to the present invention includes carrying out the hydrogenation step and desorption step on an alloy with the above-mentioned composition.",
"The conditions for the hydrogenation step are as stated above.",
"In detail.",
"The desorption step is, for example, carried out in an atmosphere with hydrogen pressure less than 10−1 Pa. And it is good if the temperature during the desorption step is, for example, 1023-1173 K. The hydrogen pressure in this document is, unless specified, the hydrogen partial pressure.",
"Accordingly, as long as during the various steps the hydrogen partial pressure is within the specified values, it is acceptavle to have a mixture with inert gasses.",
"The above-mentioned processing times for the various steps are besed on the processing amount of a single batch.",
"For example, if the processing amount for a single batch were 10 kg, it is best to carry out the hydrogenation step on the order of 360-1800 minutes, and the desorption step o the order of 30-180 minutes.",
"Other than that, the HDDR process itself is on in detail in the above-mentioned Japanese Examined Patent Publication No.",
"7-68561 and it would be best to consult that as appropriate.",
"The magnet powder obtained by this HDDR processing method has industrial significance as isotropic magnet powder.",
"This magnet powder has, for example, the superior magnetic characteristics of iHc of 0.8-1.7 (MA/m) and (BH)max of 60-120 (kJ/m3).",
"(2)d-HDDR Processing Method The d-HDDR process related to the present invention includes carrying out the low-temperature hydrogenation step, high-temperature hydrogenation step, first evacuation step and second evacuation step on an alloy with the above-mentioned composition.",
"Specifically, the first step or the low-temperature hydrogenation step is a step where hydrogen is sufficiently occluded into alloy (the RFeB-type alloy).",
"The second step or the high-temperature hydrogenation step is a step where hydrogen and alloy (the RFeB-type alloy) react slowly.",
"At this time, the crystals of the Fe2B phase, which is the anisotropy direction transcription phase, precipitate predominantly uni-axially.",
"In the third step, or the first evacuation step, while the Fe2B crystal direction remains the same, RFeB crystals precipitates.",
"The forth step, or the second evacuation step is a step where the remaining hydrogen in the RFeB-type alloy is removed.",
"The low-temperature hydrogenation step is, for example, carried out in an atmosphere with hydrogen pressure of 30-200 kPa.",
"The second evacuation step is, for example, carried out in an atmosphere with hydrogen pressure not more than 10−1 Pa, and with a temperature, for example, on the order of 1023-1173 K. In case of unifying the second evacuation step and the diffusion heat process step, considering the upper limit temperature of the diffusion heat process step, processing temperature of 1023-1123 K is favorable.",
"Here The desorption process is composed by a combination of the first evacuation step and the second evacuation step.",
"The above-mentioned processing times for the various are based on the processing amount of a single batch.",
"For example, if the processing amount of a single batch were 10 kg, it is best to carry out the low-temperature hydrogenation step for not shorter than 30 minutes, the high-temperature hydrogenation step on the order of 360-1800 minutes, the first evacuation step on the order of 10-240 minutes and the second evacuation step on the order of 10-120 minutes.",
"Other than that, the d-HDDR process itself is reported in the above-mentioned Japanese Unexamined Patent Publication No.",
"2001-76917 and it would be best to consul that as appropriate.",
"The magnet powder obtained via this d-FDDR processing method is an anisotropic magnet powder showing superb magnetic characteristics.",
"These characteristics are, for example, iHc of 0.8-1.7 (MA/m) and (BH)max of 190-290 (kJ/m3).",
"For an alloy for the HDDR process and the d-HDDR process, a coarsely crushed ingot in a dry or wet mechanical crusher (jaw crusher, disc mill, ball mill, vibrating mill, jet mill, etc.)",
"can be also used.",
"(3)Bonded Magnet and its Production Method This bonded magnet is obtained by carrying out a mixing step, where the above-mentioned isotropic magnet powder or anisotropic magnet powder is mixed with a binder, and a formation step, where the mixed powder obtained from the mixing step is formed.",
"The binder can be a binder like the above-mentioned organic binder or a metal binder, or others.",
"Resin-type organic binders are the most common.",
"The resins used in resin binders can be a thermo-setting resin or a thermo-plastic resin.",
"If this type of binder is used, in addition to the above-mentioned mixing step, it is good to carry out a kneading step where the magnet powder and resin binder are kneaded.",
"The above-mentioned molding step can be compression molding, injection molding, extruction molding, etc.",
"In the case that anisotropic magnet powder is used for the magnet powder, the anisotropic magnet powder should be formed in a magnetic field.",
"Furthermore, in the case of using heat-hardening resin as the resin binder, a hardening (curing) step should be carried out during or after the formation step.",
"B.",
"EXAMPLES The following examples are offered so as to explain the present invention in detail.",
"Examples 1 Sample No.",
"1-5 (1)Production of Anisotropic Magnet Powder {circle over (1)} By weighing raw material alloys or raw material elements and then melt-casting via high-frequency melt furnace, 100 kg of the raw material alloy ingot (an ingot fot bonded magnet) for anisotropic magnet powder was produced.",
"The composition of the ingot was Nd: 12.5%, b: 6.4%, Ga: 0.3%, Nb: 0.2%, the remainder: Fe (unit: at %, and so forth).",
"To this alloy ingot, heat processing of 1423 K (1140 C) ×40 hours was applied in an argon atmosphere, and the structure of alloy ingot was homogenized.",
"Additionally, this homogenizing heat treated alloy ingot was coarsely crushed into not more than 10 mm on its average particle size, using jaw crusher.",
"{circle over (2)} To thus obtained 10 kg of the RFeB-type alloy (coarsely crushed powder), first, the low-temperature hydrogenation step, the high-temperature hydrogenation step and the first evacuation step in the d-HDDR process was applied.",
"That is to say, each sample ingot was subject to sufficient hydrogen absorption (low-temperature hydrogenation step) under a hydrogen atmosphere at a hydrogen pressure of 100 kPa at room temperature.",
"Next, heat treatment (high-temperature hydrogenation step) was carried out for 8 hours at 1113 K and a hydrogen pressure of 35 kPa under a hydrogen atmosphere.",
"Following that, heat treatment (first evacuation step) was carried out for 150 minutes at 1113 K and 0.1-20 kPa hydrogen pressure under a hydrogen atmosphere.",
"{circle over (3)} By mixing the obtained RFeB-type alloy after the first evacuation step (RFeBHx-type powder) with each of three kinds of the La-type powder shown FIG.",
"1 to prepare the La-type mixed powder (mixing step), then heated at 1073 K for 3 hours (diffusion heat treatment step).",
"At that time, a vacuum atmosphere of not more than 10−1 Pa was created by use of rotary pump or diffusion pomp (second evacuation step).",
"In the present example, these mixing step, the diffusion heat treatment step and the second evacuation step were conducted in the unified manner.",
"Thereafter it was cooled and an anisotropic magnet powder with not more than 212 μm of the average grain size was obtained (Sample No.",
"1-5).",
"The terminal composition of the obtained anisotropic magnet powder are also shown in table 1.The La-type powders shown in FIG.",
"1 were produced as follows.",
"First, according to the desired composition, the raw material was weighed and 3 kg of melt-cast ingot was prepared.",
"This ingot was hydrogen crushed (HD) under a hydrogen atmosphere (room temperature×0.1 MPa).",
"Consequently, by making the crushed powder finer by use of a vibration mill, the La-type powder (a hydride) was achieved with an average particle diameter of 10 μm.",
"This is the same for the La-type powder shown in table 2 and table 4.Values displayed in [the La-type powder] column in each figure represents composition ratios of the La-type powder, for example, (La50Nd50)80Co20 represents that it is composed of 80% (La50Nd50)80Co and of 20% single substance of Co (the unit is at %).",
"{circle over (4)} As comparison example, next, three kinds of anisotropic magnet powder were prepared.",
"That is, as compared with the above-mentioned examples, the one with 3 at % of La additive (Sample No.",
"C1), the one heat treated at 1173 K in the diffusion heat treatment step (Sample No.",
"C2) and the one with no addition of La (Sample No.",
"C3).",
"The used La-type powders were displayed together in table 1.",
"(2)Production Method of Bonded Magnet Using the above-mentioned various kinds of anisotropic magnet powder, the following bonded magnets were produced.",
"First, each magnet powder is mixed with epoxy resin (3 wt %) dissolved beforehand in butane.",
"Then bonded-magnet-use pellets are produced by volatilizing the butane under vacuum.",
"This pellets were aligned under a 2.5 T magnetic field, and made into bonded magnets with a shape 7 mm cubed by heat-compression molding.",
"This heat-compression molding was carried out under a condition of 150° C.×9 ton.",
"(3) Magnetic Measurement of the Magnet Powder and Bonded Magnet {circle over (1)} Magnetic measurement was carried out for the various obtained magnet powders.",
"For the measurement of iHc, an ordinary BH tracer could not be used, so the iHc was measured in the following way.",
"First, the magnet powder is classified into grain diameter between 75-106 μm.",
"Using this classified magnet powder, (BH)max and iHc are measured after forming so as to achieve a demagnetization coefficient of 0.2 and after magnetization at 4.57 MAm−1 after alignment in a magnetic field.",
"These results are brought together and shown in Table 1.",
"{circle over (2)} Additionally, the maximum energy products (BH)max, the remanent magnetic flux density Br and intrinsic coercivity iHc were measured with a BH tracer.",
"The “-” in Table 1 indicates when the values were extremely undesirable which is no worth investigating.",
"These results are brought together and shown in Table 1.",
"{circle over (3)} Furthermore, the permanent demagnetization ratio was measured for each bonded magnet.",
"The permanent demagnetization ratio is the ratio of a bonded magnet's initial magnetic flux and the difference between the initial magnetic flux and the magnetic flux after remagnetization after being held for 1000 hours in an air atmosphere at 353 K (80° C.), 373 K (100° C.) or 392 K (120° C.) (an so forth in following examples).",
"Here the magnetization is carried out at 1.1 MA/m (45 kOe).",
"A fluxmeter was used to measure the magnetic flux.",
"The permanent demagnetization ratios gathered in this manner are brought together in Table 1.",
"(4)Evaluation {circle over (1)} The following can be noticed from Table 1.First of all, bonded magnets of Examples to which a proper amount of La is added, in any cases, in comparison with bonded magnets of Comparison Examples, exhibits smaller values of the permanent demagnetization ratio.",
"Above all, in the case that the La-type powder includes not only La, but also Nd or Dy such as bonded magnets of Sample No.",
"3 or Sample No.",
"4, due to a multiplier effect of the both, even small value of the permanent demagnetization ratio was revealed.",
"In addition, in any bonded magnets of Sample No.",
"1-5, in spite of an addition of La, (BH)max exhibited excellent values about 157 kJ/m3.This was on the same level as bonded magnet of Sample No.",
"3 where no La is added.",
"Indeed, when the amount of La exceeds 1 at % such as bonded magnet of Sample No.",
"C1, not only the magnetic characteristics but also the permanent demagnetization ratio deteriorates.",
"If the processing temperature of the diffusion heat process step exceeds 1123 K such as Sample No.",
"C2, both of the magnetic characteristics and the permanent demagnetization ratio considerably deteriorates.",
"This can be considered that because crystal grain growth of a main phase R2Fe14B occurs, causing a decrease of iHc.",
"Example 2 Sample No.",
"6 An alloy ingot composed of Nd: 12%, B: 9.0%, Ga: 0.4%, Nb: 0.1% and the remainder: Fe was produced in the same manner as Example 1, and the homogenizing heat treatment of 1393 K×20 hours was applied.",
"Hereafter, in the same manner as Example, the homogenizing heat treated alloy ingot was coarsely crushed, applying the d-HDDR process and the diffusion heat process step, an anisotropic magnet powder (Sample No.",
"6) and a bonded magnet were produced.",
"Here an amount of the La diffusion is 0.2 at %.",
"The used La-type powder, the terminal composition of the obtained anisotropic magnet powder and its magnetic characteristics, as well as the magnetic characteristics and the permanent demagnetization ratio of the obtained bonded magnet were brought together and shown in Table 2.As Comparison Example, a bonded magnet produced from anisotropic magnet powder with no La additive (Sample No.",
"C4) was prepared.",
"As comparing the both bonded magnet, although the magnetic properties were on the same level due to a small amount of La, bonded magnet of Sample No.",
"6 as compares with bonded magnet of Sample No.",
"C4, exhibits considerably deteriorated permanent demagnetization ratio.",
"In particular, observing the permanent demagnetization ratio after maintaining at high-temperature range not lower than 373 K, it can be seen that the degree is large.",
"Example 3 Sample No.",
"7 An alloy ingot composed of Nd: 12.5%, B: 6.4%, Ga: 0.3%, Nb: 0.2%, La: 0.4% and the remainder: Fe was produced in the same manner as Example 1, and by employing the same-conditioned as Example 1 homogenizing heat treatment and the d-HDDR process, an anisotropic magnet powder (Sample No.",
"7) was produced.",
"Unlikely the case of Example 1, mixing of La-type powder or diffusion heat treatment were not performed.",
"Using these obtained anisotropic magnet powder, a bonded magnet was produced in the same manner as Example 1.Also, as Comparison Example, an alloy ingot composed of Nd: 12.5%, B: 6.4%, Ga: 0.3%, Nb: 0.2%, the remainder: Fe and with no La, was produced in the same manner as Example 1, and by employing the same-conditioned as Example 1 homogenizing heat treatment and the d-HDDR process, an anisotropic magnet powder (Sample No.",
"7) was produced.",
"Needless to say, the diffusion heat treatment etc.",
"was not performed, either.",
"Using these obtained anisotropic magnet powder (Sample No.",
"C5), a bonded magnet was produced in the same manner as Example 1.The terminal composition and magnetic characteristics of anisotropic magnet powder related to Sample No.",
"7 and Sample No.",
"C5, as well as magnetic characteristics and permanent demagnetization ratio of the associated bonded magnet powder were brought together and shown in Table 3.As comparing the both, although the magnetic properties merely deteriorate due to a content of La, the permanent demagnetization ratio considerably deteriorates.",
"In particular, it can be seen that the permanent demagnetization ratio after maintaining at equal of or higher temperature than 373 K deteriorates considerably.",
"By the way, as clearly seen from comparison between bonded magnet of Sample.",
"No.",
"1 and bonded magnet of Sample No.",
"7, despite the almost the same composition, it can be seen that magnetic characteristics and the permanent demagnetization ratio are more superior in bonded magnet powder of Sample No.",
"1.That is to say, rather than having La included initially in the magnet alloy, it is more favorable to have La diffuse into, or onto the surface of the magnet powder by the later diffusion heat treatment step.",
"Example 4 Sample No.",
"8 Using the same alloy ingot as Example 1, after employing the same homogenizing heat treatment and the coarse crushing, the HDDR process was applied in stead of the d-HDDR process.",
"That is to say, under a hydrogen atmosphere at 1093 K and with hydrogen pressure of 100 kPa, the heat process was performed (the hydrogenation step).",
"Following to this, maintained under a vacuum atmosphere of not more than 10−1 kPa created by use of rotary pump or diffusion pomp at the same temperature (1093 K) for 60 minutes (the desorption step).",
"Thus an isotropic magnet powder with not more than 100 μm of average grain size was produced.",
"With this obtained magnet powder, the La-type powder etc.",
"displayed in Table 4 was mixed and the diffusion heat process was carried out (diffusion heat process step).",
"This heat process condition was the same as the case of Example 1.Thus, an isotropic magnet powder related to the present Example was obtained (Sample No.",
"8).",
"As Comparison Example, an isotropic magnet powder as obtained via the HDDR process without mixing with the above-mentioned La-type powder (Sample No.",
"C6), was prepared.",
"Also, as a reference example, an isotropic magnet powder produced by the rapid quenching method, using the above-mentioned alloy ingot (Reference Sample), was prepared.",
"Using each of the obtained isotropic magnet powder, bonded magnet was produced in the same manner as Example 1.The magnetic characteristics and the permanent demagnetization ratio of each bonded magnet, together with the terminal composition and the magnetic characteristics of isotropic magnet powder are shown in Table 4.Comparing bonded magnet of Sample No.",
"8 and bonded magnet of Sample No.",
"6, although magnetic characteristics merely decreases due to the La diffuse, the permanent demagnetization ratios considerably decreases at both temperature ranges.",
"As stated above, it was revealed that bonded magnet composed of magnet powder on which a proper amount of La is included or diffused, considerably decreases the permanent demagnetization ratio while scarcely deteriorating the magnetic characteristics.",
"This tendency is true of an isotropic magnet powder and an anisotropic magnet powder.",
"Here it was also revealed that, rather than having La included in the raw material magnet alloy, having La diffuse into, or onto the surface of the RfeB-type powder by the later diffusion heat process step, deteriorates the permanent demagnetization ratio more considerably.",
"TABLE 1 Anisotrpic magnet powder Isotrpic magnet powder Permanent demagnetization Utilized Mgnetic properties Mgnetic properties ratio (%) La-type Sample Terminal composition (BH)max Br iHc (BH)max Br iHc 353K × 373K × 393K × magnet No.",
"(at %) (kJm.3) (T) (MAm.1) (kJm.3) (T) (MAm.1) 1000 hr 1000 hr 1000 hr powder Exam- 1 Fe—12.3Nd—6.4B— 302.3 1.33 0.94 156.8 0.98 0.93 5.2 9.7 15.4 La80Co20Hx ple 0.3Ga—0.2Nb— 0.1Co—0.4La 2 Fe—12.3Nd—6.4B— 306.3 1.34 0.98 157.8 0.98 0.96 5.8 10.1 15.7 La80Co20Hx 0.3Ga—0.2Nb— 0.05La 3 Fe—12.8Nd—6.3B— 302.3 1.32 1.02 156.8 0.98 1.00 4.6 8.6 13.4 (La50Nd50)80Co20Hx 0.3Ga—0.2Nb— 0.1Co—0.3La—0.4Dy 4 Fe—12.1Nd—6.2B— 302.3 1.33 1.32 156.8 0.98 1.31 2.0 4.1 7.2 (La50Dy50)80Co20Hx 0.3Ga—0.2Nb— 0.1Co—0.4La 5 Fe—12.4Nd—6.3B— 310.3 1.34 0.99 156.7 0.98 0.98 6.9 11.8 17.2 La80Co20Hx 0.3Ga—0.2Nb— 0.01La Com- C1 Fe—12.1Nd—6.1B— 222.7 1.10 0.43 115.4 0.81 0.42 13.4 20.5 — La80Co20Hx parison 0.3Ga—0.2Nb— 0.2Co—2.8La Exam- C2 Fe—12.7Nd—6.2B— 79.5 0.82 0.26 46.2 0.67 0.25 — — — La80Co20Hx ple 0.3Ga—0.2Nb— 0.1Co—0.4La C3 Fe—12.5Nd—6.2B— 310.3 1.34 0.97 161.6 0.99 0.96 8.5 19.7 30.5 None 0.3Ga—0.2Nb TABLE 2 Anisotrpic bonded magnet Anisotrpic magnet powder Permanent demagnetization Utilized Mgnetic properties Mgnetic properties ratio (%) La-type Sample Terminal composition (BH)max Br iHc (BH)max Br iHc 353K × 373K × 393K × magnet No.",
"(at %) (kJm.3) (T) (MAm.1) (kJm.3) (T) (MAm.1) 1000 hr 1000 hr 1000 hr powder Exam- 6 Fe—12.1Nd—9.0B—0.4Ga— 286.4 1.30 0.97 144.1 0.91 0.95 5.5 9.9 15.9 La80Co20Hx ple 0.1Nb—0.1Co—0.2La Com- C4 Fe—12.0Nd—9.0B— 294.3 1.31 1.00 149.6 0.93 0.98 7.4 18.5 27.7 None parison 0.4Ga—0.1Nb Exam- ple TABLE 3 Anisotrpic bonded magnet Anisotrpic magnet powder Permanent demagnetization Utilized Mgnetic properties Mgnetic properties ratio (%) La-type Sample Terminal composition (BH)max Br iHc (BH)max Br iHc 353K × 373K × 393K × magnet No.",
"(at %) (kJm.3) (T) (MAm.1) (kJm.3) (T) (MAm.1) 1000 hr 1000 hr 1000 hr powder Exam- 7 Fe—12.5Nd—6.4B—0.3Ga— 302.0 1.32 0.92 152.8 0.95 0.90 6.3 10.4 18.3 Added to the ple 0.3Nb—0.4La raw material alloy Com- C5 Fe—12.5Nd—6.4B— 303.0 1.33 0.99 157.6 0.98 0.98 8.7 18.9 29.6 None parison 0.3Ga—0.2Nb Exam- ple TABLE 4 Isotrpic bonded magnet Isotrpic magnet powder Permanent demagnetization Utilized Mgnetic properties Mgnetic properties ratio (%) La-type Sample Terminal composition (BH)max Br iHc (BH)max Br iHc 353K × 373K × 393K × magnet No.",
"(at %) (kJm.3) (T) (MAm.1) (kJm.3) (T) (MAm.1) 1000 hr 1000 hr 1000 hr powder Exam- 8 Fe—12.1Nd—6.2B— 115.0 0.78 1.36 80.4 0.65 1.35 1.8 3.7 6.8 (La50Dy50)80Co20Hx ple 0.3Ga—0.2Nb— 0.1Co—0.4La—0.4Dy Com- C6 Fe—12.5Nd—6.4B— 117.0 0.77 1.39 83.6 0.68 1.39 5.2 9.7 12.5 None parison 0.3Ga—0.2Nb (HDDR) Exam- ple Refer- Fe—12.5Nd—6.4B— 111.0 0.85 0.76 77.2 0.68 0.76 2.0 4.0 7.0 None ence 0.3Ga—0.2Nb (quenching Sample sodification method)"
]
] | Patent_10204426 |
[
[
"Methods for producing a light emitting semiconductor body with a liminescence converter element",
"The invention describes two methods of fabricating semiconductor components in which a luminescence conversion element is applied directly to the semiconductor body (1).",
"In the first method, a suspension (4) containing a bonding agent and at least one luminescent material (5) is applied to the semiconductor body (1) in layers.",
"In the next step the solvent escapes, leaving only the luminescent material (5) with the bonding agent on the semiconductor body.",
"In the second method, the semiconductor body (1) is provided with a layer (6) of bonding agent to which the luminescent material (5) is applied directly."
],
[
"1.A method for fabricating a light-radiating semiconductor component that includes an electrically contacted semiconductor body (1), mounted on a substrate element (2), and a luminescence conversion element, and that comprises at least one luminescent material (5) and is deposited on said semiconductor body (1), characterized by the steps of fabricating said semiconductor body (1), mounting on said substrate element (2) and electrical contacting, applying in layers, to at least one surface of said semiconductor body (1), a suspension (4) that contains butyl acetate as solvent and into which at least one bonding agent and said at least one luminescent material (5) are introduced, drying the semiconductor component, leaving essentially said luminescent material (5) on said semiconductor body (1).",
"2.A method for fabricating a light-radiating semiconductor component that includes an electrically contacted semiconductor body (1), mounted on a substrate element (2), and a luminescence conversion element, and that comprises at least one luminescent material (5) and is deposited on said semiconductor body (1), characterized by the steps of fabricating said semiconductor body (1), mounting on said substrate element (2) and electrical contacting, applying a layer (6) of bonding agent to at least one surface of said semiconductor body, applying said least one luminescent material (5) to said at least one surface of said semiconductor body.",
"3.The method of claim 2, characterized in that epoxy resin, acrylic resin or silicone is used as the bonding agent (6).",
"4.The method of claim 2 or 3, characterized in that said at least one luminescent material (5) is scattered, blown or dusted on.",
"5.The method of any of claims 1 to 4, characterized in that inorganic phosphors, Ce- or Tb-activated garnets, alkaline earth sulfides or organic dyes are used as said luminescent material (5).",
"6.The method of any of claims 1 to 5, characterized in that said luminescent material (5) contains YAG:Ce, ThYAG:Ce, GdYAG:Ce, GdThYAG:Ce or mixtures based thereon, Al optionally being replaced at least partially by Ga or In.",
"7.The method of any of claims 1 to 6, characterized in that the average grain size of said at least one luminescent material (5) is 10 μm.",
"8.The method of any of claims 1 to 7, characterized by casting of the component in epoxy resin or acrylic resin.",
"9.The method of any of claims 1 to 8, characterized in that the central wavelength of the radiation emitted by said semiconductor body (1) during operation is less than 460 nm.",
"10.The method of any of claims 1 to 9, characterized in that the color of the radiation emitted by said semiconductor body (1) during operation and the color of the light emitted by said at least one luminescent material are mutually complementary, thereby producing the effect of white light.",
"11.The use of a plurality of components fabricated according to any of claims 1 to 10 in an LED lighting unit.",
"12.The use of a plurality of components fabricated according to any of claims 1 to 10 in an LED lighting unit, in which the components fabricated according to any of claims 1 to 10 are in a matrix type of arrangement.",
"13.The use of a component fabricated according to any of claims 1 to 10 as a light source in an imaging optics."
],
[
"The invention relates to a method for fabricating a light-radiating semiconductor body with a luminescence conversion element as recited in the preambles to claims 1 and 2.Light-radiating semiconductor components with luminescence conversion elements are known, for example, from WO 97/50132.These arrangements include a semiconductor body that transmits light (primary light) during operation and a luminescence conversion element that converts some of this light to another wavelength range (fluorescent light).",
"The color effect of the light emitted by such a semiconductor component is achieved by additive color mixing of primary and fluorescent light.",
"The luminescence conversion element can be inserted after the semiconductor body in a number of ways.",
"In many embodiments, the luminescence conversion element is composed of luminescent materials that are embedded in a casting compound surrounding the semiconductor body.",
"These arrangements have the disadvantage that the luminescent materials are spatially inhomogeneously distributed, due to their having undergone sedimentation inside the casting compound.",
"Furthermore, the sources of primary light—the semiconductor body—and of fluorescent light—the casting compound containing the luminescent material—generally vary in size and shape, causing the color effect to be spatially inhomogeneous and rendering optical images subject to severe chromatic errors.",
"A further disadvantage is that the color effect depends on the optical wavelength in the casting compound, so that production-induced variations in the thickness of the casting compound lead to different color effects.",
"Moreover, if it is necessary for the color effect to be consistent in different viewing directions, the shaping of the components is limited disadvantageously by the fact that the optical wavelength in the casting compound has to be nearly the same for all the desired viewing directions.",
"The above-cited document specifies further that the luminescent material can also be applied directly to the semiconductor body.",
"This arrangement circumvents the aforesaid disadvantages.",
"The object underlying the present invention is to develop a method by which luminescent material can be applied directly to a semiconductor body.",
"This object is achieved by a method as recited in claim 1 or 2.Advantageous improvements of the invention are the subject matter of claims 3 to 12.According to the invention, in the first method step the semiconductor body is fabricated by a usual method, mounted on a substrate and provided with contacts.",
"In the second step, the luminescent material is suspended, together with a bonding agent, in a solvent containing butyl acetate.",
"This suspension is applied to the semiconductor body in layers.",
"In a third step the component is dried, allowing the solvent to escape and leaving the luminescent material with the bonding agent on the semiconductor body.",
"A further method according to the invention comprises, in a first method step, fabricating the semiconductor body, mounting it on a substrate and providing it with contacts.",
"In the second step, the semiconductor body is coated with a bonding agent.",
"In the third step, the luminescent material is applied to this layer of bonding agent.",
"In a preferred embodiment of this method, epoxy resin, acrylic resin or silicone is used as the bonding agent.",
"These materials are often employed in the production of light-emitting diodes and can therefore be used cost-effectively in the methods according to the invention.",
"In an especially preferred embodiment of the latter method, the luminescent material is scattered over the bonding agent.",
"This method makes it possible to apply a uniform, well-proportioned layer of luminescent material.",
"Alternatively, the luminescent material can be blown or dusted on.",
"The luminescent material used is preferably an inorganic phosphor such as garnet doped with rare earths, especially Ce; alkaline earth sulfides, thiogallates, aluminates and orthosilicates.",
"Efficient luminescent materials in these cases are compounds that conform to the formula A3B5O12:M (provided that they are not unstable under ordinary production and operating conditions).",
"In this formula, A denotes at least one element of the group Y, Lu, Sc, La, Gd, Th and Sm; B at least one element of the group Al, Ga and In; and M at least one element of the group Ce and Pr, preferably Ce.",
"The following have proven to be especially efficient luminescent materials: YAG:Ce compounds (Y3Al5O12:Ce3+), TbYAG:Ce compounds ((TbxY1-x)3Al5O12:Ce3+, 0<x<1) and GdYAG:Ce compounds ((GdxY1-x)3Al5O12:Ce, 0<x<1) as well as mixed crystals formed therefrom, such as, for example, GdTbYAG:Ce ((GdxTbyY1-x-y)3Al5O12; Ce3+).",
"Al in these cases can be replaced at least partially by Ga or In.",
"The following compounds are also suitable: SrS:Ce3+, Na, SrS:Ce3+, Cl, SrS:CeCl3, CaS:Ce3+ and SrSe:Ce3+.",
"A luminescent material with an average grain size of 10 μm can be used to particular advantage in both of the methods according to the invention.",
"In prior-art methods, the grain size is much smaller and is kept to a minimum to prevent sedimentation of the luminescent material within the casting compound.",
"Disadvantageously, however, light scattering on the particles of luminescent material increases with diminishing grain size, thereby causing an overall decrease in luminescence conversion efficiency.",
"This disadvantage is avoided with the methods of the invention.",
"In an advantageous improvement of the methods according to the invention, the production process is extended to include casting of the component in epoxy or acrylic resin.",
"In a particularly advantageous manner, semiconductor bodies that radiate light with a central wavelength of less than 460 nm can be used in the methods of the invention.",
"The use of such semiconductor bodies with the above-described components of prior art is not expedient, since light in this wavelength range damages the casting compound, especially commercially available epoxy resins, causing it to age very rapidly.",
"This disadvantage does not occur with the methods of the invention, since some of the radiation is converted directly at the semiconductor body, thereby reducing the proportion of short-wave radiation in the casting compound.",
"In addition, the power density of the radiation in the casting compound is lower because the casting is farther away from the radiation-emitting region of the semiconductor body.",
"Especially advantageously, the methods of the invention are suited for the fabrication of white-light-emitting diodes as described in the above-cited document.",
"In this case, the luminescent material and the semiconductor body are adapted to each other so that the primary-light and fluorescent-light colors are mutually complementary.",
"The effect of white light is elicited by additive color mixing.",
"A plurality of components produced by a method according to the invention can be combined into larger lighting units.",
"The methods of the invention advantageously permit the manufacture of components that are small in volume and thus have a higher luminance, since no casting compound is needed.",
"Larger lighting units, optionally with a matrix-type arrangement of their components, are distinguished by especially high luminances.",
"Especially advantageously, components fabricated according to the invention are suitable for use as light sources in imaging lens systems.",
"Since primary and fluorescent light are radiated from volumes that are closely adjacent spatially and are roughly the same size, the chromatic distortions caused by such a lens system are much smaller than those that occur with light sources according to the aforesaid prior art.",
"Further features and advantages will emerge from the following description of two exemplary embodiments, provided in conjunction with FIGS.",
"1 to 4.In the figures: FIG.",
"1 is a schematic diagram of a first embodiment of a method according to the invention, FIG.",
"2 is a schematic diagram of a second embodiment of a method according to the invention, FIG.",
"3 schematically depicts the radiation characteristic of a component fabricated according to a method of the invention, and FIG.",
"4 schematically depicts the radiation characteristic of a component of prior art.",
"FIG.",
"1 illustrates the fabrication of a semiconductor body with a directly applied luminescence conversion element according to the method of claim 1.In the first method step, FIG.",
"1a, the semiconductor body 1 is fabricated by a usual production method.",
"The semiconductor body 1 is mounted on a substrate 2 and provided with contacts 3.The subsequent steps do not impose any fundamental limitations on the production process.",
"In the second method step, FIG.",
"1b, a thin layer of a suspension 4 of a luminescent material 5 in butyl acetate is applied to at least one surface of the semiconductor body 1.The bonding agent used can be, for example, PERENOL 45 (Henkel).",
"The luminescent material 5 is present in this suspension in a high concentration, with a volume ratio in excess of 40%.",
"The layer of suspension can be produced by being sprayed or dripped on.",
"In the latter case, the quantity of droplets is apportioned to yield a uniformly thin layer that encases the free surfaces of semiconductor body 1.The luminescent material 5 used can be, for example, Ce- or Th-activated yttrium aluminum garnet or a modification of said garnet.",
"If the suspension layer 4 is applied by a spraying process, it is also possible to cover only subareas of the surface of the semiconductor body with the suspension 4.In addition, rheological additives and wetting agents can be added to the suspension 4 to produce the most uniform possible layer.",
"In the next, third method step, the component is dried, during which process the solvent—butyl acetate in FIG.",
"1c—evaporates, so that only the luminescent material 5 with the bonding agent remains on the semiconductor body (FIG.",
"1d).",
"FIG.",
"2 shows the fabrication of a semiconductor body with a directly applied luminescence conversion element according to the method of claim 3.In the first method step according to FIG.",
"2a, here again, the semiconductor body 1 is fabricated, mounted on a substrate 2 and provided with contacts 3.In the second step, a thin layer of epoxy resin 6 is sprayed onto this semiconductor body as a bonding agent (FIG.",
"2b).",
"In contrast to the case with components of prior art, this layer 6 does not serve as a casting compound or a casing, but only as an adhesive for the luminescent material 5 that is to be applied.",
"In the third method step, as shown in FIG.",
"2c, the luminescent material 5 is scattered on.",
"After this step, the luminescent material 5 adheres in a thin, uniform layer to the surface of semiconductor body 1 (FIG.",
"2d).",
"Once the epoxy resin has set, further process steps can follow, for example casting of the component or insertion into a lighting matrix.",
"FIG.",
"3 shows the radiation characteristic of a component 7 fabricated according to a method of the invention.",
"In the case of components 7 according to the invention, the light cones of primary light 8 and of fluorescent light 9 are radiated from nearly identical volumes and therefore largely coincide.",
"By way of comparison, FIG.",
"4 shows the radiation characteristic of a component 10 of prior art.",
"Such prior-art components have a primary light cone 11, emitted from the semiconductor body, that is concentrated on the optical axis.",
"The fluorescent light 12 is radiated from the entire casting compound, making the fluorescent light cone 12 much more divergent than the primary light cone 11.Sighting along the optical axis toward such a component 10 of prior art when it is operating reveals that in the center, the radiated mixed light is altered in the direction of primary color, and in the outer area it is rimmed by a circular border that has the color of the fluorescent light.",
"By contrast, a component 7 fabricated by a method according to the invention produces a spatially uniform mixed-color effect."
]
] | Patent_10204576 |
[
[
"Method And Apparatus For Processing Electronic Documents",
"A computer-implemented method of generating an input to be used by a classifying apparatus based on an electronic document comprising a plurality of elements, said method comprising: analyzing said electronic document to obtain one or more of said elements together with information about their corresponding position in said document; generating an electronic layout document to be used as said input of said classifying apparatus, said electronic layout document comprising: a representation of a plurality of said elements obtained in said analyzing step together with information representing their absolute and/or relative position in said electronic document."
],
[
"1.A computer implemented method of generating an input to be used by a classifying apparatus based on an electronic document comprising a plurality of elements, said method comprising: analyzing said electronic document to obtain one or more of said elements together with information about their corresponding position in said document; generating an electronic layout document to be used as said input of said classifying apparatus, said electronic layout document comprising: a representation of a plurality of said elements obtained in said analyzing step together with information representing their absolute and/or relative position in said electronic document.",
"2.The method of claim 1, wherein said layout document comprises: a representation of a first one of said plurality elements of said text document together with information representing its absolute and/or relative position in said electronic document, and a representation of others of said plurality elements of said text document together with information representing their absolute and/or relative position in said electronic document, said other elements lying within a predefined or user defined area neighbouring and/or surrounding said first element.",
"3.The method of claim 1 or 2, further comprising: searching for elements meeting a certain searching criterion to obtain candidates for elements which with respect to their informational content fall into a certain category, and generating a layout document for one or more of said candidate elements.",
"4.The method of claim 3, wherein said step of searching candidates further comprises one or more of the following: searching for elements in said document which match a certain format criterion; searching for words in said document which match a certain string comparison criterion; performing a fault tolerant word search; performing a search for an element which has a predefined relative position with respect to a found candidate; performing a database search for searching elements which match with words stored in a database.",
"5.The method of one of claims 1 to 4, further comprising one or more of the following: representing the position of an element in said layout document by a corresponding character sequence based on a predefined position coding scheme representing elements which have a recognizable predefined format through a character sequence based on a predefined format coding scheme; representing elements which have a recognizable meaning through a character sequence based on a predefined meaning coding scheme.",
"6.The method according to claim 5, wherein said recognized format comprises one or more of the following: vertical and/or horizontal lines in said document; floating point numbers; dates; integer numbers; postal ZIP codes.",
"7.A method according to any of the preceding claims, wherein the area taken into account for generating said layout document comprises one or more geometrical areas the dimensions of which can be predefined or can be set by the user.",
"8.The method of any of claims 1 to 7, further comprising: using said electronic layout document as an input for a classifying apparatus for training said classifying apparatus or for evaluating said input through said classifying apparatus.",
"9.A method for extracting from an electronic document one or more elements which belong to a certain predefined category, said method comprising: searching for candidate elements in said document based on one or more predefined search criteria; for each candidate element obtained from said searching step, generating a layout document according to any of the preceding claims; and judging if said candidate belongs to said category based on the informational content of said layout document.",
"10.A method for training a classifying apparatus to train said apparatus for recognizing whether an element of a document belongs to a certain category or not, said method comprising: searching for candidate elements in said document based on one or more predefined search criteria; for each candidate element obtained from said searching step, generating a layout document according to any of the preceding claims; and inputting said layout together with information as to whether said candidate belongs to the certain category or not into the classifying apparatus to train said classifying apparatus.",
"11.The method of claim 9 or 10, wherein said classifying apparatus is a neural network.",
"12.An apparatus for generating an input to be used by a classifying apparatus based on an electronic document comprising a plurality of elements, said apparatus comprising: an analyzor analyzing said electronic document to obtain one or more of said elements together with information about their corresponding position in said document; a generator generating an electronic layout document to be used as said input of said classifying apparatus, said electronic layout document comprising: a representation of a plurality of said elements obtained in said analyzing step together with information representing their absolute and/or relative position in said electronic document.",
"13.The apparatus of claim 12, wherein said layout document comprises: a representation of a first one of said plurality elements of said text document together with information representing its absolute and/or relative position in said electronic document, and a representation of others of said plurality elements of said text document together with information representing their absolute and/or relative position in said electronic document, said other elements lying within a predefined or user defined area neighbouring and/or surrounding said first element.",
"14.The apparatus of claim 12 or 13, further comprising: a searcher searching for elements meeting a certain searching criterion to obtain candidates for elements which with respect to their informational content fall into a certain category, and said generator generating a layout document for one or more of said candidate elements.",
"15.The apparatus of claim 14, wherein said searcher searching for candidates further comprises one or more of the following: an element searcher searching for elements in said document which match a certain format criterion; a word searcher searching for words in said document which match a certain string comparison criterion; a word searcher performing a fault tolerant word search; an element searcher performing a search for an element which has a predefined relative position with respect to a found candidate; a database searcher performing a database search for searching elements which match with words stored in a database.",
"16.The apparatus of one of claims 12 to 15, wherein said layout generator further is adapted to one or more of the following when generating said layout document representing the position of an element in said layout document by a corresponding character sequence based on a predefined position coding scheme representing elements which have a recognizable predefined format through a character sequence based on a predefined format coding scheme; representing elements which have a recognizable meaning through a character sequence based on a predefined meaning coding scheme.",
"17.The apparatus according to claim 16, wherein said recognized format comprises one or more of the following: vertical and/or horizontal lines in said document; floating point numbers; dates; integer numbers; postal ZIP codes.",
"18.The apparatus according to any of claims 12 to 17, wherein the area taken into account for generating said layout document comprises one or more geometrical areas the dimensions of which can be predefined or can be set by the user.",
"19.The apparatus of any of claims 12 to 9, further comprising: a classifying apparatus using said electronic layout document as an input for training said classifying apparatus or for evaluating said input through said classifying apparatus.",
"20.An apparatus for extracting from an electronic document one or more elements which belong to a certain predefined category, said apparatus comprising: a searcher searching for candidate elements in said document based on one or more predefined search criteria; a generator for for each candidate element obtained from said searching step, generating a layout document according to any of the preceding claims; and a judger judging if said candidate belongs to said category based on the informational content of said layout document.",
"21.A a classifying apparatus trainable to recognize whether an element of a document belongs to a certain category or not, said apparatus comprising: a searcher searching for candidate elements in said document based on one or more predefined search criteria; a generator generating for each candidate element obtained from said searcher a layout document according to any of the preceding claims; and means for inputting said layout together with information as to whether said candidate belongs to the certain category or not into the classifying apparatus to train said classifying apparatus.",
"22.The apparatus of claim 20 or 21, wherein said classifying apparatus is a neural network.",
"23.A computer program comprising computer executable program code, said program code being adapted to cause said computer to carry out any of the methods according to one of claims 1 to 11.24.A data structure to be used as an input in a classifying apparatus, said data structure being derived by carrying out any of the methods according to cone of claims 1 to 11."
],
[
"<SOH> FIELD OF THE INVENTION <EOH>The present invention is related to a method and an apparatus for processing electronic documents, in particular for extracting certain elements from electronic text documents."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention according to one aspect provides a method and an apparatus for generating a layout document representing an element of the text document which can be used as an input for a classifying apparatus.",
"Due to the particular type of the layout document generated according to this aspect of the present invention, the classifying apparatus is able to carry out an improved classification of a text element represented by the layout document.",
"Thereby an improved extraction of certain text elements from text documents becomes possible.",
"According to one aspect of the present invention a layout document is generated based on elements of an electronic text document, the layout containing a representation of elements of said document together with representations of their corresponding position.",
"By generating such a layout document which can be used an an input for a classifying apparatus such as a neural network, it becomes possible to evaluate the elements of a document together with their geometrical context (their neighbouring/surrounding) elements and to thereby make use not only of the format or the content of an element itself but also of other information to evaluate whether the element belongs to a certain category or not.",
"Taking into account not only an element or its format itself, but rather also its surrounding area, respectively the elements contained in the surrounding area, a classifying apparatus receives further hints as to whether a text element belongs to a certain category or not.",
"Those further hints given by the surrounding area and the text elements contained therein can be recognized or learned by a classifying apparatus, such as a neural network, and a thereby trained neural network can provide an improved classification and consequently an improved extraction of elements of text documents.",
"According to a further aspect of the present invention layouts are generated for a plurality of elements which belong to a certain category, and the so generated layouts are then used to train the classifying apparatus to recognize elements of this category.",
"Preferably the classifying apparatus is a neural network which is trained by the layouts generated for a plurality of elements, and by inputting to the apparatus in the training phase whether the elements for which the layouts have been generated belong to a certain category or not.",
"A so trained neural network or classifying apparatus can then further be used for classifying unknown text elements and for performing an extraction of elements from unknown texts.",
"According to a further aspect of the present invention a classifying apparatus which has been trained is used to evaluate whether an unknown element belongs to a certain category or not based on a layout document generated for this element, to thereby extract elements from a document which belong to a certain category.",
"According to a further aspect of the present invention, candidates are identified which according to a certain search criterion could possibly belong to the category to which the extracted data should belong.",
"Preferably a search criterion can be a format of an element, a word search criterion, a fault tolerant word search criterion, or a combination of such criteria.",
"For each of those candidates then there can be generated a layout document based on the candidate itself, its position in the electronic document, and based on other elements of the electronic document and their position in said document.",
"Preferably those elements are taken into account when generating the layout document which lie within one or more predefined areas, preferably next to or surrounding the candidate.",
"Based on such a layout document then it is judged whether the candidate actually belongs to the desired category or not.",
"According to a preferred embodiment of the present invention, the decision whether said candidate belong to the desired category is made by use of a classifying apparatus which is preferably a neural network.",
"The neural network may have been trained by using layout documents of candidates and by giving further to the neural network as an input whether those candidates belong to the desired category or not.",
"According to a further preferred embodiment, the decision whether a candidate belongs to the desired category or not is made by using a method or an apparatus as disclosed in European patent application having the application number 99108354.4, having been filed on Apr.",
"28, 1999, the priority of which is claimed for the present application, and which is incorporated herein by reference."
],
[
"RELATED APPLICATIONS The present application is related to the European patent application having the application number 99108354.4, having been filed on Apr.",
"28, 1999, the priority of which is claimed for the present application, and which is incorporated herein by reference.",
"FIELD OF THE INVENTION The present invention is related to a method and an apparatus for processing electronic documents, in particular for extracting certain elements from electronic text documents.",
"DESCRIPTION OF THE RELATED ART Nowadays electronic document processing (EDP) becomes more and more important in order to cope with the huge volume of documents which have to be handled by such entities like large corporations, administration offices, or the like.",
"It is common nowadays to have documents in an electronic form, which may be the result of a scanning process and thereafter an optical character recognition (OCR) process to convert written documents into an electronic form.",
"If a large volume of such documents has to be handled, e.g.",
"in order to store specific data contained therein in an ordered manner like in e.g.",
"a database, then it is desirable that particular pieces of information (elements of the document), such as e.g.",
"the date of birth, the place of birth, or the like, can be extracted from such electronic documents in an automatic manner.",
"If for example a company wishes to automatically process a large volume of curriculum vitaes, then it could be desirable to extract those data in an automatic manner from the electronic documents.",
"Also for other purposes, like e.g.",
"for extracting accounting information for ERP systems, the extraction of data from text documents could be desirable.",
"There are many applications which can be imagined, for which it could be desirable to have certain text information or text elements, or certain numbers or number information extracted from a text document.",
"The text document can e.g.",
"be any document containing certain data of interest which should be extracted since they belong to a certain category of information which should be extracted.",
"Conventional extraction systems which can extract specific data from electronic text documents operate such that they search a fixed position in a document where it is assumed that the desired data is located.",
"Such kind of search algorithms do not perform very well, since they highly depend on documents which have a predefined format where the desired data is always located at the same place.",
"It is therefore an object of the present invention to improve the conventional methods of extracting certain pieces of data from text documents.",
"SUMMARY OF THE INVENTION The present invention according to one aspect provides a method and an apparatus for generating a layout document representing an element of the text document which can be used as an input for a classifying apparatus.",
"Due to the particular type of the layout document generated according to this aspect of the present invention, the classifying apparatus is able to carry out an improved classification of a text element represented by the layout document.",
"Thereby an improved extraction of certain text elements from text documents becomes possible.",
"According to one aspect of the present invention a layout document is generated based on elements of an electronic text document, the layout containing a representation of elements of said document together with representations of their corresponding position.",
"By generating such a layout document which can be used an an input for a classifying apparatus such as a neural network, it becomes possible to evaluate the elements of a document together with their geometrical context (their neighbouring/surrounding) elements and to thereby make use not only of the format or the content of an element itself but also of other information to evaluate whether the element belongs to a certain category or not.",
"Taking into account not only an element or its format itself, but rather also its surrounding area, respectively the elements contained in the surrounding area, a classifying apparatus receives further hints as to whether a text element belongs to a certain category or not.",
"Those further hints given by the surrounding area and the text elements contained therein can be recognized or learned by a classifying apparatus, such as a neural network, and a thereby trained neural network can provide an improved classification and consequently an improved extraction of elements of text documents.",
"According to a further aspect of the present invention layouts are generated for a plurality of elements which belong to a certain category, and the so generated layouts are then used to train the classifying apparatus to recognize elements of this category.",
"Preferably the classifying apparatus is a neural network which is trained by the layouts generated for a plurality of elements, and by inputting to the apparatus in the training phase whether the elements for which the layouts have been generated belong to a certain category or not.",
"A so trained neural network or classifying apparatus can then further be used for classifying unknown text elements and for performing an extraction of elements from unknown texts.",
"According to a further aspect of the present invention a classifying apparatus which has been trained is used to evaluate whether an unknown element belongs to a certain category or not based on a layout document generated for this element, to thereby extract elements from a document which belong to a certain category.",
"According to a further aspect of the present invention, candidates are identified which according to a certain search criterion could possibly belong to the category to which the extracted data should belong.",
"Preferably a search criterion can be a format of an element, a word search criterion, a fault tolerant word search criterion, or a combination of such criteria.",
"For each of those candidates then there can be generated a layout document based on the candidate itself, its position in the electronic document, and based on other elements of the electronic document and their position in said document.",
"Preferably those elements are taken into account when generating the layout document which lie within one or more predefined areas, preferably next to or surrounding the candidate.",
"Based on such a layout document then it is judged whether the candidate actually belongs to the desired category or not.",
"According to a preferred embodiment of the present invention, the decision whether said candidate belong to the desired category is made by use of a classifying apparatus which is preferably a neural network.",
"The neural network may have been trained by using layout documents of candidates and by giving further to the neural network as an input whether those candidates belong to the desired category or not.",
"According to a further preferred embodiment, the decision whether a candidate belongs to the desired category or not is made by using a method or an apparatus as disclosed in European patent application having the application number 99108354.4, having been filed on Apr.",
"28, 1999, the priority of which is claimed for the present application, and which is incorporated herein by reference.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a computer system which can be used to implement an embodiment according to the present invention; FIG.",
"2 illustrates an example for a text document from which elements are to be extracted; FIG.",
"3 shows an example of a working document crated from a text document; FIG.",
"4 shows an example of a user interface for the definition of he layout area; FIG.",
"5a shows an example for a layout area; FIG.",
"5b shows an example for a layout document; FIG.",
"6 shows an example of a coding scheme for the coding of a candidate box; FIG.",
"7 shows an example for the coding of layout document element positions; FIG.",
"8 shows an example of a learning phase of a classifying apparatus; FIG.",
"9 shows an example of an extraction phase of a classifying apparatus.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention may be implemented by a computer system as shown in FIG.",
"1.FIG.",
"1 schematically shows the configuration of a computer system to be used in connection with the preferred embodiment of the present invention.",
"The computer 100 contains a CPU 110, a memory 120, and an I/O-unit 130.The computer 100 is capable of executing programs by carrying out computer instructions through CPU 110 which the CPU fetched from memory 120 and which may have been stored in a storage device 150 such as a CD-ROM or a floppy disk.",
"The I/O-unit 130 is connected to a keyboard 160 and a mouse 170 to enable a user to input data to the computer, and it is further connected to a printer 180 to output documents as hardcopies.",
"Computer 100 is further connected to a display unit 140 such as a monitor.",
"It is to be understood that the computer configuration shown in FIG.",
"1 is an exemplary configuration only, and other computer configurations like parallel processing computers, neural network computers having dedicated hardware, or any other computer systems capable of carrying out the method explained below can be used in connection with the present invention.",
"The present invention will hereinafter be described in connection with the extraction of a date of birth from a curriculum vitae as shown in FIG.",
"2.It will readily be understood by the skilled person that the description of the present invention in connection with the extraction of a date of birth from a curriculum vitae is intended for exemplary purposes only, and that the same method and apparatus as described hereinafter can be applied for any other text documents from which certain pieces of information are to be extracted, such as for example to extract an account number from an accounting form sheet, to extract prices from invoices, to extract values indicating a stock pile in a factory from corresponding sheets, etc.",
"The curriculum vitae is stored in a computer or on a data carrier in electronic form, it may have been the result of an editing using a word processor, or the electronic document may be the result of a scanning process and a subsequent optical character recognition process.",
"Instead of a curriculum vitae any document may be used from which an element having a specific meaning or falling into a certain category is to be extracted.",
"At first the electronic document is analyzed to obtain the individual elements out of which it is composed.",
"“Element” here means any sequence of characters which is separated from other elements by a delimiter, such as a blank, a tabulator, an underscore, or by any other data element which is to be interpreted as delimiting one element from another.",
"The most simple way of splitting a text into individual elements is by identifying those textual parts as elements which are separated from each other by any empty space (a blank), however, depending on the purpose of the analysis also further criteria may be taken into account, such as the already mentioned underscore, a hyphen, a carriage return, or other elements of the electronic document which may be regarded as delimiting one element from another.",
"Another criterion which could be taken into account when identifying individual elements could be the geometrical distance between individual characters.",
"For example, there could be defined a threshold value beyond which a distance between two characters is to be interpreted such that the two characters are different elements.",
"In the present example we assume that an element is any single character or sequence of characters separated from other “elements” by a blank.",
"In the present example of a text document as shown in FIG.",
"2, the first two elements would be “curriculum” and “vitae”, other elements would be “Tel:”, “Fax:”, etc., as will be readily apparent to the skilled person.",
"The elements are identified by e.g.",
"a parser which just searches for blanks.",
"Apart from obtaining the elements themselves there is obtained their corresponding position in the document, e.g.",
"by calculating the x- and y-coordinates where each element is located in the document.",
"The position will later be used for generating the layout document.",
"After having identified the individual elements of the electronic text document, those elements are stored in a so-called “working document”.",
"In the working document each element which has been identified is stored together with information about its position in the electronic document.",
"For example, the element “curriculum” may be stored together with its x- and y-coordinates identifying its position in the electronic document.",
"The working document is a convenient tool for storing all elements which have been identified together with their corresponding position so that for the generation of the layout document which is explained later in detail reference can be made to the working document.",
"An example of a working document generated from any text document is shown in FIG.",
"3.The tags Tag1, Tag2, etcetera contain the position information of the corresponding elements.",
"This information may be represented in any form, e.g.",
"by directly storing the x- and y-coordinates of the elements in the tags.",
"The elements in FIG.",
"3 may be for example the individual words identified in a text document, or any other character sequences as identified through the method explained before as elements, and the tags then contain information about the position of those elements, such as where with respect to their x- and y-coordinates they are located.",
"The tags may also further comprise indications of the style of the elements, their font, whether they are underlined or not, or any similar information.",
"For example, for a bold faced element the corresponding tag may comprise the character sequence “bf” representing that the element is in bold faced characters, another character sequence may represent that the element is underlined, or the like.",
"The position of an element may represent for example the center of gravity of an element calculated based on its individual pixel values, or it may represent any other geometrical information representing the location of the element.",
"For example, a box may be constructed surrounding the element, and the average between the maximum and minimum x-coordinates of the box may be taken as the x-coordinate for the position, and the average of the maximum and the minimum y-position of the box may be used as the y-coordinate of the element when representing its position in the text through a corresponding tag in the working document.",
"The working document contains a list of the identified elements together with tags indicating their respective position and possibly also further information as mentioned before, such as further information like about the fonts of the elements, their style, whether they are underlined or not, etcetera.",
"In this way the working document is created and as containing a list of the individual elements of the electronic text document together with their corresponding position an possibly also other information.",
"Also non-textual elements may be incorporated into the working document, such as horizontal or vertical lines or grids contained in the electronic document, which then are also stored in the working document in a form representing their position and their shape (horizontal, vertical, line, grid, or the like) according to a coding scheme.",
"E. g. a horizontal line may be represented in a working document by character sequence AAAA, a vertical line may be represented by character sequence BBBB, each then followed by a tag indicating the position of the line.",
"The so created working document may then be used for identifying candidate elements which could possibly be the element to be extracted.",
"For that purpose the working document (or possibly also the “source document” based on which the working document is generated) is parsed to identify those elements which meet a certain search criterion, such as a format criterion.",
"In this step of extracting a candidate all elements are analyzed to find possible candidates for the desired elements to be extracted.",
"Preferably not only individual elements are searched but also combinations of elements so that the method can cope with spaces between the individual elements.",
"For example, when searching for a banking account number which is presumed to have eight digits, a search may be carried out for a number which has eight digits which may either be represented as “99999999” or as “999 999 99” or as “9 9 9 9 9 9 9 9”, or any other combination.",
"Searching for such a banking account number may therefore for example be carried out by searching for a number having eight digits.",
"Depending on the informational content which the element to be extracted should have, another format may be used as the search criterion.",
"Possible search criteria are searching for regular expressions (such as a format search searching for a certain format, like a character string, a sequence of numbers, possibly also requiring a certain total number of digits), or the like.",
"Another search criterion could be that a search is performed for a simple predefined element, by carrying out a string comparison.",
"For example a search may be performed for the word “birth”, and each element meeting that search criterion would then show up as a candidate.",
"Another possible search criterion could be to use a so called designator search, which means that a element is searched which is at a certain position (left/right/above/below) with respect to a candidate found by another search criterion.",
"For example, if a search criterion would be to search for the word “birth”, then a designator search could be performed for the element located right to the element “birth”, and in this case the resulting candidate would be the element located right to the element “birth”.",
"In the example of FIG.",
"2, with such a designator search the element “May 5, 1960” would show up as a candidate.",
"Another search criterion could be to carry out a search for all elements which are also present in a database.",
"The search for candidates preferably is fault tolerant in the way that prefixes/suffixes can be ignored, in order to ignore typical errors from optical character recognition, or to be able to ignore such elements like “,” and “:”.",
"For example, in the case of FIG.",
"2 a word search could be performed for the word “birth” by using such a fault tolerant search, for example by using a wildcard.",
"A search would then be performed for the element “birth*” so that the element “birth:” would show up as a candidate.",
"From the designator search the actual date located right to the element “birth” could then be obtained as a candidate.",
"Depending on the manner the candidate search is performed, more or less candidates for the elements to be extracted are identified.",
"Other search methods could for example include a trigram search, which means that combinations of three characters are searched for.",
"This is also a method of carrying out a fault tolerant search, if for example a misspelling occurs in a candidate, then a trigram search could nevertheless obtain such a candidate since several character sequences contained in the candidate would be recognized as correct trigrams.",
"Another fault tolerant search method would be to use the Levenshtein distance, which is a representation of the number of key strokes necessary on a keyboard to change one character sequence into another one.",
"Based on the Levenshtein distance also a fault tolerant search could be performed.",
"Preferably the candidate search is performed by searching the workin document for elements which match the used search criterion.",
"Thereby the analysis of the document into elements which has already been carried out can be used.",
"In principle, however, a search for candidates can also be carried out directly on the text document.",
"The search is directed to obtain candidate elements which could possibly contained the information which is searched for.",
"It is readily apparent that depending on the information which is searched for the search criteria have to be adapted accordingly.",
"If an account number is searched, then preferably a format criterion is used which makes use of the possibly known number format of the account number, to the contrary, if a place of birth is searched for, then searching for character strings is more promising then searching for numbers.",
"The adaption of the search criteria (format search, word search, database search, designator search, etc.",
"or a combination of them) to the particular piece of information which is searched can be chosen by the skilled person depending on the particular circumstances.",
"If the found candidates are to be used in a training procedure for a classifying apparatus as will be described later in more detail, then it is preferable if they are somehow indicated or displayed to the user an if the user is then able to confirm whether the found candidates match with the searched information or not.",
"Thereby the classifying apparatus then can be trained as will be explained later.",
"Displaying the candidates can be e.g.",
"done by highlighting them in the searched text document, and to then enable the user to confirm or to discard them e.g.",
"by a mouse click.",
"The format search or fault tolerant element search provides candidates for elements to be extracted.",
"The result of the candidate search is already quite good in terms of correctness since it is based on inherent properties of the elements which are searched, such as their format or their actual informational content.",
"The candidates then can however be further evaluated with respect to whether they belong to a certain category or not by taking into account elements other than the candidates as well, as will be explained in the following.",
"For each of the candidates then there is created a so-called layout document containing not only a representation of the candidate and its position in the electronic document, but also of other elements surrounding said candidate element and their corresponding position.",
"Therefore the layout document is an electronic representation of the candidate and its position in the electronic document itself, as well as of other elements in the electronic document and their corresponding position.",
"Preferably a layout document generated for a certain candidate is generated for a certain area surrounding said candidate.",
"This area (or a corresponding plurality of areas) can either be predefined or they may be defined by a user.",
"An example for the definition of such a surrounding area through a user interface is shown in FIG.",
"4.FIG.",
"4 shows how in total four boxes surrounding said candidate can be defined by a user.",
"A first box surrounds the candidate in all directions, a second box represents the neighbourhood to the left of the candidate, a third box represents the neighbourhood to the right of the candidate, and a fourth box represents the neighbourhood above the candidate.",
"Optionally also a further box representing the neighbourhood below the candidate may be used.",
"The user can dimension the size of the boxes e.g.",
"by inputting values representing their size in dots per inch or in any other unit like e.g.",
"pixels, mm, or the like.",
"In the example of FIG.",
"4 the size of the boxes can be dimensioned by the user, however, they may also be predefined.",
"The area for generating the layout document can be defined by the user depending on the specific category of element a user wishes to extract.",
"For generating the layout document all the elements which with respect to their position in the electronic document fall into the boxes defining the area of the layout document are taken into account for generating the layout document.",
"For that purpose reference can be made to the working document in which all elements are stored together with their corresponding positions.",
"In the following it is assumed that the process of obtaining candidate elements has returned the element May 5, 1960 of the document of FIG.",
"2 as a candidate.",
"This can e.g.",
"the result of a format search searching for a combination of three individual elements in series, and where the three elements should contain two integer numbers (representing day and year) and a further number or word representing the month.",
"The search result would then be the series of the three elements.",
"Also other search criteria can be imagined leading to May 6, 1960 as a candidate, such as a designator search which searches for three elements next to the element “birth”, then also resulting in May 6, 1960 as being output as a candidate.",
"Any other searches for regular expressions could also result in a candidate as May 6, 1960, such as a search for a regular expression which contains three elements, whereas two of the three elements are numbers and the third element is a word or a number, and where one of the numbers lies within a range between 1 and 31.It is readily apparent to a skilled person that many definitions of search criteria are possible which could lead to candidates for an piece of information being a “date”.",
"After one or more candidates have been obtained through a search procedure as explained above, for each of the candidates there is created a layout document which is a representation of the candidate as well of ist surrounding area.",
"To create the layout document the elements which lie within an area to be used for the generation of the layout document are at first identified and then based on these elements the layout document is created.",
"It contains a representation of the candidate as well as of the elements lying within this area together with the corresponding position of those elements.",
"FIG.",
"5a shows an example for a layout area in case of the text document of FIG.",
"2.The candidate here is “May 5, 1960”, and the dashed line in FIG.",
"5a defines the layout area surrounding the candidate.",
"All elements of the document of FIG.",
"2, respectively of FIG.",
"5a which fall into this area are used for generating the layout document.",
"The area shown in FIG.",
"5a may be the result of a user definition using an interface like the one of FIG.",
"4, or it may also be predefined.",
"An example for the layout document generated for the candidate “May 5, 1960” and the corresponding layout area as show in the example of FIG.",
"5a is shown in FIG.",
"5b.",
"The first line of the layout document corresponds to the element “May 5, 1960” itself.",
"It is represented in the layout document by the character sequence “DDMMYY”, since according to the particular implementation of the present embodiment it is recognized that its format corresponds to a “date”.",
"However, it is not necessary but only a preferable option when generating the layout document that a recognizable element the format of which can be recognized is replaced in the layout document by a corresponding representation of said format, like here by DDMMYY as a representation of the format “date”.",
"The character sequence to the right of the sequence “DDMMYY” represents the position of this element in the electronic document, as will be explained later in more detail.",
"The first line of the layout document shown in FIG.",
"5b therefore corresponds to the candidate element “May 5, 1960”.",
"The position of the candidate in the electronic document shown in FIG.",
"2 and also its size is represented by the character sequence “MXMYWLHM”, as will become more clear from the following explanation.",
"To further explain how the position of the candidate element in the electronic document is represented in the layout document through the character sequence “MXMYWLHM”, reference is made to FIG.",
"6.FIG.",
"6 shows a so-called candidate box which means the bounding rectangle of the candidate element.",
"Depending of the size of the candidate element (e.g.",
"depending on the font) also the size of the candidate box varies and can be represented in the layout document using the coding scheme for the box size as schematically illustrated in the righthand part of FIG.",
"6.Based on this coding scheme the box size is coded as “WLHM” which means that the candidate box has a “large width” (WL) and “medium height” (HM), as can be seen from FIG.",
"6.This coding sequence leads then to the last four characters WLHM in the first line of the layout document of FIG.",
"5b.",
"It is readily understood that which actual values are represented by which coding sequence, in other words which values are to be coded as “small” and which as “large” depend on the particular implementation and are a mere matter of choice to the skilled person.",
"Therefore, as can be seen from the first line of the layout document shown in FIG.",
"5b, not only the position of the candidate box (representing the position of the candidate itself) in the document is coded as will be explained later, but also the size of the candidate box (representing the size of the candidate).",
"The representation of the size of the candidate box through a corresponding coding sequence is schematically illustrated on the bottom part of the right hand side of FIG.",
"6.A candidate box which has a small candidate with in X-direction is coded as “WS” (for “width small”), a medium sized candidate box is coded as “WM” (for “width medium”), a candidate box having a large extension into the X-direction is coded as “WL” (for “width large”), and an extra large candidate box having an extra large size into the X-direction is coded as “X” (for “width extra large”).",
"Similarly, which values are to be assigned which coding sequences are a matter of choice to the skilled person.",
"Similarly to the width also the height of the candidate box is coded by one of the sequences “HS”, “HN”, “HL”, or by “HX”.",
"For the present case of FIG.",
"5b with the candidate May 5, 1960, the candidate box is coded as “WLHM”, which means that it has a large extension into the X-direction and a medium sized extension into the Y-direction.",
"The position of the candidate box in X- and Y-direction is coded as schematically illustrated in the lefthand part of FIG.",
"6.For that purpose certain areas of the document shown in FIG.",
"2 are assigned certain coding sequences, as shown in FIG.",
"6 on the lefthand part.",
"Depending on the area in which the candidate box is located, the X- and Y-position of the candidate box are coded either as “LL”, “MX”, “RR” (for the X-position), and as “TT”, “MY”, or “BB” for the Y-position).",
"In the present case of FIG.",
"5a for the candidate May 5, 1960 the candidate box with respect to its location in X-direction is medium, which means that it is not very far to the right of the document and not very far to the left of the document, but rather in the middle of the document with respect to its X-position.",
"Such a location is coded by the character sequence “MX”, as can be seen from the lefthand part of FIG.",
"6.The Y-position of the candidate box is coded by the sequence “MY”, since it is with respect to its Y-position relatively in middle of said document.",
"From that the position coding “MXMY” as shown in the first line of the layout document can be derived from the candidate box.",
"Combining the representation of the format representation of the candidate, the position of the candidate box and the sizie of the candidate box results in the character sequence shown in the first line of FIG.",
"5b It is to be understood that the coding shown in FIG.",
"6 for the candidate box is only exemplary and other codes, other assignments between position and code, and other splittings of the document into corresponding areas can be used as well.",
"The granularity of the size and the position of the candidate box may be finer or coarser than in FIG.",
"6 depending on the particular implementation as will be easily recognized by the skilled person.",
"Similarly, the coding sequences used herein are just arbitrary, here “LL” means just “to the very left”, “MX” means “rather in the middle in X-direction”, and “RR” means “at the very right of the document (in X-position)”.",
"Similarly, “TT” means “at the very top”, “MY” means “rather in the middle”, and “BB” means “at the very bottom of the document with respect to Y-direction”.",
"However, other coding sequences could be used as well as will be recognized by the skilled person.",
"Also, instead of DDMMYY other character sequences could be used to represent the recognized format of a “date”.",
"After having coded the candidate box as explained above the other elements which fall into the area of the layout document as explained with respect to FIG.",
"5a then also are coded and incorporated into the layout document.",
"The layout document as shown in FIG.",
"5b has been created based on a area which is shown in FIG.",
"5a by the dashed line.",
"As already explained before, the surrounding area may be set differently to a smaller area, depending on the user preferences and on the computing workload which can be processed by the computer in use, and it may of course also be set larger.",
"Therefore the layout are used herein is to be understood as an exemplary example only, and other area definitions could be used as well.",
"Of course, the larger the area used the more information is contained in the layout document created from this area and therefore it is possible that with an increased area the accuracy of the further evaluation of the layout document increases.",
"This may, however, depend on the particular implementation and on the particular purpose, and with small layout areas good results may be obtained as well.",
"The second line of the layout document of FIG.",
"5b is a representation of the Fax number 07029 8125 shown in FIG.",
"5a and falling into the layout area.",
"Since according to the particular implementation of the present embodiment it is recognized that the two elements 07029 and 8125 falling into the layout area consist of interger numbers, they are represented in the layout document by a coding sequence assigned to the representation of integer numbers, namely IIQQ.",
"The second and the third line of the layout document shown in FIG.",
"5d represent the area code 07029 and the number 8125, respectively.",
"The coding sequence IIQQ representing an integer then is respectively followed by a coding sequence representing the relative position of the integer in the text document of FIG.",
"2 with respect to the candidate element.",
"For coding the relative position any coding scheme can be used, the particular one used herein is schematically illustrated in FIG.",
"7.For the purpose of coding discrete ranges of distances corresponding to relative positions in the X- and the Y-directions are assigned corresponding coding sequences, like “NR” for near, “FF” for far, “HEE” for being at an equal position in horizontal direction, “VFF” for being at an equal position in the vertical direction, and so on.",
"The particular coding scheme is illustrated in FIG.",
"7 but it will be understood that this is a mere example and can very easily be modified.",
"E.g.",
"the coding sequences can be different, the partitioning into discrete ranges can be different, the number of ranges, an so on.",
"From FIG.",
"7 in connection with FIG.",
"5b it will be understood that the second line of the layout document of FIG.",
"5b is based on the fact that the area code 07029 is near to the left (LNR) and near above (ANR) the candidate box which leads to a position coding sequence LNRANR as shown in the second line of FIG.",
"5b as appended to the integer code IIQQ.",
"Since the number 8125 is horizontally equal but near above the candidate this leads to the third line in FIG.",
"5b which reads IIQQHEEANR.",
"The remaining three elements “date”, “of”, and “birth:” falling into the layout area are represented in the last three lines of the layout document of FIG.",
"5b together with their corresponding position coding sequences which will be readily understood in connection with FIG.",
"7.All elements are vertically equal to the candidate (VEE) but at different horizontal distances from the candidate.",
"It will be readily apparent that instead of the relative position coding also absolute positions of the elements within the layout area could be used for generating the layout document.",
"Furthermore it is also possible that when generating the layout document other such elements for which the format is recognizable, not only for example such as if the element has the format of a “date”, are coded in the layout document by a corresponding coding sequence.",
"While this is here only shown for the date in the first line and the integers in the second and third line of the layout document, such a replacement can also be made for other recognizable elements such as postal ZIP codes (which could be recognized from a database query) and which could be represented by a certain character sequence such as ZZZ, or the like.",
"The corresponding recognition can either be based on a format recognition, on a data base query (where e.g.",
"all postal ZIP codes are stored).",
"As explained above, a layout document is generated which contains information about the candidate itself, its position in the document, and furthermore information about other elements of the document and their position in the document.",
"The position information is in the present example represented by replacing coordinate values by character sequences representing a position according to a certain coding scheme which is used to define locations or areas into which the electronic document is partitioned for coding purposes and which have assigned corresponding character codes.",
"Similarly, number codes can be used as well for coding the positions of the elements of said electronic document.",
"Any coding scheme which represents the position and/or the format of the elements can is be used for the generation of the layout document.",
"The layout document may also contain additional information about non-textual elements of the document to be analyzed, such as lines, or grids in the document.",
"This information also can be easily obtained through a geometrical analysis of the document, and then lines or grids present in a document can be coded in the layout document through corresponding coding sequences, preferably also by representing their corresponding position, possibly also their style and further information.",
"Preferably the coding scheme used for the generation of the layout document contains a position coding based on having assigned discrete areas of location corresponding position codes as explained before.",
"Further preferably style or format information which can be recognized such as the format or style of elements also is represented in the layout document through corresponding coding sequences.",
"It is, however, possible to use only some of those elements of a coding scheme to generate a layout document.",
"The position indicated in the layout document may be a representation of the geometrical position based on coordinate values, such as the x and y coordinate values explained before.",
"It is, however, also possible that the position information for an element in the layout document represents the relative position between the candidate and this element, such as the number of elements occurring between this element and the candidate.",
"Thereby it also becomes possible to code the relative position between the candidate and other elements in the layout area through the distance between them through the number of words occurring between them.",
"Such a coding scheme could e.g.",
"be useful if the text document to be processed actually has not much of an own layout, such as an e mail message.",
"Alternatively, however, for an e-mail a virtual layout could be calculated and used for the further processing instead of the relative position of the elements as explained before.",
"The more information is present in the layout document about the candidate and its surrounding elements, the more accurate the layout document and the subsequent processing result can be.",
"However, the more sophisticated the layout document is, the more processing power is needed to create the layout document and to further process it to make the decision, therefore, depending on the desired accuracy of the decision procedure the user or a programmer may choose the area for generating the layout document as well as the information to be used when generating the layout document.",
"Hereinbefore the obtaining of candidates and the subsequent generation of a layout document for a candidate have been explained.",
"If it is now desired for example that a certain piece of information, namely the date of birth is to be extracted from the document of FIG.",
"2, then a candidate search is performed on document to obtain candidates for a date.",
"In the case of FIG.",
"2 a regular expression or format search as explained before would return two candidates which according to the used search criterion could be the date of birth, namely May 5, 1960 and May 17, 1979.For both candidates then there will be generated a layout document as explained before and this layout document is inputted to a classifying apparatus which has been trained to recognize the layout documents generated from actual dates of birth contrary to the layout documents generated from dates which, however are not the dates of birth.",
"Such a recognition becomes possible since the layout document generated from a date of birth contains further hints which make it possible to recognize them as the layout documents from dates of birth rather than any other dates.",
"It is e.g.",
"often the case that the word “birth” occurs in the neighbourhood of the date of birth, and by having a layout document where this term is included there is a further hint that this is the layout document generated from a date of birth.",
"Similarly, other elements occurring in the neighbourhood of the date of birth may also be interpreted as a hint, like the term “place” or the term “of” as is the case in the example of FIG.",
"5b.",
"However, if e.g.",
"several dates of birth are arranged in a column of a table, the header of the column containing the term “birth”, then by coding the position of the term “birth” as explained before this may be used by a classifying apparatus as a hint that the dates in this column are actually dates of birth.",
"In general, the surrounding area or the neighbourhood of a candidate for which a layout document is generated can be used as a hint for the actual informational content of such a candidate by a classifying apparatus.",
"By taking into account the hints given by such a surrounding area or neighbourhood a set of candidates extracted from a document according to any search criteria can be evaluated further as to whether those candidates actually contain the information which is looked for.",
"Of course, the layout document can also directly be generated for all elements of a text document and then each element can be evaluated based on the so generated layout document as to whether it belongs to a certain desired category or not.",
"However using a candidate search first reduces the computational costs which would arise if a layout document would have to be generated for each element of the text document.",
"In the following the extraction process and the training process using a classifying apparatus will be explained in more detail.",
"After the layout document has been generated, it may be used for training a neural network or any other computerized system which can decide whether a certain document belongs to a certain category or class or not.",
"For that purpose, the layout documents of candidates are input to the neural network or any other decision apparatus (classifying apparatus) together with the information whether the layout document corresponds to a correct candidate or not, which means whether the candidate has the desired informational content or not.",
"A training of such a neural network is schematically illustrated in FIG.",
"8.An electronic document is analyzed as explained above to obtain elements and of a text document and their corresponding positions.",
"Preferably then therefrom a text-based document, a working document is created.",
"Then a filtering is performed to obtain therefrom a set of candidates which could possibly match with a desired category.",
"Preferably the obtained set is corrected, either based on a manual input by the user or automatically, e.g.",
"by checking whether an obtained candidate has a probability of correctness beyond a certain threshold.",
"For a manual correction in the training phase the candidates can be highlighted in the document and the user can then for some or all of them confirm whether they are correct ones or not.",
"The aforementioned manual or automatic selection of correct results then leads to a set of correct results and to a set of wrong results.",
"For each of the elements of the set of the correct results and for each of the elements of the set of the wrong results then layout documents are generated.",
"Thereafter the layout documents generated for the set of wrong results and the ones generated for the set of correct results are used to train the neural network.",
"If no candidate is recognized at all, then the user may also choose himself a candidate, highlight it (e.g.",
"by the mouse) and use it as a training input.",
"An extraction process using a network which has been trained as shown in FIG.",
"8 is shown in FIG.",
"9.A set of candidates is obtained similarly to FIG.",
"8, for each of them a layout document is generated as explained before.",
"The layouts then are used as inputs for the trained neural network which then decides whether the candidates belong to the desired category or not.",
"An output of the network may consist in the correctly extracted candidates, or e.g.",
"also in a weight weighing the probability of correctness for each candidate.",
"The extracted candidates may also directly be imported or exported into another electronic document, such as a database, an MS-Excel file, a table, a Word document or any other document suitable for further electronic processing, or the like.",
"The extraction process including the identification of the candidates and the generation of the layout document can be carried out as explained in detail above.",
"For all found candidates then the corresponding generated layout document is input to a classifying or decision apparatus not necessarily though preferably being a neural network, and then for each candidate a decision is made whether it belongs to the correct category or not.",
"A particularly suitable apparatus for classifying the generated layout documents as to whether they belong to the desired category or not is disclosed in European patent application 99108354.4, the whole content of which is incorporated hereinto by reference.",
"The apparatus disclosed therein is able to classify text documents by representing them as vectors, where the values of the vector components corresponds to the frequency with which a certain word or term occurs in the document.",
"Such a vector representing a document spans up a n-dimensional vector space, and several documents together also span up a certain vector space.",
"The classification is performed by calculating a hyperplane which separates the vector space into at least two sub spaces, thereby a classification into as many classes as sub spaces are present can be performed.",
"A learning or training process consists in building up the vector space and the corresponding separating hyperplane for a set of training documents, and an unknown document then can be classified by calculating whether the corresponding vector falls into one or the other sub space.",
"Since with the method described hereinbefore in detail it is possible to represent elements of a text document through a layout document which gives hints about their surrounding areas, and since the layout document itself again is a text document, the classifying apparatus disclosed in the aforementioned European patent application can be used for classifying purposes.",
"A preferable implementation of the apparatus for classification disclosed in the patent application consists in a neural network, such as in a Perceptron.",
"Further details as to how the decision apparatus may be implemented can be taken from this application and will therefore not be outlined in further detail herein.",
"However, it is to be understood that any other neural network or any computer method or apparatus which is capable of evaluating (classifying) documents with respect to whether they belong to a certain category or not can be used for training layout documents and then for making the decision whether a candidate (or ist corresponding layout document) has to be regarded as correctly extracted or not.",
"It should further be understood that also any other layout document presentations can be used in connection with the present invention, not only those layout documents where the positions are represented by character sequences.",
"It is for example also very well possible that the positions are coded by absolute numbers representing the positions (coordinates) or by angles and distances (polar coordinates).",
"It will be understood by the skilled person that the aforementioned detailed description only illustrates an exemplary embodiment of the present invention, other embodiments being well within the reach of the general knowledge of the skilled person.",
"It is further readily apparent to the skilled person that the method of the present invention may be implemented by any computer system, by any general purpose computer, or by any other dedicated hardware carrying out a method as explained before.",
"An apparatus according to the present invention therefore may consist in any computer system carrying out the method of the present invention, whereas the apparatus may for example consist in a computer system as shown in FIG.",
"1.As far as certain apparatus elements or apparatus components are mentioned herein or in the appended claims, they may be implemented by a computer or part of a computer which carry, embody, or carry out programs or parts of computer programs.",
"As far as the present invention is related to a computer program or a computer program product, it will be apparent to the skilled person that any data carrier or any computer element such as a memory, a transmission line, or the like, which can embody computer program instructions, can form an embodiment of the present invention in so far as they may embody computer program instructions which enable a computer to carry out a method according to the present invention.",
"The skilled person will also recognize that many computer programs can be written which work according to the principles set forth hereinbefore, so that any computer programs working according to the method of the invention as described herein are to be regarded as falling under the scope of the present invention.",
"Moreover, a data structure representing the structure of a layout document as described can also form an embodiment of the present invention, independent whether it is incorporated or embodied on a storage medium, a data carrier, a transmission line, a memory such as a ROM, a RAM, or the like.",
"Furthermore, the present invention may be used in a client server architecture, which means that parts of a computer program implementing the present invention may be executed at a server and other parts may be executed at a client.",
"As far as apparatus components are mentioned in the description before or in the appended claims, they may be realized either by a computer carrying out a computer program or certain program instructions, or they may be implemented by any dedicated hardware performing the function of that component, such as an electronic circuit, a special purpose computer, or the like.",
"Further modifications and applications of the present invention will be apparent to the skilled reader, and it will be understood that the present application has been explained by means of exemplary embodiments which are not to be understood as limiting the scope of the present invention.",
"In particular, it is to be understood that the example of extracting a date of birth is just an exemplary example, and the method explained hereinbefore can be used for extracting any information element which belongs to a certain category from a text document, as will be readily apparent to the skilled reader."
]
] | Patent_10204756 |
[
[
"Apparatus and method for water treatment by adsorption",
"The present invention relates to an apparatus and method for removal of arsenic or other metal salts from drinking water.",
"The invention uses a bed of granulated ferric hydroxide to adsorp the metal in a pressurized adsorption chamber.",
"The bed is sized to about 10 m3 and run with an EBCT of about 3 minutes which has been found to give an unprecedently long bed life of upto 200,000 bed volumes of treated water."
],
[
"1.An apparatus for removing metals from water comprising: an adsorption chamber including a bed adapted to absorb metals, the bed having a bed height ranging from about 0.5 m to about 2.0 m, the bed comprising a ferric material, the ferric material comprising a granular ferric material having a grain density ranging from about 1 kg/dm3 to about 2 kg/dm3; a water inlet connectable to a first water supply on one side of the bed; a water outlet on the other side of the bed; a backwash inlet connectable to a second water supply on the said other side of the bed; wherein the apparatus is structured for normal operations and backwash operations, during normal operations, the apparatus is adapted for absorption of metals onto the bed as water flows from said one side to said other side, and the apparatus is structured for backwash operations as water flows from the backwash inlet to said one side of the bed and the empty bed contact time (EBCT) is between 1 and 6 minutes.",
"2.The apparatus according to claim 1, wherein the bed has a bed volume capacity of at least 10,000 bed volumes of water to be treated during normal operations.",
"3.The apparatus according to claim 1, wherein the bed has a bed volume capacity of at least 60,000 bed volume of water to be treated during normal operations.",
"4.The apparatus according to claim 1, wherein the bed has a bed volume capacity of at least 30,000 bed volume of water to be treated during normal operations between backwash operations.",
"5.The apparatus according to claim 1, wherein the bed has a bed volume capacity ranging from about 5 bed volumes of water to about 10 bed volumes of water during backwash operations.",
"6.The apparatus according to claim 1 wherein the apparatus is arranged for backwash operation having a flow rate of less than 50 m/h.",
"7.The apparatus according to claim 1 wherein the bed is arranged for backwash operation having a flow rate of less than 30 m/h.",
"8.The apparatus according to claim 1 wherein the apparatus is structured such that a forward flush immediately precedes a backwash operation.",
"9.The apparatus according to claim 8, wherein the apparatus is structured such that the forward flush comprises less then about 10 bed volumes of water.",
"10.The apparatus according to claim 8 wherein the apparatus is structured such that the forward flush comprises flushing water from the second supply from the said one side of the bed to the said other side of the bed.",
"11.The apparatus according to claim 8, wherein the apparatus is structured such that the forward flush has a flow rate of less than 30 m/h.",
"12.The apparatus according to claim 8, wherein the apparatus is structured such that the forward flush has a flow rate of less than 25 m/h.",
"13.The apparatus according to claim 1 wherein the adsorption chamber further comprises a base arranged to support the bed.",
"14.The apparatus according to claim 13, wherein the base includes one or more openings.",
"15.The apparatus according to claim 9 wherein the base comprises a wedgewire material.",
"16.The apparatus according to claim 1 wherein the granular ferric material has an average diameter of less than 2 mm and more than 0.25 mm, 10% or less of the granules have a diameter greater than 2 mm and 5% or less of the granules have a diameter less than 0.25 mm.",
"17.The apparatus according to claim 1, wherein the granular ferric material has an average diameter of about 0.8 mm.",
"18.The apparatus according to claim 1 wherein the granular ferric material is a granulated natural material.",
"19.The apparatus according to claim 1 wherein the granular ferric material comprises ferric hydroxide.",
"20.The apparatus according to claim 1 wherein the grain density ranges from about 1.5 kg/dm3 to about 1.7 kg/dm3.21.The apparatus according to claim 1 wherein the grain density is about 1.58 kg/dm3.22.The apparatus according to claim 21 wherein the average bulk density at 45% water content is at least 1.1 g/cm3 and no more than 1.4 g/cm3.23.The apparatus according to claim 21 wherein the average bulk density at 45% water content is within a range of about 1.2 g/cm3 to 1.3 g/cm3.24.The apparatus according to claim 21 wherein the average bulk density at 45% water content is approximately 1.25 g/cm3.25.The apparatus according to claim 1 wherein the material has a specific surface of about 1.6×105 m2/dm3.26.The apparatus according to claim 1 wherein the material comprises at least 50% by weight iron.",
"27.The apparatus according to claim 1 wherein the bed includes a layer of gravel of greater average diameter than the ferric material.",
"28.The apparatus according to claim 1 where the metal is selected from the group consisting of arsenic; copper; cadmium, nickel; chromium; silver; lead; molybdenum; manganese; and mixtures thereof.",
"29.The apparatus according to claim 1 wherein the bed has a bed volume and the bed is structured such that the ferric material is replaced after at least 100,000 bed volumes of water have been treated in normal operation.",
"30.The apparatus according to claim 1 wherein the bed has a bed volume and the bed is structured such that the ferric material is replaced after at least 120,000 bed volumes of water have been treated in normal operation.",
"31.The apparatus according to claim 1 wherein the bed has a bed volume and the bed is structured such that the ferric material is replaced after at least 150,000 bed volumes of water have been treated in normal operation.",
"32.The apparatus according to claim 1 wherein the pH in the bed of the adsorption chamber is within the range of 7.0 and 8.5 during normal operation.",
"33.The apparatus according to claim 1 wherein the pH in the bed of the adsorption chamber is within the range of 7.5 and 8.0 during normal operation.",
"34.The apparatus according to claim 1 wherein the pH in the bed of the adsorption chamber is within the range of 7.8 during normal operation.",
"35.The apparatus according to claim 1 wherein the bed is at least 1 m in height and the bed has a volume of about 10 m3.36.The apparatus according to claim 1 wherein the apparatus is arranged to provide a conditioning cycle when new bed material is added to the adsorption chamber, wherein the conditioning cycle comprises an initial bed stratification backwash, a conditioning backwash and a final conditioning forward flush.",
"37.The apparatus according to claim 36 wherein the initial bed stratification backwash comprises about 4 bed volumes of water at about 30 m/h.",
"38.The apparatus according to claim 36 wherein the final conditioning backwash comprises a backwash cycle as defined in the preceding claims.",
"39.The apparatus according to claim 36 wherein the conditioning forward flush comprises a forward flush as defined in the preceding claims.",
"40.The apparatus according to claim 1 wherein the apparatus is arranged to provide a loading backwash of about 10 bed volumes of water at less than 10 m/h e.g.",
"about 5 m/h, when loading the bed material into the adsorption chamber.",
"41.The apparatus according to claim 1 wherein the apparatus includes a plurality of similar adsorption chambers having shared first water supplies and second water supplies.",
"42.The apparatus of claim 41 wherein one of the adsorption chambers is undergoing any backwash cycle or forward flush at any one time.",
"43.The apparatus according to claim 41 wherein the adsorption chambers comprise a first group and a second group and the first water supply is arranged to supply water at different rates to the first and second groups.",
"44.The apparatus according to claim 1 wherein the bed is structured such that the rate of water through the bed in normal operation is less than 50 m/h.",
"45.The apparatus according to claim 1 wherein the bed is structured such that the rate of water through the bed in normal operation is about 25 m/h.",
"46.The apparatus according to claim 1 wherein the EBCT in normal operation is about 3 minutes.",
"47.An apparatus for removing metals from water comprising: an adsorption chamber including a bed adapted to absorb arsenic, the bed having a bed height ranging from about 0.5 m to about 2.0 m, the bed comprising a ferric material, the ferric material comprising a granular ferric material having a grain density ranging from about 1 kg/dm3 to about 2 kg/dm3, the bed comprising a bed volume capacity of at least 60,000 bed volumes of water to be treated during normal operations between backwash operations; a water inlet connectable to a first water supply on one side of the bed; a water outlet on the other side of the bed; a backwash inlet connectable to a second water supply on the said other side of the bed; wherein the apparatus is structured for normal operations and backwash operations, during normal operations, the apparatus is adapted for absorption of metals onto the bed as water flows from said one side to said other side, and the apparatus is structured for backwash operations as water flows from the backwash inlet to said one side of the bed and the empty bed contact time (EBCT) is between 1 and 6 minutes.wherein the.",
"48.A process for removing metal from water comprising: a) providing a bed of ferric material having a bed height of between 0.5 and 2.0 m wherein the ferric material comprises a granular ferric material having a grain density between 1 to 2 kg/dm3; b) in normal operation, supplying water from a first water supply from one side of the bed to the other side of the bed such that metal is adsorbed from the water onto the bed; c) in backwash operation supplying water from a second water supply from said other side of the bed to said one side of the bed so as to remove contaminating material from the bed without substantially disrupting the media; wherein in normal operation, the empty bed contact time for said water is between 1 and 6 minutes.",
"49.The process according to claim 48 wherein at least 10,000 bed volumes of water are treated in normal operation between backwash operations.",
"50.The process according to claim 48 wherein at least 30,000 bed volumes of water are treated in normal operation between backwash operations.",
"51.The process according to claim 48 wherein 60,000 bed volumes of water are treated in normal operation between backwash operations.",
"52.The process according to claim 48 wherein the backwash operation comprises about 10 bed volumes of water.",
"53.The process according to claim 48 wherein the backwash operation comprises about 5 bed volumes of water.",
"54.The process according to claim 48 wherein the backwash operation has a flow rate of less than 50 m/h.",
"55.The process according to claim 48 wherein the backwash operation has a flow rate of less than 30 m/h.",
"56.The process according to claim 48 wherein a forward flush immediately precedes a backwash operation.",
"57.The process according to claim 56 wherein the forward flush comprises about 10 bed volumes of water.",
"58.The process according to claim 56 wherein the forward flush comprises about 5 bed volumes of water.",
"59.The process according to claim 56 wherein the forward flush comprises flushing water from the second supply from the said one side of the bed to the said other side of the bed.",
"60.The process according to claim 56 wherein the forward flush has a flow rate of less than 30 m/h.",
"61.The process according to claim 56 wherein the forward flush has a flow rate of less than 25 m/h.",
"62.The process according to claim 48, wherein the bed is supported on a base.",
"63.The process according to claim 62 wherein the base includes openings.",
"64.The process according to claim 62, wherein the base comprises a wedgewire material.",
"65.The process according to claim 48, wherein the granular ferric material has an average diameter of less than 2 mm and more than 0.25 mm, less than 10% of the granules have a diameter greater than 2 mm and less than 5% of the granules have a diameter less than 0.25 mm.",
"66.The process according to claim 48 wherein the granular ferric material has an average diameter of about 0.8 mm.",
"67.The process according to claim 48 wherein the granular ferric material is a granulated natural material.",
"68.The process according to claim 48 wherein the material comprises ferric hydroxide.",
"69.The process according to claim 48 wherein the grain density is between 1.5 to 1.7 kg/dm3.70.The process according to claim 69 wherein the grain density is about 1.58 kg/dm3.71.The process according to claim 69 wherein the average bulk density at 45% water content is at least 1.1 g/cm3 and/or no more than 1.4 g/cm3.72.The process according to claim 69 wherein the average bulk density at 45% water content is within a range of from about 1.2 to about 1.3 g/cm3.73.The process according to claim 69 wherein the average bulk density at 45% water content is about 1.25 g/cm3.74.The process according to claim 48 wherein the granular ferric material has a specific surface of about 1.6×105 m2/dm3.75.The process according to 48, wherein the granular ferric material comprises at least 50% by weight iron.",
"76.The process according to claim 48, wherein the bed includes a layer of gravel of greater average diameter than the ferric material.",
"77.The process according to claim 48 wherein the metal removed from the water is selected from the group consisting of: arsenic; copper; cadmium; nickel; chromium; silver; lead molybdenum and mercury.",
"78.The process according to claim 48 wherein the ferric material is replaced after at least 100,000 bed volumes of water have been treated in normal operation.",
"79.The process according to claim 48 wherein the ferric material is replaced after at least 120,000 bed volumes of water have been treated in normal operation.",
"80.The process according to claim 48 wherein the ferric material is replaced after at least 150,000 bed volumes of water have been treated in normal operation.",
"81.The process according to claim 48 wherein the pH in the bed of the adsorption chamber is normal operation is within a range of about 7.0 to about 8.5.82.The process according to claim 48 wherein the pH in the bed of the adsorption chamber is normal operation is within a range of about 7.5 to 8.0.83.The process according to claim 48 wherein the pH in the bed of the adsorption chamber is normal operation is about 7.8.84.The process according to claim 48, wherein the bed height is at least 1 m. 85.The process according to claim 48 wherein the process further comprises a conditioning cycle when new bed material is added to process, wherein the conditioning cycle comprises an initial bed stratification backwash, a final conditioning backwash and a final conditioning forward flush.",
"86.The process according to claim 85 wherein the initial bed stratification backwash comprises about four bed volumes of water at about 30 m/h.",
"87.The process according to claim 85 wherein the final conditioning backwash comprises a backwash cycle as defined in any of the preceding claims.",
"88.The process according to claim 85 wherein the conditioning forward flush comprises a forward flush as herein before defined in any of the preceding claims.",
"89.A process for removing arsenic from water comprising: a) providing a bed of ferric material having a bed height of between 0.5 and 2.0 m wherein the ferric material comprises a granular ferric material having a grain density between 1 to 2 kg/dm3; b) in normal operation, supplying water from a first water supply from one side of the bed to the other side of the bed such that metal is adsorbed from the water onto the bed wherein 60,000 bed volumes of water are treated in normal operation between backwash operations; c) in backwash operation supplying water from a second water supply from said other side of the bed to said one side of the bed so as to remove contaminating material from the bed without substantially disrupting the media; wherein in normal operation, the empty bed contact time for said water is between 1 and 6 minutes; (d) replacing the ferric material after at least 120,000 bed volumes of water have been treated in normal operation.",
"90.A process for removing metal from water comprising: a) providing a bed of ferric material having a bed height of between 0.5 and 2.0 m wherein the ferric material comprises a granular ferric material having a grain density between 1 to 2 kg/dm3; b) in normal operation, supplying water from a first water supply from one side of the bed to the other side of the bed such that metal is adsorbed from the water onto the bed wherein 60,000 bed volumes of water are treated in normal operation between backwash operations; c) in backwash operation supplying water from a second water supply from said other side of the bed to said one side of the bed so as to remove contaminating material from the bed without substantially disrupting the media; wherein in normal operation, the empty bed contact time for said water is between 1 and 6 minutes; (d) replacing the ferric material after at least 120,000 bed volumes of water have been treated in normal operation; (e) performing a conditioning cycle when new bed material is added to process, wherein the conditioning cycle comprises an initial bed stratification backwash, a final conditioning backwash and a final conditioning forward flush."
],
[
"<SOH> BACKGROUND <EOH>Ground water represents an important source of drinking water but has been found to contain dissolved metal ions such as arsenic, copper, nickel, chromium, lead, cadmium, molybdenum, silver, mercury and manganese, often at undesirable levels.",
"For example, recent regulations have stipulated that the level of arsenic should be less than 10 μg/l.",
"Arsenic and other metals may be removed from water by using one or more of the following methods; (i) adsorption by activated aluminium, (ii) nanofiltration, (iii) in a clarification/filtration plant using overdosing coagulation; or (iv) ion exchange.",
"Each of these processes has disadvantages.",
"Activated aluminium has a limited adsorption capacity.",
"Nanofiltration allows comparatively slow processing rates.",
"The construction of a coagulation plant has a high capital cost due to e.g.",
"the large amount of land required and high operating costs."
],
[
"<SOH> SUMMARY <EOH>The present invention seeks to provide an advantageous apparatus and method for the removal of metals.",
"According to a first aspect of the present invention there is provided an apparatus for removing metals from water comprising an adsorption chamber including a bed having a bed height and comprising a ferric material, a water inlet connectable to a first water supply on one side of the bed, a water outlet on the other side of the bed and a backwash inlet connectable to a second water supply on the said other side of the bed, the apparatus being arranged for normal and backwash operations, in normal operation arsenic is adsorbed onto the bed from water flowing from the said one side to said other side, and in backwash operation water flows from the backwash inlet to said one side of the bed, the bed is between 0.5 m and 2.0 m, preferably at least 1.0 m, and the empty bed contact time (EBCT) is between 1 and 6 minutes.",
"According to a second aspect of the present invention there is provided a process for removing metal from water comprising the steps of: a) providing a bed of ferric material having a bed height of between 0.5 and 2.0 m; b) in normal operation supplying water from a first water supply from one side of the bed to the other side of the bed such that arsenic is adsorbed from the water onto the bed; c) in backwash operation supplying water from a second water supply from said other side of the bed to said one side of the bed so as to remove contaminating material from the bed without substantially disrupting the media; wherein in normal operation, the empty bed contact time for said water is between 1 and 6 minutes.",
"The present inventors have found that using a bed of ferric adsorption media allows increased metal adsorption when compared to activated aluminium (wt/wt) with the consequential reduction in the cost associated therewith.",
"Surprisingly, given the fragile nature of the bed, a long interval between backwashing cycles is possible without clearly increasing the differential pressure across the bed and/or without significant reduction in the adsorption rate and capacity of the bed.",
"Furthermore the bed of adsorption material required by the present invention is made from a material which is relatively fragile in nature and so has been proportioned to allow the bed to be formed by educting the material into the adsorber without the bed material being disrupted.",
"Yet the bed maintains sufficient size to allow the treatment of commercial volumes of water.",
"Surprisingly, it has been found that the adsorption bed of the invention can treat an unprecedently large volume of water before replacement i.e.",
"when it reaches the allowed/set limit for the metal being removed from the water, e.g.",
"in the case of arsenic the treated water having 10 μg/l arsenic.",
"The bed size in conjunction with the EBCT provide an adsorber with a high capacity, yet the bed size is not so great to cause logistical problems in maintaining the bed.",
"This is important as the apparatus may well be in a remote location where complicated and/or frequent maintenance would greatly increase costs for the process.",
"The adsorption bed of the present invention should not need to be replaced more frequently than once per annum which is considered to be commercially acceptable given the cost of the material.",
"Preferred features of the invention have been given in the dependent claims.",
"The preferred features have each been found to improve the cost efficiency of the plant and may be used together or individually.",
"The use of a ferric adsorption material was also found to allow relatively high rates of metal adsorption and a reduction in the capital expenditure required to build a plant when compared to a sludge plant.",
"Ferric oxide and hydroxide containing natural materials have been found to be both relatively cheap to use and to give satisfactory adsorption efficiencies e.g.",
"the ability to adsorb large quantities of arsenic, typically 1.3 g to 3.5 g of arsenic is removed per Kg of Ferric Hydroxide.",
"The use natural material containing these two compounds has been found to give high economy and high adsorption.",
"Advantageously according to the first and second aspects of the present invention a layer of gravel separates the media from the support base.",
"Gravel has been found to be an economic inert material to use which is widely available.",
"Advantageously according to the first, second and third aspects of the invention the pH use is in the range of 6.5 to 8.5, particularly 7.0 to 8.0 and more particularly, 7.8 during normal operation.",
"The adsorption of arsenic is satisfactory within this PH range and normally this means that no adjustment to the PH is required for processing greatly reducing the cost and complexity of the operating condition which latter advantage is very important given the plant is normally unmanned."
],
[
"This application is submitted under 35 USC 371 as the national stage application of International application number: PCT/GB01/00822 filed Feb. 26, 2001 claiming priority to GB 0004579.9 filed Feb. 25, 2000.FIELD OF INVENTION The present invention relates to a method and apparatus for arsenic or other metal removal from water, in particular but not exclusively, potable ground water.",
"BACKGROUND Ground water represents an important source of drinking water but has been found to contain dissolved metal ions such as arsenic, copper, nickel, chromium, lead, cadmium, molybdenum, silver, mercury and manganese, often at undesirable levels.",
"For example, recent regulations have stipulated that the level of arsenic should be less than 10 μg/l.",
"Arsenic and other metals may be removed from water by using one or more of the following methods; (i) adsorption by activated aluminium, (ii) nanofiltration, (iii) in a clarification/filtration plant using overdosing coagulation; or (iv) ion exchange.",
"Each of these processes has disadvantages.",
"Activated aluminium has a limited adsorption capacity.",
"Nanofiltration allows comparatively slow processing rates.",
"The construction of a coagulation plant has a high capital cost due to e.g.",
"the large amount of land required and high operating costs.",
"SUMMARY The present invention seeks to provide an advantageous apparatus and method for the removal of metals.",
"According to a first aspect of the present invention there is provided an apparatus for removing metals from water comprising an adsorption chamber including a bed having a bed height and comprising a ferric material, a water inlet connectable to a first water supply on one side of the bed, a water outlet on the other side of the bed and a backwash inlet connectable to a second water supply on the said other side of the bed, the apparatus being arranged for normal and backwash operations, in normal operation arsenic is adsorbed onto the bed from water flowing from the said one side to said other side, and in backwash operation water flows from the backwash inlet to said one side of the bed, the bed is between 0.5 m and 2.0 m, preferably at least 1.0 m, and the empty bed contact time (EBCT) is between 1 and 6 minutes.",
"According to a second aspect of the present invention there is provided a process for removing metal from water comprising the steps of: a) providing a bed of ferric material having a bed height of between 0.5 and 2.0 m; b) in normal operation supplying water from a first water supply from one side of the bed to the other side of the bed such that arsenic is adsorbed from the water onto the bed; c) in backwash operation supplying water from a second water supply from said other side of the bed to said one side of the bed so as to remove contaminating material from the bed without substantially disrupting the media; wherein in normal operation, the empty bed contact time for said water is between 1 and 6 minutes.",
"The present inventors have found that using a bed of ferric adsorption media allows increased metal adsorption when compared to activated aluminium (wt/wt) with the consequential reduction in the cost associated therewith.",
"Surprisingly, given the fragile nature of the bed, a long interval between backwashing cycles is possible without clearly increasing the differential pressure across the bed and/or without significant reduction in the adsorption rate and capacity of the bed.",
"Furthermore the bed of adsorption material required by the present invention is made from a material which is relatively fragile in nature and so has been proportioned to allow the bed to be formed by educting the material into the adsorber without the bed material being disrupted.",
"Yet the bed maintains sufficient size to allow the treatment of commercial volumes of water.",
"Surprisingly, it has been found that the adsorption bed of the invention can treat an unprecedently large volume of water before replacement i.e.",
"when it reaches the allowed/set limit for the metal being removed from the water, e.g.",
"in the case of arsenic the treated water having 10 μg/l arsenic.",
"The bed size in conjunction with the EBCT provide an adsorber with a high capacity, yet the bed size is not so great to cause logistical problems in maintaining the bed.",
"This is important as the apparatus may well be in a remote location where complicated and/or frequent maintenance would greatly increase costs for the process.",
"The adsorption bed of the present invention should not need to be replaced more frequently than once per annum which is considered to be commercially acceptable given the cost of the material.",
"Preferred features of the invention have been given in the dependent claims.",
"The preferred features have each been found to improve the cost efficiency of the plant and may be used together or individually.",
"The use of a ferric adsorption material was also found to allow relatively high rates of metal adsorption and a reduction in the capital expenditure required to build a plant when compared to a sludge plant.",
"Ferric oxide and hydroxide containing natural materials have been found to be both relatively cheap to use and to give satisfactory adsorption efficiencies e.g.",
"the ability to adsorb large quantities of arsenic, typically 1.3 g to 3.5 g of arsenic is removed per Kg of Ferric Hydroxide.",
"The use natural material containing these two compounds has been found to give high economy and high adsorption.",
"Advantageously according to the first and second aspects of the present invention a layer of gravel separates the media from the support base.",
"Gravel has been found to be an economic inert material to use which is widely available.",
"Advantageously according to the first, second and third aspects of the invention the pH use is in the range of 6.5 to 8.5, particularly 7.0 to 8.0 and more particularly, 7.8 during normal operation.",
"The adsorption of arsenic is satisfactory within this PH range and normally this means that no adjustment to the PH is required for processing greatly reducing the cost and complexity of the operating condition which latter advantage is very important given the plant is normally unmanned.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described with reference to the accompanying drawings, in which: FIG.",
"1 is a schematic illustration of a arsenic adsorption plant according to the present invention; FIG.",
"2 shows a vertical cross sectional view through an adsorber according to FIG.",
"1; and FIG.",
"3 shows a horizontal cross sectional view of an adsorber according to FIG.",
"1.DETAILED DESCRIPTION OF DRAWINGS The present invention is exemplified with the following description and drawings which show an arsenic adsorption plant.",
"The invention is not limited to arsenic adsorption, but this is the preferred embodiment of the invention.",
"The metal can be selected from the group consisting of arsenic; copper; cadmium, nickel; chromium; silver; lead; molybdenum; manganese; and mixtures thereof.",
"FIG.",
"1 shows a plant according to the present invention which has five adsorbers 10 operating in parallel.",
"Although the present invention can function with a single adsorber it is preferred, the reasons which will become apparent below, to have a plurality of adsorbers 10.Water to be treated flows from water inlet 12 through inlet line 14 to the inlet 13 of each of the adsorbers 10.The water supplied normally comes from a ground water source.",
"The inlet line 14 has an inlet branch 16 to each of the adsorbers 10.Each inlet branch 16 includes an inlet control valve 18 which is controllable to allow water flow into the particular adsorber 10 to which respective branch 16 extends.",
"The water inlet 12 may be diverted around the plant via a dedicated bypass line.",
"In the illustrated embodiment the water inlet 12 is supplied from a mixing tank (not shown) upstream of the water inlet 12.Each inlet control valve 18 is arranged to allow a predetermined rate of water flow into the respective adsorber 10.Water flow through inlet branch 16 will be prevented in certain instances, for example during backwashing cycles of the adsorber 10 and commissioning cycles of the adsorber 10 as explained in more detail below.",
"In other cases the inlet control valve 18 is adjusted to provide a proportion of the water to be treated into the adsorber 10.Each adsorber 10 has a water outlet 20 opening into an outlet line 22 via a respective outlet branch line 24.Each outlet branch line 24 is provided with an outlet branch valve 25 between the adsorber outlet 20 and the outlet line 22.The illustrated plant is also provided with backwash and conditioning wash cycles for backwashing and conditioning washes of the adsorber 10.In the illustrated embodiment the water is arranged to flow substantially in the reverse direction through each adsorber 10 in backwashing cycles.",
"In the illustrated embodiment, the inlets for the backwashing and conditioning washes are formed from the adsorbers outlets 20 and the outlet for the backwashing and conditioning water is formed through the water inlet 13 in the adsorber 10.Thus, the inlet/outlet 13,20 in the adsorber 10 perform multiple functions and this is advantageous in reducing the required ports into the adsorber 10.This is preferred in the present invention though of course separate inlet/outlets could be provided for in each washing cycle or the backwashing and conditioning washes may have a separate inlet and outlets compared to the normal wash cycles which may be advantageous where the flow rate and volumes between backwashing and normal operation are particularly different.",
"In the illustrated embodiment the backwash inlet line 30 is fed from backwash pumps 32.The backwash line 30 has backwash inlet line branches 34 to each adsorber 10.The backwash line 30 is also used for conditioning washes.",
"Each backwash inlet line branch 34 opens into the respective outlet branch line 24 between the adsorber outlet 20 and the outlet branch line valve 25.The backwash inlet line branch 34 has a backwash inlet line valve 36 immediately before opening into the outlet line branch 24.This valve 36 prevents water flow through the backwash when the inlet line branch 34 is in normal operating cycle; and, as will be apparent operates in conjunction with outlet line branch valve 25 to allow water flow through the backwash line 30, 34 in backwashing and in conditioning cycles whilst at the same time valve 25 prevents flow through outlet branch line 24 to the outlet line 22.In a backwashing cycle and conditioning wash cycles, water is outlet from the adsorber 10 through the water inlet 13.A backwash-outlet line 38 is linked to each adsorber via backwash outlet line branch 37.The inlet control valve 18 is provided in the inlet line branch 16 which is closed during backwashing and conditioning cycles to prevent water flow towards the water inlet 12.A corresponding backwash outlet branch valve 39 is provided in the backwash-outlet branch 37 which prevents flow through this line 37 during normal cycles of the apparatus, but allows water flow through this line 37 during backwash and conditioning wash cycles.",
"The inlet line branch 16 opens into the backwash outlet branch line 37 between the valve 19 and the inlet 13.The apparatus is arranged for backwash operation having a flow rate of less than 50 m/h, preferably less than 30 m/h.",
"Each backwash-outlet line branch 37 opens into the backwash outlet line 38 which feeds either into a holding tank 40 or directly to a waste outlet, e.g.",
"a sewer 42.In backwashing cycles, the apparatus is normally designed to feed into the holding tank 40 which is provided as a settlement tank to allow particulate matter washed out of the adsorbers 10 to settle.",
"The tank 40 is provided with a top water draw-off device and the cleaned water from which outputs to the water outlet 42.A pump can be provided to pump the water from out of the holding tank 40 if desired.",
"Preferably, a forward flush immediately precedes a backwash operation.",
"Tthe apparatus is structured such that the forward flush comprises less then about 10 bed volumes of water and comprises flushing water from the second supply from the said one side of the bed to the said other side of the bed.",
"The height of the bed is between 0.5 m and 2.0 m, preferably at least 1.0 m. Preferably, the forward flush has a flow rate of less than 30 m/h, more preferably, the forward flush has a flow rate of less than 25 m/h.",
"In the conditioning washing cycles of the apparatus, the conditioning wash waters are directed directly to the outlet 42 through a filter arrangement 44.In conditioning washes, considerably more particular matter is washed out of the adsorbers and the use of the filter cartridges 46 therefore removes this particulate matter before the water is outlet to waste.",
"There are of course requirements on the quality of water allowed to pass to waste.",
"Use of the cartridge filter then means that the cartridge can then be removed after a conditioning cycle and disposed of separately.",
"It has an advantage in so far as the holding tank volume can be greatly reduced if it does not have to be used for conditioning washes as well as back washes.",
"Preferably, the conditioning cycle comprises an initial bed stratification backwash, a final conditioning backwash and a final conditioning forward flush.",
"In another aspect, the initial bed stratification backwash comprises about 4 bed volumes of water at about 30 m/h.",
"Exploded cross-sections of the adsorbers 10 are shown in FIG.",
"2.From FIG.",
"2 it is clear that in the illustrated embodiment the adsorber 10 comprises a substantially cylindrical casket with domed closures at the top and bottom.",
"The adsorber 10 has an inlet 13 and an outlet 20 through which water is pumped through the chamber.",
"In normal cycle, water enters through the inlet 13, passes down the chamber to the outlet 20.In backwashing and conditioning cycles water mainly through the outlet 20 and exits via the inlet 13, though of course in forward flush modes water travels in the same direction as the normal cycle.",
"The adsorber 10 includes a base 62 which supports a media column 64.The base is located above the outlet 20.The column 64 extends from the base 62 to just below the inlet 13.The media column 64 comprises a thin layer of gravel 68 over which particulate adsorption media 66 rests.",
"The adsorption media 66 must adsorb arsenic thereonto from water.",
"In the illustrated embodiment the adsorption media 66 is a particulate ferric oxide material produced from a naturally mined ore.",
"This has been treated to have an average diameter of less than 2 mm and more than 0.25 mm.",
"In the illustrated embodiment, less than 10% of the granules have a diameter greater than 2 mm and less than 5% of the granules have a diameter of less than 0.25 mm.",
"The average diameter is approximately 0.8 mm.",
"In the granulization process of the ferric material, the grain density is adjusted to between 1 and 2 kg/dm3 and is preferably within a range of about 1 .kg/dm3 to 1.7 kg/dm3 and, more preferably in the region of 1.58 kg/dm3.The average bulk density at 45% water content should be above 1.1 g/cm3, but no more than 1.4 g/cm3.The bulk density in the illustrated embodiment is controlled to 1.25 g/cm3.The granules are controlled to have a particular surface about 1.6×10−5 m2/dm3.The natural material contains at least 50% by weight iron.",
"The granulated ferric material of this nature has a very high adsorption of arsenic which allows unprecedently long bed lifes.",
"Also through the control of the properties as described above, the material is substantially more robust than untreated material which allows it to be handled and used.",
"The untreated material has been found to smash when pouring into adsorbers and can not form a useable media bed.",
"Alternatively the granulated ferric material is formed synthetically.",
"The synthetic material will be treated to produce similar robustness and surface features to allow the high adsorption and handability of the naturally formed material.",
"The naturally produced material is slightly preferred at present in view of the cost of a naturally produced material compared to a synthetic material.",
"However, it is expected that synthetic manufacturing processes will be able to produce a synthetic material at an acceptable cost for commercial situations, even though they may be about 20% also more expensive than a naturally formed material.",
"The advantages of a synthetic material are that the adsorption capacity may be even higher than a naturally produced material in view of the purity of the synthetic constituents.",
"Table 1 shows the constituents of a preferred synthetic material which may be used as the adsorption media 66.One preferred process comprises the step of replacing the ferric material after at least 120,000 bed volumes of water have been treated in normal operation.",
"The gravel base layer 68 is put into the media column on the base 62 in order to prevent the granulated material 66 being washed from the adsorber 10 through the base 62.The base 62 must be provided with through openings 63 in order to allow backwashing flow and thus the small granular size ferric material may be washed through these openings 63.This immediate layer 68 of gravel prevents such loss.",
"Of course, other supports could be used instead of gravel.",
"The base 62 may be provided with nozzle openings 63 on a platform or alternatively may comprise a header and lateral pipework arrangement fitted with either mesh sleeves or nozzles.",
"Turning now to FIG.",
"2, it will be clear that there are several ports into the adsorber 10.There is a manway 72 located about half way down the chamber.",
"This is provided to allow access to the interior of the chamber.",
"There are sight glasses 74, 76 located to allow visual inspection of the interior of the chamber.",
"Main port 78 is provided to allow the media to be poured into the chamber from the top thereof.",
"There is also a main outlet port 80 provided to allow access to the underside of the nozzle plate, next thereto is a smaller port 82 to act as a drain outlet.",
"Above the main inlet port 78 a davit 90 is provided which is used when filling the chamber with the media columns 64.The adsorber 10 is supported on four legs 88.Each of the adsorbers 10 are substantially the same as in the illustrated embodiment, though sizes and detailed construction may vary in particular installations.",
"It is preferred to have a plurality of adsorbers, rather than a single larger adsorber.",
"The plurality of adsorbers allows one of the adsorbers to be taken offline, e.g.",
"for backwashing and refilling of media column 64, whilst the other adsorbers take the flow which would normally be processed by the adsorber taken out of line.",
"The present invention relates to an apparatus and method for removal of arsenic or other metal salts from drinking water.",
"The invention uses a bed of granulated ferric hydroxide to adsorp the metal in a pressurized adsorption chamber.",
"The bed has a bed volume capacity of at least 10,000 bed volumes of water to be treated during normal operations.",
"Alternatively, the bed volume is at least 30,000 bed volumes and preferably, 60,000 bed volumes of water to be treated during normal operations between backwash operations.",
"During backwash operations, the bed has a bed volume capacity ranging from about 5 bed volumes of water to about 10 bed volumes of water.",
"The bed is sized to about 10 m3 and run with an EBCT of about 3 minutes which has been found to give an unprecedently long bed life of upto 200,000 bed volumes of treated water."
]
] | Patent_10204998 |
[
[
"Method for accommodating large movements in a mooring system",
"A method of operating a mooring system exemplified by active mooring devices having attractive attachment elements fixable to a ship's hull.",
"Each mooring device includes active means for moving the attachment element vertically and in the horizontal plane, and the method involves repositioning the attachment elements in a stepwise manner.",
"The mooring devices also includes a seal for a vaccum attachment element."
],
[
"1-19.",
"(canceled) 20.A method of operating a mooring system, the mooring system comprising at least a first and a second mooring robot, each mooring robot having a robot arm with an attachment element for releasably fastening to a surface, the first and second robots each having first and second attachment elements respectively, wherein the operating method incorporates co-ordinated stepwise movements to re-position each attachment element between respective spaced apart starting and finishing positions, in which positions the attachment elements are fastened to the surface, the method comprising the steps: (a) firstly, while maintaining the second attachment element in its respective starting position, releasing the first attachment element from the surface; (b) secondly, while maintaining the second attachment element in its respective starting position, actuating the first mooring robot to move the first attachment element and re-fasten the first attachment element in its respective finishing position; (c) thirdly, while maintaining the first attachment element in its respective finishing position, releasing the second attachment element from the surface; and (d) fourthly, while maintaining the first attachment element in its respective finishing position, actuating the second mooring robot to move the second attachment element and re-fasten the second attachment element in its respective finishing position.",
"21.The method of claim 20, wherein the mooring robots are mounted to a fixed dock.",
"22.The method of claim 20, wherein the mooring robots are mounted to a floating dock.",
"23.The method of claim 20, wherein said surface is part of a freeboard of a ship's hull.",
"24.The method of claim 20, wherein the mooring robots are mounted to a floating vessel.",
"25.The method of claim 20, wherein each mooring robot provides means for at least two-dimensional movement for positioning the attachment element.",
"26.The method of claim 20, wherein each attractive element is pivotally fixed to a mooring robot providing three-dimensional translational movement, the mooring robot allowing external forces to displace the moored object, and the attachment element engaged therewith, by a distance in the horizontal plane from a selected moored position; wherein, separate from its structural components, the mooring robot comprises resilient restorative means which provide a restorative force acting to restore the attachment element to the selected moored position.",
"27.The method of claim 26, wherein the three dimensional translational movement comprises movement of the mooring robot about two substantially perpendicular axes of rotation and translational movement along a translational axis arranged substantially perpendicular to the plane of the two axes of rotation.",
"28.The method of claim 26, wherein each attractive element is pivotally fixed to a mooring robot providing three-dimensional translational movement, the mooring robot allowing external forces to displace the moored object, and the attachment element engaged therewith, by a distance in the horizontal plank from a selected moored position; wherein, separate from its structural components, the mooring robot comprises resilient restorative means which provide a restorative force acting to restore the attachment element to the selected moored position.",
"29.The method of claim 20, wherein the stepwise movement is performed in the vertical direction.",
"30.The method of claim 20, wherein the stepwise movement is performed in the horizontal direction.",
"31.The method of claim 20, wherein in addition to the first and second mooring robots at least two additional mooring robots are employed, the attachment element of each additional mooring robot remaining fastened to the surface throughout the stepwise movement of the first and second robots.",
"32.The method of claim 28, wherein in addition to the first and second mooring robots at least two additional mooring robots are employed, the attachment element of each additional mooring robot remaining fastened to the surface throughout the stepwise movement of the first and second robots.",
"33.The method of claim 20, wherein each attachment element comprises an array of vacuum cups, each vacuum cup having a circumferential seal including a circumferential seal member of substantially constant cross-section, said member being mountable in a support frame rigidly fixed to the attachment element, the seal member being of elastomeric material and comprising: a first sealing face which has an arcuate portion between first and second sealing edges wherein partial deformation of the said first sealing face adjacent the first edge is required before the said second sealing edge contacts the surface.",
"34.The method of claim 26 wherein each attachment element comprises an array of vacuum cups, each vacuum cup having a circumferential seal including a circumferential seal member of substantially constant cross-section, said member being mountable in a support frame rigidly fixed to the attachment element, the seal member being of elastomeric material and comprising: a first sealing face which has an arcuate portion between inner and outer edges wherein partial deformation of the said first sealing face is required before the said inner sealing edge contacts the surface.",
"35.The method of claim 32 wherein each attachment element comprises an array of vacuum cups, each vacuum cup having a circumferential seal a circumferential seal member of substantially constant cross-section, said member being mountable in a support frame rigidly fixed to the attachment element, the seal member being of elastomeric material and comprising: a first sealing face which has an arcuate portion between inner and outer edges wherein partial deformation of the said first sealing face is required before the said inner sealing edge contacts the surface."
],
[
"<SOH> BACKGROUND ART <EOH>One disadvantage of traditional mooring is the necessity to constantly adjust the mooring lines, particularly when a ship is secured to a fixed dock.",
"This adjustment is to account for movement of the ship in response to winds, shifting tides, the addition or removal of cargo, and the like.",
"The combination of high tidal movements and variations in ship displacement due to loading can result in a considerable vertical movement having to be accommodated by the mooring system.",
"With a mooring device such as that described in the co-pending application based upon New Zealand Patent application No.",
"501395 (which specification is incorporated herein by reference), a vacuum attachment cup assembly is fixed to the ship's hull.",
"Mechanical means limits movement of mooring robot up and down over the full extent of the relative vertical travel.",
"This possible movement necessitates a larger working area, with consequent complication and increased cost.",
"Japanese patent abstract publication no.",
"58206478 describes a mooring device and a method of changing the position of a vacuum cup fastening the device to the hull.",
"When the device reaches the limits of its vertical travel the negative pressure in the vacuum cup is raised to a degree permitting the cup to slide without releasing from the hull.",
"At its limits of travel this passive method therefore offers greatly reduced mooring forces, making the moored vessel vulnerable to failure of the mooring in adverse conditions of weather and current.",
"The seal of the vacuum cup also suffers from abrasion when the cup slides down the hull in this manner and so to avoid regular sliding movement during operation the mooring device is provided with increased mechanical travel in the vertical direction, with consequent added complication and expense.",
"It is an object of the present invention to provide a mooring system and method of operating a mooring system for accommodating a large relative vertical movement of a ship when docked.",
"It is a further objective of the present invention to provide a mooring system and method and system for accommodating a large relative vertical movement of a ship when docked which overcomes the problems of the prior art.",
"A still further object of the present invention is the provision of a seal for use in an attachment element for use on a mooring robot.",
"It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.",
"Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: FIG.",
"1 is a plan view of a pair of mooring robots, being a first preferred arrangement for performing the stepwise movement method according to the present invention; FIG.",
"2 is a front elevation illustrating the vertical travel of the vacuum cups of the mooring robots according to FIG.",
"1 ; FIG.",
"3 is front elevation of the vacuum cups of FIG.",
"2 at an intermediate stage in the stepping movement of the present invention; FIG.",
"4 is a sectional view of a vacuum cup provided with a seal according to the present invention in a released position, and FIG.",
"5 is a sectional view of a vacuum cup provided with a seal according to the present invention fully engaged with a hull surface.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to mooring devices for mooring vessels and, more particularly to a method and mooring system for accommodating large relative movements between two objects moored or secured together.",
"BACKGROUND ART One disadvantage of traditional mooring is the necessity to constantly adjust the mooring lines, particularly when a ship is secured to a fixed dock.",
"This adjustment is to account for movement of the ship in response to winds, shifting tides, the addition or removal of cargo, and the like.",
"The combination of high tidal movements and variations in ship displacement due to loading can result in a considerable vertical movement having to be accommodated by the mooring system.",
"With a mooring device such as that described in the co-pending application based upon New Zealand Patent application No.",
"501395 (which specification is incorporated herein by reference), a vacuum attachment cup assembly is fixed to the ship's hull.",
"Mechanical means limits movement of mooring robot up and down over the full extent of the relative vertical travel.",
"This possible movement necessitates a larger working area, with consequent complication and increased cost.",
"Japanese patent abstract publication no.",
"58206478 describes a mooring device and a method of changing the position of a vacuum cup fastening the device to the hull.",
"When the device reaches the limits of its vertical travel the negative pressure in the vacuum cup is raised to a degree permitting the cup to slide without releasing from the hull.",
"At its limits of travel this passive method therefore offers greatly reduced mooring forces, making the moored vessel vulnerable to failure of the mooring in adverse conditions of weather and current.",
"The seal of the vacuum cup also suffers from abrasion when the cup slides down the hull in this manner and so to avoid regular sliding movement during operation the mooring device is provided with increased mechanical travel in the vertical direction, with consequent added complication and expense.",
"It is an object of the present invention to provide a mooring system and method of operating a mooring system for accommodating a large relative vertical movement of a ship when docked.",
"It is a further objective of the present invention to provide a mooring system and method and system for accommodating a large relative vertical movement of a ship when docked which overcomes the problems of the prior art.",
"A still further object of the present invention is the provision of a seal for use in an attachment element for use on a mooring robot.",
"It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.",
"Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.",
"DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided a seal for a vacuum attachment element, which element can be secured against a surface, said seal comprising a circumferential seal member of substantially constant cross-section, said member being mountable in a support frame rigidly fixed to the attachment element, the seal member being of elastomeric material and including: a first sealing face which has an arcuate portion between inner and outer edges, wherein partial deformation of the said first sealing face is required before the said inner sealing edge contacts the surface.",
"According to another aspect of the present invention there is provided a seal for an attachment element substantially as described above, wherein the attachment element is part of a mooring robot.",
"According to another aspect of the present invention there is provided a seal for an attachment element substantially as described above, wherein the mooring robot releasably fastens to the surface, being a surface of a first moveable object, the mooring robot being mountable to a second object, said first object moving in response to the application of external forces, relative to the second object, which movement moves the first object from a pre-determined operating position, of the type as described in the co-pending application based upon New Zealand Patent application No.",
"501395.According to a still further aspect of the present invention there is provided a method of operating a mooring system, which system includes at least a first and second mooring robot, each mooring robot having a robot arm with at least one attachment element for releasable engagement with a surface, wherein the operating method involves stepwise movements to re-position the attachment elements between respective starting and a finishing positions in which positions all attachment elements are fastened to the surface, the method including the steps: (a) with respect the first mooring robot, releasing all respective first attachment elements from engagement with the surface; (b) moving all said first attachment elements, by operation of the first mooring robot, and re-fastening said elements in the respective finishing position on the surface; (c) with respect to the second mooring robot, releasing all respective second attachment elements from engagement with the surface; and (d) moving all said second attachment elements, by operation of the second mooring robot, and re-fastening the said elements in the respective finishing position on the surface.",
"According to a still further aspect of the present invention there is provided a method of operating a mooring system, substantially as described above, including the steps: limits of vertical travel before the stepwise movement is initiated.",
"Alternatively, the stepwise movement may be performed in the horizontal direction for providing movement of the ship in the fore-and-aft direction.",
"According to a still further aspect of the present invention there is provided a method of operating a mooring system, substantially as described above, wherein the method is performed with a mooring system which includes mooring robots as described in New Zealand Patent application No.",
"501395.Four mooring robots, in first and second pairs are employed, the first pair performing the stepwise movement while the second pair remains fastened to the ship.",
"Alternatively both the first and second pairs may perform the stepwise movement together.",
"According to a still further aspect of the present invention there is provided a method of operating a mooring system, substantially as described above, wherein the attachment element is an array of vacuum cups, each vacuum cup having a seal as described according to the first aspect above.",
"It will be appreciated that one of the cups of the each mooring robot is sufficient to hold that portion of the ship moored, during the operation of the above described method.",
"Thus very large vertical movements of a vessel can be accommodated, without the need to re-moor a vessel and without risking the security of the mooring system.",
"BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: FIG.",
"1 is a plan view of a pair of mooring robots, being a first preferred arrangement for performing the stepwise movement method according to the present invention; FIG.",
"2 is a front elevation illustrating the vertical travel of the vacuum cups of the mooring robots according to FIG.",
"1; FIG.",
"3 is front elevation of the vacuum cups of FIG.",
"2 at an intermediate stage in the stepping movement of the present invention; FIG.",
"4 is a sectional view of a vacuum cup provided with a seal according to the present invention in a released position, and FIG.",
"5 is a sectional view of a vacuum cup provided with a seal according to the present invention fully engaged with a hull surface.",
"BEST MODES FOR CARRYING OUT THE INVENTION Referring to FIG.",
"1 of the drawings, a device for performing the method of the present invention comprises the first preferred embodiment of a mooring system 500, as described in the co-pending PCT application based upon New Zealand Patent application No.",
"501395 is illustrated in plan view.",
"The description of the mooring robot and mooring system in the co-pending application is hereby incorporated by reference.",
"Other preferred embodiments (not illustrated) include a mooring system 500 wherein mooring robots 100 are fixed to the ship S allowing the ship S to be readily fastened to a bearing plate fixed to the dock 50 or to another ship S. It will be appreciated, however, that this as well as other robot type mooring devices may be employed for performing the method of the present invention.",
"In the following description 100a, 100b have been used to refer to two specific examples of the mooring robot 100.FIG.",
"1 shows a first mooring robot 100a and a second mooring robot 100b fixed to the dock 50 for mooring a ship S. The mooring system 500 includes at least two pairs of mooring robots 100a, 100b at spaced positions along a mooring face of the dock 50.Each mooring robot 100 has two separate vacuum cups 1 pivotally fixed to a robot arm 10 and permitting accurate positional control of the vacuum cups 1 in three dimensions.",
"The method of operating the mooring system 500 providing a stepwise movement is described below with reference to FIG.",
"2.To accommodate a ship S falling or rising relative to the dock 50 (FIG.",
"1), the vacuum attachment cups 1 fixed to the hull are raised or lowered respectively.",
"It will be appreciated, however, that the same stepwise movement method applies to other relative movement such as moving the vacuum attachment cups 1 from side-to-side in the longitudinal direction, so the following description should not be seen a limiting.",
"Before mooring the ship S, each vacuum attachment cup 1 is initially free (FIGS l and 4).",
"From initial engagement each cup 1 moves through partial engagement (not shown) to complete engagement (FIG.",
"5) wherein both the seal 60 and the abutment member 61 are fully compressed.",
"Referring to FIGS.",
"2 & 3, the vacuum attachment cups 1 of both mooring robots (100a, 100b) are fixed to the hull at approximately the same height H2 and the mooring robots (100a, 100b) are able to accommodate a limited degree of vertical travel either side of height H2, between an upper limit of travel at height H1 and a lower limit of travel at H3.The heights H1, H2, H3 are absolute heights relative to the fixed dock 50.When the controls (not shown) of the mooring system 500 detects a requirement to raise the mooring robots 100, due to a mooring robot (100a, 100b) approaching the limit of its downward travel H3 (through either a falling tide or the addition of cargo) the stepwise movement of the vacuum attachment cups 1 is then initiated.",
"FIG.",
"3 shows an intermediate stage during the process of raising the vacuum cups 1 from height H3 to height H4.The vacuum cups 1 of the first mooring robot 100a have been released and the vacuum cups 1 raised to height H4.Before moving the vacuum attachment cups 1 they are completely released from engagement with the hull (to a position as shown in FIG.",
"4) thereby allowing the movement to be completed more quickly, as is desired.",
"Next the vacuum cups 1 of the first mooring robot 100a are fully engaged (FIG.",
"5).",
"On indication of complete engagement, the second mooring robot 100b is also raised to height H4 in the same manner.",
"A first preferred embodiment of a seal 60 according to the present invention is shown in FIG.",
"6.The seal 60 provides a continuous seal around the circumference of each vacuum cup 1, to which it is rigidly fixed.",
"The seal 60 is made from elastomeric material, preferably neoprene.",
"It includes a first arcuate sealing face 62 between an inner sealing edge 63 and on outer sealing edge 61.The seal 60 is optionally used to form the perimeter of each vacuum cup 1 used in the method of the present invention.",
"However, it will be appreciated by those skilled in the art that other seals may also be used without departing from the scope of the inventive method.",
"This configuration of the seal 60 allows it to absorb irregularities in the surface to which cup 1 is attached.",
"During engagement of the seal 60, an initial seal is attained with partial deformation of the outer sealing edge 61 at the partial engagement stage (nor shown) before the inner sealing edge 61 contacts the hull of the ship S. With this seal 60 there has been found to be a predictable relationship between the amount of deformation at the partial engagement stage and the vacuum applied to the vacuum cups 1.In the partial engagement stage the arcuate face 62 is readily adapted for sliding engagement with the hull of a ship S or another surface.",
"The above method of operating a mooring system has been described with reference to vessel moored to a dock, which may be either fixed or floating.",
"However, it will be appreciated that the dock may be replaced by a vessel (so that there is vessel to vessel docking and relative movement).",
"Also, it will be appreciated that the mooring system, described herein as affixed to the dock, may be fixed to the vessel.",
"The operation is the same except that the surface is a surface affixed to the dock.",
"Also, the above method of operating a mooring system has been described with reference to vessel moored to a dock.",
"It will, however, be appreciated that another type of vessel or object may be moved relative to a second object, for example under water, etc without departing from the scope of the invention.",
"Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof."
]
] | Patent_10220010 |
[
[
"Method for reversing the immunosuppressive effects of the melanoma inhibitory activity \"mia\"",
"A method for stimulating immune cells and/or the immune system, and/or reducing invasion and/or metastasis of tumor cells by inhibiting expression and/or functional activity of “Melanoma Inhibitory Activity” MIA."
],
[
"1.A method for stimulating immune cells and/or the immune system, and/or reducing invasion and/or metastasis of tumor cells by inhibiting expression and/or functional activity of “Melanoma Inhibitory Activity” MIA.",
"2.The method according to claim 1, wherein the inhibition of the expression and/or functional activity of MIA is achieved by using at least one nucleic acid molecule or derivative thereof, 3.The method according to claim 2 wherein the at least one nucleic acid molecule is an oligonucleotide, an antisense nucleic acid and/or a ribozyme.",
"4.The method according to claim 1, wherein the inhibition of the synthesis and/or function of MIA is achieved using a molecule comprising the antisense sequences SEQ ID No.",
"1 to 3 or SEQ ID No.",
"10 to 39 or parts of the sequences having at least 8 nucleotides.",
"5.The method according to claim 3 wherein the antisense and/or ribozyme molecule is derived by synthesising a sequence wholly or partially complementary to MIA mRNA and testing for inhibitory activity of MIA.",
"6.The method according to claim 3 wherein the antisense and/or ribozyme molecule is integrated into a DNA delivery system comprising viral and/or non-viral vectors together with lipids selected from the group of anionic lipids, cationic lipids, non-cationic lipids and mixtures thereof.",
"7.The method according to claim 3 wherein the antisense and/or ribozyme molecule is modified at one or more of the sugar moieties, the bases and/or the internucleotide linkages and/or by coupling the antisense and/or ribozyme molecule to an enhancer of uptake and/or inhibitory activity.",
"8.The method according to claim 1 wherein the inhibition of the expression and/or functional activity of MIA is achieved using peptides and/or proteins.",
"9.The method of claim 8 wherein the peptides and/or proteins comprise the sequences SEQ ID No.",
"40 to 63 and analogs or derivatives thereof.",
"10.The method according to claim 8 wherein the peptide and/or protein is derived by screening an expression library and testing the expression products for inhibitory activity of MIA.",
"11.The method according to claim 8 wherein the peptide and/or protein is derived by screening randomly synthesised peptides and/or proteins for inhibitory activity of MIA.",
"12.The method according to claim 1, wherein the inhibition of the expression and/or the function activity of MIA is achieved using an inhibitor of low molecular weight.",
"13.The method of claim 12 wherein the inhibitor of low molecular weight is selected from compounds having any on of the structures 1 to 492 of FIG.",
"1 to 42 or comprise any of these structures as substructures, or parts of the structures 1 to 492 comprising at least an aromatic system and an amid bond.",
"14.The method according to claim 12 wherein the inhibitor of low molecular weight is obtainable by combinatorial chemistry and testing the products for inhibitory activity of MIA.",
"15.The method according to claim 1, wherein the inhibition the expression and/or functional activity of MIA is achieved using DNA or RNA derivatives including aptamers and/or spiegelmers that bind to MIA.",
"16.The method according to claim 1 wherein the inhibition of MIA is achieved using antibodies or antibody fragments, such as Fab-fragments, single chain antibody or combinations thereof.",
"17.The method according to claim 16 wherein the antibody or antibody fragments, such as Fab-fragments, single chain antibody or combinations thereof are obtainable by screening antibody libraries and testing the expression products for inhibitory activity of MIA.",
"18.The method according to any of the claims 1 to 17 wherein additionally an immunostimulatory agent, such as cytokines and/or inhibitors of the expression and/or function of interleukin-10 and/or transforming growth factor beta (TGF-β) and/or Prostaglandin B2 and/or receptors for Prostaglandin E2 and/or inhibitors of VEGF.",
"19.A composition comprising a molecule or a combination of molecules for inhibiting the synthesis and/or function of MIA 20.A composition according to claim 19 wherein the molecule is selected from the oligonucleotides having any one of the SEQ.",
"ID.",
"No.",
"1 to 3 and SEQ ID No.",
"10 to 39 or parts of these sequences having at least 8 nucleotides.",
"21.A medicament comprising an inhibitor of the synthesis and/or function of MIA.",
"22.A medicament comprising an inhibitor of the synthesis and/or function of MIA combined with an immunostimulatory agent.",
"23.The use of the composition according to claim 19 for the preparation of a medicament for the prevention or the treatment of neoplasms, infections and/or immunosuppressive disorders."
],
[
"The polypeptide “Melanoma Inhibitory Activity”, MIA, was discovered in 1989 as a factor that inhibits growth of melanoma tumor cells.",
"The antiproliferative action of MIA was also demonstrated in other tumor cells and Peripheral Blood Mononuclear Cells.",
"Thus, CANCER RES.",
"49; 5358-63, Bogdahn et al, (1989) demonstrated a very strong tumor cell growth-inhibiting effect of a factor called melanoma inhibitory activity (MIA).",
"Three active fraction pools, named MIA-I, MIA-II and MIA-III were identified.",
"Tumor stem cell colony formation was reduced by an astonishing 99.890/0 e.g.",
"by MIA-II.",
"CANCER RES 50; 6981-86, Weilbach et al.",
"(1990) further demonstrated that MIA inhibits cell proliferation by prolonging of the S-Phase and arrest of the cells in the G2 compartment.",
"PROC.AACR 40; 79, Jachimczak et al.",
"(1999) further extended these observations to Peripheral Blood Mononuclear Cells (PBMCs), although to only a slight degree: “IL-2-stimulated PBMC proliferation has been only slightly inhibited by addition of MIA”.",
"CANCER RES.",
"54; 5695-5701, Blesch et al.",
"(1994) identified MIA as a 131-amino acid precursor, processed into a mature 107-amino acid protein.",
"This publication confirmed that MIA acts as a potent tumor cell growth inhibitor for malignant melanoma cell and further extended this observation to other neuroectodermal tumors and concluded that “ .",
".",
".",
"MIA .",
".",
".",
"might be attractive as a future antitumor therapeutical substance.” Controversial data were obtained regarding correlation of MIA expression with melanoma progression.",
"CANCER RES.",
"57; 3149-53, Bosserhoff et al.",
"(1997) and ANTICANCER RES.",
"19; 2691-3, Bosserhoff et al.",
"(1997) found enhanced MIA levels in 13-23% of stage I and II melanomas, but in 100% of stage III or stage IV disease.",
"In contrast, CANCER RES.",
"55; 6237-43, van Groningen et al.",
"(1995) found MIA mRNA expression in non metastasising cell lines and an inverse correlation of MIA mRNA expression with pigmentation in melanoma metastasis lesions, but notably expression was found to be absent in highly metastasising cell lines.",
"Furthermore, CLIN.",
"CANCER RES.",
"5; 1099-105, Muhlbauer et al.",
"(1997) concluded that “ .",
".",
".",
"MIA amplification seems to be of little value as a surrogate marker for clinical staging or the detection of metastatic disease.” Surprisingly while the above literature suggested that MIA had the potential as a therapeutic agent to treat melanoma patients it was now found that in contrast MIA is a potent immunosuppressive factor and that agents inhibiting expression and/or function of MIA have therapeutic potential for treatment of neoplasms and immunosuppression.",
"The present invention therefore pertains to a method for stimulating immune cells and/or the immune system, and/or reducing invasion and/or metastasis of tumor cells by inhibiting expression and/or functional activity of “Melanoma Inhibitory Activity” MIA.",
"According to the invention the stimulation of the immune system is preferably achieved by inhibiting expression and/or functional activity of “Melanoma Inhibitory Activity” MIA in combination with enhancing expression in target cells and/or target pathogens of the molecules listed under a) to m); alternatively by vaccination with DNA and/or RNA coding for all or part of the molecules listed under a) to m) and/or polypeptides contained in the molecules listed under a) to m); by transfection of an organism and/or transfecting the target cells and/or target pathogens with genes coding for the molecules listed under a) to m); by applying to an organism and/or to the target cells and/or target pathogens the molecules listed under a) to m); and/or by enhancing the synthesis and/or function of molecules stimulating and/or enhancing and/or upregulating and/or positively regulating the immune response with molecules including the molecules listed under a) to m), wherein a) represents molecules selected from the group comprising chemokines, including lymphotactin and/or immune cell attracting factors; b) represents elements selected from the group comprising viruses and/or parts of viruses, including adeno viruses, papilloma viruses, Epstein-barr-Viruses, viruses that are non-pathogenic including Newcastle-Disease virus, Cow-pox-virus; c) represents molecules selected from the group comprising autologous and/or heterologous MHC-molecules; d) represents molecules selected from the group comprising molecules involved in antigen processing; e) represents molecules selected from the group comprising molecules involved in antigen presentation; f) represents molecules selected from the group comprising molecules involved in mediating immune-cell effects; g) represents molecules selected from the group comprising molecules involved in mediating immune cell cytotoxic effects; h) represents moledules selected from the group comprising molecules involved in antigen transportation; i) represents molecules selected from the group, comprising co-stimulatory molecules; j) represents molecules selected from the group comprising peptides enhancing recognition by immune cell and/or cytotoxic effects of immune cells; k) represents molecules selected from the group comprising peptides containing one or more amino acids differing between a protein in the target cell from the other cells within an organism including, but not limited to antigens, specific for melanoma cells and/or melanocytes and/or breast cells and/or breast cancer cells; according to the invention the inhibition of the syntheses and/or function of MIA is achieved by using molecule of group 1) wherein l) represents molecules selected from the group comprising the peptides according to j) being peptides containing one or more mutations and/or amino acid substitutions of the ras protein amino, the p53 protein, the EGF receptor protein, fusion peptides and/or fusion proteins, the retinoblastoma protein, peptides containing one or more mutations and/or amino acid substitutions and/or amino acid substitutions caused by gene rearrangements and/or gene translocations, peptides containing one or more mutations and/or amino acid substitutions of proteins coded by oncogenes and/or protooncogenes, proteins coded by anti-oncogenes and/or tumor suppressor genes; peptides derived from proteins differing in the target cell by one or amino acids from the proteins expressed by other cells in tile same organism, peptides derived from viral antigens and/or coded by viral nucleic acids, peptides derived from proteins over expressed in the target cell compared to a normal cell and combinations thereof m) tumor cell extracts and/or tumor cell lysates and/or adjuvants.",
"In a preferred embodiment of the invention the inhibition of the expression and/or functional activity of MIA is achieved by using at least one nucleic acid molecule, peptide, protein or low molecular weight substance.",
"Preferably, the nucleic acid molecule is an oligo- or polynucleotide molecule, in particular an antisense molecule and/or ribozyme.",
"Methods for preparing effective antisense oligonucleotides are known to those skilled in the art.",
"A preferred method is disclosed in WO 99/63975, incorporated by reference.",
"The inhibition of the synthesis and/or function of MIA is preferably achieved by using at molecules comprising the following antisense sequences: MIA-2841-W GTC AGG AAT CGG GAG (Seq.",
"ID No 1) MIA-1278-W CTT GGA GAA GAG ATA C (Seq.",
"ID No 2) MIA-2842-W TGC CTC CCC AGA AG (Seq.",
"ID No 3) as well as the following sequences (Seq.",
"ID No 10-39): AGCCATGGAGATAG CAGCCATGGAGATAG ACAGCCATGGAGATAG CACAGCCATGGAGATAG CCACAGCCATGGAGAT GCCATGGAGATAGG AGCCATGGAGATAGG CAGCCATGGAGATAGG ACAGCCATGGAGATAGG CATGGAGATAGGGT CATGGAGATAGGGTG CATGGAGATAGGGTGG ATGGAGATAGGGTG ATGGAGATAGGGTGG ATGGAGATAGGGTGGC ATGGAGATAGGGTGGCT GGAGATAGGGTGGC GGAGATAGGGTGGCT GAAATAGCCCAGGC GAAATAGCCCAGGCG GAAATAGCCCAGGCGAG GGAAATAGCCCAGG GGAAATAGCCCAGGC GTCTTCACATCGAC GTCTTCACATCGACT GTCTTCACATCGACTT GTCTTCACATCGACTTT GTCTTCACATCGACTTTG GTCTTCACATCGACTTTG CCATTTGTCTGTCTTCAC or parts of the sequences having at least 8 nucleotides.",
"Methods for synthesizing further antisense oligonucleotides are for example disclosed in WO 98/33904 of the same applicant.",
"Preferably, the antisense and/or ribozyme molecule is derived by synthesising a sequence wholly or partially complementary to MIA mRNA and testing for inhibitory activity of MIA.",
"According to the invention the antisense and/or ribozyme molecule is for example integrated into a DNA delivery system, comprising viral and/or non-viral vectors together with lipids selected from the group of anionic lipids, cationic lipids, non-cationic lipids and mixtures thereof.",
"In a preferred embodiment of the invention the nucleic acid molecules contain flanking sequences and/or vector sequences and/or sequences enhancing the expression and/or transfection of the nucleic acid molecules.",
"In a further preferred embodiment of the invention the nucleic acid molecules are part of one or more vectors and/or viral sequences and/or viral vectors.",
"According to the invention it is preferred that the antisense and/or ribozyme molecule is modified at one or more of the sugar moieties, the bases and/or the internucleotide linkages as well as the phosphate moieties.",
"For example, the modification of the oligonuclecotides, ribozymes and/or nucleic acids comprises modifications such as phosphorothioate (S-ODN) internucleotide linkages, methylphosphonate internucleotide linkages, phosphoramidate linkages, peptide linkages, 2′-O-alkyl modifications of the sugar, in particular methyl, ethyl, propyl, butyl and the like, 2′-methoxyethoxy modifications of the sugar and/or modifications of the bases.",
"The various modifications may be combined in an oligo- or polynucleotid.",
"In a further preferred embodiment of the invention the oligonucleotides, ribozymes and/or nucleic acids are coupled to or mixed with folic acid, hormones, steroid hormones such as oestrogene, progesterone, corticosteroids, mineral corticoids, peptides, proteoglycans, glycolipids, phospholipids and derivatives therof.",
"Inhibition of the expression and/or functional activity of MIA can also be achieved using peptides or proteins.",
"The peptide and/or protein that can be used in the method of the invention can be obtained by screening an expression library and testing the expression products for inhibiting expression and/or functional activity of MIA.",
"Alternatively, a synthetic peptide and/or protein can be obtained by screening randomly synthesised peptides and/or polypeptides for inhibiting expression and/or functional activity of MIA.",
"Suitable peptides binding to MIA are for example the following peptides (SEQ ID No.",
"40-63): VPHIPPN MPPTQVS QMHPWPP QPPFWQF TPPQGLA IPPYNTL AVRPAPL GAKPHPQ QQLSPLP GPPPSPV LPLTPLP QLNVNHQARADQ TSASTRPELHYP TFLPHQMHPWPP VPHIPPNSMALT RLTLLVLIMPAP RKLPPRPRR VLASQIATTPSP TPLTKLPSVNHP PPNSFSSAGGQRT EQDSRQGQELTKKGL ETTIVITWTPAPR TSLLISWDAPAVT NSLLVSWQPPRAR and proteins or peptides comprising the forgoing peptides and analogs or derivatives of these peptides.",
"EMBO J.",
"20; 340-349, Stoll et al.",
"(2001) disclose the three-dimensional structure of human MIA, thus allowing the computational construction and synthesis of specific peptides binding to MIA.",
"In a further embodiment of the invention, the inhibition of expression and/or functional activity of MIA as well as of the expression of the MIA gene and/or MIA mRNA is achieved by using an inhibitor of low molecular weight which can for example be obtained by combinatorial chemistry and testing the products for inhibiting expression and/or functional activity of MIA [Fernandes, P. B. in Curr.",
"Opin.",
"Chem.",
"Biol.",
"1998; 2 (5): 597-803 Technological advances in high-throughput screening].",
"Low molecular weight molecules (small molecules) as used herein are molecules having up to 100 carbon atoms in combination with further atoms such as N, S, O, P and the like.",
"Suitable small molecules can also be identified using computational methods.",
"Methods for computational construction are for example disclosed in Murcko, M. A. , Caron, P. R., Charifson, P. S. (1999), Structure-based drug design, Annual Reports in Medicinal Chemistry, vol.",
"34, Academic Press, San Diego, 1999.Suitable compounds are for example structures 1 to 492 identified in FIGS.",
"1 to 42.Also structures, which comprise structures 1 to 492 as substructures are useful in the present invention.",
"Also parts and/or substructures of the structures 1 to 492 are useful in the present invention, as long as they comprise at least an aromatic system and an amid-bond.",
"In a further embodiment of the invention, the inhibition of expression and/or functional activity MIA is achieved by using DNA or RNA derivatives including aptamers and/or spiegelmers that bind to MIA.",
"Inhibition of expression and/or functional activity of MIA is can also be achieved by using antibodies or antibody fragments, such as Fab-fragments, single chain antibodies or combinations thereof.",
"These molecules can be identified and obtained by screening antibody libraries and testing the expression products for inhibiting expression and/or functional activity of MIA.",
"Any of the foregoing elements, molecules or substances can be combined with an immunostimulatory agent, for example cytokines and/or inhibitors of the expression and/or function of interleukin-10 and/or transforming growth factor beta (TGF-β) and/or Prostaglandin B2 and/or receptors for Prostaglandin E2 and/or inhibitors of VEGF.",
"The present invention is also concerned with a composition for the manufacturing of a medicament comprising a molecule or a combination of molecules which is able to inhibit the expression and/or functional activity of MIA.",
"The resulting medicament comprising an inhibitor of the expression and/or functional activity of MIA is also subject of the present invention.",
"The medicament of the invention may be combined with an immunostimulatory agent.",
"Any of the foregoing elements, molecules or substances can be employed for the preparation of a medicament for the prevention or the treatment of neoplasms, infections and/or immunosuppressive disorders.",
"Preferably, both, the inhibitor of MIA expression and/or functional activity is applied locally to a tumor or other pathologically affected site or organ and may also is applied systemically (e.g.",
"i.v.",
"or s.c. or orally).",
"The present invention is also related with the use of a method for stimulating the immune system by inhibiting expression and/or functional activity of “Melanoma Inhibitory Activity” (MIA) in combination with the use of methods and/or molecules enhancing the immune response against diseased cells or pathogens, methods and/or molecules enhancing immunogenicity of target cells and/or target pathogens and/or immunostimulatory molecules, comprising cytokines including interleukins, including IL-1, IL-2, IL-4, IL-12, IL-18, such cytokines being applied systemically to an organism including man or being applied locally e.g.",
"to certain regions or organs or parts of organ or compartments of a body and/or enhancing expression of cytokines in target cells or pathogens by stimulating their expression and/or by transfecting expression Systems into the target cell or target pathogen, capable of expressing these cytokines and/or chemokines attracting immune cells including lymphotactin, such chemokines being applied systemically to an organism including man or being applied locally e.g.",
"to certain regions or organs or parts of organ or compartments of a body and/or enhancing expression of chemokines in target cells or pathogens by stimulating their expression and/or by transfecting expression systems into die target cell or target pathogen, capable of expressing these chemokines and/or peptides and/or DNA and/or RNA molecules and or other antigens that are found in tumor cells and/or pathogens, but not in normal cells and/or enhancing expression of peptides and/or antigens that are found in tumor cells and/or pathogens, but not in normal cells and/or tumor cell extracts and/or tumor cell lysates and/or adjuvants.",
"The method of the present invention is especially useful for the treatment of 1.Solid tumors, e.g.",
"cancer of the skin (including melanoma), head and neck cancer, sarcoma (including osteosarcoma and chondrosarcoma), retinoblastoma, breast cancer, ovarian cancer, small-cell bronchogenic/lung carcinoma, non-small-cell bronchogenic/lung carcinoma, esophageal cancer, colon carcinoma, colorectal carcinoma, gastric cancer, small intestine carcinoma, liver carcinoma, carcinoma of the kidney, pancreas carcinoma, gallbladder cancer, cervical carcinoma, endometrial cancer, mesothelioma, prostate carcinoma, testicular carcinoma, brain tumor 2.Leukemia, e.g.",
"myeloid leukemia (acute and chronic), acute lymphoblastic leukemia (ALL), Non-Hodgkin Lymphoma, Hodgkin-Lymphoma 3.Degenerative disorders, e.g.",
"arthritis, degeneration/injury of cartilage and bone 4.Immunosuppressive diseases e.g.",
"HIV infection, myelosuppressive diseases, ataxia-telangiectasia, DiGeorge syndrome, Bruton disease, congenital agammaglobulinemia, combined immunodeficiency disease, Wiscott-Aldrich syndrome, complement deficiencies, leukopenia.",
"EXAMPLES Example 1 Allogenic anti-glioma LAK immune response was strongly inhibited by exogenous addition of MIA.",
"To study the effects of MIA upon cytotoxic T-lymphocytes (CTL) and Lymphokine Activated Killer cells (LAK cells) a CARE-LASS assay has been employed (Lichtenfels, R., Biddison, W. E., Schulz, H., Vogt, A.",
"B. and R. Martin.",
"CARE-LASS (calcein-release assay), an improved fluorescence based test system to measure cytotoxic lymphocyte activity J. Immunol.",
"Meth., 172: 227-239, 1994).",
"Briefly, glioma cells were harvested, washed in 5% FCS/PBS solution and resuspended at 10 Mio cells/ml in 5% FCS/FBS.",
"Calcein-AM was added to a final concentration of 25 μM (Molecular Probes, USA).",
"The cells were labeled for 30 min at 37° C. then washed twice in 5%/FCS/PBS, adjusted to 1 Mio cells/ml and loaded into 96-well U-shaped microtiter plates at the final concentration of 0.1 Mio/100 μL/1 well (Nunc, Denmark).",
"To measure cytotoxic activity of effector cells pretreated with MIA (f.c.",
"500 ng/ml), wells were loaded with 100 μL of CTL and LAK cells to produce the desired E:T ratios of 1:10 and 1:100.To measure spontaneous release and total release of calcein, wells were preloaded with 100 μL 5% FCS/PBS or 100 μL lysis buffer (50 nM sodium-borate, 0.1% Triton®×100, pH 9.0) respectively.",
"After incubating the plate for 4 h at 37° C. the supernatants (50 μL) were transferred into new wells and measured using an automated fluorescence scanner (Titertek Fluoroskan II, Germany).",
"The percent cytotoxicity was determined from the following equation: F / CTL assay - F spontaneous release F total lysis - F spontanous release × 100 = % cytotoxicity Results: MIA inhibited autologous and allogenic LAK, cytotoxicity against malignant glioma ceil lines (HTZ-17, -243, -374, -375) up to 40% a compared to controls.",
"Example 2 Furthermore, inhibition of MIA synthesis in MIA-secreting melanoma cells enhanced autologous LAK and CTL activity.",
"To study the effects of MIA upon cytotoxic T-lymphocytes (CTL) and Lymphokine Activated Killer cells (LAK cells) a CARE-LASS assay has been employed as described above in Example 1.Briefly, melanoma cells were harvested, washed in 5% FCS/PBS solution and resuspended at 10 Mio cells/ml in 5% PCS/PBS.",
"Calcein-AM was added to a final concentration of 25 μM (Molecular Probes, USA).",
"The cells were labeled for 30 min at 37° C., then washed twice in 5% FCS/PBS, adjusted to 1 Mio cells/ml and loaded into 96-well U-shaped microtiter plates at the final concentration of 0.1 Mio/100 μl/1 well (Nunc, Denmark).",
"To measure cytotoxic activity of effector cells pretreated with MIA-antisense oligonucleotides (f.c.",
"1-5 μM), wells were loaded with 100 μl of CTL and LAK cells at E T ratios of 1:10 and 1:100.To measure spontaneous release and total release of calcein, wells were preloaded with 100 μl 5% FCS/PBS or 100 μl lysis buffer (50 nM sodium-borate, 0.1% Triton®×100, pH 9.0) respectively.",
"After incubating the plate for 4 h at 37° C. the supernatants (50 μL) were transferred into new wells and measured using an automated fluorescence scanner (Titertek Fluoroskan II, Germany).",
"The cytotoxicity was determined according to the equation described in Example 1.Results Inhibition of endogenous MIA synthesis by specific phosphorothioate antisense oligonucleotides in human, MIA-secreting melanoma cell lines (GI and HW) was enhanced by up to 20% autologous LAK cytotoxicity compared to untreated MIA-producing melanoma cell lines.",
"Active sequences inhibiting MIA expression were MIA-2841-W GTC AGG AAT CGG GAG (Seq.",
"ID No 1) MIA-1278-W CTT GGA GAA GAG ATA C (Seq.",
"ID No 2) MIA-2842-W TGC CTC CCC AGA AG (Seq.",
"ID No 3) Less active or inactive sequences inhibiting MIA expression were MIA-2843-N CAG TGG GAG TAG AAA TC (Seq.",
"ID No 4) MIA-2844-N GGT GAG TGG GAG TAG (Seq.",
"ID No 5) MIA-0202-N ATG GTG AGG AAT CG (Seq.",
"ID No 6) MIA-1277-N GAA TGG TCA GGA ATG G (Seq.",
"ID No 7) MIA-2328-N CAT GGT GGA GTG TG (Seq.",
"ID No 8) Example 3 Furthermore, peptides inhibiting MIA activity in MIA-secreting melanoma cells also enhanced autologous LAK activity by up to 30%.",
"Example 4 Inhibition of endogenous MIA synthesis by specific phosphorothioate antisense oligonucleotides in human, MIA-secreting melanoma as well as breast cancer cell lines strongly reduced their migration activity, as well as increasing their adhesion to matrices, both showing a strong inhibitory effect of MIA inhibitors on tumor invasion and metastasis.",
"Example 5 Inhibition of endogenous MIA synthesis by specific phosphorothioate antisense oligonucleotides in human, MIA-secreting melanoma cell lines (GI and HW) in combination with application of the cytokines IL-12, IL-4, IL-18 and/or antisense oligonucleotides specific for TGF-B increased the autologous LAK cytotoxicity even further compared to inhibition of endogenous MIA synthesis by specific phosphorothioate antisense oligonucleotides alone in MIA-producing tumor cell lines.",
"Example 6 Inhibition of endogenous MIA synthesis by a transfecting vector expressing the antisense sequence (SEQ.",
"ID No 9): GGCAGGGCCAGCGGTAGGCTGAGCTCACTGGCAGTAGAAATCCCATTTGT CTGTCTTCACATCGACTTTGCCAGGTTTCAGGGTCTGGTCCTCTCGGACA ATGCTACTGGGGAAATAGCCCAGGCGAGCAGCCAGATCTCCATAGTAATC TCCCTGAACGCTGCCTCCCCAGAAGAGCCGCCCACGGCCCTTCAGCTTGG AGAAGACATACACCACTTGGCCCCGGTGAATGGTCAGGAATCGGCAGTCG GGGGCCATGTAGTCCTGAAGGGCCACAGCCATGGAGATAGGGTGGCTGCA CTCCTGGTCCGCACACAGCTTCCGGTCAGCCAGCTTGGGCATAGGACCAC CCCTGACACCAGGTCCGGAGAAGGCAGACAGCAAGATGATGACACCAAGG CACACCAGGGACCGGGCCATCGTGGACTGTGAGCAAGAGAGTGAGCAAGG GGGTGCTGG or parts of this sequence in human MIA-secreting melanoma as well as breast cancer cell lines strongly reduced their tumor invasion and metastasis in scid-mice and nude mice.",
"Example 7 Inhibition of tumor invasion and metastasis was increased by a combination of inhibitors of MIA with inhibitors of VEGF or TGF-β."
]
] | Patent_10220265 |
[
[
"Waxes for producing printing inks",
"The invention relates to mixtures for producing printing inks, wherein the mixtures comprise a homopolymerizate or copolymerizate of C2-C18-α-olefins, which are produced by means of metallocene catalysis, and of decomposing waxes, which are prepared from polyolefins of longer chain lengths produced by means of metallocene catalysis, and one or more additional additive."
],
[
"1.A method for preparing a printing ink comprising the step of adding to the printing ink a mixture comprising: a) homopolymer or copolymer of C2-C18 μ-olefins, prepared using metallocene catalysis, and also degradation waxes, prepared from longer-chain polyolefins produced using metallocene catalysis, with one or more further additives selected from the group consisting of b) polytetrafluoroethylene having a molecular weight (Mn) of between 30 000 and 2 000 000 g/mol, c) thermoplastic PTFE having a molecular weight (Mn) of between 500 000 and 10 000 000 g/mol, whose particle size is situated in the range 1-100 μm, d) amide waxes prepared by reacting ammonia or ethylenediamine with saturated and unsaturated fatty acids, e) montan waxes, including acid waxes and ester waxes having a carbon chain length of the carboxylic acid of from C22 to C36, f) natural plant waxes, g) reaction products of sorbitol with saturated and/or unsaturated fatty acids and/or montanic acids, h) synthetic hydrocarbons, i) paraffins and microcrystalline waxes obtained in the course of petroleum refining, j) polar polyolefin waxes prepared by oxidizing ethylene or propylene homopolymer and copolymer waxes or grafting them with maleic anhydride, k) polyamides whose particle size is situated in the range 1-100 μm, l) polyolefins or copolymers thereof of high or low density having molecular weights (Mn) of between 10 000 and 1 000 000 g/mol whose particle size is situated in the range 1-100 μm, m) agents which lower the surface tension of liquids (wetting agents).",
"2.The method as claimed in claim 1, wherein constituent a) is an ethylene homopolymer or copolymer wax.",
"3.The method as claimed in claim 1, wherein constituent a) is a propylene homopolymer or copolymer wax.",
"4.The method as claimed in claim 1, wherein the polyolefin waxes have a molecular weight distribution Mw/Mn<5.5.The method as claimed in claim 1, wherein the polyolefin waxes have a melt viscosity of from 5 to 100 000 mPas.",
"6.The method as claimed in claim 1, wherein the polyolefin waxes have a dropping point of from 70 to 165° C. 7.The method as claimed in claim 1, wherein the fraction of the further additive b) through m) is 1-99% by weight based on the overall mass of the mixture.",
"8.The method as claimed in claim 1, wherein the waxes are used in micronized form.",
"9.A prinking ink prepared in accordance with the method of claim 1.10.A method for preparing a printing ink comprising the step of adding to the printing ink a mixture comprising: a) at least one degradation wax, prepared from longer-chain polyolefins produced using metallocene catalysis, with one or more further additives selected from the group consisting of b) polytetrafluoroethylene having a molecular weight (Mn) of between 30 000 and 2 000 000 g/mol, c) thermoplastic PTFE having a molecular weight (Mn) of between 500 000 and 10 000 000 g/mol, whose particle size is situated in the range 1-100 μm, d) amide waxes prepared by reacting ammonia or ethylenediamine with saturated and unsaturated fatty acids, e) montan waxes, including acid waxes and ester waxes having a carbon chain length of the carboxylic acid of from C22 to C36, f) natural plant waxes, g) reaction products of sorbitol with saturated and/or unsaturated fatty acids and/or montanic acids, h) synthetic hydrocarbons, i) paraffins and microcrystalline waxes obtained in the course of petroleum refining, j) polar polyolefin waxes prepared by oxidizing ethylene or propylene homopolymer and copolymer waxes or grafting them with maleic anhydride, k) polyamides whose particle size is situated in the range 1-100 μm, l) polyolefins or copolymers thereof of high or low density having molecular weights (Mn) of between 10 000 and 1 000 000 g/mol whose particle size is situated in the range 1-100 μm, m) agents which lower the surface tension of liquids (wetting agents).",
"11.A prinking ink made in accordance with the method of claim 1.12.A method for preparing a printing ink comprising the step of adding to the printing ink a mixture comprising: a) at least one homopolymer or copolymer of C2-C18 α-olefins, prepared using metallocene catalysis, with one or more further additives selected from the group consisting of b) polytetrafluoroethylene having a molecular weight (Mn) of between 30 000 and 2 000 000 g/mol, c) thermoplastic PTFE having a molecular weight (Mn) of between 500 000 and 10 000 000 g/mol, whose particle size is situated in the range 1-100 μm, d) amide waxes prepared by reacting ammonia or ethylenediamine with saturated and unsaturated fatty acids, e) montan waxes, including acid waxes and ester waxes having a carbon chain length of the carboxylic acid of from C22 to C36, f) natural plant waxes, g) reaction products of sorbitol with saturated and/or unsaturated fatty acids and/or montanic acids, h) synthetic hydrocarbons, i) paraffins and microcrystalline waxes obtained in the course of petroleum refining, j) polar polyolefin waxes prepared by oxidizing ethylene or propylene homopolymer and copolymer waxes or grafting them with maleic anhydride, k) polyamides whose particle size is situated in the range 1-100 μm, l) polyolefins or copolymers thereof of high or low density having molecular weights (Mn) of between 10 000 and 1 000 000 g/mol whose particle size is situated in the range 1-100 μm, m) agents which lower the surface tension of liquids (wetting agents).",
"13.A printing ink made in accordance with the process of claim 12."
],
[
"The present invention relates to the use of polyolefin waxes prepared by means of metallocene catalysts, in combination with PTFE, amide wases, montan waxes, natural plant waxes, sorbitol esters, synthetic hydrocarbon waxes, microcrystalline and macrocrystalline paraffins, polar polyolefin waxes, polyamides, polyolefins and/or wetting agents, as an additive component in printing inks.",
"The function of waxes in printing inks is to increase the resistance of printed products to abrasion, scuffing and scratching.",
"The waxes are usually used in the form of solvent dispersions or solvent pastes or else in solid micronized form.",
"Micronization is done either by milling on appropriate mills or spraying from the melt, in each case with subsequent classification if desired.",
"The average particle sizes required are generally below 10 μm.",
"To date, waxes from different kinds of preparation processes have been used for this application.",
"Besides the thermal degradation of high molecular mass polyolefins or free-radical polymerization of ethylene at high pressures and temperatures, a common method of preparing waxes is by homopolymerization or copolymerization of ethylene or propylene using Ziegler-Natta catalysts comprising a titanium compound as the catalytically active species, as disclosed, for example, in DE-A-1 520 914.EP-A-0 890 619 discloses how in particular the use of metallocene catalyst systems for the preparation of polyolefin waxes leads to materials which when used inprinting inks bring about improved scuff protection effects.",
"The use of the straight polyolefin waxes prepared by means of metallocene catalysis in printing inks covers the basic requirements in relation to enhanced scuff protection relative to the original ink.",
"Over and above this, however, there are applications which demand specifically improved scuff protection or high surface lubricity or good overprintability, e.g., when printing abrasive, matt-coated papers, or in the packaging printing sector.",
"It has surprisingly now been found that polyolefin waxes, prepared using metallocene catalysts and combined with additives, meet this heightened profile of requirements in a particular way.",
"The present invention provides for the use of mixtures of a) homopolymer or copolymer of C2-C18 α-olefins, prepared using metallocene catalysis, and also degradation waxes, prepared from longer-chain polyolefins produced using metallocene catalysis, with one or more further additives selected from the group consisting of b) polytetrafluoroethylene having a molecular weight (Mn) of between 30 000 and 2 000 000 g/mol, c) thermoplastic PTFE having a molecular weight (Mn) of between 500 000 and 10 000 000 g/mol, whose particle size is situated in the range 1-100 μm, d) amide waxes prepared by reacting ammonia or ethylenediamine with saturated and unsaturated fatty acids, e) montan waxes, including acid waxes and ester waxes having a carbon chain length of the carboxylic acid of from C22 to C36, f) natural plant waxes, g) reaction products of sorbitol with saturated and/or unsaturated fatty acids and/or montanic acids, h) synthetic hydrocarbons, i) paraffins and microcrystalline waxes obtained in the course of petroleum refining, j) polar polyolefin waxes prepared by oxidizing ethylene or propylene homopolymer and copolymer waxes or grafting them with maleic anhydride, k) polyamides whose particle size is situated in the range 1-100 μm, l) polyolefins, such as, for example, polyethylene, polypropylene or copolymers thereof of high or low density having molecular weights (Mn) of between 10 000 and 1 000 000 g/mol whose particle size is situated in the range 1-100 μm, m) agents which in general lower the surface tension of liquids (wetting agents), for preparing printing inks.",
"Suitable polyolefin waxes a) are homopolymers of ethylene or propylene, or copolymers of ethylene with one or more 1-olefins, in particular propylene.",
"1-Olefins used are linear or branched olefins having 2-18 carbon atoms, preferably 3-6 carbon atoms.",
"The 1-olefins may carry an aromatic substitution.",
"Examples of these 1-olefins are ethylene, propylene, 1-butene, 1-hexene, 1-octene and 1-octadecene, and also styrene.",
"Preference is given to homopolymers of ethylene or propylene or copolymers of ethylene with propylene or 1-butene.",
"Where copolymers are used, their ethylene content is preferably 70-99.9% by weight, especially 80-99% by weight.",
"In a further preferred embodiment, the waxes have a molecular weight distribution Mw/Mn<5.Their melt viscosity is preferably between 5 and 100 000 mPas.",
"Particularly preferred polyolefin waxes are those having a dropping point of between 90 and 165° C., in particular between 100 and 160° C., a melt viscosity at 140° C. (polyethylene waxes) or at 170° C. (polypropylene waxes) of between 10 and 10 000 mPas, in particular between 50 and 5 000 mPas, and a density at 20° C. of between 0.85 and 0.96 g/cm3.In preferred embodiments, additive b) comprises polytetrafluoroethylene having a molecular weight of between 100 000 and 1 000 000 g/mol.",
"In preferred embodiments, additive c) comprises thermoplastic polytetrafluoroethylene with particle sizes in the range from 3 to 30 μm.",
"In preferred embodiments, additive d) comprises amide waxes preparable by reacting ammonia or ethylenediamine with stearic acid, tallow fatty acid, palmitic acid or erucic acid.",
"Additive e) comprises montan waxes, including acid waxes and ester waxes having a carbon chain length of the carboxylic acid of from C22 to C36.The ester waxes comprise preferably reaction products of the montanic acids with monohydric or polyhydric alcohols having 2 to 6 carbon atoms, such as ethanediol, 1,3-butanediol or 1,2,3-propanetriol, for example.",
"In one preferred embodiment, additive f) comprises carnauba wax.",
"In preferred embodiments, additive g) comprises reaction products of sorbitol with stearic acid, tallow fatty acid, palmitic acid or erucic acid.",
"In preferred embodiments, additive h) comprises Fischer-Tropsch waxes.",
"Additive i) preferably comprises paraffins with dropping points between 48 and 65° C., and microcrystalline waxes with dropping points between 75 and 95° C. Additive j) comprises polar polyolefin waxes preparable by oxidizing ethylene or propylene homopolymer and copolymer waxes or grafting them with maleic anhydride.",
"For this purpose, particular preference is given to starting from polyolefin waxes having a dropping point of between 90 and 165° C., in particular between 100 and 160° C., a melt viscosity at 140° C. (polyethylene waxes) or at 170° C. (polypropylene waxes) of between 10 and 10 000 mPas, in particular between 50 and 5 000 mPas, and a density at 20° C. of between 0.85 and 0.96 g/cm3.Additive j) preferably comprises polyamide 6, polyamide 6,6, and polyamide 12.In a further preferred embodiment, the particle size of the polyamides is in the range 3-30 μm.",
"Additive k) preferably comprises polyolefins, such as, for example, polyethylene, polypropylene or copolymers thereof of high or low density having molecular weights (Mn) of between 15 000 to 500 000 g/mol.",
"In a further preferred embodiment the particle size is 3-30 μm.",
"Additive m) comprises amphiphilic compounds which in general lower the surface tension of liquids, such as, for example, alkyl ethoxylates, fatty alcohol ethoxylates, alkylbenzenesulfonates or betaines.",
"The mixing ratio of constituent a) to constituents b) to m) may be varied in the range from 1 to 99% by weight a) through to from 1 to 99% by weight b) to m), preferably between 5 and 50%.",
"Where a mixture of two or more of the constituents b) to m) is used, the amount indicated applies to the sum of the amounts of these constituents.",
"In one preferred embodiment, the above-described waxes are used in micronized form for the purpose of the invention.",
"The metallocene catalysts for preparing polyolefin waxes are chiral or nonchiral transition metal compounds of the formula M1Lx.",
"The transition metal compound M1Lx includes at least one central metal atom M1 to which is attached at least one π ligand, e.g., a cyclopentadienyl ligand.",
"Substituents as well, such as halogen, alkyl, alkoxy or aryl groups, may be attached to the central metal atom M1.M1 is preferably an element from main group III, IV, V or VI of the Periodic Table of the Elements, such as Ti, Zr or Hf.",
"By cyclopentadienyl ligands are meant unsubstituted cyclopentadienyl radicals and substituted cyclopentadienyl radicals such as methylcyclopentadienyl, indenyl, 2-methylindenyl, 2-methyl-4-phenylindenyl, tetrahydroindenyl or octahydrofluorenyl radicals.",
"The π ligands may be bridged or unbridged, the possibilities encompassing single and multiple bridges and extending to bridges via ring systems.",
"The term metallocene also embraces compounds containing more than one metallocene fragment, known as polynuclear metallocenes.",
"These metallocenes may have any desired substitution patterns and bridging variants.",
"The individual metallocene fragments of such polynuclear metallocenes may be either identical to or different from one another.",
"Examples of such polynuclear metallocenes are described, for example, in EP-A-0 632 063.Examples of general structural formulae of metallocenes, and of their activation with a cocatalyst, are given, inter alia in EP-A-0 571 882.Hereinbelow, the polyolefin waxes from metallocene catalysis are referred to as component 1, and the additives b) to m) as component 2.The mixtures may be prepared by conjointly milling the two components or by mixing the components in liquid-melt phase beforehand and then spraying or milling the mixture.",
"EXAMPLES TABLE 1 Physical properties of the test polyolefin waxes Melt viscosity Dropping Mn Density Type mPas point ° C Mw/Mn g/mol g/cm3 Wax 1 Metallocene ethylene 350 at 140° C. 124 2.4 990 0.965 homopolymer wax Wax 2 Metallocene propylene 40 at 170° C. 135 2.1 1 870 0.880 homopolymer wax Comparative Ethylene homopolymer 300 at 140° C. 125 2.8 1 500 0.970 sample 1 wax, prepared using Ziegler-Natta catalyst On the basis of wax 1 and wax 2 and also comparative wax 1, the following mixtures were prepared: TABLE 2 Mixtures of polyolefin waxes with additives Mixing ratio Code Type Parts by weight Mixture 1 M1 Wax 1 93 PTFE 7 Mixture 2 M2 Comparative wax 1 93 PTFE 7 Mixture 3 M3 Wax 1 60 Paraflint H2 40 Mixture 4 M4 Comparative wax 1 60 Paraflint H2 40 Mixture 5 M5 Wax 1 50 Sorbitan tristearate 50 Mixture 1 M1 Wax 1 93 PTFE 7 Mixture 6 M6 Comparative wax 1 50 Sorbitan tristearate 50 Mixture 7 M7 Wax 1 50 Sorbitan trimontanate 50 Mixture 8 M8 Comparative wax 1 50 Sorbitan trimontanate 50 Mixture 9 M9 Wax 1 50 Ethylenebisstearoylamide 50 Mixture 10 M10 Comparative wax 1 50 Ethylenebisstearoylamide 50 Mixture 11 M11 Wax 1 80 Polar polyethylene wax 20 Mixture 12 M12 Comparative wax 1 80 Polar polyethylene wax 20 To prepare the mixtures, the pulverulent starting substances were first of all premixed and then comminuted on a fluidized-bed opposed-jet mill from Hosokawa Alpine AG to an average particle size of less than 10 μm.",
"The particle size is measured by the laser diffraction method in an instrument from Malvern.",
"The waxes may be added to the printing ink as a dry powder or, preferably, as a dispersion in binder solution or solvent.",
"Printing Ink Preparation Examples 1) Offset Ink The mixtures M1, M2, M3, M4, M9 and M10 were incorporated at 1.5% by weight into an offset ink (Novaboard® cyan 4 C 86, BASF Drucksysteme GmbH) with intensive stirring using a dissolver and subsequent homogenization on a triple-roll mill.",
"A test print (Prüfbau multipurpose test printing machine, System Dr. Dürner) was produced on paper of the Phoenomatt® type, 115 g/m2 (Scheufelen GmbH+Co KG), and the scuffing behavior was investigated on a scuff testing instrument (Prüfbau Quartant scuff tester) at a scuffing load of 48 g/cm2, scuff rate 15 cm/sec.",
"The parameter assessed was the intensity of the ink transferred to the test sheet after 50, 100 and 200 scuff cycles (strokes) (color difference according to DIN 6174, measured with Hunterlab D 25-2, Hunter) and the damage to the printed image.",
"TABLE 3 Result of testing in an offset ink with wax incorporated as micropowder Color difference Damage to Example Particle 100 200 the printed No.",
"size d50 μm strokes strokes image 1 Comparative — 15.5 18.3 Yes without wax 2 M1 8.0 1.2 1.8 No 3 M2 8.5 2.0 2.4 No 4 M3 7.8 3.5 4.2 No 5 M4 8.0 5.2 7.6 Yes 6 M9 6.5 3.2 4.2 No 7 M10 6.7 5.8 8.3 Yes The waxes of the invention result in a lower color difference and thus in improved abrasion resistance.",
"2.Gravure Ink The mixtures M3, M4, M5, M6, M7 and M8 were incorporated at 1% by weight into a publication gravure ink (type RR Grav red, Siegwerk Fabenfabrik) with intensive stirring using a dissolver.",
"A test print (gravure test printing instrument LTG 20, Einlehner Prüfmaschinenbau) was produced on paper of the Allgäu type, 60 g/m (G. Haindl'sche Papierfabriken KG), and was tested as for the offset ink example.",
"TABLE 4 Result of testing in an gravure ink with wax incorporated as micropowder Dynamic Particle Color difference friction Example size 100 strokes coefficient No.",
"μm Masstone Halftone μ 8 Comparison — 16.7 15.1 0.53 without wax 9 M3 (invent.)",
"7.5 2.9 1.8 0.33 10 M4 (compar.)",
"7.6 3.4 2.2 0.35 11 M5 (invent.)",
"8.5 2.0 1.1 0.27 12 M6 (compar.)",
"8.5 2.5 1.7 0.28 13 M7 (invent.)",
"8.4 2.6 1.2 0.26 14 M8 (compar.)",
"8.3 3.1 1.8 0.27 The waxes of the invention result in a lower color difference and thus in improved abrasion resistance.",
"Also found, surprisingly, was the reduction in the sliding friction when sorbitan esters were added.",
"3) Flexographic Ink The mixtures M3, M4, M5, M6, M7, M8, M11 and M12 were incorporated at 1% by weight into an aqueous flexographic ink with intensive stirring using a dissolver.",
"The flexographic printing ink had the composition 35% by weight Synthacryl® SW 175, 20% by weight Hostapermblau® B2G, 45% by weight water.",
"A test print was prepared by applying the ink with a wet film thickness of 6 μm using a wire-wound coating bar to paper of type Allgäu 80 g/m (G. Haindl'sche Papierfabriken KG) and was tested as for the set ink example.",
"TABLE 5 Result of testing in a flexographic ink with wax incorporated as micropowder Color Dynamic Example Particle size difference friction No.",
"μm 50 strokes coefficient μ 15 Comparison — 7.4 0.37 without wax 16 M3 (invent.)",
"7.5 3.8 0.23 17 M4 (compar.)",
"7.6 4.4 0.25 18 M5 (invent.)",
"8.5 3.0 0.18 19 M6 (compar.)",
"8.5 3.7 0.19 20 M7 (invent.)",
"8.4 2.8 0.16 21 M8 (compar.)",
"8.3 3.1 0.17 22 M11 (invent.)",
"8.6 3.1 0.23 23 M12 (compar.)",
"8.5 3.9 0.24 The waxes of the invention result in a lower color difference and thus in improved abrasion resistance.",
"Also found, surprisingly, was the reduction in the sliding friction when sorbitan esters were added."
]
] | Patent_10220411 |
[
[
"Model transition sensitivity analysis system and method",
"A method and system for analyzing an infectious disease uses computer based simulation engines.",
"The method and system utilizes at least two computer-based simulation engines of the transmission of the infectious disease.",
"The transmission of the infectious disease is analyzed as a function of the first and second computer-based simulation engines."
],
[
"1.A method for analyzing an infectious disease using computer based simulation engines, including the steps of: simulating transmission of the infectious disease using a first computer-based simulation engine; simulating the transmission of the infectious disease using a second computer-based simulation engine; and, analyzing the transmission of the infectious disease as a function of the first and second computer-based simulation engines.",
"2.A method, as set forth in claim 1, wherein the step of analyzing transmission of the infectious disease includes the step of observing the transmission of the infectious disease.",
"3.A method, as set forth in claim 1, including the step of observing the transmission of the infectious disease using geographic information system software.",
"4.A method, as set forth in claim 1, including the step of determining an impact of the computer-based simulation engines on the analysis of the transmission of the infectious disease.",
"5.A method, as set forth in claim 1, including the step of making decisions related to controlling the transmission of the infectious disease.",
"6.A method, as set forth in claim 1, wherein the first and second computer-based simulation engines use a common set of foundation classes.",
"7.A method, as set forth in claim 1, including the step of transitioning between the first and second computer-based simulation engines.",
"8.A method, as set forth in claim 1, wherein the first computer-based simulation engine is one of a deterministic compartmental engine, a deterministic ODE engine, a stochastic discrete individual engine in continuous time without retention of individual histories, and a stochastic engine with retention of individual histories.",
"9.A method, as set forth in claim 8, wherein the second computer-based engine is one of a deterministic compartmental engine, a deterministic ODE engine, a stochastic discrete individual engine in continuous time without retention of individual histories, and stochastic engine with retention of individual histories.",
"10.A method, as set forth in claim 1, wherein the first and second computer-based simulation engines include a model of disease progression.",
"11.A method, as set forth in claim 1, wherein the first and second computer-based simulation engines include a model of infection force.",
"12.A method, as set forth in claim 1, wherein the first and second computer-based simulation engines include a model of sub-populations.",
"13.A method, as set forth in claim 1, wherein the first and second computer-based simulation engines include a model of mixing.",
"14.A method, as set forth in claim 1, including the step of transiting between the first and second simulation engines using a common framework.",
"15.A method for analyzing an infectious disease using computer based simulation engines, including the steps of: simulating transmission of the infectious disease using a deterministic compartmental engine; simulating the transmission of the infectious disease using a deterministic ODE engine; simulating transmission of the infectious disease using a stochastic discrete individual engine in continuous time without retention of individual histories; simulating the transmission of the infectious disease using a stochastic engine with retention of individual histories; and, analyzing the transmission of the infectious disease as a function of the simulation engines.",
"16.A method for analyzing an infectious disease using computer based simulation engines, including the steps of: simulating transmission of the infectious disease using a first computer-based simulation engine; simulating the transmission of the infectious disease using a second computer-based simulation engine, wherein the first and second simulation engines include a model of infection force, a model of sub-populations, and a model of mixing; transiting between the first and second simulation engines using a common framework; and, analyzing the transmission of the infectious disease as a function of the first and second computer-based simulation engines.",
"17.A system for reviewing and analyzing transmission of an infectious disease, comprising: an input device for inputting data related to the infectious disease by a user; a first computer-base simulation engine of the infectious disease for simulating transmission of the infectious disease as a function of the data input by the user; a second computer-based simulation engine of the infectious disease coupled to the first computer-based simulation engine for simulating transmission of the infectious disease; and, a visual display coupled to the first and second computer-based simulation engines for displaying results of the first and second computer-based simulation engines to the user.",
"18.A system, as set forth in claim 17, wherein the system includes geographic information system software which is adapted to combine the results of the first and second computer-based simulation engines and display information on the visual display to the user.",
"19.A system, as set forth in claim 18, wherein the geographic information system is used to observe the transmission of the infectious disease.",
"20.A system, as set forth in claim 18, wherein the geographic information system is used to determine an impact of the computer-based simulation engines on the analysis of the transmission of the infectious disease.",
"21.A system, as set forth in claim 17, wherein the system is used to make decisions related to controlling the transmission of the infectious disease.",
"22.A system, as set forth in claim 17, wherein the first and second computer-based simulation engines use a common set of classes.",
"23.A system, as set forth in claim 17, wherein the system is adapted to transition between the first and second computer-based simulation engines.",
"24.A system, as set forth in claim 17, wherein the first computer-based simulation engine is one of a deterministic compartmental engine, a deterministic ODE engine, a stochastic discrete individual engine in continuous time without retention of individual histories, and a stochastic engine with retention of individual histories.",
"25.A system, as set forth in claim 24, wherein the second computer-based engine is one of a deterministic compartmental engine, a deterministic OCE engine, a stochastic discrete individual engine in continuous time without retention of individual histories, and a stochastic engine with retention of individual histories.",
"26.A system, as set forth in claim 17, wherein the first and second computer-based simulation engines include a model of infection force.",
"27.A system, as set forth in claim 17, wherein the first and second computer-based simulation engines include a model of sub-populations.",
"28.A system, as set forth in claim 17, wherein the first and second computer-based simulation engines include a model of mixing.",
"29.A system, as set forth in claim 17, including means for transiting between the first and second simulation engines using a common framework.",
"30.A system for reviewing and analyzing transmission of an infectious disease, comprising: an input device for inputting data related to the infectious disease by a user; a deterministic compartmental engine of the infectious disease for simulating transmission of the infectious disease as a function of the data input by the user; a deterministic OCE engine of the infectious disease for simulating transmission of the infectious disease as a function of the data input by the user; a stochastic discrete individual engine in continuous time without retention of individual histories of the infectious disease for simulating transmission of the infectious disease as a function of the data input by the user; a stochastic engine with retention of individual histories for simulating transmission of the infectious disease as a function of the data input by the user; and, a visual display coupled to the first and second computer-based simulation engines for displaying results of the first and second computer-based simulation engines to the user.",
"31.A system for reviewing and analyzing transmission of an infectious disease, comprising: an input device for inputting data related to the infectious disease by a user; a first computer-based simulation engine of the infectious disease for simulating transmission of the infectious disease as a function of the data input by the user; a second computer-based simulation engine of the infectious disease coupled to the first computer-based simulation engine for simulating transmission of the infectious disease, wherein the first and second simulation engines include a model of infection force, a model of sub-populations, and a model of mixing; means for transiting between the first and second simulation engines using a common framework; and, a visual display coupled to the first and second computer-based simulation engines for displaying results of the first and second computer-based simulation engines to the user.",
"32.A system, as set forth in claim 17, wherein the first and second computer-based simulation engines include a model of disease progression."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates generally to infectious disease analysis, and more particularly, to a system and method for infectious disease model transition sensitivity analysis.",
"2.Description of the Prior Art Effective infectious disease analysis, surveillance and control depend on informed decisions by public health authorities and infection control professionals.",
"Because infection transmission systems are complex and non-linear, these professionals often rely on models to help them understand the complexities involved in their decisions.",
"They use models to predict the course of epidemics, evaluate the efficacy of interventions, allocate resources efficiently, determine what drug and vaccine designs will be most effective, and in general assess how to best limit the spread of infectious diseases.",
"The goal of modeling is to formulate models that capture relevant aspects of system behavior while maintaining simplicity and ease of understanding.",
"This is accomplished via abstraction and simplification.",
"Models are abstractions.",
"Reality is highly complex and this complexity is made tractable by abstracting reality to represent important components within a mathematical form (the model type).",
"Because they are abstractions, all models are “wrong” since they do not fully represent reality and thus cannot capture all behaviors of the modeled system.",
"As abstractions, models are useful only when they capture aspects of a system's structure and behavior deemed relevant to a specific problem.",
"Models are simplifications.",
"Simplification of complex reality is required in order to formulate understandable mathematical models.",
"Hence there is a dynamic and a tension between simplification and the ability of models to adequately represent system behaviors.",
"Models rely on assumptions.",
"Simplification is accomplished by making restrictive assumptions, and many such assumptions are intrinsic to model type.",
"Thus one of the most important problems in modeling is to determine the sensitivity of model results and decisions based on those results to assumptions of model type and complexity.",
"How can we determine whether an abstraction is appropriate, and how do we know when reality is oversimplified?",
"These two issues underlie all mathematical models, and are the fundamental questions answered by MTSA.",
"When formulating models, critical choices are made regarding model type and complexity.",
"Model type is the mathematical approach used to represent a system, for example, an ordinary differential equation model versus a discrete event model; or a deterministic model versus a stochastic model.",
"Model complexity is determined by the amount of abstraction and simplification employed during model construction.",
"A growing body of work demonstrates that choice of model type and complexity has substantial impacts on simulation results and on disease control decisions.",
"Despite this, most analyses assess sensitivity only to a model's parameter values.",
"Sensitivity to model type an complexity assumptions is difficult because of the lack of model transition sensitivity analysis (MTSA) software.",
"This new term describes the analysis of how sensitive infection control decisions are to model type and complexity assumptions.",
"In the absence of MTSA software it is almost impossible for decision makers to assess whether a proposed course of action is a good choice, or instead is highly sensitive to model type and complexity and is therefore an artifact of model selection.",
"To support MTSA we need simulation tools that support a variety of model types, provide a common us interface, and that provide seamless transition from one form of model to another.",
"Infection transmission system models play an important role in the cost/benefit analysis of alternative approaches to reducing the risk of infection from an infectious disease such as Waterborne Cryptosporidia.",
"Cryptosporidia is transmitted via animal reservoirs; within families, between unrelated individuals, are by ingestion of contaminated water.",
"These different transmission modes can strongly influence the growth, behavior and dynamics of Cryptosporidia epidemics in human populations.",
"The Environmental Protection Agency expends significant resources to reduce the risk of Cryptosporidia infection among the immuno-compromised.",
"EPA Interventions are expensive and include installing an ozonation process at water plants and the installation of water filters in the homes of individuals at high risk of death from Cryptosporid infection.",
"The failure to reach the correct decision thus involves enormous human as well as economic costs.",
"Model Transition Sensitivity Analysis will substantially improve our ability to accurately decide whether water sanitation to control Cryptosporidia transmission should be directed at water treatment plants or at households of high-risk individuals such as those suffering from HIV infection.",
"Influenza immunization policy is founded on an understanding of influenza transmission systems.",
"Current immunization efforts focus on high-risk individuals (e.g.",
"the young, the elderly, al those prone to life-threatening pulmonary infections such as pneumonia).",
"However, data on influenza transmission shows that transmission probabilities in families are considerably below the levels needs to sustain transmission in a population.",
"What then accounts for epidemic spread?",
"One hypothesis is that great variability contagiousness accounts for high transmission levels in settings like schools and low transmission settings like households.",
"This strongly suggests that immunization strategy should focus on highly transmitting individuals to control the epidemic; as well as on high-risk individuals to reduce mortality.",
"The design of studies to effectively evaluate alternative immunization policies is a difficult undertaking and requires Model Transition Sensitivity Analysis to assure the stability and accuracy the results.",
"Recent studies of HIV demonstrated a substantially increased probability of transmission between both homosexual and heterosexual partners when the infected partner was in the stage of primary viremia, and that this increased transmission probability is caused by elevated virus particle concentrations in the blood and semen.",
"This observation suggests an intervention strategy that focuses on reducing serum virus particle concentrations during primary viremia.",
"Model-based analyses demonstrate that such a strategy would be highly effective in controlling the HIV epidemic (Koopman, Jacquez et al.",
"1997).",
"This strategy requires only a reduction in virus particle concentrations over the relatively brief stage of primary viremia in order to achieve a dramatic decrease in the total number of individuals infected over the course of the epidemic.",
"Model Transition Sensitivity Analysis holds the promise of guiding drug and vaccine design decisions by more accurately evaluating the benefits and effectiveness of vaccination protocols in populations against the efficacy of drugs and vaccines in individuals.",
"The present invention is aimed at one or more of the problems as set forth above."
],
[
"<SOH> SUMMARY OF THE INVENTION AND ADVANTAGES <EOH>In one aspect of the present invention, a method for analyzing an infectious disease using computer based simulation engines, is provided.",
"The method included the steps of simulating transmission of the infectious disease using a first computer-based model and simulating the transmission of the infectious disease using a second computer-based model.",
"The method further includes the steps of analyzing the transmission of the infectious disease as a function of the first and second computer-based simulation engines.",
"In another aspect of the present invention, a system for reviewing and analyzing transmission of an infectious disease, is provided.",
"The system includes an input device for inputting data related to the infectious disease by a user and a visual display for displaying information to the user.",
"The system further includes first and second computer-based simulation engines for modeling the transmission of the infectious disease.",
"The present invention is embodied in software tools for assessing the impacts of model assumptions on decisions regarding the analysis, surveillance, and control of infectious diseases.",
"Most analyses consider sensitivity only to changes in a model's parameter values, and ignore how assumptions of model form (e.g.",
"deterministic vs. stochastic, ODE vs. discrete individual) impact results and concomitant decisions.",
"The present invention enables analysis of sensitivity to model type and complexity, as well as to parameter values.",
"Second, it will implement multiple, e.g., four, simulation engines in a common framework that empowers decision-makers to conduct model transition sensitivity analyses.",
"Third, it will develop software that interfaces with a Geographic Information System (GIS) and spatial analysis tools that will make the handling of geographic and social dimensions tractable within infection transmission system analyses.",
"The present invention insures that unrealistic model assumptions don't lead to bad decisions.",
"Model transition sensitivity analysis will greatly enhance our ability to make sound disease surveillance and control decisions by systematically relaxing the assumptions on which models are based.",
"This project will put in place the methods and software to fully exploit this substantial opportunity."
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates generally to infectious disease analysis, and more particularly, to a system and method for infectious disease model transition sensitivity analysis.",
"2.Description of the Prior Art Effective infectious disease analysis, surveillance and control depend on informed decisions by public health authorities and infection control professionals.",
"Because infection transmission systems are complex and non-linear, these professionals often rely on models to help them understand the complexities involved in their decisions.",
"They use models to predict the course of epidemics, evaluate the efficacy of interventions, allocate resources efficiently, determine what drug and vaccine designs will be most effective, and in general assess how to best limit the spread of infectious diseases.",
"The goal of modeling is to formulate models that capture relevant aspects of system behavior while maintaining simplicity and ease of understanding.",
"This is accomplished via abstraction and simplification.",
"Models are abstractions.",
"Reality is highly complex and this complexity is made tractable by abstracting reality to represent important components within a mathematical form (the model type).",
"Because they are abstractions, all models are “wrong” since they do not fully represent reality and thus cannot capture all behaviors of the modeled system.",
"As abstractions, models are useful only when they capture aspects of a system's structure and behavior deemed relevant to a specific problem.",
"Models are simplifications.",
"Simplification of complex reality is required in order to formulate understandable mathematical models.",
"Hence there is a dynamic and a tension between simplification and the ability of models to adequately represent system behaviors.",
"Models rely on assumptions.",
"Simplification is accomplished by making restrictive assumptions, and many such assumptions are intrinsic to model type.",
"Thus one of the most important problems in modeling is to determine the sensitivity of model results and decisions based on those results to assumptions of model type and complexity.",
"How can we determine whether an abstraction is appropriate, and how do we know when reality is oversimplified?",
"These two issues underlie all mathematical models, and are the fundamental questions answered by MTSA.",
"When formulating models, critical choices are made regarding model type and complexity.",
"Model type is the mathematical approach used to represent a system, for example, an ordinary differential equation model versus a discrete event model; or a deterministic model versus a stochastic model.",
"Model complexity is determined by the amount of abstraction and simplification employed during model construction.",
"A growing body of work demonstrates that choice of model type and complexity has substantial impacts on simulation results and on disease control decisions.",
"Despite this, most analyses assess sensitivity only to a model's parameter values.",
"Sensitivity to model type an complexity assumptions is difficult because of the lack of model transition sensitivity analysis (MTSA) software.",
"This new term describes the analysis of how sensitive infection control decisions are to model type and complexity assumptions.",
"In the absence of MTSA software it is almost impossible for decision makers to assess whether a proposed course of action is a good choice, or instead is highly sensitive to model type and complexity and is therefore an artifact of model selection.",
"To support MTSA we need simulation tools that support a variety of model types, provide a common us interface, and that provide seamless transition from one form of model to another.",
"Infection transmission system models play an important role in the cost/benefit analysis of alternative approaches to reducing the risk of infection from an infectious disease such as Waterborne Cryptosporidia.",
"Cryptosporidia is transmitted via animal reservoirs; within families, between unrelated individuals, are by ingestion of contaminated water.",
"These different transmission modes can strongly influence the growth, behavior and dynamics of Cryptosporidia epidemics in human populations.",
"The Environmental Protection Agency expends significant resources to reduce the risk of Cryptosporidia infection among the immuno-compromised.",
"EPA Interventions are expensive and include installing an ozonation process at water plants and the installation of water filters in the homes of individuals at high risk of death from Cryptosporid infection.",
"The failure to reach the correct decision thus involves enormous human as well as economic costs.",
"Model Transition Sensitivity Analysis will substantially improve our ability to accurately decide whether water sanitation to control Cryptosporidia transmission should be directed at water treatment plants or at households of high-risk individuals such as those suffering from HIV infection.",
"Influenza immunization policy is founded on an understanding of influenza transmission systems.",
"Current immunization efforts focus on high-risk individuals (e.g.",
"the young, the elderly, al those prone to life-threatening pulmonary infections such as pneumonia).",
"However, data on influenza transmission shows that transmission probabilities in families are considerably below the levels needs to sustain transmission in a population.",
"What then accounts for epidemic spread?",
"One hypothesis is that great variability contagiousness accounts for high transmission levels in settings like schools and low transmission settings like households.",
"This strongly suggests that immunization strategy should focus on highly transmitting individuals to control the epidemic; as well as on high-risk individuals to reduce mortality.",
"The design of studies to effectively evaluate alternative immunization policies is a difficult undertaking and requires Model Transition Sensitivity Analysis to assure the stability and accuracy the results.",
"Recent studies of HIV demonstrated a substantially increased probability of transmission between both homosexual and heterosexual partners when the infected partner was in the stage of primary viremia, and that this increased transmission probability is caused by elevated virus particle concentrations in the blood and semen.",
"This observation suggests an intervention strategy that focuses on reducing serum virus particle concentrations during primary viremia.",
"Model-based analyses demonstrate that such a strategy would be highly effective in controlling the HIV epidemic (Koopman, Jacquez et al.",
"1997).",
"This strategy requires only a reduction in virus particle concentrations over the relatively brief stage of primary viremia in order to achieve a dramatic decrease in the total number of individuals infected over the course of the epidemic.",
"Model Transition Sensitivity Analysis holds the promise of guiding drug and vaccine design decisions by more accurately evaluating the benefits and effectiveness of vaccination protocols in populations against the efficacy of drugs and vaccines in individuals.",
"The present invention is aimed at one or more of the problems as set forth above.",
"SUMMARY OF THE INVENTION AND ADVANTAGES In one aspect of the present invention, a method for analyzing an infectious disease using computer based simulation engines, is provided.",
"The method included the steps of simulating transmission of the infectious disease using a first computer-based model and simulating the transmission of the infectious disease using a second computer-based model.",
"The method further includes the steps of analyzing the transmission of the infectious disease as a function of the first and second computer-based simulation engines.",
"In another aspect of the present invention, a system for reviewing and analyzing transmission of an infectious disease, is provided.",
"The system includes an input device for inputting data related to the infectious disease by a user and a visual display for displaying information to the user.",
"The system further includes first and second computer-based simulation engines for modeling the transmission of the infectious disease.",
"The present invention is embodied in software tools for assessing the impacts of model assumptions on decisions regarding the analysis, surveillance, and control of infectious diseases.",
"Most analyses consider sensitivity only to changes in a model's parameter values, and ignore how assumptions of model form (e.g.",
"deterministic vs. stochastic, ODE vs. discrete individual) impact results and concomitant decisions.",
"The present invention enables analysis of sensitivity to model type and complexity, as well as to parameter values.",
"Second, it will implement multiple, e.g., four, simulation engines in a common framework that empowers decision-makers to conduct model transition sensitivity analyses.",
"Third, it will develop software that interfaces with a Geographic Information System (GIS) and spatial analysis tools that will make the handling of geographic and social dimensions tractable within infection transmission system analyses.",
"The present invention insures that unrealistic model assumptions don't lead to bad decisions.",
"Model transition sensitivity analysis will greatly enhance our ability to make sound disease surveillance and control decisions by systematically relaxing the assumptions on which models are based.",
"This project will put in place the methods and software to fully exploit this substantial opportunity.",
"BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: FIG.",
"1 is block diagram of a system for analyzing the transmission of an infectious disease, according to an embodiment of the present invention; FIG.",
"2 is a flow diagram of a method for analyzing the transmission of an infectious disease, according to an embodiment of the present invention; FIG.",
"3 is a block diagram of a class diagram according to an embodiment of the present invention; FIG.",
"4 is a block diagram of a state diagram according to an embodiment of the present invention; and FIG.",
"5 is a block diagram of a system for analyzing the transmission of an infectious disease, according to another embodiment of the present invention.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT With reference to the drawings an in operation, the present invention provides a system 100 and method 200 for review and analyzing transmission of an infectious disease is provided.",
"With specific reference to FIG.",
"1, the system 100 is preferably embodied in software running on a computer 102, e.g., a general purpose computer.",
"The computer 102 includes a display 104, such as a cathode-ray tube (CRT) device or a flat panel display, and an input device 106, such as a keyboard, mouse, and/or microphone.",
"The computer 102 has stored thereon at least two computer-based simulation engines 108 for modeling the transmission of the infectious disease, e.g., Cryptosporidia, influenza, or HIV.",
"Preferably, the system 100 includes first, second, third, and fourth computer-based simulation engines 108A, 108B, 108C, 108D.",
"Data regarding the infectious disease is input by a user 110.The user 110 inputs parameters to the computer-based simulation engines and reviews the results displayed on the visual display 104.Preferably, the system 100 utilizes geographic information system (GIS) software to display, organize and assist in the analysis of the model results.",
"Suitable GIS software is available from ESRI of Redlands, Calif. under the name “ArcView GIS”.",
"The GIS software is adapted to combine the results of the first and second computer-based models and display information on the visual display to the user; to observe the transmission of the infectious disease; and to determine an impact of the computer-based simulation engines on the analysis of the transmission of the infectious disease.",
"Based on the results displayed on the visual display 104, the user 110 is able to make decisions related to controlling the transmission of the infectious disease.",
"In the preferred embodiment, the computer-based simulation engines 108 are written in the C++ program language.",
"The simulation engines are constructed using a common set of classes.",
"This enables the system 100 to transition between the computer-based simulation engines 108 quickly and easily in order to facilitate analyzing the effect of the type and complexity of the engine has on decision made by the user 110.The engines 108 are different in type and/or complexity.",
"As discussed below in the preferred embodiment, the models are one of the following types: a deterministic compartmental engine; a deterministic ODE engine; a stochastic discrete individual engine in continuous time without retention of individual histories; and a stochastic engine with retention of individual histories.",
"For example, of the system 100 includes four computer-based simulation engines.",
"Each engine is one of the types identified above and may differ by type and/or complexity.",
"With specific, reference to FIG.",
"2, a method 200 for analyzing an infectious disease using computer based engine simulations is provided.",
"The method includes the steps of : simulating transmission of the infectious disease using a first computer-based simulation engine (first process step 202); simulating the transmission of the infectious disease using a second computer-based simulation engines (second process step 204); and, analyzing the transmission of the infectious disease as a function of the first and second computer-based simulation engines (third process step 206).",
"Specific requirements are formulated for the simulation engines and for components of the model complexity to be transited by the software.",
"These requirements will determine: simulation engines for modeling infection transmission systems; spatial methods for identifying geographic subpopulations for modeling purposes; components of model complexity that can be relaxed across simulation engines; and, functionality of the system 100, including visualization techniques, and the user interface.",
"Infectious disease models and simulation approaches for discrete and stochastic methods, as implemented in ODE and individual-level models, are well developed and are expected to carry over with little modification into the new MTSA software.",
"Other features may be incorporated into the simulation engines with substantial benefits in disease control, including: Incorporate spatial and social dimensions GIS interface and multivariate spatial analysis methods of identifying geographic subpopulations Relax model type and complexity assumptions Common framework for transiting across simulation engines.",
"Geography enters into infectious disease processed in several ways.",
"First, individuals, groups and populations have geographic locations and spatial extent, and these may be static or change through time.",
"Second, contact networks have geographic projections defined by the locations where infection events take place and by the spatial paths traveled by the infectious agent.",
"The importance of geography varies from system to system.",
"Water-borne diseases such as Cryptosporidia have transmission modes mediated by water flow.",
"In these instances the map of the water distribution system is critical to our understanding of disease spread.",
"In some diseases social and behavioral factors are important determinants of disease transmission, and the ma of the contact network has both social and spatial dimensions.",
"Preferably, one or more of the simulation engines 108 include: (1) the representation and identification of individuals and populations in geographic space, and (2) the representation of contact networks as maps incorporating both spatial and social dimensions.",
"Preferably, one or more of the simulation engines 108 will also include: Spatially agglomerative clustering: statistical methods and software for identifying geographic subpopulations based on multivariate characteristics including ethnicity, socioeconomic status, and race.",
"The technique of spatially agglomerative clustering is particularly well suited to the identification of groups and populations for incorporation into infection transmission models.",
"Space-time information systems: a space-time data model that provides building blocks for constructing object models customized for specific applications.",
"The space-time data model is implemented at two levels: the data structure level and the application level.",
"Objects defined at the data structure level are the foundation upon which application-specific objects are built.",
"This model extends the diad (what, where) used in conventional GIS to the triad (what, where, when necessary for infectious disease modeling.",
"Object identifies the modeled entity (i.e.",
"a person or population); space-time coordinate is a spatio-temporal location (e.g.",
"Latitude, Longitude Altitude, Date); and attributes are observations on variables describing the modeled entity and it environment (e.g.",
"disease status, socioeconomic status or SES, exposed, vaccinated, etc.).",
"The attributes are defined at the application level (see below) by extending the object definitions provided by the MTSA programming tool.",
"This provides a powerful mechanism for designing custom objects that retain full functionality provided by the MTSA foundation library.",
"With reference to FIGS.",
"3 and 4, class diagrams 300 and state diagrams 400 are used to represent higher-level perspectives at the application level.",
"During implementation, classes in the Class Diagram 300 are constructed from object represented at the data structure level.",
"Hence, State and Class Diagrams 300, 400 are typically application specific.",
"The diagrams in FIGS.",
"3 and 4 have been designed for an infectious disease, but are generally enough to represent chronic disease as well.",
"The State Diagram 400 represents a subject's disease susceptibility with the five states ‘at-risk’ 402, disease initiation’ 404, ‘disease detection’ 406, ‘immunity’ 408, and ‘death’ 410.These states are attributes of the object used to model subjects.",
"The class diagram shows the objects ‘subject’ 302 and ‘population’ 304, and the classes ‘risk factor’ 306, ‘space’ 308, and ‘time’ 310.Class and object relationships are shown as diamonds and include ‘exposure’ 312, ‘confounding’ 314, ‘inclusion’ 316, ‘contiguous’ 318 and ‘containment’ 320.Lines indicate logical connections and class relationships (the relationship exposure’ and the class ‘Time’ appear twice to avoid crossing lines).",
"This class diagram 300 was constructed using the Unified Modeling Language.",
"For example, a Population is comprised of n subjects, and is indicated by the ‘1 to many’ relationship.",
"The attribute birth rate, immigration rate, emigration rate and so on are related both to a Population, a Time (duration) and a geographic Space (spatial extent).",
"This class diagram is the basis of the system 100 object model, and has several advantages: It records information on individuals and populations through time, enabling the tracking a individual history and model results.",
"It indexes spatial and temporal location, tracking information required to make geographic maps of contact networks and disease maps.",
"It handles spatial, temporal and social dimensions necessary for transmission system modeling.",
"It is general and flexible, and uses inheritance to reduce overhead at the application level.",
"A link to GIS software provides a seamless mechanism for integrating the software into spatial decision support systems.",
"This is accomplished using common GIS file formats and through an ArcView link.",
"This leverages ArcView's relational data base management (RDBM) capabilities an provides a mechanism (the common link to ArcView) for exchanging data with existing spatial analysis software.",
"The link to ArcView will be accomplished as an ArcView extension.",
"Extensions are a convenient way to add functionality to ArcView.",
"Extensions add new buttons and menu choices to ArcView, and allow it to display, utilize and prepare additional data formats.",
"The extension will be created as an Avenue script that is then saved as an extension.",
"This extension will add an ‘MTSA’ speed button to ArcView.",
"Clicking on this button allows the user to define data sets, variables and fields to share with our software (e.g.",
"establish a database connection), and to then run MTSA.",
"The key to model transition sensitivity analysis is the ability to relax model complexity assumptions while transiting across simulation engines.",
"Four simulation engines types have been identified which represent the range of model types and complexity used t model infectious disease transmission systems.",
"Engine I: Deterministic compartmental models formulated as ODEs where each compartment represents a fraction of a population.",
"These assume point-time contacts in large, thoroughly mixed populations.",
"Engine II: Deterministic ODE models with compartments representing transmission units like sexual couples or families as well as individuals.",
"This relaxes the assumption in the previous engine that contacts have no duration.",
"Engine III: Stochastic discrete individual models in continuous time without retention of individual histories.",
"Stochasticity can be simulated in either individuals or populations depending on which unit maximizes computational efficiency while preserving simulation faithfulness to model assumptions Unlike forms I and II, this engine can capture stochastic effects attributable to small mixing units.",
"Engine IV: Stochastic models with retention of individual histories.",
"The retention of past history by individual agents facilitates thorough exploration of model behavior, and allows past events to influence current behavioral tendencies as represented by model parameters.",
"With reference to FIG.",
"5, a system 500 for analyzing the transmission of an infectious disease embodied in a model transition sensitivity system (MTSA) software application 502.The MTSA software application 502 is indicated by the large box “MTSA software” and is comprised of modules for preparing input 504, conducting simulations 506, and for handling results 508.A decision maker 510 is represented by the stick figure to the left of the MTSA software box 502.Input on model type and complexity is provided by the decision maker 510 and by an external Geographic Information System 512 that defines geography, spatial sub-populations, and mixing sites.",
"Results are passed to the decision maker 510 as graphics and tabular numerical results comparing and contrasting how changes in model type and complexity impact model outputs.",
"These results may also be imported to external decision support tools 512 (e.g.",
"spreadsheets) for cost/benefit analysis.",
"The decision maker 510 evaluates assumptions regarding model type and complexity by determining how these assumptions impact model outputs and the decisions drawn from those outputs, resulting in a model transition sensitivity analysis.",
"MTSA software 502 includes the ability to traverse models of varying complexity.",
"They almost certainly include the four structural components models of disease progression, models of the force of infection, sub-populations, and mixing.",
"Models of Disease Progression: A basic structural component is a model or models of disease progression.",
"“Model or models” is used because the model of disease progression may differ for some subgroups.",
"This is important because we need to model the stages of development of the disease in individuals that influence transmission.",
"Thus the infectivity of infecteds generally changes as a disease progresses.",
"In addition, contact rates are important for transmission and may change as an illness progresses.",
"A model of disease progression is needed for each population subgroup.",
"Models of the Force of Infection: For each model of disease progression, we need a model of the force of infection.",
"This requires that we define the contact rates of different subgroups, the mixing between them and the probability of transmission per contact for the different subgroups.",
"That in effect gives model of transmission of the disease to incorporate in the model of disease progression for each subgroup.",
"Subpopulations: Real populations are not homogeneous and the members of different subgroups do n mix randomly with all other individuals.",
"Thus a population has in it subgroups that differ in ethnicity, religion and socio-economic status and these differences influence the rates of transmission of diseases.",
"Two major ways are, the extent of mixing between subgroups as compared with within subgroup mixing, and the probability of transmission per contact may differ because of particular group practices or customs, for example, in food preparation.",
"Population subgroups can also be define by geographic location and such subgroups intersect with subgroups defined by socio-economic and ethnic status.",
"Furthermore, the subgroups relevant for transmission of one disease may differ from those important in the transmission of a different disease.",
"Consequently, one has to examine the issue of definition of important subgroups for each particular disease.",
"One of the more difficult problems in modeling transmission of diseases is to model to mixing processes that lead to disease-transmitting contacts between individuals.",
"The most commonly used assumption is that individuals make contacts at random with others in the population.",
"In terms of contacts between subgroups that is commonly called proportional mixing.",
"There are a number of other more complicated ways to model mixing that allow for the possibility of varying within group contact rates as compared with contact rates with other groups.",
"Two of the most versatile are called preferred mixing and structured mixing.",
"In preferred mixing, one can reserve an arbitrary fraction of a group contacts for within-group mixing; all non-reserved contacts are then subject to proportional mixing.",
"Structured mixing takes into account that most contacts occur in or are initiated in particular gathering places or activities.",
"An arbitrary fraction of a group's contacts is allocated to each activity; the mixing at each activity is then assumed to be a proportional mixing.",
"These complexity components are preferably formalized within a unifying object model that constitutes the framework for transiting from one simulation engine to another.",
"This abstraction is designed specifically for translating model inputs to meet the specific input requirements of each simulation engine, and provides paths for navigating between the simulation engines in order to relax complexity assumptions.",
"This object model will be constructed using Object-Oriented Analysis and Design (OOA&D).",
"OOA&D is a relatively new technique that achieve unprecedented programming efficiency.",
"The era of monolithic ‘spaghetti code’, inherently difficult to maintain, translate and program, is coming to an end.",
"The simulation engines 108 are constructed using classes whose relationships define the system architecture.",
"The object design is critical to project execution, because it directly impacts the long-term sustainability of the end product and because an efficient and clear design dramatically streamlines all programming tasks Obviously, many modifications and variations of the present invention are possible in light of the above teachings.",
"The invention may be practiced otherwise than as specifically described within the scope of the appended claims, wherein that which is prior art is antecedent to the novelty set forth in the “characterized by” clause.",
"The novelty is meant to be particularly and distinctly recited in the “characterized by” clause whereas the antecedent recitations merely set forth the old and well-known combination in which the invention resides.",
"These antecedent recitations should be interpreted to cover any combination in which the incentive novelty exercises its utility.",
"In addition, the reference numerals in the claims are merely for convenience and are not to be read in any way as limiting."
]
] | Patent_10220874 |
[
[
"Ergonomic Chair",
"A work station chair is enabled to shift in response to a person leaning forward in the chair when in a working mode and leaning back in a rest position.",
"The chair includes a seat, a backrest and supportive structure.",
"The seat is resiliently supported on the supportive structure and moveably along an arc track which is mounted on the supportive structure and has a radius generated from the ankle of the person sitting on the chair.",
"The backrest is resiliently supported on the seat and moveable along an arc track which is mounted to the seat and has a radius generated from H-point that is a natural pivot point of the torso and thigh lines of the person.",
"The chair provides a combined tilting movement of the person's body about the ankle point and the H-point when the person shifts his or her gravity to reduce adverse static postural loads and forces which are responsible for the fatigue and biomechanical dysfunction.",
"It is also easy to adjust simultaneously the chair and the backrest for different weight loads of persons, which can be done by the person while sitting on the chair and reaching for an adjustment knob in front of the chair."
],
[
"1.A chair passively adjustable in response to shift center of gravity of a person sitting on the chair, comprising: a supportive structure adapted to support the chair and the weight of the person; a seat adjustably mounted on the supportive structure and pivotable in a vertical plane about an ankle point of the person; a backrest adjustably mounted on the seat and pivotable in the vertical plane about a nature pivot point of the torso and thigh lines of the person; resilient means for resiliently supporting the respective seat and backrest in a position in which the person sitting erectly or forwardly for work, and permitting the seat and backrest to pivot in response to the shift center of gravity of the person in rest; and positive adjustment mechanism mounted on a front of the chair and associated with the resilient means for positively adjusting the resilience of the resilient means by the person while sitting on the chair.",
"2.A chair as claimed in claim 1 wherein the resilient means comprises a first support end pivotally mounted on the supportive structure, a second support end pivotally mounted on the backrest, and a third support end pivotally mounted to the positive adjustment mechanism and slidable relative to the seat.",
"3.A chair as claimed in claim 2 wherein the resilient means comprises a first resilient device for supporting the seat and a second resilient device for supporting the backrest, one end of the first resilient device and one end of the second resilient device being connected to a common point of the positive adjustment mechanism, the common point being moveable to pre-compress the first and second resilient devices simultaneously.",
"4.A chair as claimed in claim 3 wherein the positive adjustment mechanism comprises a threaded connection assembly to convert a rotation to the movement of the common point.",
"5.A backrest structure for a chair comprising: a curved track in a fixed relation to a seat of the chair; a carriage slidably mounted on the curved track; a backrest mounted on the carriage; an resilient support mechanism provided for resiliently supporting the carriage and backrest with respect to the seat so that the carriage is biased uppermost to support the backrest in an erect position, and adapted to slide down along the curved track and tilt the backrest rearwards and downwardly in response to a rearward shift of the weight of a person sitting on the chair and leaning against the backrest.",
"6.A backrest structure as claimed in claim 5 wherein the curved track is a circular arc having a radius in a vertical plane generated from a point which substantially matches a natural pivot point of the torso and thigh lines of the person sitting on the chair.",
"7.A backrest structure as claimed in claim 5 wherein adjustment means are provided for adjusting the resilient support mechanism such that the backrest is enabled to be maintained in the erect position in response to different body loads of persons utilising the chair.",
"8.A backrest structure as claimed in claim 7 wherein the resilient support member includes a pair of compressible spring rods positioned at sides of the chair, each being anchored at one end to the carriage and at the other end to the adjustment means positioned on a front end of the seat.",
"9.A backrest structure as claimed in claim 8 wherein the adjustment means comprise a threaded bolt rotatably mounted on the front end of the seat, an adjustment bar threadedly connected to the threaded bolt and associated with the other end of the spring rod so that the spring rod is pre-compressed when the threaded bolt is rotated.",
"10.A backrest structure as claimed in claim 9 wherein ball bearings are provided between the carriage and the curved track.",
"11.A chair comprising: a seat and a base of the chair for supporting the seat; a supportive structure adapted to support the chair and the weight of a person sitting on the chair; a first curved track mounted on the supportive structure; a first carriage movably mounted on the first curved track and attached in fixed relation to the base so that the base is moveable relative to the supportive structure; a first resilient support mechanism provided for resiliently supporting the base relative to the supportive structure, the first carriage being biased to support the seat in a substantially horizontal position; a backrest of the chair; a second curved track mounted to the base; a second carriage movably mounted on the second curved track, and associated in fixed relation with the backrest so that the backrest is moveable relative to the base; an second resilient support mechanism provided for resiliently supporting the second carriage and backrest with respect to the base, the second carriage being biased uppermost to support the backrest in an erect position; and whereby the first and second carriages are enabled to slide down along the respective first and second curved tracks and tilt the respective seat and backrest rearwards and downwardly in response to a rearward shift of the weight of a person sitting on the chair and leaning against the backrest.",
"12.A chair as claimed in claim 11 wherein the first curved track is a circular arc having a radius in a vertical plane generated from a point which is close to a natural pivot point of the ankle joint of the person sitting on the chair.",
"13.A chair as claimed in claim 11 wherein the second curved track is a circular arc having a radius in a vertical plane generated from a point which substantially matches a natural pivot point of the torso and thigh lines of the person sitting on the chair.",
"14.A chair as claimed in claim 11 wherein adjustment means are provided for adjusting the resilience of the first and second resilient support mechanisms in response to different weight loads of persons utilising the chair.",
"15.A chair as claimed in claim 14 wherein the first resilient support mechanism includes a pair of compressible gas cylinder assemblies, each being anchored at one end to the supportive structure and at the other end to the adjustment means mounted on a front end of the base.",
"16.A chair as claimed in claim 15 wherein the second resilient support mechanism includes a pair of compressible spring rods, each being anchored at one end to the second carriage and at the other end to the adjustment means.",
"17.A chair as claimed in claim 16 wherein the adjustment means comprises a threaded bolt rotatably mounted to the front end of the base and an adjustment bar threadedly connected to the threaded bolt and associated with the other ends of the gas cylinder assemblies and the spring rods, so that the gas cylinder assemblies and spring rods are pre-compressible when the threaded bolt is rotated.",
"18.A chair as claimed in claim 17 wherein the base includes a seat plate on top of which the seat is attached, two side plates extending downwards from two sides of the seat plate respectively, and a slot being defined in each side plate at a front end, the slots receiving a cylinder support rod which is laterally extending therethrough and slidable along the slot, the cylinder support rod being attached to the adjustment bar and connected to the respective other ends of the gas cylinder assemblies and the spring rods so that the respective other ends of the cylinder assemblies and the spring rods are moveable along the slot in response to the rotation of the threaded bolt.",
"19.A chair as claimed in claim 18 wherein the supportive structure includes a vertical stem rotatably received in a sleeve mounted to the first curved track.",
"20.A chair as claimed in claim 19 wherein ball bearings are provided between the first carriage and the first curved track, and between the second carriage and the second curved track."
],
[
"<SOH> BACKGROUND ART <EOH>The demands of the seated work position mandate the user to accommodate a range of postural adjustments from the slightly rearward reclined rest position through to the forward hunched task posture.",
"Passive automatic adaptation or adjustment of the seat support system is required if the natural balance and equilibrium of the body's support is to be maintained.",
"Failure to maintain the body's equilibrium and structural balance will result in the creation of adverse, static postural loads and forces responsible for fatigue and biomechanical dysfunction so common in today's seated society.",
"There have been many attempts to better design a seating arrangement for persons working at a desk or computer terminal.",
"Such ergonomic chairs are described, for instance, in U.S. Pat.",
"No.",
"4,650,249 which issued on Mar.",
"17, 1987 to Cerber; U.S. Pat.",
"No.",
"4,738,487 which issued on Apr.",
"19, 1988 to Shalinsky et al.",
"; U.S. Pat.",
"No.",
"5,048,893 which issued on Sep. 17, 1991 to Cowan et al.",
"and Applicant's Canadian patent application serial no.",
"2,116,079 which was filed on Feb. 21, 1994 and laid-open on Aug. 23, 1994 in the inventors' names of Cowan et al.",
"It has been found that when a person leans forward to work or back in a rest position, there is a movement combination of the person's body pivoting about the ankles of the person with the person's upper body pivoting about a center called the “H-point” which is a natural pivoting point of the torso and thigh lines.",
"The H-point is defined in SAE standard J826.Although most chairs described in the above prior art provide reasonable adjustment in the fore and aft directions and allow for tilting of the seat, they do not provide, except in Canadian patent application serial no.",
"2,116,079, the combined movement of seats and backrests pivoting about the respective ankle point and the H-point and, therefore, result in a compromise in terms of vertical adjustment.",
"An upward movement of any part of the chair will jeopardize the body's equilibrium and structural balance.",
"Cowan et al.",
"describes, in Canadian patent application no.",
"2,116,079, a work station chair having a seat passively pivotable about the ankle of the person sitting on the chair and a backrest passively pivotable about the H-point.",
"A cable system is provided for positive adjustment of resiliency of the pivoting movement of the seat and backrest for different weight loads of persons sitting on the chair.",
"However, the H-point is physically required in the chair, which is a pair of pivoting pins attached to two arm support posts respectively.",
"Such a configuration limits the application of the H-point backrest because the position of the H-point is always above the seat and cannot be attached to the supportive structure under the seat.",
"Therefore, an improvement is desirable.",
"A positive adjustment mechanism simpler in structure and easier for use is also desired to replace the cable system which has to be adjusted in an inconvenient rear position."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Having thus generally described the nature of the invention, reference will now be made to the preferred embodiment thereof and the accompanying drawings, in which: FIG.",
"1 is a side view of a chair in accordance with the preferred embodiment of the invention; FIG.",
"2 is a partial side view of a frame structure of the chair in FIG.",
"1 ; FIG.",
"3 is a top end front perspective view of the frame structure in FIG.",
"2 ; FIG.",
"4 a is a side view of a backrest moving rail; FIG.",
"4 b is a perspective view of the backrest moving rail in FIG.",
"4 a , showing the grooves for bearing balls; FIG.",
"5 is a partial cross-sectional view of the backrest rail assembly taken from lines 5 - 5 in FIG.",
"3 ; FIG.",
"6 is a perspective view of the backrest balls cage; FIG.",
"7 is a perspective view of a positive adjustment assembly for adjusting the resilience of the seat and the backrest of the chair; and FIG.",
"8 is a partial side view of the frame structure in FIG.",
"2 , showing the positive adjustment in response to the different weight loads of persons utilizing the chair.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to a sitting unit such as a chair and, more particularly, to a work station chair which can shift in response to different positions of a person sitting on the chair, leaning forward in work mode and leaning back in a rest position.",
"BACKGROUND ART The demands of the seated work position mandate the user to accommodate a range of postural adjustments from the slightly rearward reclined rest position through to the forward hunched task posture.",
"Passive automatic adaptation or adjustment of the seat support system is required if the natural balance and equilibrium of the body's support is to be maintained.",
"Failure to maintain the body's equilibrium and structural balance will result in the creation of adverse, static postural loads and forces responsible for fatigue and biomechanical dysfunction so common in today's seated society.",
"There have been many attempts to better design a seating arrangement for persons working at a desk or computer terminal.",
"Such ergonomic chairs are described, for instance, in U.S. Pat.",
"No.",
"4,650,249 which issued on Mar.",
"17, 1987 to Cerber; U.S. Pat.",
"No.",
"4,738,487 which issued on Apr.",
"19, 1988 to Shalinsky et al.",
"; U.S. Pat.",
"No.",
"5,048,893 which issued on Sep. 17, 1991 to Cowan et al.",
"and Applicant's Canadian patent application serial no.",
"2,116,079 which was filed on Feb. 21, 1994 and laid-open on Aug. 23, 1994 in the inventors' names of Cowan et al.",
"It has been found that when a person leans forward to work or back in a rest position, there is a movement combination of the person's body pivoting about the ankles of the person with the person's upper body pivoting about a center called the “H-point” which is a natural pivoting point of the torso and thigh lines.",
"The H-point is defined in SAE standard J826.Although most chairs described in the above prior art provide reasonable adjustment in the fore and aft directions and allow for tilting of the seat, they do not provide, except in Canadian patent application serial no.",
"2,116,079, the combined movement of seats and backrests pivoting about the respective ankle point and the H-point and, therefore, result in a compromise in terms of vertical adjustment.",
"An upward movement of any part of the chair will jeopardize the body's equilibrium and structural balance.",
"Cowan et al.",
"describes, in Canadian patent application no.",
"2,116,079, a work station chair having a seat passively pivotable about the ankle of the person sitting on the chair and a backrest passively pivotable about the H-point.",
"A cable system is provided for positive adjustment of resiliency of the pivoting movement of the seat and backrest for different weight loads of persons sitting on the chair.",
"However, the H-point is physically required in the chair, which is a pair of pivoting pins attached to two arm support posts respectively.",
"Such a configuration limits the application of the H-point backrest because the position of the H-point is always above the seat and cannot be attached to the supportive structure under the seat.",
"Therefore, an improvement is desirable.",
"A positive adjustment mechanism simpler in structure and easier for use is also desired to replace the cable system which has to be adjusted in an inconvenient rear position.",
"DISCLOSURE OF THE INVENTION It is an object of the present invention to provide an improved work chair of the type described above but without the disadvantages mentioned hereinabove.",
"It is another object of the present invention to allow the user to passively maintain the natural lordotic curvatures and integrated biomechanical relationship of the spine, pelvis and lower limbs in a balanced dynamic equilibrium while in the seated posture either for work or in rest.",
"It is a further object of the present invention to provide a chair which provides a passive adjustment combination of a seat thereof pivoting about an ankle point of the user with a backrest pivoting about a natural pivot point of the torso and thigh lines of the user in response to a shift in gravity of the user sitting on the chair, the respective pivot points being virtually required in the chair structure.",
"It is yet a further object of the present invention to provide a positive adjustment mechanism which is simple in structure and easy for use to adjust the resiliency of the seat and backrest in their passive adjustment.",
"In a general term, the present invention is used to provide a chair passively adjustable in response to the shift center of gravity of a person sitting on the chair, comprising a supportive structure adapted to support the chair and the weight of the person; a seat adjustably mounted on the supportive structure and pivotable in a vertical plane about an ankle point of the person; a backrest adjustably mounted on the seat and pivotable in the vertical plane about a nature pivot point of the torso and thigh lines of the person; resilient means for resiliently supporting the respective seat and backrest in a position in which the person sitting erectly or forwardly for work, and permitting the seat and backrest to pivot in response to the shift center of gravity of the person for rest; and a positive adjustment mechanism mounted on a front of the chair and associated with the resilient means for positively adjusting the resilience of the resilient means by the person while sitting on the chair.",
"The resilient means preferably comprises a first support end pivotly mounted on the supportive structure, a second support end pivotally mounted on the backrest, and a third support end pivotally mounted to the positive adjustment mechanism and slidable relative to the seat for adjusting the resilience of the resilient means.",
"In accordance with one aspect of the invention, a backrest structure for a chair comprising a curved track in fixed relation to a seat of the chair; a carriage slidably mounted on the curved track; a backrest mounted on the carriage; a resilient support mechanism provided for resiliently supporting the carriage and backrest with respect to the seat so that the carriage is biased uppermost to support the backrest in an erect position, and adapted to slide down along the curved track and tilt the backrest rearwards and downwardly in response to a rearward shift of the weight of a person sitting on the chair and leaning against the backrest.",
"The curved track is preferably a circular arc having a radius in a vertical plane generated from a point which substantially matches a natural pivot point of the torso and thigh lines of the person sitting on the chair so-called H point.",
"In a more specific embodiment of the present invention, the chair further includes: a supportive structure adapted to support the chair and the weight of a person sitting on the chair, a curved track mounted on the supportive structure, a carriage slidably mounted on the curved track and attached in a fixed relationship to a base of the seat so that the base is moveable relative to the supportive structure, and a resilient support mechanism is provided for resiliently supporting the base relative to the supportive structure so that the carriage is biased to support the seat on the base in a substantially horizontal position and is enabled to slide down along the curved track and tilt the seat rearwards and downwardly in response to a rearward shift of the weight of the person sitting on the chair.",
"The curved track is a circular arc having a radius in a vertical plane generated from a point which is close to a natural pivot point of the ankle joint of the person.",
"The chair structure according to the present invention is simple and applicable to different styles of work station chairs, such as chairs with or without arm support.",
"The chairs are comfortable and reduce adverse static postural loads and forces, especially in reclined position when angle between torso and thigh is open which are responsible for the fatigue and biomechanical dysfunction.",
"It is easy to adjust the chairs for different weight loads of persons, which can be done by the person while sitting on the chair and reaching for an adjustment knob in front of the chair and under the seat pan.",
"Other features and advantages of the invention will be apparent from the description of a preferred embodiment given hereinafter.",
"BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the nature of the invention, reference will now be made to the preferred embodiment thereof and the accompanying drawings, in which: FIG.",
"1 is a side view of a chair in accordance with the preferred embodiment of the invention; FIG.",
"2 is a partial side view of a frame structure of the chair in FIG.",
"1; FIG.",
"3 is a top end front perspective view of the frame structure in FIG.",
"2; FIG.",
"4a is a side view of a backrest moving rail; FIG.",
"4b is a perspective view of the backrest moving rail in FIG.",
"4a, showing the grooves for bearing balls; FIG.",
"5 is a partial cross-sectional view of the backrest rail assembly taken from lines 5-5 in FIG.",
"3; FIG.",
"6 is a perspective view of the backrest balls cage; FIG.",
"7 is a perspective view of a positive adjustment assembly for adjusting the resilience of the seat and the backrest of the chair; and FIG.",
"8 is a partial side view of the frame structure in FIG.",
"2, showing the positive adjustment in response to the different weight loads of persons utilizing the chair.",
"MODE FOR CARRYING OUT THE INVENTION Referring now to the drawings and, in particular, to FIG.",
"1, there is shown a chair 10 having an upstanding post 12 and a base 14 mounted with rollers 16, as seen in typical work station chairs.",
"The chair 10 also includes a seat portion 18 having a seat 20 that is fixed on carriage 30 to support the weight of the person sitting on the chair, and a backrest portion 22 having a backrest 24 that is mounted to the seat portion 18 for supporting the upper body of the person.",
"The seat portion 18 is rotatable in a horizontal plane about an axis of the post 12 and pivotable in a vertical plane about an ankle point A of the person while the backrest portion 22 is pivotable in the vertical plane about a natural pivot point H is at the intersection of the torso and thigh lines of the person.",
"Having thus structured, the chair 10 is enabled to move and be oriented in any direction as well as provide passive adjustment in response to shift in the weight of the person between a position in which the person sits erectly or leans forward for work and another position in which the person tilts and leans rearwardly in rest.",
"The activation of the mechanism is a result of the combination of the body of the person pivoting about the ankle point A with the upper body of the person pivoting about the H point.",
"The structural details of the chair 10 is now described with reference to FIGS.",
"2 and 3 in which a frame structure of the chair 10 is shown.",
"The seat portion 18 further includes a seat plate 26 which is rectangular, and two side plates 28 extend downwards from two sides of the seat plate 26, respectively.",
"A carriage 30 is fixed to the undersurface of the seat plate 26 and to the rear ends of the two side plates 28, and rolls on a track member 32.The track member 32 is mounted to the sleeve 34 by side plates 36.The track member 32 is a circular arc track having a predetermined radius, and is welded or otherwise fixed to the mounting sleeve 34 through a pair of sleeve side plates 36 in such a position that the central point of the circular arc track 32 is close to the ankle point A of the person sitting on the chair after the chair installation is completed.",
"The sleeve 34 is mounted to the rotatable cylinder of the post 12.The sleeve 34 and the post 12 assembly may include bearings and height adjustment structures which are not shown, but are typical in the existing work station chairs in the market, and are well known by those skilled in the art.",
"A similar carriage and track assembly is used for the backrest of the chair and, therefore, the structural details of the carriage and the track assembly will be described below when the backrest structure is described.",
"The seat portion 18 is resiliently supported by two compressible gas cylinders 38.Each gas cylinder 38 includes a piston rod 40 axially extending from the cylinder 38, terminating at a piston rod end 42.A rear cylinder support rod 44 is supported on the lower end of the sleeve side plates 36, extending transversely with two ends protruding outwardly from the respective sleeve side plates 36.The rear cylinder support rod 44 is perpendicular to the piston rods 40 and each end of the rear cylinder support rod 44 is rotatably received in a concave surface on the piston rod end 42 at either side of the seat portion 18.A cylinder end, not shown, has a similar concave surface to rotatably receive a front cylinder support rod 46 which is parallel to the rear cylinder support rod 44, and adjustably supported by a positive adjustment assembly 48 mounted on the front end of the seat portion 18.Having this arrangement, the seat portion 18 and the weight load of the person sitting on the seat are supported by the post 12 through the gas cylinders 38 when the carriage 30 under the load rolls down along the track member 32 and the gas cylinder 38 is compressed to a certain extent.",
"The resilient force caused by the compressed gas cylinders 38 balances the load.",
"This will be a normal position for a person sitting on the chair in an erect position, as shown in FIG.",
"1.It is noted that the front cylinder support rod 46 is supported on the seat portion 18 and the position thereof relative to the seat portion is not changed in the passive adjustment when the gas cylinder 38 is compressed by the weight load.",
"However, the front cylinder support rod 46 is enabled to be changed in positions relative to the seat portion 18 for different weight loads of persons utilizing the chair when the positive adjustment assembly 48 is adjusted.",
"The detail of the positive adjustment assembly 48 and its operation will be described hereinafter.",
"The seat portion 18 further includes a pair of support side plates 50 and a rear end plate 52.The support side plates 50 are welded or otherwise connected to the rear side of the carriage 30 and the undersurface of the seat plate 26, and interconnected by the rear end plate 52 at the rear ends to form a rigid frame structure of the seat portion 18, providing a base for attachment of the backrest portion 22.A front plate 54 having an L-shape in cross-section is attached to the front end of the seat plate 26 and between two side plates 28 to provide a structural support for the attachment of the positive adjustment assembly 48.A plurality of apertures 56 are defined in the respective seat plate 26, support side plates 50 and the rear end plate 52 to reduce the weight of the frame structure.",
"Mounting bores, not shown, are provided in the seat plate 26 for mounting the seat 20.The backrest portion 22 includes a carriage 58 which is attached to an adapter plate 60.The adapter plate 60 in turn supports a pair of mounting brackets 62 of a fork type which are well known and adapted to support the backrest 24.Any other arrangement can be used to attach the backrest 24.The carriage 58 rolls on a track member 64 which has a circular arc in the vertical plan and terminates at an end plate 66 to stop the downward movement of the carriage 58 relative to the track member 64 at the lowest extremity.",
"The track member 64 is supported on the two support side plates 50 by the end plate 66 and a pair of attachment strips 68.The circular arc of the track member 64 has a predetermined radius and the track member 64 is attached to the seat portion 18 of the chair in such a position that the radius center of the circular arc of the track member 64 substantially matches the natural pivot point H of the torso and thigh lines of the person sitting on the chair after the installation of the chair is completed, as shown in FIG.",
"1.The backrest portion 22 is resiliently supported by a pair of compressible spring rods 70 positioned at the respective sides.",
"Each spring rod 70 is provided with a telescoping rod (not shown) extending through a spiral spring 72 which is pre-compressed between the two ends 74 of the spring rod.",
"The telescoping rod inside the spiral spring 72 permits the spring rod 70 to be compressible and prevents the spiral spring 72 from losing stability and buckle.",
"The front end 74 of the spring rod 70 is provided with a concave surface for rotatably receiving the front cylinder support rod 46 which has its two respective ends protruding outwardly from a pair of slots 76 defined in the respective side plates 28.Similarly, the rear end 74 of the spring rods 70 has a concave surface for rotatably receiving a spring rod support pin 78 which extends perpendicularly to the spring rods 70 and is supported by a pair of brackets 80 at the respective sides of the carriage 58 such that when the person sitting on the chair leans backwards and downwardly, the backrest 24 pivots about the H-point (shown in FIG.",
"1) and the whole backrest portion 22 slides along the track member 64, compressing the spring 70 until the spring force balances a component of the weight load of the person supported by the backrest 24.The carriage and track member assemblies for passively adjustable support of the seat or backrest can be configured in various structures, such as described in the Applicant's Canadian Patent Application No.",
"2,116,079.Another example is described in FIGS.",
"4a, 4b, 5 and 6 according to the preferred embodiment of the invention.",
"These drawings only show the details of the carriage 58 and track members 64 of the backrest portion 22 in order to avoid redundancy.",
"However, the structural features are similarly applied to the carriage 30 and track member 32 assembly of the seat portion 18, except different radii of curvature are required to meet the different requirements of the pivoting points A and H. In FIGS.",
"4a and 4b, a main moving rail 82 is a steel plate formed generally in a circular arc having a radius of curvature generated from the H point when it is installed to the chair.",
"The main moving rail 82 includes two grooves 84 at two sides acting as bearing ball races.",
"The two sides of the steel plate of the main moving rail 82 are bent to form two side flanges 86 to weld a claw rail 88 (see FIGS.",
"3 and 5) at each side.",
"A plastic ball cage 90 is provided to hold two rows of bearing balls at each side in position within the carriage 58.A perspective view of the ball cage 90 is shown in FIG.",
"6.The cage 90 has a radius of curvature similar to the main moving rail 82, and is restrained within the carriage 58 by any means well known to the person skilled in the art.",
"The two rows of bearing balls roll on two respective ball races defined by two respective grooves 94 on each side of the track member 64.FIG.",
"7 illustrates the positive adjustment assembly 48 which includes a threaded bolt 96 (the threads not shown) that are threadedly connected to an adjustment bar 98.Two adjustment bar side plates 100 are welded, or otherwise attached to the two sides of the adjustment bar 98, and each adjustment bar side plate 100 has an aperture for snugly receiving the front cylinder support rod 46.The threaded bolt 96 is rotatably supported at one end by a bracket, not shown, which is fixed to the top on the inner side of the front plate 54 and at the other end is rotatably supported by the shorter portion 102 of the L-shaped front plate 54 (see FIG.",
"2) so that the bolt 96 is rotatable but prevented from axial movement relative to the front plate 54.The front cylinder support rod 46 is slidably received by the two slots 76 which are parallel to the threaded bolt 96 and defined in the side plates-28 to prevent the adjustment bar 98 from rotation with the bolt 96 so that the front cylinder support rod 46 is moved forward or backward along the slots 76 when the bolt 96 is rotated.",
"A knob 104 is mounted to a head portion (not shown) of the threaded bolt 96 which extends outwardly from the shorter section 102 of the L-shaped front plate 54 for the person to conveniently make simultaneous adjustment of the chair and the backrest resiliency while still sitting on the chair.",
"In operation, the person using the chair assumes a working position as shown in FIG.",
"1.In this position, the person is upright or leaning forward over a work table.",
"In such a case, the center of gravity of the person is over the post 12 or forward thereof.",
"The gas cylinders 38 should be sufficient to maintain the seat 20 in a substantially horizontal position and thus the carriage 30 is in its upper position such as shown in FIGS.",
"1, 2 and 8.The weight of the person using the chair will effect the equilibrium of the carriage 30 on the track member 32.In this position, the chair functions as normal work station chair and the backrest 24 receives no or little component of the weight load.",
"The carriage 58 is also in its upmost position.",
"When the person leans back in a rest position, the center of gravity shifts rearwardly relative to the point A to a point where the action moment overcomes the resiliency moment of the gas cylinders 38 to move the front cylinder support rod 46 to the point L1 (see FIG.",
"2), for example.",
"The lower end of the carriage 30 begins to move downwardly following the arcuate path of the track member 32 to the point L2 so that the seat portion 18 tilts backwards and downwardly in an angle B about the ankle point A.",
"Meanwhile, a component of the weight load of the person acts on the backrest 24 to an extent in which the action moment overcomes the resilience of the spring 70 to move the spring rod support pin 78 downwardly and forwardly along the track member 64 until the carriage 58 is stopped by the track and plate 66, as an extreme example.",
"The backrest 24 tilts backwardly and downwardly together with the backrest portion 22 over an angle C about the H point.",
"The combination of the tilting movement of the seat 20 with the tilting movement of the backrest 24 is illustrated in FIG.",
"1.A vertical line P passing through the H point represents an erect position of the person sitting on the chair.",
"When the person leans back to a rest position as described above, the seat 20 with the whole seat portion 18 tilts over the angle B about the ankle point A so that the body of the person pivots down over the angle B about the ankle point A and therefore the natural pivot point H of the torso and thigh lines of the person moves to H1 and the line P moved to P1, no longer being vertical.",
"Because of the tilting movement of the backrest 24 with the whole backrest portion 22, the line P1 further pivots rearwardly and downwardly over the angle C about the pivoting point H1 to a position shown as P2 which represents the position of the upper body of the person in the rest position with the open angle of the torso and thighs.",
"In the combination of the two tilting movements of the chair, there are no parts of the chair having an upwardly moving component which usually exists with most chairs in the prior art and causes the person utilizing the chair to be in an unnatural and uncomfortable position when the feet lose contact with the floor and pressure is applied to the underside of the thighs.",
"FIG.",
"8 illustrates the function of the positive adjustment assembly 48 used in this embodiment.",
"The position for lighter persons is shown in FIG.",
"8 in full lines.",
"When a heavier person is to use the chair, the knob 104 is rotated so as to move the front cylinder support rod 46 along the slots 76 toward the extreme position shown in broken lines or intermediate positions therebetween.",
"Both spring rods 70 and the gas cylinders 38 are further compressed relative to their positions shown in full lines.",
"Being thus pre-compressed, the spring rods 70 and gas cylinders 38 are harder and will balance a more heavier weight load of a person in a normal work position.",
"Because of the orientation of the slots, the gas cylinders 38 are not compressed as much as the spring rod 70 when they are adjusted to the position shown in broken lines.",
"However, the gas cylinders 38 in the position shown in broken lines are further away from the ankle point A, thereby increasing the resistance to downward movement of the carriage 30 because a spring force will produce a bigger moment of force about point A to balance the action moment produced by a heavier weight load of a person utilizing the chair when the acting point of the same spring force is moved farther away from the pivoting point A.",
"Modifications and improvements to the above-described embodiment of the invention may become to those scaled in the art.",
"The foregoing description is intended to be exemplary rather than limiting.",
"The scope of the invention is therefore intended to be limited solely by the scope of the appended claims."
]
] | Patent_10221186 |
[
[
"Compounds for pdt",
"A tetrakis(hydroxyphenyl)chlorin, bacteriochlorin or isobacteriochlorin, derivatised at one or more of the hydroxy groups by addition reaction with a diisocyanate, diisothiocyanate or isocyanate-isothiocyanate at one isocyanate or isothiocyanate group thereof, the other isocyanate or isothiocyanate group being itself derivatised by addition reaction with the hydroxy group of an w-alkylated or acylated poly(alkylene oxide) or to a hydroxy group of a link residue itself carrying a residue of such poly(alkylene oxide)."
],
[
"1.A tetrakis(hydroxyphenyl)chlorin, bacteriochlorin or isobacteriochlorin, derivatised at one or more of the hydroxy groups by addition reaction with a diisocyanate, diisothiocyanate or isocyanate-isothiocyanate at one isocyanate or isothiocyanate group thereof, the other isocyanate or isothiocyanate group being itself derivatised by addition reaction with the hydroxy group of an ω-alkylated or acylated poly(alkylene oxide) or to a hydroxy group of a link residue itself carrying a residue of such poly(alkylene oxide).",
"2.A tetrakis(hydroxyphenyl) chlorin, bacteriochlorin or isobacteriochlorin of formula (12), (13) or (14) or imino tautomer thereof, wherein n=1 to 3 and each substituent R the same or different may be a hydroxyl (OH) group itself free or substituted with a C1 to C12 alkyl or acyl group or otherwise derivatised, but in at least one and desirably more than one instance is of formula (15); where: (i) each X, the same or different is O, S; (ii) Y is O (carbamate or thiocarbamate link); (iii) A is a hydrocarbon group containing 2 to 40 carbon atoms, preferably 4 to 20 carbon atoms and very preferably 6 carbon atoms (this group may be branched or unbranched, cyclic or acyclic, saturated or unsaturated, aliphatic or aromatic); (iv) B is an optional group ((CH2)p—O)q where p=1 to 4; q=0,1 (v) D is a poly(alkylene oxide), preferably polyethylene glycol, with an average molecular weight of at least 200 and not more than 40,000, preferably 750 to 20,000 and very preferably 2,000 to 5,000 Da; (vi) E is an alkyl or acyl group containing 1 to 12 carbon atoms, preferably a methyl group; and any pharmaceutically acceptable derivative of such chlorin, bacteriochlorin or isobacteriochlorin as a salt with a mineral or other acid or a metal complex or a hydrate or other solvate.",
"3.The compounds 7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy polyethylene glycol (average MW=2000) via biscarbamate linkages derived from hexane-1,6-diisocyanate; 7,8,17,18-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=2000) via bis carbamate linkages derived from hexane-1,6-&isocyanate; 7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=5000) via bis carbamate linkages derived from hexane-1,6diisocyanate; 7,8,17,1 8-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl) porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=5000) via bis carbamate linkages derived from hexane-1,6 isocyanate 4.A method of preparation of a medicament for photodynamic therapy of cancer tumour or other diseased tissue, and such therapy itself, wherein use is made of a chlorin, bacteriochlorin, isobacteriochlorin or any other pharmaceutically acceptable compound as in claim 1 or 2.5.The use of compounds according to claim 3 where conditions include, but are not limited to, Barrett's Oesophagus, age-related macular degeneration, keratinosis, diabetic neuropathy, peripheral vascular disease, coronary artery disease, and disorders arising from bacterial or viral infections."
],
[
"<SOH> BACKGROUND <EOH>Photodynamic therapy (PDT) involves the administration of a photosensitising agent for localisation in target diseased tissue followed by irradiation of the target tissue containing the compound with light of a specific and appropriate wavelength.",
"The resulting photoactivated compound, in the presence of oxygen, leads to necrosis of the tissue.",
"The success of this modality is dependent on administration of a compound that is selectively retained in tumour tissue as compared to normal tissue.",
"Thus, on irradiation of the tumour with light of the photoactivating wavelength, the amount of damage caused by necrosis is proportionately higher than that in normal tissue.",
"However, some normal tissue damage typically occurs and one specific side effect seen with the use of many photosensitisers is redness and swelling of the skin on subsequent exposure to normal lighting levels and particularly sunlight.",
"Such side effects are minimised by keeping the patients in subdued light for a prolonged period after treatment, consequently restricting their quality of life.",
"A more efficient delivery of the photosensitiser into tumour tissue, thus providing a much higher tumour to normal tissue ratio of drug concentration could dramatically reduce the potential for skin side effects with this treatment.",
"A group of photosensitising agents have previously been the subject of patents EP 0 337 601 and U.S. Pat.",
"No.",
"4,992,257.These compounds are dihydroporphyrins (chlorins) (1) and the corresponding tetrahydroporphyrins (bacteriochlorins) (2) and (3) of the formulae: wherein each n=1 to 3 and each substituent, R, the same or different, is a hydroxyl (OH) group, each itself free or substituted with an alkyl or acyl group.",
"Salts, internal salts, metal complexes or hydrates or other solvates of the compounds are also covered.",
"The above formulae, it will be appreciated, represent particular tautomers among various possibilities including chlorins as shown below (represented without meso Phenyl groups): The invention covers all tautomers of the above compounds and is not limited to those shown in diagrams.",
"Published Proposals Modification of compounds by PEGylation, that is the direct or indirect attachment of polyethylene glycol chains (PEG), and in principle other poly(alkylene oxide) chains, is known to introduce useful properties.",
"PEG is non-toxic, imparts good water solubility to drug molecules and alters the biodistribution, which can result in a favourable pharmacokinetic profile.",
"The general topic of polyether substituted anti-tumour agents is described in DKFZ's specification PCT EP 91/00992 (WO 91/18630).",
"No particular attention is given to the selection of the linkage between the polyether chain and the anti-tumour agent, the only example disclosed being a triazine introduced by initial activation of the polyether with cyanuric chloride.",
"More recently, DKFZ have described a method for the production of chlorins and bacteriochlorins containing a polyether (WO 98/01156).",
"The method involves initial attachment of the polyether to the porphyrin with subsequent reduction to the corresponding chlorins and bacteriochlorins.",
"Again, no particular attention is given to the nature of the linkage between the polyether and the anti-tumour agent, the only example disclosed being an amide link.",
"PEGylation of compounds (1), (2) and (3) via triazine, ether and ester linkages has been previously reported by us in PCT GB 95/00998 (WO 95/29915).",
"However, lability of the ester linkages and significant difficulties in the scale up of the triazine and ether linked moieties severely limits the practical utility of these compounds.",
"Enzon have also reported polyether compositions containing isocyanate and/or isothiocyanate groups for covalent attachment to bioeffecting substances such as peptides or chemotherapeutics (WO 94/04193).",
"However, in relation to isocyanates and isothiocyanates, coverage is directed to compounds in which bioeffecting substances are attached to both ends of the polyether chain.",
"Outside the PDT field, hexane-1,6-diisocyanate has been used to link PEG to atropine (Zalipsky et al, Eur.",
"Polym.",
"J.",
"1983, 19(12), 1177-1183) and to 5-fluorouracil (Ouchi et al, Drug Design and Discovery 1992, 9, 93-105).",
"Bayer (U.S. Pat.",
"No.",
"4,684,728) have reported a process for improving the solubility in water of a sparingly soluble biologically active compound by reaction to form a derivative carrying the active moiety, a linking group such as an optionally substituted diisocyanate group and a polyether chain.",
"No mention is made of any benefit to the therapeutic profile of such compounds other than the ease of formulation and administration of a water soluble compound.",
"Discussion of Present Work Advances in photodynamic therapy for clinical disease treatment, particularly cancer, depend on developing improved photosensitisers.",
"The desired characteristics of an ideal photosensitiser include selective diseased tissue localisation, activation at long wavelengths so that maximum depth of tissue penetration is shown, and high efficiency as sensitisers.",
"From a formulation and administration point of view, water solubility is also a beneficial attribute.",
"Photodynamic therapy is a dual therapy, which consists of the combined action of photosensitiser and light.",
"In clinical practice the drug is first administered, and then activated by light some time later.",
"The time period between administering the drug and applying the light is called the drug-light interval.",
"It is desirable to apply light at a time when the photosensitiser has accumulated maximally in the target tissue and has been eliminated from the normal surrounding tissue.",
"The principal factor determining the drug-light interval is the drug pharmacokinetic profile which itself varies between every tissue.",
"The drug-light interval has to be suitable for clinical practice.",
"From a clinical standpoint, pharmacokinetics which give a maximum drug concentration in a tumour as soon as possible, for example from a few hours to at most 3 days, together with rapid elimination from the body thereafter, would be ideal.",
"This would allow flexibility in scheduling treatment.",
"In European Patent Specification 0 337 601 (U.S. Pat.",
"No.",
"4,992,257), the applicants disclose compounds with many of the desired characteristics, particularly an extremely high photo efficiency, that is to say the ability to generate free radical species such as singlet oxygen through the absorption of light.",
"The long absorption wavelengths of the molecules, e.g.",
"at 652 nm and 734 nm, penetrates tissue efficiently and thus the sensitisers can be used to treat deep tumours.",
"However, the disclosed compounds do not fulfil all the requirements equally well.",
"A residual disadvantage is the degree of normal tissue photosensitivity, particularly of the skin, that occurs following administration of the sensitiser.",
"This arises from unwanted deposition of the sensitiser in the skin and other normal tissue and is a consequence of imperfect tumour targeting by the drug.",
"The skin photosensitivity can last up to 4 weeks depending on the drug dose administered.",
"At the usual clinical dose of 0.15 mg kg −1 skin sensitivity of, for example, m-THPC (a tetraphenyl chlorin derivative in which each phenyl group carries a m-hydroxy group) lasts for 2-3 weeks.",
"This limits the patient's freedom and is an undesirable restriction.",
"We have sought ways of overcoming normal tissue photosensitivity, particularly of the skin, by converting m-THPC to a polyethylene glycol derivative.",
"This ‘PEGylation’ profoundly alters the bodily distribution in a favourable way by increasing tumour targeting, and at the same time reducing uptake to the skin.",
"The distribution of the compound is altered by PEGylation, due to hydrogen bonding of water to oxygen on the polyethylene glycol chains when the compound is injected into the blood.",
"A ‘water envelope’ forms around the photosensitiser and prevents the compound sticking to the endothelium of blood vessel walls and in turn passing into the surrounding tissue including the skin.",
"This favours uptake to the tumour through the enhanced permeability and retention (EPR) effect.",
"Tumours have a disturbed vasculature and lymphatic drainage, leading to increased accumulation of substances such as drugs in the tumour compared to normal tissue (R. Duncan and F. Spreafico, Clin.",
"Pharmacokinet.",
"1994, 27, 290-306).",
"This effect can be enhanced with higher molecular weight compounds.",
"The net effect of PEGylation is that the compound is favourably redirected from the skin and other normal tissues towards the tumour, thus reducing the degree of skin sensitivity.",
"It has been confirmed experimentally, for example, that PEGylation can produce a favourable tissue re-distribution.",
"This was shown in a mouse experimental model in which an outstanding difference between muscle and tumour photosensitivity during PDT was found for the triazine-linked derivative (Grahn et al., Proc.",
"SPE 1997, 3191, 180-6).",
"Three days after the PEGylated m-THPC was administered it was found that the muscle was no longer photosensitive, while the tumour retained its maximum sensitivity to light for at least 15 days after drug administration.",
"This presented ample time for tumour-selective treatment, but did indicate the less desirable characteristic of tumour persistence with this derivative.",
"Other work previously performed with triazine-linked PEG derivatives [applicants PCT Patent specification WO 95/29915, (PCT/GB95/00998)] confirmed that the pharmacokinetics with the triazine linkage were rather too prolonged for routine clinical use.",
"In particular, excretion from the liver was very slow indeed, which is undesirable pharmaceutically.",
"An alternative linker to the triazine molecule was sought, including a glycidic ether with amino PEG and also an hexylbiscarbamate linker.",
"The PEGylated derivatives of m-THPC with triazine and carbamate links have very different and unexpected pharmacokinetics to each other and m-THPC.",
"The triazine-linked compound (SPC 0038B) is excreted from the liver more slowly than m-THPC, while the carbamate derivative (SPC 0172) is excreted in a comparable period to m-THPC.",
"The solubilities of the compounds, and hence ease of pharmaceutical preparation, were enhanced to levels of up to 52 mg/mL in water, compared to m-THPC, which is insoluble in aqueous solvents.",
"The linker group should provide a stable point of PEG attachment, permitting reasonable in vivo circulation and should be available via a practical and robust synthesis.",
"It should not, however, affect the PDT efficiency of the drug molecule.",
"The method of Zalipsky was modified and utilised for a two step synthesis from the chlorin and bacteriochlorin molecules to their PEGylated derivatives.",
"Analysis of these compounds using gel permeation chromatography (GPC) allowed separation of lesser PEGylated forms, but high performance liquid chromatography (HPLC) proved superior with separation between the peaks of 0.8 min.",
"Reaction products were also analysed by UV/Visible spectroscopy, which gave a quantitative measurement of molecular weight (mw) using the formula: in-line-formulae description=\"In-line Formulae\" end=\"lead\"?",
"Apparent mw=mw (chlorin)× A (1%, 1 cm) (chlorin)/ A (1%, 1 cm) (PEG chlorin).",
"in-line-formulae description=\"In-line Formulae\" end=\"tail\"?",
"The applicants work has thus built on previous work in trying to develop an ideal sensitiser.",
"Unpredictably, the carbamate-linked polyethylene glycol derivatives have an excellent and preferred pharmacokinetic profile from a clinical point of view and exhibit less potential to cause cutaneous photosensitivity.",
"Furthermore, studies in Balb/c mice bearing colo26, a murine colorectal cancer, show that the photodynamic effect of the carbamate-linked derivative in tumour is maximum at 2 days and that it has the same PDT activity as m-THPC itself This is considerably more active than the triazine-linked compound.",
"Thus overall, the PEGylated carbamate-linked compound appears to add new features, which enhance the desired characteristics of the sensitiser.",
"The Invention The present invention summarised below and set out in the claims thus concerns the derivatisation or partial derivatisation of the phenolic groups of compounds of formulae (1), (2) and (3) with poly(alkylene oxides) using a carbamate or thiocarbamate link: (X═O, S) formed by addition reaction of the compounds with an isocyanate (—N═C═O) or isothiocyanate (—N═C═S) group of a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate, the poly(alkylene oxide) chain being attached directly or indirectly by addition at the other group and its terminal hydroxyl group being etherified or esterified with for example a C 1-12 alkyl or acyl group of which methyl is the most preferred.",
"The reactions, which may be carried out in any convenient order, result in compounds of formulae: and imino-tautomers thereof wherein n=1 to 3 and R′ may be the same or different, is a hydroxyl (—OH) group, each itself free or substituted with an alkyl or acyl group, but in at least one, preferably more than one instance is as follows: where: (i) each X, the same or different, is O, S; (ii) Y is O (carbamate or thiocarbamate link); (iii) A is a hydrocarbon group containing 2 to 40 carbon atoms, preferably 4 to 20 carbon atoms and very preferably 6 carbon atoms.",
"This group may be branched or unbranched, cyclic or acyclic, saturated or unsaturated, aliphatic or aromatic; (iv) B is an optional group ((CH 2 ) p —O) q where p=1 to 4; q=0,1; (v) D is a poly(alkylene oxide), preferably polyethylene glycol, with an average molecular weight of at least 200 and not more than 40,000, preferably 750 to 20,000 and very preferably 2,000 to 5,000 Da; (vi) E is an alkyl or acyl group containing 1 to 12 carbon atoms, preferably a methyl group.",
"In any of the above compounds derivatives such as salts with mineral acids (e.g.",
"hydrochlorides, sulphates), internal salts, metal complexes (e.g.",
"with Zn, Ga), or hydrates and other solvates may be formed.",
"Suitable diisocyanates include butane-1,4-diisocyanate, hexane-1,6-diisocyanate, octane-1,8-diisocyanate, dodecane-1,1 2-iisocyanate, 2-methylpentane-1,5-diisocyanate, toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, cyclohexane-trans-1,4diisocyanate, dicyclohexylmethane-4,4′-diisocyanate, diphenylmethane-3,4′-diisocyanate, xylene diisocyanate and 2,4,4-trimethylhexylmethylene diisocyanate.",
"Corresponding diisothiocyanates and isocyanate-isothiocyanates are also appropriate.",
"The most preferred linker is hexane-1,6-diisocyanate.",
"Chemistry Compounds of types (12), (13) and (14) may be prepared in a two step process.",
"(i) activation of poly(alkylene oxide) by reaction with a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate (e.g.",
"hexane-1,6-diisocyanate) in a suitable inert, anhydrous solvent (e.g.",
"toluene) with or without a catalyst (e.g.",
"dibutyl tin dilaurate), with or without a tertiary organic base (e.g.",
"triethylamine) at a temperature between 0 and 110° C. (ii) coupling of the activated poly(alkylene oxide) to the reduced porphyrin in a suitable inert solvent (e.g.",
"toluene) with or without a catalyst (e.g.",
"dibutyl tin dilaurate), with or without a tertiary organic base (e.g.",
"triethylamine) at a temperature between 0 and 110° C. Synthesis of compounds may also be achieved by reversing the order of the steps, namely activation of the reduced porphyrin by reaction with the diisocyanate, diisothiocyanate or isocyanate-isothiocyanate followed by coupling with the poly(alkylene oxide).",
"However, the former approach is preferred.",
"Routes of Administrations By parenteral or any other suitable route in per se known manner.",
"Pharmaceutical Presentations Any suitable presentation as known in the field, including, but not limited to: i) injectable solution ii) freeze dried powder for reconstitution and injection iii) infusion solution for addition to saline or other vehicle iv) tablet or capsule for oral administration.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF INVENTION The invention relates to poly(alkylene oxide) substituted photosensitising compounds and to their use in photodynamic therapy of cancerous and other diseased tissues.",
"BACKGROUND Photodynamic therapy (PDT) involves the administration of a photosensitising agent for localisation in target diseased tissue followed by irradiation of the target tissue containing the compound with light of a specific and appropriate wavelength.",
"The resulting photoactivated compound, in the presence of oxygen, leads to necrosis of the tissue.",
"The success of this modality is dependent on administration of a compound that is selectively retained in tumour tissue as compared to normal tissue.",
"Thus, on irradiation of the tumour with light of the photoactivating wavelength, the amount of damage caused by necrosis is proportionately higher than that in normal tissue.",
"However, some normal tissue damage typically occurs and one specific side effect seen with the use of many photosensitisers is redness and swelling of the skin on subsequent exposure to normal lighting levels and particularly sunlight.",
"Such side effects are minimised by keeping the patients in subdued light for a prolonged period after treatment, consequently restricting their quality of life.",
"A more efficient delivery of the photosensitiser into tumour tissue, thus providing a much higher tumour to normal tissue ratio of drug concentration could dramatically reduce the potential for skin side effects with this treatment.",
"A group of photosensitising agents have previously been the subject of patents EP 0 337 601 and U.S. Pat.",
"No.",
"4,992,257.These compounds are dihydroporphyrins (chlorins) (1) and the corresponding tetrahydroporphyrins (bacteriochlorins) (2) and (3) of the formulae: wherein each n=1 to 3 and each substituent, R, the same or different, is a hydroxyl (OH) group, each itself free or substituted with an alkyl or acyl group.",
"Salts, internal salts, metal complexes or hydrates or other solvates of the compounds are also covered.",
"The above formulae, it will be appreciated, represent particular tautomers among various possibilities including chlorins as shown below (represented without meso Phenyl groups): The invention covers all tautomers of the above compounds and is not limited to those shown in diagrams.",
"Published Proposals Modification of compounds by PEGylation, that is the direct or indirect attachment of polyethylene glycol chains (PEG), and in principle other poly(alkylene oxide) chains, is known to introduce useful properties.",
"PEG is non-toxic, imparts good water solubility to drug molecules and alters the biodistribution, which can result in a favourable pharmacokinetic profile.",
"The general topic of polyether substituted anti-tumour agents is described in DKFZ's specification PCT EP 91/00992 (WO 91/18630).",
"No particular attention is given to the selection of the linkage between the polyether chain and the anti-tumour agent, the only example disclosed being a triazine introduced by initial activation of the polyether with cyanuric chloride.",
"More recently, DKFZ have described a method for the production of chlorins and bacteriochlorins containing a polyether (WO 98/01156).",
"The method involves initial attachment of the polyether to the porphyrin with subsequent reduction to the corresponding chlorins and bacteriochlorins.",
"Again, no particular attention is given to the nature of the linkage between the polyether and the anti-tumour agent, the only example disclosed being an amide link.",
"PEGylation of compounds (1), (2) and (3) via triazine, ether and ester linkages has been previously reported by us in PCT GB 95/00998 (WO 95/29915).",
"However, lability of the ester linkages and significant difficulties in the scale up of the triazine and ether linked moieties severely limits the practical utility of these compounds.",
"Enzon have also reported polyether compositions containing isocyanate and/or isothiocyanate groups for covalent attachment to bioeffecting substances such as peptides or chemotherapeutics (WO 94/04193).",
"However, in relation to isocyanates and isothiocyanates, coverage is directed to compounds in which bioeffecting substances are attached to both ends of the polyether chain.",
"Outside the PDT field, hexane-1,6-diisocyanate has been used to link PEG to atropine (Zalipsky et al, Eur.",
"Polym.",
"J.",
"1983, 19(12), 1177-1183) and to 5-fluorouracil (Ouchi et al, Drug Design and Discovery 1992, 9, 93-105).",
"Bayer (U.S. Pat.",
"No.",
"4,684,728) have reported a process for improving the solubility in water of a sparingly soluble biologically active compound by reaction to form a derivative carrying the active moiety, a linking group such as an optionally substituted diisocyanate group and a polyether chain.",
"No mention is made of any benefit to the therapeutic profile of such compounds other than the ease of formulation and administration of a water soluble compound.",
"Discussion of Present Work Advances in photodynamic therapy for clinical disease treatment, particularly cancer, depend on developing improved photosensitisers.",
"The desired characteristics of an ideal photosensitiser include selective diseased tissue localisation, activation at long wavelengths so that maximum depth of tissue penetration is shown, and high efficiency as sensitisers.",
"From a formulation and administration point of view, water solubility is also a beneficial attribute.",
"Photodynamic therapy is a dual therapy, which consists of the combined action of photosensitiser and light.",
"In clinical practice the drug is first administered, and then activated by light some time later.",
"The time period between administering the drug and applying the light is called the drug-light interval.",
"It is desirable to apply light at a time when the photosensitiser has accumulated maximally in the target tissue and has been eliminated from the normal surrounding tissue.",
"The principal factor determining the drug-light interval is the drug pharmacokinetic profile which itself varies between every tissue.",
"The drug-light interval has to be suitable for clinical practice.",
"From a clinical standpoint, pharmacokinetics which give a maximum drug concentration in a tumour as soon as possible, for example from a few hours to at most 3 days, together with rapid elimination from the body thereafter, would be ideal.",
"This would allow flexibility in scheduling treatment.",
"In European Patent Specification 0 337 601 (U.S. Pat.",
"No.",
"4,992,257), the applicants disclose compounds with many of the desired characteristics, particularly an extremely high photo efficiency, that is to say the ability to generate free radical species such as singlet oxygen through the absorption of light.",
"The long absorption wavelengths of the molecules, e.g.",
"at 652 nm and 734 nm, penetrates tissue efficiently and thus the sensitisers can be used to treat deep tumours.",
"However, the disclosed compounds do not fulfil all the requirements equally well.",
"A residual disadvantage is the degree of normal tissue photosensitivity, particularly of the skin, that occurs following administration of the sensitiser.",
"This arises from unwanted deposition of the sensitiser in the skin and other normal tissue and is a consequence of imperfect tumour targeting by the drug.",
"The skin photosensitivity can last up to 4 weeks depending on the drug dose administered.",
"At the usual clinical dose of 0.15 mg kg−1 skin sensitivity of, for example, m-THPC (a tetraphenyl chlorin derivative in which each phenyl group carries a m-hydroxy group) lasts for 2-3 weeks.",
"This limits the patient's freedom and is an undesirable restriction.",
"We have sought ways of overcoming normal tissue photosensitivity, particularly of the skin, by converting m-THPC to a polyethylene glycol derivative.",
"This ‘PEGylation’ profoundly alters the bodily distribution in a favourable way by increasing tumour targeting, and at the same time reducing uptake to the skin.",
"The distribution of the compound is altered by PEGylation, due to hydrogen bonding of water to oxygen on the polyethylene glycol chains when the compound is injected into the blood.",
"A ‘water envelope’ forms around the photosensitiser and prevents the compound sticking to the endothelium of blood vessel walls and in turn passing into the surrounding tissue including the skin.",
"This favours uptake to the tumour through the enhanced permeability and retention (EPR) effect.",
"Tumours have a disturbed vasculature and lymphatic drainage, leading to increased accumulation of substances such as drugs in the tumour compared to normal tissue (R. Duncan and F. Spreafico, Clin.",
"Pharmacokinet.",
"1994, 27, 290-306).",
"This effect can be enhanced with higher molecular weight compounds.",
"The net effect of PEGylation is that the compound is favourably redirected from the skin and other normal tissues towards the tumour, thus reducing the degree of skin sensitivity.",
"It has been confirmed experimentally, for example, that PEGylation can produce a favourable tissue re-distribution.",
"This was shown in a mouse experimental model in which an outstanding difference between muscle and tumour photosensitivity during PDT was found for the triazine-linked derivative (Grahn et al., Proc.",
"SPE 1997, 3191, 180-6).",
"Three days after the PEGylated m-THPC was administered it was found that the muscle was no longer photosensitive, while the tumour retained its maximum sensitivity to light for at least 15 days after drug administration.",
"This presented ample time for tumour-selective treatment, but did indicate the less desirable characteristic of tumour persistence with this derivative.",
"Other work previously performed with triazine-linked PEG derivatives [applicants PCT Patent specification WO 95/29915, (PCT/GB95/00998)] confirmed that the pharmacokinetics with the triazine linkage were rather too prolonged for routine clinical use.",
"In particular, excretion from the liver was very slow indeed, which is undesirable pharmaceutically.",
"An alternative linker to the triazine molecule was sought, including a glycidic ether with amino PEG and also an hexylbiscarbamate linker.",
"The PEGylated derivatives of m-THPC with triazine and carbamate links have very different and unexpected pharmacokinetics to each other and m-THPC.",
"The triazine-linked compound (SPC 0038B) is excreted from the liver more slowly than m-THPC, while the carbamate derivative (SPC 0172) is excreted in a comparable period to m-THPC.",
"The solubilities of the compounds, and hence ease of pharmaceutical preparation, were enhanced to levels of up to 52 mg/mL in water, compared to m-THPC, which is insoluble in aqueous solvents.",
"The linker group should provide a stable point of PEG attachment, permitting reasonable in vivo circulation and should be available via a practical and robust synthesis.",
"It should not, however, affect the PDT efficiency of the drug molecule.",
"The method of Zalipsky was modified and utilised for a two step synthesis from the chlorin and bacteriochlorin molecules to their PEGylated derivatives.",
"Analysis of these compounds using gel permeation chromatography (GPC) allowed separation of lesser PEGylated forms, but high performance liquid chromatography (HPLC) proved superior with separation between the peaks of 0.8 min.",
"Reaction products were also analysed by UV/Visible spectroscopy, which gave a quantitative measurement of molecular weight (mw) using the formula: Apparent mw=mw (chlorin)×A(1%, 1 cm) (chlorin)/A(1%, 1 cm) (PEG chlorin).",
"The applicants work has thus built on previous work in trying to develop an ideal sensitiser.",
"Unpredictably, the carbamate-linked polyethylene glycol derivatives have an excellent and preferred pharmacokinetic profile from a clinical point of view and exhibit less potential to cause cutaneous photosensitivity.",
"Furthermore, studies in Balb/c mice bearing colo26, a murine colorectal cancer, show that the photodynamic effect of the carbamate-linked derivative in tumour is maximum at 2 days and that it has the same PDT activity as m-THPC itself This is considerably more active than the triazine-linked compound.",
"Thus overall, the PEGylated carbamate-linked compound appears to add new features, which enhance the desired characteristics of the sensitiser.",
"The Invention The present invention summarised below and set out in the claims thus concerns the derivatisation or partial derivatisation of the phenolic groups of compounds of formulae (1), (2) and (3) with poly(alkylene oxides) using a carbamate or thiocarbamate link: (X═O, S) formed by addition reaction of the compounds with an isocyanate (—N═C═O) or isothiocyanate (—N═C═S) group of a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate, the poly(alkylene oxide) chain being attached directly or indirectly by addition at the other group and its terminal hydroxyl group being etherified or esterified with for example a C1-12 alkyl or acyl group of which methyl is the most preferred.",
"The reactions, which may be carried out in any convenient order, result in compounds of formulae: and imino-tautomers thereof wherein n=1 to 3 and R′ may be the same or different, is a hydroxyl (—OH) group, each itself free or substituted with an alkyl or acyl group, but in at least one, preferably more than one instance is as follows: where: (i) each X, the same or different, is O, S; (ii) Y is O (carbamate or thiocarbamate link); (iii) A is a hydrocarbon group containing 2 to 40 carbon atoms, preferably 4 to 20 carbon atoms and very preferably 6 carbon atoms.",
"This group may be branched or unbranched, cyclic or acyclic, saturated or unsaturated, aliphatic or aromatic; (iv) B is an optional group ((CH2)p—O)q where p=1 to 4; q=0,1; (v) D is a poly(alkylene oxide), preferably polyethylene glycol, with an average molecular weight of at least 200 and not more than 40,000, preferably 750 to 20,000 and very preferably 2,000 to 5,000 Da; (vi) E is an alkyl or acyl group containing 1 to 12 carbon atoms, preferably a methyl group.",
"In any of the above compounds derivatives such as salts with mineral acids (e.g.",
"hydrochlorides, sulphates), internal salts, metal complexes (e.g.",
"with Zn, Ga), or hydrates and other solvates may be formed.",
"Suitable diisocyanates include butane-1,4-diisocyanate, hexane-1,6-diisocyanate, octane-1,8-diisocyanate, dodecane-1,1 2-iisocyanate, 2-methylpentane-1,5-diisocyanate, toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, cyclohexane-trans-1,4diisocyanate, dicyclohexylmethane-4,4′-diisocyanate, diphenylmethane-3,4′-diisocyanate, xylene diisocyanate and 2,4,4-trimethylhexylmethylene diisocyanate.",
"Corresponding diisothiocyanates and isocyanate-isothiocyanates are also appropriate.",
"The most preferred linker is hexane-1,6-diisocyanate.",
"Chemistry Compounds of types (12), (13) and (14) may be prepared in a two step process.",
"(i) activation of poly(alkylene oxide) by reaction with a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate (e.g.",
"hexane-1,6-diisocyanate) in a suitable inert, anhydrous solvent (e.g.",
"toluene) with or without a catalyst (e.g.",
"dibutyl tin dilaurate), with or without a tertiary organic base (e.g.",
"triethylamine) at a temperature between 0 and 110° C. (ii) coupling of the activated poly(alkylene oxide) to the reduced porphyrin in a suitable inert solvent (e.g.",
"toluene) with or without a catalyst (e.g.",
"dibutyl tin dilaurate), with or without a tertiary organic base (e.g.",
"triethylamine) at a temperature between 0 and 110° C. Synthesis of compounds may also be achieved by reversing the order of the steps, namely activation of the reduced porphyrin by reaction with the diisocyanate, diisothiocyanate or isocyanate-isothiocyanate followed by coupling with the poly(alkylene oxide).",
"However, the former approach is preferred.",
"Routes of Administrations By parenteral or any other suitable route in per se known manner.",
"Pharmaceutical Presentations Any suitable presentation as known in the field, including, but not limited to: i) injectable solution ii) freeze dried powder for reconstitution and injection iii) infusion solution for addition to saline or other vehicle iv) tablet or capsule for oral administration.",
"PREPARATIVE EXAMPLES Example 1 7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy polyethylene glycol (average MW=2000) via biscarbamate linkages derived from hexane-1,6-diisocyanate.",
"[Compound (12); n=1: meta substitution on all aryl groups; X═O; A=(CH2)6; Y═O; q=0; D=PEG (average MW=2000); E=CH3] Part 1: Preparation of Activated mPEG (ω-methoxy Polyethylene Glycol) A solution of mPEG (average MW=2000, 40 g) in toluene was dried azeotropically for 4 h and added dropwise over a 2 h period to a mixture of anhydrous toluene (100 mL), hexane-1,6-diisocyanate (16.2 mL) and dibutyl tin dilaurate (0.5 mL).",
"After standing overnight under anhydrous conditions, the product was precipitated by the addition of hexane (200 mL).",
"The solid was collected by filtration, reprecipitated from toluene/hexane and dried under vacuum.",
"This yielded the product as a white powder (38 g).",
"Analysis by non-aqueous titration gave an isocyanate assay of 95% of theory.",
"Molecular weight as determined by titration of NCO groups: mPEG2000-hexylcarbamateisocyanate 2160 (requires 2168).",
"IR (nujol, cm−1) 3300 (NH); 2250 (NCO); 1715, 1535 (HN—COO); 1110 (CH2OCH2); AδH (CCl4) 1.3-1.6 (m, CH2), 3.6 (s, OCH2).",
"This material was used immediately in the second part of the synthesis.",
"Part 2: Coupling of Activated mPEG to m-THPC A mixture of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (300 mg) and activated mPEG (as prepared in Part 1) (8.95 g) in anhydrous toluene was stirred overnight under nitrogen at 30-60° C. HPLC analysis on an aliquot indicated >95% tetraPEGylation.",
"The product was precipitated by the addition of hexane to the stirred contents at room temperature.",
"The solid was collected by filtration, washed with hexane and dried under vacuum The product was then purified by reverse phase chromatography, eluting with methanol/water.",
"After removal of the methanol under reduced pressure, the solution was freeze dried to yield the product as a dark brown solid.",
"Example 2 7,8,17,18-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=2000) via bis carbamate linkages derived from hexane-1,6-diisocyanate.",
"[Compound (13); n=1; meta substitution on all aryl groups; X═O; A=(CH2)6; Y═O; q=0; D=PEG (average MW=2000); E=CH3] By replacing 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with 7,8,17,18-tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin and toluene with 1,4-dioxan as solvent in Example 1, Part 2, the title compound was prepared as a brown powder.",
"Example 3 7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=5000) via bis carbamate linkages derived from hexane-1,6-diisocyanate.",
"[Compound (12); n=1; meta substitution on all aryl groups; X═O; A=(CH2)6; Y═O; q=0; D=PEG (average MW=5000); E=CH3] By replacing mPEG (average MW=2000) with mPEG (average MW=5000) in Example 1, Part 1, [Molecular weight as determined by titration of NCO groups: mPEG5000-hexylcarbamateisocyanate 4944 (requires 5168)] the title compound was prepared as a brown powder.",
"Example 4 7,8,17,18-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatised with ω-methoxy poly(ethylene glycol) (average MW=5000) via bis carbamate linkages derived from hexane-1,6diisocyanate.",
"[Compound (13); n=1; meta substitution on all aryl groups; X═O; A=(CH2)6; Y═O; q=0; D=PEG (average MW=5000); E=CH3] By replacing mPEG (average MW=2000) with mPEG (average MW=5000) in Example 1, Part 1 and 7,8-dihydro-5, 10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with 7,8,17,18-tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin in Example 1, Part 2, the title compound was prepared as a brown powder.",
"Biology In the following account of work done: SPC 0172 is carbamate-linked PEG2000 m-THPC, compound name tetrakis (6′-(methoxy PEG 2000 carbamate)1′-isocyanate hexamethylene) derivative of 7,8dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin.",
"This compound is shown in example 1.SPC 0038B is a corresponding triazine compound, tetrakis (ω-methoxypolyethylene glycol [MW=2000] triazine of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin.",
"m-THPC is temoporfin or 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, the basis of SPC 0172 and SPC 0038B.",
"Spectroscopic Properties of SPC 0172 SPC 0172 has a very similar absorption spectrum to that of SPC 0038B which in turn was similar to that of m-THPC (absorption peaks for m-THPC in the 500-700 nm range at 516, 542, 594 and 650 nm).",
"FIG.",
"1 (Absorbance, A against wavelength, λ) shows the UV-visible absorbance spectra of SPC 0172 and SPC 0038B in 0.25 μM aqueous solution measured using a Hitachi U3000 spectrophotometer.",
"SPC 0172 has a somewhat lower molar extinction coefficient at the red peak than does m-THPC (ca 22,000 L mol−1 cm−1 compared to ca 30,000 L mol−1 cm−1).",
"The fluorescence emission spectrum in ethanol of SPC 0172 is very similar to those of SPC 0038B and m-THPC in the same solvent.",
"All three compounds show a fluorescence emission peak between 655-660 nm when excited by light in the Soret band region (405 nm).",
"m-THPC fluorescence is severely quenched in aqueous solution owing to the formation of aggregates.",
"Both SPC 0038B and SPC 0172 fluoresce more weakly in aqueous solution than in ethanol (47% and 36% respectively) indicating that some aggregation or other conformational change takes place when they are dissolved in water.",
"Both the PEGylated photosensitisers exhibit greatly improved solubility in water (at least 50 mg/ml) compared to m-THPC, which is insoluble.",
"Uptake by Tumour Cells in Culture Colo26 mouse colorectal tumour cells were grown in culture using standard methods.",
"Confluent monolayers of the cells kept at 37° C. in the dark were exposed to 1.5 μM m-THPC, SPC 0038B or SPC 0172 added to the incubation medium for periods of between 3 and 72 hours.",
"At the end of the set time period, the culture medium containing the added photosensitiser was removed and the cells washed with cold phosphate buffered saline.",
"The cells were freed from the culture flask by treatment with trypsin solution.",
"The viable cell count was determined using a standard haemocytometer and the photosensitiser extracted from the cells by treatment with methanol:DMSO (4:1, v:v).",
"Cell extracts were frozen in liquid nitrogen and stored at −70° C. for later analysis.",
"The photosensitiser content of the cell extract was determined by direct fluorescence using standard curves of photosensitiser and correcting for extraction efficiency from the cell suspension.",
"The uptake of the three photosensitisers is shown in FIG.",
"2 temoporfin uptake by tumour cells in culture, FIG.",
"3 SPC0172 uptake by tumour cells in culture and FIG.",
"4 SPC0038B uptake by tumour cells in culture, all at 37° C. (millions of molecules per cell, m against time t (hours)).",
"The amount of photosensitiser is expressed as the number of molecules of photosensitiser (in millions) present within a single cell at each time point The data in FIGS.",
"2 to 4 represents the mean value obtained from two experiments and the bars represent the range.",
"The uptake of m-THPC is rapid, reaching a peak around 24 hours after which the photosensitiser content of the cells declines.",
"The uptake of SPC 0038B, in contrast is very slow, with a barely detectable increase in cell drug concentration at 72 hours compared with 3 hours of incubation.",
"After 72 hours the cell photosensitiser content is about a factor of 100 less than that of m-THPC.",
"The uptake of SPC 0172 shows the same slower uptake as does SPC 0038B, uptake being relatively linear up to the final measurement point at 72 hours of incubation.",
"However, the amount of photosensitiser taken up is greater.",
"After 72 hours of incubation there is about 5 times as much SPC 0172 in each cell as SPC 0038B, but again this is much less than the peak cellular m-THPC content.",
"The mechanism of photosensitiser uptake by cells remains a subject of study.",
"In the case of small hydrophobic sensitisers such as m-THPC, which are generally presented to cells bound to protein or lipoprotein in biological systems, a role for specific lipoprotein receptors has been implicated.",
"The uptake mechanisms of photosensitiser PEG conjugates have received much less attention, although fluorescence microscopy studies show that the initial intracellular distribution is limited to foci in the cytoplasm in the vicinity of the plasma membrane suggesting that endocytosis and sequestration in vesicles plays a part.",
"One way in which uptake mechanisms may be studied is by determining the influence of temperature.",
"In general, active (energy-dependent) uptake processes are inhibited in cells maintained at 4° C. compared to those at 37° C. Passive (energy-independent) uptake processes are much less temperature-dependent.",
"The effect of temperature on uptake of the three photosensitisers at 1.5 μM is shown in Table 1.TABLE 1 Effect of temperature on photosensitiser uptake by Colo26 cells 6 hour uptake 24 hour uptake Rate of uptake 6-24 h (millions of molecules (millions of molecules (thousands of molecules per cell) per cell) per cell per hour) Compound 4° C. 37° C. 4° C. 37° C. 4° C. 37° C. m-THPC 0.82 79.2 1.33 208 28 7184 SPC 0038B 0.56 0.82 0.64 0.86 4 2 SPC 0172 1.08 1.12 1.78 2.08 39 53 It can be seen that at 4° C. the uptake of m-THPC is inhibited by a factor of over 250.Such strong temperature dependence suggests that the uptake of this compound occurs by an active, energy-dependent process.",
"The uptake of SPC 0038B appears to be twice as great in the cold than at 37° C. whilst that of SPC 0172 was reduced on cooling to about 75% of that at 37° C. The uptake of both PEGylated photosensitisers therefore was relatively unaffected by temperature compared to the effect with m-THPC.",
"This suggests that SPC 0172 and SPC 0038B are taken up into tumour cells by means of an energy-independent mechanism.",
"SPC 0172 is taken up more efficiently by tumour cells through this mechanism than SPC 0038B.",
"Pharmacokinetic Characteristics An assessment of the relative pharmacokinetic characteristics has been made by comparison of the plasma concentration-time profiles for SPC 0172, SPC 0038B and m-THPC in mice bearing a subcutaneously implanted tumour.",
"Adult female Balb/c mice bearing the syngeneic colo26 tumour implanted subcutaneously were produced using methods previously described (Ansell et al.",
"Lasers in Medical Science 1997, 12, 336-41).",
"Groups of mice of weight 19-22 g were given the photosensitisers at a dose of 0.88 μmol kg−1 (equivalent to 0.6 mg kg−1 in the case of m-THPC) by injection into the tail vein.",
"The solutions for injection of each compound were prepared at a concentration of 0.352 μmol ml−1 so that a typical 20 g mouse would be injected with a volume of 50 μl.",
"For M-THPC, which is insoluble in water, the injection solution was prepared using a PEG400: ethanol:water vehicle (30:20:50, w/w), whilst SPC 0172 and SPC 0038B were prepared in water for injection.",
"Animals were sacrificed at 6, 24, 72 and 192 hours after injection of each photosensitiser.",
"Immediately after sacrifice, blood was obtained by cardiac puncture, and centrifuged at 13,000×g for three minutes.",
"The resulting supernatant (blood plasma) was aspirated and stored at −70° C. for subsequent analysis.",
"The photosensitiser content of each sample was measured from the fluorescence of an extract obtained using methanol:DMSO (3:5, v:v).",
"A 50 μl portion of each extract was transferred to a disposable fluorescence cuvette and the fluorescence determined using an excitation wavelength of 418 nm and emission wavelength of 650 nm with an eight second response setting.",
"The assay was calibrated using stock solutions of each compound in methanol, further diluted in methanol:water (1:1, v/v) and extracted with methanol:DMSO as described above.",
"For each photosensitiser the fluorescence yield was found to be linear up to a sample concentration of 200 nmoles per ml.",
"The measurement protocol was designed to dilute each sample to a sufficient extent to avoid interference by endogenous chromophores (absorbance of samples <<0.1 at the excitation wavelength).",
"The results obtained are presented in FIG.",
"5 (nano-moles per ml, n against time, t (hours)) plasma photosensitiser concentrations following injection of 0.88 nanomoles of each photosensitiser per g live weight.",
"The data show that higher plasma levels of SPC 0038B occur in these mice compared to either m-THPC or SPC 0172 given at equimolar doses intravenously.",
"It is also clear that the plasma concentrations of both m-THPC and SPC 0172 diminish to background levels more rapidly than SPC 0038B.",
"In effect SPC 0038B persists longer and exhibits a longer terminal elimination phase from plasma compared to either SPC 0172 or m-THPC.",
"The data indicate a fundamental difference in the plasma concentration-time profile for both PEGylated photosensitisers, with SPC 0172 showing lower absolute levels and more rapid elimination.",
"Other tissues were taken from the mice at the same timepoints as the blood samples and treated using a similar extraction and fluorescence analysis method to determine photosensitiser levels.",
"Levels in liver mapped the same trends as seen in plasma for the three photosensitisers FIG.",
"6 (nano-moles per g wet weight, w against time, t (hours)) shows liver photosensitiser concentrations following injection of 0.88 nanomoles of each photosensitiser per g live weight.",
"Once again liver levels for SPC 0038B were higher and more persistent whereas levels for SPC 0172 were lower and eliminated more quickly.",
"Levels in skin are presented in FIG.",
"7 (nano-moles per g wet weight, w against time, t (hours)), skin photosensitiser concentrations following injection of 0.88 nanomoles of each photosensitiser per g live weight.",
"Relatively high concentrations of m-THPC occurred in skin 24 hours after administration falling to lower levels from 72 hours onwards.",
"SPC 0172 concentrations in skin were low throughout the 6-192 hour measurement period suggesting a lower potential to cause cutaneous photosensitivity.",
"Skin concentrations of SPC 0038B were higher than those of SPC 0172 or m-THPC between 72 and 192 hours suggesting a higher potential to cause cutaneous photosensitivity during this period.",
"The selectivity for tumour specific tissue distribution was also evaluated in this work.",
"Tissue selectivity was evaluated as the tumour:muscle concentration ratio over the duration of the observation period (4-192 hours post-administration).",
"This ratio provided a direct measure of the normal tissue to tumour tissue differential in concentrations and allows selection of an optimal treatment time for PDT that minimises the potential to cause collateral damage of normal tissues.",
"The results obtained are presented in FIG.",
"8 (Tumour:muscle ratio, r against time, t (hours)), Tumour:muscle concentration ratio of the three photosensitisers.",
"The results indicate an optimal tumour:muscle concentration ratio for SPC 0172, and hence tumour PDT, occurs at 72 hours post-administration.",
"The ratio value of about 7.5 with SPC 0172 was similar to that that obtained with SPC 0038B at the same timepoint and 2-3 fold higher than that observed with m-THPC.",
"Both PEGylated photosensitisers therefore exhibit improved tumour targeting in comparison to m-THPC.",
"In comparison to SPC 0172, the favourable tumour: muscle ratio for SPC 0038B does not decline as rapidly beyond 72 hours suggesting a broader window for tumour PDT but also the less attractive feature of persistent PDT bioactivity in the target tissue.",
"SPC 0172 exhibits the most appropriate profile for tumour PDT of these two PEGylated photosensitisers with a single optimum at 72 hours and low skin levels suggesting a reduced potential to cause cutaneous photosensitivity.",
"Published work (Grahn et al, Proc.",
"SPIE 1997, 3191, 180-6) with SPC 0038B in this model support the observation that this photosensitiser is eliminated more slowly than m-THPC.",
"This work also showed that SPC 0038B concentrations measured in muscle peaked at or before 72 hours after injection whilst those in tumour peaked between 72 and 144 hours, after which they declined.",
"An optimal drug-light treatment interval for tumour PDT specific tissue necrosis later than 72 hours post-administration was therefore indicated.",
"Allied measurements of tumour bioactivity showed sustained PDT tumour necrosis from SPC 0038B occurred for PDT treatment times ranging from 4-15 days.",
"This report also confirms the persistence of liver concentrations of SPC 0038B.",
"These data further illustrate a phenomenon of persistent tumour levels and sustained potential for PDT-mediated tumour necrosis with SPC 0038B that are sub-optimal characteristics of an ideal PDT agent.",
"Cutaneous Photosensitivity Cutaneous photosensitivity was determined in adult female Balb/c mice, held under standard conditions and allowed access to food and water ad libitum.",
"Groups of mice of weight 19-22 g were given the photosensitisers at a dose of 0.88 μmol kg−1 by injection into the tail vein.",
"The solutions for injection of each compound were prepared at a concentration of 0.352 μmol ml−1 so that a typical 20 g mouse would be injected with a volume of 50 μl.",
"Each mouse was weighed immediately before injection and the injected volume adjusted to give the correct dose.",
"For m-THPC the injection solution was prepared using the standard PEG400: ethanol:water vehicle, whilst SPC 0172 and SPC 0038B were prepared in water for injection.",
"At 24 or 72 hours after photosensitiser injection one ear of each mouse was irradiated with full-spectrum xenon light from a 1 KW clinical photo-irradiator (Model UV-90, Applied Photophysics, London).",
"The light was delivered to the ear using a 7 mm diameter light guide (Serial SU3) placed lightly against the ear.",
"The light dose given was 40 Joules per cm2, which was achieved by exposing the ear for 63 seconds to 245 mW of light from the light guide having a contact area of 0.384 cm2.The light guide filtered infra-red and ultra-violet light, so as to minimise heating of the ear and damage resulting from ultra-violet light irradiation.",
"Animals treated with light at the 24 or 72 hour drug-light intervals were killed at 48 or 24 hours respectively after irradiation of the ears.",
"The oedema of the irradiated and control ears was assessed by measuring ear thickness.",
"The thickness of the ear was measured at three sites on the upper third of the ear using a fixed-force micrometer with a vernier-interpolated resolution of 2 μm and an accuracy of 10 μm (Neill instruments).",
"The results indicate that ear swelling or thickness, as a surrogate endpoint for cutaneous photosensitivity, is lower with SPC 0172 than with SPC 0038B or m-THPC following light irradiation either at 24 or 72 hours after photosensitiser administration (Table 2).",
"SPC 0038B exhibited superiority over m-THPC to cause less photosensitivity at the 24 hour drug-light interval but this difference was negated even reversed by the 72 hour drug-light interval.",
"These observations match the trends in skin concentrations of these photosensitisers noted earlier.",
"The indication therefore is that SPC 0172 is superior to SPC 0038B or m-THPC in terms of potential to cause cutaneous photosensitivity.",
"TABLE 2 Differences in ear thickness (irradiated-control) in μm for mice injected with 0.88 μmol kg−1 of each photosensitiser 24 or 72 hours before light irradiation.",
"24 h drug-light interval 72 h drug-light interval Photosensitiser Mean SEM 1,2 N Mean SEM 1,2 N SPC 0038B 25 7 (16 to 38) 3 4 6 (−2 to 15) 3 SPC 0172 2 (22; −18) 2 0 (8; −8) 2 m-THPC 41 25 (8 to 90) 3 2 7 (−12 to 13) 3 1 Standard error of mean.",
"2 Range or individual values provided in parentheses.",
"Tumour PDT Activity Information on the tumour PDT activity have been obtained in the aforementioned mouse model bearing the syngeneic colo26 tumour implanted subcutaneously at the top of the left hind leg (approximately 1 cm lateral to the spine).",
"The tumours were used after 12 to 16 days when they had reached an average diameter of 8 to 10 mm.",
"For drug injection and irradiation animals were sedated with Hypnorm diluted in water (1:3).",
"SPC 0172 demonstrated tumour necrosis at 1 day after drug injection which increased in extent at two days post-injection (Table 3).",
"The effect observed at two days post-injection comprised full tumour necrosis, an effect equivalent to that observed with m-THPC at half the dose in the same timescale.",
"Examination of the treated sites with SPC 0172 suggested that tumour necrosis was accompanied by only limited damage of the surrounding skin and underlying muscle, particularly at the two day drug-light interval.",
"Further experiments using the same dose of SPC 0172 and a light dose of 20 J cm−2 were carried out at a range of drug-light intervals.",
"Full thickness tumour damage was observed at all drug-light intervals up to 72 hours.",
"Comparative data on SPC 0038B at its optimal drug-light interval of 72 hours in this model show 10-20 fold less potency on a dose level and degree of tumour necrosis basis in comparison to SPC 0172 and m-THPC.",
"The results suggest that the in-vivo potency of SPC 0172 as a tumour PDT agent is similar to that of m-THPC at its optimal dose (0.88 μmol kg−1) whereas SPC 0038B is somewhat less potent.",
"TABLE 3 Tumour necrosis induced in the mouse implanted colorectal tumour model by the 3 photosensitisers Light applied Biological Drug-light at 652 nm (J effect Drug Dose interval cm−1) at 100 (mm of tumour Compound (μmol kg−1) (hours) mW cm−1 necrosis) SPC 0172 1.76 24 20 3.8 ± 1.3 SPC 0172 1.76 48 10 Full thickness (>6 mm) SPC 0038B 4.4 72 5 3.3 ± 0.7 1 m-THPC 0.88 24 5 5.8 ± 0.4 m-THPC 0.88 48 5 5.6 ± 0.8 1 Taken from Grahn et al Proc.",
"SPIE 1997, 3191, 180-6.Conclusion In summary, PEGylated photosensitisers exhibit improved water solubility compared to the parent photosensitiser m-THPC making them far more pharmaceutically acceptable for parenteral administration.",
"They are taken up by tumour cells in a different energy-independent mechanism compared to m-THPC in which SPC 0172 is more efficient than SPC 0038B.",
"Improved tumour targeting over m-THPC was achieved by these PEGylated molecules with SPC 0172 showing a superior pharmacokinetic profile compared to SPC 0038B with a peak in tumour: normal tissue ratio within 72 hours of administration and more rapid elimination from the tissues and plasma.",
"A lower potential to cause cutaneous photosensitivity was evident with SPC 0172 over SPC 0038B or m-THPC and a similar potency to m-THPC for tumour necrosis following PDT by SPC 0172 was shown whereas SPC 0038B was less active.",
"It is therefore apparent that carbamate-linked PEGylated photosensitisers as evidenced by SPC 0172 are more ideal tumour PDT agents than triazine-linked PEGylated photosensitisers as evidenced by SPC 0038B, and possess improved features in comparison to m-THPC."
]
] | Patent_10221218 |
[
[
"Method for producing an electric machine subassembly produced according to said method, and electric machine with said subassembly",
"The invention relates to a method for the manufacture of an assembly of an electric machine with the steps of: manufacturing at least one portion of a coil of a rotor or stator of an electric machine; positioning the at least one portion of the coil in a casting mould, whose cavity shape reproduces at least a portion of the assembly; filling the casting mould with pulverised soft iron particles with a thermoplastic synthetic coating; heat treatment of the filled casting mould until the synthetic coating of the soft iron particles softens; allowing the filled casting mould to cool; and removing the assembly from the casting mould."
],
[
"1.A method for the manufacture of an assembly of an electric machine with the following steps: manufacturing of at least one portion of a winding of a rotor or stator of an electric machine; positioning of the at least one portion of the winding in a casting mould, whose cavity shape reproduces at least a portion of the assembly; filling of the casting mould with pulverised soft iron particles with a thermoplastic synthetic coating; heat treatment of the filled casting mould until the synthetic coating of the soft iron particles softens; allowing the filled casting mould to cool; and removing the assembly from the casting mould.",
"2.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein the soft iron particles filled into the casting mould are mechanically compacted prior to the heat treatment.",
"3.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein a recess is formed in the soft iron particles filled into the casting mould, for the accommodation of an electronic circuit.",
"4.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein the heat treatment step is performed in such a manner that the insulation of the coil is not damaged.",
"5.The method for the manufacture of an assembly of an electric machine according to claim 4, wherein the heat treatment is performed at approx.",
"250°-350° C., until the soft iron particles are surrounded by a uniformly melted synthetic coating, without the iron particles coming into a significant magnetically (or electrically) conductive connection with each other.",
"6.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein at least one portion of the coil is manufactured from aluminium or copper.",
"7.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein the coil is manufactured by casting.",
"8.The method for the manufacture of an assembly of an electric machine according to claim 1, wherein the coil is manufactured by punching recesses from a circular cylindrical tube of aluminium or copper.",
"9.An assembly for an electric machine, wherein a coil is at least partially surrounded by iron, characterised in that the iron is formed by interconnected soft iron particles with a thermoplastic synthetic coating.",
"10.An electric machine with a stator and a rotor, characterised in that the stator and/or the rotor is formed by an assembly according to claim 9."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to a method for the manufacture of an assembly of an electric machine and an assembly which can be manufactured to this method, as well as an electric motor comprising such an assembly.",
"Definitions The term “assembly of an electric machine” as used herein is to be understood as covering both a stator of an electric machine and, under certain conditions which will be described later, a rotor.",
"An electric machine in this context is both an electric motor as well as an electric generator, regardless of whether the electric machine is designed as a rotary machine or, for example, as a linear motor.",
"State of the Art In the state of the art it is known to build the magnetically conductive parts of an electric machine (in particular of an alternating current machine) of laminations because of the otherwise occurring eddy currents.",
"For the laminated cores, typically single or double-sided insulated sheet panels of a thickness between 0.35 mm and 1.5 mm of dynamo sheet metal (hot rolled electric-quality sheet) are cut to correspondingly shaped strips.",
"From these strips stator and rotor laminations with respective recesses are punched, whereby it is aimed at producing as little scrap as possible.",
"The stator and rotor laminations manufactured in this manner are stacked upon each other and under pressure united to laminated cores in which the recesses of the individual laminations form grooves for the stator or rotor windings to be subsequently installed.",
"These stator or rotor windings are preassembled and insulated preformed coils which are inserted into the open grooves.",
"In the case of closed grooves the winding wires must be threaded in from the face of the laminated cores so that a corresponding coil is created.",
"The above described approach has been developed in several detail aspects.",
"In the manufacture of electric machines it has, in principle, been the proven and employed method for many decades and has not seen any fundamental changes.",
"The above explanation, however, also shows that the manufacture is very expensive, that punching scrap is produced in the punching operation of the laminations, and that the installation operation of the windings or winding wires, respectively, into the groove is very time consuming.",
"Moreover, so-called winding overhangs (portions of the windings which protrude beyond the face of the laminated cores) are inevitable, which do not contribute effectively to the performance of the electric machine.",
"A development originating from a completely different field consists of providing finely pulverised soft iron particles with a thermoplastic coating of a synthetic material.",
"Particularly well suited for this purpose is an iron powder where the iron particles with a diameter of approx.",
"20-150 μm have a coating of polyethylene of approx.",
"2-10 μm.",
"Such a powder is, for example, described in SAE Technical Paper Series, “P/M Cores for Pulsed DC Ignition Systems”, David E. Gay, International Congress & Exposition, Detroit, Mich., Feb. 24-27, 1997.The materials described therein are suited for the implementation of the invention.",
"It is, however, also possible to use corresponding materials with similar properties from other manufacturers for the inventive method.",
"The inventors have found that such a material, in a surprising manner, is excellently suited to revolutionise the concept of the manufacturing process for electric machines or their assemblies, respectively, with the potential to manufacture electric machines, whose electric properties, in some cases, are even better than those of conventional electric machines which are comparable with respect to size, type of construction, etc.",
"Problem on Which the Invention is Based Starting from the initially described approach for the manufacture of an electric machine, the invention is based on the problem to fundamentally simplify this operation in order to render it significantly more economic.",
"The inventive solution of this object consists in a method for the manufacture of an assembly of an electric machine with the following steps: manufacturing of at least one portion of a winding of a rotor or stator of an electric machine; positioning of the at least one portion of the winding in a casting mould, whose cavity shape reproduces at least a portion of the assembly; filling of the casting mould with pulverised soft iron particles with a thermoplastic synthetic coating; heat treatment of the filled casting mould until the synthetic coating of the soft iron particles softens; allowing the filled casting mould to cool; and removing the assembly from the casting mould.",
"This completely novel way of manufacturing electric machines or their components, respectively, has a number of significant advantages: Obviously, the expensive assembly step of placing the winding into the grooves of the laminations is omitted.",
"In addition, assembly and alignment of the laminations relative to each other is no longer necessary.",
"Furthermore, the winding can virtually be completely surrounded by the soft iron particles, so that no winding overhangs develop.",
"In addition, the wires of the winding are not accommodated in preformed grooves.",
"Rather, the soft iron particles adhere directly to the wires of the winding, so that virtually no air gap exists between the wires of the winding and the moulded body of soft iron particles (except the material thickness of the synthetic coating of the soft iron particles).",
"detailed-description description=\"Detailed Description\" end=\"lead\"?",
"Advantageous Developments of the Invention In an embodiment of the inventive method the soft iron particles which have been introduced into the casting mould are mechanically compacted to such an extent before the heat treatment, that air pockets are no longer present within the soft iron particle powder or at the interfaces between the powder and the coil(s).",
"This can be achieved by shaking, vibrating of the casting mould, compressing by means of a male mould, or the like, as well as by combinations of the individual above mentioned methods.",
"In a further embodiment of the inventive method, a recess is formed in the soft iron particles in the casting mould before or during the heat treatment, into which an electronic circuit (e.g.",
"a control circuit or a sensor assembly) for the electric machine is installed.",
"The ends of the coil(s) may end in this recess so that the electronic circuit can be directly connected with them.",
"The heat treatment step is performed in such a manner that the insulation of the winding wire (of paint, synthetic material, enamel, or ceramic material) is not damaged.",
"Preferably, the heat treatment is performed at approx.",
"2500-350° C., until the soft iron particles are surrounded by a uniformly melted synthetic coating, without the iron particles coming into a significant magnetically (or electrically) conductive connection with each other.",
"In a preferred embodiment of the inventive method, which is particularly suited for the manufacture of a rotor of an asynchronous motor, the coil forming the squirrel-cage rotor is made by casting.",
"Alternatively, the coil can be manufactured by punching recesses from a circular cylindrical tube of aluminium or copper.",
"In both cases, the coil which is manufactured in this manner is subsequently surrounded by the soft iron particles and heat treated.",
"The invention also relates to an assembly for an electric machine, wherein one coil is at least partially surrounded by iron, with the iron being formed by soft iron particles interconnected by a thermoplastic synthetic coating.",
"The invention finally relates to an electric machine with a stator and/or a rotor which is formed by such an assembly.",
"In a preferred embodiment of the invention, prior to compacting and heat treating a shaft is installed in the rotor and in the powder, with the shaft comprising an external polygon or projections, in order that the shaft be accommodated secured against rotation and captively after compacting and the heat treatment (and cooling down) in the rotor assembly.",
"It is understood from the above description that other electric components with inductive components, too, for example reactors or transformers can be manufactured in accordance with this novel method.",
"detailed-description description=\"Detailed Description\" end=\"tail\"?"
],
[
"BACKGROUND OF THE INVENTION The invention relates to a method for the manufacture of an assembly of an electric machine and an assembly which can be manufactured to this method, as well as an electric motor comprising such an assembly.",
"Definitions The term “assembly of an electric machine” as used herein is to be understood as covering both a stator of an electric machine and, under certain conditions which will be described later, a rotor.",
"An electric machine in this context is both an electric motor as well as an electric generator, regardless of whether the electric machine is designed as a rotary machine or, for example, as a linear motor.",
"State of the Art In the state of the art it is known to build the magnetically conductive parts of an electric machine (in particular of an alternating current machine) of laminations because of the otherwise occurring eddy currents.",
"For the laminated cores, typically single or double-sided insulated sheet panels of a thickness between 0.35 mm and 1.5 mm of dynamo sheet metal (hot rolled electric-quality sheet) are cut to correspondingly shaped strips.",
"From these strips stator and rotor laminations with respective recesses are punched, whereby it is aimed at producing as little scrap as possible.",
"The stator and rotor laminations manufactured in this manner are stacked upon each other and under pressure united to laminated cores in which the recesses of the individual laminations form grooves for the stator or rotor windings to be subsequently installed.",
"These stator or rotor windings are preassembled and insulated preformed coils which are inserted into the open grooves.",
"In the case of closed grooves the winding wires must be threaded in from the face of the laminated cores so that a corresponding coil is created.",
"The above described approach has been developed in several detail aspects.",
"In the manufacture of electric machines it has, in principle, been the proven and employed method for many decades and has not seen any fundamental changes.",
"The above explanation, however, also shows that the manufacture is very expensive, that punching scrap is produced in the punching operation of the laminations, and that the installation operation of the windings or winding wires, respectively, into the groove is very time consuming.",
"Moreover, so-called winding overhangs (portions of the windings which protrude beyond the face of the laminated cores) are inevitable, which do not contribute effectively to the performance of the electric machine.",
"A development originating from a completely different field consists of providing finely pulverised soft iron particles with a thermoplastic coating of a synthetic material.",
"Particularly well suited for this purpose is an iron powder where the iron particles with a diameter of approx.",
"20-150 μm have a coating of polyethylene of approx.",
"2-10 μm.",
"Such a powder is, for example, described in SAE Technical Paper Series, “P/M Cores for Pulsed DC Ignition Systems”, David E. Gay, International Congress & Exposition, Detroit, Mich., Feb. 24-27, 1997.The materials described therein are suited for the implementation of the invention.",
"It is, however, also possible to use corresponding materials with similar properties from other manufacturers for the inventive method.",
"The inventors have found that such a material, in a surprising manner, is excellently suited to revolutionise the concept of the manufacturing process for electric machines or their assemblies, respectively, with the potential to manufacture electric machines, whose electric properties, in some cases, are even better than those of conventional electric machines which are comparable with respect to size, type of construction, etc.",
"Problem on Which the Invention is Based Starting from the initially described approach for the manufacture of an electric machine, the invention is based on the problem to fundamentally simplify this operation in order to render it significantly more economic.",
"The inventive solution of this object consists in a method for the manufacture of an assembly of an electric machine with the following steps: manufacturing of at least one portion of a winding of a rotor or stator of an electric machine; positioning of the at least one portion of the winding in a casting mould, whose cavity shape reproduces at least a portion of the assembly; filling of the casting mould with pulverised soft iron particles with a thermoplastic synthetic coating; heat treatment of the filled casting mould until the synthetic coating of the soft iron particles softens; allowing the filled casting mould to cool; and removing the assembly from the casting mould.",
"This completely novel way of manufacturing electric machines or their components, respectively, has a number of significant advantages: Obviously, the expensive assembly step of placing the winding into the grooves of the laminations is omitted.",
"In addition, assembly and alignment of the laminations relative to each other is no longer necessary.",
"Furthermore, the winding can virtually be completely surrounded by the soft iron particles, so that no winding overhangs develop.",
"In addition, the wires of the winding are not accommodated in preformed grooves.",
"Rather, the soft iron particles adhere directly to the wires of the winding, so that virtually no air gap exists between the wires of the winding and the moulded body of soft iron particles (except the material thickness of the synthetic coating of the soft iron particles).",
"Advantageous Developments of the Invention In an embodiment of the inventive method the soft iron particles which have been introduced into the casting mould are mechanically compacted to such an extent before the heat treatment, that air pockets are no longer present within the soft iron particle powder or at the interfaces between the powder and the coil(s).",
"This can be achieved by shaking, vibrating of the casting mould, compressing by means of a male mould, or the like, as well as by combinations of the individual above mentioned methods.",
"In a further embodiment of the inventive method, a recess is formed in the soft iron particles in the casting mould before or during the heat treatment, into which an electronic circuit (e.g.",
"a control circuit or a sensor assembly) for the electric machine is installed.",
"The ends of the coil(s) may end in this recess so that the electronic circuit can be directly connected with them.",
"The heat treatment step is performed in such a manner that the insulation of the winding wire (of paint, synthetic material, enamel, or ceramic material) is not damaged.",
"Preferably, the heat treatment is performed at approx.",
"2500-350° C., until the soft iron particles are surrounded by a uniformly melted synthetic coating, without the iron particles coming into a significant magnetically (or electrically) conductive connection with each other.",
"In a preferred embodiment of the inventive method, which is particularly suited for the manufacture of a rotor of an asynchronous motor, the coil forming the squirrel-cage rotor is made by casting.",
"Alternatively, the coil can be manufactured by punching recesses from a circular cylindrical tube of aluminium or copper.",
"In both cases, the coil which is manufactured in this manner is subsequently surrounded by the soft iron particles and heat treated.",
"The invention also relates to an assembly for an electric machine, wherein one coil is at least partially surrounded by iron, with the iron being formed by soft iron particles interconnected by a thermoplastic synthetic coating.",
"The invention finally relates to an electric machine with a stator and/or a rotor which is formed by such an assembly.",
"In a preferred embodiment of the invention, prior to compacting and heat treating a shaft is installed in the rotor and in the powder, with the shaft comprising an external polygon or projections, in order that the shaft be accommodated secured against rotation and captively after compacting and the heat treatment (and cooling down) in the rotor assembly.",
"It is understood from the above description that other electric components with inductive components, too, for example reactors or transformers can be manufactured in accordance with this novel method."
]
] | Patent_10221816 |
[
[
"Molecular weight markers for western blot",
"A method for the preparation of molecular weight standards having peroxidase activity which can be directly identified at the time of the detection of the antigen in all western blot techniques based on the use of peroxidase."
],
[
"1.A method per the preparation of molecular weight standards for western blot, comprising the conjugation of two or more (poly)peptides, which can be the same or different, at least one of them having peroxidase activity, by means of a cross-linker.",
"2.The method as claimed in claim 1, further comprising the separation of the conjugation products according to their molecular weights.",
"3.A method as claimed in claims 1-2, wherein the (poly)peptide having peroxidase activity is selected from the group consisting of heme proteins/peptides.",
"4.A method as claimed in claim 3, wherein said (poly)peptide having peroxidase activity is selected from the group of cytochrome C, microperoxidase, lactoperoxidase and horseradish peroxidase.",
"5.A method as claimed in claims 1-2, wherein the protein having no peroxidase activity is selected from the group of lysozyme, carbonic anhydrase, ovalbumin and serum albumin.",
"6.A method as claimed in any one of the preceding claims, wherein the cross linker is selected from irreversible bifunctional cross-linkers.",
"7.Molecular weight standards for western blot obtainable by the method of claims 1-6.8.Molecular weight standards as claimed in claim 7, selected from the group consisting of: cytochrome C conjugate, microperoxidase and lysozyme conjugate, carbonic anhydrase and microperoxidase conjugate, cytochrome C and ovalbumin conjugate, cytochrome C and serum albumin conjugate.",
"9.A kit for the preparation of the molecular weight standards as claimed in claims 7 and 8, comprising, in separate containers, the (poly)peptide having peroxidase activity and the cross-linker, and optionally the protein having no peroxidase activity and the incubation buffer.",
"10.The use of a (poly)peptide having peroxidase activity for the preparation of a molecular weight standard for western blot.",
"11.The use as claimed in claim 10, wherein said (poly)peptide having peroxidase activity is selected from heme (poly)peptides.",
"12.The use of small size (poly)peptides having peroxidase activity as protein markers in those techniques that require enzyme labeling."
],
[
"<SOH> TECHNOLOGICAL BACKGROUND <EOH>Since the introduction of western blot technique, radioisotope-labelled antibodies have been progressively replaced by the enzyme-labelled antibodies, mainly for their easier handling and shorter detection time.",
"At present, the most popular methods for western blot detection of proteins are based on peroxidase conjugates (antibodies, protein A, protein G, avidin, streptavidin, and the like) whose enzymatic activity is revealed by an appropriate substrate, either chromogenic or chemiluminescent.",
"In common laboratory procedure, the molecular weight standards used in western blot, after transfer on membrane and staining, are marked by pencil to exactly attribute the corresponding bands.",
"This procedure is also followed when using pre-stained standards, as the colors are generally altered or attenuated at the end of the western blot procedure, and therefore some bands disappear or are hardly detectable on the membrane.",
"Moreover, when using chemiluminescent methods, all these standards have to be marked again on the autoradiography film, to avoid uncertain determination of the detected band.",
"Molecular weight standards directly detectable on the membrane exist, but they require specific conditions or treatments.",
"For example, biotin-conjugated standards are only detectable when biotin-(strept)avidin systems are used.",
"A set of standards sharing a common epitope, detectable by a specific antibody included within the secondary antibody solution, is commercially available (Santa Cruz Biotechnology, Santa Cruz, Calif., U.S.A.).",
"However, this method implies purchasing both markers and secondary antibodies from a unique manufacturer.",
"No standards detectable by the peroxidase enzymatic activity directly in the membrane used for western blot are currently available.",
"Methods for the detection of heme-proteins, after SDS-PAGE and transfer onto a nitrocellulose filter, based on the use of peroxidase substrates, both chromogenic and chemiluminescent, have been reported (Dorward D. W., 1993, Anal.",
"Biochem.",
"209:219; Vargas C. et al., 1993, Anal.",
"Biochem.",
"209:323).",
"Furthermore, electrophoresis standards obtained by polymerization of a protein that gives raise to a discrete series of homopolymers are commercially available (Sigma, U.S.A., catalogue A9392, H2757, H2507, P8906)."
],
[
"The present invention generally relates to the techniques for the separation and detection of proteins by electrophoresis and western blot.",
"More particularly, the invention relates to a method for the preparation of molecular weight markers (standards) for use in western blot.",
"TECHNOLOGICAL BACKGROUND Since the introduction of western blot technique, radioisotope-labelled antibodies have been progressively replaced by the enzyme-labelled antibodies, mainly for their easier handling and shorter detection time.",
"At present, the most popular methods for western blot detection of proteins are based on peroxidase conjugates (antibodies, protein A, protein G, avidin, streptavidin, and the like) whose enzymatic activity is revealed by an appropriate substrate, either chromogenic or chemiluminescent.",
"In common laboratory procedure, the molecular weight standards used in western blot, after transfer on membrane and staining, are marked by pencil to exactly attribute the corresponding bands.",
"This procedure is also followed when using pre-stained standards, as the colors are generally altered or attenuated at the end of the western blot procedure, and therefore some bands disappear or are hardly detectable on the membrane.",
"Moreover, when using chemiluminescent methods, all these standards have to be marked again on the autoradiography film, to avoid uncertain determination of the detected band.",
"Molecular weight standards directly detectable on the membrane exist, but they require specific conditions or treatments.",
"For example, biotin-conjugated standards are only detectable when biotin-(strept)avidin systems are used.",
"A set of standards sharing a common epitope, detectable by a specific antibody included within the secondary antibody solution, is commercially available (Santa Cruz Biotechnology, Santa Cruz, Calif., U.S.A.).",
"However, this method implies purchasing both markers and secondary antibodies from a unique manufacturer.",
"No standards detectable by the peroxidase enzymatic activity directly in the membrane used for western blot are currently available.",
"Methods for the detection of heme-proteins, after SDS-PAGE and transfer onto a nitrocellulose filter, based on the use of peroxidase substrates, both chromogenic and chemiluminescent, have been reported (Dorward D. W., 1993, Anal.",
"Biochem.",
"209:219; Vargas C. et al., 1993, Anal.",
"Biochem.",
"209:323).",
"Furthermore, electrophoresis standards obtained by polymerization of a protein that gives raise to a discrete series of homopolymers are commercially available (Sigma, U.S.A., catalogue A9392, H2757, H2507, P8906).",
"DISCLOSURE OF THE INVENTION It has now been found that the peroxidase activity can be retained and, at least partially, stabilized against denaturation by conjugation of two or more proteins having peroxidase activity or of proteins having peroxidase activity with other proteins without peroxidase activity.",
"This allowed to prepare molecular weight standards which can directly be detected on the membrane used for western blot by reaction with a suitable peroxidase substrate.",
"Therefore, the invention relates to a method for the preparation of molecular weight standards for use in western blot, comprising the conjugation of two or more (poly)peptides, that can be the same or different, at least one of which has peroxidase activity, by a cross-linker, and, optionally, the subsequent separation of the conjugation products so as to cover the desired molecular weight range.",
"“Conjugation” herein means the link of two or more (poly)peptides by means of a suitable compound (cross-linker) capable of covalently binding the two (poly)peptides.",
"Said cross-linkers can be selected from the group consisting of all the irreversible bifunctional cross-linkers.",
"Disuccinimidyl suberate (DSS) or its water-soluble analog disuccinimidyl glutarate (DSG) are preferably used.",
"“Proteins having peroxidase activity” herein preferably means the proteins or (poly)peptides having an heme group.",
"Cytochrome C, microperoxidase, lactoperoxidase and horseradish peroxidase are non-limitative examples of proteins having peroxidase activity which can be used according to the invention.",
"On the other hand, “protein without peroxidase activity” can be any non-heme protein with known molecular weight having constant, reproducible migration under the different conditions used for the electrophoresis, and capable of being easily and stably conjugated with the above mentioned cross-linker.",
"The non-heme protein is preferably selected from the group consisting of lysozyme, carbonic anhydrase, ovalbumin and serum albumin.",
"Conjugation can be either single, i.e.",
"two equal or different proteins are joined, or multiple, i.e.",
"more than two proteins may be variously joined by means of the cross-linker.",
"Conjugation multiplicity can be adjusted by varying the conditions of the reaction between the cross-linker and the protein or mixture of different proteins, particularly the reaction time or the reagent concentration.",
"After completion of the reaction, a mixture of products with different conjugation multiplicities will be obtained.",
"The conjugation provides different products, the amount of each being inversely related to its molecular weight.",
"The product mixture can be used either as such or separated into its different components.",
"In this way, standards covering various molecular weight ranges can be prepared.",
"In order to define more accurately the molecular weight, for each preparation a calibration may be performed by SDS-PAGE and/or western blot or with a commercial or laboratory standard consisting of proteins with known molecular weight.",
"It should however be considered that western blot is not an analytical technique for the determination of molecular weight.",
"Therefore, standards whose molecular weight is not precisely determined can be used, as it is commonly the case with commercial pre-stained standards, where conjugation with the chromophore molecule induces an alteration in the electrophoretic migration.",
"The standards, after electrophoretic separation and transfer on the western blot membrane, can be directly detected on the membrane by reaction with a chromogenic substrate or on the autoradiography film, when using chemiluminescent substrates.",
"Examples of conventional chromogenic and chemiluminescent substrates are: 4-chloro-1-naphthol and diaminobenzidine (chromogenic); ECL (Amersham), Supersignal (Pierce) and DuoLux (Vector) (chemiluminescent).",
"Since cross-linking can prevent the complete protein denaturation under SDS-PAGE conditions, resulting in unpredictable migration, the apparent M.W.",
"of the labeled proteins should preferably be estimated by calibration with commercial markers.",
"Furthermore, in the case of conjugation products between different proteins, the protein having peroxidase activity should preferably have low molecular weight, to increase the conjugation (peroxidase/protein) ratio thus providing better detection of the standards.",
"Microperoxidases are preferred, such as microperoxidase MP-11, a cytochrome C peptide having 11 amino acids, or microperoxidases MP-9 and MP-8, respectively nona- or octapeptide, described by Plattner et al., 1977, Histochemistry 53:223-242, or by Harbury et al., 1960.J.",
"Biol.",
"Chem., 235:3649-3655, or by Kraehenbuhl et al., 1974, J. Exp.",
"Med., 139:208-223; furthermore, MP-6 and MP-17, having 6 and 17 amino acids respectively, (Spee et al, 1996, Eur.",
"J. Biochem.",
"241:215-220), can be used.",
"As a rule, any peptide derived from peroxidase proteins by proteolysis or synthesis can be used, as far as it retains its enzymatic activity.",
"In principle, the molecular weight standards of the invention proved to be stable after treatment in SDS-PAGE and western blot, although the peroxidase activity is better preserved under mild denaturing conditions, avoiding high-temperature.",
"A further aspect of the invention relates to a kit for the preparation of molecular weight standards including, in separate containers, the protein/peptide having peroxidase activity, the cross-linker and optionally the protein having no peroxidase activity, the incubation buffer and the buffer to stop the reaction.",
"The buffers can be Tris or lysine buffer, lysine buffer being preferred in that it provides a better protein-membrane interaction.",
"The effectiveness of small (poly)peptides having peroxidase activity as protein markers allows their use in place of more hindering peroxidase proteins.",
"Secondary antibodies, avidin or streptavidin usually employed in western blot, E.L.I.S.A, immunocytochemistry and immunohistochemistry, are examples of molecules that can preferably be conjugated with the microperoxidase.",
"The resulting conjugate will have a remarkably reduced size and a higher per-molecule number of markers, thus increasing its efficiency.",
"Therefore, according to a further aspect, the invention relates to the use of conjugation products prepared with small size (polypeptides having peroxidase activity, preferably cytochrome C and microperoxidase, in those techniques where enzyme labeling is required.",
"DESCRIPTION OF THE FIGURE lane 1: (from bottom to top) cytochrome C monomer, dimer and trimer; lane 2: as in lane 1, after separating and mixing the oligomers to give bands of comparable intensity; lane 3: lysozyme conjugated with microperoxidase (from bottom to top) monomer, dimer, trimer; lane 4: carbonic anhydrase conjugated with microperoxidase; lane 5: (from bottom to top) cytochrome C monomer, dimer and trimer as in lane 2; ovalbumin conjugated with cytochrome C; bovine serum albumin conjugated with cytochrome C. The following examples illustrate the invention in greater detail.",
"EXAMPLES Example 1 ˜13 to 40 kDa range (cytochrome C oligomers) Material: 20 mg cytochrome C buffer A: 10 mM NaPi pH 7.4, 150 mM NaCl buffer B: IM DL-lysine in buffer A buffer C: 40 mM Tris-Cl pH 7.4, 300 mM NaCl solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).",
"1.Dissolve cytochrome C in 0.18 ml of buffer A.",
"2.Add 0.02 ml of solution D. 3.Stir 1 h at room temperature.",
"4.Add 0.02 ml of buffer B.",
"If a set with bands having the same intensity is desired, continue with the subsequent steps, otherwise directly skip to step 7.5.Load on a Superdex 75 column (10×300 mm) or on a column with similar characteristics, equilibrated and eluted in buffer C at a 0.5 ml/min flow rate, separately recovering the eluted peaks.",
"6.Mix each peak in amounts inversely proportional to the chromatographic peak area.",
"7.Aliquot and freeze at a temperature below −20° C. Example 2 ˜60 to 130 kDa range (cytochrome C conjugates) Material: 1 mg cytochrome C 3.3 mg ovalbumin 10 mg bovine serum albumin buffer A: 10 mM NaPi pH 7.4, 150 mM NaCl buffer B: 1M DL-lysine in buffer A solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).",
"1) Dissolve cytochrome C in 1 ml of buffer A.",
"2) Dissolve ovalbumin in 0.8 ml of buffer A and add 0.1 ml of solution from step 1.3) Dissolve bovine serum albumin in 0.8 ml of buffer A and add 0.1 ml of solution from step 1.4) Add 0.1 ml of solution D to the solutions prepared at steps 2 and 3.5) Stir the solutions from step 4 for 1 h at room temperature.",
"6) Add 0.1 ml of buffer B.",
"7) Aliquot and freeze at temperature below −20° C. Example 3 ˜15 to 45 kDa range (microperoxidase MP-11 conjugates) Material: 1 mg microperoxidase MP-11 10 mg lysozyme 5 mg carbonic anhydrase buffer A: 10 mM NaPi pH 7.4, 150 mM NaCl buffer B: 1M DL-lysine in buffer A solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).",
"1.Dissolve microperoxidase in 1 ml of buffer A.",
"2.Dissolve lysozyme in 0.8 ml of buffer A and add 0.1 ml of solution from step 1.3.Dissolve carbonic anhydrase in 0.8 ml of buffer A and add 0.1 ml of solution of step 1.4.Add 0.1 ml of solution D to the solutions prepared at steps 2 and 3.5.Stir the solutions from step 4 for 1 h at room temperature.",
"6.Add 0.1 ml of buffer B.",
"7.Aliquot and freeze at temperature below −20° C. EXAMPLE 4 Western Blot Duplicated samples of unconjugated cytochrome C, horseradish peroxidase, lactoperoxidase, and of conjugated proteins (with both cytochrome C and MP-11) were mixed with reducing Laemmli sample buffer: the first duplicate was heated for 5 min at 95° C., the second at 40° C., then the samples were run in parallel with Broad Molecular Weight Markers (Bio-Rad, Hercules, Calif., U.S.A.) on 15% mini-SDS-PAGE following standard procedure.",
"After run, the gel was blotted onto 0.45 μm nitrocellulose sheets by a semi-dry cell.",
"Membranes were washed with 20 mM TrisCl pH 7.4, 150 mM NaCl (TBS), blocked 1 h in 3% (w/v) bovine serum albumin in TBS (BT), then incubated in BT containing 0.05% (v/v) Tween-20 either 2 h or overnight to simulate different western blot procedures.",
"After washing in TBS containing 0.25% Tween-20 and further washing in TBS, the peroxidase activity was revealed.",
"Cytochrome C was also treated with DSS without adding any other protein, and subjected to gel-filtration on a Superdex 75 (1×30 cm) column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated and eluted in 40 mM Tris-Cl pH 7.4, 300 mM NaCl, at 0.5 ml/min flow: each eluted peak was recovered separately.",
"Cytochrome C and MP-11 conjugates were found to retain peroxidase activity at the end of the western blot procedure, as summarized in Table 1A-B.",
"Unconjugated peroxidase proteins were found to be more sensitive to high temperature treatment, which drastically decreased their enzymatic activity: cross-linker treatment is in fact likely to stabilize the protein structure against denaturation.",
"TABLE 1 A. Cytochrome C conjugates Cytochrome C mg/ml Approximate Protein and 100 10 0.1 protein/cytochrome source KDa Mg/ml (a) C molar ratio Cytochrome C 12.4 100 + (Sigma C.2506) 10 + Ribonuclease I A 13.7 1 − 10 (Pharmacia 27-0323-01) Ovalbumin 45.3.3 + 10 (Sigma A-7642) Bovine serum 67.10 + 20 albumin (Bio-Rad 500-0007) B. MP-11 microperoxide conjugates MP-11 (Sigma M-6756) 1.8 kDa Approximate 0.1 mg/ml Protein/MP-11 Protein and source KDa Mg/ml (a) Molar ratio Lysozyme 14.3 10 + 12 (Sigma L-6876) Carbonic anhydrase 29 5 + 3 (Sigma C-2273) (a) the sign + o − indicates whether after western blot procedure the peroxidase activity of the conjugate was detectable or not, respectively When cytochrome C was treated with the cross-linker, three oligomers were generated (Figure, lanes 1 and 2) which were easily separated by gel filtration.",
"The electrophoretic mobility suggests their identification as monomers, dimers and trimers (Table 2).",
"The amount of each conjugate and of the cytochrome C oligomers was adjusted so as to make the signal intensity of each band as homogeneous as possible.",
"Mixing the three species cytochrome C, ovalbumin- and serum albumin-cytochrome C, provided a valuable series of molecular weight standards (Figure, lane 5).",
"The conjugation of MP-11 with lysozyme produced three species of lysozyme oligomers, all of them MP-11 conjugates (Table 1B and Figure, lane 3), monomers, dimers and trimers (Table 2).",
"Conjugation of MP-11 with carbonic anhydrase produced a single band (lane 4).",
"TABLE 2 Calibrated molecular weights of the conjugates KDa Cytochrome C, monomer 12.9 Cytochrome C, dimer 28.4 Cytochrome C, trimer 38.6 Lysozyme-MP-11, monomer 14.8 Lysozyme-MP-11, dimer 30.7 Lysozyme-MP-11, trimer 45.5 Carbonic anhydrase-MP11 31.5 Ovalbumin-cytochrome C 58.9 Bovine serum albumin-cytochrome C 131"
]
] | Patent_10221854 |
[
[
"Circuit arrangement for operating electric or electronic components in a motor vehicle having an electric system comprising two voltages",
"The invention relates to a circuit arrangement for operating electric or electronic components in a motor vehicle with a two-voltage onboard network, with a direct current/direct current converter which comprises at least one input terminal, at least one output terminal and one ground terminal, with the input terminal being adapted to receive an input switching signal between a first voltage level and a ground level, and the output terminal being adapted to emit an output switching signal between a second voltage level, different from the first voltage level, and the ground level, the signal characteristic of which essentially follows the characteristic of the input switching signal, with the voltage converter with its input, output, and ground terminals being arranged in a housing which comprises a socket and which corresponds to a relay with respect to the dimensions and the positions of the input, output, and ground terminals at the socket."
],
[
"1.A circuit arrangement for operating electric or electronic components in a motor vehicle with a two-voltage onboard network, with a direct current/direct current converter (30) which comprises at least one input terminal (12a′), at least one output terminal (18′) and one ground terminal (12b′), with the input terminal (12a′) being adapted to receive an input switching signal between a first voltage level and a ground level, and the output terminal (18′) being adapted to emit an output switching signal between a second voltage level, different from the first voltage level, and the ground level, the signal characteristic of which essentially follows the characteristic of the input switching signal, with the voltage converter with its input, output, and ground terminals being arranged in a housing (10) which comprises a socket and which corresponds to a relay with respect to the dimensions and the positions of the input, output, and ground terminals at the socket.",
"2.The circuit arrangement according to claim 1, wherein the direct current/direct current converter is adapted to convert a voltage of approx.",
"12-14 V, which is applied at the input terminal (12a′) to a voltage of approx.",
"42 V, which is provided at the output terminal (18′).",
"3.The circuit arrangement according to claim 1, wherein the direct current/direct current converter (30) is adapted to convert a voltage of approx.",
"42 V, which is applied at the input terminal (12a′) to a voltage of approx.",
"12-14 V, which is provided at the output terminal (18′).",
"4.The circuit arrangement according to claim 2 or 3, with a supply voltage terminal for a supply voltage (approx.",
"12-14 V or approx.",
"42 V, respectively) which corresponds to the level of the input switching signal.",
"5.The circuit arrangement according to claim 1, wherein one or each power semiconductor device of the direct current/direct current converter is arranged in a heat conductive contact with inductive components containing iron of the direct current/direct current converter.",
"6.The circuit arrangement according to claim 1, wherein electronic or electric components of the direct current/direct current converter are electrically and mechanically connected with each other via load-carrying lines.",
"7.The circuit arrangement according to one of the previous claims, wherein the direct current/direct current converter (30) comprises: at least two half-bridge circuits (H1, H2, H3) formed by two semiconductor devices (S11, S12; S21, S22; S31, S32) connected in series, wherein the respective two power semiconductor devices (S11, S12; S21, S22; S31, S32) are connected with a control circuit (ECU) which is adapted to switch the two power semiconductor devices (S11, S12; S21, S22; S31, S32) to connect the two power semiconductor devices forward and reverse in an antiphase manner, with a first terminal of an inductor (L1, L2, L3) being connected electrically conductive with the centre of each half-bridge circuit (H1, H2, H3), second terminals each of the inductors (L1, L2, L3) being connected electrically conductive with each other, and the inductors (L1, L2, L3) being connected magnetically conductive with each other by a magnetic coupling element (T), and with the control circuit (ECU) being adapted to drive the half-bridge circuits (H1, H2, H3) in such a manner that voltage is applied to only one of the inductors (L1, L2, L3).",
"8.The circuit arrangement according to claim 7, in whose direct current/direct current converter (30) a predetermined number n half-bridge circuits (H1, H2, H3) is connected with the control circuit (ECU) and with the centre of each half-bridge circuit (H1, H2, H3) a first terminal of one of a predetermined number n inductors (L1, L2, L3) is connected electrically conductive, and the second terminals of each inductor (L1, L2, L3) are connected electrically conductive with each other, with the magnetic coupling element (T) being preferably a ferrite-containing component which couples the n inductors (L1, L2, L3) with each other.",
"9.The circuit arrangement according to claim 7 or 8, in whose direct current/direct current converter (30) a further inductor (L4) is connected in series at the electric connecting point of the second terminals of the inductors (L1, L2, L3).",
"10.The circuit arrangement according to claim 7 or 9, in whose direct current/direct current converter (30) a smoothing capacitor (C1, C2) each is arranged in parallel to the half-bridge arrangements and at the terminal remote from the half-bridge arrangements of the inductors (L1, L2, L3) or of the further inductor (L4), respectively."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to a circuit arrangement for the operation of electric or electronic components in a motor vehicle with a two-voltage onboard network.",
"In the field of motor vehicles, relays are employed which are of compact construction, reliable function, and robustness.",
"In particular, relays with corresponding housing dimensions and pin assignments are employed in motor vehicles, with the relays being normally inserted in plug-in sockets, in order to be easily replaceable for remedial purposes.",
"In the following, the terms “direct current/direct current converter” and “direct voltage/direct voltage converter” will be used as synonyms in the sense of a dc/dc converter, in which an input voltage of a first level is converted to an output voltage of a second level."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWING <EOH>FIG.",
"1 shows a schematic illustration of a circuit arrangement of a relay in a two-voltage onboard network in a conventional circuitry.",
"FIG.",
"2 shows a schematic illustration of a circuit arrangement of a relay in a two-voltage onboard network according to the invention.",
"FIG.",
"3 shows a schematic illustration of a circuit arrangement of a voltage converter for an inventive circuit arrangement according to FIG.",
"2 .",
"FIG.",
"4 a shows a schematic side view of a magnetic coupling element with three inductors for the circuit arrangement according to FIG.",
"3 .",
"FIG.",
"4 b shows a schematic plan view of the magnetic coupling element according to FIG.",
"4 a.",
"FIG.",
"5 shows a schematic characteristic of drive signals for the circuit arrangement of the voltage converter according to FIG.",
"3 .",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"BACKGROUND OF THE INVENTION The invention relates to a circuit arrangement for the operation of electric or electronic components in a motor vehicle with a two-voltage onboard network.",
"In the field of motor vehicles, relays are employed which are of compact construction, reliable function, and robustness.",
"In particular, relays with corresponding housing dimensions and pin assignments are employed in motor vehicles, with the relays being normally inserted in plug-in sockets, in order to be easily replaceable for remedial purposes.",
"In the following, the terms “direct current/direct current converter” and “direct voltage/direct voltage converter” will be used as synonyms in the sense of a dc/dc converter, in which an input voltage of a first level is converted to an output voltage of a second level.",
"STATE OF THE ART Due to the increasing electrification of motor vehicles, in particular of passenger motor vehicles, where an ever increasing power has to be made available for electric loads, the development is heading towards the introduction of a second onboard network with a higher voltage (e.g.",
"42 V) in addition to the currently most commonly used 12 V onboard network.",
"This, however, has the consequence that the cabling expenditure increases considerably, because both voltages must be available at virtually all sites in the motor vehicle.",
"Moreover, the quantity of loads with higher operating voltage, which has to be manufactured, is probably not as high as the number of loads with conventional operating voltage (12 V), so that the manufacturing costs of loads with higher operating voltage are relatively high.",
"Thus, there is definitely a need to operate loads with the one operating voltage by means of drive signals with the other operating voltage.",
"On the other hand, the line cross-section for supply lines to loads with low voltage and high current consumption is to be dimensioned relatively large so that the space requirement of the cable trees in the motor vehicle is higher with the low operating voltage (12-14 V) than with a higher operating voltage (42 V).",
"The desire for a higher operating voltage (42 V) results from this circumstance.",
"DE 40 41 220 A1 describes a current supply for motor vehicles, wherein a voltage-regulated DC/DC converter arrangement at the input side at which the low voltage battery voltage is applied, maintains a medium voltage bus at a stabilised medium voltage.",
"The medium voltage bus serves as the energy supply of loads which are to be operated by means of the medium voltage or by means of a high voltage.",
"Medium voltage loads are immediately supplied with energy via the medium voltage bus, while high voltage loads each are supplied by a high voltage converter which is arranged between the DC/DC converter arrangement and the relevant load.",
"The DC/DC converter arrangement, in particular, serves to transform low voltage onboard voltages for motor vehicles of approx.",
"12 V to a higher, stabilised, medium voltage of 150 V. The medium voltage applied to the medium voltage bus is brought up to the respective required high voltage between approx.",
"5 and 30 kV by the high voltage converters of conventional construction.",
"DE 40 05 809 A1 discloses a regulator module with a standard relay or connector housing and a circuit arrangement disposed therein, whose connecting elements are routed out of the housing.",
"The connecting elements are, in particular, arranged in accordance with a grid dimension specified for the standard housing and, especially, in accordance with a standard for automotive relays.",
"In this manner it is possible to insert the regulator module into a standardised plug-in socket for relays.",
"By a corresponding minor adaptation of the wiring of a relay which is generally arranged adjacent to the regulator module, said module can be driven be a regulated voltage.",
"Hence, the regulator module is not used in order to replace a relay.",
"The standardised plug-in socket is rather used for arranging the regulator module which serves to control one or several relays.",
"Accordingly, it is necessary to provide a further plug-in socket adjacent to the plug-in sockets which are required for the existing relays, or to remove a relay in order to provide a free plug-in socket.",
"DE 44 19 005 A1 describes an electronic load interrupter switch for motor vehicles, where an electronic circuit-breaker and integrated drive electronics are arranged on a wiring carrier.",
"External terminals of the wiring carrier are formed as a plug-in contact part typical for motor vehicles.",
"In this manner it is possible to directly replace conventional electromechanical load interrupter switches by this circuit-breaker without requiring a modification of the external wiring of the load interrupter switch.",
"If the drive electronics comprises an integrated clock generator, the load interrupter switch can be used as an electronic flasher unit.",
"Parts of the wiring carrier can be formed as low-resistance resistors which serve for current monitoring and can have a current-limiting function.",
"The load interrupter switch described therein merely serves as a replacement for conventional electromechanical load interrupter switches (relays), with the signals, currents, and voltages received and output by the load interrupter switch essentially corresponding to those of a relay to be replaced.",
"From DE 196 00 074 A1, a vehicle onboard network is known which in addition to the conventional onboard network voltage of approx.",
"12 V provides a further voltage for powerful electric loads.",
"The higher voltage which can be up to four times the conventional onboard network voltage is generated by means of a parallel connection of several chopper stages.",
"The supply of powerful electrical loads is effected in the conventional manner in that a power electronic circuit comprising the chopper stages is inserted between a vehicle battery and a generator of conventional construction, and this is connected conventionally with the loads by means of cable trees.",
"In this manner the maximum current in the lines of the cable trees and, with the load output remaining constant, their ohmic losses are reduced, which is the reason why lines of smaller cross-section can be used.",
"PROBLEMS ON WHICH THE INVENTION IS BASED With this situation at hand, the invention is based on the problem to provide a possibility for reducing the wiring and connecting expenditure in motor vehicles with two-voltage onboard networks and to realise it by economic means.",
"INVENTIVE SOLUTION The inventive solution of this object consists in a circuit arrangement for operating electric or electronic components in a motor vehicle with a two-voltage onboard network, with a direct current/direct current converter which comprises at least one input terminal, at least one output terminal and one ground terminal, with the input terminal being adapted to receive an input switching signal between a first voltage level and a ground level, and the output terminal being adapted to emit an output switching signal between a second voltage level, different from the first voltage level, and the ground level, the signal characteristic of which essentially follows the characteristic of the input switching signal, with the voltage converter with its input, output, and ground terminals being arranged in a housing which comprises a socket and which corresponds to a relay with respect to the dimensions and the positions of the input, output, and ground terminals at the socket.",
"ADVANTAGES OF THE INVENTION This embodiment essentially allows to maintain the previously used wiring or cabling, respectively, of the motor vehicle electric/electronic system also in the case of vehicles with a two-voltage onboard network.",
"The space requirement is virtually the same because in the previously used wiring or cabling, respectively, of the motor vehicle electric/electronic system relays are also provided between the respective switching element (e.g.",
"on/off switch for the rear window heater).",
"Usually, the relays of the motor vehicle electric/electronic system of a function group (starter; lighting, signalling system; blower, ventilation, heater; distributor injection pump, etc.)",
"are combined and arranged in a close spatial relationship to one another.",
"The invention makes it possible to employ externally (dimensions, pin assignment, etc.)",
"corresponding components instead of the previous relays, which effect a voltage conversion to the respective voltage level.",
"Another advantage is that trouble shooting and maintenance of a motor vehicle electric/electronic system equipped with such circuit arrangements are particularly simple because they are carried out in the same way as the replacement of relays inserted in sockets.",
"ADVANTAGEOUS DEVELOPMENTS OF THE INVENTION In a first embodiment of the invention the direct current/direct current converter is adapted to convert a voltage of approx.",
"12-14 V, which is applied at the input terminal to a voltage of approx.",
"42 V, which is provided at the output terminal.",
"In a second embodiment of the invention the direct current/direct current converter is adapted to convert a voltage of approx.",
"42 V, which is applied at the input terminal to a voltage of approx.",
"12-14 V, which is provided at the output terminal.",
"In an embodiment of the invention, the converter circuit which is required for this comprises an oscillator which can be switched on and off with an oscillation frequency of approx.",
"20 kHz to at least approx.",
"2 MHz, and a power output stage coupled with the oscillator's output, downstream of which a rectifier is connected.",
"A transformer can be connected either upstream or downstream of the power output stage, if an electrical isolation is deemed to be necessary.",
"In this case, however, it is preferred that the input side of the power output stage is connected with the secondary side of the transformer because then the power need not be transferred via the transformer so that this can be implemented with a small size.",
"In embodiments with a common ground potential, however, no electrical isolation takes place so that the transformer is omitted.",
"If the electrical power fed into the input terminal of the circuit arrangement is not sufficient for providing the power required for the respective load at the output terminal, a supply voltage terminal for a supply voltage (approx.",
"12-14 V or approx.",
"42 V, respectively) which corresponds to the level of the input switching signal is provided in an embodiment of the inventive circuit arrangement.",
"This voltage is then converted by the converter circuit to the respective other voltage level and provided at the output terminal in accordance with the characteristic of the voltage at the input terminal.",
"In an embodiment of the invention the power semiconductor devices are arranged in a heat conductive contact with inductive components of the direct current/direct current converter, containing iron, in order to avoid separate heat sinks for the power semiconductor devices.",
"As inductive components containing iron, transformers, reactors, or other inductive coupling elements provided with one or several windings and made from iron sheet or from ferrite or the like are taken into consideration.",
"In this way, the relatively voluminous iron body of, for example, a reactor or a transformer at the same time has as second function that of a heat dissipating element.",
"In particular for applications with relatively short-time loading (e.g.",
"flasher lamp, stop lamp, audible signalling device or the like) this provides an opportunity for very compact circuit arrangements with a relatively high power.",
"In a preferred embodiment of the invention the use of a printed board or card (printed circuit) is dispensed with, also for the sake of volume saving.",
"Electric components of the direct current/direct current converter are electrically and mechanically connected with each other via load-carrying lines instead.",
"In order to still realise an adequate mechanical strength and insusceptibility against external influences (moisture, condensed water, dust, etc.",
"), the entire circuit is additionally encapsulated with a synthetic resin.",
"According to a preferred embodiment of the invention a direct current/direct current converter for use in the above described circuit arrangement comprises at least two half-bridge circuits formed by two semiconductor devices connected in series, wherein the respective two power semiconductor devices are connected with a control circuit which is adapted to connect the two power semiconductor forward and reverse in an antiphase manner, with a first terminal of an inductor being connected electrically conductive with the centre of each half-bridge circuit, second terminals each of the inductors being connected electrically conductive with each other, and the inductors being connected magnetically conductive with each other by a magnetic coupling element, and with the control circuit being adapted to drive the half-bridge circuits in such a manner that voltage is applied to only one of the inductors.",
"With this circuit a predetermined number n half-bridge circuits is connected with the control circuit and with the centre of each half-bridge circuit a first terminal of a predetermined number n inductors is connected electrically conductive and the second terminals of each inductor are connected electrically conductive with each other, with the magnetic coupling element being preferably a ferrite-containing component which couples the n inductors with each other.",
"Provided the number of the half-bridge arrangements or the inductors, respectively, in the magnetic coupling element equals the transmission ratio of the input to the output voltage, virtually no filter elements or the like are required for smoothing the voltage.",
"For the reduction of possibly occurring voltage peaks, however, another inductor may be connected in series at the electrical connection point of the second terminals of the inductors.",
"For further smoothing and for the compensation of load variations an additional smoothing capacitor each can be arranged at the terminal parallel to the half-bridge arrangements and at the terminal remote from the half-bridge arrangements of the inductors or the further inductor, respectively.",
"Further properties, characteristics, advantages, and possible modifications of the invention will become apparent from the following description of the drawing in which embodiments of the invention are illustrated.",
"BRIEF DESCRIPTION OF THE DRAWING FIG.",
"1 shows a schematic illustration of a circuit arrangement of a relay in a two-voltage onboard network in a conventional circuitry.",
"FIG.",
"2 shows a schematic illustration of a circuit arrangement of a relay in a two-voltage onboard network according to the invention.",
"FIG.",
"3 shows a schematic illustration of a circuit arrangement of a voltage converter for an inventive circuit arrangement according to FIG.",
"2.FIG.",
"4a shows a schematic side view of a magnetic coupling element with three inductors for the circuit arrangement according to FIG.",
"3.FIG.",
"4b shows a schematic plan view of the magnetic coupling element according to FIG.",
"4a.",
"FIG.",
"5 shows a schematic characteristic of drive signals for the circuit arrangement of the voltage converter according to FIG.",
"3.DETAILED DESCRIPTION OF THE DRAWING FIG.",
"1 is a schematic representation of a circuit arrangement of a relay 10 of a two-voltage onboard network.",
"In the relay 10, its first terminal 12a of a coil 12 is connected with a first voltage of a lower level (approx.",
"12-14 V) via an on/off switch 14, and its second terminal 12b of the coil 12 is connected with ground.",
"A third terminal 16 and a fourth terminal 18 of the relay 10 are respectively connected with a normally open switching element 20 whose contact is closed in the operating condition of the relay 10 and is opened in the rest condition of the relay 10.It is understood that in lieu of the illustrated normally open switching element 20 normally closed, two-way contact elements with neutral position, or multiple contact elements can also be included in the relay 10.The third terminal 16 of the relay 10 is connected with a second voltage which has a higher level (approx.",
"42 V), while its fourth terminal 18 is connected with a first terminal 22 of a load 24.A second terminal 26 of the load 24 is connected with ground.",
"Upon an operation of the on/off switch 14 current flows through the coil 12 of the relay 10 so that the normally open switch element 20 changes from its open to its closed position and the load 24 is connected with the second voltage which has a higher level (approx.",
"42 V).",
"In FIG.",
"2 an embodiment of the invention is schematically shown, where in a housing with the same dimensions, properties, and pin assignments or terminal layout, respectively, etc.",
"as those of the relay 10 in FIG.",
"1 a direct current/direct current converter 30 is arranged which comprises an input terminal 12a′, an output terminal 18′, and a ground terminal 12b′.",
"The first terminal 12a′ leads to an input of the direct current/direct current converter 30 and is connected with a first voltage with a lower level (approx.",
"12-14V) via an on/off switch 14′.",
"The second terminal 12b′ is the ground terminal of the direct current/direct current converter 30 and is connected with ground in the same manner as the terminal 12b in FIG.",
"1.At the output terminal 18′ of the direct current/direct current converter 30 it provides an output voltage with the on/off switch 14′ operated, which—against ground—lies on a higher lever (e.g.",
"42 V) than the first voltage with a lower level (approx.",
"12-14 V).",
"For direct current/direct current converters 30, in particular, where the output at the output terminal 18′ is to be higher than the input power at the input terminal 12a′, the direct current/direct current converter 30 in an embodiment of the invention which is not illustrated is provided with an additional supply voltage terminal for a supply voltage (approx.",
"12-14 V or approx.",
"42 V, respectively) corresponding to the input switching signal.",
"Without discussing further details of the circuit arrangement already here, the power semiconductor devices of the direct current/direct current converter 30 in an embodiment are arranged in heat conductive contact with iron containing inductive components of the direct current/direct current converter.",
"For purposes of space/volume saving, the electronic components of the direct current/direct current converter 30 in the inventive circuit arrangement are connected with each other both electrically and mechanically via load-carrying lines.",
"FIG.",
"3 is a schematic representation of a circuit arrangement of a voltage converter for an inventive circuit arrangement according to FIG.",
"2, wherein a voltage with a lower level (approx.",
"12-14 V) is applied at the input side, while at the output side a voltage with a higher level (approx.",
"42 V) is provided by this inventive circuit arrangement.",
"The circuit arrangement as specified herein has been described in conjunction with the application of the invention according to FIG.",
"2.It is, however, also possible to use this circuit arrangement, if required, with higher input power or output, respectively, so as to be advantageous for other purposes or fields of application.",
"At the input side of the inventive circuit arrangement a direct voltage of 12-14 V is applied.",
"This direct voltage is applied in parallel at the three inductors L1, L2, L3 whose input side terminals are connected with each other.",
"These inductors L1, L2, L3 are of identical inductance (L1=L2=L3) and are connected with each other magnetically conducting by a magnetic coupling element T (see FIGS.",
"4a, 4b).",
"The magnetic coupling element T is a ferrite containing component which couples the three inductors L1, L2, L3 with each other.",
"The shown embodiment is a ferrite core with three legs K1, K2, K3 each of which being surrounded by one of the three inductors L1, L2, L3.At their respective faces, the three legs K1, K2, K3 are connected by one joke J1, J2 each (see FIGS.",
"4a, 4b).",
"It is understood that the ferrite core as a whole can be an integral part or can be formed as an EI, M, or L-shaped iron core.",
"This magnetic coupling element T acts as a hybrid transformer which does not store electric energy.",
"FIG.",
"4b also shows the sense of winding for the three inductors L1, L2, L3.The inventive circuit arrangement comprises three half-bridges H1, H2, H3 connected in parallel, each of which being formed by two power semiconductor devices S11, S12; S21, S22; S31, S32 being connected in series between the output voltage (42 V) and ground.",
"The power semiconductor devices are preferably power MOSFET's or IGBT's wherein one diode D each is additionally connected in parallel in the reverse direction (as shown by way of example for the power semiconductor device S31 only).",
"At the centre tap of each of the three half-bridges H1, H2, H3 the respective other terminal of one of the inductors L1, L2, L3 is connected.",
"The three half-bridges H1, H2, H3 or the six power semiconductor devices S11, S12; S21, S22; S31, S32, respectively, of FIG.",
"3 are driven by an electronic control circuit ECU via six control lines a, a\\; b, b\\; c, c\\ in such a manner that current flows off of only one of the three inductors L1, L2, L3 at a time.",
"The magnetic coupling element T is thereby subjected to a complete magnetisation stroke so that no premagnetisation occurs.",
"The characteristic of the drive signals on the six control lines a, a\\; b, b\\; c, c\\ is illustrated in FIG.",
"5.The electronic control circuit ECU can be realised as a three-place shift register with inverted and non inverted outputs x, x\\, through which a 1-0-0 sequence with a corresponding shift cycle passes.",
"It must be ensured that the inverted and non inverted outputs x, x\\ do not “overlap” in time.",
"Rather a dead time (e.g.",
"some 100 ns) which is adapted to the switching behaviour of the six power semiconductor devices S11, S12; S21, S22; S31, S32 must be maintained between the level changes with the inverted and the non inverted outputs x, x\\.",
"In order to minimise voltage peaks at the output side of the circuit arrangement a further inductor L4 is provided which is connected in series with the three inductors L1, L2, L3 at the input side.",
"In addition, at the output side in parallel to the half-bridge arrangements and at the terminal remote from the half-bridge arrangements of the further inductor L4, a smoothing capacitor C1 or C2, respectively, each can be arranged at the output side or at the input side, respectively.",
"In lieu of the illustrated three-phase embodiment of the circuit arrangement, it is also possible to utilise only one or two phases.",
"In these cases, however, the expenditure for smoothing in order to achieve an essentially constant output voltage is higher.",
"As described above, the ferrite core can be used as a heat sink for the six power semiconductor devices S11, S12; S21, S22; S31, S32.In the above described embodiment of the inventive circuit arrangement of the voltage converter, an input/output voltage ratio of 1:3 (14 V:42 V) is realised.",
"This is obtained from the pulse duty factor of the drive signals on the six control lines a, a\\; b, b\\; c, c\\.",
"The invention is, of course, not limited to the conversion ratio of 1:3.It is also possible to realise a higher or also a lower conversion ratio 1:n. In this case, the pulse duty factor (pulse:total time of a period) is also to be selected as 1:n. Moreover, it is recommended to also select an n-phase design of the circuit arrangement (n half-bridges with corresponding control lines from the electronic control circuit ECU, n inductors, etc.",
"), in order to keep the above described expenditure for voltage smoothing small, or to realise a high voltage constancy at load changes, respectively.",
"Moreover, there is the possibility to realise a conversion ratio different from 1:n, where “gaps” occur between the individual pulses (see FIG.",
"5).",
"This, however, requires a control circuit different from the above described control circuit ECU, in the form of the three-place circulating register with inverted and non-inverted outputs x, x\\.",
"In this case, the above described further inductance L4 and correspondingly dimensioned smoothing capacitors C1 or C2, respectively, are mandatory for energy storage.",
"In order to reliably isolate the output side (also in the case of a defect of one or several of the six power semiconductor devices S11, S12; S21, S22; S31, S32) from the input side, two further power semiconductor devices in the form of n-channel power MOSFET's S40, S41 are provided at the output side, which are connected in such a manner that their parasitic diodes D40, D41 are oriented against one another.",
"These two power semiconductor devices S40, S41 are also driven by the control circuit ECU (with no input voltage applied) in such a manner that the input side is isolated from the output side."
]
] | Patent_10221980 |
[
[
"Process for forming plastic, apparatuses for forming plastic,and articles made therefrom",
"Process and apparatus for forming plastics by heating an open mold and contacting the open mold with plastic particulate material to form a melted skin on the mold, and resulting articles made therefrom.",
"Single skin molded articles may be multi-layered with or without paint on the surface.",
"Multiple skin molded articles may be made by complementary male and female molds, brought together after the skins are made on the individual male and female molds, and may include sandwiched layers of plastic filler or expandable foam filled centers, with or without various inserts or reinforcements.",
"Multi-layer composite articles are made with inserts and/or reinforcements that may be embedded in and surrounded by expandable foam plastic, if used as a middle layer.",
"Resulting articles include a pick-up truck bed box, an industrial tabletop, a series of modular housing panels, an airplane cockpit door, and material handling pallets, among others."
],
[
"1.A process for forming plastic into a predetermined shape from a plastic particulate material having a melting point, said process comprising: providing an open mold made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said mold being formed into a predetermined shape with at least a face portion to impart a desired shape; heating at least the face portion of the open mold to an elevated temperature of from about 100° C. to about 865° C. such that the mold is at a higher temperature than the melting point of the plastic particulate material; contacting the open mold to the plastic particulate material while the plastic particulate is at least at room temperature for a dwell time period of from about one second to about 30 minutes, said dwell time period being pre-selected to melt the plastic particulate into a skin of a desired thickness of from about 0.001 cm to about 3.0 cm prior to ceasing contact of the open mold with the plastic particulate.",
"2.The process of claim 1, further comprising cooling the mold to release the resulting article.",
"3.The process of claim 1, further comprising providing a second heated mold, said second heated mold being complementary to the first heated mold, and said molds being adapted to be placed together to form a double skinned article.",
"4.The process of claim 3, further comprising a step of placing a reinforcement between the first and second heated molds, such that the reinforcement is embedded between the heated double skins.",
"5.The process of claim 1, wherein the step of contacting the open mold to a plastic particulate material is accomplished by contacting with a polyolefin.",
"6.A process for forming plastic into a predetermined shape from a plastic particulate material having a melting point, said process comprising: providing male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; heating said male and female molds at least on their face portions to an elevated temperature of from about 100° C. to about 865° C. such that the mold is at a temperature above the melting point of the plastic particulate; contacting the male and female molds with the plastic particulate while the plastic particulate is at least at room temperature for a dwell time period of from about 1 second to about 30 minutes, said dwell time period being pre-selected to melt the plastic particulate into a molded skin of a plastic article having a skin of a desired thickness of from about 0.001 cm to about 3.0 cm prior to ceasing contact with the plastic particulate; bringing together the complementary face portions of the heated male and female molds to adhere the two skins of plastic articles to each other such that an article is formed having a molded surface on both sides.",
"7.The process of claim 6, further comprising a step of placing a reinforcement between said male and female molds prior to the step of bringing together the male and female molds and adhering the skins together.",
"8.The process of claim 6, wherein the plastic particulate at least at room temperature is at an elevated temperature under the melting point of the plastic particulate.",
"9.The process of claim 6, wherein said heating of the molds is accomplished by heating them to an elevated temperature of from about 160° C. to about 220° C. 10.The process of claim 6, wherein the dwell time period is from about three to about 10 minutes.",
"11.The process of claim 6, wherein the step of bringing together the male and female molds is accomplished by spacing them apart.",
"12.A process for forming a plastic composite into a predetermined shape from a plastic particulate material and a plastic filler material, each plastic material having a respective melting point, said process comprising: providing male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; heating at least the face portions of the male and female complementary molds to an elevated temperature of from about 130° C. to about 865° C. which is above the melting points of the respective plastic particulate material and the plastic filler material; contacting the face portion of the complementary molds to the plastic particulate material resulting in two melted plastic skins on the surfaces of the male and female molds, said contacting occurring while the plastic particulate is at least at room temperature for a dwell time period of from about 1 second to about 30 minutes, said dwell time period being pre-selected to melt the plastic particulate into a layer of a desired thickness prior to ceasing contact with the plastic particulate; spacing apart the two complementary male and female molds from one another at a predetermined distance and placing plastic filler material between the male and female molds such that the plastic filler material may be sandwiched between the male and female molds to form a double skinned composite having a plastic filler material between the two skins, with an overall predetermined thickness of the composite; and cooling at least one of the male and female molds to loosen and remove the entire resultant double skinned, filled composite article.",
"13.The process of claim 12, wherein the step of heating the molds is accomplished by elevating the temperature to about 160° C. to about 220° C. 14.The process of claim 12, further comprising a step of placing a reinforcement between said male and female molds prior to the step of bringing together the male and female molds and adhering the skins together.",
"15.The process of claim 12, further comprising inserting an expandable plastic filler material between the heated molds after the skins have been formed to form an expanded plastic center.",
"16.A process for forming a plastic composite into a predetermined shape from a plastic particulate material and an expandable plastic filler material, each having a respective melting point, said process comprising: providing male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; providing a means for heating the male and female complementary molds to an elevated temperature of from about 100° C. to about 865° C. which is above the melting points of the respective plastic particulate material and the expandable plastic filler material, such that contacting the complementary molds to the plastic particulate material will result in a melted plastic layer on the surface contacted; heating said complementary male and female molds by said heating means at least on their face portions to a temperature above the melting point of the plastic particulate, and then contacting the face portion of the two molds with plastic particulate while the plastic particulate is at least at room temperature for a dwell time period preselected to melt the plastic particulate into a skin layer of a desired thickness on the face portion, prior to ceasing contact with the plastic particulate; and holding the two complementary male and female molds apart from one another at a predetermined distance and placing expandable plastic filler between the two heated complementary molds such that the expandable plastic filler material is sandwiched and expanded between the male and female molds to a thickness determined by the distance the molds are apart.",
"17.An apparatus for forming plastic into a predetermined shape from a plastic particulate material having a melting point, said apparatus comprising: an open mold made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said mold being formed into a predetermined shape with at least a face portion to impart the desired shape; said complementary male and female molds being heatable at least on their face portions to a temperature above the melting point of the plastic particulate, for contacting with the plastic particulate while the plastic particulate is at least at room temperature for a dwell time period preselected to melt the plastic particulate into a layer of a desired thickness prior to ceasing contact with the plastic particulate.",
"18.An apparatus for forming plastic into a predetermined shape from a plastic particulate material having a melting point, said apparatus comprising: a pair of male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; said complementary male and female molds being heatable at least on their face portions to a temperature above the melting point of the plastic particulate, for contacting with the plastic particulate while the plastic particulate is at least at room temperature for a dwell time period preselected to melt the plastic particulate into a layer of a desired thickness prior to ceasing contact with the plastic particulate.",
"19.An apparatus for forming a plastic composite into a predetermined shape from a plastic particulate material and a plastic filler material, each having a respective melting point, said apparatus comprising: a pair of male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; means for heating the male and female complementary molds to an elevated temperature of from about 100° C. to about 865° C. which is above the melting points of the respective plastic particulate material and the plastic filler material, such that contacting the heated complementary molds to the plastic particulate material results in a melted plastic skin layer on the surface contacted; said complementary male and female molds being heatable at least on their face portions to a temperature above the melting point of the plastic particulate, for contacting with the plastic particulate while the plastic particulate is at least at room temperature for a dwell time period preselected to melt the plastic particulate into a layer of a desired thickness prior to ceasing contact with the plastic particulate; and a spacer for holding the two complementary male and female molds apart from one another at a predetermined distance such that the plastic filler material may be sandwiched between the male and female molds to a thickness determined by the spacer.",
"20.An apparatus for forming a plastic composite into a predetermined shape from a plastic particulate material and an expandable plastic filler material, each having a respective melting point, said apparatus comprising: male and female complementary molds made of a material selected from the group consisting of aluminum, aluminum alloys, kirksite, metals, ceramics, cermets, high temperature plastics, formable heat resistant materials and combinations thereof, said molds being formed into a predetermined shape with at least a face portion on each of said complementary male and female molds to impart a desired shape; means for heating the male and female complementary molds to an elevated temperature of from about 100° C. to about 865° C. which is above the melting points of the respective plastic particulate material and the expandable plastic filler material, such that contacting the complementary molds to the plastic particulate material results in a melted plastic layer on the surface contacted; said complementary male and female molds being heatable at least on their face portions to a temperature above the melting point of the plastic particulate, for contacting with the plastic particulate while the plastic particulate is at least at room temperature for a dwell time period preselected to melt the plastic particulate into a layer of a desired thickness prior to ceasing contact with the plastic particulate; and a spacer for holding the two complementary male and female molds apart from one another at a predetermined distance such that the expandable plastic filler material may be sandwiched and expanded between the male and female molds to a thickness determined by the spacer.",
"21.A plastic pick-up truck bed box for installing on the chassis of a pick-up truck, comprising: spaced apart male and female plastic skins, each being shaped over an open mold and both being adapted to fit onto the chassis of the pick-up truck, said male and female skins being formed of a polyolefinic material, each having a thickness of from about 0.001 cm to about 1.0 cm; a low density expandable plastic material sandwiched between said male and female skins, having a thickness of from about 1.0 cm to about 5.0 cm; a steel mesh reinforcement shaped like the molds for the male and female skins, said steel reinforcement being inserted between the male and female molds, embedded into and surrounded by the expandable plastic, such that the pick-up truck box is reinforced by the strength of the steel; and load rails inserted into the female mold prior to the expandable plastic material, said load rails being located at the bottom of the resulting truck box, being surrounded by the plastic skins and the expandable plastic, such that the plastic pick-up truck bed box is adapted for easy mounting onto a pick-up truck chassis.",
"22.A plastic industrial tabletop to be mounted on table legs, comprising: spaced apart male and female plastic skins, each being shaped over an open mold and both being adapted to fit onto table legs, said male and female skins being formed of a polyolefinic material, each having a thickness of from about 0.001 cm to about 1.0 cm; a low density expandable plastic material sandwiched between said male and female skins, having a thickness of from about 1.0 cm to about 5.0 cm; and a steel mesh reinforcement shaped like the molds for the male and female skins, said steel reinforcement being inserted between the male and female molds, embedded into and surrounded by the expandable plastic, such that the tabletop is reinforced by the strength of the steel.",
"23.A plastic airplane cockpit door, comprising: spaced apart male and female plastic skins, each being shaped over an open mold and both being adapted to fit into an airplane cockpit, said male and female skins being formed of a polyolefinic material, each having a thickness of from about 0.001 cm to about 1.0 cm; a low density expandable plastic material sandwiched between said male and female skins, having a thickness of from about 1.0 cm to about 5.0 cm; a sheet of Kevlar inserted between the male and female plastic skins and being encapsulated and surrounded by the expandable plastic material; and a steel mesh reinforcement shaped like the molds for the male and female skins, said steel reinforcement being inserted between the male and female molds, embedded into and surrounded by the expandable plastic, such that the airplane cockpit door is reinforced by the strength of the steel and made bullet-proof by the Kevlar sheet inclusion.",
"24.A plastic modular housing panel, comprising: spaced apart male and female plastic skins, each being shaped over an open mold and both being adapted to fit into together with other modular housing panels, said male and female skins being formed of a polyolefinic material, each having a thickness of from about 0.001 cm to about 1.0 cm; a low density expandable plastic material sandwiched between said male and female skins, having a thickness of from about 1.0 cm to about 5.0 cm; and thermally insulating material inserted between the male and female plastic skins and being encapsulated and surrounded by the expandable plastic material.",
"25.A lightweight plastic material handling pallet, comprising: spaced apart male and female plastic skins, each being shaped over an open mold and both being adapted to support industrial material loads, said male and female skins being formed of a polyolefinic material, each having a thickness of from about 0.001 cm to about 1.0 cm; a low density expandable plastic material sandwiched between said male and female skins, having a thickness of from about 1.0 cm to about 5.0 cm; and steel cone reinforcements embedded into and surrounded by the expandable plastic material, said reinforcements located between the male and female molds, thereby lending strength and durability to the pallet."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>This patent application generally relates to the forming of plastic and, more specifically, relates to the forming of plastic using a heated mold in contact with plastic particles, whether they be in the form of powder, resins, pellets or the like.",
"Although conventional methods of forming plastics are good, there is always room for improvement.",
"There are many ways to make plastics, but there are few ways to make plastic articles which are lightweight, strong, fire retardant, bullet proof, insulative, impact resistant, as well as having a decorative, textured or functional skin, or made with plastic in a single layer on a heated mold.",
"Furthermore, there are few ways taught in the prior art of embedding articles within the plastic, in order to either reinforce the article or to change its properties.",
"Moreover, there are even fewer ways known in the art for including various materials throughout the body of an article without having seams, including multiple layer structures and various materials dispersed throughout the surface of the article.",
"Although it is known to put inserts into injection molded plastic articles, the present inventors do not know of any other low temperature, low pressure methods which can completely suspend an insert, reinforcement, foam core or other sandwiched material within the plastic material itself.",
"It would be advantageous for such a method of forming plastic, as well as one to utilize such relatively low temperatures, ambient pressures and the ability to use inexpensive and easily machined molds which will last for an entire production of an article.",
"Of course, it would also be advantageous for such a method to be capable of using recycled materials.",
"Such a new method of forming plastic would be usable for a huge multitude of applications, including, but certainly not limited to: automotive and industrial vehicle components; modular housing panels; airplane components; consumer and industrial furniture such as tables, tabletops and the like; doors; windows; material handling pallets and other articles; consumer goods; industrial articles; marine applications and boat hulls; molds and components, including seawalls, boat hulls and the like; medical apparatuses and other applications; scaffolding and other building construction articles; sea containers; railroad containers; composite wheels for trains, vehicles and food shipping containers including food containers of all sizes and shapes, just to name some of the applications.",
"Each of these applications will include various forms of the plastic articles, including various materials sandwiched between two or more skins in order to produce the desired material properties.",
"One of the largest applications for the present invention and technology is the creation of automobile vehicle components, including pick-up truck boxes, roof components, underbody components, and the like.",
"In an industry which traditionally used steel for its components, the automotive vehicle manufacturers in Detroit and abroad are seeking lightweight plastic components for their vehicles because the new stricter fuel economy regulations are forcing them to rethink how they manufacture vehicles.",
"Environmentally friendly politicians in various governments, including Washington, D.C., are backing regulations which will press the automotive industry hard into developing more fuel-efficient vehicles.",
"Currently, the best selling vehicles in the United States are heavy trucks and sport utility vehicles, all of which have poor fuel economy due to their massive size and incredibly high weights.",
"For most of these vehicles that weigh 4,000 to 6,500 pounds, normal side roads with a gross weight limit of one and a half tons will crack under a sustained weight such as these vehicles.",
"The easiest way to achieve a more fuel-efficient vehicle is to reduce the weight.",
"One way to reduce the weight is to remove various steel portions and replace them with lightweight, strong plastic, such as the topic of the present invention.",
"The Corporate Average Fuel Economy, or “CAFE”, is increasingly putting demands on the automotive industry because of the growing evidence of the vehicle pollution-caused greenhouse effect and other environmental maladies.",
"The change to plastic components has huge implications for the American automotive industry which is already facing pinched profits and dented sales with the slow economy.",
"The Big 3 automakers in the United States say that tougher mileage rules, particularly for sport utility vehicles, could cost each of the companies several billion dollars over the next few years and would seriously hurt their profits.",
"In one answer to meeting the new CAFE standards, some of the automobile manufacturers have pledged to launch the new highly efficient hybrid vehicles, which are part electric and part gasoline having much lower pollution emissions.",
"However, it is widely acknowledged that the new hybrid technology alone will not suffice to achieve the CAFE standards which are on the horizon.",
"Automakers admit that they will have to start selling many more lighter-weight vehicles, such as compact sport utility vehicles, to meet new fuel economy standards.",
"Of the 11.4 million vehicles which Detroit automakers sold last year, 59 percent were trucks or SUVs.",
"And of America's top ten best selling trucks, sport utility vehicles and mini-vans, all fall far short of the current gas standards, as measured by their combined average highway and city miles per gallon.",
"The current CAFE standard for trucks is 20.7 miles per gallon, although most of the trucks and SUVs have fuel efficiencies from 13 mpg to 18 mpg.",
"As the advent of the electric hybrid vehicles are not the only solution to the automakers complying with CAFE standards, it becomes clear that improving gas mileage of sport utility vehicles and trucks is the easy target.",
"It is an easy target because the SUVs and trucks are very heavy and they get the worst fuel economy of almost any vehicle on the road.",
"For example, the Ford Motor Company's Excursion SUV only achieves 13 miles per gallon for city driving.",
"The current predictions by Detroit automakers is that if the current proposed boosted truck standards of 24 mpg, at an increase of 3.3 miles per gallon from present 20.7 mpg standards, this would cause the automakers to lose sales of over 1 million large pick-ups and sport utilities.",
"And, as always, the industry warns that they won't be the only one hurt.",
"They say that the price added onto vehicles to make them more fuel-efficient will cost our economy close to 300,000 jobs.",
"The standard solution in Japan to increasing the fuel mileage of vehicles is, industry wide, to improve small cars and their gas mileage, to sacrifice horse power at the expense of fuel efficiency, particularly for sport utility vehicles, to boost fuel-efficiency research and development in order to develop fuel-efficient engines for cars and SUVs, as well as to curb production of the largest SUVs which are the main culprits in lowering Detroit's average fuel efficiency for trucks.",
"Therefore, it would be of a great advantage to significantly reduce the weight of pick-up trucks and sport utility vehicles, thereby boosting the fuel economy and thereby alleviating the payment of fines for exceeding CAFE standards.",
"It would be most advantageous, and most environmentally friendly, to make plastic vehicle components.",
"Although the use of conventional plastics is a huge advancement, we may take this trend a step further by using biodegradable plastics or those made from renewable sources, such as the polylactic acid polymer being proposed by Cargill Dow and the National Renewable Energy Laboratory.",
"The new corn-based biodegradable polymer is called polylactic acid (PLA), and may be utilized in various components of the present invention.",
"Furthermore, plastics were made for automobiles as early as the 1920s by Henry Ford, from plastics made from the hemp plant.",
"In one prototype made in accordance with the present invention, a Dodge Dakota truck had its steel truck box bed replaced with a plastic truck box made in accordance with the present invention.",
"The weight savings was 95 pounds.",
"This is incredible considering the fact that Chrysler has been asking its suppliers to shave ounces off of the parts that they are supplying.",
"To save nearly 100 pounds per vehicle will have an enormous effect on the gas consumption and fuel economy.",
"Furthermore, due to the extreme light weight, a dump truck configuration is possible, as well as a scissors-jack man lift configuration.",
"As an impact-resistant material may be utilized, the truck box bed can handle slow accidents with minimal damage to the pickup truck box.",
"In addition, it would be of great advantage to be able to embed conduits and/or wire harnesses directly into the plastic truck box bed itself, thereby allowing a plug-in operation once the pickup truck box has been placed on the chaise.",
"It would also be advantageous to provide a method for forming plastic for such a pickup truck box that would allow heavy metal components to extend therefrom in order to give a mounting device to the chaise of the vehicle, such as is possible with the present invention.",
"In yet another embodiment, it would be advantageous to be able to easily and inexpensively form modular housing panels which can be clipped together and caulked in place to make rapid housing.",
"For instance, there are currently over 1.5 million Afghanistan refugees caused by war in their country.",
"Other third-world nations would be excellent candidates for such modular housing components.",
"There is a long felt need for a cheap, lightweight and inexpensive, insulated clip together housing component which can be manufactured on site, as well as manufactured in a plant back at a home base and then shipped to the location itself.",
"As one may be aware, Rubbermaid Corporation of Ohio in the United States makes many little work sheds and garden sheds for use in a back yard, although these sheds are not suitable for human living conditions.",
"However, those sheds are made by injection molding which does not lend itself well to even larger products, and the molds are extremely expensive for ones of that size to be used for production.",
"It would be a great advantage to utilize very inexpensive molds, recycled materials and insulation which can be embedded within a plastic composite article such that a useful modular house can be made in a very short period of time.",
"It would further be useful to be able to embed conduits and/or electrical wires themselves directly into the plastic panels.",
"Plug-in devices could extend from the panels and the panels could be directly plugged into one another, or if conduits were embedded into the modular housing components, then electrical wires could be run as easily and quickly as they are in conventional homes by licensed electricians.",
"By setting up an exterior generator station, an entire complex of clipped together houses could be rapidly outfitted with electric heat, electric lights and cooking devices.",
"Sewage treatment could also be molded into the panels, such as the electric incinerating toilets commonly used in Japan and on boats.",
"The insulated panels could have R-values of up to 100 or more, making electric heat feasible.",
"This would remove any temptation to heat with dangerous carbon monoxide gas-generating heating devices, such as kerosene lamps, fires, and the like.",
"In the event that a plastic modular housing panel is made in accordance with the present invention, it is also possible for windows and doors to be molded into the panels, as well as toilet incinerator outlet stacks and roofing components.",
"In addition to large panels being made from the plastic, it would also be advantageous to provide various smaller components, similar to clipped together “logs” in order to allow for the greatest number of configurations for the modular house itself.",
"It would also be advantageous to provide methods, apparatuses and articles for a multitude of other applications, too numerous to mention here."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Therefore, in accordance with the above objects and advantages, the present invention discloses a new method for forming plastic into either a single skin configuration or a multiple skin configuration, usually two skins, which may also have contained therebetween either an expandable plastic material, reinforcements for strengthening the plastic article, other filler materials, or combinations thereof.",
"In addition to the materials which can be incorporated into the middle layer between two skins, the present invention also discloses the use of many embedded articles to be placed between the two skins, whether they are completely embedded into the article, or whether portions of them are allowed to extend therethrough outside the molded article, i.e.",
"for purposes such as mounting brackets, electrical wires, and the like.",
"Furthermore, the present invention lends itself to the manufacture of profile plastics, including hard and soft combinations such that a component can have a hard backing layer, with a foamed or expandable plastic front layer, such as would be useful for dashboards and automotive vehicle components.",
"As inserts can be placed into or onto the mold due to its low temperature, even things such as ashtrays and carpet could be placed into the mold or could be placed onto the hot plastic after it has been molded onto, for example, the male or female mold.",
"In this embodiment, the carpeting would be melded into the hot plastic, such that the carpet would never come off.",
"Multiple layers are also capable of being made through the methods in accordance with the present invention, including, but not limited to, in-mold paints.",
"For instance, if the mold could be electrostatically charged, a powder coat paint could be first contacted with the heated mold, and then could cure at its proper temperature while the heated mold is accepting its contact with plastic particulates for producing a skin on top of the powder coated paint.",
"Other multi-layer concepts are envisioned by the present inventors which may also include reinforcements or other materials to be sandwiched between multiple skins of plastic such as made by the multiple mold configuration.",
"For example, heated male and female complementary molds can each have a skin formed on their complementary face portions, followed by an expandable or foamable plastic being sprinkled onto either of the molds.",
"In addition, a reinforcement, such as a metal wire mesh, may be shaped into the appropriate shape and inserted between the two skins.",
"The two skins can then be spaced apart from one another such that the expandable foam will expand to the predetermined thickness, thereby embedding and surrounding the metal mesh which has been placed between the two skins.",
"This configuration, i.e.",
"the sandwich with the reinforcement therebetween, is capable of adding structural strength while maintaining a lightweight and inexpensive plastic configuration, which is much more light weight than steel.",
"Therefore, in accordance with the present invention, there are numerous important embodiments, including, but not limited to, an open mold made of aluminum or other suitable material which can be worked to impart a desired shape, heated and then contacted with plastic particulate to melt the plastic particulate onto the mold itself, thereby producing a plastic article.",
"In another embodiment, male and female complementary molds made of similar materials can be heated on their face portions to a temperature above the melting point of a plastic particulate into which it comes in contact, and then the male and female articles can be held pressed or held together to form a double-skinned article.",
"In yet another embodiment, a double-skinned article can be manufactured using the male and female complementary molds from above, with the introduction of a plastic filler material onto one of the molds prior to holding the molds together, such that there is a “sandwich” which is formed from these plastic composites.",
"In yet a further embodiment, the double-skinned embodiment further comprises an expandable plastic filler material which will give a double-skinned plastic article with an expanded plastic filler material therebetween.",
"A predetermined thickness for the expandable plastic is created by holding the male and female molds at a predetermined distance apart.",
"In yet another embodiment, reinforcements can be embedded into the plastic filler material or into the expandable plastic filler material such that when the expandable material is heated and expanded up around the reinforcement, the reinforcement is embedded into and surrounded by the expandable plastic filler material.",
"In yet still another embodiment of the present invention, mounting brackets, wiring harnesses, and/or other desired materials may be encapsulated within the plastic composite article itself or may be inserted into the mold prior to the two skin molds being placed in close proximity to one another, such that the plastic skin and the filler material can embed and encapsulate the mounting brackets, wiring harnesses or the like.",
"In addition, apparatuses for accomplishing these types of articles and processes are also disclosed, including a trunion design for moving and tipping the male and female mold sections to produce articles.",
"Robots may be utilized to load reinforcements between the male and female molds prior to the filler material being melted or expanded.",
"A vacuum apparatus for filling/emptying the plastic particulate into and around the mold is also disclosed incorporating a vacuum system and a blow bag for removing the excess plastic particulate once a desired skin thickness has been achieved.",
"Further, plastic particulate from additional blower bags may be connected to the vacuum system in order to form layers of various other materials.",
"In yet one more embodiment of the present invention, there are disclosed various particular articles which are made by the process of the present invention, including, but not limited to, automotive components, industrial tabletops, airplane components, modular housing panels and components, material handling pallets, and many other applications which will be described hereinbelow or which will become obvious to one of ordinary skill in the art.",
"Therefore, in accordance with the present invention, there is disclosed new processes for forming plastic, apparatuses for carrying out those processes, and articles which are made therefrom.",
"For understanding the present invention, we refer the reader to the following detailed description, taken in conjunction with the accompanying drawings and the accompanying text."
],
[
"CROSS REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S.",
"Provisional Application No.",
"60/270,321 filed on Feb. 5, 2001, U.S.",
"Provisional Application No.",
"60/300,874 filed on Jun.",
"25, 2001, and U.S.",
"Provisional Application No.",
"60/346,336 filed on Jan. 7, 2002.BACKGROUND OF THE INVENTION This patent application generally relates to the forming of plastic and, more specifically, relates to the forming of plastic using a heated mold in contact with plastic particles, whether they be in the form of powder, resins, pellets or the like.",
"Although conventional methods of forming plastics are good, there is always room for improvement.",
"There are many ways to make plastics, but there are few ways to make plastic articles which are lightweight, strong, fire retardant, bullet proof, insulative, impact resistant, as well as having a decorative, textured or functional skin, or made with plastic in a single layer on a heated mold.",
"Furthermore, there are few ways taught in the prior art of embedding articles within the plastic, in order to either reinforce the article or to change its properties.",
"Moreover, there are even fewer ways known in the art for including various materials throughout the body of an article without having seams, including multiple layer structures and various materials dispersed throughout the surface of the article.",
"Although it is known to put inserts into injection molded plastic articles, the present inventors do not know of any other low temperature, low pressure methods which can completely suspend an insert, reinforcement, foam core or other sandwiched material within the plastic material itself.",
"It would be advantageous for such a method of forming plastic, as well as one to utilize such relatively low temperatures, ambient pressures and the ability to use inexpensive and easily machined molds which will last for an entire production of an article.",
"Of course, it would also be advantageous for such a method to be capable of using recycled materials.",
"Such a new method of forming plastic would be usable for a huge multitude of applications, including, but certainly not limited to: automotive and industrial vehicle components; modular housing panels; airplane components; consumer and industrial furniture such as tables, tabletops and the like; doors; windows; material handling pallets and other articles; consumer goods; industrial articles; marine applications and boat hulls; molds and components, including seawalls, boat hulls and the like; medical apparatuses and other applications; scaffolding and other building construction articles; sea containers; railroad containers; composite wheels for trains, vehicles and food shipping containers including food containers of all sizes and shapes, just to name some of the applications.",
"Each of these applications will include various forms of the plastic articles, including various materials sandwiched between two or more skins in order to produce the desired material properties.",
"One of the largest applications for the present invention and technology is the creation of automobile vehicle components, including pick-up truck boxes, roof components, underbody components, and the like.",
"In an industry which traditionally used steel for its components, the automotive vehicle manufacturers in Detroit and abroad are seeking lightweight plastic components for their vehicles because the new stricter fuel economy regulations are forcing them to rethink how they manufacture vehicles.",
"Environmentally friendly politicians in various governments, including Washington, D.C., are backing regulations which will press the automotive industry hard into developing more fuel-efficient vehicles.",
"Currently, the best selling vehicles in the United States are heavy trucks and sport utility vehicles, all of which have poor fuel economy due to their massive size and incredibly high weights.",
"For most of these vehicles that weigh 4,000 to 6,500 pounds, normal side roads with a gross weight limit of one and a half tons will crack under a sustained weight such as these vehicles.",
"The easiest way to achieve a more fuel-efficient vehicle is to reduce the weight.",
"One way to reduce the weight is to remove various steel portions and replace them with lightweight, strong plastic, such as the topic of the present invention.",
"The Corporate Average Fuel Economy, or “CAFE”, is increasingly putting demands on the automotive industry because of the growing evidence of the vehicle pollution-caused greenhouse effect and other environmental maladies.",
"The change to plastic components has huge implications for the American automotive industry which is already facing pinched profits and dented sales with the slow economy.",
"The Big 3 automakers in the United States say that tougher mileage rules, particularly for sport utility vehicles, could cost each of the companies several billion dollars over the next few years and would seriously hurt their profits.",
"In one answer to meeting the new CAFE standards, some of the automobile manufacturers have pledged to launch the new highly efficient hybrid vehicles, which are part electric and part gasoline having much lower pollution emissions.",
"However, it is widely acknowledged that the new hybrid technology alone will not suffice to achieve the CAFE standards which are on the horizon.",
"Automakers admit that they will have to start selling many more lighter-weight vehicles, such as compact sport utility vehicles, to meet new fuel economy standards.",
"Of the 11.4 million vehicles which Detroit automakers sold last year, 59 percent were trucks or SUVs.",
"And of America's top ten best selling trucks, sport utility vehicles and mini-vans, all fall far short of the current gas standards, as measured by their combined average highway and city miles per gallon.",
"The current CAFE standard for trucks is 20.7 miles per gallon, although most of the trucks and SUVs have fuel efficiencies from 13 mpg to 18 mpg.",
"As the advent of the electric hybrid vehicles are not the only solution to the automakers complying with CAFE standards, it becomes clear that improving gas mileage of sport utility vehicles and trucks is the easy target.",
"It is an easy target because the SUVs and trucks are very heavy and they get the worst fuel economy of almost any vehicle on the road.",
"For example, the Ford Motor Company's Excursion SUV only achieves 13 miles per gallon for city driving.",
"The current predictions by Detroit automakers is that if the current proposed boosted truck standards of 24 mpg, at an increase of 3.3 miles per gallon from present 20.7 mpg standards, this would cause the automakers to lose sales of over 1 million large pick-ups and sport utilities.",
"And, as always, the industry warns that they won't be the only one hurt.",
"They say that the price added onto vehicles to make them more fuel-efficient will cost our economy close to 300,000 jobs.",
"The standard solution in Japan to increasing the fuel mileage of vehicles is, industry wide, to improve small cars and their gas mileage, to sacrifice horse power at the expense of fuel efficiency, particularly for sport utility vehicles, to boost fuel-efficiency research and development in order to develop fuel-efficient engines for cars and SUVs, as well as to curb production of the largest SUVs which are the main culprits in lowering Detroit's average fuel efficiency for trucks.",
"Therefore, it would be of a great advantage to significantly reduce the weight of pick-up trucks and sport utility vehicles, thereby boosting the fuel economy and thereby alleviating the payment of fines for exceeding CAFE standards.",
"It would be most advantageous, and most environmentally friendly, to make plastic vehicle components.",
"Although the use of conventional plastics is a huge advancement, we may take this trend a step further by using biodegradable plastics or those made from renewable sources, such as the polylactic acid polymer being proposed by Cargill Dow and the National Renewable Energy Laboratory.",
"The new corn-based biodegradable polymer is called polylactic acid (PLA), and may be utilized in various components of the present invention.",
"Furthermore, plastics were made for automobiles as early as the 1920s by Henry Ford, from plastics made from the hemp plant.",
"In one prototype made in accordance with the present invention, a Dodge Dakota truck had its steel truck box bed replaced with a plastic truck box made in accordance with the present invention.",
"The weight savings was 95 pounds.",
"This is incredible considering the fact that Chrysler has been asking its suppliers to shave ounces off of the parts that they are supplying.",
"To save nearly 100 pounds per vehicle will have an enormous effect on the gas consumption and fuel economy.",
"Furthermore, due to the extreme light weight, a dump truck configuration is possible, as well as a scissors-jack man lift configuration.",
"As an impact-resistant material may be utilized, the truck box bed can handle slow accidents with minimal damage to the pickup truck box.",
"In addition, it would be of great advantage to be able to embed conduits and/or wire harnesses directly into the plastic truck box bed itself, thereby allowing a plug-in operation once the pickup truck box has been placed on the chaise.",
"It would also be advantageous to provide a method for forming plastic for such a pickup truck box that would allow heavy metal components to extend therefrom in order to give a mounting device to the chaise of the vehicle, such as is possible with the present invention.",
"In yet another embodiment, it would be advantageous to be able to easily and inexpensively form modular housing panels which can be clipped together and caulked in place to make rapid housing.",
"For instance, there are currently over 1.5 million Afghanistan refugees caused by war in their country.",
"Other third-world nations would be excellent candidates for such modular housing components.",
"There is a long felt need for a cheap, lightweight and inexpensive, insulated clip together housing component which can be manufactured on site, as well as manufactured in a plant back at a home base and then shipped to the location itself.",
"As one may be aware, Rubbermaid Corporation of Ohio in the United States makes many little work sheds and garden sheds for use in a back yard, although these sheds are not suitable for human living conditions.",
"However, those sheds are made by injection molding which does not lend itself well to even larger products, and the molds are extremely expensive for ones of that size to be used for production.",
"It would be a great advantage to utilize very inexpensive molds, recycled materials and insulation which can be embedded within a plastic composite article such that a useful modular house can be made in a very short period of time.",
"It would further be useful to be able to embed conduits and/or electrical wires themselves directly into the plastic panels.",
"Plug-in devices could extend from the panels and the panels could be directly plugged into one another, or if conduits were embedded into the modular housing components, then electrical wires could be run as easily and quickly as they are in conventional homes by licensed electricians.",
"By setting up an exterior generator station, an entire complex of clipped together houses could be rapidly outfitted with electric heat, electric lights and cooking devices.",
"Sewage treatment could also be molded into the panels, such as the electric incinerating toilets commonly used in Japan and on boats.",
"The insulated panels could have R-values of up to 100 or more, making electric heat feasible.",
"This would remove any temptation to heat with dangerous carbon monoxide gas-generating heating devices, such as kerosene lamps, fires, and the like.",
"In the event that a plastic modular housing panel is made in accordance with the present invention, it is also possible for windows and doors to be molded into the panels, as well as toilet incinerator outlet stacks and roofing components.",
"In addition to large panels being made from the plastic, it would also be advantageous to provide various smaller components, similar to clipped together “logs” in order to allow for the greatest number of configurations for the modular house itself.",
"It would also be advantageous to provide methods, apparatuses and articles for a multitude of other applications, too numerous to mention here.",
"SUMMARY OF THE INVENTION Therefore, in accordance with the above objects and advantages, the present invention discloses a new method for forming plastic into either a single skin configuration or a multiple skin configuration, usually two skins, which may also have contained therebetween either an expandable plastic material, reinforcements for strengthening the plastic article, other filler materials, or combinations thereof.",
"In addition to the materials which can be incorporated into the middle layer between two skins, the present invention also discloses the use of many embedded articles to be placed between the two skins, whether they are completely embedded into the article, or whether portions of them are allowed to extend therethrough outside the molded article, i.e.",
"for purposes such as mounting brackets, electrical wires, and the like.",
"Furthermore, the present invention lends itself to the manufacture of profile plastics, including hard and soft combinations such that a component can have a hard backing layer, with a foamed or expandable plastic front layer, such as would be useful for dashboards and automotive vehicle components.",
"As inserts can be placed into or onto the mold due to its low temperature, even things such as ashtrays and carpet could be placed into the mold or could be placed onto the hot plastic after it has been molded onto, for example, the male or female mold.",
"In this embodiment, the carpeting would be melded into the hot plastic, such that the carpet would never come off.",
"Multiple layers are also capable of being made through the methods in accordance with the present invention, including, but not limited to, in-mold paints.",
"For instance, if the mold could be electrostatically charged, a powder coat paint could be first contacted with the heated mold, and then could cure at its proper temperature while the heated mold is accepting its contact with plastic particulates for producing a skin on top of the powder coated paint.",
"Other multi-layer concepts are envisioned by the present inventors which may also include reinforcements or other materials to be sandwiched between multiple skins of plastic such as made by the multiple mold configuration.",
"For example, heated male and female complementary molds can each have a skin formed on their complementary face portions, followed by an expandable or foamable plastic being sprinkled onto either of the molds.",
"In addition, a reinforcement, such as a metal wire mesh, may be shaped into the appropriate shape and inserted between the two skins.",
"The two skins can then be spaced apart from one another such that the expandable foam will expand to the predetermined thickness, thereby embedding and surrounding the metal mesh which has been placed between the two skins.",
"This configuration, i.e.",
"the sandwich with the reinforcement therebetween, is capable of adding structural strength while maintaining a lightweight and inexpensive plastic configuration, which is much more light weight than steel.",
"Therefore, in accordance with the present invention, there are numerous important embodiments, including, but not limited to, an open mold made of aluminum or other suitable material which can be worked to impart a desired shape, heated and then contacted with plastic particulate to melt the plastic particulate onto the mold itself, thereby producing a plastic article.",
"In another embodiment, male and female complementary molds made of similar materials can be heated on their face portions to a temperature above the melting point of a plastic particulate into which it comes in contact, and then the male and female articles can be held pressed or held together to form a double-skinned article.",
"In yet another embodiment, a double-skinned article can be manufactured using the male and female complementary molds from above, with the introduction of a plastic filler material onto one of the molds prior to holding the molds together, such that there is a “sandwich” which is formed from these plastic composites.",
"In yet a further embodiment, the double-skinned embodiment further comprises an expandable plastic filler material which will give a double-skinned plastic article with an expanded plastic filler material therebetween.",
"A predetermined thickness for the expandable plastic is created by holding the male and female molds at a predetermined distance apart.",
"In yet another embodiment, reinforcements can be embedded into the plastic filler material or into the expandable plastic filler material such that when the expandable material is heated and expanded up around the reinforcement, the reinforcement is embedded into and surrounded by the expandable plastic filler material.",
"In yet still another embodiment of the present invention, mounting brackets, wiring harnesses, and/or other desired materials may be encapsulated within the plastic composite article itself or may be inserted into the mold prior to the two skin molds being placed in close proximity to one another, such that the plastic skin and the filler material can embed and encapsulate the mounting brackets, wiring harnesses or the like.",
"In addition, apparatuses for accomplishing these types of articles and processes are also disclosed, including a trunion design for moving and tipping the male and female mold sections to produce articles.",
"Robots may be utilized to load reinforcements between the male and female molds prior to the filler material being melted or expanded.",
"A vacuum apparatus for filling/emptying the plastic particulate into and around the mold is also disclosed incorporating a vacuum system and a blow bag for removing the excess plastic particulate once a desired skin thickness has been achieved.",
"Further, plastic particulate from additional blower bags may be connected to the vacuum system in order to form layers of various other materials.",
"In yet one more embodiment of the present invention, there are disclosed various particular articles which are made by the process of the present invention, including, but not limited to, automotive components, industrial tabletops, airplane components, modular housing panels and components, material handling pallets, and many other applications which will be described hereinbelow or which will become obvious to one of ordinary skill in the art.",
"Therefore, in accordance with the present invention, there is disclosed new processes for forming plastic, apparatuses for carrying out those processes, and articles which are made therefrom.",
"For understanding the present invention, we refer the reader to the following detailed description, taken in conjunction with the accompanying drawings and the accompanying text.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1a is a side elevational view of the apparatus for utilizing an open mold in accordance with the present invention; FIG.",
"1b is a side elevational view of the mold with a plastic skin adhered thereto; FIG.",
"1c is a perspective view of a plastic molded article of the present invention after it is removed from the mold; FIG.",
"2a is a side elevational view of a cutaway portion of a double skinned article made in accordance with one of the embodiments of the present invention having an expanded foam in the center of the article; FIG.",
"2b is a side elevational view of a cutaway portion of a double skinned article with an expanded foam center and a wire mesh reinforcement embedded and surrounded therein; FIG.",
"3a illustrates both a male and female mold in contact with plastic particulate; FIG.",
"3b illustrates side views of the male and female molds with plastic skins thereon; FIG.",
"3c shows the skinned male and female molds in complementary positions; FIG.",
"4 is a perspective view of a flat panel made with various materials over various portions of the article; FIG.",
"5 is a schematic of the basic process steps used in one of the embodiments of the present invention for making a pick up truck bed box; FIG.",
"6 is an illustration of the pre-formed male and female steel wire mesh reinforcements to be embedded into the pick up truck bed box; FIG.",
"7 shows the vacuum and blower lines used to fill and remove the plastic particulate material; FIG.",
"8 shows the load rails being inserted into the mold; FIG.",
"9 shows the trunion for rotating and tipping the excess material out of the mold container; FIG.",
"10 shows the female mold for the pick-up truck bed box; FIG.",
"11 shows the male mold for the pick-up truck bed box; FIG.",
"12 shows a test tool design with a male and female cavity and core molds; FIG.",
"13 shows a cavity mold for the test tool; FIG.",
"14 shows a core mold for the test tool; FIG.",
"15 shows the cavity side with a screen; FIG.",
"16 shows the core side with a screen; FIG.",
"17 shows a core with the broad cone reinforcements; FIG.",
"18 shows plugs to mount into the core to receive cone reinforcements; FIG.",
"19 is a perspective view of a van roof with the dimpled surface effect; FIG.",
"20 is a side cutaway view of the energy absorbing embodiment of the present invention; FIG.",
"21 is a side perspective view of an industrial tabletop made with the present invention; FIG.",
"22 is a front elevational view of an airplane cockpit door made with the present invention; and FIG.",
"23 is a front elevational view of a modular housing panel with electrical conduits running horizontally and vertically.",
"DETAILED DESCRIPTION OF THE DRAWINGS In accordance with the present invention, there are disclosed various processes for forming plastic, the apparatuses which are useful for performing those processes, and certain articles made therefrom.",
"Needless to say, the scope of the invention will be determined by the claims and shall not be otherwise limited.",
"As with all new materials and forming technologies, the number of applications and permutations of those applications are so numerous, they cannot all be mentioned here.",
"However, in the spirit of providing the best mode and detailed description of many of the embodiments, the following description will be broken down into paragraphs, beginning with a generalized description of the technology, followed by specific applications and their descriptions.",
"I.",
"General Description In general, the present invention and process can be most basically described as the use of at least one single or a set of molds which is heated and contacted with at least a polyolefinic plastic particulate material.",
"T polyolefin material may be in the form of powder, pellets, resin, shavings, or whatever.",
"The contacting acts to melt the plastic particles into a formed article against the shape of the heated mold.",
"The thickness of the article is determined by the length of time the heated mold comes into contact with the plastic.",
"For example, a heated mold elevated to a temperature of from approximately 100° C. to 865° C. can be placed in contact with a powdered polyethylene material and will achieve a plastic skin thickness of approximately 1 mm for every minute that the mold is contacted with the plastic.",
"For most of the applications described hereinbelow, it is most advantageous to have the plastic formed on the mold of a thickness from about 1 mm thick to about 10 mm thick, requiring a contact dwell time of between about 1 minute and 10 minutes.",
"If other polyolefin materials are utilized, such as plastic pellets, which are much less expensive than ground plastic powder, the contact time must be adjusted accordingly.",
"Specific times will be described hereinbelow with regards to specific applications and specific materials.",
"Although a single mold can produce a single piece of a plastic article merely by contacting the heated mold into a reservoir of plastic, it is further envisioned that a sandwich-type of composite material can be made by making both male and female mold portions, forming “skins” on each of the molds, and placing materials in between the two skins in a clamshell-type configuration with a filler or foaming plastic in between.",
"Generally, the expandable foam is activated by the residual heat from the molds.",
"In the event that male and female mold skins are utilized, any type of reinforcing material or desired insert may be sandwiched between the two skins and may be fully surrounded by the filler or expandable foam plastic.",
"For example, to add structural strength, it is envisioned that a whole host of reinforcements may be used.",
"Especially strong is a metal mesh inserted between the two skins along with expandable plastic material which will attach the two skins to one another, while embedding the steel mesh there between.",
"In yet another reinforcement embodiment, a sheet of Kevlar, a registered trademark of DuPont Corporation of Wilmington, Del., can be introduced between the skins and within the foamed plastic in order to provide a bulletproof door, for example, for airplane cockpit door applications.",
"Small individual wire mesh cones may be utilized for superior strength.",
"Furthermore, crumbed tire may be incorporated into the center of the male and female mold skins in order to make it nailable for modular housing applications.",
"If it is desired that the plastic article needs to be cut to shape, then the insert/reinforcement material sandwiched between the male and female mold skins may be made of small particles such that the article can be machined or cut.",
"Any of the inserts or reinforcements may be pre-treated to aid in the adhesion between layers, or to help prevent the insert or reinforcement from cutting or shearing the foamed plastic that encases it, when under load.",
"Such pre-treatments may include power-coating a wire mesh with a compatible epoxy resin; or applying a sulfonating technique to individual particulates, such as tire crumb or other recycled materials, to enhance their adhesion; or plating and/or depositing certain metallic or non-metallic coatings onto the insert/reinforcement to enhance adhesion; or even structural treatments such as sandblasting, surface grinding, tackifying with chemical treatments or the like; or the application of heat treatments such as annealing and/or quenching to change the surface properties; or the application of magnetic fields; or by forming an easy-to-adhere-to surface by forming or etching the insert/reinforcement to resemble reticulated foam by increasing the surface area.",
"Furthermore, it is envisioned by the present inventors that multiple layer structures can be formed by first making a male or female mold skin, followed by making a second male or female mold skin, and then a third complementary and mating male or female mold section can be formed.",
"Each of these forms can be placed one on top of the other and heated with a filler material or foamable plastic in between, or with other materials which will melt and attach the skins altogether.",
"Because the present process is done at a relatively low temperature, i.e.",
"slightly higher than that of the melting point of the plastic particulate being contacted with the heated mold, the mold itself will last a long time.",
"In conventional injection molding, the plastic must be elevated in temperature to over 1,000° F., and commonly up to 1,500° F. in the worm screw before it is injected into the mold.",
"With the combined effect of these high temperatures and high pressures used, the mold rapidly degrades.",
"Also, the present invention is done in ambient pressure, rather than the many tons of pressure required by injection molding machines.",
"Of special interest to all manufacturers, is the fact that the molds which can be used in the present invention may be made of pure aluminum or inexpensive and recyclable aluminum alloys such as kirksite which are cheap to make and easy to machine.",
"Because of the low temperature and low pressure application, the molds do not degrade as they do in injection molding.",
"For example, a mold used to make the entire truck bed box would cost more than a million dollars for a typical injection mold production mold, while the present invention mold can be made for less than one-tenth of that price.",
"This factor alone will encourage new products because of the lower necessary up-front costs.",
"In that regard, the following description of the general article construction is disclosed, and will be followed by the various process embodiments for manufacturing articles in accordance with the present invention, and then by specific embodiments for various applications.",
"Of course, the scope of the present invention is not to be limited to the specific applications promulgated herewith, but rather will be limited by the claims when they are filed.",
"II.",
"General Article Construction With reference to FIGS.",
"1a-1c, there is shown an illustration of a very general article and the respective process for manufacturing articles in accordance with the present invention, generally denoted by the numeral 10.Mold 12 is shown as being formed to make a plate article with raised edges.",
"Mold 12 is to be heated to an elevated temperature of greater than the melting point of the plastic particulates 16 held within container 14.As seen in FIG.",
"1b, an article 20 is formed once heated mold 12 has contacted plastic particulate 16 for a sufficiently long time to achieve the desired thickness of article.",
"Thereafter, the heated mold can be purposely cooled, or allowed to cool, as illustrated in FIG.",
"1c and article 20 can be easily removed from mold 12.If heating and cooling lines are used in carrier 18 or in mold 12 itself, then cooling fluids could be run through the lines, which would automatically contract the mold as it got cooler, pulling mold 12 away from formed article 20, which would remain relatively hot when compared to a cooler mold.",
"Generally, the plastic particulate material may be powder, pellets, resin, or any other form of plastic, including sheets or blocks, and they may either be at room temperature, or at an elevated temperature, depending upon the application as will be seen further hereinbelow.",
"Preferred plastics include HDPE, LDPE, polyethylene, polypropylene, polyurethane, or other widely used plastic resins.",
"Environmentally friendly plastics, such as polylactic acid may also be utilized, or other plastics made from renewable sources including the plastic made by Cargill Dow from corn and its husks, or plastics made from the hemp plant.",
"Mold 12 may be heated in a number of ways, including, but not limited to, heater lines in the mold itself for conducting hot water, oil or gas; a heat dissipative material attached to the mold itself or a backplate on the mold, such as mold carrier 18 of FIGS.",
"1a-1c; the mold might be heated in an oven to a predetermined temperature prior to contacting the plastic; the mold might be heated with heater torches or direct flame application; the mold might be heated with Infra-Red lamps or other light energy; the mold might be heated with microwave energy or other radio frequency energy; the mold might be heated with plasma heat generated by a plasma generator; the mold might be heated by thermoelectric devices in or on the mold itself; the mold might be heated uniformly over the surface to achieve a uniform coating of melted plastic or it might be selectively heated over portions of the surface so that multiple materials can be sequentially melted next to one another or spaced apart; the mold may be heated to a first temperature and contacted with a first material, and then may be heated or cooled to a second temperature to melt a second material; or the mold may be heated by any conventional means for heating a mold.",
"Furthermore, combinations of these techniques may prove to be helpful, including heating the mold in an oven while also heating the mold with microwave or other radio frequency energy.",
"It is also envisioned that the plastic particulate material may be heated to a near melting point temperature before contacting it with a heated mold.",
"The plastic particulate may be held in a container waiting to receive the mold, or it may find utility in a heated or unheated fluidized bed of the plastic particulate.",
"Thus, submerging the heated mold into the fluidized bed would contact the heated mold with the fluidized particulates.",
"The fluidized bed could be fluidized with gases other than air such as nitrogen, helium, sulfur-containing gases, etc., in order to impart a surface effect once the plastic melts and sticks to the heated mold.",
"If a different gas was utilized, any number of surface effects could be experienced, which might help with adhesion of later layers, or could help with “sealing” the plastic once it was formed into an appropriate shape.",
"Possible gas applications would include the use of a sulfur-containing gas to effect a sulfonation of the plastic in order to prevent chemical migration through the plastic, the use of an inert gas such as argon or neon to cause a peening, annealing or quenching effect of the plastic without effecting any surface chemistry reactions at such elevated temperatures; a nitrogen-containing gas to prevent oxidation of the surface; a fluoride or other halogen-containing gas to effect electrical conductivity changes on the surface of the resultant article; hydrogen or helium gas may be used to encourage thermal transfers through the plastic if the article is a relatively thick or bulky piece; or various acidic or basic gas compositions to impart a particular predetermined pH on the surface of the article.",
"Moreover, it is also envisioned that an initial layer of viscous plastic may be imparted on the bare surface of the heated mold 12 by contacting with a finely ground powdered plastic first to form a first “sticky” surface prior to contacting with heavier plastic particulates in order to provide an adhesion layer for subsequent contact with other, possibly less expensive plastics.",
"This viscous layer may be accomplished by contacting the mold with a finely powdered plastic first, or by using heated plastic particulates, or heated and finely ground plastic material combined.",
"In addition, a different type of plastic may first be used, one which exhibits greater flow and adhesion with the mold material, followed by the bulk plastic material.",
"For certain applications, it may be advantageous for the adhesion of a first plastic, one that is relatively expensive, to be followed up with at least one more layer of inexpensive plastic.",
"This way, an article can have the desired strength from a bulk or recycled plastic, while the skin can be made of an expensive material with decorative features or colors.",
"Color can be blended right into the underlying materials so that any scratches or minor surface blemishes will be indistinguishable from the surface, alleviating the necessity for repairs.",
"The inner layer(s) of material may also be selected to impart strength, heat insulation, fire retardation, energy dispersion qualities such as impact or bullet resistance, or filling with various materials to achieve certain other qualities, such as the inclusion of crumbed tire to give a spongy center, or one that can be easily cut, scored or nailed.",
"Insulation materials may be included for modular housing panels.",
"Looking next to FIG.",
"2a, there is shown a multilayer structure made in accordance with a preferred embodiment of the present invention which is generally denoted by the numeral 30.First and second plastic skins 32 and 34, respectively, are individually formed on separate heated complementary male and female molds, and then a foamable or expandable plastic 36 may be placed between the two skins and heated to expand and adhere to the two plastic skins, forming a lightweight, but very strong, article suitable for many applications.",
"Air pockets 37 are formed as a consequence of the expansion of expandable plastic 36, available from numerous plastic resin suppliers.",
"An especially desirable expandable plastic is available from Equistar Corporation of Cincinnati, Ohio.",
"As will be discussed below, any number of porous sheets, wire meshes, or other inserts and/or reinforcements can be loaded onto the first male skin mold prior to the placement of the foamable or expandable plastic and prior to the second female skin mold being put into place over the first skin mold.",
"Generally, it is most advantageous for the expandable or foamable plastic to be activated by the heat which is imparted by the two heated male and female molds as they are held together in a spaced apart relation with the foamable plastic and/or any desired reinforcements in between.",
"Once the expandable plastic is expanded due to the heat imparted from the first and second molds, any insert or reinforcement which was placed between the molds is encapsulated and sandwiched into the article 30 structure.",
"Looking now to FIG.",
"2b, there is shown again a multi-layer structure generally denoted by numeral 30 having a reinforcing wire mesh 38 shown embedded and encapsulated within expanded plastic 36, and between first and second plastic skins 32 and 34.Numerous other inserts and/or reinforcements may be encapsulated between the top and bottom skins, including, but not limited to, wire meshes for strength, metal bars and mounting pieces which are to extend outwardly from the skin to facilitate mounting to other fixtures, Kevlar material may be sandwiched to render the piece bulletproof, such as for airplane cockpit doors, or fire retardant materials may be used as sheets to prevent burn-through.",
"Other material properties can be exhibited by inclusion into the plastic skins of magnetic materials, ceramic powders or whiskers for heat and flame resistance, chemically resistant materials, thermoelectric materials, colored pigments, tough plastics for impact resistance and energy dispersion, anti-microbial chemicals on the surface, enzymes for different purposes, among others.",
"Virtually anything can be encapsulated in the expandable plastic, and it will be kept encapsulated until a total rupture of the multilayer structure occurs.",
"The only restriction is that the insert or reinforcement will experience an elevated temperature due to the heated molds which can melt or deform certain types of material.",
"The inserts can be entirely encapsulated, or only partially encapsulated such that portions of the insert can extend outwardly from the plastic article.",
"This will enable the plastic article to have mounting bars encapsulated by the plastic, with mounting bar portions extending outside the article to be mounted on, for example, a metal truck chassis frame by bolting or otherwise fastening the mounting bars to the chassis.",
"Furthermore, the insert may be a heat resistant or insulative piece which can contact a metal frame, without dissipating the heat to the plastic article, and alleviating a fear of melting.",
"As seen in FIGS.",
"3a-3c, there is shown a method of making a double skinned article, such as a pick-up truck bed box or housing module.",
"Heated male mold 40 is placed into a box 42 containing plastic powder or pellets 44 or the plastic particulates may be blown into the box after the mold is in the box.",
"A skin 50 forms on top of the mold, as shown in FIG.",
"3b.",
"The female mold 46 is shown filled with plastic particulate matter 48, and a second skin 52 is formed on the inside of female mold 46.Thereafter, the excess plastic particulates are removed by dumping or vacuuming, an expandable foam is distributed between the molds, and the male mold is placed within the female mold, or vise versa, and held at a predetermined distance apart so that the expandable plastic 54 can be expanded between the two molds with their respective skins.",
"The expandable plastic can “foam up” until it fills the cavity created by the two mold pieces.",
"If the molds are secured to one another while leaving a one inch (1″) space between them, a one inch expansion will occur.",
"If, on the other hand, the mold pieces are maintained six inches (6″) apart, then the expansion layer will be six inches thick.",
"As described above, any desired inserts and/or reinforcements may be placed between the two molds, along with the expandable plastic, before they are placed together and the heat from the molds heat up the expandable foamable plastic to make it expand.",
"Once the expandable plastic sets, it will encapsulate the insert/reinforcement within the skins and will secure the insert/reinforcement from any side-to-side motion, especially if the insert/reinforcement has any surface contour or porosity so that the expandable plastic will surround the insert and hold it in place.",
"The inventors have found that gravity alone is a sufficient force to hold the two molds together, held apart by spacers, and the residual heat from the mold is sufficient to kick off the expandable foam plastic such that it will expand.",
"In the event of using this technology for a pick-up truck bed box, it is envisioned that the wiring harness can be embedded into the truck bed box itself, with the electrical connectors extending outwardly from the box, ready to be plugged into the electrical connections coming out of the back of the truck.",
"The wiring components can be laid onto the male mold before the female mold is laid over top of it, and before the expandable plastic is subjected to heat, causing it to expand and encapsulate the wiring components right into the truck bed box itself, while allowing the connectors to hang loose, ready to be assembled into the truck.",
"In the alternative, a conduit could be embedded into the plastic truck box to allow for wiring to be fed therethrough.",
"The outer skins of the truck bed box can be molded to perfection with color so that painting of the truck bed box is unnecessary.",
"Other applications for the present technology will be discussed below, and the appropriate configuration and insert/reinforcement for each application will be discussed.",
"The inventors also envision that the mold itself can be made of an electrically conductive material.",
"This electrically conductive mold can be charged to attract fine plastic particles, melt them on the surface, and form a thin-skinned part to be removed after cooling.",
"This is also suitable for use with electrostatic powder coat paints.",
"For example, a mold can be electrically charged and sprayed with a releasable powder coat paint resin first, then heated and cured while using the curing heat to heat the mold and then contacting with plastic particulates which will adhere to the paint, to a desired thickness.",
"Upon cooling, the newly formed article will “pop” out of the mold with a freshly cured paint job thereon.",
"It is also envisioned by the present inventors that varying materials can be used across the surface of a formed article, as shown in FIG.",
"4, with the article being generally denoted by the numeral 60, with top and bottom skins 62 and 64 respectively, and having various materials 66 and 68, respectively.",
"This is accomplished by heating various portions of the mold and contacting with different materials.",
"Preferably, heater lines can be incorporated into the mold in separate sections.",
"For instance, the mold could first be heated in the regions of area 68, and then contacted with a first material.",
"Then, the mold would be cooled in those areas, such that it would not melt plastic, although the remainder of the mold, the top skin 62 could be heated so that a second material would be melted against its surface.",
"Likewise with the material region 66 as shown in FIG.",
"4., which could remain cool during the first two procedures, but would be heated by itself later on and then contacted with a third material.",
"Other means are envisioned for only heating certain portions of the mold, while controlling which portions will have different plastics adhered to those various portions.",
"In addition, once the multi-material layer has been formed, the double skin, or sandwich concept described hereinabove, may come into play in order to form a foamed or reinforced article from a multiple material skin.",
"Therefore, the various material configurations, layers and inserts/reinforcements envisioned, among others, are described.",
"There are many more configurations which will become apparent as we discuss some of the most pertinent applications hereinbelow.",
"III.",
"Various Process Embodiments Now that we have discussed the actual structure of a portion of an article made in accordance with the present invention, we will turn to the various methods of contacting the powder to the mold, so that the mold can melt the plastic and form it to its ultimate shape.",
"Because one of the most pressing applications is for automotive vehicle components, the basic tip molding process embodiment of the present invention will be discussed now with respect to a polyethylene pick-up truck bed box.",
"As shown in FIG.",
"5, there is a production method for manufacturing the truck box in accordance with the present invention by using an upper and lower line generally denoted by the numeral 70.There are two molds shown, top and bottom 72 and 74, which represent the male and female molds being covered with melted plastic.",
"The mold is heated by any of the acceptable methods described above, which may include placing in an oven, heating with torches, or by utilizing lines within the mold to contain hot water, oil or gas.",
"In the case of the male mold, the heated mold is placed within a box 76 capable of holding the mold and containing enough plastic particulate to cover the male mold.",
"In the preferred embodiment, while this is going on, the female mold 74 is heated and then filled with the desired plastic particulate 78, and both are allowed to remain in contact with the heated molds for approximately six to eight minutes to achieve a polyethylene truck bed box skin of about three millimeters (3 mm) thick.",
"Then, the molds are either tipped upside down to dust off the excess plastic particulate or the excess is vacuumed out of the box by vacuum hoses 80.Load rails 82 and a steel wire mesh reinforcement screen 84 is laid onto the top of the male mold as seen in FIG.",
"6.This wire mesh 84 adds strength and impact resistance to the truck bed box once manufactured.",
"A second wire mesh 84 may be especially useful, and would be placed in the female mold after the excess plastic has been removed.",
"Thus, a set of complementary wire mesh reinforcements 84 can be encapsulated between the double skins.",
"After expandable plastic has been placed on the male mold, the two pieces are then slid into and over one another and the expandable plastic is heated by the residual heat in the hot molds and the expandable plastic “blows” and expands to fill the cavity which has been pre-set by the distance that the male and female molds have been held apart.",
"Then, the mold is cooled, and the part is popped out.",
"In this embodiment, and as shown in FIG.",
"7, it is envisioned that having a vacuum portal 90 attached to the bottom of the mold will aid in the removal of any loose plastic particulate after the desired thickness has been achieved.",
"As shown in FIG.",
"8, load rails 82, or any other desirable mounting means, may be lowered into the bottom of the female mold 74.That way there will be steel mounting rails 82 extending from the bottom of the truck bed box, so that mounting will be easily achieved on the truck chassis.",
"It is also envisioned that there could be vacuum lines 92 and hoses attached to the top and bottom of the mold-containing box or into a cap to be placed over the female mold, and those vacuum lines 92 could also be a means for delivering the plastic particulate 78 onto the top of the mold.",
"Whether male or female, the plastic particulate is allowed to sit for an appropriate resident time, and then vacuumed out from vacuum portals 90 located in the bottom.",
"The plastic particulate materials could be cycled in and out of the molds.",
"For example, vacuum line 92 could be used to blow in the plastic, and then vacuum portal 90 could be used to vacuum out the particulate after it has contacted the heated mold for a sufficient length of time.",
"Or, the same lines could be used to blow in and vacuum the plastic.",
"Further, the vacuum lines could be valved to different bags filled with different materials to achieve a multi-layer article.",
"The particulate would then be the moving part, not the mold.",
"This would allow the heated molds to remain stationary, thereby alleviating the need for tipping over the mold, and would require the same amount of time for filling and emptying the molds.",
"Furthermore, multiple plastic sources would be much simpler due to the ability of picking up any plastic particulates, including different materials for multiple layers, or different regions with varying materials.",
"Looking now to FIG.",
"9, there is shown another embodiment of a trunion 100 used by the present invention for “tipping” the loaded mold(s) in order to empty out the excess plastic particulate after the appropriate time for melting has taken place.",
"A cradle 102 is incorporated into the apparatus and is shown for tipping the mold 104 about a pivot 106, effecting the tip molding method of FIG.",
"5.FIG.",
"10 illustrates the preferred embodiment for the lower side of the truck bed mold 74, while FIG.",
"11 shows the upperside of the truck bed mold 72.FIG.",
"12 shows a truck bed test tool design 110 for making test “plaques” on a rotating stand 112.The molds are made with a cavity and a core design.",
"The mold is heated with electrical cartridges 118 by controllers (not shown) and the test plaque is cooled for release by mold waterlines 114.The mold is attached to a rotating stand 112 so that the excess plastic particulate can be “tipped” out.",
"Rotation about a pivot 116 is utilized to “tip” out the excess.",
"Cavity 120 is shown above the core 122.FIG.",
"13 shows the test device of FIG.",
"12 with the cavity and the waterlines 114 in conjunction with the waterlines and the heaters 118, in more detail.",
"Thermocouples 119 are shown.",
"This mold configuration may generally be applied to other relatively flat double-skinned items, such as the modular housing units.",
"Looking next to FIG.",
"14, a core 122 is shown with an insert block 124 and reinforcing cones 126 where they are located.",
"The “cones” 126 are located at the places where the load might be the greatest in the resulting article, thereby requiring the most reinforcement.",
"FIG.",
"15 shows a screen die 130, within the cavity 120, while FIG.",
"16 shows the same screen die 130, but from the core 122 side.",
"FIG.",
"17 shows yet another embodiment with a redesigned core insert to incorporate a larger cone design 132 for additional strength.",
"FIG.",
"18 shows mold plugs 140 which will be mounted to create the indentations for receiving a cone reinforcement once the male and the female molds are together with the reinforcements being held therebetween.",
"Although the plastic particulate may be powder, pellets, resin, sheets, blocks, or any other commercially available form of plastic, it may be any suitable polyolefinic chemical composition, so long as it melts at a reasonable temperature.",
"The plastic may contact the heated mold by any number of methods, including, but not limited to, spraying, either manually, robotically or through spray bars; dumping plastic over the mold and containing the over-dumped amount in a container with the heated mold inside (in the case of a male mold), or it may be dumped or sprayed directly into a female mold.",
"The plastic can be distributed with a shaker arm or may be done manually.",
"Or, the blown in/vacuumed out method as described earlier may be most advantageous in which the plastic may also be blown into a container with the heated male mold inside, or may be blown into the cavity directly, as created by a female mold.",
"In either event, the excess plastic may be vacuumed out of the box or the mold, or the excess may be “tipped” out by rotating the mold to drop the excess plastic from the heated mold.",
"Yet another embodiment for the process may use a fluidized bed to contact a heated mold with plastic particulate.",
"Although most easily accomplished if the plastic is in the form of powder, the present inventors also envision that the fluidized bed could use pellets after a first layer of powder is melted onto the mold.",
"A fluidized bed configuration may also use the vacuum concept discussed above for introducing the plastic, as well as for flowing and removing the plastic.",
"Still yet another embodiment for contacting the plastic to the heated mold may include the use of a heated, electrically charged mold coming into contact with an electrically charged plastic which is sprayed toward or onto the surface, and held on the surface of the mold.",
"This electrostatic method may require further layering to achieve a perfectly painted surface once the article is removed from the mold.",
"Since the mold pieces can be “clam-shelled” together after the skin has been formed, this electrostatic method may be able to make very thin skins for the production of thinner, more delicate, articles.",
"For adhesion, the electrostatic method may require the use of an epoxy resin, as is usually used with powder coat paints, known well in the art.",
"However, it is believed that combining the traditional epoxy spraying with heating the electrically charged mold and contacting it with electrically charged plastic particulate is a novel method.",
"Then, when the part is released from the mold, either the heat from the mold will cure the resin paint, or it can be heated even further to impart a beautifully cured painted surface, just like powder coated paint.",
"Or, the plastic particulate could be in the form of a powder that is somewhat electrically charged, and it could be attracted to the heated mold by the electrically charged heated mold.",
"A fine powder would be able to be sprayed on, or used in a fluidized bed, as described above.",
"A heavier, coarser plastic particulate may be utilized in order to save money on the powder.",
"In this instance, it may be advantageous to incorporate a thin layer of finely ground powder material prior to contacting with the coarser material, in order to encourage a thin, tacky layer of plastic to build up first on the mold, making it easier for the coarse material to heat and “stick” to the mold.",
"True to electrostatic coating, a finer plastic powder which is electrically charged could be attracted to the mold, and then heated while the powder is being held in place by electricity, in order to melt the plastic and form a thin-skinned article.",
"Once the skins have been formed by the electrostatic method, the male and female portions can be “clam-shelled” together and any other inserts and/or reinforcements may be utilized in conjunction with expandable plastic therebetween, similar to the description above.",
"Now we turn our attention to additional materials, inserts and/or other reinforcements which may be useful in strengthening the plastic forms.",
"Additional materials may render them fire resistant, or as thick or thin as needed.",
"Although this is not an all inclusive list, the following additions are specifically envisioned for various applications: metal screens, grids and meshes, either bare or coated, such as with powder coating, as well as screens, grids and meshes that may be welded or secured with adhesives to prevent lateral shearing motion; thermoelectric devices for heating and/or cooling; slag, lava, and other construction materials to act as heat resistant fillers, fiberglass whether in the form of mesh, woven or non-woven for strength; whisker-filled particulates; conduits or pipelines used for cooling the center of the mold, i.e.",
"pins placed in the mold; electrical wires or conduits placed in the center to house electrical wires; foamed or solid ceramics for adding tensile strength without weight; a pre-formed foam core with a higher melting temperature; metallic structures, such as metal mesh reinforcing cones or other high-rising embeddable structures to add strength; low density stones or other naturally occurring low density materials; wood in any shape to be used for reinforcements or to add strength without adding much weight; metal mounting or securing reinforcements, including metal bars and mounting plates for mounting purposes; whiskers of various glasses such as fiberglass; Kevlar to impart impact and energy dispersion; fire retardant materials; anti-microbial agents to be placed near the surface for alleviating germ transfer; chemical treatments at the surface to reduce chemical interactions with materials being contained within the articles; and any other desirable insert.",
"In addition to the above described inner workings of articles made in accordance with the present invention, certain surface effects can be molded directly into the article itself.",
"One embodiment of the present invention which deals with surface effects only is illustrated in FIG.",
"19, showing a raised vehicle roof generally denoted by the numeral 200 having dimples 202 to allow air flow 204 to glide thereover.",
"The dimples 202 are similar to the dimples on a golf ball which improve the aerodynamics of a large vehicle roof.",
"In the instance of a large van or mini-van, the effect could be significant.",
"The dimples 202 could occur over the entire surface, or may be located in the front only.",
"In addition, it is envisioned that the dimples may be of the same or of varying sizes across the surface.",
"Because the popularity of raised roof vans is gaining, as they improve headroom in vans, the added height of the raised roof vans increases the frontal area, thereby increasing overall drag for the vehicle.",
"This has a negative effect on the vehicle fuel economy.",
"The proposed dimpling positively effects the fuel economy of such vehicles by interrupting the attached flow of air, while the laminar flow of air becomes turbulent flow.",
"It is known that turbulent air flow on the back side or downward side of center of the vehicle will reduce the vehicle aerodynamic drag.",
"This embodiment may also be used on side panels, or any other surface which needs to have its drag reduced.",
"In addition to the other disclosed insert or reinforcement materials, a zigzagged metal wire reinforcement may be embedded into the skins of polyolefin material for energy absorbing applications such as knee restraints, dashboards and the like.",
"FIG.",
"20 illustrates how the wire is anchored in place within the polyolefin skin.",
"Especially useful will be a hard side to support the load while the other side may be soft to permit controlled distortion on impact.",
"It is especially useful if low density plastic foam is used between the two skins.",
"FIG.",
"20 generally denotes the energy absorbing article as 220, and includes a zigzagged wire reinforcement 222 embedded between inner and outer skins 224 and 226 respectively.",
"Low density foamed plastic 230 is shown in the center, surrounding the wire reinforcement 222.As can be seen from the drawing, an impacting force 240 would have its energy absorbed by crushing the wire reinforcement 222, which will collapse upon impact, and smashing the low density foam.",
"In addition, certain energy dispersive materials such as Kevlar or certain energy dispersive foam pre-forms may be embedded into the plastic article to absorb and/or disperse energy.",
"Such an energy absorbing combination would allow lighter-weight components to be used in vehicles, while maintaining the structural strength needed for safety.",
"This material composite can be used for high impact resistance bumpers or front end modules, in addition to interior components.",
"In this invention, the most preferred embodiment of the pick-up truck box includes a high density polyethylene truck box having an embedded welded steel wire grid.",
"Having been powder coated, and thereafter formed into a pre-formied piece that can be dropped into the mold after the two skins are formed, the wire grid is inserted before the skins are “clam shelled” together.",
"It has been discovered that by encapsulating the wire mesh pre-form between the plastic skins, the coefficient of linear thermal expansion of the combined materials becomes that of the mold material.",
"Complementary male and female wire grids may include raised, cone-shaped portions for additional reinforcement and strength.",
"This type of configuration also finds special utility with regards to vehicle roof components, pillar sections, underbody components, wheel well covers, large battery trays and floor sections.",
"The long glass fibers used in prior art manufacturing techniques generally exhibit a strength of about 3×106 PSI modulus, while the composite with metal reinforcement of the present invention exhibits the modulus of the metal itself, generally on the order of 30×106 PSI modulus, or about ten (10) times stronger.",
"Each of the reinforcements listed above provide additional modulus strength to the plastic composite article.",
"In that regard, if the modulus strength of metal is necessary, then it is useful to utilize a metal reinforcement.",
"On the other hand, if an impact resistant part is required, then the reinforcement could be a memory plastic foam that will “bounce back” after the impact.",
"If the desired article needs to bend around a corner, then the skin material and the reinforcement would be selected accordingly.",
"When making a multi-layer article in accordance with the present invention, the male and female skinned molds or “clamshells” may be allowed to lay on top of one another, or the two “clamshells” may be clamped together if gravity alone is insufficient to handle the job.",
"In that case, the clamping may be accomplished via hydraulics, pneumatics, hydropneumatics, or electrically.",
"If an expandable foam center is to be used, the molds may be clamped together while being held apart at a pre-determined distance in order to determine the thickness of the part.",
"However, in general, the weight of the top mold will hold it down onto the bottom.",
"Cooling of the heated mold may be accomplished by various means, including, but not limited to utilizing heating/cooling lines within the mold itself; moving the entire plastic/mold assembly into a freezer or refrigerator or some other climate controlled room.",
"Thermoelectric devices may be used in the mold to cool.",
"Once cooled, the plastic article generally pops off the heated mold and does so easily.",
"The cooling configuration could also be in the form of pins that can be inserted within the mold after the heating takes place, and the pins could be refrigerated themselves, or could contain lines that will cool the mold.",
"These pins could be easily removed from the mold so that the next cycle of the mold could be a heated cycle (with heater lines already in the mold-just turned off during the cooling phase).",
"Therefore, the process and articles made in accordance with the present invention have been described.",
"Now we turn to specific applications, and the configuration of some of the individual manufactured articles.",
"IV.",
"Specific Applications Specific applications for the above invention may include, but are certainly not limited to: automotive and industrial vehicle components such as pick-up truck bed boxes, SUV roofs, underbody constructions, and wheel wells; modular housing panels; airplane components; consumer and industrial furniture; doors; windows, material handling pallets and other articles; consumer goods; industrial articles; marine applications and boat hulls; molds and components, including seawalls, boat hulls and the like; medical apparatuses and other applications; scaffolding and other building construction articles; sea containers; railroad containers; composite wheels for trains and vehicles; children's toys; military applications such as floating bridges and personnel transport vehicle components; playground equipment; aerospace applications; roofing components; rot-free lumber and decking without arsenic; farming and agricultural applications; industrial machinery pads; dunnage; and food shipping containers including food containers of all sizes and shapes, just to name some of the applications.",
"Each of these applications will include various forms of the plastic articles, including various materials sandwiched between two or more skins in order to produce the desired material properties.",
"A.",
"Pick-Up Truck Bed Box A pick-up truck bed box is disclosed which includes two outer skins and an expandable plastic therebetween, with reinforcing screens and cones for structural strength as shown in the Figures.",
"The preferred production method is the “tipping” method described hereinabove.",
"The pick-up truck bed box is one of the preferred embodiments of the present invention and is manufactured pursuant to the method disclosed hereinabove with reference to FIG.",
"5, among others.",
"This is a configuration which utilizes two complementary male and female exterior skins, having an inner portion of expandable or low density foam plastic.",
"In addition to the filled skins, two pre-formed steel wire mesh grid reinforcements are embedded and surrounded within the low density foamed inner plastic center.",
"These steel wire mesh grid reinforcements have been made of a welded mesh configuration that has been previously powder coated to aid in adhesion.",
"The mesh reinforcements have been pre-formed to be shaped like each of the male and female molds, and are therefore easy to drop into or onto their respective molds prior to holding the molds together to effect the foaming of the center plastic.",
"Wire mesh cone reinforcements are also incorporated at strategic locations to further enhance the strength of the resulting article.",
"The reinforcements can easily be placed by robots once the skins of plastic have been melted onto the male and female molds and the excess plastic particulate has been removed.",
"Then the two heated and skinned molds can be brought together, one resting on the other, and the residual heat from the molds will make the expandable foam plastic expand until it reaches the two outer skins, thereby embedding and encapsulating the two wire mesh reinforcements and the cone reinforcements forever in place within the truck bed box.",
"B.",
"Industrial Tabletops An industrial tabletop is disclosed which utilizes two polyethylene skins with an expandable plastic, reinforced with a metal mesh screen, or not, to form a table top which is commonly used as a banquet table for catering in hotels, restaurants and the like.",
"Such an industrial tabletop made in accordance with the methods, processes and apparatuses of the present invention would be made by complementary male and female molds which can be used to form plastic skins of any suitable polyolefin, such as high density polyethylene, with an expanded plastic center.",
"The expandable plastic is preferably the low density polyethylene foamable plastic from Equistar Corporation of Cincinnati, Ohio.",
"Depending upon the usage of the tabletop, whether it be a hotel banquet table, or a medical table, the weight requirements may necessitate the use of an optional metal mesh matrix insert/reinforcement to be placed in the middle of the two skins.",
"Table leg mounting portions such as mounting rails, may be inserted into the mold such that certain portions of the mounting rails extend outwardly from the molded article, such that table legs can be easily attached.",
"This yields a strong yet lightweight tabletop, one which will easily attach to table legs.",
"In addition, the tabletop can have a surface texture formed directly into the mold, and various colors of plastic can be used to simulate wood, stone, or any other desirable look for the top.",
"C. Airplane Cockpit Doors An airplane cockpit door may be made from the present invention, utilizing a double skin configuration with an energy dispersive expandable foam, preferably containing sheets of Kevlar in the middle of the layers, to render the doors bulletproof, and to prevent a hijacker from kicking down the door.",
"Steel mesh reinforcements may be utilized to provide the necessary strength, while allowing the entire configuration to be lightweight enough for use on an airplane.",
"Similar to the pick up truck box configuration, there are two skins with an expandable foam plastic center having at least a Kevlar sheet embedded into the foam plastic center to render it bulletproof.",
"Steel reinforcing inserts may be utilized around the door latch and hinges to add strength against someone trying to get into the cockpit of the plane.",
"The Kevlar sheet may be coated with a fine layer of plastic or resin prior to its insertion in order to prevent it from shearing out of the plastic foamed center during impact.",
"Furthermore, the Kevlar material can be permanently attached to a steel mesh cage which can be incorporated into the center of the cockpit door composition, to prevent it from coming loose during impact, such as when it is fired on by a bullet.",
"Other inserts and reinforcements may be necessary in order to form an entirely useful airplane cockpit door.",
"D. Modular Housing Panels and Components Modular housing panels can easily be manufactured using the present invention incorporating a double skin configuration with insulative materials in the expandable plastic portion of the panels.",
"The expandable plastic center portion can be mixed with sawdust, foam beads, hollow spheres of plastic, or any other suitable insulating material that can withstand the heat from the expansion of the foamable plastic center, while retaining its insulating properties.",
"When the panels are clipped or caulked together, the insulating properties of the filler material yield a home that is resistant to winter temperatures outside.",
"It is envisioned that standard sizes and shapes of the panels could be molded to create a standard three to four meter wide box ready to have floor and roof sections attached thereto.",
"There are also T-shaped connectors and L-shaped connectors in order to fit the panels together into desirable configurations.",
"The panels may have a single set of electrical wires embedded therein, or they may have a conduit running vertically and one running horizontally to thread the necessary electrical wires therethrough.",
"Electric lights could be mounted or attached to the vertical conduit, while the supply and ground wires could be threaded horizontally to an electric source outside the “house”, whether it be to a generator or a source of electricity.",
"The panels can be molded into log-like strips as well.",
"They can be made to snap together and then caulked to form an “instant” house.",
"This will be most useful for wartime refugees entering into cold countries.",
"Instead of setting up tents, these panels can be snapped together.",
"The wiring for electricity can be embedded right into the panels, so that they can have instant electricity for warmth and food preparation.",
"Recreational use for “camping” can also be envisioned E. Material Handling Pallets Pallets used in industrial material handling are made with a double skin configuration, with an expandable reinforced core, preferably including metal cone structures to make a very durable, lightweight, and inexpensive pallet that can be made with recycled materials.",
"The other applications are too numerous to mention in detail here, although it must be stated that various combinations and permutations of the present invention may be utilized for all the applications mentioned, as well as for ones which were not mentioned.",
"The present invention may be incorporated into the manufacture of so many articles, it would be impossible to list them all here.",
"INDUSTRIAL APPLICABILITY This invention finds industrial applicability in the formation of various articles from plastic particulate materials.",
"It is especially useful in the manufacture of automotive components, modular housing panels, industrial tabletops and furniture, and airplane components."
]
] | Patent_10239039 |
[
[
"Electronic apparatus",
"In the structure of an electronic apparatus, in which cooling of an heat-generating element is achieved through circulation of a liquid, in particular, for providing the structure of being high in cooling performance and reliability, wherein a heat-radiation pipe 9 is connected to a heat-radiation plate 10 disposed in a rear surface of a display 2, while thermally connecting a water-cooling jacket 8 with the heat-generating element 7, thereby circulating a coolant liquid between the water-cooling jacket 8 and the heat-radiation pipe 9 by means of a liquid driving device 11.",
"The water-cooling jacket 8 can be formed in one body of a jacket base and a flow passage therein through the die-cast forming thereof, or can be constructed in one body with the water-cooling jacket and the flow passage of piping, through connection between the jacket base and the metal pipe."
],
[
"1.An electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein said heat-receiving member has a metal plate being thermally connected with said heat-generating element, and a flow passage for said coolant liquid is formed within an inside of said metal plate.",
"2.An electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein a flow passage of said heat-receiving member is formed with a portion of a pipe constructing a flow passage within which said coolant liquid circulates.",
"3.An electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein said heat-receiving member has a metal plate being thermally connected with said heat-generating element, and said metal base is thermally connected with a portion of a pipe within which said coolant liquid circulates.",
"4.An electronic apparatus, as described in the claim 3, wherein said metal base and the portion of the pipe, within which said coolant liquid circulates, are connected through a grease or an adhesive of thermo-conductive property.",
"5.An electronic apparatus, as described in the claim 3, wherein said metal base and the portion of the pipe, within which said coolant liquid circulates, are formed in one body.",
"6.An electronic apparatus, as described in the claim 3, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in loop-like, and is thermally connected with said metal base.",
"7.An electronic apparatus, as described in the claim 3, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in a loop-like shape, directing from a center to an outer periphery thereof, by roughly bringing a central position of the loop in coincident with that of said heat-generating element, so that said coolant liquid is directed from the center to the outer periphery in direction of circulation.",
"8.An electronic apparatus, as described in the claim 3, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in such a loop-like shape, that flows within flow passages are directed opposing to each other in direction thereof, and is thermally connected with said metal base.",
"9.An electronic apparatus, as described in the claim 1, wherein said heat-generating elements are disposed in a plural number thereof, and those plural number of the heat-generating elements are thermally connected with said heat-receiving member.",
"10.An electronic apparatus, as described in the claim 2, wherein said heat-generating elements are disposed in a plural number thereof, and those plural number of the heat-generating elements are thermally connected with said heat-receiving member.",
"11.An electronic apparatus, as described in the claim 3, wherein said heat-generating elements are disposed in a plural number thereof, and those plural number of the heat-generating elements are thermally connected with said heat-receiving member.",
"12.An electronic apparatus, as described in the claim 1, wherein said heat-receiving member is cooled by means of a fan.",
"13.An electronic apparatus, as described in the claim 2, wherein said heat-receiving member is cooled by means of a fan.",
"14.An electronic apparatus, as described in the claim 3, wherein said heat-receiving member is cooled by means of a fan."
],
[
"<SOH> BACKGROUND ART <EOH>Conventional arts can be seen in, for example, Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994), and Japanese Patent Laying-Open No.",
"Hei 7-142886 (1995), etc.",
"In the Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994), for example, is shown the structure of an electronic apparatus, being made up with a main housing accommodating a wiring or circuit board therein, on which a heat-generating element is mounted, and a display housing, having a display panel and being attached onto the main housing rotatably, wherein a water-cooling jacket attached onto the heat-generating element, a heat-radiation pipe, and a liquid driving mechanism are connected with one another through flexible tubes.",
"Further, in the Japanese Patent Laying-open No.",
"Hei 7-142886 (1995), there is shown an example, in which the housing is made of a metal, for example, in the structure shown in the Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994).",
"In those examples, heat generated in the heat-generating element is transferred to the water-cooling jacket, and then the heat is transferred from the water-cooling jacket to the heat-radiation pipe by driving a liquid by means of a liquid driving mechanism, thereby being radiated into the air outside.",
"With such the electronic apparatuses, as being represented by a portable personal computer, etc., an increase of high heat generation by the heat-generating element (i.e., a semiconductor element) is remarkable accompanying with an improvement in performances thereof.",
"On the other side, miniaturization or small-sizing and/or thinning in sizes of the housing is still desired or demanded, so as to be fit to be carried with.",
"Any one of those known prior arts mentioned above has the structure, so that the heat generated in the heat-generating element is transferred to the display side, thereby to be irradiated thereon, with respect to the high heat generation of the heat-generating element.",
"The transfer of heat from the heat-generating element to the display side is carried out through driving a liquid between both sides.",
"The heat transfer by means of the liquid is very preferable in efficiency, and it is suitable for the heat transfer from the element generating heat at high temperature.",
"However, cooling cannot be obtained fully upon the heat-generating element, when the efficiency is bad in the heat transfer from the heat-generating element to the liquid, even if being good in efficiency of the heat transfer by means of the liquid.",
"It is also necessity to take in the consideration, reduction of the liquid within a system, due to penetration or permeation of liquid from the water-cooling jacket itself or a piping system thereof, and corrosion of the water-cooling jacket or the like, as well.",
"However, in the conventional arts mentioned above, the consideration is not fully taken into about the structure of the water-cooling jacket for dissolving those drawbacks mentioned above."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a perspective view of an electronic apparatus, according to a first embodiment of the present invention; FIGS.",
"2 ( a ) and 2 ( b ) are a front view and a A-A cross-section view of a water-cooling jacket applied in the electronic apparatus, according to the above-mentioned first embodiment of the present invention, for showing details thereof; FIGS.",
"3 ( a ) and 3 ( b ) are a front view and a B-B cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a second embodiment of the invention, and FIG.",
"3 ( c ) a front view of a variation thereof, for showing details thereof; FIG.",
"4 is a perspective view of an electronic apparatus, according to a third embodiment of the present invention; FIGS.",
"5 ( a ) and 5 ( b ) are partial cross-section views of a water-cooling jacket applied in an electronic apparatus, according to a fourth embodiment of the present invention; FIG.",
"6 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a fifth embodiment of the present invention; FIG.",
"7 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a sixth embodiment of the present invention; FIG.",
"8 is a front view of a water-cooling jacket applied in an electronic apparatus, according to a seventh embodiment of the present invention; FIG.",
"9 is a front view of a water-cooling jacket applied in an electronic apparatus, according to an eighth embodiment of the present invention; FIG.",
"10 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a ninth embodiment of the present invention; FIG.",
"11 is a front view of a water-cooling jacket and a fan applied in an electronic apparatus, according to a tenth embodiment of the present invention; and FIG.",
"12 is a front view of a water-cooling jacket and a fan applied in an electronic apparatus, according to an eleventh embodiment of the present invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to an electronic equipment or apparatus, having a device for cooling a semiconductor elements generating heat therefrom, with utilizing a circulating liquid therein.",
"BACKGROUND ART Conventional arts can be seen in, for example, Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994), and Japanese Patent Laying-Open No.",
"Hei 7-142886 (1995), etc.",
"In the Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994), for example, is shown the structure of an electronic apparatus, being made up with a main housing accommodating a wiring or circuit board therein, on which a heat-generating element is mounted, and a display housing, having a display panel and being attached onto the main housing rotatably, wherein a water-cooling jacket attached onto the heat-generating element, a heat-radiation pipe, and a liquid driving mechanism are connected with one another through flexible tubes.",
"Further, in the Japanese Patent Laying-open No.",
"Hei 7-142886 (1995), there is shown an example, in which the housing is made of a metal, for example, in the structure shown in the Japanese Patent Laying-Open No.",
"Hei 6-266474 (1994).",
"In those examples, heat generated in the heat-generating element is transferred to the water-cooling jacket, and then the heat is transferred from the water-cooling jacket to the heat-radiation pipe by driving a liquid by means of a liquid driving mechanism, thereby being radiated into the air outside.",
"With such the electronic apparatuses, as being represented by a portable personal computer, etc., an increase of high heat generation by the heat-generating element (i.e., a semiconductor element) is remarkable accompanying with an improvement in performances thereof.",
"On the other side, miniaturization or small-sizing and/or thinning in sizes of the housing is still desired or demanded, so as to be fit to be carried with.",
"Any one of those known prior arts mentioned above has the structure, so that the heat generated in the heat-generating element is transferred to the display side, thereby to be irradiated thereon, with respect to the high heat generation of the heat-generating element.",
"The transfer of heat from the heat-generating element to the display side is carried out through driving a liquid between both sides.",
"The heat transfer by means of the liquid is very preferable in efficiency, and it is suitable for the heat transfer from the element generating heat at high temperature.",
"However, cooling cannot be obtained fully upon the heat-generating element, when the efficiency is bad in the heat transfer from the heat-generating element to the liquid, even if being good in efficiency of the heat transfer by means of the liquid.",
"It is also necessity to take in the consideration, reduction of the liquid within a system, due to penetration or permeation of liquid from the water-cooling jacket itself or a piping system thereof, and corrosion of the water-cooling jacket or the like, as well.",
"However, in the conventional arts mentioned above, the consideration is not fully taken into about the structure of the water-cooling jacket for dissolving those drawbacks mentioned above.",
"DISCLOSURE OF THE INVENTION An object is, therefore, according to the present invention, to provide an electronic apparatus, equipped with a water-cooling jacket having a good heat-transfer efficiency from the heat-generating element to a coolant liquid, and being high in a reliability thereof, in particular, in relation to the corrosion, the liquid penetration or permeation, and liquid leakage, etc.",
"The object mentioned above, according to the present invention, can be achieved by an electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein said heat-receiving member has a metal plate being thermally connected with said heat-generating element, and a flow passage for said coolant liquid is formed within an inside of said metal plate.",
"Also, the object mentioned above, according to the present invention, can be achieved by an electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein a flow passage of said heat-receiving member is formed with a portion of a pipe constructing a flow passage within which said coolant liquid circulates.",
"Further, the object mentioned above, according to the present invention, can be achieved by an electronic apparatus, comprising: a heat-receiving member being thermally connected with a heat-generating element; a heat-radiation member being connected with said heat-receiving member; and a liquid driving means being connected with said heat-receiving member and said heat-radiation member, being received within a casing, in which a coolant liquid is circulated by said liquid driving means between said heat-receiving member and said heat-radiation member, wherein said heat-receiving member has a metal plate being thermally connected with said heat-generating element, and said metal base is thermally connected with a portion of a pipe within which said coolant liquid circulates.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein said metal base and the portion of the pipe, within which said coolant liquid circulates, are connected through a grease or an adhesive of thermo-conductive property.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein said metal base and the portion of the pipe, within which said coolant liquid circulates, are formed in one body.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in loop-like, and is thermally connected with said metal base.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in a loop-like shape, directing from a center to an outer periphery thereof, by roughly bringing a central position of the loop in coincident with that of said heat-generating element, so that said coolant liquid is directed from the center to the outer periphery in direction of circulation.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein the portion of the pipe, within which said coolant liquid circulates, is formed in such a loop-like shape, that flows within flow passages are directed opposing to each other in direction thereof, and is thermally connected with said metal base.",
"Also, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein said heat-generating elements are disposed in a plural number thereof, and those plural number of the heat-generating elements are thermally connected with said heat-receiving member.",
"And, the object mentioned above, according to the present invention, can be achieved by the electronic apparatus, as described in the above, wherein said heat-receiving member is cooled by means of a fan.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view of an electronic apparatus, according to a first embodiment of the present invention; FIGS.",
"2(a) and 2(b) are a front view and a A-A cross-section view of a water-cooling jacket applied in the electronic apparatus, according to the above-mentioned first embodiment of the present invention, for showing details thereof; FIGS.",
"3(a) and 3(b) are a front view and a B-B cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a second embodiment of the invention, and FIG.",
"3(c) a front view of a variation thereof, for showing details thereof; FIG.",
"4 is a perspective view of an electronic apparatus, according to a third embodiment of the present invention; FIGS.",
"5(a) and 5(b) are partial cross-section views of a water-cooling jacket applied in an electronic apparatus, according to a fourth embodiment of the present invention; FIG.",
"6 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a fifth embodiment of the present invention; FIG.",
"7 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a sixth embodiment of the present invention; FIG.",
"8 is a front view of a water-cooling jacket applied in an electronic apparatus, according to a seventh embodiment of the present invention; FIG.",
"9 is a front view of a water-cooling jacket applied in an electronic apparatus, according to an eighth embodiment of the present invention; FIG.",
"10 is a partial cross-section view of a water-cooling jacket applied in an electronic apparatus, according to a ninth embodiment of the present invention; FIG.",
"11 is a front view of a water-cooling jacket and a fan applied in an electronic apparatus, according to a tenth embodiment of the present invention; and FIG.",
"12 is a front view of a water-cooling jacket and a fan applied in an electronic apparatus, according to an eleventh embodiment of the present invention.",
"BEST MODE FOR CARRYING OUT THE INVENTION An electronic apparatus, such as a so-called personal computer, includes a notebook-type personal computer, which is portable, and a desktop-type personal computer, which is mainly used on a desk.",
"In each of those personal computers, being demanded to be high and large in processing speed and capacity thereof every year, temperature of the heat generation of a CPU, i.e., the semiconductor element, comes up to be higher, as a result of those requirements.",
"And it is expected that this tendency will continue further in future.",
"On the contrary to this, in general, the personal computers are of an air-cooling type by means of a fan or the like, at the present situation.",
"This air-cooling type has a limit in the capacity of heat-radiation, and there is a possibility that it cannot follow the heat-radiation of the CUP, which is on the tendency of high heat-generation as was mentioned in the above.",
"However, it may be possible to treat with, by making the fan rotating with higher speed and/or large in the size thereof, but it brings about an effect against for low-noise generation and/or light-weighting, therefore it is not a realistic solution.",
"On the other hand, as a method for heat-radiation, to be replaced with heat-radiation of the conventional air-cooling type, there is a device for cooling the CPU by circulating a cooling medium or coolant, such as water, etc.",
"Such the cooling device is mainly used in a cooling system for use in a large-scaled computer, being located in a company or bank, etc., and wherein cooling water is circulated compulsively by means of a pump and is cooled down by a refrigerator for exclusive use thereof, thereby being large in the scale or sizes.",
"Accordingly, such the cooling device by means of the water as was mentioned above is unable, at all, to be mounted into the notebook-type personal computer, which is moved or carried with frequently, and into the desk-top personal computer, which also may be moved due to re-arrangement in an office, etc., even if this cooling device can be made small in sizes, for example.",
"Then, as was in the conventional arts mentioned above, various devices are studied, for achieving the cooling by means of the water, which can be mounted onto a small-sized personal computer, however the temperature of heat-generation by the semiconductor element was not so high as in the recent year, at the time when such the conventional arts were made and filed as the patent applications, therefore no personal computer equipped with such the water-cooling device comes out, as an actual product available on markets, until up to now.",
"On the contrary to this, according to the present invention, it is possible to achieve small-sizing of the water-cooling device, greatly, by building up the housing defining an external form of the computer main body, made of aluminum alloy or magnesium alloy, etc., being superior in heat-radiation, thereby enabling to mount the water-cooling device into the personal computer.",
"By the way, in order to cool down the semiconductor element within a limited space, such as inside the personal computer, it is necessary to perform cooling with a liquid medium (coolant) of a limited amount.",
"Accordingly, it is an important object for the water-cooling device to transfer heat of the semiconductor element to a water-cooling jacket without waste.",
"If assuming that heat of the semiconductor element cannot be transferred to the liquid medium fully, the cooling of the semiconductor element cannot be carried out sufficiently, thereby bringing about a probability of causing thermal runaway thereof, depending on a case.",
"Then, according to the present invention, as a result of studying the water-cooling jacket of high heat-transfer efficiency, it is possible to obtain a water-cooling jacket, which will be explained in the following.",
"Hereinafter, explanation will be made on various embodiments according to the present invention, by referring to the drawings attached.",
"FIG.",
"1 shows a perspective view of an electronic apparatus, as a first embodiment according to the present invention.",
"In FIG.",
"1, the electronic apparatus is constructed with a main case 1 and a display case 2 having a display thereon, and those cases 1 and 2 are rotatable with each other through a hinge.",
"On the main case 1 are provided a keyboard 3, a wiring or circuit board 4, on which a plural number of elements are mounted, a hard-disc drive 5, an auxiliary memory device (for example, a floppy-disc drive, a CD drive, etc.)",
"6, a battery 13, etc.",
"On the wiring or circuit board 4, there is mounted a semiconductor element, such as a central processing unit 7 (hereinafter, being described by “CPU”) having a large heat-generation amount, in particular.",
"The CPU 7 is attached with a water-cooling jacket 8 thereon.",
"The CPU 7 and the water-cooling jacket 8 are connected with each other, through a soft thermo-conductive material (i.e., a mixture of thermo-conductive fillers of aluminum oxide within Si robber, for example).",
"On a rear surface (an inner side surface of the case) of the display case 2 are provided a metal heat-radiator plate 10, on which a heat-radiation pipe 9 (being made of metal, such as copper, stainless, etc.)",
"is connected.",
"In an upper portion of the rear surface in the display case 2 is provided a tank 14, being connected with the heat-radiation pipe 9, and in more detail, being provided on the way of flow passage thereof.",
"The tank 14 has such a capacity, so as to secure an amount of a liquid therein, being necessary for cooling within a circulation flow passage, even if it is lessened due to permeation thereof, or the like.",
"However, by making the display case 2 itself of a metal, such as aluminum alloy, magnesium alloy, etc., it may be possible to connect the heat-radiation pipe 9 directly on the display case 2, without the heat-radiation plate 10.And, a pump 11, as a liquid driving means, is provided within the main case 1.The water-cooling jacket 8, the heat-radiation pipe 9 and the pump 11 are connected with flexible tubes 12, so that a coolant contained in an inside thereof can be circulated by means of the pump 11.The flexible tubes 12 may be used, for example, at least only in a part of the hinge portion 15.Thus, using metal pipes in piping between the water-cooling jacket 8 and the hinge portion 15, between the pump 11 and the hinge portion 15, and between the pump 11 and the water-cooling jacket 8, while connecting the metal pipe and the heat-radiation pipe 9 by the flexible tuber 12, at least only in the portion of the hinge portion 15 can make the ratio of the metal piping portion as large as possible, occupying in the entire piping.",
"With this, it is possible to meet the requirement of open-close of the display portion around the hinge portion, as well as, to suppress the permeation of water from the pipe.",
"The piping system in this case is built up, by connecting the water-cooling jacket, the flexible tube, the metal pipe, the flexible tube of the hinge portion, the heat-radiation pipe, the flexible tube of the hinge portion (the metal pipe, and the flexible tube), the pump, the flexible tube (the metal pipe, and the flexible tube), and the water-cooling jacket (the elements within the parentheses can be added).",
"In each of connecting portions thereof are applied an appropriate joint and a clamping band (in plate-like, or coil spring-like in the shape) for preventing from slipping off.",
"Further, the connecting portion may be coated with a resin for the purpose of protection from leakage of water.",
"However, as the material of the flexible tubes 12 is applied a butyl rubber, etc., such as, being less in permeation of water therethrough.",
"FIGS.",
"2(a) and 2(b) are a front view and a partial cross-section view thereof, for showing the details of the water-cooling jacket.",
"In the structure shown FIGS.",
"2(a) and 2(b), a flow passage 21 is formed within a base 22, which is made from a metal block, and is sealed with a cover 24 through an O-ring 23.As the material of the base 22 is adopted (pure) aluminum, being superior in thermo-conductivity and forming thereof, and after the forming, it is treated with an anti-corrosion processing, such as an alumite processing (or anodizing process of aluminum), etc.",
"The water-cooling jacket and the heat-generating element 7 are connected with each other, through a soft thermo-conductive material or member 16.It is preferable to make an outer size of the water-cooling jacket larger than that of the heat-generating element 7, and to make an area 21a forming the flow passage within the inside of the O-ring groove as large as possible.",
"The base 22 is formed, together with the flow passage, the O-ring groove, and inlet and outlet ports 31 and 32, within the base, in one body, through the die-cast forming, etc.",
"Through the die-cast forming, the flow passage 21 can be formed with miniaturizing the width thereof to a limit through the die-cast forming, thereby enabling to ensure a surface area of the flow passage large.",
"Accordingly, it is possible to improve the performance of heat transfer to the liquid flowing within the flow passage 21.Further, with miniaturizing the width of the flow passage 21, it is possible to form the flow passage 21, to have the necessary and sufficient surface area within the area, nearly equal to that of the heat-generating element 7 in the base 22, thereby reducing the thermal resistance accompanying with enlargement of the area from the heat-generating element to the area where the flow passage is formed.",
"At the same time, also small-sizing of the water-cooling jacket can be obtained.",
"FIGS.",
"3(a) and 3(b) are a front view and a partial cross-section view of the water-cooling jacket, according to other (i.e., a second) embodiment of the present invention, and FIG.",
"3(c) a front view of a variation thereof.",
"In those FIGS.",
"3(a) and 3(b), the water-cooling jacket is built up with a flow passage made by winding or drawing a metal pipe 26, and it is bonded onto a base 25 of metal (for example, of aluminum, copper, etc.)",
"metallically, as shown by a bonding portion 27, through the soldering or the silver-alloy brazing, in the structure thereof.",
"After being thermally conducted to the base 25, heat of the heat-generating element diffuses within the base 25, thereby conducted to the metal pipe 26, and it is thermally conducted into a liquid coming through an interior wall of the metal pipe 26.In this instance, it is also contributed by thermal conduction in a peripheral direction within the wall of the metal pipe 26.Accordingly, the thermal resistance from the heat-generating element up to the liquid within the flow passage can be made small, by bonding the base 25 within the flow passage and the metal pipe, metallically, and also by bringing the metal pipe 26 to be thick in the thickness thereof while making up the metal pipe 26 of a material, being superior in thermal conductivity, such as copper, etc.",
"In case of forming the winding flow passage from the metal pipe 26, it is necessary to wind or bend the pipe at the minimum-bending radius, so as not to be bent or folded down.",
"Accordingly, in order to keep length of the flow passage within the base as long as possible (i.e., to make the number of turns large), as shown in FIG.",
"3(c), it is also possible to bend or wind the metal pipe at the minimum bending radius being greater than 180°, so as to make the wound portions neighboring contact with.",
"In the present embodiment, since the structure has no such seal portion, comparing to that shown in FIGS.",
"2(a) and 2(b) mentioned above, reduction of the liquid will not occur due to the leakage of the liquid and/or the water permeation in the seal portion.",
"Further, applying the material of anti-corrosion to the metal pipe 26, in the similar manner as in the heat-radiation pipe 26, negates a necessity of taking the corrosion onto the base 25 into the consideration.",
"Also, elongating the metal pipe 26 makes up the flow passage of the water-cooling jacket, thereby bringing it to be used in common, as a part of the piping of a system as a whole, further enables a system, being less in the liquid leakage and the water permeation therein.",
"This embodiment will be shown in FIG.",
"4.FIG.",
"4 is a perspective view of an electronic apparatus, according a third embodiment of the present invention, in particular, in a case that flexible tubes are provided only in the hinge portions of the both cases in the structure thereof.",
"In FIG.",
"4, a portion of piping of the entire cooling system is constructed by elongating the metal pipe 26 of the flow passage, thereby to be used in the water-cooling jacket 8.Thus, in the piping between the pump 11 and the water-cooling jacket and between the water-cooling jacket 8 and the hinge portion, the metal pipe 26 is used for building up the flow passage of the water-cooling jacket.",
"In the hinge portion, between the metal pipe 26 and the heat-radiation pipe 9, and between the pump 11 and the heat-radiation pipe 9 are connected by means of the flexible tubes 12.In the connection portion are provided joints or clamping band 35a-35d for preventing from slipping off, appropriately.",
"According to the present embodiment, since the ratio can be made large of the metal piping portion occupying in the entire piping large, therefore it is possible to build up a piping system being less in the leakage of the liquid and the water.",
"FIGS.",
"5(a) and 5(b) and FIG.",
"6 are cross-section views for showing other (i.e., a fourth and a fifth) embodiments, according to the present invention, being similar to that shown in FIG.",
"3 mentioned above.",
"In the embodiments shown in FIGS.",
"5(a) and 5(b) and FIG.",
"6, a groove 28 is formed in the base 25 made of metal (such as, aluminum, copper, etc.)",
"along with the shape of the metal pipe 26 wound around (for example, through the die-cast forming, etc.",
"), thereby to be fitted with the metal pipe 28 within an inside thereof.",
"In contact portion between the metal pipe 26 and the groove 28 of the base 25 is filled up with a grease or/and an adhesive of high thermo-conductivity.",
"In particular, in the embodiments shown in FIG.",
"5(b) and FIG.",
"6, the contact area thereof is widen or expanded for the purpose of increasing the heat-conduction efficiency between the base 25 and the metal pipe 26, respectively, much higher than that in the structure shown in FIG.",
"5(a), and in particular, in FIG.",
"5(b), the groove 28 is deepen to be filled up with a grease or/and an adhesive of high thermo-conductivity therebetween.",
"In FIG.",
"6, the metal pipe 26 is positioned between the bases 25, in each of which is formed a groove of a half (½) depth of the diameter of metal pipe 26, thereby being attached therebetween.",
"According to those embodiments, an advantage can be obtained that the water-cooling jacket can be manufactured with a low cost, comparing to that of metallically bonding between the metal pipe 26 and the base 25 (see FIG.",
"3, for example).",
"However, because of use of the grease or/and the adhesive in the contact portion between the metal pipe 26 and the base 25, it is inferior in the performance of the thermo-conductivity.",
"On the contrary thereto, an embodiment shown in FIG.",
"7 is provided for dissolving this disadvantage.",
"In the embodiment shown in a partial cross-section view of FIG.",
"7, as a sixth embodiment according to the present invention, the metal pipe 26, being wound around in a predetermined shape in advance, is cast into when forming the metal base 25 made of, such as aluminum, through the die-cast.",
"With this method, since the metal pipe 26 and the base 25 are in contact with, completely, therefore high thermo-conductivity can be obtained without using such the grease or/and the adhesive of high thermo-conductivity.",
"FIG.",
"8 is a front view of the water-cooling jacket, according to further other (i.e., a seventh) embodiment of the present invention.",
"The embodiment shown in FIG.",
"8 is constructed in combination of with the metal pipe and the metal base, in the similar manner of the embodiments shown in FIGS.",
"3 to 7 mentioned above.",
"The structure shown in FIG.",
"8 is that for the purpose of increasing the thermo-conductivity, from the heat-generating element 7 to the coolant, by making the length of flow passage (i.e., the surface area between the liquid flowing in a inside) as long as possible.",
"The metal pipe 26 is formed in loop-like, and thereby being connected with the metal base 25.As a method for connection, the similar one can be applied to, shown in FIGS.",
"3 to 7 mentioned above.",
"With the present embodiment, since it is enough to make a loop radius at the center thereof being equal or larger that the minimum curvature radius to the bending, therefore the pipe can be disposed within a surface of the base 25 with high efficiency, and thereby elongating the length of flow passage.",
"Also, disposing the central position of the loop roughly coincident with that of the heat-generating element 7, as well as, bringing the flow of liquid into a direction from 32 to 31 of the pipe, the liquid is supplied to the central portion of the heat-generating element 7 where it comes to be the highest in temperature, and therefore enabling the cooling with high efficiency.",
"On the other hand, the pipe 32 at an inlet side comes across the loop portion of the pipe.",
"Therefore, the height in the flow passage portion of the water-cooling jacket must be two (2) times large as the diameter of the pipe.",
"On the contrary to this, an embodiment shown in FIG.",
"9 is formed with a pipe in such a loop shape, that the height is same at the inlet side and the outlet side of the flow passage.",
"Thus, FIG.",
"9 shows a front view of the water-cooling jacket, according to other (i.e., an eight) embodiment of the present invention.",
"According to that shown in FIG.",
"9, the pipe 26 is disposed in an ellipse-like within the base 25, differing from that shown in FIG.",
"8, and this enables the length of flow passage to be long, as well as, to direct the flow directions in opposite within the pipes neighboring to each other.",
"Accordingly, it is possible to obtain a high cooling efficiency.",
"Furthermore, the water-cooling jacket can be made thin in the thickness, since the pipe can be so disposed that, the height of the flow passage is same at the inlet side and the outlet side thereof.",
"However, the pipe 26 may be formed, as is shown in FIG.",
"10, i.e., nearly equal to a square or quadrangular in the cross-section shape thereof, being flat on both upper and lower surfaces of the pipe through the press, thereby being connected on the base 25 of a flat plate, directly.",
"In FIG.",
"10, further a plate 25a is provided in an upper portion, thereby fixing the pipe 26, by putting it between the base 25 and the plate 25a.",
"FIG.",
"11 is a front view for showing other (i.e., a tenth) embodiment according to the present invention, being similar to that shown in the above-mentioned FIG.",
"9.In FIG.",
"11, the pipe 26 is elongated, which makes up the water-cooling jacket (being similar to the embodiment shown in FIG.",
"9 mentioned above) for cooling the heat-generating element 7, so as to be connected with a second base plate 33.This second base plate 33 is in contact with a second heat-generating element 34, thereby cooling down the second heat-generating element 34 with the similar structure (it may be any structure of the embodiments mentioned above) and the mechanism (or the manner) of cooling the heat-generating element 7.Further, the base plate 25 and the second base plate 33 may be formed in one body, to be connected with the plural number of heat-generating elements 7 and 34.According to the present structure, the plural number of heat-generating elements can be cooled by the flow passage formed in one body.",
"FIG.",
"12 is a front view for showing the water-cooling jacket, according to further other (i.e., an eleventh) embodiment of the present invention.",
"In FIG.",
"12, with the water-cooling jacket for cooling the heat-generating element 7 is further added a compulsive air-cooling structure, by means of a fan 37, in the combination therewith.",
"On the base plate 25 in contact with the heat-generating element 7, a fin 36 is attached, and further provided the fan 37.This fan 37 sucks the air from the above plane (in the direction perpendicular to the sheet surface of the drawing), for example, and discharges the air from a side surface of the fan 37 towards the fin 36.Also, the fin 36 may be formed on the plate 25a, which holds the pipe 26 therebetween as shown in FIG.",
"10 mentioned above, thereby cooling the pipe 26 directly and compulsively.",
"According to the present embodiment, although the case is shown where the plural number of heat-generating elements 7 and 34 are cooled down, by using the base plates 25 and 33, separately, in the similar manner to the embodiment shown in FIG.",
"11 mentioned above, however it may be the structure without providing such the second base plate 26, or may be the structure, in which the cooling by means of the fan 37 is combined with the structure for cooling the plural number of the heat-generating elements 7 and 34 on the base plate 25 and the second base plate 33, which are formed in one body.",
"According to the present embodiment, since further the compulsive cooling by means of the fan is added to the cooling of thermally transferring the heat of the heat-generating element through circulation of the coolant liquid to the heat-radiation plate, to radiate heat therefrom, therefore high performance of cooling can be obtained.",
"However, all the embodiments shown in FIGS.",
"1 to 12 mentioned above show the case of being applied only to the notebook-type personal computer, however they may be applied to a computer of other type than this and/or other electronic apparatuses or appliances, as well.",
"As was mentioned in the above, the heat generated from the heat-generating element is transferred to the coolant liquid flowing within the water-cooling jacket, and is radiated from the heat-radiation plate provided on a rear surface of the display through the surface of the display case into the air outside, during the time when it passes through the heat-radiation pipe.",
"With this, the coolant liquid lowered in temperature thereof is sent out to the water-cooling jacket, again, through the liquid driving device.",
"The heat-conducting route from the heat-generating element to the liquid includes: the heat conduction from the heat-generating element to the water-cooling jacket; the heat diffusion into the area where the flow passage is formed within the base; and the heat conduction from the area where the flow passage is formed to the liquid flowing within the flow passage.",
"The die-cast forming of the flow passage within the water-cooling jacket enables the flow passage to be formed within the base, being formed in an area nearly equal to the heat-generating element and having a sufficient surface area, thereby reducing the thermal resistance accompanying with an increase of area in the area where the flow passage is formed within the jacket base.",
"Also, connection of the jacket base and the metal pipe for forming the flow passage can be obtained in the piping, thereby forming the flow passage in the water-cooling jacket by means of the metal pipe, can achieves the structure without no seal portion therein, i.e., no permeation of the liquid.",
"In the case of the present structure, with using the pipe of a metal material of anti-corrosion in the flow passage of the piping, it is also possible to suppress the corrosion in the water-cooling jacket.",
"INDUSTRIAL APPLICABILITY As was fully mentioned in the above, according to the present invention, it is possible to provide an electronic apparatus equipped with a water-cooling jacket, being superior in thermal conduction efficiency from the heat-generating element to the coolant liquid and being high in reliability to the corrosion, the liquid permeation, and the liquid leakage, as well."
]
] | Patent_10239156 |
[
[
"Composite building components",
"A structural insulated panel having a core (40) of expanded polystyrene sandwiched between, and bonded to, two facings (52).",
"The facings are attached to faces of the core formed by moulding.",
"Preferably the core is an expanded polymer moulding and the preferred polymer is polystyrene.",
"The panel is useful as a building component."
],
[
"1.A structural insulated panel having a core of expanded polystyrene sandwiched between, and bonded to, two facings, the facings being attached to faces of the core formed by moulding.",
"2.A panel as claimed in claim 1 wherein the core is an expanded polystyrene moulding.",
"3.A panel as claimed in claim 2 wherein the core is formed by expansion of polystyrene cells in a mould in such that any variations in density are minimal.",
"4.A panel as claimed in any preceding claim wherein the expanded polystyrene is produced by pre-expanding polystyrene, maturing the pre-expanded polystyrene and then expanding the pre-expanded polystyrene and then expanding the pre-expanded matured polystyrene in steam.",
"5.A panel as claimed in any preceding claim wherein the panel dimensions are 1.2 metres wide, 0.2 metres thick and 2.4 metres high/long.",
"6.A panel as claimed in any preceding claim wherein the facings are made from cementitious board, plywood, gypsum/textile composite board or OGB.",
"7.A panel as claimed in any preceding claim wherein the core comprises two mirror image halves.",
"8.A panel as claimed in claim 7 wherein each mirror image half of the core is provided with male/female location means for engagement of the two halves.",
"9.A panel as claimed in any preceding claim wherein the core includes at least one passageway.",
"10.A panel as claimed in claim 9 wherein there is a matrix of passageways positioned such that each passageway will align with, and be capable of connection to, a passageway of an adjacent such panel.",
"11.A panel as claimed in any preceding claim wherein an organic non-solvent, moisture controlled penetrative adhesive or glue is employed for bonding the parts of the panel together.",
"12.A panel as claimed in any preceding claim including recesses along the edges of oppositely facing surfaces of the core for receiving joining elements for connecting the panel to another such panel.",
"13.Use of an individual moulding of expanded polystyrene as a core in a structural insulated panel in which the core is sandwiched between, and bonded to, two facings.",
"14.A method of manufacturing a structural insulated panel comprising forming an expanded polystyrene core with at least two opposite faces produced by moulding and bonding facings to the two moulded faces.",
"15.A method as claimed in any preceding claim wherein the step of forming an expanded polystyrene core comprises pre-expanding polystyrene beads by heating the beads and providing steam thereto, cooling and drying the pre-expanded beads, maturing the pre-expanded beads and then further expanding the pre-expanded and matured beads with steam in a mould.",
"16.A method as claimed in claim 15 wherein the mould used for further expansion of the pre-expanded and matured beads comprises a two part mould defining a mould cavity, each part being connected to a steam source, wherein the surfaces of the mould cavity are provided with a multiplicity of steam injection points.",
"17.A method as claimed in either claim 15 or claim 16 wherein the mould is an hermaphrodite mould.",
"18.A method as claimed in claim 17 wherein the mould is shaped to provide each half of the core with male/female location means.",
"19.A method as claimed in any one of claims 15 to 18 wherein the mould is shaped to form recesses along the edges of oppositely facing surfaces of the core.",
"20.A method as claimed in any one of claims 15 to 19 wherein the mould is shaped to form at least one passageway in the core.",
"21.A method as claimed in any one of claims 14 to 20 wherein the bonding of parts of the panel is carried out with an organic non-solvent, moisture controlled penetrative adhesive or glue.",
"22.A method of constructing a building comprising using a panel as claimed in any one of claims 1 to 12."
],
[
"This invention relates to composite building components, primarily but not exclusively for use in the construction of buildings such as houses and more particularly to composite building components which are generically known as structural insulated panels or SIPs.",
"Typically, SIPs incorporate a relatively flat plastics foam core of rectangular shape sandwiched between, and bonded to, two relatively thin, high strength, rectangularly shaped facings to form a laminated sandwich SIPs have been in use for many years and have become well established in the construction industry, particularly in the USA, as an alternative to traditional brick/block cavity walls and the framed panel inner skin and outer skin brick/block cavity walls of timber frame buildings.",
"The foam cores of SIPs provide thermal and acoustic insulation which are superior to those of conventional brick or timber built houses, are resistant to moisture, shock, impact and fire and avoid the need for a water vapour barrier (house wrap).",
"Moreover, SIPs are lightweight and easy to manipulate and a single SIP takes the place of many masonry blocks or building bricks, thereby decreasing construction time and reducing material costs.",
"The foam cores also permit passageways or conduits for supply lines such as electrical wires to be cut in the fully formed foam cores in the factory, prior to assembly of the SIPs on site which further decreases construction time.",
"Instead of being applied separately in a second stage, it is advantageous to manufacture walls, floors and roofs incorporating insulation material as part of the building (or individual module structure).",
"Such modules or panels known as SIPs have been used in the United States for over 50 years.",
"These SIPs vary in thickness from say 50 mm up to 300 mm and to comply with common international building component dimensions, a typical wall or floor SIP would be 2.4 metres×1.2 metres and the thickness would depend on the particular application, load bearing qualities and thermal insulation requirements.",
"In the USA, the most popular SIPs comprise an expanded polystyrene (EPS) core faced on its inner and outer surfaces respectively with two facing sheets of say 9 mm to 15 mm thick of OSB (oriented Strand Board) or ply wood or in some cases cementitious board.",
"These SIP building components have been successfully used, extensively over the last 50 years in the US in the construction of houses (usually single storey).",
"Typically, the SIPs were supported from a base length of timber fixed to a suitable foundation and joined by timber splines or so called biscuits to each other to form the walls of the building.",
"When larger roof and wall loadings were required, the SIP modules were reinforced by incorporating a 2×4 inch timber reinforcing post within the SIP or in some cases these timber elements were used to connect the individual SIPs to each other.",
"This method results in a SIP wall reinforced with timber frame elements.",
"Timber frame elements suffer from dry rot due to poor circulation of fresh air around the timber elements.",
"The use of timber elements can also give rise to “cold spots” which reduce thermal efficiency.",
"Therefore with any SIP for use with timber frame elements there is a need for adequate air circulation around the timber elements.",
"It has been ascertained that an SIP foam core has several important functions.",
"The core has to be stiff enough to keep the distance between the facings constant and must also be so rigid in sheer that the facings do not slide over each other and to prevent buckling.",
"If the core is weak in sheer, the facings do not co-operate and the SIP sandwich will lose its stiffness.",
"It has also been ascertained that the foam core has to fulfil further complex demands, namely strength in different directions and low density (economics) and also has other special demands with regard to buckling, insulation, moisture absorption, ageing, resistance etc.",
"For example, the facings are required to transmit the compressive loads down to the foundation and the adhesive used to bond the facings to the core must be sufficiently strong to resist sheer and transmit load between the core and the facings.",
"The most practical and economical solution initially found to manufacture an SIP giving the requisite load bearing strength and insulation qualities, was an SIP panel utilising a core of high density (HD) extruded polystyrene (XPS).",
"Testing XPS proved that this material had the necessary qualities and the synergy required to construct an SIP panel capable of sustaining compressive loading as would be found in a typical three storey housing structure.",
"Testing was carried out at the Building Research Establishment (BRE) and it was proven that these XPS cored SIPs faced with ply wood facings were capable of sustaining phenomenal loads.",
"However, further investigation showed that the initial advantages of XPS were overweighed by XPS being considerably more expensive, both in terms of manufacturing and capital expense which rendered XPS uneconomical to use in an SIP composite building component.",
"Further consideration was therefore made of the SIP products as manufactured and used in the USA, particularly because SIPs utilising cut EPS cores have obtained approvals (ASTM) in the USA and general acceptance for use as load bearing building panels in construction.",
"The loading figures achievable for EPS core material in use in the USA for SIPs are common knowledge.",
"The minimum SIP core thickness typically used for SIPS in the USA is 150 mm whereas in Europe cores of 50 mm thickness could be described as commonplace.",
"Cut EPS is typically in the region of three times less expensive than XPS and moulded polyurethane foam which is also used as a core in a SIP system already on the UK market.",
"Urethane cores are dangerous in that they give off poisonous fumes when burned and so were not considered.",
"Also, plans are afoot globally to ban the use of urethane in composite building components because urethane used in buildings, particularly houses, is no longer considered to be an environmentally responsible material.",
"The beauty of using cut EPS for the core material of SIPs is that EPS is not only cheap to manufacture but is universally regarded as an environmentally responsible construction material.",
"This is because EPS does not contain harmful fibres, represents an efficient use of natural resources which saves energy and conserves resources through its manufacture, use and disposal.",
"EPS does not contain or release compounds harmful to the ozone layer such as CFCs or HCFC's and its manufacture and use represents no danger to health.",
"EPS insulation, in particular, has an invaluable role to play in helping to achieve dramatic reductions in energy use and reducing emissions that contribute to the greenhouse effect EPS can be, and is being, recycled and the EPS industry is also leading the way in terms of developing a range of waste management solutions to ensure maximum recovery of waste.",
"Further, EPS manufacture by the well known three stage process comprising pre-expansion, maturing and final block moulding is already proven and is capable of economically producing gigantic blocks of EPS of up to 20 metres long, 6 metres wide and 4 metres thick which is then cut into smaller sizes by the standard hot wire technique depending upon their intended purpose, such as cores for SIPs.",
"The raw material from which EPS is made is in the form of free flowing, lightweight and cellular beads made from styrene monomers derived from ethylene and benzene, themselves derived from crude oil.",
"The beads contain an expansion agent, usually pentane, and have the appearance of granulated sugar.",
"The raw material, which is available in various grades and can be described generally as regular and fire retardent types, is delivered in this form to the manufacturing plant in either 600 or 1000 kg ‘octabins’ or in a bulk carrier for transfer to storage silos, the latter being more economical.",
"In the first, pre-expansion, stage, the polystyrene beads are pre-expanded to 20-40 times their original volume by heating to a temperature of about 1000° C., using steam as the heat carrier, in an enclosed vessel known as a pre-expander.",
"In pre-expansion, the volume of the polystyrene beads is increased and their bulk density changes accordingly—e.g.",
"from 620 kg/cu.",
"metre to 20 kg/cu.",
"metre if the moulded density of the foamed material is to be 20 kg/cu.",
"metre.",
"Following pre-expansion, the beads are cooled and dried before being stored to mature.",
"After pre-expansion the beads have a partial vacuum and this is equalised by allowing air to diffuse through the beads.",
"The beads are matured over around 24 hours.",
"The density of the foamed block moulding produced from the beads is therefore practically the same because in final forming the block mould is completely filled with beads.",
"This second stage of maturing is required because, after cooling, the pre-expanded beads are initially still sensitive to pressure, and time must be allowed for them to acquire adequate strength.",
"This happens by diffusion of air into the foam cells until the reduced pressure resulting from cooling and expanding agent condensation has been compensated.",
"Accordingly, the pre-expanded beads are generally dropped straight out of the expander into a fluidised bed drier in which warm air from 25° to 35° C. is blown in through the base of the drier.",
"Fluidised bed driers operate continuously but must be designed with sufficient length to ensure adequate drying.",
"The residence time of the expanded beads in the fluidised bed should be 1 to 5 minutes depending on their moisture content.",
"After drying, the freshly pre-expanded beads are transferred to a maturing silo.",
"Whilst maturing some expanding agent (pentane) escapes and this cuts down the foam pressure decay time required in moulding.",
"In the third and final block moulding/secondary expansion stage, the pre-expanded and matured beads are further expanded with steam in the mould until they fuse together to form a moulded block.",
"Although polystyrene can also be expanded with other heat sources, e.g.",
"with boiling water, hot air and other gases, steam has decisive advantages because:—it is a highly efficient heat transfer medium; its temperature at atmospheric pressure is close to the softening point of polystyrene; it is readily available; and it helps in the actual expansion process.",
"Polystyrene is highly permeable to steam (water vapour) and as soon as the expanding agent starts to expand the beads, steam permeates into the newly formed cells.",
"The steam pressure inside the cells thus balances the pressure of the steam surrounding the beads which can expand against virtually no resisting force.",
"This permits expansion of the beads to low densities.",
"The mould for the production of block polystyrene foam for use in producing SIP cores normally consists of two parts defining a mould cavity that produces the shape of the finished moulding with each mould part being bolted onto a steam chamber.",
"Steam is introduced into the mould cavity through a multiplicity of special core vents or jets, usually made from aluminium alloy.",
"The spacing and number of core vents and the total vent area is important to guarantee proper filling (with no back pressure), steaming, cooling, and consequently the quality of the mouldings.",
"Ease of cleaning and maintenance of the core vents is an important feature for efficient operation.",
"The mould parts typically are closed using hydraulic pressure and the pre-expanded beads are blown into the closed mould using air injectors with the air escaping via the steam nozzles or special vents.",
"For large block moulds, for producing gigantic EPS blocks, which are of simple design, steam is supplied via the steam chambers through the multiplicity of steam jets or vents in the mould walls.",
"The block mould is completely filled with the matured pre-expanded beads which are, in effect closed polystyrene cells, and then steamed.",
"As a result of the renewed heating to temperatures between 110° and 120° C., further expansion of the beads takes place but is confined to filling up the free volume of the mould cavity which compresses beads together because being contained by the mould they cannot expand freely and therefore creates internal pressure in the mould cavity.",
"The beads fuse together along their boundary faces to form a moulded block.",
"After a cooling (pressure reduction) period, usually using a vacuum to remove any moisture, the moulded block is dimensionally stable and can be released from the mould.",
"Any remaining expanding agent (pentane gas) is expended during moulding so that the moulded block does not contain any residual expanding agent.",
"Investigations were carried out into American production methods for SIPs using EPS for the core material and the quality control procedure, and the material consistency was found to be seriously lacking and would not comply with typical current British & European quality control assurance schemes (BS5750, ESO 9000 and 9002).",
"Detailed testing of the lamination of SIPS using EPS faced with OSB, ply wood and cementitious board were carried out.",
"It was found, however, that spasmodically the SIP panel would be prone to collapse in the process of manufacture, usually when the panels were placed in vacuum press for curing of the adhesive.",
"Detailed examination of the EPS core materials showed that whilst this material was manufactured to BS 3837/BS4370 and BS4735 and the overall density of say a 2.4×12×20 cm panel showed the material was correct, if the panel was cut into segments however there was seen to be significant variations in density across the panel.",
"Panel samples were purchased from numerous UK EPS block manufacturers and sample weight tests showed significant variation in density from panel to panel and also segment tests showed significant density variations across individual panels.",
"It was realised that with such density variations and poor quality control methods, EPS manufactured and as supplied in the UK market would be totally unsuitable for the manufacture of SIPs for use in housing.",
"There was therefore a need to devise some new form of manufacturing process for the cores of SIPs that enabled the density of the finished product to be controlled so that it could be held within exacting standards.",
"Another disadvantage of cores cut from EPS blocks, is that judder which occurs during hot wire cutting of the EPS block causes the formation of ridges and indentations in the surfaces of the cut EPS cores.",
"In order to provide the precise surface tolerances required for the core surfaces that are bonded to the facings, the cut cores are passed through a planar thicknesser.",
"This process produces waste EPS, another disadvantage.",
"It is known that when cycle crash helmets are moulded as individual items it is possible to control the density and quality within defined limits and apply stringent quality controls, thus ensuring that this vital piece of head protection will meet the necessary British Standards tests.",
"The present invention involves using moulding to manufacture expanded polymer cores for SIPs as individual quality controlled items.",
"It has been found feasible to apply quality control procedures to produce a moulded expanded polymer product capable of complying with the exacting criteria of the insulating core material of a SIP.",
"Specifically, it has been found possible by moulding polymers in a quality controlled environment to ensure that density variations do not exceed permitted amounts.",
"In one aspect, the present invention resides in a structural insulated panel having a core of an expanded polystyrene moulding sandwiched between, and bonded to, two facings, facings being attached to faces of the core formed by moulding.",
"The core is preferably formed by expansion of polystyrene cells in a mould such that any variations in density are minimal and/or the core is of sufficiently uniform density to permit load bearing of the panel without the need for additional structural supporting elements.",
"Moulded cores of expanded polystyrene have been made that exhibit a density variation of as low as up to/down to ±2.0% as compared with the large density variations in cores cut from gigantic blocks.",
"Moulded cores in accordance with the invention are calculated to be 40% stronger than has hitherto been possible and have improved u-values.",
"In a still further aspect the invention resides in an individual moulding of expanded polymer for use as a core in a structural insulated panel in which the core is sandwiched between, and bonded to, two facings.",
"The invention also resides in methods of manufacturing any of the structural insulated panels defined above.",
"Hereinafter the expanded polymer will be referred to as XPS.",
"Significant advantages result from the invention.",
"Firstly, the use of additional structural members of timber etc., in particular beyond the bottom story is avoided and thermal bridging within a building made form such laminated composite building components is minimised, thereby raising thermal efficiency.",
"The structural insulated composite building components rely on the compression strength (core strength) of the component without the use of timber.",
"A building can be produced, in particular a house, in which not only the traditional cavity wall and brick construction are replaced but also joist and floorboard floors and timber trussed roofing systems are replaced.",
"This is all for a fraction of the cost of these traditional systems.",
"There are therefore significant technical advantages over other competing products, notably urethane cored composite building component structures, timber frame, and some concrete or steel framed structures.",
"Building costs are reduced and construction is facilitated by means of a preferred embodiment of the invention in which the basic moulded core structural insulated panel is 1.2 metres (1200 mm) wide, 0.2 metres (200 mm) thick and 2.4 metres (2400 mm) high/long and 2.88 square metres in area.",
"It has been calculated that it takes 334 standard bricks to produce a normal cavity wall construction (one brick thick and two half brick skins) of the same area.",
"This is clearly a major leap forward in terms of on-site productivity.",
"To aid in flexibility of building, there is also envisaged blocks that are 0.6 metres and 0.3 metres wide, 2.75 and 3 metres high (3 meters is storey high) and 50 mm, 75 mm, 150 mm, 250 mm and 300 mm thick.",
"The reinforcing facings need to be tough and to this end, facings of cementitious board, plywood, gypsum/textile composite board or OSB (oriental strand board) are preferred.",
"In order to ensure that the steam carries to all parts of the mould and ensure minimum variations in density, all surfaces of the mould are provided with a multiplicity, e.g.",
"thousands, of small steam injection points.",
"By providing all surfaces of the mould with a multiplicity of small steam injection points, the moulded core structural insulated panel of the invention is strong, free of noxious gases, and thus is suitable for its main purpose as an environmentally responsible low cost structural building component.",
"Preferably, each moulded core is individually moulded in a full sized mould which provides a stronger core than that cut from a block.",
"This is because the core has an integral surrounding skin of well-fused, denser cells.",
"In a preferred embodiment, which facilitates moulding and the obtaining of full thickness dimensions (at least 200 mm), as well as having other advantages, the core is made in two mirror image halves that are moulded in what is called an hermaphrodite mould so that two mould halves taken from the same mould can be bonded together to complete a two piece core.",
"Each mirror image half is provided with male/female location means, preferably in the form of complementary projections and recesses with each half being provided with both complimentary projections and recesses so that it is a simple matter to turn one half through 180° and engage the projections and recesses of one half with the complimentary recesses and projections of the other half.",
"A given strength can thus be obtained with individually moulded cores at a lower density than with cut blocks.",
"This saving is estimated at approximately 10% for densities of 25 kg/cu.mtr.",
"and higher.",
"Accordingly individually moulded cores exhibit a lower density gradient than large cut blocks, especially at higher densities that always show considerable gradation in density across the thickness.",
"The certre of a gigantic moulded block is of significantly lower density than the overall density.",
"It is, therefore, necessary to mould blocks at a higher density than is actually required in order to make sure that the centres of the blocks reach the required density.",
"This problem does not occur with moulded cores and this factor gives a further saving of 8% to 10%.",
"Since no density gradient is present, the moulded core weight and hence the product quality, are more consistent.",
"In order substantially to facilitate the supply of services in a building utilising moulded core structural insulated panels, preferably, the mould is provided with inserts which form hidden passageways or conduits in the ultimate moulded core which are suitable for accommodating any form of supply line but in particular electrical wires and cables.",
"In addition to electricity, conduits may be provided for gas, communications, water, ventilation and other usages.",
"A matrix of passageways can be formed in this way to satisfy all necessary service requirements which are aligned as between adjacent panels both side by side and one above the other.",
"Moreover, the positions of the matrix of passageways in relation to the dimensions of the core, can be so arranged that when one panel turned onto one of its sides of lesser width to form the wall beneath a window for example, the passageways in the adjacent panels will still be in alignment.",
"Whilst a cut core would loose some of its strength by the removal of material for supply passageways, e.g.",
"0.1% this does not happen with two part moulded cores because the passageways will be lined with a skin of fused cells that is integral with the surrounding skin of fused cells.",
"It has been found that an organic non-solvent, moisture controlled penetrative adhesive or glue e.g.",
"MCPU, is the most effective, not only for bonding the facings together, but also the two part core pieces when the core is moulded in two parts.",
"Such an adhesive is stronger than the building component itself because it penetrates between the closed cells.",
"With two piece cores, the penetration of the adhesive in this way forms a layer of adhesive which extends between the cells of each moulded piece, thereby preventing the formation of a plane of separation between the two pieces and forming a bond that lasts as long as the foam cores.",
"It has been ascertained that the moulded foam core and reinforcing facings glued together is comparable with an I-beam but is stronger than steel.",
"The foam core is the equivalent of the I-beam web and the facings are the equivalents of the I-beam flanges.",
"Whilst the strength of the panel is more than sufficient for normal building structures, because of its composite nature it is possible to increase the strength still further by adding a layer of, for example, a textile or fibre cloth to the interior surface of one or both facings.",
"Adding such a layer or layers may have effects other than or in addition to increasing strength depending on the properties of the material.",
"As one example, fire retardant properties may be increased.",
"In another example, a textile layer may have ceramics embedded in it for security reasons or a thin electricity conducting wire entwined therein which could allow for heat flow and so obviate the need to put in under floor heating.",
"In a still further example a metal weave web or hurrican fencing could be used not only to add great strength but also to act as a security barrier giving an indication if it is cut.",
"In the embodiment where the core is formed in two parts, an additional layer may be provided between the core halves as well as, or in addition to, between the core and one or both facings.",
"Moulded expanded polystyrene cores in accordance with the invention are so remarkably strong in compression that the structural insulated panels require no further input in terms of structural elements.",
"There are no timber beams, steelwork etc.",
"Initial tests indicate that structural insulated panels in accordance the invention might well be approved to build up to six floors and even ten floors high without further structural elements, hence opening up a potential further market in commercial construction.",
"A number of other components which will be used in the building of a house.",
"These include a ring beam, of the same basic material, which adds horizontal stability and acts as a lintel over doors and windows, a box beam for extending panel spans by adding rigidity to lengths, a corner section and a seismic joint, again made from the same basic materials.",
"Intermediate floors, roofs etc.",
"may all made from these basic components in a factory environment, and the large pieces are simply assembled on site.",
"Once assembled, the whole can then be clad in local materials (brick tiles, stone, timber, rendering etc).",
"The surface of moulded cores has a better appearance than that of hot swire cut cores of which the appearance has been marred due to hot wire cutting judder and this could be used to impart a quality image by moulding-in trade names or marks.",
"A further contribution to the good surface appearance is the fact that normally a low pentane grade material can be used which consists of smaller beads than the block moulding equivalent.",
"Exact dimensions are obtained since they are determined by the mould dimensions.",
"The accuracy obtained is, therefore, much higher than in the case of block-cutting.",
"It can be said that a design disadvantage with moulded cores is that the range of sizes offered must be limited, since mould costs are high and the mould changing time is long compared to the resetting of a hot-wire cutter.",
"However, thickness adjustment can be easily achieved by the incorporation of spacers between the mould surfaces.",
"For effective insulation and structural connection, the structural insulated panels have to be provided with a system to eliminate the formation of gaps in the insulation caused by shrinkage or thermal contraction.",
"In the case of cores cut from the block, this requires an extra, thus costly, operation by grinding, planing or milling.",
"Moulded core panels, however, can be provided with special features, thus eliminating secondary operations to which reference will now be made.",
"For example, recesses may be moulded in along the edges of the opposite facing surfaces of the cores by means of inserts in the mould so that the aligned recesses of adjacent cores assembled to form a wall of a building for example may receive respective elongate elements in the form of strips, known as “biscuits”, for use in joining adjacent cores together without thermal bridging.",
"To keep the facings and the core cooperating with each other, the joints between the facings and the core must be able to transfer the shear forces between the faces and the core.",
"The joints must be able to carry shear and tensile stresses.",
"It's hard to specify the demands on the joints.",
"A simple rule is that the joints should be able to take up the same shear stress as the core.",
"The biscuit/recess joints guard against such problems occurring.",
"Whilst cut recesses would cause the core to lose loose some of its strength by the removal of material, the moulding process in two individually moulded part cores causes the recesses to be lined with a skin of fused cells that is integral with the surrounding skin of fused cells, like the service line passageways, to prevent any loss of strength.",
"Moulded two part EPS cores 200 mm wide can be produced to the requisite dimensions in a core moulding machine at a density of 24 kg per cm and a flexural strength of 400 kn/m2.To achieve the desired flexural strength using cores cut from the block, it would be necessary to use block material expanded at a minimum density of 35 kg.",
"per cm.",
"As previously stated, the density across the block would vary considerably and therefore it would be impossible to implement accurate quality control procedures.",
"Accuracy of hot wire cutting would not give the dimensional tolerance required and the percentage waste ratio would climb dramatically.",
"The invention also comprehends methods of constructing buildings using any of the structural insulated panels defined hereinabove and to buildings constructed of such panels and/or in accordance with the method.",
"The advantages of moulded core structural insulated panels made according to the present invention are manifold particularly for the preferred EPS embodiment and are as follows:— Cost effective—as compared with any other conventional building system.",
"Mechanical Strength—Trials on this style of construction material show it to be far superior in all performance criteria to brick, timber or concrete structures of comparable size.",
"A finished building, e.g.",
"a house, will also be earthquake and hurricane proof.",
"Workable—Using standard tools can be adapted to suit specific customer requirements.",
"Multi-skilled Constructors—once certification is achieved the buildings/houses can be constructed by relatively low skilled (or multi-skilled) workforce readily available.",
"Hidden Utilities—provision is easily made during moulding for power, communications cables, water pipes etc.",
"to be completely hidden by engineering them directly into the moulded cores at the outset, thereby solving the conduit problems.",
"This eliminates all types of costs relating to adding utilities after construction of the walls and is a considerable improvement on the American SIPs referred to previously in which conduits for service supply lines are cut into the already formed core which takes time and results in waste polystyrene and can cause core weakening.",
"Weather Strength—new, old and damaged components will meet the highest standards of resistance against wind, rain, snow, sun and frost.",
"Fire Resistance—of the two major constituents of the EPS moulded core structural insulated panels, one is nonflammable and has a two-hour fire rating and the other is self-extinguishing.",
"Neither gives off toxic fumes during a fire.",
"Thus a home can be built with out there being any combustible materials whatsoever.",
"Moisture Resistance—the EPS moulded core structural insulated panels are not susceptible to damage by water from blocked gutters, breached damp proof course, leaking pipes, rain exposure, etc.",
"Noise Attenuation—the use of high-density core material and the thickness of the walls formed from the EPS moulded core structural insulated panels components will give outstanding noise attenuation performance.",
"Vibration through the panels is virtually impossible.",
"Long Life—the life of a brick and mortar house is around 100 years.",
"Beyond that a major expense is required to keep it in good order.",
"The design life of the EPS moulded core composite component homes will be targeted as 200 years.",
"Information from the USA rates their SIP constructions relying on additional structural support from timber elements as having a 300-year life.",
"Thermal Performance—It is considered that EPS moulded core structural insulated panels will be the best thermally performing building material in the world.",
"The u value, a measure of thermal resistance of a material, of the moulded core panel remains constant throughout the life of the component.",
"Readily Available Materials—all the main components of the EPS moulded core structural insulated panels will be available as commodity items or will be manufactured in-house.",
"Resistance to Organisms—none of the EPS moulded core structural insulated panels is prone to attack from insects, rodents, fungus or rot.",
"If a particular problem exists in a certain part of the world, the product can readily accept fungicides, insecticides etc to resolve these issues.",
"Toxicity—the materials from which the EPS moulded core structural insulated panels are made contain no toxins, carcinogens or odours.",
"EPS itself can actually be used in certain food grade applications.",
"Maintainability—there is no requirement for ongoing maintenance.",
"The EPS moulded core structural insulated panels are resilient and will resist minor impact damage, e.g.",
"from a slow moving vehicle.",
"For serious impact damage, the building can be readily repaired using replacement panels.",
"Additions—the form of construction lends itself very well to extensions for additional rooms, bedrooms, garages etc.",
"as the family grows.",
"This fits well with many cultures where family dwellings start small and grow as funds and demands so dictate.",
"Technically Approved—thinner SIPs than the moulded core composite building components are well accepted in the United States.",
"Tests that have already been carried out by BRE show that the EPS moulded core structural insulated panels exceed racking resistance requirements for both stiffness and strength given in BS5268:part 6: Section 6.1 to resist wind and vertically imposed loads in domestic buildings.",
"Environmentally Friendly—the materials from which the EPS moulded core structural insulated panels are made are environmentally friendly.",
"They offer substantial energy savings; over 80% of the components (by volume) can be recycled; and 100% of each component can be used in a power plant as fuel, thereby utilising the energy expended in its production: so it is energy efficient In order that the invention may be more fully understood, some embodiments thereof will now be described, by way of example, with reference to the accompanying drawings in which:— FIGS.",
"1 and 2 are photographs of the raw polystyrene material and pre-expanded polystyrene beads respectively used for manufacturing ESP moulded cores of a structural insulated panel made by a method illustrated in FIGS.",
"3 and 4; FIGS.",
"3 and 4 are schematic drawings illustrating one method of manufacturing a structural insulated panel (SIP) having a custom made/individually EPS moulded two part core and reinforcing facings, in accordance with one embodiment of the invention; FIG.",
"5 is a perspective view of a two part hermaphrodite mould for manufacturing EPS moulded cores of which two such cores form a two part core in the structural insulated panel made in the method of FIGS.",
"3 and 4; FIG.",
"6 is a perspective view of the lower part of the hermaphrodite mould of FIG.",
"5; FIGS.",
"7, 8 and 9 are a side elevation, bottom plan view and top plan view respectively of one EPS moulded core part made in the mould of FIGS.",
"5 and 6; FIG.",
"10 is a cross-section taken along the line A-A of FIG.",
"8 of two EPS moulded core parts made in the mould of FIGS.",
"5 and 6, positioned one above the other in vertical alignment; FIG.",
"11 shows the two EPS moulded core parts of FIG.",
"10 glued together to form a two part EPS moulded core: FIG.",
"12 is detail view to an enlarged scale of one part of the two part EPS moulded core of FIG.",
"11; FIG.",
"13 is a perspective view of a structural insulated panel comprising the two part EPS moulded core of FIGS.",
"11 and 12 sandwiched between, and laminated by gluing to, two facings; FIG.",
"14 is a perspective view of a part of a corner structural insulated panel comprising the two part moulded core of FIGS.",
"11 and 12 sandwiched between, and laminated by gluing to, four facings; FIGS.",
"15 and 16 are enlarged detail views of two adjacent structural insulated panels showing one method of joining the two panels together, for example to form a section of a wall of a building, just before and before and after joining together, FIG.",
"17 is perspective view of with parts cut away of a wall section comprising three adjacent structural insulated panels joined together in the manner shown in FIGS.",
"15 and 16; FIG.",
"18 is an exploded perspective view of a plurality of two part EPS moulded core structural insulated panels showing how the panels are joined together to form a wall of a building; FIG.",
"18a is diagrammatic view of the wall of a building formed of the joined together panels of FIG.",
"18; FIG.",
"19 is an exploded perspective view of a plurality of two part EPS moulded core structural insulated panels having window and door apertures and showing how the panels are joined together to form a wall of a building; FIG.",
"20 is a perspective view from the front of a building, with the front removed, to show the interior and of which the walls, floors and roof are made from two part EPS moulded core structural insulated panels according to the invention; FIGS.",
"21 and 22 are cross sectional and front elevational views respectively of a seismic joint joining together two part EPS moulded core structural insulated panels according to the invention and forming a floor and the walls of a building and which may be used to join the first floor to the walls of the building of FIG.",
"20 to each other; FIGS.",
"23 to 25 are part cross-sectional views of the components of a box beam using two part EPS moulded core structural insulated panels according to the invention; FIG.",
"26 is a part cross-sectional view of a box beam assembled from the components of FIGS.",
"23 to 25; FIG.",
"27 is a part perspective view of a one-piece individually EPS moulded core or use in making a structural insulated panel in accordance with another embodiment of the invention; FIGS.",
"28 and 29 are enlarged detail views of two adjacent structural insulated panels using the core of FIG.",
"27 showing one method of joining the two panels together for example to form a section of a wall of a building, just before and before and after joining together, FIG.",
"30 is a part perspective view of a one-piece individually EPS moulded core made of expanded polystyrene for use in making a structural insulated panel in accordance with a further embodiment of the invention; and FIGS.",
"31 and 32 show graphs.",
"In the drawings the same reference characters have been used to designate the same or similar parts.",
"Referring to FIGS.",
"1 to 6 of the drawings, a low pentane grade polystryrene raw material, which consists of smaller free flowing beads 1 than the block moulding equivalent from which conventional EPS cores are made, is stored in a storage container 3 shown in FIG.",
"4 from whence it is subjected to the three stage process involving pre-expansion, cooling and maturing and moulding/secondary expansion.",
"The raw polystyrene beads 1 are fed to the first, pre-expansion, stage 5 where the beads 1 are pre-expanded to 20-40 times their original volume by heating to a temperature of about 100° C., using steam as the heat carrier in the manner previously described herein.",
"The pre-expanded beads which are indicated by the reference 6 in FIG.",
"2 are cooled and dried in a fluidised bed dryer 7 (FIG.",
"4) before being stored to mature in storage silos 8, as what are, in effect, closed cells, again as previously described herein.",
"The third and final moulding/secondary expansion stage 9 (FIG.",
"3) comprises an hermaphrodite mould 10 having two mould parts 10a and 10b as will be apparent from FIGS.",
"5 and 6.The walls of the mould parts 10a and 10b define a multiplicity of nozzles or vents 12 and air injectors (not shown) for a purpose to be described.",
"The mould part 10a defines a mould cavity that is formed with a peripheral recess (not visible) which accommodates a correspondingly shaped mould insert (not shown) that projects into the mould cavity during moulding.",
"The mould part 10b is formed with a grid 14 (see FIG.",
"6) of interconnecting longitudinally and transversely extending channels 16 and 18 respectively which are in alignment with respective slots 16a and 18a in the walls of the mould part 10a which slots and channels accommodate a correspondingly shaped grid mould insert when the mould is hydraulically or pneumatically closed to commence a moulding operation.",
"Additionally, the mould part 10b is provided with complimentary male/female locating means constituted by three projections 20 towards one end (the right hand end, as illustrated in FIG.",
"6) of the mould part 10b and three identically positioned complimentary recesses 22 toward the other end (the left-hand end as illustrated in FIG.",
"6) of the mould part 10b.",
"The pre-expanded and matured beads 6 are blown from the storage silos 8 into the mould cavity in the mould part 10a of the closed mould 10, using air injectors (not shown) with the air escaping via the nozzles or vents 12.Each mould part 10a, 10b is provided with its own bolted on steam chamber (not shown) which is in communication with the nozzles or vents 12 through which steam is introduced into the pre-expanded and matured bead 6 filled mould cavity in the mould part 10a of the closed mould 10.In the closed mould 10, the beads 6 are heated to temperatures between 110° and 120° C. and are further expanded with steam which is confined to filling up the free volume of the mould cavity which compresses beads together because, being contained by the mould, they cannot expand freely.",
"This, therefore, creates internal pressure in the mould cavity so that the beads fuse together along their boundary faces, assisted by any residual stickiness of the circumference of the individual cells due to the heating to form an individually (custom) EPS moulded shaped core part.",
"After a cooling (pressure reduction) period, usually using a vacuum to remove any moisture, the moulded core part is dimensionally stable and can be released from the mould 10.The moulded core part is indicated by the reference 24 and is illustrated in FIGS.",
"7 to 9.Any remaining expanding agent (pentane gas) is expended during moulding so that the moulded core part 24 does not contain any residual expanding agent.",
"The individually (custom) moulded shaped EPS core part 24 part has a surrounding skin 26, as shown in FIG.",
"12 and a grid of moulded, skin covered channels.",
"Only the channel 18b is visible in FIG.",
"12.The spacing and number of nozzles or vents 12 and the total nozzle/vent area ensures that the steam reaches all parts of the mould cavity and thus provides moulded core parts 24 of which the density is substantially uniform in that it does not vary up or down more than ±2.0%.",
"Referring more particularly to FIGS.",
"7 to 9, the surface 28, which is the upper surface as illustrated in FIGS.",
"7 and 9 of the individually moulded core part 24, has a peripheral recess 30 therein, i.e.",
"a recess that extends all the way around its periphery.",
"This peripheral recess 30 is formed by the mould insert in the recess in the mould part 10a and which projects into the mould cavity during moulding.",
"A grid 14a of longitudinally and transversely extending channels 16b and 18b respectively are formed in the surface 32 by the mould insert grid that occupies the grid 14 of channels 16 and 18 and slots 16a and 16b during moulding.",
"Also, it will be appreciated from FIGS.",
"7 and 8 that the three projections 20 and three identically positioned complimentary recesses 22 of the mould part 10b are responsible for forming the three recesses 20a and complimentary projections 22a in the undersurface 32, as illustrated, of the moulded core part 24.When two (mirror image) moulded core parts or halves 24 have been produced in the mould 10 and successively demoulded, they are conveyed to an adhesive coating stage 34 (FIG.",
"3) where their surfaces 32 are coated with an MCPU adhesive.",
"Then, the two adhesive coated core parts 24 are conveyed to a pressing and setting stage 36 (FIG.",
"3) where one core part 24 is turned through 180° relative to the other core part 24 to occupy the positions shown in FIG.",
"10.In this position, the purpose of the complimentary projections 22a and recesses 20a will readily become apparent.",
"This is because at the left hand end as illustrated, the recesses 20a of the upper core part 24 align with the projections 22a of the lower core part 24 and at the right hand end as illustrated, the projections 22a of the upper core part 24 align with the recesses 20a of the lower core part 24.The transverse channels 18b of the upper and lower core parts 24 as well as the longitudinal channels (not visible) are also aligned.",
"Thus, when the upper and lower core parts 24 are pressed together at the pressing and setting stage 36 to adhere the one to the other as shown in FIG.",
"1.The aligned complimentary projections 22a and recesses 20a inter-engage precisely to locate the two core parts 24 with respect to each other and the aligned channels 16b, 18b form a matrix of passageways 38 for service lines.",
"Once the adhesive has set, a two part custom moulded core 40 is produced which is conveyed to a quality check and assurance stage 42, as shown in FIG.",
"3.The adhesive penetrates into the interstices between the closed cells of the two mould parts 24 to form a layer which is not shown in FIG.",
"12 and extends between the two mould parts 24 so that there is no plane of separation between the two mould parts.",
"Indeed the bond made by the adhesive layer is stronger than the EPS material of the moulded parts 24.The next stage which is indicated by the reference 46 in FIG.",
"3 involves the application of an MCPU adhesive to one surface of each of two panel facings, e.g.",
"of OSB, ply wood or cementious board.",
"The adhesive coated surfaces of the facings are then conveyed to a stage 48 (FIG.",
"3) where they are applied carefully to the oppositely facing surfaces 28 of the moulded core 40.To ensure long lasting adhesion under load bearing conditions, the moulded two part core 40 with its applied facings is conveyed to a pressing and setting/curing stage 49 (FIGS.",
"3 and 4) where a mechanically or pneumatically operated press is used.",
"A completed structural insulated panel (SIP) 50 and which is illustrated in FIG.",
"13 has a core 40 sandwiched between, and adhesively bonded to, two facings 52.FIG.",
"14 shows a corner SIP 50 which, because the core 40 actually forms the corner, is virtually moisture in-penetrable as compared to conventional SIP corners formed by abutting separate SIPs against each other.",
"It will be seen in each case that the recesses 30 are disposed inwardly of the facings which define with the core 40, a slot 30a for a purpose to be described with reference to FIGS.",
"15 to 17.Referring to FIG.",
"15, the slots 30a receive strips which are called biscuits 54 which may be adhered to those parts of the core 40 and facings defining the slots 30a to join adjacent SIPs 50 together, as shown in FIGS.",
"16 and 17.Additionally, the abutting faces of adjacent SIPs 50 may be adhered together, optionally as shown in FIG.",
"16 by forming adhesive receiving channels 56 therein so that in FIG.",
"16 there is shown a longitudinally extending bead of adhesive 56a occupying the channels 56.The longitudinally and transversely extending passageways 38 for supply lines can be seen in FIG.",
"17.FIG.",
"18 shows how SIPs 50 may be assembled to form a wall of a building which is shown completed in FIG.",
"18a, as indicated by the reference 57 by the use of biscuits 54 in the manner shown in FIGS.",
"15 to 17 and by extending the facings 52 upwards beyond the cores 40 to provide top channels 60 for elongate elements 58.It will be seen that the upper SIPs 50 have been shaped to fit with an unshown pitched roof.",
"In FIG.",
"19, apertures 62 for doors and windows are cut in SIPs 50 forming a wall 64 and are provided with respective frames 66 that fit in channels 60 formed by extending the facings 52 beyond the cores 40.The SIPs 50 are supported on a foundation 68 by means of an elongate sole plate element 58 engaging in a channel 60 in each SIP 50.The building 70 illustrated in FIG.",
"20 is a two storey (floor) building with a foundation (ground floor) 72, walls 74, first floor 76, roofs 78 and a roof supporting beam 80 acting as an I-beam in which the core 40 is the equivalent of the I-beam web and the facings 52 are the equivalents of the I-beam flanges, are of SIPs 50.The first floor 76 may be joined to the wall SIPs 50 by means of the joint 90 illustrated in FIGS.",
"21 and 22 to which reference will now be made.",
"The joint 90 comprises a channel element 91 supporting the second storey wall on the first floor 76 with a dowel element 92 extending through the channel element 91 and into the cores 40 of the SIPs 50 of the first floor and ground floor walls.",
"The joint 90 has a capping 93 that fits over the projecting part 94 of the first floor 76.Referring to FIGS.",
"23 to 25, there is shown the elements of an SIP having cores 40, facings 52 and biscuits 54 that are adhered together into a box beam which is shown assembled and indicated by the reference 100 in FIG.",
"26.The box beam 100 is utilised for extending SIP spans by adding rigidity to lengths.",
"An I-beam such as is mentioned in the preceding paragraph can be substituted for the box beam 100 as required by load demands.",
"The embodiment of core 40a shown in FIG.",
"27 differs from the two part core 40 of the previous drawings in that the core 40a is a one-piece custom made individually moulded EPS block type core having a maximum thickness of 100 mm.",
"As will be apparent from FIGS.",
"28 and 29, two adjacent SIP's 50 are joined together in a similar manner as described with reference to FIGS.",
"15 and 16 for the SIPs 50 with the two-part cores 40 except that there are no channels 56 which receive an adhesive bead 56a.",
"The core 40a will be made in a mould that functions in the same way as the mould 10 and the upper mould part will have a recess for receiving a complimentary mould insert to produce the recess 30.Except for the recesses for mould inserts, the simple individually moulded EPS block core 40b of FIG.",
"30 may be made in such a mould.",
"The cores 40a and 40b are sandwiched between and bonded to unshown facings 52 to produce an SIP 50.In FIG.",
"31 there are two graphs which illustrate a comparison between cores that are rigid and weak in shear respectively.",
"In the upper graph, the trace shows that the core tested is rigid in shear, i.e.",
"a two part moulded core 40 of substantially uniform density, and is the acceptable deflection for use in an SIP to be placed in long term compressive loading such as when used in the wall of a building.",
"On the other hand in the lower graph, the core tested is weak in shear.",
"i.e.",
"a core of variable (low) density such as that cut from an EPS block because the trace shows bad deflection which would be an undesirable quality for use in an SIP to be placed in long term compressive loading such as when used in the wall of a building.",
"Some typical values of flexural strength of moulded EPS cores versus those of cores cut from EPS block are set out in the graph shown in FIG.",
"32 and are self evident.",
"Core shrinkage is in the order of 0.5-0.6%, this value being obtained after two or three months.",
"Prototype testing shows representative results according to the following Table which are given purely by way of example to enable the invention to be more readily understood.",
"TABLE Panel Panel stiffness Estimate stiffness in of in stiff- strength Average failure Failure Vertical ness cycle, cycle, panel load, load, Panel load Rstiff Rstr stiffness, Fmax,ext Fmax ref.",
"(kN) (N/mm) (N/mm) R (N/mm) (kN) (kN) Test series 1 MPR1 0 4613 5101 4857 25 33.59 MPR2 0 5010 5063 5023 32 47.54 MPR3 0 4294 5418 4856 42 39.54 MPR4 0 2951 4986 3968 40 34.44 MPR5 0 4225 5677 4951 40 38.80 MPR8 5 6558 7136 6847 60 45.02 MPR7 5 6987 6983 6975 48 44.78 MPR8 5 5058 5821 5439 44 54.02 MPR9 5 5861 7176 6519 46 49.28 MPR10 5 5521 7627 6574 48 48.03 Test series 2 MIP1 0 3543 3894 3718 36 26.46 MIP2 0 642 877 759 10 5.70 MIP3 0 392 553 472 8 6.00 Various modifications may be made to the embodiments described without departing from the inventive concepts defined in the introductory portion of this specification.",
"For example, the EPS moulded cores may be cut to smaller sizes of rectangular shape or different shapes depending upon their location and/or application (see FIG.",
"18 for example) either before or after bonding of the facings 52.In such instances, it may be necessary, depending upon load requirements, to provide the cut surface of an EPS moulded core with a facing such as a biscuit to restore any losses in strength that might conceivably occur."
]
] | Patent_10239276 |
[
[
"Device for adjusting the components of a chair",
"The invention relates to a device for adjusting a first component of a chair, in particular an office chair, with respect to a second component of the chair, the said device being provided with a latching unit by means of which one of the two components can be arranged in different positions with respect to the other component.",
"The latching unit has a guide path which is arranged on the first component.",
"A latching element is provided on the other component, it being possible for the latching element to be arranged in the guide path in latching positions which predetermine the positions of one component with respect to the other component.",
"In order to obtain a functionally reliable latching procedure using a device of this type, it is proposed that the guide path (40) is of essentially groove-shaped design at least in the region of the latching positions, and the latching element (45) is guided in the groove-shaped guide path (40)."
],
[
"1.Device for adjusting a first component of a chair, in particular an office chair, with respect to a second component of the chair, the said device being provided with a latching unit by means of which one of the two components can be arranged in different positions with respect to the other component, the latching unit having a self-contained guide path which is arranged on the first component and on the other component a latching element being provided which has a latching pin (47) and can be arranged in the guide path in latching positions which predetermine the positions of one component with respect to the other component, the guide path (40) being of essentially groove-shaped design at least in the region of the latching positions, and the latching pin of the latching element (45) being guided in the groove-shaped guide path (40), characterized in that the latching element is a lever (45) which can pivot freely around a spindle body (40).",
"2.Device according to claim 1, characterized in that the guidance between the latching element (45) and the guide path (40) has just one degree of freedom and the latching positions of the latching element (45) are arranged in a latching section (41′) of the guide path (40), the latching element (45) beina guided in an essentially form-fitting manner in the latching section (41′).",
"3.Device according to one or both of the preceding claims, characterized in that the groove-shaped guide path (40) is provided with a return section (41″) 2nd a latching section (41′), and the return section (41″) and the latching section (41′) are connected to each other by two deflecting paths (42, 48), as a result of which the guide path is self-contained.",
"4.Device according to one or more of the preceding claims 2 to 3, characterized in that the latching segment (41′) has a plurality of latching positions of the latching segment, which positions are connected to one another in each case by essentially identical segments of the guide path (40).",
"5.Device according to one or more of the preceding claims, characterized in that the guide path (40) is arranged on a guide-path element (35′) which is connected to one of the components, and this component is guided rectilinearly on the other component of the chair.",
"6.Device according to claim 5, characterized in that the segments have a doubly curved profile.",
"7.Device according to one or more of the preceding claims, characterized in that it is provided for adjusting the height of one component with respect to the other component.",
"8.Device according to claim 7, characterized in that the latching element (45) is arranged on a support of the chair and the guide path (40) is arranged on a backrest which is moveable with respect to the support.",
"9.Device according to one or more of claims 4 to 8, characterized that the two sections (41′, 41″) are spaced apart from one each other by means or an intermediate piece (36) which is orientated in the longitudinal direction of the said sections, a plurality of deflecting toes (38 and 38.1 to 38.4) which are spaced apart from each other being integrally formed on the intermediate piece (36) on the side which faces the latching section (41′).",
"10.Device according to claim 9, characterized in that the deflecting toes (38 and 38.1 to 38.4) are designed and integrally formed on the intermediate piece (36) in such a manner that the latching pin (47) can be brought automatically into engagement with the particular latching stop (44 and 44.1 to 44.4).",
"11.Device according to claims 1 and 5, characterized in that the spindle body (46) of the freely pivotable lever (45) is arranged opposite the guide-path element (35′).",
"12.Device according to claims 1 and 3, characterized in that the pivotable lever (45) is substantially shorter than the longitudinal extent of the return section (41″)."
],
[
"The invention relates to a device for adjusting one component of a chair, in particular an office chair, with respect to another component of the chair, the device being provided with a latching unit by means of which one of the two components can be arranged in positions, which can be changed in latching steps, with respect to the other component, the latching unit having a guide path which is arranged on one of the two components and on the other component a latching element is provided which can be arranged in the guide path in latching positions which predetermine positions of one component with respect to the other component.",
"In the case of chairs, it is often desirable to be able to arrange certain components, for example the backrest of a chair, in different positions.",
"By this means, the intention is to be able to adapt the chair to the users body so as to make an ergonomically favourable seat position possible for the user.",
"In this connection, the publication WO-A 98/09553 discloses a device for regulating the height of the backrest, in which the backrest which can be adjusted in height relative to a seat plate is arranged on a fixed support element.",
"The support element comprises a guide part which is orientated in the vertical direction and in profile cross section is of approximately U-shape design, a grid rail which is arranged in the said guide part and a sliding rail which is guided displaceably in the guide part and also a spring element which can be brought into engagement with it.",
"The spring element as two contact arms which can be brought into engagement with the two toothed arrangements of the grid rail, which are arranged lying opposite the spring element and are each open on one side, the said contact arms, when the backrest is raised, latching in any desired position into the toothed arrangement as a consequence of the spring-elastic restoring force.",
"With this device, the backrest can also be adjusted upwards from the particular position.",
"In particular due to the relatively great length of the contact arms and their undefined movement options there is the risk of them being easily deformed and/or of the ends of the contact spring changing their position.",
"Both possibilities, however, mean that satisfactory guidance of the contact arms in the toothed arrangement can no longer be ensured and adjustment of the backrest is no longer possible.",
"The invention is therefore based on the object of improving a device of the type mentioned in the introduction to the effect that a functionally reliable latching procedure between two components of the chair, in particular of a backrest with respect to a seat body, can be provided using relatively simple structural means.",
"In the case of a device according to the prechraracterizing clause of Claim 1, this object is achieved in that the guide path is of essentially groove-shaped design at least in the region of the latching positions, and the latching element is guided in the groove-shaped guide path.",
"Since the latching element itself does not have to be acted upon by a force, but is intended to be forcibly guided by the profile of the guide path, a particularly functionally reliable guidance of components of a chair is possible.",
"In addition, unlike in WO-A 98/09553, just one latching element is required in devices according to the invention.",
"The reduction to fewer moveable components has a positive effect on the functional reliability of the device and makes possible a compact arrangement and simple installation.",
"It has proven expedient if the groove of the guide path is of a width which permits only slight play of the latching element with regard to directions transverse to the course of the guide path.",
"At least in the region of the latching section, the groove should therefore be only slightly wider then a width of the latching element.",
"This means that the guidance between the latching element in the guide path with regard to the instantaneous direction of relative movement between the latching element in the guide path has essentially just one degree of freedom, namely the possibility of relative movement of the latching element in the guide path in the direction of the course of the guide path.",
"The latching element can therefore be guided in an essentially form-fitting manner, as a result of which long lasting reliable guide elements can be achieved in a structurally particularly simple manner.",
"In a further preferred embodiment of the invention, the latching element can be designed as a pivotable lever which has a latching pin which is arranged in the guide path.",
"The pivot-mounting enables, in a structurally particularly simple and functionally reliable manner, the latching element to be guided in the guide path and thus relative movement to be produced between the latching element and the guide-path element.",
"It is particularly preferred if the device according to the invention is used for adjusting the height of a backrest of an office chair.",
"However, it is equally possible to use the device according to the invention to make it possible for other chair components to be adjusted, for example an arm rest of a chair to be adjusted with respect to the seat surface.",
"Other examples include adjustment of the seat depth of the backrest with respect to the seat surface or else adjustment of the seat height of the sea surface with respect to a base of the chair.",
"Further preferred embodiments of the invention emerge from the dependent claims.",
"The invention is explained in greater detail with reference to an exemplary embodiment which is illustrated schematically in the figures, in which: FIG.",
"1 shows an office chair, which is illustrated in schematic view, having a backrest unit formed from a backrest support and a height-adjustable backrest; FIG.",
"2 shows the backrest unit illustrated in a view according to the arrow direction X sketched in FIG.",
"1, with the backrest support and the backrest; FIG.",
"3 shows the backrest unit illustrated in plan view and in section according to the line III-III sketched in FIG.",
"2; FIG.",
"4 shows the backrest unit illustrated in section according to the line IV-IV sketched in FIG.",
"3; and FIG.",
"5 shows a section according to the line V-V in FIG.",
"4 with a latching unit which is arranged, for example, on the backrest support and is intended for adjusting the height of the backrest.",
"FIG.",
"1 shows a chair which is illustrated in a schematic view, in particular an office chair 10 which can be adjusted in respect of height and inclination in a manner which is not illustrated.",
"The office chair 10 has a base frame 15 which is provided, for example, with a plurality of supporting arms 11 and castors 12 amounted thereon and has a central vertical pillar 14 and a gas-filled compression spring 13 arranged therein.",
"Mounted at the upper end of the tubular vertical pillar 14 is a first supporting body 20 which is operatively connected to the gas-filled compression spring 13 and designed in the form of a cantilever in the direction towards a knee side of the chair.",
"Furthermore, a second supporting body 19 which is arranged on the first supporting body 20 can be seen, as can a backrest unit which is arranged thereon and is denoted in its entirety by 75.The second supporting body 19 is mounted on the first supporting body by means of a rotating/sliding bearing 20′.",
"Two bearing brackets 16 and 17, which are arranged spaced apart from each other transverse to the seat direction, are respectively fastened in the knee-side region of the first supporting body 20 and in the backrest-side region of the second supporting body 19.The bearing brackets 16 and 17 are used for holding and fastening a seat body 18 (illustrated schematically) by means of rotating bearings 16′ and 17′ which are respectively arranged on the knee side and backrest side and by means of which a respective pivot axis is formed.",
"The seat body 18, the first supporting body 20, the second supporting body 19 and the backrest unit 75 which is arranged on the latter can therefore be pivoted with respect to one another, as a result of which different inclinations of the seat body 18 and the backrest unit 75 can be achieved.",
"The backrest unit 75, which is illustrated schematically in FIG.",
"1, comprises a backrest support 30 which is arranged and fastened on the second supporting body 19, and a backrest 25 which is arranged on the backrest support.",
"By means of an appropriately designed latching unit 35, the backrest 25 can be adjusted in height, for example starting from a first basic position illustrated in FIG.",
"1, stepwise in latched steps relative to the seat body 16 in the arrow direction H and can be returned back into the basic position rectilinearly in one step over the entire adjusting distance in accordance with the arrow direction H′.",
"However, in another embodiment which is not illustrated, provision could also be made for the backrest 25 to be guided from an upper position stepwise in latched steps according to the arrow direction h′ in the direction of the seat body 18 and for it to be returned from the lower position rectilinearly into the upper position in the arrow direction H. FIG.",
"2 shows the backrest unit in a view according to the arrow direction X sketched in FIG.",
"1.This illustration shows the backrest support 30 which is arranged on the second supporting body 19 and is arranged with an upper subsection in a recess 24 of approximately pocket-shaped design in the backrest 25.In the exemplary embodiment which is illustrated, the backrest 25 is in engagement with the latching unit 35, which is arranged in the backrest support 30 and in the backrest 25 and is illustrated schematically in FIGS.",
"1 and 2, in such a manner that raising of the backrest 25 causes the latching unit to be adjusted stepwise relative to the seat body 18 in the arrow direction H and to be returned from an upper end position into a lower one in the arrow direction H′.",
"In FIG.",
"3, an exemplary embodiment of the backrest unit 75 is Illustrated in section and in plan view according to the line III-III sketched in FIG.",
"2.The backrest support 30, a subsection of the backrest 25, a plate-shaped supporting part 50 which is designed as a retaining element and is connected to the backrest 25, and a guide-path element 35′ arranged on the supporting part 50 and forming part of the latching unit 35 can be seen here.",
"The backrest unit 75, together with the individual elements 25, 30, 35 and 50, is explained in greater detail below.",
"In the exemplary embodiment illustrated in FIG.",
"3, the backrest 25 comprises a back wall 23 and an end wall 22 spaced apart therefrom.",
"The end wall 22 is divided into two subsections 22′ and 22″ so as to form the pocket-shape recess 24.The sub sections 22′ and 22″ are connected to the back wall 23 in the region of the recess 24 by means of walls 21 and 21 ′ which are integrally formed on them.",
"The recess 24 which is arranged on the end side 22 of the back rest 25 is open downwards towards the seat surface 18 and is used to hold the supporting part 50 and the backrest support 30 which is partially arranged therein.",
"The backrest support 30 which his illustrated in FIG.",
"3 and is of approximately U-shaped design in profile cross section has an end wall 31 and limbs 26 and 26′ arranged laterally thereon.",
"Provided on the mutually facing inner sides of the two limbs 26 and 26′ are respective grooves 27 and 27′ which essentially run rectilinearly and are bounded by first webs 28, 28′ and second webs 29, 29′ which are arranged parallel to one another.",
"The supporting part 50 which is illustrated in profile cross section in FIG.",
"3 and is arranged in the recess 24 of the backrest 25 comprises a plate 51 which is provided on one side with a plurality of ribs 52 which are spaced apart from one another and are connected to the backrest 25.Integrally formed on the two opposite ends of the plate 51 are respective webs 53 and 53′ which are or L-shaped or backwards L-shaped design in profile cross section and respectively engage in one of the two grooves 27, 27′.",
"A groove 55 or a web 32 of the backrest support 30 is provided on the plate 51 of the supporting part 50, between two webs 54 and 54′ on the side which faces the inner side of the end wall 31.The webs 53, 53′, 32 guide the backrest 25 longitudinally in the grooves 27, 27′ D 55 in its adjustment direction.",
"Regarding a Width of the supporting part 50, the guide-path element 35′ is integrally formed approximately in the centre of the supporting part and on its side facing the backrest support 30.Furthermore, a lever arm 45, which has a spindle body 45 at one end and a latching pin 47 at the other end, is arranged on the backrest support 30 opposite the guide-path element 35′ as latching element.",
"The lever arm 45 is mounted in the backrest support 30 in a manner such that it can pivot freely by the spindle body 46 around its (imaginary axe) Axis Y (see also FIG.",
"4).",
"The lever arm is operatively connected to the guide-path element by means of the latching pin 46 which engages in the guide-path element 35′ (FIGS.",
"3, 4).",
"In FIG.",
"4, the backrest unit 75 is illustrated in section according to the line IV-IV sketched in the region of the recess 24 in FIG.",
"3.The pivoting lever 45 which is mounted with the spindle body 46 on the backrest support 30 and engages in a groove-shape guide path 40 by means of the latching pin 47 can also be seen here.",
"FIG.",
"5 shows in more detail the guide-path element 35′ which is illustrated in sectional view according to the incepting line V-V in FIG.",
"4 and, in the exemplary embodiment which is illustrated, is arranged on the wall 51 of the supporting part 50.FIG.",
"5 furthermore shows the pivoting lever 45 which is mounted on the spindle body 46 together with the latching pin 47 which is arranged on the said lever and is positioned in a latching stop 44.2 of the guide path 40.FIG.",
"5 furthermore schematically illustrates, by means of the arrows 1 and 2 and the “dash-dotted” lines which are not numbered, the sequence or movement of the latching pin 47 caused by adjusting the height of the backrest 25.The guide-path element 35′ is bounded laterally by the first webs 33 and 33′ (FIG.",
"3) and at the upper and lower end by second webs 34 and 34′ (FIG.",
"4).",
"Furthermore, the guide-path element 35′ is provided with the self-contained, groove-shaped guide path 40.The guide path 40 has a latching section 41′ and a return section 41″ which are connected to each other at their upper and lower ends by means of likewise groove-shaped deflecting paths 42 and 48 to form a self-contained path.",
"Except for the return section 42, a width of the guide path 40 is only slightly larger than the diameter of the latching pin.",
"In addition, as can be seen from FIG.",
"5, the pivotable lever arm 45 of the latching element is substantially shorter than the longitudinal extent of the return section (41″).",
"The latching section 41′ and the return section 41″ are arranged spaced apart from each other and functionally separated from each other by means of an intermediate piece 36 which is orientated parallel to the longitudinal extent of the two sections 41′ and 41″.",
"The latching section 41′ of the groove-shaped guide path 40 is provided, on the side which faces the web 33′, with a plurality of latching stops 44, 44.1-44.5 which are arranged spaced apart from one another and are connected to one another by a plurality of groove-shaped curved paths having a double curve.",
"In the exemplary embodiment illustrated in FIG.",
"5, the latching section 41′ comprises the latching stops which are arranged one behind another the longitudinal course of the latching section and are denoted by 44; 44.1; 44.2; 44.3; 44.4 and 44.5, and also the curved path which connect the said latching stops to one another and are denoted by 43; 43.1; 43.2; 43.3 and 43.4.Each curved path 43, 43.1-43.5 has a projection 39, 39.1-39.4 which is formed on he web 33′ and in incliner in the adjustment direction.",
"Between the respective latching stop 44, 44.1-44.5 and the respective projection 39, 39.1-3.9.4 the latching pin executes with respect to the guide-path element 35′ a curved relative movement which is orientated in the clockwise direction in the guide-path 40.Each segment, corresponding to one latching step, of the latching section 41′ has a deflecting toe 38; 38.1; 38.2; 38.3 and 38.4 between a projection and the next latching stop 44.1-44.5 in each case.",
"The deflecting toes are integrally formed on the intermediate piece 36.The contour of the deflecting toes 38; 38.1; 38.2; 38.3 and 38.4 in each case ends an anticlockwise, second, curved relative movement of the latching pin 47 with respect to the guide-path element 35′.",
"An end piece of each deflecting toe is inclined in the manner of a ramp towards the adjustment direction H. The lever 45 therefore pivots in two opposite directions during a latching step.",
"Starting from a latching stop, during movement towards the respectively next latching stop the latching pin 47 is first of all situated on one side of the line 60 along which the pivoting axis Y moves during an adjustment movement of the backrest 25.A projection 39, 39.1-39.4 of a segment of the latching section 41′ causes the latching pin 47 then to pivot by means of a pivoting movement around the axis Y onto the other side of the line 60.The approximately S-shaped profile of each segment subsequently leads to an opposite pivoting movement of the latching pin 47 back to the original side of the line 60.As soon as the latching pin strikes against the deflecting toe 38, 38.1-38.4 of a segment of the latching section 41′, the deflecting-toe contour, which rises in a ramped-like manner counter to the adjustment direction, causes the adjustment movement in the adjustment direction of the backrest to stop.",
"By means of a slight movement of the backrest counter to the adjustment movement, the Latching pin can be transferred, by continuing its pivoting movement, into the next latching 44.1-44.5.This is indicated in FIG.",
"5 by the arrow, 61 which reproduces the relative movement, corresponding to this, of the latching pin from the particular deflecting toe to the next latching stop.",
"By this means, the backrest has taken up a further intermediate position in which it has been adjusted upwards by one latching step.",
"In this position, in which the latching pin is fixed in the latching stop 44, 44.1-44.5, in an undercut formed by the projection 39, 39.1-39.4, the backrest remains on the latching pin 47 because of its own weight.",
"FIG.",
"5 shows this by way of example for the latching stop 44.2 which defines an intermediate position between a lower latching position (predetermined by the latching stop 44) and an upper latching position (predetermined by the latching stop 44.5) of the backrest.",
"Starting from the latching stop 44.5, the latching pin can be transferred via the deflecting section 48, the return section 41″ and the further deflecting section 42 back to the beginning of the latching section 41′, namely the latching stop 4A (arrow 2).",
"During this movement, the backrest is displaced from its upper latching position to its lower latching position.",
"In another exemplary embodiment which is not illustrated it would also be possible to arrange the guide-path element on the backrest support and the lever on the backrest.",
"The effect which could be achieved by this is that it would be possible for the backrest to be displaced downwards in latching steps and to be returned in a single step from its lowest position into its upper starting position.",
"The guide-path element 35′ which is described above in conjunction with the individual figures and the arrangement thereof on a backrest or on a backrest support is not restricted to the exemplary embodiments which have been illustrated and described.",
"Further expedient embodiments and arrangements are also possible without departing from the basic concept of the invention."
]
] | Patent_10240160 |
[
[
"Optical device for simultaneous multiple measurement using polarimetry and spectrometry and method for regulating/monitoring physical-chemical and biotechnical processes using said device",
"The invention relates to a device, especially an optical flow-through measuring cell, for the combined use of spectrometry and polarimetry for simultaneously measuring multiple variables in physical-chemical and biotechnical processes, with multiple optical layer thicknesses at the same time.",
"Spectrometry can be used to detect dissolved substances in the medium flowing through the cell in the ultraviolet range (UV), the visible range (light) and the near infrared range (NIR) of electromagnetic radiation, in particular."
],
[
"1-32.",
"(canceled) 33.A flow-through measuring cell having an oblong measuring body (2) and a base structure (1) surrounding the measuring body (2) in a lengthwise direction, wherein the base structure (1) comprises an inlet connection element and an outlet connection element for the liquid to be measured and liquid-tight guides (17) on both longitudinal ends of the base structure (1) for one or more rods (16) extending crosswise or lengthwise to the base structure (1) and to the measuring body (2), for providing a continuously variable optical path length.",
"34.The flow-through measuring cell according to claim 1, in which the measuring body (2) is made of a transparent material for measuring.",
"35.The flow-through measuring cell according to claim 1, in which the measuring body (2) exhibits a round cross section with two plane-parallel surfaces in a lengthwise direction on the outer sides.",
"36.The flow-through measuring cell according to claim 1, in which the measurement body (2) exhibits a square cross section (11).",
"37.The flow-through measuring cell according to claim 1, in which the base structure (1) exhibits adapter receptacles (6, 6′).",
"38.The flow-through measuring cell according to claim 1, in which the base structure (1) and the measuring body (2) are designed together as a reciprocal, exchangeable module (14).",
"39.The flow-through measuring cell according to claim 1, in which the base structure (1) and the measuring body (2) at each end exhibit a closing component (7).",
"40.The flow-through measuring cell according to claim 7, in which one or more optical windows (4) are placed in the closing component.",
"41.The flow-through measuring cell according to claim 1, in which the base structure (1) is equipped with one or several tempering units (12).",
"42.The flow-through measuring cell according to claim 1, in which the base structure (1) exhibits one or several tempering channels (13).",
"43.The use of a flow-through measuring cell according to claim 1 for regulating and monitoring physical-chemical and biotechnical processes.",
"44.The flow-through measuring cell according to claim 2, in which the measuring body (2) exhibits a round cross section with two plane-parallel surfaces in a lengthwise direction on the outer sides.",
"45.The flow-through measuring cell according to claim 2, in which the measuring body (2) exhibits a square cross section (11).",
"46.The flow-through measuring cell according to claim 2, in which the base structure (1) exhibits adapter receptacles (6, 6′).",
"47.The flow-through measuring cell according to claim 3, in which the base structure (1) exhibits adapter receptacles (6, 6′).",
"48.The flow-through measuring cell according to claim 2, in which the base structure (1) and the measuring body (2) are designed together as a reciprocal, exchangeable module (14).",
"49.The flow-through measuring cell according to claim 3, in which the base structure (1) and the measuring body (2) are designed together as a reciprocal, exchangeable module (14).",
"50.The flow-through measuring cell according to claim 2, in which the base structure (1) and the measuring body (2) at each end exhibit a closing component (7).",
"51.The flow-through measuring cell according to claim 2, in which the base structure (1) is equipped with one or several tempering units (12).",
"52.The flow-through measuring cell according to claim 2, in which the base structure (1) exhibits one or several tempering channels (13)."
],
[
"The invention relates to a device, in particular an optical-flow-through measuring cell for the combined use of spectrometry and polarimetry for simultaneously determining multiple variables in physical-chemical and biotechnical processes.",
"Spectrometry can be used, in particular, in the ultraviolet range (UV), the visible range (light), and the near infrared range (NIR) of electromagnetic radiation to detect dissolved substances in the medium flowing through.",
"In monitoring and regulating physical-chemical and biotechnical processes, for example in chemistry, pharmacy, biotechnology, environmental technology, and medicine, properties of substances in solution must often be continuously and quantitatively recorded without delay; the concentration and/or the optical activity may be the variables to be measured or recorded, among other things.",
"In chemical analysis as well as process regulation, such as for example, chemical conversion and the regulation of biological processes in bioreactors, properties of substances in solution (for example, the concentration and/or optical activity) must often be continuously and quantitatively recorded without delay.",
"A principal possibility of the technical embodiment of such measuring tasks exists in the continued withdrawal and return of the material being measured, as well as a measurement in the created “bypass flow” or “measurement cycle” with the optical analytical method in the flow-through measuring cells.",
"For certain analytical methods, for example, chromatographic methods, which always work with optically clear media, a measurement in the entire liquid medium, i.e., in the main stream (here the eluate), can also be imperative.",
"Optical measuring devices containing the flow-through measuring cells, as well as modular flow-through measuring cells for optical measurements, have long been known and exist in great numbers with varying developments.",
"However, these flow-through measuring cells are primarily conceived for a single, special measuring task, so that a combination of different measuring methods necessarily exists in a series connection of various measuring devices or measurement systems.",
"As a result, it is not possible to conduct simultaneous measurements of different variables in the same sample.",
"Moreover, liquid increments in the particular measurement sections are often clearly mixed with one another, and this results in a decrease of the separation efficiency of analytical methods, for example.",
"A summation of such effects by adding several measurement sections is therefore very disadvantageous.",
"There may be a further problem when the bypass flow consists of a sterile liquid (for example, from a bioreactor): The more mechanical connections there are in the measurement cycle, the greater the danger of bacterial contamination.",
"Moreover, it would be advantageous if the measuring cell, as well as the measuring system (electronics, radiation sources, detectors, etc.",
"), is arranged spatially separate from one another so that it is possible to measure at variously adjustable temperatures and at the overpressure in the cell even in areas exposed to a risk of explosion and/or in areas under strong electromagnetic influence.",
"German Patent Application 199 11 265.7 (filing date: Mar.",
"13, 1999) describes a device, using polarimetry and IR spectrometry, though specifically geared for measuring the glucose concentration in tissue fluids, with no simultaneous measurement of variables being possible in a wide spectrometric and polarimetric area.",
"The task of the present invention, therefore, is to develop a device, in particular-a flow-through measuring cell for combined optical measurements in liquid material being measured via spectrometry and polarimetry that allow quantitative measurements practically without delay.",
"The measurement device together with the modular units that are necessarily connected thereto on one side, as well as measuring system (electronics, radiation sources, detectors, etc.)",
"on the other side, should preferably be arranged spatially separate from one another.",
"As a spectrometric measuring method, the so-called UV spectrometry (wavelength range (Δλ): from 0.2 to 0.4 μm, UV: ultraviolet radiation), the light spectrometry (wavelength range (Δλ): from 0.4 to 0.8 μm), and the NIR spectrometry (wavelength range (Δλ): from 0.8 to 2.5 μm, NIR: near infrared radiation) should be applicable, that is, measurements either in one, two, or even all three wavelength ranges simultaneously and/or measurements at several wavelengths in one or all mentioned wavelength ranges.",
"In addition, there-should be an option to set different, continuously variable optical path lengths (film thicknesses).",
"Polarimetry should preferably be executable with light, at least with two different optical path lengths, without having to modify the cell.",
"Moreover, it should be possible to temper the cell and to use it in overpressure operation.",
"The above-mentioned task is achieved, according to the invention, using a device (cell) according to the following descriptions and the (main) claim 1.Advantageous forms of embodiment are specified in the sub-claims.",
"The device or cell according to the invention is preferably long and provided with optical devices for guiding measuring light beams for polarimetry.",
"A measuring light beam may run lengthwise, and/or another measuring light may run crosswise through the device, in particular the cell.",
"The combination of lengthwise and crosswise arrangement of the polarimetric working beams is particularly preferred (devices 3.1, 3.2).",
"The relation of the optical path lengths of the measuring beams then depends on the dimensions of the base (cell), namely the diameter (in particular, the inner diameter, for example, of the cell) in relation to the length, and is 1:1 to 1:50, preferably more than 1:1, in particular, 1:2 to 1:40 or 1:11 to 1:30, and particularly preferred, 1:2 to 1:10, in particular, 1:10.Because of the chosen difference of the optical path lengths, dissolved, optically active substances may surprisingly be measured in a large area of concentration in one and the same device (cell).",
"All optical devices used for the polarimetrical analysis do not change the polarization state of the measuring beam.",
"There may be optical devices (3′) for the spectrometric measurement, together with the above mentioned combination of the polarimetric devices, e.g., crosswise to the base axis, preferably through suitable adapter receptacles, which establish the measurement sections for the spectrometric measurements.",
"In this embodiment, their optical path lengths (thickness of layer) are thus equal to the inner diameter of the base.",
"It is also preferred if the polarimetric device(s) and the spectrometric device(s) are arranged crosswise to the base, e.g., if outlet connection elements are available lengthwise.",
"The optical devices 3′ may alternatively be positioned in the longitudinal direction and optionally also in the transverse-direction, as described below, and/or arranged on adapter receptacles through guides with glass rods.",
"This correspondingly increases the number of possible optical path lengths.",
"The optical device(s) for polarimetry is thus arranged crosswise to the base.",
"The arrangement of the optical devices is consequently variable and may be structured depending on the application requirement.",
"The device according to the invention may preferably exhibit cell windows, which in particular consist of radioparent material, for example out of quartz, which has good optical transparency for a wide range—from UV to NIR—of electromagnetic beams.",
"The beam coupling and uncoupling may take place via conductors, in particular fiber optics, with polarized optic light guides preferably being used for the polarimetric analysis and fiber optics made of quartz preferably being used for the spectrometric analysis.",
"A spatial separation of the signal receiver and signal processing system from the cell may consequently be achieved, in particular.",
"The device (cell) according to the invention for spectrometric and polarimetric optical measurements in the liquid material for measurement consequently includes a base 1, a measuring system, and optical devices, with an optical device being arranged to guide the polarimetric measuring light lengthwise to the base and an optical device being arranged to guide the polarimetric measuring light crosswise to the base, as well as one or several other optical devices to lead spectrometric working beams lengthwise and/or crosswise to the base.",
"An optical device includes two identical parts, each of which, for example, exhibits a collimator and/or focuser and/or optical neutral filter and/or optical interference filter and/or polarizer.",
"These are known mechanisms for optical devices, as described, for example, in the NAUMANN SCHRÖDER book, Bauelemente der Optik [Optical Elements].",
"The measuring system includes, in particular, the electronics, radiation sources, signal processing systems, and detectors.",
"The optical devices are preferably connected with the measuring system via conductors, in particular via polarized fiber optic light guides for polarimetry, and via fiber optics for spectrometry.",
"A spatial separation of measuring system and base is consequently achieved with the corresponding advantages.",
"It is furthermore preferred for the base 1 or the device to contain a measuring body 2, in particular a tubular profile measuring body, preferably out of a material radioparent for measuring, preferably out of quartz.",
"Alternatively, a glass tube may also be chosen.",
"The measuring body, in particular the tubular profile measuring body (profile measurement tube), may exhibit a round cross section 2-with two plane-parallel surfaces in a lengthwise direction on the outer sides or a square cross section 11 or another suitable shape, such as a polygon.",
"In particular, the optical devices for the spectrometric devices 3′ may be arranged via adapter receptacles 6, 6′ thus at least 1×2, and altogether as many as there are optical devices present, preferably 1 to 10, thus 1×2 to 1×10 adapter receptacles.",
"The adapter receptacles thus represent, for example, guide bushs with cylindrical cross section.",
"The number of optical devices depends on the dimension of the base, in particular, its length.",
"It is also preferred for the adapter receptacles 6, 6′ to be arranged parallel to the surface normals of the plane-parallel surfaces of the measuring body 2 or of the square measuring body 11.Alternatively, the adapter receptacle(s) 6, 6′ for accommodating the spectrometric measurement beams can be situated on glass rods 16 over guides 15, whereby in particular the the rods 16 being of a material radioparent for measuring, such as quartz.",
"In this arrangement, crosswise to the base, the optical path length (thickness of layer d) is changeable in a continuously variable manner for the spectrometric measurement in the range of 0 mm up to the inner diameter of the base.",
"In particular, the guides are liquid-tight and the glass rods hold the adapter receptacles at one end and rise up at the other end into the graduated tube.",
"In the device according to the invention, the base 1 and the measuring body 2 or 11 may be designed as a reciprocally exchangeable module 14.The modules are advantageously of different lengths so that different optical lengths of path are possible for polarimetry in a lengthwise direction.",
"It is also preferred for the rotational axis of the optical device 3.1 to be arranged parallel to the surface normals of the front surfaces of the base structure 1.The devices 3.2 as well as the devices 3′ are preferably arranged crosswise, in particular vertical thereto.",
"Alternatively, angles may also not be equal to 0°(based on the surface normals), insofar as optically and physically possible within the bounds.",
"The device according to the present invention may exhibit optical devices, in particular to guide spectrometric measurement beams in the wavelength range from UV to NIR, and preferably devices to guide polarimetric measurement beams in the visible spectral range.",
"Furthermore, the base 1 and the measuring body 2, 11, in particular, may at each end exhibit a (cell-)closing component 7.This may exhibit the inlet or outlet connection elements 5, preferably on the side.",
"Alternatively, the connection elements 5 may also be arranged in a lengthwise direction.",
"The optical devices are then arranged crosswise for polarimetry as well as for spectrometry.",
"Moreover, one or several optical (cell-)window(s) 4 may be put in the (cell-)closing component 7, the window having a rotational axis congruent to the rotational axis of the (cell-)closing component.",
"The (cell-)window(s) is/are preferably made of a material radioparent for measuring, such as quartz.",
"An optical device for polarimetry 3.1 placed in the (cell-)closing component 7 is preferred, the rotational axis of the optical device being congruent to the rotational axis of the (cell-)closing component.",
"Furthermore, the guides 17 may be congruent to the rods 18 or be incorporated around the rotational axis of a (cell-)closing component 7, which is arranged at each end of the base 1 and of the measuring body 2.These glass rods 18 as well as the above-mentioned glass rods 16 preferably made of a material radioparent for measuring, such as quartz, and adjustable to one another with a radiopaque outer surface.",
"As mentioned, adapter receptacles for spectrometric devices 3′ may be arranged on these glass rods.",
"This allows the optical path length to be changed in a lengthwise direction in a continuously variable manner for the spectrometric measurement.",
"In such an arrangement, the spectrometric device is in a lengthwise direction, and if necessary, in a crosswise direction, and the polarimetric device is in a crosswise direction to the base.",
"It is furthermore preferred for the base 1 and the measuring body 2 or 11 to exhibit a (cell-)closing component 7 at each end and for an inlet or outlet connection element 5 to be incorporated into the front of the (cell-)closing component 7.In this embodiment, the optical devices are situated in the transverse direction.",
"The configuration of the other parts, such as adapted receptacles, guides, modules, etc.",
"is similar to that described above.",
"As mentioned, it is particularly preferred for the optical devices, particularly 3.1 and 3.2, for polarimetry to be linked to the measuring system via an optic light guide 8.The optic light guides 8 are preferably connected to the device via couplers 9.The optical device for the spectrometric measurement, particularly 3′, may preferably be connected directly to the measuring system via conductors, in particular fiber optics 10, which are in particular made of a material radioparent for measuring, such as quartz.",
"A spatial separation between the measurement device and the measurement system is consequently possible, the measurement system including electronics, radiation sources, signal processing systems, and detectors, such as a generally known polarimeter or spectrometer.",
"Especially articularly preferred is a device in which the measurement body, in particular, the profile measurement body, exhibits dimensions of no more than a 15 mm diameter, in particular, 0.5 to 12 mm, and a length of 1 to 750 mm, in particular, 300 mm, for example.",
"The base 1 of the device according to the invention may furthermore be equipped with one or two lateral tempering units 12, or alternatively, with one or several tempering channels 13.The device according to the present invention is thus temperable, in particular, and also usable even in overpressure, with measurements being simultaneously possible in different wavelength ranges, in particular, in a continuously variable manner.",
"The device according to the invention is thus suitable, in particular, for controlling and monitoring physical-chemical processes, such as chromatographys and purification of stereospecific substances as well as biotechnical processes, such as bioreactors, by coupling the device in a suitable manner with the process to be monitored or regulated.",
"This may take place through a process control center, for example.",
"The previously described features and advantages of the invention are illustrated using the following detailed description of the attached drawings.",
"Shown are: FIG.",
"1, A schematic representation of an embodiment of a device according to the invention FIG.",
"2, A schematic representation of another embodiment of the measuring body 2 FIG.",
"3 a schematic representation of another embodiment of the base structure 1 FIG.",
"4, a schematic representation of another embodiment of the base structure 1 FIG.",
"5, a schematic representation of another embodiment of the device according to the invention FIG.",
"6, a schematic representation of another embodiment of the device according to the invention FIG.",
"7, a schematic representation of another embodiment of the device according to the invention FIG.",
"8, a schematic representation of another embodiment possibility of the (cell-)closing component 7 FIG.",
"9, a schematic representation of another embodiment of the device according to the invention.",
"FIG.",
"1 shows the profile of an example of the device according to the invention (flow-through measuring cell).",
"It essentially consists of a base 1, which here surrounds a tubular profile measuring body 2 made of quartz.",
"This round measuring body has two plane-parallel surfaces (spanner opening) in a lengthwise direction on the outside, whose surface normals are parallel to the rotational axis of the adapter receptacles 6 (Section A-A′) and to the rotational axis of the optical devices 3.2 for polarimetry to be carried out crosswise.",
"There are as many adapter receptacles as the length of the structure allows.",
"The base is provided with closing components 7 at the axial ends.",
"An inlet or outlet connection element 5 each are incorporated into these on the sides.",
"The rotational axis of the closing component (cell closing component), the rotational axis of the optical cell window 4, and the rotational axis of the optical devices 3.1 for polarimetry in a lengthwise direction are congruent.",
"The coupling or uncoupling of the measurement light for polarimetry takes place via the polarization-receiving glass optic light guides 8, which may be connected directly to the cell via couplings 9.The coupling or decoupling of the measuring beam for spectrometry takes place via fiber optics 10 made of quartz, whose ends are directly connected with the optical devices 3′ for the spectrometric analysis, which may be introduced into the adapter receptacles.",
"FIG.",
"2 shows a design of the measuring body similar to that in FIG.",
"1, but in which the profile measurement body 11 made of quartz has a square cross section instead of the previously described.",
"The optical path length (film thickness) here is constant over the entire beam width.",
"FIG.",
"3 shows a design similar to that in FIG.",
"1, but in which the profile measurement body 11 made of quartz has a square cross section, and there are other adapter receptacles 6′ for optical devices for the spectrometric analysis in the base that are perpendicular to the adapter receptacles 6 (Section A-A′) in FIG.",
"1.The number of adapters, and consequently, the number of the “measuring” wavelengths, may be increased in this manner.",
"FIG.",
"4a shows a design similar to that in FIG.",
"1, but in which the base is connected to a tempering unit 12 (e.g., Peltier elements) on one or on both sides.",
"FIG.",
"4b: instead of the Peltier elements, channels 13 may run in the base, through which a tempered medium flows so that the device (cell) may be brought to a desired temperature.",
"FIG.",
"5 shows embodiments similar to that in FIG.",
"1, but in which the base 1 and measuring body 2 are combined in a “module” 14 and are replaceable, so that different optical path lengths, in a lengthwise direction for polarimetry may be realized.",
"FIG.",
"6 shows a design similar to that in FIG.",
"1, but in which the optical path length (film thickness d) is more or less changeable in a continuously variable manner in a certain range (0 mm up to the inner diameter of the measurement body).",
"Liquid-tight guides 15 for glass rods 16 have been incorporated perpendicularly into the base 1 and into the profile measuring body 2, instead of the two opposing adapter receptacles 6 for optical devices for the spectrometric analysis.",
"In a preferred embodiment, the rods are made of quartz.",
"The rods are movable against each other with a radiopaque outer surface.",
"Placed on the outer end of each rod is an adapter receptacle 6 for the optical device.",
"FIG.",
"7 shows a design similar to that in FIG.",
"1, but in which the optical path length (film thickness d) for the spectrometric analysis is more or less changeable in a continuously variable manner in an enhanced range (0 mm up to the length of the measuring body).",
"Instead of the two optical devices 3.1 for polarimetry in a lengthwise direction, liquid-tight guides 17 for glass rods 18 are incorporated lengthwise to the base structure 1.In a preferred embodiment, the rods are made of quartz.",
"The rods are movable against each other with a radiopaque outer surfaces.",
"Placed on the outer end of each rod is an adapter receptacle 6 for the optical device for the spectrometric analysis.",
"FIG.",
"8 shows a design similar to that in FIG.",
"7, but in which several guides 17 for glass rods 18 are incorporated.",
"The number of the “measuring” wavelengths can thus be increased.",
"FIG.",
"9 shows a design similar to that in FIG.",
"1, but in which the optical devices 3.1 for polarimetry in a lengthwise direction, as well as the guides 17 for the glass rods 18 (as per FIG.",
"7), are missing.",
"Instead, the inlet or outlet connection element 5, congruent to the rotational axis of the (cell closing) component 7."
]
] | Patent_10240165 |
[
[
"Attenuation material",
"An attenuation material and a method of making such an attenuation material.",
"The attenuation material includes a matrix of elastomer having mixed therewith particulate vulcanised rubber and particulate magnetisable material, the mixture being substantially free of sublimable foaming agents."
],
[
"1.An attenuation material comprising a mixture, the mixture comprising a matrix of elastomer having mixed therewith a particulate vulcanised rubber and a particulate magnetisable material, the mixture being substantially free of sublimable foaming agents.",
"2.The attenuation material according to claim 1, wherein the mixture further comprises a filler material.",
"3.The attenuation material according to claim 2, wherein the filler material comprises at least one of particulate carbon black, particulate clay, particulate limestone, particulate silica and finely divided paper.",
"4.The attenuation material according to claim 2 or 3, wherein the filler material comprises a finely divided form having a particulate size in the range of from about 50 to about 500 microns.",
"5.The attenuation material according to claim 3, wherein the particulate silica comprises waste silica.",
"6.The attenuation material according to claim 1, wherein the magnetisable material comprises a ferrous material.",
"7.The attenuation material according to claim 1, wherein the magnetisable material comprises at least one of millscale, copper silicate and Fe3O4.8.The attenuation material according to claim 1, wherein the magnetisable material comprises a finely divided form.",
"9.The attenuation material according to claim 8, wherein the magnetisable material has a particle size of about 300 microns.",
"10.The attenuation material according to claim 1, wherein the magnetisable material and a filler are present in a ratio of about 4 to 1 by weight.",
"11.The attenuation material according to claim 1, wherein the vulcanised rubber comprises discrete particles.",
"12.The attenuation material according to claim 1, wherein the vulcanised rubber is substantially free of reaction with the elastomer when the elastomer is cured.",
"13.The attenuation material according to claim 1, wherein the particulate vulcanised rubber is obtained cryogenically, by comminution of waste rubber compounds.",
"14.The attenuation material according to claim 13, wherein the waste rubber compounds comprise at least one of natural rubber and SBR.",
"15.The attenuation material according to claim 1, wherein the vulcanised rubber comprises comminuted rubber tires, the comminuted rubber tires comprising at least one of waste and worn tires.",
"16.The attenuation material according to claim 1, wherein the vulcanised rubber is in a finely divided form.",
"17.The attenuation material according to claim 16, wherein the vulcanised rubber has a particle size of less than about 500 microns.",
"18.The attenuation material according to claim 1, further comprising at least one fire retardant material.",
"19.The attenuation material according to claim 1, further comprising an adhesion enhancing material.",
"20.The attenuation material according to claim 19, wherein the adhesion enhancing material is added in the ratio of about 50 parts per 100 of the matrix of elastomer, by weight.",
"21.The attenuation material according to claim 1, wherein the matrix of elastomer includes at least one of the following batch ingredients: (A) KELTAN; (B) SPHERON 5000A Medium Fine Carbon Black; (C) Zinc Oxide Active; (D) Stearic Acid; (E) PEG 4000; (F) Britomya BSH 20; (G) Flexon 815 Liquid; (H) KEZADOL GR; (I) RHENOGRAN 580 Sulphur; (J) RHENOGRAN TMTD 80 Tetramethylthiuram Disulphide; (K) RHENOGRAN MBT 80 Mercaptobenzothiazole; and (L)RHENOGRAN MBTS 80 Benzothiazyl Disulphide.",
"22.The attenuation material according to claim 1, wherein the elastomer is substantially free of at least one of a natural rubber and an SBR.",
"23.The attenuation material according to claim 1, wherein the elastomer comprises at least one of polyolefin type rubber and EPDM.",
"24.The attenuation material according to claim 1, wherein the matrix of elastomer, the vulcanised rubber and the magnetisable material are present in a ratio in a range of from about 100:20:20 to about 100:50:50.25.An attenuation material comprising a composition comprising at least one of matrix A, matrix B, matrix C and matrix D, wherein matrix A, matrix B, matrix C and matrix D comprise: Matrix A Matrix B Matrix C Matrix D Keltan 314 2.4000 2.4000 2.4000 2.700 Keltan 4903 9.6000 9.6000 9.6000 10.6000 Zinc Oxide Active 0.3500 0.3500 0.3500 0.3900 Stearic Acid 1800 0.1200 0.1200 0.1200 0.1300 PEG 4000 0.1200 0.1200 0.1200 0.1300 TE 80 Powder 0.2000 — — — STUKTOL W816 0.2000 — — — Baco Superfine 7 14.8000 — — — Spheron 5000A 2.0000 16.8000 16.8000 18.7000 Britomya BSH 20 4.8000 4.8000 5.3000 Flexon 815 Liquid 8.8000 8.8000 8.800 9.800 KEZADOL GR 0.5000 0.5000 0.500 0.5600 Rhenogran CTP80(PVI) 0.0900 0.0900 0.090 0.1000 RHENOGRAN 580 0.1100 0.1100 0.1100 0.1200 RHENOGRAN 0.8800 0.0800 0.0800 0.0900 TMTD 80 RHENOGRAN MBT 80 0.0270 0.0270 0.0270 0.0300 RHENOGRAN 0.0900 0.0900 0.0900 0.1000 MBTS 80 XETAL Filler A 13.0000 13.0000 13.000 9.7000 Vulcanised Rubber XETAL Filler B Mill 21.6000 30.3000 13.00 9.7000 scale, copper silicate or Fe3O4 Total Weight (kg) 78.8871 87.1871 69.8871 68.1500 26.A method of manufacturing a material suitable for use as an attenuation material, the method comprising: 1) providing a mixture comprising a matrix of elastomer having mixed therewith particulate vulcanised rubber and particulate magnetisable material, the mixture being substantially free of sublimable foaming agents; and 2) forming the mixture into at least one sheet.",
"27.The method according to claim 26, wherein at least the magnetisable material and the vulcanised rubber are mixed together prior to further mixing with the elastomer, prior to curing or vulcanisation of the elastomer.",
"28.The method according to claims 26 or 27, wherein the further mixing is typically carried out in a mixer, the mixer comprising a Banbury rotor type mixer.",
"29.The method according to claim 28, wherein the mixer has a rotor speed in the range of from about 60 to about 100 rpm.",
"30.The method according to claim 26, wherein the vulcanised rubber is not added to the elastomer after the addition of the magnetisable material.",
"31.The method according to claim 26, wherein the mixture in step 1) is mixed for about 4 to about 12 minutes.",
"32.The method according to claim 26, wherein the mixing is at a temperature from about 80° C. to about 110° C. 33.The method according to claim 26, wherein the magnetisable material and a filler are mixed prior to addition of the elastomer.",
"34.The method according to claim 26, wherein the sheet in step 2) is formed by passing the mixture produced in step 1) through a dump mill, then onto a sheeting mill and finally onto a three bowl calender for sheeting off.",
"35.The method according to claim 26, further comprising the step of curing the mixture formed in step 1) after the mixture has been formed into the sheet.",
"36.The method according to claim 35, wherein the curing takes place in at least one of a line oven, a microwave oven, or an autovac oven.",
"37.The method according to claim 35, wherein the curing is at a temperature in the range from about 155° C. to about 165° C. 38.The method according to claim 26, wherein the sheet has a thickness of from about 0.2 mm to about 6 mm, when in an unexpanded form.",
"39.The method according to claim 26, wherein the sheet formed in step 2) may be laminated to at least one of paperboard, plasterboard, and woven fabrics.",
"40.The method according to claim 26, wherein the material is magnetised.",
"41.A fire retardant body comprising the attenuation material according to claims 1 or 25, and a fire retardant.",
"42.A laminate material comprising the attenuation material according to claims 1 or 25, having bonded thereto a second material, the second material comprising at least one of steel, high performance polymers, PET and aluminium, wherein the high performance polymers comprise polyamides.",
"43.The attenuation material according to claim 2 or 3, wherein the filler material comprises a finely divided form having a particulate size of about 120 microns.",
"44.The attenuation material according to claim 4, wherein the particulate silica comprises waste silica.",
"45.The attenuation material according to claim 6, wherein the magnetisable material is coated with copper.",
"46.The attenuation material according to claim 8, wherein the magnetisable material has a particle size in the range of from about 25 to 400 microns.",
"47.The attenuation material according to claim 16, wherein the vulcanised rubber has a particle size of less than about 120 microns.",
"48.The attenuation material according to claim 23, wherein the polyolefin type rubber comprises an ethylene propylene rubber.",
"49.The method according to claim 28, wherein the mixer has a rotor speed of about 80 rpm.",
"50.The method according to claim 26, wherein the mixture in step 1) is mixed for about 8 minutes.",
"51.The method according to claim 26, wherein step 1) further comprises the matrix of elastomer having mixed therewith a filler.",
"52.The method according to claim 26, wherein at least the magnetisable material, the vulcanised rubber, and a filler are mixed together prior to further mixing with the elastomer, prior to curing or vulcanisation of the elastomer.",
"53.The method according to claim 26, wherein the vulcanised rubber is not added to the elastomer after the addition of the magnetisable material and a filler.",
"54.The attenuation material according to claim 5, wherein the waste silica is from power station lakes.",
"55.The attenuation material according to claim 19, wherein the adhesion enhancing material comprises neoalkoxy zirconate.",
"56.The attenuation material according to claim 24, wherein the magnetisable material comprises at least one of millscale, copper silicate and Fe3O4.57.The method according to claim 30, wherein the vulcanised rubber is added to the elastomer before addition of the magnetisable material.",
"58.The method according to claim 30, wherein the vulcanised rubber is added to the elastomer at substantially the same time as the magnetisable material.",
"59.The method according to claim 44, wherein the waste silica is from power station lakes.",
"60.The method according to claim 53, wherein the vulcanised rubber is added to the elastomer before addition of the magnetisable material and the filler.",
"61.The method according to claim 53, wherein the vulcanised rubber is added to the elastomer at substantially the same time as the magnetisable material and the filler."
],
[
"The present invention is concerned with an attenuation material and a method of manufacture of such a material.",
"It is a general desire to improve the environment through noise reduction.",
"Noise pollution is an environmental problem in many industries.",
"For example noise is a problem in military vehicles, consumer vehicles, factories, and also in communal buildings.",
"It is therefore an aim of the present invention to alleviate some of the problems identified above.",
"It is a further aim of the present invention to provide an attenuation material which can be used, for example, for acoustic insulation and noise reduction, or for reduction of interference of radio frequency (RF) waves.",
"It is a further aim of the present invention to provide a method of making an attenuation material which can be used, for example, for noise reduction and acoustic insulation, or for reduction of interference of radio frequency (RF) waves.",
"It is a further aim of the present invention to provide a laminated panel suitable for use as an attenuation panel.",
"It is yet a further aim of the present invention to provide an attenuation material which is fire resistant.",
"Therefore, according to a first aspect of the present invention, there is provided an attenuation material which includes a matrix of elastomer having mixed therewith particulate vulcanised rubber and particulate magnetisable material, the mixture being substantially free of sublimable foaming agents.",
"Advantageously, the mixture further includes a filler material, such as particulate carbon black, clay or limestone or finely divided paper.",
"However, particulate silica is preferred.",
"It is preferred that the filler material, such as silica, is in finely divided form, preferably having a particle size in the range of about 50 to 500 microns.",
"A preferred particle size is about 120 microns.",
"When silica is used, it may advantageously be waste silica, such as, for example, waste silica from skimmings from power station lakes.",
"The magnetisable material and filler are preferably in a ratio of about 4 to 1 by weight.",
"The, magnetisable material may be a ferrous material, which may, in some embodiments, be coated with copper; this embodiment is particularly preferred when the material is to be used in a Faraday screen.",
"The magnetisable material for use according to the invention may be mill scale (which is preferred), any ferrous oxide material (such as Fe3O4) or copper silicate.",
"Typically, the particulate magnetisable material is in finely divided form, preferably having a particle size in the range of about 25 to 400 micron.",
"Further preferably, the particulate magnetisable material has a particle size of about 300 micron.",
"The particulate vulcanised rubber is preferably present in the form of discrete particles.",
"The vulcanised rubber should therefore be such that it is substantially free of reaction with the elastomer when the latter is cured.",
"It is preferred that the particulate vulcanised rubber is obtained cryogenically, by comminution of waste rubber compounds (for example, from waste tyres or the like).",
"Such waste rubber compounds generally include natural rubber and/or SBR.",
"The vulcanised rubber may advantageously include comminuted rubber tyres such as waste or worn tyres.",
"Disposal of waste or worn tyres would previously be in landfill sites.",
"Therefore, according to a further aspect of the present invention there is provided a use for waste tyres.",
"The vulcanised rubber is preferably in finely divided form.",
"It is further preferred that the vulcanised rubber has a particle size of less than about 500 microns, further preferably less than about 120 microns.",
"Typically, the attenuation material further includes at least one fire retardant material.",
"Preferably, the attenuation material further includes an adhesion enhancing material such as neoalkoxy zirconate, which is present so as to promote adhesion of the attenuation material to a number of materials (for example, steel, high performance polymers such as polyamides, PET and aluminium).",
"It is further preferred that the neoalkoxy zirconate is added in the ratio of about 1 to 10 parts per 100 of the elastomer, by weight.",
"A preferred ratio is 5 parts per 100 of the elastomer, by weight.",
"It is preferred that the matrix of elastomer includes at least one of the following batch ingredients: KELTAN (a Product of DSM-NV Ethylene Propylene Rubber) SPHERON 5000A Medium Fine Carbon Black Zinc Oxide Active Stearic Acid PEG 4000 (polyethylene glycol soap) Britomya BSH 20 (plasticiser) Flexon 815 Liquid (plasticiser) KEZADOL GR (processing aid) RHENOGRAN 580 Sulphur (vulcanising agent) RHENOGRAN TMTD 80 Tetramethylthiuram Disulphide (vulcanising agent) RHENOGRAN MBT 80 Mercaptobenzothiazole (vulcanising agent) RHENOGRAN MBTS 80 Benzothiazyl Disulphide (vulcanising agent) Preferred solid mixes for use according to the invention are set out in the following Table 1.TABLE 1 Matrix A Matrix B Matrix C Matrix D Keltan 314 2.4000 2.4000 2.4000 2.700 Keltan 4903 9.6000 9.6000 9.6000 10.6000 Zinc Oxide Active 0.3500 0.3500 0.3500 0.3900 Stearic Acid 1800 0.1200 0.1200 0.1200 0.1300 PEG 4000 0.1200 0.1200 0.1200 0.1300 TE 80 Powder 0.2000 — — — STUKTOL W816 0.2000 — — — Baco Superfine 7 14.8000 — — — Spheron 5000A 2.0000 16.8000 16.8000 18.7000 Britomya BSH 20 4.8000 4.8000 5.3000 Flexon 815 Liquid 8.8000 8.8000 8.800 9.800 KEZADOL GR 0.5000 0.5000 0.500 0.5600 Rhenogran CTP80 (PVI) 0.0900 0.0900 0.090 0.1000 RHENOGRAN 580 0.1100 0.1100 0.1100 0.1200 RHENOGRAN 0.8800 0.0800 0.0800 0.0900 TMTD 80 RHENOGRAN MBT 80 0.0270 0.0270 0.0270 0.0300 RHENOGRAN 0.0900 0.0900 0.0900 0.1000 MBTS 80 XETAL Filler A 13.0000 13.0000 13.000 9.7000 Vulcanised Rubber XETAL Filler B Mill 21.6000 30.3000 13.00 9.7000 scale, copper silicate or Fe3O4 Total Weight (kg) 78.8871 87.1871 69.8871 68.1500 The elastomer is preferably of a type different to that normally employed in tyres, the latter (as mentioned above generally including natural rubber and/or SBR).",
"It is particularly preferred that the elastomer should be a polyolefin type rubber, such as an ethylene-propylene rubber or EPDM.",
"In a preferred embodiment, the matrix of elastomer, the vulcanised rubber, mill scale or silica or Fe3O4 are preferably present in the following ratios: Mill Scale/ Matrix of Vulcanised Copper Silicate/ Elastomer Rubber Fe3O4 100 20 20 100 25 25 100 30 30 100 35 35 100 40 40 100 45 45 100 50 50 According to a second aspect of the present invention there is provided a method of manufacturing a material suitable for use as an attenuation material, which method includes: a) providing a mixture comprising a matrix of elastomer having mixed therewith particulate vulcanised rubber and particulate magnetisable material, the mixture being substantially free of sublimable foaming agents; and b) forming the mixture into at least one sheet.",
"The solid ingredients (including at least the magnetisable material and vulcanised rubber, and filler material, when present), may be mixed together prior to further mixing with the elastomer, prior to curing or vulcanisation of the latter.",
"The latter further mixing is typically carried out in a mixer such as a Banbury rotor type mixer.",
"The rotor speed of the mixer is typically in the range of 60 to 100 rpm, preferably about 80 rpm.",
"The particulate vulcanised rubber may be added to the elastomer before addition of the other solid ingredients, or with the other solid ingredients.",
"Typically the mixing of the solid ingredients with the elastomer is for about 4 to 12 minutes, preferably for about 8 minutes; such mixing is typically at a temperature of about 80° C. to 110° C. The magnetisable material and the filler may be mixed prior to addition to the elastomer material in step b).",
"The sheet formed in step b) is typically permitted to cure.",
"The sheet obtained in step b) may be formed by passing the mixture produced in step a) through a dump mill, then onto a sheeting mill and finally onto a three bowl calender for sheeting off.",
"Advantageously, the thickness of the sheets may be varied.",
"A preferred thickness of the sheets is from about 0.2 mm up to about 6 mm, when in an unexpanded form.",
"The method typically includes a further step c) after step b) whereby the mixture is cured.",
"The attenuation level of the material may depend on the thickness of the resulting sheet.",
"The sheets may be used for a wide variety of uses.",
"Typical uses include in buildings (traditionally built buildings with cavity space and roof trusses can be lined with the material according to the present invention), refitting of buildings (for example, existing buildings that have changed uses to pubs, clubs, discos etc), building door linings, blinds, in road, rail or air transport (for example as a lining material inside cabins, carriages, tunnels and barriers on roads) and in general household and industrial machinery.",
"Advantageously, the method may further include the step of curing the elastomer matrix after step b).",
"The material may be cured in a number of ways, for example, in line ovens or microwave ovens, however an autovac oven is preferred.",
"It is preferred that curing is at a temperature in the range of 155° C. to 165° C. A lower temperature range will permit curing of the elastomer matrix; however, this will incur a longer cycle time.",
"Advantageously, the sheet formed in step (b) may be laminated to paperboard, plasterboard, woven fabrics or the like.",
"According to a further embodiment of the present invention, it is preferred that at least one fire retardant is added to the material prior to step b).",
"According to yet a further embodiment of the present invention, it is preferred that the material is magnetised to provide hysteresis characteristics.",
"The material is typically magnetised after step b).",
"Advantageously a magnetic screen created around the material provides attenuation of electromagnetic induction and radio frequency (RF) waves.",
"The screen is silk screen printed inductive ink with apertures to screen RF frequencies.",
"According to yet a further embodiment of the present invention, an adhesion enhancing agent such as neoalkoxy zirconate may be added to the mixture prior to or during step (b) so as to promote adhesion of the material to a number of materials (for example, steel, high performance polymers such as polyamides, PET and aluminium).",
"It is preferred that the adhesion enhancing agent is added in the ratio of about 1 to 10 (preferably 5) parts per 100, by weight of the material made.",
"According to a further aspect of the present invention, there is provided laminate which includes attenuation material according to the first aspect of the present invention, having bonded thereto a second material such as, for example, steel, high performance polymers such as polyamides, PET and aluminium."
]
] | Patent_10240298 |
[
[
"Organic field emission device and emission device",
"The present invention provides an organic electroluminescence light emitting device capable of preventing a color (hue) of light and a luminous efficiency from being varied depending on a concentration of a luminous material contained in a light emission layer and an operational condition such as an applied voltage, thereby exhibiting a high luminance, a high performance, and a stable, reliability.",
"The light emitting device includes an anode (6), a hole transport layer (2), a light emission layer (3), an electron transport layer (4), or includes an anode (6), a hole transport layer (2), and a light emission layer (4) serving as an electron transport layer.",
"A light emission region is formed by a mixed layer made from a luminescent material and a charge injection accelerating material.",
"The luminescent material exhibits, in a state held as a single thin film between the anode (6) and the cathode (7), electroluminescence light emission when a DC voltage is applied thereto and has a charge transport characteristic.",
"The charge injection accelerating material is different from the luminous material and has a charge transport characteristic capable of accelerating injection of charges in the luminescent material.",
"The light emission region exists not only at an interface with an adjacent layer or its vicinity but also over a specific thickness region in the layer thickness direction."
],
[
"1.An organic electroluminescence light emitting device, in which an organic layer having a light emission region is provided between an anode and a cathode, characterized in that at least one of layers constituting said organic layer is composed of a mixed layer made from a luminescent material and a charge injection accelerating material, wherein said luminescent material exhibits, in a state held as a single thin film between said anode and said cathode, electroluminescence light emission when a voltage is applied thereto and has a charge transport characteristic, and said charge injection accelerating material has a charge transport characteristic capable of accelerating injection of charges in said luminescent material; and said at least one of layers constituting said organic layer has a light emission region existing not only at an interface with an adjacent layer or its vicinity but also over a specific thickness region from said interface or its vicinity in the layer thickness direction.",
"2.An organic electroluminescence light emitting device according to claim 1, wherein said light emission region exists over the whole thickness of said mixed layer.",
"3.An organic electroluminescence light emitting device according to claim 2, wherein on the basis of an intensity of light emitted from each or both of an interface between said mixed layer and an electron transport layer adjacent thereto and an interface between said mixed layer and a hole transport layer adjacent thereto, an intensity of light emitted from a position being equidistant from said both interfaces is in a range of 25% or more.",
"4.An organic electroluminescence light emitting device according to claim 1, wherein a concentration range of said luminous material on the basis of a mole number of said charge injection accelerating material in said mixed layer is in a range of 5 to 90 mole percent.",
"5.An organic electroluminescence light emitting device according to claim 1, wherein when said luminous material is configured as a luminous material having at least an electron transport characteristic, said charge injection accelerating material is configured as a charge injection accelerating material having an electron and/or hole transport characteristic.",
"6.An organic electroluminescence light emitting device according to claim 1, wherein when said luminous material is configured as a luminous material having at least a hole transport characteristic, said charge injection accelerating material is configured as a charge injection accelerating material having a hole and/or electron transport characteristic.",
"7.An organic electroluminescence light emitting device according to claim 1, wherein an energy level of a highest occupied molecular orbital of said charge injection accelerating material is equal to or deeper than that of said luminous material, and/or an energy level of a lowest unoccupied molecular orbital of said charge injection accelerating material is equal to or shallower than that of said luminous material.",
"8.An organic electroluminescence light emitting device according to claim 7, wherein an energy level of a lowest unoccupied molecular orbital of a hole transport layer adjacent to said mixed layer is shallower than that of each of said luminous material and said charge injection accelerating material.",
"9.An organic electroluminescence light emitting device according to claim 7, wherein the same material as an electron transport material forming an electron transport layer adjacent to said mixed layer is used as said charge injection accelerating material.",
"10.An organic electroluminescence light emitting material according to claim 7, wherein the same material as a hole transport material forming a hole transport layer adjacent to said mixed layer is used as said charge injection accelerating material.",
"11.An organic electroluminescence material according to claim 7, wherein both the same material as an electron transport material forming an electron transport layer adjacent to said mixed layer and the same material as a hole transport material forming a hole transport layer adjacent to said mixed layer are used as said charge injection accelerating material.",
"12.A light emitting apparatus using an organic electroluminescence light emitting device described in any one of claims 1 to 11.13.A light emitting apparatus according to claim 12, wherein said light emitting apparatus is configured as a display in which said organic electroluminescence light emitting device is used as at least part of a pixel."
],
[
"<SOH> BACKGROUND ART <EOH>Displays have a major role in our daily living, for example, in the forms of television receivers, computer monitors, and portable information terminals.",
"With advance of the Internet, displays as human interfaces have become increasingly important.",
"These displays have been required to have a screen being comfortable for eyes to see and ensuring a definition high enough to allow clear viewing, and to have a resolution and a responsiveness high enough to allow clear, clean viewing of moving pictures without delay.",
"Organic EL devices using organic compounds as luminescent materials exhibit a wide viewing angle, a high contrast, and an excellent visibility.",
"Another advantage of organic EL devices is that since the devices emit spontaneous light, they do not require any backlight unlike liquid crystal, to thereby realize thinning of the size and reduction in weight, and also realize reduction in power consumption.",
"Organic EL devices using organic compounds as luminescent materials are further advantageous in that they are operable with a low DC voltage at a high response speed, have a resistance against vibration, and are usable in a wide range of temperatures.",
"Organic EL devices, therefore, have become a focus of attention as the next generation display devices, and some of them have already begun to be put into market.",
"Organic electroluminescence light emitting devices using organic luminescent materials have a configuration that an organic electroluminescence layer containing an organic luminescent material is sandwiched between an anode and a cathode, wherein at least one of the anode and the cathode has a light permeability.",
"In these devices, light emission occurs when a DC voltage is applied between the anode and the cathode.",
"FIGS.",
"10 and 12 show examples of related art organic electroluminescence light emitting devices (organic EL devices).",
"FIG.",
"10 shows a related art organic electroluminescence light emitting device having a single-hetero structure, wherein an anode 6 made from a light permeable ITO (Indium Tin Oxide) or the like, an organic layer 15 a composed of a hole transport layer 2 and an electron transport layer 4 , and a cathode 7 are sequentially stacked on a substrate 10 made from light permeable glass or the like, and the stacked structure thus formed on the substrate 10 is sealed with a protective layer 14 .",
"In this device, when a DC voltage supplied from a power source 20 is applied between the anode 6 and the cathode 7 , light 5 having a specific wavelength is emitted from an interface between the hole transport layer 2 and the electron transport layer 4 .",
"FIG.",
"11 shows another organic electroluminescence light emitting device having a double-hetero structure, wherein a light permeable anode 6 , an organic layer 15 b composed of a hole injection layer 1 , a hole transport layer 2 , a light emission layer 3 , and an electron transport layer 4 , and a cathode 7 are sequentially stacked on a light permeable substrate 10 , and the stacked structure formed on the substrate 10 is sealed with a protective layer 14 .",
"It is to be noted that the hole injection layer is not necessarily provided.",
"In this case, when a DC voltage supplied from a power source 20 is applied between the anode 6 and the cathode 7 , holes injected from the anode 6 reach the light emission layer 3 via the hole transport layer 2 , and electrons injected from the cathode 7 reach the light emission layer 3 via the electron transport layer 4 .",
"As a result, hole-electron recombination occurs in the light emission layer 3 , to produce single exciton, from which light 5 having a specific wavelength is emitted.",
"FIG.",
"12 is a view showing a configuration of a flat display using the above-described organic electroluminescence light emitting device.",
"As shown in this figure, for a full-color display, an organic layer 15 ( 15 a , 15 b ) allowing emission of light of three primary colors of red (R), green (G), and blue (B) is disposed between a cathode 7 and an anode 6 .",
"In general, the cathode 7 is composed of cathode stripes 7 , and the anode 6 is composed of anode stripes 6 , wherein the cathode stripes 7 and the anode stripes 6 are arranged to cross each other.",
"A signal voltage is selectively applied from a luminance signal circuit 24 to one of the cathode stripes 7 and a signal voltage is selectively applied from a shift register integrated control circuit 25 to one of the anode stripes 6 , whereby a portion (pixel) of the organic layer, located at a position where the selected cathode stripe 7 and the selected anode stripe 6 cross each other, emits light.",
"The light emission layer can be made from one kind or two or more kinds of materials selected from various materials.",
"As one example of a device structure, for example, of a type shown in FIG.",
"10 , characterized by including a light emission layer made from two or more kinds of materials, a two-layer structure having an electron transport layer 4 containing a luminescent material so as to serve as a light emission layer and a hole transport layer 2 has been reported by C. W. Tang, S. A. VanSlyke, and C. H. Chen in J. of Appl.",
"Phys.",
"65-9, 3610-3616 (1989).",
"This device structure has been also disclosed in Japanese Patent Laid-open No.",
"Sho 63-264692.This known technique is intended to provide an electroluminescence light emitting device capable of obtaining an optical output in a wider wavelength range at a lower applied voltage and exhibiting a high stability.",
"In this device, as a mean for realizing such characteristics, a light emission layer is composed of an organic host material capable of sustaining injection of both holes and electrons, and a fluorescent material capable of emitting light in response to hole-electron recombination.",
"The hue of light emitted from the light emission layer is modified by doping a slight amount of such a fluorescent material in the light emission layer.",
"The minimum amount, being enough to achieve the above-described effect, of the fluorescent material varies depending on specific selection of the host material and the fluorescent material; however, according to the above-described known technique, it is described in the specification of the patent document that in any case, it is not required to use the fluorescent material in an amount of about 10 mole percent or more on the basis of the mole number of the host material, and more specifically, it is seldom required to use the fluorescent material in an amount of 1 mole percent or more.",
"In the specification of the patent document, a hue as a function of a concentration of a fluorescent material is shown in Table II which summarizes the results of Examples 7-13.In this table, it is shown that assuming that a luminous efficiency of an EL device including a light emission layer containing no fluorescent material is taken as 1.0, a luminous efficiency of the same EL device as that described above except that the light emission layer containing 4.4 mole percent of a fluorescent material exemplified by 4-(dicyanomethylene)-2-methyl-6-[2-(9-julolidyl)ethenyl]-4H-thiopyran is reduced to 0.14.It is also shown that the emission wavelength (535 nm) of the EL device including the light emission layer containing no fluorescent material becomes longer with the increase in concentration of the fluorescent material, and the emission wavelength of the EL device reaches 690 nm when the concentration of the fluorescent material is 4 mole percent.",
"As compared with an EL device including a light emission layer made from a single luminescent material, the EL device produced by the above-described cited technique has some advantages.",
"One of the advantages is to easily change the emission wavelength.",
"For the EL device including the light emission layer made from a single luminescent material, in order to change the emission color, it is required to change a chemical structure of the luminescent material, and therefore, in order to introduce each substitutional group for changing the emission wavelength, it is required to synthesize a new material.",
"On the contrary, according to the known technique, the emission wavelength can be changed by doping a slight amount of a fluorescent material in the host material.",
"Another advantage is that a fluorescent material having neither film formability nor charge transport characteristic can be used.",
"The known technique, however, has important problems from the viewpoint of practical use.",
"One of the problems is that since the hue and the luminous efficiency are greatly varied depending on a variation in concentration of the fluorescent material as described in the specification of the patent document, it is difficult to perform quality control for keeping the characteristics in the production process.",
"Another problem is that since the hue is varied depending on an applied voltage or current, it is difficult to stably control the hue in the case where the device is applied as an element of a display.",
"In the device having the structure shown in FIG.",
"10 or 11 , holes and electrons are concentrated at an interface between the organic layers 2 and 4 or its vicinity, or concentrated at an interface between the organic layers 2 and 3 and its vicinity, or an interface between the organic layers 3 and 4 and its vicinity, and consequently, light emission occurs at the interface or its vicinity, causing deterioration of the device, thereby shortening the service life of the device.",
"In other words, the device having the above structure is poor in reliability.",
"To be more specific, for the device having the structure shown in FIG.",
"11 , if the light emission layer 3 is made from a single material having an electron transport characteristic, as shown in FIG.",
"13 illustrating energy levels of the layers constituting the device, a region (light emission region) in which hole-electron recombination efficiently occurs is concentrated in the vicinity of an interface, adjacent to a hole transport layer, of the light emission layer; and if the light emission layer 3 is made from a single material having a hole transport characteristic, as shown in FIG.",
"14 illustrating energy levels of layers constituting the device, a light emission region is concentrated in the vicinity of an interface, adjacent to an electron transport layer, of the light emission layer.",
"Taking into account the above-described technical background, the present invention has been made, and an object of the present invention is to provide an organic electroluminescence light emitting device capable of preventing a color (hue) of light and a luminous efficiency from being varied depending on a concentration of a luminous material contained in a light emission layer and an operational condition such as an applied voltage, thereby exhibiting a high luminance, a high performance, and a stable, high reliability."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.",
"1 is a diagram showing an energy level of each layer and a mechanism of light emission for an organic electroluminescence light emitting device of the present invention; FIG.",
"2 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for another organic electroluminescence light emitting device of the present invention; FIG.",
"3 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for a further organic electroluminescence light emitting device of the present invention; FIG.",
"4 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for still a further organic electroluminescence light emitting device of the present invention; FIG.",
"5 is a diagram showing an emission spectrum of a comparative organic electroluminescence light emitting device; FIG.",
"6 is a diagram showing an emission spectrum of an organic electroluminescence light emitting device of the present invention; FIG.",
"7 is a schematic sectional view of an organic electroluminescence light emitting device used for measuring a light emission region; FIG.",
"8 is a spectral diagram showing a relationship between an actually measured emission spectrum and an emission spectrum estimated from a light emission distribution in a light emission layer; FIG.",
"9 is a spectral diagram showing a light emission distribution in a light emission layer; FIG.",
"10 is a schematic sectional view of an essential portion of a related art organic electroluminescence light emitting device; FIG.",
"11 is a schematic sectional view of an essential portion of another related art organic electroluminescence light emitting device; FIG.",
"12 is a view showing a configuration of a full-color flat display using related art organic electroluminescence light emitting devices; FIG.",
"13 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for a related art organic electroluminescence light emitting device; and FIG.",
"14 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for another related art organic electroluminescence light emitting device.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to an organic electroluminescence light emitting device (organic EL device) including an organic layer having a light emitting region between an anode and a cathode, and to a light emitting apparatus, such as a display device, using the same.",
"BACKGROUND ART Displays have a major role in our daily living, for example, in the forms of television receivers, computer monitors, and portable information terminals.",
"With advance of the Internet, displays as human interfaces have become increasingly important.",
"These displays have been required to have a screen being comfortable for eyes to see and ensuring a definition high enough to allow clear viewing, and to have a resolution and a responsiveness high enough to allow clear, clean viewing of moving pictures without delay.",
"Organic EL devices using organic compounds as luminescent materials exhibit a wide viewing angle, a high contrast, and an excellent visibility.",
"Another advantage of organic EL devices is that since the devices emit spontaneous light, they do not require any backlight unlike liquid crystal, to thereby realize thinning of the size and reduction in weight, and also realize reduction in power consumption.",
"Organic EL devices using organic compounds as luminescent materials are further advantageous in that they are operable with a low DC voltage at a high response speed, have a resistance against vibration, and are usable in a wide range of temperatures.",
"Organic EL devices, therefore, have become a focus of attention as the next generation display devices, and some of them have already begun to be put into market.",
"Organic electroluminescence light emitting devices using organic luminescent materials have a configuration that an organic electroluminescence layer containing an organic luminescent material is sandwiched between an anode and a cathode, wherein at least one of the anode and the cathode has a light permeability.",
"In these devices, light emission occurs when a DC voltage is applied between the anode and the cathode.",
"FIGS.",
"10 and 12 show examples of related art organic electroluminescence light emitting devices (organic EL devices).",
"FIG.",
"10 shows a related art organic electroluminescence light emitting device having a single-hetero structure, wherein an anode 6 made from a light permeable ITO (Indium Tin Oxide) or the like, an organic layer 15a composed of a hole transport layer 2 and an electron transport layer 4, and a cathode 7 are sequentially stacked on a substrate 10 made from light permeable glass or the like, and the stacked structure thus formed on the substrate 10 is sealed with a protective layer 14.In this device, when a DC voltage supplied from a power source 20 is applied between the anode 6 and the cathode 7, light 5 having a specific wavelength is emitted from an interface between the hole transport layer 2 and the electron transport layer 4.FIG.",
"11 shows another organic electroluminescence light emitting device having a double-hetero structure, wherein a light permeable anode 6, an organic layer 15b composed of a hole injection layer 1, a hole transport layer 2, a light emission layer 3, and an electron transport layer 4, and a cathode 7 are sequentially stacked on a light permeable substrate 10, and the stacked structure formed on the substrate 10 is sealed with a protective layer 14.It is to be noted that the hole injection layer is not necessarily provided.",
"In this case, when a DC voltage supplied from a power source 20 is applied between the anode 6 and the cathode 7, holes injected from the anode 6 reach the light emission layer 3 via the hole transport layer 2, and electrons injected from the cathode 7 reach the light emission layer 3 via the electron transport layer 4.As a result, hole-electron recombination occurs in the light emission layer 3, to produce single exciton, from which light 5 having a specific wavelength is emitted.",
"FIG.",
"12 is a view showing a configuration of a flat display using the above-described organic electroluminescence light emitting device.",
"As shown in this figure, for a full-color display, an organic layer 15 (15a, 15b) allowing emission of light of three primary colors of red (R), green (G), and blue (B) is disposed between a cathode 7 and an anode 6.In general, the cathode 7 is composed of cathode stripes 7, and the anode 6 is composed of anode stripes 6, wherein the cathode stripes 7 and the anode stripes 6 are arranged to cross each other.",
"A signal voltage is selectively applied from a luminance signal circuit 24 to one of the cathode stripes 7 and a signal voltage is selectively applied from a shift register integrated control circuit 25 to one of the anode stripes 6, whereby a portion (pixel) of the organic layer, located at a position where the selected cathode stripe 7 and the selected anode stripe 6 cross each other, emits light.",
"The light emission layer can be made from one kind or two or more kinds of materials selected from various materials.",
"As one example of a device structure, for example, of a type shown in FIG.",
"10, characterized by including a light emission layer made from two or more kinds of materials, a two-layer structure having an electron transport layer 4 containing a luminescent material so as to serve as a light emission layer and a hole transport layer 2 has been reported by C. W. Tang, S. A. VanSlyke, and C. H. Chen in J. of Appl.",
"Phys.",
"65-9, 3610-3616 (1989).",
"This device structure has been also disclosed in Japanese Patent Laid-open No.",
"Sho 63-264692.This known technique is intended to provide an electroluminescence light emitting device capable of obtaining an optical output in a wider wavelength range at a lower applied voltage and exhibiting a high stability.",
"In this device, as a mean for realizing such characteristics, a light emission layer is composed of an organic host material capable of sustaining injection of both holes and electrons, and a fluorescent material capable of emitting light in response to hole-electron recombination.",
"The hue of light emitted from the light emission layer is modified by doping a slight amount of such a fluorescent material in the light emission layer.",
"The minimum amount, being enough to achieve the above-described effect, of the fluorescent material varies depending on specific selection of the host material and the fluorescent material; however, according to the above-described known technique, it is described in the specification of the patent document that in any case, it is not required to use the fluorescent material in an amount of about 10 mole percent or more on the basis of the mole number of the host material, and more specifically, it is seldom required to use the fluorescent material in an amount of 1 mole percent or more.",
"In the specification of the patent document, a hue as a function of a concentration of a fluorescent material is shown in Table II which summarizes the results of Examples 7-13.In this table, it is shown that assuming that a luminous efficiency of an EL device including a light emission layer containing no fluorescent material is taken as 1.0, a luminous efficiency of the same EL device as that described above except that the light emission layer containing 4.4 mole percent of a fluorescent material exemplified by 4-(dicyanomethylene)-2-methyl-6-[2-(9-julolidyl)ethenyl]-4H-thiopyran is reduced to 0.14.It is also shown that the emission wavelength (535 nm) of the EL device including the light emission layer containing no fluorescent material becomes longer with the increase in concentration of the fluorescent material, and the emission wavelength of the EL device reaches 690 nm when the concentration of the fluorescent material is 4 mole percent.",
"As compared with an EL device including a light emission layer made from a single luminescent material, the EL device produced by the above-described cited technique has some advantages.",
"One of the advantages is to easily change the emission wavelength.",
"For the EL device including the light emission layer made from a single luminescent material, in order to change the emission color, it is required to change a chemical structure of the luminescent material, and therefore, in order to introduce each substitutional group for changing the emission wavelength, it is required to synthesize a new material.",
"On the contrary, according to the known technique, the emission wavelength can be changed by doping a slight amount of a fluorescent material in the host material.",
"Another advantage is that a fluorescent material having neither film formability nor charge transport characteristic can be used.",
"The known technique, however, has important problems from the viewpoint of practical use.",
"One of the problems is that since the hue and the luminous efficiency are greatly varied depending on a variation in concentration of the fluorescent material as described in the specification of the patent document, it is difficult to perform quality control for keeping the characteristics in the production process.",
"Another problem is that since the hue is varied depending on an applied voltage or current, it is difficult to stably control the hue in the case where the device is applied as an element of a display.",
"In the device having the structure shown in FIG.",
"10 or 11, holes and electrons are concentrated at an interface between the organic layers 2 and 4 or its vicinity, or concentrated at an interface between the organic layers 2 and 3 and its vicinity, or an interface between the organic layers 3 and 4 and its vicinity, and consequently, light emission occurs at the interface or its vicinity, causing deterioration of the device, thereby shortening the service life of the device.",
"In other words, the device having the above structure is poor in reliability.",
"To be more specific, for the device having the structure shown in FIG.",
"11, if the light emission layer 3 is made from a single material having an electron transport characteristic, as shown in FIG.",
"13 illustrating energy levels of the layers constituting the device, a region (light emission region) in which hole-electron recombination efficiently occurs is concentrated in the vicinity of an interface, adjacent to a hole transport layer, of the light emission layer; and if the light emission layer 3 is made from a single material having a hole transport characteristic, as shown in FIG.",
"14 illustrating energy levels of layers constituting the device, a light emission region is concentrated in the vicinity of an interface, adjacent to an electron transport layer, of the light emission layer.",
"Taking into account the above-described technical background, the present invention has been made, and an object of the present invention is to provide an organic electroluminescence light emitting device capable of preventing a color (hue) of light and a luminous efficiency from being varied depending on a concentration of a luminous material contained in a light emission layer and an operational condition such as an applied voltage, thereby exhibiting a high luminance, a high performance, and a stable, high reliability.",
"DISCLOSURE OF INVENTION The present invention has been made to provide a new, effective configuration of a light emission layer in order to achieve the above object, and according to the present invention, there is provided an organic electroluminescence light emitting device, in which an organic layer having a light emission region is provided between an anode and a cathode, characterized in that at least one of layers constituting the organic layer is composed of a mixed layer made from a luminescent material and a charge injection accelerating material, wherein the luminescent material exhibits, in a state held as a single thin film between the anode and the cathode, electroluminescence light emission when a voltage is applied thereto and has a charge transport characteristic, and the charge injection accelerating material has a charge transport characteristic capable of accelerating injection of charges in the luminescent material; and the at least one of layers constituting the organic layer has a light emission region existing not only at an interface with an adjacent layer or its vicinity but also over a specific thickness region from the interface or its vicinity in the layer thickness direction.",
"The present invention also provides a light emitting apparatus using such an organic electroluminescence light emitting device.",
"The organic electroluminescence light emitting device of the present invention typically includes an anode, a hole transport layer, a light emission layer, an electron transport layer, or includes an anode, a hole transport layer, and a light emission layer serving as an electron transport layer.",
"A light emission region of the device is formed by a mixed layer made from a luminescent material and a charge injection accelerating material.",
"The luminescent material exhibits, in a state held as a single thin film between the anode and the cathode, electroluminescence light emission when a DC voltage is applied thereto and has a charge transport characteristic.",
"The charge injection accelerating material is different from the luminous material and has a charge transport characteristic capable of accelerating injection of charges in the luminescent material.",
"The light emission region exists not only at an interface with an adjacent layer or its vicinity but also over a specific thickness region in the layer thickness direction.",
"Accordingly, a region in which light is emitted is not limited to the above-described interface or its vicinity but can be spread in a wide range of the light emission layer.",
"This means that even if the performance of a portion located in the layer thickness direction is deteriorated, light emission occurs a portion above or below the deteriorated portion, so that light emission occurs substantially over a specific thickness region in the light emission layer.",
"As a result, it is possible to significantly improve the service life of the device.",
"The luminous material used for the above-described known technique (Japanese Patent Laid-open No.",
"Sho 63-264692) exhibits light emission resulting from fluorescence.",
"On the other hand, the luminous material used for the present invention exhibits, in a state held as a single thin film between an anode and a cathode, electroluminescence light emission by itself when a DC voltage is applied thereto.",
"In other words, the luminous material used for the present invention has not only electroluminescence light emission characteristic but also a charge transport characteristic, and therefore, does not require a material equivalent to the host material (used in the known technique) for keeping injection of holes and electrons in the light emission layer, and further the luminous material does not emit light in response to hole-electron recombination but emits light by performing hole-electron recombination by itself.",
"Accordingly, a hue and a luminous efficiency of the device of the present invention is not varied so much depending on a concentration of the luminous material, with a result that the device of the present invention can obtain a stable hue.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 is a diagram showing an energy level of each layer and a mechanism of light emission for an organic electroluminescence light emitting device of the present invention; FIG.",
"2 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for another organic electroluminescence light emitting device of the present invention; FIG.",
"3 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for a further organic electroluminescence light emitting device of the present invention; FIG.",
"4 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for still a further organic electroluminescence light emitting device of the present invention; FIG.",
"5 is a diagram showing an emission spectrum of a comparative organic electroluminescence light emitting device; FIG.",
"6 is a diagram showing an emission spectrum of an organic electroluminescence light emitting device of the present invention; FIG.",
"7 is a schematic sectional view of an organic electroluminescence light emitting device used for measuring a light emission region; FIG.",
"8 is a spectral diagram showing a relationship between an actually measured emission spectrum and an emission spectrum estimated from a light emission distribution in a light emission layer; FIG.",
"9 is a spectral diagram showing a light emission distribution in a light emission layer; FIG.",
"10 is a schematic sectional view of an essential portion of a related art organic electroluminescence light emitting device; FIG.",
"11 is a schematic sectional view of an essential portion of another related art organic electroluminescence light emitting device; FIG.",
"12 is a view showing a configuration of a full-color flat display using related art organic electroluminescence light emitting devices; FIG.",
"13 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for a related art organic electroluminescence light emitting device; and FIG.",
"14 is a schematic diagram showing an energy level of each layer and a mechanism of light emission for another related art organic electroluminescence light emitting device.",
"BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention; the above-described thickness region is preferably specified such that on the basis of an intensity of light emitted from each or both of an interface between the mixed layer and an electron transport layer adjacent thereto and an interface between the mixed layer and a hole transport layer adjacent thereto, an intensity of light emitted from a position being equidistant from the both interfaces is in a range of 25% or more.",
"A concentration range of the luminous material on the basis of a mole number of the charge injection accelerating material having a charge transport characteristic capable of accelerating injection of charges in the luminous material in the light emission layer is preferably in a range of 5 to 90 mole percent (about 5 to 90 wt %), more preferably, in a range of 10 to 90 mole percent (about 10 to 90 wt %).",
"On the contrary, according to the above-described known technique (Japanese Patent Laid-open No.",
"Sho 63-264692), it is described that the minimum ratio of the fluorescent material is varied depending on the specific selection of the host material and the fluorescent material; however, in any case, it is not required to use the fluorescent material in an amount of about 10 mole percent or more on the basis of the mole number of the host material, and more specifically, it is seldom required to use the fluorescent material in an amount of 1 mole percent or more.",
"Therefore, the ratio of the fluorescent material is substantially not overlapped to the preferred concentration range of the luminous material used for the present invention.",
"Further, according to the present invention, a hue of light is not varied depending on a change in the applicable concentration of the luminous material.",
"This is basically different from the known technique intended to positively change a hue of light by changing the concentration of the fluorescent material.",
"The charge injection accelerating material having a charge transport characteristic capable of accelerating injection of charges in the light emission layer for acceleration light emission due to hole-electron recombination in the luminous material is preferably specified as follows: (1) an energy level of a lowest unoccupied molecular orbital (LUMO) of the charge injection accelerating material is equal to or smaller (shallower) than that of the luminous material, or an energy level of a highest occupied molecular orbital (HOMO) of the charge injection accelerating material is equal to or larger (deeper) than that of the luminous material; or (2) the energy level of the lowest unoccupied molecular orbital (LUMO) of the charge injection accelerating material is equal to or smaller (shallower) than that of the luminous material, and the energy level of the highest occupied molecular orbital (HOMO) of the charge injection accelerating material is equal to or larger (deeper) than that of the luminous material.",
"If the above energy condition (1) is satisfied, the same material as a material forming the electron transport layer can be used as a material having the charge transport characteristic capable of accelerating injection of charges in the luminous material, and the same material as a material forming the hole transport layer can be used as a material having the charge transport characteristic capable of injection of charges in the luminous material.",
"If the above energy condition (2) is satisfied, both the same material as a material forming the electron transport layer and the same material as a material forming the hole transport layer can be used as the material having the charge transport characteristic capable of accelerating injection of charges in the luminous material.",
"Further, an energy level of the lowest unoccupied molecular orbital (LUMO) of the hole transport layer adjacent to the mixed layer may be shallower than that of each of the luminous material and the charge injection accelerating material.",
"The organic electroluminescence light emitting device of the present invention does not make use of a mechanism that either or both of electrons injected from the cathode and holes injected from the anode are concentrated at the interface between the organic layers, to cause hole-electron recombination, resulting in light emission, but make use of a mechanism that the luminous material efficiently acts as a trap for electrons and holes in the material having the charge transport characteristic in the light emission layer, to cause hole-electron recombination in the luminous material, resulting in light emission.",
"The organic electroluminescence light emitting device of the present invention is thus characterized in that the light emission region is not concentrated in the vicinity of the interface between the organic layers but can exist over a wide range in the light emission layer, so that as compared with the case where a luminous material singly exists in a light emission layer, it is possible to significantly improve the reliability of the device.",
"This means that according to the light emitting device of the present invention, it is possible to prevent occurrence of an inconvenience that the concentrated charge density in a light emission layer of a device accelerates deterioration of the device.",
"In the case (A) where a light emission layer 3 is made from a single material and the material has an electron transport characteristic as shown in FIG.",
"13, a region (light emission region) in which recombination 8 of holes and electrons efficiently occurs is concentrated in the vicinity of an interface of a portion in the light emission layer-3 with a hole transport layer 2.Meanwhile, in the case (B) where a light emission layer 3 is made from a single material and the material has a hole transport characteristic as shown in FIG.",
"14, a light emission region is concentrated in the vicinity of an interface of a portion in the light emission layer 3 with an electron transport layer 4.On the contrary, according to the present invention, in the case (C) where a light emission layer is formed by mixing a luminous material having an electron transport characteristic in a charge injection accelerating material 18 having an electron transport characteristic as shown in FIG.",
"1 and in the case (D) where a light emission layer is formed by mixing a luminous material having a hole transport characteristic in a charge injection accelerating material 18 having a hole transport characteristic as shown in FIG.",
"2, a region (light emission region) in which recombination 19 of holes and electrons efficiently occurs is somewhat spread inwardly from the interface into the light emission layer (within a specific thickness region in the layer thickness direction).",
"Further, in the case (E) where a light emission layer is formed by mixing a luminous material having a hole transport characteristic in a charge injection accelerating material 18 having an electron transport characteristic as shown in FIG.",
"3 and in the case (F) where a light emission layer is formed by mixing a luminous material having an electron transport characteristic in a charge injection accelerating material 18 having a hole transport characteristic as shown in FIG.",
"4, the density of electrons and holes is not concentrated only in the vicinity of an interface between the organic layers but is widely dispersed in the light emission layer (within a further enlarged thickness region), so that recombination 19 of holes and electrons occurs in the wide range dispersed in the light emission layer.",
"Accordingly, deterioration of the device does not occur at a concentrated portion in the light emission layer but occurs in a wide range in the light emission layer, with a result that the service life of the device is prolonged and thereby the reliability of the device is enhanced.",
"The distribution of a light emission region in the light emission layer is varied depending on a voltage and a current applied to the device and a luminance; however, at an effective available luminance necessary for a display or the like using the device, the light emission region in the light emission layer is preferably specified such that on the basis of an intensity of light emitted from each or both of an interface between the mixed layer and an electron transport layer adjacent thereto and an interface between the mixed layer and a hole transport layer adjacent thereto, an intensity of light emitted from a position being equidistant from the both interfaces, that is, from a central portion of the light emission layer is in a range of 25% or more.",
"By making effective use of the whole region of the light emission layer as the light emission region as described above, the reliability of the device can be significantly improved.",
"To improve the reliability of the device by not concentrating the light emission region only in the vicinity of the interface but widely dispersing the light emission region in the light emission layer as described above, in the above-described case (E), the charge injection accelerating material may have not only the electron transport characteristic but also the hole transport characteristic, and the luminous material may have not only the hole transport characteristic but also the electron transport characteristic.",
"Further, in the above-described case (F), the charge injecting accelerating material may have not only the hole transport characteristic but also the electron transport characteristic, and the luminous material may have not only the electron transport characteristic but also the hole transport characteristic.",
"In addition, the organic electroluminescence light emitting device of the present invention may be used as at least part of each of pixels of a display.",
"Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.",
"First, examples of device structures to each of which the organic electroluminescence light emitting device of the present invention is applied.",
"FIG.",
"10 shows the above-described device structure including the anode 6, the hole transport layer 2, the light emission layer 4 serving as an electron transport layer, and the cathode 7, and FIG.",
"11 shows the above-described device structure including the anode 6, the hole injection layer 1 (provided as needed), the hole transport layer 2, the light emission layer 3, the electron transport layer 4, and the cathode 7.The organic electroluminescence light emitting device of the present invention is applicable to each of these device structures; however, as shown in FIGS.",
"1 to 4, according to the present invention, a charge (electron or hole) injection accelerating material is mixed in the light emission layer.",
"When a voltage is applied between the anode 6 and the cathode 7 of the device having the above device structure, a current flows therebetween, to allow light emission from the light emission layer of the device.",
"A substrate 10 for supporting the organic electroluminescence light emitting device is in contact with the anode 6 or the cathode 7.A light permeable substrate made from glass, quartz, or plastic, a substrate having no light permeability in a visible light region such as silicon, or a substrate on the surface of which a thin film transistor circuit is formed is used in accordance with an application of the device.",
"Light emitted from the light emission layer 4 serving as an electron transport layer or the light emission layer 3 can be emerged to the outside by giving light permeability to at least one of the anode 6 and the cathode 7.The anode 6 may be made from ITO (Indium Tin Oxide), a metal such as SnO2, Cr, Pd, In, Au, W, or Ni, or an alloy containing one of the metals.",
"The cathode 7 may be made from an active metal such as Li, Mg or Ca, or an alloy containing Ag, Al, or In and one of the active metals.",
"In addition, the cathode 7 may be of a stacked structure of these metal or alloy layers.",
"It is to be noted that the above materials for the anode 6 and the cathode 7 are only for illustrative purposes only, and it is to be understood that other materials may be used in accordance with an application of the device.",
"Further, to improve the service life of the organic electroluminescence light emitting device, the device may be provided with a means for cutting off permeation of water or oxygen from external.",
"The hole injection layer 1 for accelerating injection of holes may be provided between the anode 6 and the hole transport layer 2.The hole injection layer 1 may be made from, for example, a porphyrin compound described in U.S. Pat.",
"No.",
"4,720,432.To facilitate injection of holes to the light emission layer serving as an electron transport layer or the light emission layer, the hole transport may be of not a single layer but a stacked structure.",
"Each of the electron transport layer and the hole transport layer may be made from a material suitably selected from known materials.",
"To facilitate injection of electrons from the cathode 7 to the light emission layer serving as an electron transport layer and the electron transport layer, a thin film made from LiF or LiO2 may be inserted.",
"Further, a hole block layer for preventing injection of holes from the light emission layer to the electron transport layer may be provided.",
"As a useful example of a luminous material contained in the light emission layer, there is known a material having, in its molecular skeleton, a styryl or distyryl structure described in Japanese Patent Laid-open Nos.",
"Hei 11-329730, Hei 11-329731, 2000-91076, 2000-173773, 2000-12224, 2000-12225, 2000-12226, 2000-12227, 2000-12228, 2000-91073, 2000-91074, 2000-91075, 2000-173774, 2001-110570, and 2001-110571.Such a material can satisfy the prerequisite of the present invention that it has formability of an amorphous thin film and exhibits, in a state held as a single thin film between the anode and the cathode, electroluminescence light emission when a DC voltage is applied thereto.",
"As a material having a charge transport characteristic (charge injection accelerating material) capable of accelerating injection of charges in the luminous material, there may be used a material-suitably selected from known electron transport materials and hole transport materials.",
"The present invention will be hereinafter described in detail by way of, while not limited thereto, the following examples.",
"EXAMPLE 1 Concentration of Compound 1 and Luminous Efficiency In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from a styryl compound represented by a compound 1 described below and an electron transport material represented by Alq3 (tris (8-quinolinol) aluminum) described below, are produced as samples, and a relationship between a luminance of light emitted from each sample and a luminous efficiency of the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared as follows: namely, as a light emission layer of each organic electroluminescence light emitting device, a mixed layer made from a styryl compound 1 expressed by the following chemical formula, and an electron transport material Alq3 expressed by the following chemical formula was formed by a vacuum vapor-deposition process; and as the other components of this light emitting device, a glass sheet was used as a substrate, and an anode made from ITO, a hole transport layer made from α-NPD expressed by the following chemical formula, and a cathode made from Mg—Ag were each formed by a known process.",
"Device structures of the samples thus prepared are shown below.",
"It is to be noted that in each device structure, a value in the parenthesis expresses a film thickness (the same applies in the later examples).",
"Device (1-1): ITO/α-NPD [30 nm]/compound 1:Alq3=1:9 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (1-2): ITO/α-NPD [30 nm]/compound 1:Alq3=3:7 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (1-3): ITO/α-NPD [30 nm]/compound 1:Alq3=5:5 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (1-4): ITO/α-NPD [30 nm]/compound 1:Alq3=8:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [1 200 nm] Device (1-5): ITO/α-NPD [30 nm]/compound 1:Alq3=9:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] A relationship between a luminance and a luminous efficiency of each of the sample Nos.",
"Device (1-1), Device (1-2), Device (1-3), Device (1-4), and Device (1-5) is shown in Table 1.It is to be noted that the luminous efficiency is calculated as a rate of luminance to a current value.",
"Table 1 (Relationship between Concentration of Compound 1 in Light Emission Layer and Luminous Efficiency) Concentration of Compound 1 in Light 1000 Emission Layer 1 cd/m2 10 cd/m2 100 cd/m2 cd/m2 Device 10 wt % 2.35 cd/A 2.36 cd/A 2.30 cd/A 2.24 cd/A (1-1) Device 30 wt % 2.33 cd/A 2.34 cd/A 2.23 cd/A 2.09 cd/A (1-2) Device 50 wt % 2.33 cd/A 2.33 cd/A 2.32 cd/A 2.12 cd/A (1-3) Device 80 wt % 2.32 cd/A 2.33 cd/A 2.31 cd/A 2.11 cd/A (1-4) Device 90 wt % 2.31 cd/A 2.33 cd/A 2.30 cd/A 2.12 cd/A (1-5) The results show that the luminous efficiency of each of the sample Nos.",
"Device (1-1), Device (1-2), Device (1-3), Device (1-4), and Device (1-5) is high and stable irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with concentration-dependent quenching caused when a high concentration of the luminous material is contained in the light emitting device.",
"EXAMPLE 2 Concentration of Compound 1 and Luminous Efficiency In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same hole transport material α-NPD as that described in Example 1, are produced as samples, and a relationship between a luminance of light emitted from each sample and a luminous efficiency of the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1, except that a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same hole transport material α-NPD as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"[Device Structure]: Device (2-1): ITO/α-NPD [30 nm]/compound 1:α-NPD=1:9 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (2-2): ITO/α-NPD [30 nm]/compound 1:α-NPD=3:7 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (2-3): ITO/α-NPD [30 nm]/compound l:α-NPD=5:5 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (2-4): ITO/α-NPD [30 nm]/compound 1:α-NPD=8:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (2-5): ITO/α-NPD [30 nm]/compound 1:α-NPD=9:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] A relationship between a luminance and a luminous efficiency of each of the sample Nos.",
"Device (2-1), Device (2-2), Device (2-3), Device (2-4), and Device (2-5) is shown in Table 2.It is to be noted that the luminous efficiency is calculated as a rate of luminance to a current value.",
"Table 2 (Relationship between Concentration of Compound 1 in Light Emission Layer and Luminous Efficiency) Concentration of Compound 1 in Light 1000 Emission Layer 1 cd/m2 10 cd/m2 100 cd/m2 cd/m2 Device 10 wt % 2.22 cd/A 2.24 cd/A 2.30 cd/A 2.13 cd/A (2-1) Device 30 wt % 2.12 cd/A 2.15 cd/A 2.11 cd/A 2.07 cd/A (2-2) Device 50 wt % 2.11 cd/A 2.12 cd/A 2.1 cd/A 2.05 cd/A (2-3) Device 80 wt % 2.09 cd/A 2.1 cd/A 2.08 cd/A 2.01 cd/A (2-4) Device 90 wt % 2.1 cd/A 2.1 cd/A 2.07 cd/A 2.03 cd/A (2-5) The results show that the luminous efficiency of each of the sample Nos.",
"Device (2-1), Device (2-2), Device (2-3), Device (2-4), and Device (2-5) is high and stable irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with concentration-dependent quenching caused when a high concentration of the luminous material is contained in the light emitting device.",
"EXAMPLE 3 Concentration of Compound 2 and Luminous Efficiency In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from a styryl compound represented by a compound 2 described below and the same electron transport material Alq3 (tris (8-quinolinol) aluminum) as that described in Example 1, are produced as samples, and a relationship between a luminance of light emitted from each sample and a luminous efficiency of the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1, except that a mixed layer made from a styryl compound represented by a compound 2 expressed by the following chemical formula and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"[Device Structure]: Device (3-1): ITO/α-NPD [30 nm]/compound 2: Alq3=1:9 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (3-2): ITO/α-NPD [30 nm]/compound 2: Alq3=3:7 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (3-3): ITO/α-NPD [30 nm]/compound 2: Alq3=5:5 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (3-4): ITO/α-NPD [30 nm]/compound 2: Alq3=8:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (3-5): ITO/α-NPD [30 nm]/compound 2: Alq3=9:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] A relationship between a luminance and a luminous efficiency of each of the sample Nos.",
"Device (3-1), Device (3-2), Device (3-3), Device (3-4), and Device (3-5) is shown in Table 3.It is to be noted that the luminous efficiency is calculated as a rate of luminance to a current value.",
"Table 3 (Relationship between Concentration of Compound 1 in Light Emission Layer and Luminous Efficiency) Concentration of Compound 2 in Light 1000 Emission Layer 1 cd/m2 10 cd/m2 100 cd/m2 cd/m2 Device 10 wt % 2.18 cd/A 2.16 cd/A 2.12 cd/A 2.03 cd/A (3-1) Device 30 wt % 2.14 cd/A 2.13 cd/A 2.1 cd/A 2.05 cd/A (3-2) Device 50 wt % 2.13 cd/A 2.12 cd/A 2.12 cd/A 2.08 cd/A (3-3) Device 80 wt % 2.11 cd/A 2.12 cd/A 2.11 cd/A 2.1 cd/A (3-4) Device 90 wt % 2.13 cd/A 2.11 cd/A 2.13 cd/A 2.1 cd/A (3-5) The results show that the luminous efficiency of each of the sample Nos.",
"Device (3-1), Device (3-2), Device (3-3), Device (3-4), and Device (3-5) is high and stable irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with concentration-dependent quenching caused when a high concentration of the luminous material is contained in the light emitting device.",
"EXAMPLE 4 Concentration of Compound 2 and Luminous Efficiency In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from the same styryl compound 2 as that described in Example 2 and the same hole transport material α-NPD as that described in Example 1, are produced as samples, and a relationship between a luminance of light emitted from each sample and a luminous efficiency of the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1, except that a mixed layer made from the same styryl compound 2 as that described in Example 2 and the same hole transport material α-NPD as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"[Device Structure]: Device (4-1): ITO/α-NPD [30 nm]/compound 2:α-NPD=1:9 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (4-2): ITO/α-NPD [30 nm]/compound 2:α-NPD=3:7 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm].",
"Device (4-3): ITO/α-NPD [30 nm]/compound 2:α-NPD=5:5 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (4-4): ITO/α-NPD [30 nm]/compound 2:α-NPD=8:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (4-5): ITO/α-NPD [30 nm]/compound 2:α-NPD=9:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] A relationship between a luminance and a luminous efficiency of each of the sample Nos.",
"Device (4-1), Device (4-2), Device (4-3), Device (4-4), and Device (4-5) is shown in Table 4.It is to be noted that the luminous efficiency is calculated as a rate of luminance to a current value.",
"Table 4 (Relationship between Concentration of Compound 2 in Light Emission Layer and Luminous Efficiency) Concentration of Compound 2 in Light 1000 Emission Layer 1 cd/m2 10 cd/m2 100 cd/m2 cd/m2 Device 10 wt % 2.15 cd/A 2.12 cd/A 2.09 cd/A 2.04 cd/A (4-1) Device 30 wt % 2.1 cd/A 2.09 cd/A 2.07 cd/A 2.05 cd/A (4-2) Device 50 wt % 2.09 cd/A 2.07 cd/A 2.06 cd/A 2.03 cd/A (4-3) Device 80 wt % 2.08 cd/A 2.09 cd/A 2.07 cd/A 2.02 cd/A (4-4) Device 90 wt % 2.09 cd/A 2.08 cd/A 2.06 cd/A 2.00 cd/A (4-5) The results show that the luminous efficiency of each of the sample Nos.",
"Device (4-1), Device (4-2), Device (4-3), Device (4-4), and Device (4-5) is high and stable irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with concentration-dependent quenching caused when a high concentration of the luminous material is contained in the light emitting device.",
"EXAMPLE 5 Concentration of Compound 1 and Hue In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1, are produced as samples, and a relationship between a luminance of light emitted from each sample and a hue of the light emitted from the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1.Namely, like Example 1, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample Nos.",
"Device (1-1), Device (1-2), Device (1-3), Device (1-4), and Device (1-5) as those prepared in Example 1 were thus prepared.",
"A relationship between a luminance and a hue of each of the samples is shown in Table 5.Table 5 (Relationship between Concentration of Compound 1 in Light Emission Layer and Hue) Concentration of Compound 1 1 cd/m2 10 cd/m2 100 cd/m2 1000 cd/m2 Device (1-1) 10 wt % 0.634, 0.365 0.633, 0.355 0.630, 0.368 0.624, 0.375 Device (1-2) 30 wt % 0.643, 0.355 0.640, 0.359 0.638, 0.359 0.633, 0.366 Device (1-3) 50 wt % 0.642, 0.356 0.641, 0.358 0.637, 0.360 0.631, 0.368 Device (1-4) 80 wt % 0.644, 0.354 0.642, 0.357 0.637, 0.361 0.632, 0.367 Device (1-5) 90 wt % 0.643, 0.357 0.642, 0.358 0.637, 0.362 0.632, 0.367 The results show that the hues of the sample Nos.",
"Device (1-1), Device (1-2), Device (1-3), Device (1-4), and Device (1-5) are nearly equal to each other irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with a change in hue upon high luminance, which problem is liable to be caused for a usual red color light emitting device in which a pigment is doped in Alq3 or the like in a concentration as low as 2 to 3 wt % or less.",
"EXAMPLE 6 Concentration of Compound 1 and Hue In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from the same styryl compound 1 as that described in Example 2 and the same hole transport material α-NPD as that described in Example 2, are produced as samples, and a relationship between a luminance of light emitted from each sample and a hue of the light emitted from the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 2.Namely, like Example 2, a mixed layer made from the same styryl compound 1 as that described in Example 2 and the same hole transport material α-NPD as that described in Example 2 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample Nos.",
"Device (2-1), Device (2-2) Device (2-3), Device (2-4), and Device (2-5) as those prepared in Example 2 were thus prepared.",
"A relationship between a luminance and a hue of each of the samples is shown in Table 6.Table 6 (Relationship between Concentration of Compound 1 in Light Emission Layer and Hue) Concentration of Compound 1 1 cd/m2 10 cd/m2 100 cd/m2 1000 cd/m2 Device (2-1) 10 wt % 0.623, 0.376 0.622, 0.377 0.621, 0.378 0.620, 0.379 Device (2-2) 30 wt % 0.632, 0.368 0.631, 0.369 0.629, 0.370 0.627, 0.372 Device (2-3) 50 wt % 0.635, 0.365 0.635, 0.364 0.633, 0.366 0.631, 0.368 Device (2-4) 80 wt % 0.635, 0.364 0.634, 0.365 0.634, 0.366 0.632, 0.367 Device (2-5) 90 wt % 0.634, 0.365 0.634, 0.365 0.634, 0.366 0.631, 0.368 The results show that the hues of the sample Nos.",
"Device (2-1), Device (2-2), Device (2-3), Device (2-4), and Device (2-5) are nearly equal to each other irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with a change in hue upon high luminance, which problem is liable to be caused for a usual red color light emitting device in which a pigment is doped in Alq3 or the like in a concentration as low as 2 to 3 wt % or less.",
"EXAMPLE 7 Concentration of Compound 2 and Hue In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from the same styryl compound 2 as that described in Example 3 and the same electron transport material Alq3 as that described in Example 3, are produced as samples, and a relationship between a luminance of light emitted from each sample and a hue of the light emitted from the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 3.Namely, like Example 3, a mixed layer made from the same styryl compound 2 as that described in Example 3 and the same electron transport material Alq3 as that described in Example 3 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample Nos.",
"Device (3-1), Device (3-2) Device (3-3), Device (3-4), and Device (3-5) as those prepared in Example 3 were thus prepared.",
"A relationship between a luminance and a hue of each of the samples is shown in Table 7.Table 7 (Relationship between Concentration of Compound 2 in Light Emission Layer and Hue) Concentration of Compound 2 1 cd/m2 10 cd/m2 100 cd/m2 1000 cd/m2 Device (3-1) 10 wt % 0.645, 0.354 0.644, 0.355 0.643, 0.356 0.640, 0.359 Device (3-2) 30 wt % 0.651, 0.348 0.650, 0.349 0.648, 0.351 0.645, 0.354 Device (3-3) 50 wt % 0.652, 0.348 0.651, 0.350 0.650, 0.349 0.645, 0.354 Device (3-4) 80 wt % 0.652, 0.347 0.653, 0.347 0.651, 0.348 0.647, 0.352 Device (3-5) 90 wt % 0.652, 0.347 0.652, 0.347 0.651, 0.348 0.646, 0.353 The results show that the hues of the sample Nos.",
"Device (3-1), Device (3-2), Device (3-3), Device (3-4), and Device (3-5) are nearly equal to each other irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with a change in hue upon high luminance, which problem is liable to be caused for a usual red color light emitting device in which a pigment is doped in Alq3 or the like in a concentration as low as 2 to 3 wt % or less.",
"EXAMPLE 8 Concentration of Compound 2 and Hue In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from the same styryl compound 2 as that described in Example 4 and the same hole transport material α-NPD as that described in Example 4, are produced as samples, and a relationship between a luminance of light emitted from each sample and a hue of the light emitted from the sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 4.Namely, like Example 4, a mixed layer made from the same styryl compound 2 as that described in Example 4 and the same hole transport material α-NPD as that described in Example 4 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample Nos.",
"Device (4-1), Device (4-2) Device (4-3), Device (4-4), and Device (4-5) as those prepared in Example 4 were thus prepared.",
"A relationship between a luminance and a hue of each of the samples is shown in Table 8.Table 8 (Relationship between Concentration of Compound 2 in Light Emission Layer and Hue) Concentration of Compound 2 1 cd/m2 10 cd/m2 100 cd/m2 1000 cd/m2 Device (4-1) 10 wt % 0.635, 0.364 0.634, 0.365 0.633, 0.366 0.630, 0.369 Device (4-2) 30 wt % 0.643, 0.357 0.642, 0.356 0.641, 0.358 0.638, 0.361 Device (4-3) 50 wt % 0.645, 0.355 0.644, 0.355 0.643, 0.356 0.641, 0.358 Device (4-4) 80 wt % 0.645, 0.355 0.645, 0.354 0.644, 0.355 0.640, 0.359 Device (4-5) 90 wt % 0.645, 0.354 0.644, 0.355 0.643, 0.356 0.641, 0.358 The results show that the hues of the sample Nos.",
"Device (4-1), Device (4-2), Device (4-3), Device (4-4), and Device (4-5) are nearly equal to each other irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with a change in hue upon high luminance, which problem is liable to be caused for a usual red color light emitting device in which a pigment is doped in Alq3 or the like in a concentration as low as 2 to 3 wt % or less.",
"EXAMPLE 9 Stability of Device (1-3) In this example, an organic electroluminescence light emitting device using, as a light emission layer having an electron transport characteristic, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1 is produced as a sample, and a reliability of the sample is examined.",
"A sample of an organic electroluminescence light emitting device was prepared in the same manner as that described in Example 1.Namely, like Example 1, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample No.",
"Device (1-3) as that prepared in Example 1 was thus prepared.",
"The sample thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"The sample was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 1,000 hours.",
"EXAMPLE 10 Stability of Device (3-3) In this example, an organic electroluminescence light emitting device using, as a light emission layer having an electron transport characteristic, a mixed layer made from the same styryl compound 2 as that described in Example 3 and the same electron transport material Alq3 as that described in Example 3 is produced as a sample, and a reliability of the sample is examined.",
"A sample of an organic electroluminescence light emitting device was prepared in the same manner as that described in Example 3.Namely, like Example 3, a mixed layer made from the same styryl compound 2 as that described in Example 3 and the same electron transport material Alq3 as that described in Example 3 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample No.",
"Device (3-3) as that prepared in Example 3 was thus prepared.",
"The sample thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"The sample was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 4,800 hours.",
"EXAMPLE 11 Device Using Mixed Layer Made of Compound 3 and Alq3 as Light Emission Layer Having Electron Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from a styryl compound represented by a compound 3 expressed by the following chemical formula and the same electron transport material Alq3 as that described in Example 1, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a styryl compound represented by a compound 3 having the above chemical formula and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (11-1), (11-2), (11-3) (11-4), and (11-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 3 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of red, and exhibited a high luminous efficiency in a practical luminance region from 10 to 1,000 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (11-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 1,200 hours.",
"EXAMPLE 12 Device Using Mixed Layer Made from Compound 4 and Alq3 as Light Emission Layer Having Electron Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from a styryl compound represented by a compound 4 expressed by the following chemical formula and the same electron transport material Alq3 as that described in Example 1, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a styryl compound represented by a compound 4 having the above chemical formula and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (12-1), (12-2), (12-3) (12-4), and (12-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 4 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of green, and exhibited a high luminous efficiency in a practical luminance region from 10 to 1,000 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (12-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 500 hours.",
"EXAMPLE 13 Device Using Mixed Layer Made from Compound 5 and α-NPD as Light Emission Layer Having Hole Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from a styryl compound represented by a compound 5 expressed by the following chemical formula and the same hole transport material α-NPD as that described in Example 2, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (2-1) to Device (2-5) in Example 2, except that a mixed layer made from a styryl compound represented by a compound 5 having the above chemical formula and the same hole transport material α-NPD as that described in Example 2 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (13-1), (13-2), (13-3) (13-4), and (13-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 5 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue-green, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (13-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 800 hours.",
"EXAMPLE 14 Device Using Mixed Layer Made from Compound 6 and α-NPD as Light Emission Layer having Hole Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from a compound 6 expressed by the following chemical formula and the same hole transport material α-NPD as that described in Example 2 and the like, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared by forming the following layer structures on an ITO substrate by a vacuum vapor-deposition process.",
"As a feature of this example, a second electron transport layer made from BAlq (bis (8-quinolinol) aluminum derivative) expressed by the following chemical formula was additionally formed between the light emission layer and the electron transport layer.",
"[Device Structure]: Device (14-1): ITO/α-NPD [30 nm]/compound 6: α-NPD=1:9 [30 nm]/BAlq [10 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (14-2): ITO/α-NPD [30 nm]/compound 6: α-NPD=3:7 [30 nm]/BAlq [10 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (14-3): ITO/α-NPD [30 nm]/compound 6: α-NPD=5:5 [30 nm]/BAlq [10 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (14-4): ITO/α-NPD [30 nm]/compound 6: α-NPD=8:2 [30 nm]/BAlq [10 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (14-5): ITO/α-NPD [30 nm]/compound 6: α-NPD=9:1 [30 nm]/BAlq [10 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Each of the five samples emitted light of blue-green, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (14-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 700 hours.",
"EXAMPLE 15 Device Using Mixed Layer Made from Compound 7 and α-NPD as Light Emission Layer Having Hole Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from a compound 7 expressed by the following chemical formula and the same hole transport material α-NPD as that described in Example 14, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (14-1) to Device (14-5) in Example 14, except that a mixed layer made from a compound 7 having the above chemical formula and the same hole transport material α-NPD as that described in Example 14 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (15-1), (15-2), (15-3), (15-4), and (15-5) containing 10 wt %, 30 wt %, 50-wt %, 80 wt %, and 90 wt % of the compound 7 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (15-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/M2 so as to continuously emit light, the result of which showed that the half value period of luminance was 600 hours.",
"EXAMPLE 16 Device Using Mixed Layer Made from Compound 8 and α-NPD as Light Emission Layer Having Hole Transport Characteristic In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having a hole transport characteristic, a mixed layer made from a compound 8 expressed by the following chemical formula and the same hole transport material α-NPD as that described in Example 14, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (14-1) to Device (14-5) in Example 14, except that a mixed layer made from a compound 8 having the above chemical formula and the same hole transport material α-NPD as that described in Example 14 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (16-1), (16-2), (16-3), (16-4), and (16-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 8 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (16-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 750 hours.",
"EXAMPLE 17 Device Using Mixed Layer Made from Compound 9 and CBP as Light Emission Layer In this example, organic electroluminescence light emitting devices, each using, as a light emission layer, a mixed layer made from a compound 9 expressed by the following chemical formula and CBP (4,4′-N,N′-dicarbazole-biphenyl) expressed by the following chemical formula, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a compound 9 having the above chemical formula and CBP having the above chemical formula was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (17-1), (17-2), (17-3), (17-4), and (17-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 9 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (17-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 780 hours.",
"EXAMPLE 18 Device Using Mixed Layer Made from Compound 10 and CBP as Light Emission Layer In this example, organic electroluminescence light emitting devices, each using, as a light emission layer, a mixed layer made from a compound 10 expressed by the following chemical formula and the same material CBP as that described in Example 17, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a compound 10 having the above chemical formula and the same material CBP as that described in Example 17 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (18-1), (18-2), (18-3), (18-4), and (18-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 10 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue green, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (18-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 1,100 hours.",
"EXAMPLE 19 Device Using Mixed Layer Made from Compound 11 and CBP as Light Emission Layer In this example, organic electroluminescence light emitting devices, each using, as a light emission layer, a mixed layer made from a compound 11 expressed by the following chemical formula and the same-material CBP as that described in Example 17, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a compound 11 having the above chemical formula and the same material CBP as that described in Example 17 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (19-1), (19-2), (19-3), (19-4), and (19-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 11 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (19-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 650 hours.",
"EXAMPLE 20 Device Using Mixed Layer Made from Compound 12 and CBP as Light Emission Layer In this example, organic electroluminescence light emitting devices, each using, as a light emission layer, a mixed layer made from a compound 12 expressed by the following chemical formula and the same material CBP as that described in Example 17, are produced as samples, and device characteristics of each sample are examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that for preparing the sample Nos.",
"Device (1-1) to Device (1-5) in Example 1, except that a mixed layer made from a compound 12 having the above chemical formula and the same material CBP as that described in Example 17 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Five sample Nos.",
"Device (20-1), (20-2), (20-3), (20-4), and (20-5) containing 10 wt %, 30 wt %, 50 wt %, 80 wt %, and 90 wt % of the compound 12 in the mixed layer, respectively were thus prepared.",
"Each of the five samples emitted light of blue, and exhibited a high luminous efficiency in a practical luminance region from 10 to 500 cd/m2.Also, it was revealed that the sample did not exhibit any change in hue with an increase in luminance, and therefore, the sample was useful as a display device.",
"Each of the samples thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"Of these samples, the sample No.",
"Device (20-3) was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 500 cd/M2 so as to continuously emit light, the result of which showed that the half value period of luminance was 880 hours.",
"Comparative Example 1 Hue and Reliability of Light Emitting Device Using Single Layer of Compound 1 as Light Emission Layer In this comparative example, a sample of an organic electroluminescence light emitting device was produced in the same manner as that described in Example 1, except that a single layer of 100% of the styryl compound 1 described in Example 1 was used as a light emission layer, and a reliability of the sample was examined.",
"[Device Structure]: Device (1-6): ITO/α-NPD [30 nm]/compound 1 [30 nm]/Alq3 (30 nm)/Mg:Ag [200 nm] As shown in Table 9, the hue of the sample No.",
"Device (1-6) is nearly equal to each of the sample Nos.",
"Device (1-1), Device (1-2), Device (1-3), Device (1-4), and Device (1-5) prepared in Example 5.Table 9 (Relationship between Concentration of Compound 1 in Light Emission Layer and Hue in Comparative Example 1) Concentration 100 1000 of Compound 1 1 cd/m2 10 cd/m2 cd/m2 cd/m2 Device (1-6) 100 wt % 0.633, 0.632, 0.632, 0.628, 0.367 0.366 0.366 0.371 The sample No.",
"Device (1-6) thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed, and it was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 200 hours.",
"This half value life is about one-fifth of the half value life (1,000 hr) of the sample No.",
"Device (1-3) prepared in Example 9.Comparative Example 2 Hue and Reliability of Light Emitting Device Using Single Layer of Compound 2 as Light Emission Layer In this comparative example, a sample of an organic electroluminescence light emitting device was produced in the same manner as that described in Example 3, except that a single layer of 100% of the styryl compound 2 described in Example 3 was used as a light emission layer, and a reliability of the sample was examined.",
"[Device Structure]: Device (3-6): ITO/α-NPD [30 nm]/compound 2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] As shown in Table 10, the hue of the sample No.",
"Device (3-6) is nearly equal to each of the sample Nos.",
"Device (3-1), Device (3-2), Device (3-3), Device (3-4), and Device (3-5) prepared in Example 7.Table 10 (Relationship between Concentration of Compound 2 in Light Emission Layer and Hue in Comparative Example 2) Concentration 100 1000 of Compound 2 1 cd/m2 10 cd/m2 cd/m2 cd/m2 Device (3-6) 100 wt % 0.648, 0.647, 0.646, 0.641, 0.351 0.352 0.353 0.358 The sample No.",
"Device (3-6) thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed, and it was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 800 hours.",
"This half value life is about one-sixth of the half value life (4,800 hr) of the sample No.",
"Device (3-3) prepared in Example 10.EXAMPLE 21 Relationship between Concentration of Luminous Material and Device Resistance In this example, organic electroluminescence light emitting devices, each using, as a light emission layer having an electron transport characteristic, a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1, are produced as samples, and a relationship between a concentration of the compound 1 and a current density/voltage of each sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1, except that a mixed layer made from the same styryl compound 1 as that described in Example 1 and the same electron transport material Alq3 as that described in Example 1 was formed as a light emission layer by a vacuum vapor-deposition process.",
"Sample Nos.",
"Device (21-1), Device (21-2), Device (21-3), Device (21-4), and Device (21-5) were thus prepared.",
"The results are shown in Table 11.For comparison, the result of an organic electroluminescence light emitting device, which was prepared in the same manner except the use of a layer composed of only the compound 1 as the light emission layer, is listed as sample No.",
"Device (21-5) in Table 11.Table 11 (Relationship between Concentration of Compound 1 in Light Emission Layer and Device Resistance) Concentration of Compound 1 6 V 10 V Device (21-1) 5 wt % 3.7 mA/cm2 216 mA/cm2 Device (21-2) 10 wt % 11.7 mA/cm2 607 mA/cm2 Device (21-3) 20 wt % 15.7 mA/cm2 1007 mA/cm2 Device (21-4) 40 wt % 19.2 mA/cm2 1320 mA/cm2 Device (21-5) 100 wt % 30.0 mA/cm2 1810 mA/cm2 As shown in Table 11, it becomes apparent that the device resistance increases with a decrease in concentration of the compound 1.Taking into account a low voltage drive from the viewpoint of power consumption, the concentration of the compound 1 in the light emission layer is desirable to be in a range of 20 wt % or more.",
"EXAMPLE 22 Concentration of Compound 1, Luminous Efficiency, and Hue In this example, organic electroluminescence light emitting devices, each using, as a light emission layer, a mixed layer made from the styryl compound 1, the hole transport material α-NPD, and the electron transport material Alq3, which are the same as those described in Example 1, are produced as samples, and a relationship between a luminance of light, a luminous efficiency, and a hue of light of each sample is examined.",
"Samples of organic electroluminescence light emitting devices were prepared in the same manner as that described in Example 1, except that a mixed layer made from the styryl compound 1, the hole transport material α-NPD, and the electron transport material Alq3, was formed as a light emission layer by a vacuum vapor-deposition process.",
"[Device Structure]: Device (22-1): ITO/α-NPD [30 nm]/compound 1:α-NPD: Alq3=1:2:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (22-2): ITO/α-NPD [30 nm]/compound 1:α-NPD: Alq3=1:1:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (22-3): ITO/α-NPD [30 nm]/compound 1:α-NPD: Alq3=4:2:2 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm] Device (22-4): ITO/α-NPD [30 nm]/compound 1:α-NPD: Alq3=8:1:1 [30 nm]/Alq3 [30 nm]/Mg:Ag [200 nm].",
"A relationship between a luminance and a luminous efficiency of each of the sample Nos.",
"Device (22-1), Device (22-2), Device (22-3), and Device (22-4) thus prepared is shown in Table 12.It is to be noted that the luminous efficiency is calculated as a rate of luminance to a current value.",
"Table 12 (Relationship Between Concentration of Compound 1 in Light Emission Layer and Luminous Efficiency) Concentration of Compound 1 in Light 1000 Emission Layer 1 cd/m2 10 cd/m2 100 cd/m2 cd/m2 Device 20 wt % 2.11 cd/A 2.13 cd/A 2.23 cd/A 2.08 cd/A (22-1) Device 33 wt % 2.14 cd/A 2.15 cd/A 2.11 cd/A 2.07 cd/A (22-2) Device 50 wt % 2.13 cd/A 2.14 cd/A 2.10 cd/A 2.04 cd/A (22-3) Device 80 wt % 2.12 cd/A 2.11 cd/A 2.06 cd/A 2.03 cd/A (22-4) The results show that the luminous efficiency of each of the sample Nos.",
"Device (22-1), Device (22-2), Device (22-3), and Device (22-4) is high and stable irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with concentration-dependent quenching caused when a high concentration of the luminous material is contained in the light emitting device.",
"Similarly, a relationship between a luminance and a hue of each sample is shown in Table 13.Table 13 (Relationship Between Concentration of Compound 1 in Light Emission Layer and Hue) Concentration of Compound 1 1 cd/m2 10 cd/m2 100 cd/m2 1000 cd/m2 Device (22-1) 20 wt % 0.627, 0.372 0.625, 0.374 0.624, 0.375 0.621, 0.380 Device (22-2) 33 wt % 0.631, 0.369 0.630, 0.369 0.629, 0.370 0.627, 0.372 Device (22-3) 50 wt % 0.633, 0.366 0.632, 0.367 0.632, 0.367 0.631, 0.368 Device (22-4) 80 wt % 0.633, 0.366 0.633, 0.365 0.632, 0.367 0.631, 0.368 The results show that the hues of the sample Nos.",
"Device (22-1), Device (22-2), Device (22-3), and Device (22-4) are nearly equal to each other irrespective of the concentration of the luminescent material in a practical luminance region from 1 to 1000 cd/m2, and that each of the samples has no problem associated with a change in hue upon high luminance, which problem is liable to be caused for a usual red color light emitting device in which a pigment is doped in Alq3 or the like in a concentration as low as 2 to 3 wt % or less.",
"EXAMPLE 23 Stability of Device (22-3) In this example, an organic electroluminescence light emitting device using, as a light emission layer, a mixed layer made from the styryl compound 1, the hole transport material α-NPD, and the electron transport material Alq3, which are the same as those described in Example 22, is produced as a sample, and a reliability of the sample is examined.",
"A sample of an organic electroluminescence light emitting device was prepared in the same manner as that described in Example 22.Namely, like Example 22, a mixed layer made from the styryl compound 1, the hole transport material α-NPD, and the electron transport material Alq3 was formed as a light emission layer by a vacuum vapor-deposition process.",
"The same sample No.",
"Device (22-3) as that prepared in Example 22 was thus prepared.",
"The sample thus prepared was left in a nitrogen atmosphere for one month, the result of which showed that no deterioration of the sample was observed.",
"The sample was also forcibly deteriorated by continuously applying a constant current thereto with an initial luminance set to 1,000 cd/m2 so as to continuously emit light, the result of which showed that the half value period of luminance was 950 hours.",
"EXAMPLE 24 Verification 1 of Light Emission Region In this example, it is examined whether a light emission region is concentrated in the vicinity of an interface of a light emission layer or dispersed over the whole range of the light emission layer, by measuring a change in intensity of a photoluminescence spectrum (PL spectrum) of a luminescent material before and after reduction in luminance.",
"For the sample No.",
"Device (3-3) using the mixed layer containing the compound 2 and the electron transport material Alq3 at a mixing weight ratio of 1:1 as the light emission layer in Example 3 and the sample No.",
"Device (3-6) using a single layer containing only the compound 2 as the light emission layer, the intensity of the PL spectrum of the device at the time when the EL luminous intensity thereof was reduced to a half of that in an initial stage was examined.",
"As a result, for the sample No.",
"Device (3-3), when the EL luminous intensity was reduced to a half, the intensity of the PL spectrum was reduced to 50% of the initial luminance.",
"On the contrary, for the sample No.",
"Device (3-6) using the single layer containing only the compound 2 as the light emission layer, when the EL luminous intensity was reduced to a half, the intensity of the PL spectrum was reduced to 70% of the initial luminance.",
"Since the whole range of the light emission layer is optically pumped at the time of measuring the intensity of the PL spectrum, it may be considered that if the luminance of the whole range of the light emission range is reduced along with the electroluminescence (EL), the intensity of the PL spectrum is reduced at the same rate; however, if the luminance of part of the light emission layer is reduced along with electroluminescence (EL), the intensity of the PL spectrum is reduced at a rate smaller than the reduction rate of the EL luminous intensity.",
"Accordingly, it becomes apparent that for the sample No.",
"Device (3-3), the whole range of the light emission layer almost acts as the light emission region; however, for the sample No.",
"Device (3-6), only part of the light emission layer (interface between organic layers) acts as the light emission region.",
"The EL spectrum of the sample No.",
"Device (3-6) is shown in FIG.",
"5.The EL spectrum exhibits the peak value at a wavelength near 650 nm, which indicates the compound 2, and a small peak value at a wavelength near 520 nm, which indicates Alq3.This means that for the sample No.",
"Device (3-6), light emission occurs at an interface between the light emission layer containing only the compound 2 and the electron transport layer Alq3, and the light emission region is concentrated in the vicinity of the interface.",
"On the contrary, from the EL spectrum of the sample No.",
"Device (3-3) shown in FIG.",
"6, it is apparent that light emission occurs nearly in the whole range of the light emission layer and that no light emission of Alq3 occur.",
"EXAMPLE 25 Verification 2 of Light Emission Region To verify the effect of the organic electroluminescence light emitting device of the present invention, as shown in FIG.",
"7, a device was produced with a film thickness condition that a light interference effect between light emerged from a light emission interface and light reflected from an aluminum electrode 37 weakens light outputted from the device.",
"An ITO electrode 36 (thickness: 119 nm), a TNATA (4,4′,4″-tris (2-naphthylphenylamino)triphenylamine) layer 32a (thickness: 40 nm), an EL022 (triphenylamine tetramer) layer 32b (thickness: 50 nm), an Alq3 layer 33 mixed with 40% of the above-described styryl compound 1 (thickness: 50 nm), and an Alq3 layer 34 (thickness: 165 nm) were sequentially stacked, and the aluminum electrode 37 was formed thereon.",
"A relationship between an actually measured emission spectrum of this device and an emission spectrum estimated from a light emission distribution in the light emission layer of the device was examined.",
"As shown in FIG.",
"8, it becomes apparent that a light emission distribution 49 in the light emission layer corresponds to an emission spectrum of the device operated at a current density of 0.1 mA/cm2, and a light emission distribution 46 in the light emission layer corresponds to an emission spectrum of the device operated at a current density of 1 mA/cm2.The light emission distribution in the light emission layer used for calculation is shown in FIG.",
"9.Here, the superimposition of a light ray emitted and directly emerged downwardly and a light ray reflected from Al and then emerged downwardly is performed by using plane-wave approximation.",
"Concretely, a phase difference δ at the superimposition of the light rays is given by δ=2π·2L/λ+Φ where character L is an optical distance between the emission plane and Al, and character Φ is a phase shift amount due to Al reflection.",
"Accordingly, letting a complex amplitude of the light ray directly emerged be A and a complex reflection coefficient of Al be γ, a complex amplitude of the superimposed light ray is expressed by the following equation: (1+exp(iδ)γ)A By expressing the real number of this equation, the intensity can be obtained.",
"Further, by integrating the intensity with a luminous intensity distribution in each depth direction and calculating the integrated intensity for each wavelength, a spectrum of the light ray emerged to the outside can be obtained.",
"According to the above-described result, it is verified that in the light emission layer formed by mixing the luminous material having the charge transport characteristic in the charge injection accelerating material, the emission spectrum exhibits that a distribution of a light emission region having a large luminous intensity occurs at both of the interface between the hole transport layer and the light emission layer and the interface between the electron transport layer and the light emission layer.",
"According to this embodiment, the light emission distribution on the hole transport layer side increases with the increased current density.",
"The above-described embodiments and the examples according to the present invention may be variously modified without departing from the technical thought of the present invention.",
"According to the present invention, a light emission region is formed by a mixed layer made from a luminous material exhibiting electroluminescence when a voltage is applied thereto and having a charge transport characteristic and a charge injection accelerating material capable of accelerating injection of charges in the luminous material, and the light emission region exists not only at an interface with an adjacent layer or its vicinity but also over a specific thickness region in the layer thickness direction.",
"Accordingly, a region in which light is emitted is not limited to the above-described interface or its vicinity but is spread in a wide range of the light emission layer.",
"As a result, it is possible to significantly improve the service life of the device.",
"Further, it is possible to prevent the hue of light from being varied depending on the concentration of the luminous material contained in the light emission layer and on an operational condition such as an applied voltage, and hence to stabilize the performance of the device."
]
] | Patent_10240328 |
[
[
"Plasma system and method of producing a functional coating",
"A plasma system has at least one inductively coupled high-frequency plasma jet source having a burner body delimiting a plasma generating space, having an outlet orifice for the plasma jet, and a chamber communicating with the plasma jet source through the outlet orifice, having a substrate situated in the chamber, where it is exposed to the plasma jet.",
"The substrate is situated on a substrate electrode to which an electric voltage may be applied.",
"In addition, a method of producing a functional coating on the substrate using such a plasma system is also described.",
"In a preferred embodiment, during operation of the plasma system, both the plasma jet and the electric voltage on the substrate electrode are pulsed and/or a pressure gradient is maintained between the interior of the plasma jet source and the interior of the chamber."
],
[
"1-18.",
"(canceled) 19.A plasma system, comprising: at least one inductively coupled high-frequency plasma jet source including a burner body delimiting a plasma generating space; a chamber including an outlet orifice for a plasma jet, the chamber communicating with the at least one inductively coupled plasma jet source through the outlet orifice; a substrate electrode capable of receiving an electric voltage; and a substrate situated in the chamber and on the substrate electrode, the substrate being exposed to the plasma jet in the chamber.",
"20.The plasma system as recited in claim 19, further comprising: a generator to which the substrate electrode is connected, the generator applying one of an electric direct voltage and an alternating voltage having an amplitude between 10 V and 5 kV and a frequency between 0 Hz and 50 MHz to the substrate electrode.",
"21.The plasma system as recited in claim 19, further comprising: a generator to which the substrate electrode is connected, the generator applying to the substrate electrode one of an electric direct voltage and an alternating voltage having an amplitude between 50 V and 300 kV and a frequency between 1 kHz and 100 kHz.",
"22.The plasma system as recited in claim 19, wherein: the burner body includes: a coil surrounding the plasma generating space in some areas, and at least one inlet for supplying at least one of a gas and a precursor material into the plasma generating space, and a high-frequency generator is connected to the coil for igniting a plasma and for injecting an electric power into the plasma.",
"23.The plasma system as recited in claim 19, further comprising: an arrangement for periodically varying an intensity of the plasma jet of the plasma jet source.",
"24.The plasma system as recited in claim 22, wherein: the burner body is pot-shaped, the coil one of surrounds the burner body in a vicinity of the outlet orifice and is integrated into the burner body in the vicinity of the outlet orifice, an injector gas is supplied into the plasma generating space through the at least one inlet, and the burner body includes at least one second inlet for supplying at least one of a central gas and an enveloping gas into the plasma generating space, the central gas reacting with the injector gas, and the enveloping gas separating the burner body from the plasma produced therein in at least some areas and concentrically surrounding the plasma in the plasma generating space.",
"25.The plasma system as recited in claim 22, wherein: the burner body is pot-shaped, the coil one of surrounds the burner body in a vicinity of the outlet orifice and is integrated into the burner body in the vicinity of the outlet orifice, a precursor material for producing a functional coating on the substrate is supplied into the plasma generating space through the at least one inlet, and the burner body includes at least one second inlet for supplying at least one of a central gas and an enveloping gas into the plasma generating space, the central gas reacting with the injector gas, and the enveloping gas separating the burner body from the plasma produced therein in at least some areas and concentrically surrounding the plasma into the plasma generating space.",
"26.The plasma system as recited in claim 19, further comprising: a pumping device to which the chamber is connected in order to maintain at least one of: a pressure difference of more than 100 mbar between the plasma generating space and an interior of the chamber in operation of the plasma system, and a ratio of a pressure in the plasma generating space to a pressure in the interior of the chamber that is greater than 1.5.27.The plasma system as recited in claim 19, further comprising: a pumping device to which the chamber is connected in order to maintain at least one of: a pressure difference of more than 100 mbar between the plasma generating space and an interior of the chamber in operation of the plasma system, and a ratio of a pressure in the plasma generating space to a pressure in the interior of the chamber that is greater than 3.28.A method of producing a functional coating on a substrate, comprising: placing the substrate in a chamber; generating a plasma having reactive particles by a high-frequency, inductively coupled plasma jet source, the plasma emerging in the form of a plasma jet from the plasma jet source and entering the chamber connected thereto, where the plasma acts on the substrate so that the functional coating is one of produced and deposited on the substrate; and situating the substrate on a substrate electrode in order to expose the substrate to an electric voltage at least intermittently.",
"29.The method as recited in claim 28, further comprising: injecting one of a direct voltage and an alternating voltage into the substrate electrode via a generator, the one of the direct voltage and the alternating voltage having an amplitude between 10 V and 5 kV and a frequency between 0 Hz and 50 MHz.",
"30.The method as recited in claim 28, further comprising: injecting one of a direct voltage and an alternating voltage into the substrate electrode via a generator, the one of the direct voltage and the alternating voltage having an amplitude between 50 V and 300 V and a frequency between 1 kHz and 100 kHz.",
"31.The method as recited in claim 28, further comprising: varying the electric voltage over time at least one of intermittently provided with an adjustable offset voltage and pulsed with a selectable pulse-pause ratio.",
"32.The method as recited in claim 28, wherein: the electric voltage has one of a unipolar saw-tooth characteristic, a bipolar saw-tooth characteristic, a triangular characteristic, and a sinusoidal characteristic.",
"33.The method as recited in claim 28, further comprising: periodically varying an intensity of the plasma jet in an action of the plasma jet on the substrate at a frequency of 1 Hz to 10 kHz, between an adjustable upper limit and an adjustable lower limit.",
"34.The method according to claim 33, wherein: the plasma jet is periodically extinguished for an adjustable period of time.",
"35.The method as recited in claim 28, further comprising: periodically varying an intensity of the plasma jet in an action of the plasma jet on the substrate at a frequency of 50 Hz to 1 kHz, between an adjustable upper limit and an adjustable lower limit.",
"36.The method according to claim 35, wherein: the plasma jet is periodically extinguished for an adjustable period of time.",
"37.The method as recited in claim 28, further comprising: injecting an electric power of 500 W to 50 kW at a high frequency of 0.5 MHz to 20 MHz into the plasma in the plasma jet source via a coil.",
"38.The method as recited in claim 28, further comprising: injecting an electric power of 1 kW to 10 kW at a high frequency of 0.5 MHz to 20 MHz into the plasma in the plasma jet source via a coil.",
"39.The method as recited in claim 28, further comprising: discharging the plasma as the plasma jet out of the plasma jet source and into the chamber through an outlet orifice by supplying a gas at a gas flow rate of 5,000 sccm to 100,000 sccm to the plasma jet source.",
"40.The method according to claim 39, wherein: the gas includes argon.",
"41.The method as recited in claim 28, further comprising: discharging the plasma as the jet out of the plasma jet source and into the chamber through an outlet orifice by supplying a gas at a gas flow rate of 20,000 sccm to 70,000 sccm to the plasma jet source.",
"42.The method according to claim 41, wherein: the gas includes argon.",
"43.The method as recited in claim 28, further comprising: supplying at least one precursor material to at least one of the plasma through an inlet in the plasma jet source and the plasma jet through a feeding device in the chamber, the at least one precursor material in a modified form after undergoing one of a chemical reaction and a chemical activation then at least one of forming the functional coating on the substrate and being integrated into the functional coating.",
"44.The method according to claim 43, wherein: the at least one precursor material includes one of a gaseous precursor material, a microscale precursor material, a nanoscale precursor material, a suspension of the precursor material, and a reactive gas.",
"45.The method as recited in claim 43, further comprising: supplying to the plasma a carrier gas for the at least one precursor material to cause a chemical reaction with the at least one precursor material.",
"46.The method according to claim 45, wherein: the carrier gas includes one of oxygen, nitrogen, ammonia, silane, acetylene, methane, and hydrogen."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>Applying functional coatings to substrates is a widely used method of imparting desired properties to the surfaces of workpieces and/or components.",
"A conventional method of producing such functional layers is by plasma coating in a medium-high or high vacuum, which requires complex evacuation techniques and yields relatively low coating rates.",
"Therefore, this method is time-intensive and expensive.",
"Thermal plasmas in particular which allow higher coating rates in the range of mm/h to able achieved are suitable for coating substrates in the atmospheric and subatmospheric pressure range.",
"In this regard, reference is made to R. Henne, Contribution to Plasma Physics, 39 (1999) pages 385-397, for example.",
"Of the thermal plasma sources, the high-frequency inductively coupled plasma jet source (HF-ICP jet source) is especially promising, such as that described by E. Pfender and C. H. Chang “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6 th Workshop on Plasma Technology, Technical University of Illmenau, 1998.Furthermore, German Published Patent Application No.",
"199 58 474 has proposed a method of producing functional layers by using such a plasma jet source.",
"The advantages of the HF-ICP jet source include the range of operating pressures in the source, usually extending from 50 mbar to 1 bar or more, and also the great variety of materials that may be used and deposited with such a plasma jet source.",
"In particular, due to the fact that the starting materials are introduced axially into the very hot plasma jet, hard substances having a very high melting point may also be used.",
"Another advantage of the HF-ICP jet source is that it works without electrodes, i.e., contamination of the layers produced by the jet source electrode material are prevented.",
"One disadvantage of the known HF-ICP jet sources and plasma systems using such plasma jet sources is the high temperatures in the plasma jet of several thousand degrees Celsius to which the substrate that is to be coated is also exposed.",
"The choice of usable substrates is considerably restricted in this regard.",
"Another disadvantage is that to produce layer systems on the substrate, such as those currently produced by CVD methods, for example, a minimum energy of particles impinging on the substrate is often necessary.",
"This is true in particular in deposition of DLC (diamond-like carbon) coatings.",
"This minimum energy of the impinging ions is not achieved with the high-frequency inductively coupled plasma jet sources known in the past and the plasma systems equipped with them."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a plasma system having an inductively coupled HF plasma jet source and a method of producing functional coatings, the deposition of which requires a higher energy of the ions from the plasma striking the substrate than provided by conventional HF-ICP plasma jet sources.",
"In particular, the object of the present invention is to provide a plasma system and a method with which it is possible to produce hard carbon coatings, i.e., DLC layers, in a low vacuum.",
"The plasma system according to the present invention having a high-frequency inductively coupled plasma jet source and the method according to the present invention for producing a functional coating on a substrate have the advantage over the related art that they permit the production of layers and/or layer systems which could previously be produced only by CVD methods.",
"Because the pressure prevailing in the chamber in deposition of layers with HF-ICP plasma jet sources is reduced from 100 mbar to 1 bar, as is customary, to less than 50 mbar, this advantageously yields the result that a sufficient mean free path length is available to the ions present in the plasma, and thus the electric voltage supplied to the substrate electrode and thereby to the substrate connected to the substrate electrode also manifests an adequate effect with regard to the desired acceleration.",
"In addition, this pressure significantly lowers the thermal load on the substrate being coated.",
"On the other hand, it is advantageous that the plasma system according to the present invention requires only a low vacuum of less than 50 mbar, even in the chamber in which the substrate is located, to ensure adequate ion energies for the desired coating processes and/or surface modifications.",
"It is possible to reliably and quickly produce a low vacuum in the chamber of the plasma system by using conventional pumping devices, and this requires much less equipment and is less time consuming in comparison with a medium-high or high vacuum, as required for CVD methods.",
"Due to the relatively high pressure in the chamber of the plasma system, it is also possible to process workpieces made of sintered materials which release a large amount of gas.",
"It is also advantageous that due to the applied substrate electrode voltage and the selected pressure in the plasma system the reactive properties of the HF-ICP plasma are improved for producing a coating and/or achieving a surface modification on the substrate.",
"Thus, on the whole, the method according to the present invention is a high-rate deposition method which is implementable in a low vacuum in short process times, i.e., pumping times, and which is suitable for deposition, i.e., production, of coatings on all substrates that are of industrial relevance, e.g., high-grade steel, other electrically conducting materials, ceramics, etc.",
"Thus, due to the fact that the high-frequency plasma jet source and the chamber containing the substrate communicate only through the outlet orifice of the plasma jet source, it is readily possible to maintain a pressure difference between the interior of the plasma jet source and the interior of the chamber.",
"It is furthermore advantageous if the action of the electric voltage on the substrate electrode is correlated with a periodic variation in intensity of the plasma jet produced by the plasma jet source.",
"The thermal load on the substrate is further reduced in this way, and also physical disequilibrium states occur in the plasma to a great extent due to the fluctuation in intensity of the plasma jet, which is preferably also extinguished periodically, and these disequilibrium states may be used to deposit novel coatings on the substrate.",
"With regard to the choice of materials supplied to the plasma jet source, i.e., the plasma jet produced, for producing the functional coating on the substrate, there are also a great variety of options, including those proposed in German Published Patent Application No.",
"199 58 474.Other advantageous refinements of the present invention involve providing a cooling device for cooling the substrate and/or a movable mount, preferably a mount that is movable in all directions or rotatable in space, so that the substrate is easily oriented relative to the plasma jets and may also be cooled during plasma deposition if desired.",
"It is particularly advantageous if the electric voltage supplied to the substrate electrode is an electric voltage which is variable over time, in particular a pulsed electric voltage, which may also be provided with an adjustable positive or negative offset voltage and/or pulsed with a virtually freely selectable pulse-pause ratio.",
"Another parameter that is easily varied and adapted to the requirements of the individual case is also the shape of the envelope of the electric voltage that is variable over time and may have, for example, a saw-tooth, triangular or sinusoidal curve.",
"The electric voltage used may also be a direct voltage.",
"Other parameters that are easily varied with regard to the concrete signal shape of the electric voltage used include its edge steepness, its amplitude, and its frequency.",
"In addition, it should be emphasized that the variation over time in the voltage injected into the substrate electrode need not necessarily be periodic."
],
[
"FIELD OF THE INVENTION The present invention relates to a plasma system having a high-frequency inductively coupled plasma jet source and to a method of producing a functional coating on a substrate.",
"BACKGROUND INFORMATION Applying functional coatings to substrates is a widely used method of imparting desired properties to the surfaces of workpieces and/or components.",
"A conventional method of producing such functional layers is by plasma coating in a medium-high or high vacuum, which requires complex evacuation techniques and yields relatively low coating rates.",
"Therefore, this method is time-intensive and expensive.",
"Thermal plasmas in particular which allow higher coating rates in the range of mm/h to able achieved are suitable for coating substrates in the atmospheric and subatmospheric pressure range.",
"In this regard, reference is made to R. Henne, Contribution to Plasma Physics, 39 (1999) pages 385-397, for example.",
"Of the thermal plasma sources, the high-frequency inductively coupled plasma jet source (HF-ICP jet source) is especially promising, such as that described by E. Pfender and C. H. Chang “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6th Workshop on Plasma Technology, Technical University of Illmenau, 1998.Furthermore, German Published Patent Application No.",
"199 58 474 has proposed a method of producing functional layers by using such a plasma jet source.",
"The advantages of the HF-ICP jet source include the range of operating pressures in the source, usually extending from 50 mbar to 1 bar or more, and also the great variety of materials that may be used and deposited with such a plasma jet source.",
"In particular, due to the fact that the starting materials are introduced axially into the very hot plasma jet, hard substances having a very high melting point may also be used.",
"Another advantage of the HF-ICP jet source is that it works without electrodes, i.e., contamination of the layers produced by the jet source electrode material are prevented.",
"One disadvantage of the known HF-ICP jet sources and plasma systems using such plasma jet sources is the high temperatures in the plasma jet of several thousand degrees Celsius to which the substrate that is to be coated is also exposed.",
"The choice of usable substrates is considerably restricted in this regard.",
"Another disadvantage is that to produce layer systems on the substrate, such as those currently produced by CVD methods, for example, a minimum energy of particles impinging on the substrate is often necessary.",
"This is true in particular in deposition of DLC (diamond-like carbon) coatings.",
"This minimum energy of the impinging ions is not achieved with the high-frequency inductively coupled plasma jet sources known in the past and the plasma systems equipped with them.",
"SUMMARY OF THE INVENTION An object of the present invention is to provide a plasma system having an inductively coupled HF plasma jet source and a method of producing functional coatings, the deposition of which requires a higher energy of the ions from the plasma striking the substrate than provided by conventional HF-ICP plasma jet sources.",
"In particular, the object of the present invention is to provide a plasma system and a method with which it is possible to produce hard carbon coatings, i.e., DLC layers, in a low vacuum.",
"The plasma system according to the present invention having a high-frequency inductively coupled plasma jet source and the method according to the present invention for producing a functional coating on a substrate have the advantage over the related art that they permit the production of layers and/or layer systems which could previously be produced only by CVD methods.",
"Because the pressure prevailing in the chamber in deposition of layers with HF-ICP plasma jet sources is reduced from 100 mbar to 1 bar, as is customary, to less than 50 mbar, this advantageously yields the result that a sufficient mean free path length is available to the ions present in the plasma, and thus the electric voltage supplied to the substrate electrode and thereby to the substrate connected to the substrate electrode also manifests an adequate effect with regard to the desired acceleration.",
"In addition, this pressure significantly lowers the thermal load on the substrate being coated.",
"On the other hand, it is advantageous that the plasma system according to the present invention requires only a low vacuum of less than 50 mbar, even in the chamber in which the substrate is located, to ensure adequate ion energies for the desired coating processes and/or surface modifications.",
"It is possible to reliably and quickly produce a low vacuum in the chamber of the plasma system by using conventional pumping devices, and this requires much less equipment and is less time consuming in comparison with a medium-high or high vacuum, as required for CVD methods.",
"Due to the relatively high pressure in the chamber of the plasma system, it is also possible to process workpieces made of sintered materials which release a large amount of gas.",
"It is also advantageous that due to the applied substrate electrode voltage and the selected pressure in the plasma system the reactive properties of the HF-ICP plasma are improved for producing a coating and/or achieving a surface modification on the substrate.",
"Thus, on the whole, the method according to the present invention is a high-rate deposition method which is implementable in a low vacuum in short process times, i.e., pumping times, and which is suitable for deposition, i.e., production, of coatings on all substrates that are of industrial relevance, e.g., high-grade steel, other electrically conducting materials, ceramics, etc.",
"Thus, due to the fact that the high-frequency plasma jet source and the chamber containing the substrate communicate only through the outlet orifice of the plasma jet source, it is readily possible to maintain a pressure difference between the interior of the plasma jet source and the interior of the chamber.",
"It is furthermore advantageous if the action of the electric voltage on the substrate electrode is correlated with a periodic variation in intensity of the plasma jet produced by the plasma jet source.",
"The thermal load on the substrate is further reduced in this way, and also physical disequilibrium states occur in the plasma to a great extent due to the fluctuation in intensity of the plasma jet, which is preferably also extinguished periodically, and these disequilibrium states may be used to deposit novel coatings on the substrate.",
"With regard to the choice of materials supplied to the plasma jet source, i.e., the plasma jet produced, for producing the functional coating on the substrate, there are also a great variety of options, including those proposed in German Published Patent Application No.",
"199 58 474.Other advantageous refinements of the present invention involve providing a cooling device for cooling the substrate and/or a movable mount, preferably a mount that is movable in all directions or rotatable in space, so that the substrate is easily oriented relative to the plasma jets and may also be cooled during plasma deposition if desired.",
"It is particularly advantageous if the electric voltage supplied to the substrate electrode is an electric voltage which is variable over time, in particular a pulsed electric voltage, which may also be provided with an adjustable positive or negative offset voltage and/or pulsed with a virtually freely selectable pulse-pause ratio.",
"Another parameter that is easily varied and adapted to the requirements of the individual case is also the shape of the envelope of the electric voltage that is variable over time and may have, for example, a saw-tooth, triangular or sinusoidal curve.",
"The electric voltage used may also be a direct voltage.",
"Other parameters that are easily varied with regard to the concrete signal shape of the electric voltage used include its edge steepness, its amplitude, and its frequency.",
"In addition, it should be emphasized that the variation over time in the voltage injected into the substrate electrode need not necessarily be periodic.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a first embodiment of a plasma system having an ICP plasma jet source in a sectional view.",
"FIG.",
"2 shows an example of a variation in intensity of the plasma jet over time.",
"FIG.",
"3a shows a first photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3b shows a second photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3c shows a third photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3d shows a fourth photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3e shows a fifth photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3f shows a sixth photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3g shows a seventh photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"3h shows an eighth photograph of the plasma jet emerging from the plasma jet source as a function of time, the jet being pulsed according to FIG.",
"2.FIG.",
"4 shows a photograph of a plasma jet emerging from a plasma jet source at a high velocity.",
"FIG.",
"5 shows a detail of the plasma jet source according to FIG.",
"1.DETAILED DESCRIPTION The present invention is based on a high-frequency, inductively coupled plasma jet source such as that known in a similar form from E. Pfender and C. H. Chang, “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6th Workshop on Plasma Technology, Technical University of Illmenau.",
"In addition, a coating method similar to that already described in German Published Patent Application No.",
"199 58 474 is implemented with this system.",
"Specifically, FIG.",
"1 shows in detail a high-frequency, inductively coupled plasma jet source 5 having a pot-shaped burner body 25, which has on one side an outlet orifice 26, e.g., circular in design and having a diameter of 1 cm to 10 cm, provided with an orifice constrictor 22, which is preferably variably adjustable, i.e., shaped.",
"In addition, plasma jet source 5 has a coil 17 integrated into burner body 25 in the area of outlet orifice 26, e.g., a water-cooled copper coil which may also be coiled around burner body 25 as an alternative.",
"In addition, an inlet 10 in the form of a conventional injector for supplying an injector gas 11, a first cylindrical sleeve 14, and a second cylindrical sleeve 15 are provided on the side of burner body 25 facing away from outlet orifice 26.First sleeve 14 and/or second sleeve 15 are each designed to be concentric with the side wall of burner body 25, second sleeve 15 being used primarily to keep a plasma 21 produced in a plasma generating space 27 in burner body 25 away from the walls of burner body 25.To do so, an enveloping gas 13 is introduced into burner body 25 through a suitable gas inlet between first sleeve 14 and second sleeve 15, this burner body also having the function of blowing plasma 21 thus produced out of plasma jet source 5 through outlet orifice 26 in such a way as to form a plasma jet 20 which is largely bundled when it impinges on substrate 19 in a chamber 40 on a substrate carrier 18, which in the specific example also functions as substrate electrode 18 at the same time, so that a functional coating is produced and/or deposited on the substrate.",
"Enveloping gas 13 in the example presented here is argon, which is supplied to plasma jet source 5 at a gas flow rate of 5000 sccm to 100,000 sccm (standard cubic centimeters per minute), in particular 20,000 sccm to 70,000 sccm.",
"FIG.",
"1 also shows that coil 17 is electrically connected to a high-frequency generator 16 with which an electric power of 500 W to 50 kW, in particular 1 kW to 10 kW, at a high frequency of 0.5 MHz to 20 MHz is injected into coil 17, and is also input into the plasma 21 ignited and maintained in plasma generating space 27.In a preferred embodiment, high-frequency generator 16 is provided with an essentially known electric component 28, with which the intensity of plasma jet 20 in its action on substrate 19 is variable periodically at a frequency of 1 Hz to 10 kHz, in particular 50 Hz to 1 kHz, between an adjustable upper intensity limit and an adjustable lower intensity limit.",
"Plasma jet 20 is preferably also extinguished periodically for an adjustable period of time, i.e., a selectable pulse-pause ratio.",
"FIG.",
"1 also shows that a central gas 12 may be supplied through first sleeve 14 to the area between first sleeve 14 and inlet 10.This is, for example, an inert gas or an inert gas to which a gas that reacts with injector gas 11 is added.",
"A gaseous, microscale or nanoscale precursor material, a suspension of such a precursor material or a reactive gas in particular is supplied to plasma 20 through inlet 10 and/or an additional supply device located between first sleeve 14 and inlet 10, so that this reactive gas in modified form, in particular after undergoing a chemical reaction or a chemical activation, produces the desired functional coating on substrate 19 or is integrated into the functional coating there.",
"As an alternative, however, plasma 21 may also be used to merely produce a chemical modification in the surface of substrate 19, so that the desired functional coating is thereby produced on the surface of substrate 19.If a precursor material is supplied to plasma 21, i.e., plasma jet 20, preferably at the same time a carrier gas for this precursor material, in particular nitrogen and/or a reactive gas for a chemical reaction with the precursor material, in particular oxygen, nitrogen, ammonia, a silane, acetylene, methane or hydrogen is also at the same time.",
"Either inlet 10, the feeder device for supplying central gas 12 or the feeder device for supplying enveloping gas 13 is suitable for supplying these gases.",
"As an alternative or in addition, another feeder device, e.g., an injector or a gas spray, may also be provided in chamber 40 to supply a reactive gas and/or a precursor material into plasma jet 20 which is already emerging from plasma jet source 5.The precursor material used is preferably an organic compound, an organosilicon or organometallic compound, which may thus be supplied to plasma 21 and/or plasma jet 20 in a gaseous or liquid form, as microscale or nanoscale powder particles, as a liquid suspension, in particular having microscale or nanoscale particles suspended in it, or as a mixture of gaseous or liquid substances with solids.",
"Through a suitable choice of the individual gases, i.e., the reactive gases supplied and/or central gas 12 and injector gas 11 as well as the choice of precursor materials, as explained in detail in German Published Patent Application No.",
"199 58 474, e.g., a metal silicide, a metal carbide, a silicon carbide, a metal oxide, a silicon oxide, a metal nitride, a silicon nitride, a metal boride, a metal sulfide, amorphous carbon, diamond-like carbon (DLC) or a mixture of these materials in the form of a layer or a sequence of layers may be produced on substrate 19.In addition, the method proposed here is also suitable for cleaning or carbonizing or nitriding the surface of substrate 19.FIG.",
"1 also shows that substrate electrode 18 is coolable with cooling water 39 through a cooling water inlet 31, and substrate electrode 18 and thus also substrate 19 are movable in chamber 40 through an appropriate mount 32.Both mount 32 and cooling water supply 31 are electrically separated from substrate electrode 18, which receives the electric voltage, by insulation 34.Substrate 19 together with substrate electrode 18 is preferably situated on movable mount 32, in particular movable in all directions and/or rotatable in space.",
"In addition, substrate electrode 18 is electrically connected to a substrate generator 37 which provides an electric voltage to be injected into substrate electrode 18 and thereby also into substrate 19.To do so, generator lead 36 is provided between substrate generator 37 and substrate electrode 18.Specifically, substrate electrode 18 together with substrate generator 37 receives a direct electric voltage or an alternating voltage having an amplitude between 10 V and 5 kV, in particular between 50 V and 300 V, and a frequency between 0 Hz and 50 MHz, in particular between 1 kHz and 100 kHz.",
"This direct voltage or alternating voltage may also be provided continuously or intermittently with a positive or negative offset voltage.",
"The injected electric voltage is preferably an electric voltage that is variable over time, in particular a pulsed electric voltage having a pulse-pause ratio which may be selected on the basis of simple preliminary tests in the individual case, and an offset voltage which optionally also varies over time, e.g., with regard to polarity.",
"The variation in the electric voltage over time is preferably adjusted so that its envelope varies according to a unipolar or bipolar saw-tooth, triangular or sinusoidal curve.",
"Additional parameters include the amplitude and polarity of the offset voltage, the edge steepness of the individual pulses of the electric voltage injected, the frequency (carrier frequency) of this voltage and its amplitude.",
"A particularly preferred embodiment of the method according to the present invention provides for the change in intensity of plasma jet 20 via high-frequency generator 16 and electric component 28 integrated into it, which may also be designed as a separate electric component and may then be connected between coil 17 and high-frequency generator 16, in particular the pulsing of plasma jet 20 to be correlated in time with the variation in or pulsation of the electric voltage injected into substrate electrode 18.This time correlation is also preferably a pulsation (in phase opposition or with a time offset) of the intensity of plasma jet 20 with respect to the change in or pulsation of the electric voltage.",
"FIG.",
"1 also shows that a first pressure area 30 prevails in the interior of plasma jet source 5, where a pressure of 1 mbar to 2 bar prevails, in particular 100 mbar to 1 bar.",
"Then a second pressure area 33 prevails in the interior of chamber 40.In addition, conventional pumping equipment (not shown) is connected to chamber 40 to maintain the pressure difference between the first and second pressure areas 30, 33 and in particular to keep the pressure in chamber 40 less than 50 mbar, in particular between 1 mbar and 10 mbar.",
"Therefore, a pressure gradient always exists between the interior of plasma jet source 5 and the interior of chamber 40, although gas is continuously supplied to plasma jet source 5 during operation, and plasma jet source 5 and chamber 40 are connected via outlet orifice 26.The pressures are preferably selected so that the ratio of the pressure in first pressure area 30 to the pressure in second pressure area 33 is greater than 1.5, in particular greater than 3.For example, a pressure difference of more than 100 mbar is maintained between plasma generating space 27 in the interior of plasma jet source 5 and the interior of chamber 40 by a pumping device (not shown) which is connected to chamber 40.Suitable materials for substrate 19 include both electrically conducting materials and electrically insulating materials, the latter with a suitable choice of the variable voltage on the substrate electrode.",
"In addition, the reduction in thermal load on substrate 19 due to the cooling device and in particular the pulsation of plasma jet 20 results in even thermally sensitive substrates such as polymers being usable.",
"FIG.",
"2 illustrates how the intensity of plasma jet 20 is varied in accordance with the variation in the voltage supplied to coil 17, by varying the voltage supplied by high-frequency generator 16 cooperating with electric component 28 through a variation in the voltage supplied to the coil.",
"In particular, in a further refinement of FIG.",
"2, the voltage on coil 17 may also be zero temporarily, so that plasma jet 20 is extinguished in this period of time.",
"FIGS.",
"3a through 3h directly show plasma jet 20 emerging in chamber 40 from outlet orifice 26 through orifice restrictor 22.The typical distance between outlet orifice 26 and substrate 19 is 5 cm to 50 cm.",
"FIGS.",
"3a through 3h show how plasma jet 20 emerges from outlet orifice 26 at a high intensity initially according to FIG.",
"3a at time t=0, then this intensity drops significantly according to FIG.",
"3b, so that plasma jet 20 is extinguished completely shortly thereafter, then the plasma jet is reignited according to FIGS.",
"3c through 3e, and swings back briefly before then expanding continuously according to FIGS.",
"3f through 3h, so that after approx.",
"13.3 ms, the starting state according to FIG.",
"3a has almost been reached again.",
"This pulsation of plasma jet 20 according to FIGS.",
"3a through 3h is induced by a change in the HF electric power injected into coil 17.FIG.",
"4 illustrates how plasma jet 20 emerges from outlet orifice 26 at a high velocity due to a suitably high pressure difference between the interior of plasma jet source 5 and the interior of chamber 40, i.e., the pressure gradient with respect to chamber 40, as explained above, and impinges on substrate 19 with a correspondingly high velocity.",
"In particular, compression nodes 23 (Mach nodes) are clearly discernible in FIG.",
"4, indicating that the velocity of the particles in plasma jet 20 is of the same order of magnitude as the velocity of sound.",
"It may also be greater than the velocity of sound.",
"The high velocity of plasma jet 20, which is influenceable via the pressure difference, achieves the result that not only are deep cavities on the surface of substrate 19 acted upon by plasma 21, but also the diffusion interface between the surface of substrate 19 and plasma jet 20 is reduced in size, which facilitates diffusion of reactive plasma components onto the surface of substrate 19 and thus shortens and/or intensifies the required processing time of substrate 19 with plasma jet 20.FIG.",
"5 illustrates a detail from FIG.",
"1, where plasma jet source 5 is shown again on an enlarged scale.",
"In particular, the arrangement of inlet 10 and the embodiment of first sleeve 14 and second sleeve 15 are more clearly discernible."
]
] | Patent_10240474 |
[
[
"Plasma system and method of producing a functional coating",
"A plasma system has at least one inductively coupled high-frequency plasma jet source having a burner body delimiting a plasma generating space having an outlet orifice for the plasma jet, a coil surrounding the plasma generating space in some areas, an inlet for supplying a gas and/or a precursor material into the plasma generating space and a high-frequency generator which is connected to the coil for igniting the plasma and for injecting an electric power into the plasma.",
"The plasma jet source has an electric component using which the intensity of the plasma jet is variable periodically over time.",
"In addition, a method of producing the functional coating on a substrate by using this plasma system is described."
],
[
"1-17.",
"(canceled) 18.A plasma system, comprising: at least one inductively coupled high-frequency plasma jet source, including: a burner body delimiting a plasma generating space and including an outlet orifice for a plasma jet and at least one inlet orifice for supplying at least one of a gas and a precursor material into the plasma generating space, a coil surrounding the plasma generating space in some areas, and a high-frequency generator connected to the coil for igniting a plasma and for injecting an electric power into the plasma; and an electric component for periodically varying an intensity of the plasma jet over time.",
"19.The plasma system as recited in claim 18, wherein: the electric component is one of: integrated into the high-frequency generator, and connected between the coil and the high-frequency generator.",
"20.The plasma system as recited in claim 18, wherein: the burner body is designed in the form of a pot, the coil one of surrounds the burner body in the vicinity of the outlet orifice and is integrated into the burner body, an injector gas is supplied through the at least one inlet orifice into the plasma generating space, and at least one second inlet is provided for at least one of supplying a central gas that reacts with an injector gas into the plasma generating space and supplying an enveloping gas that separates the burner body from the plasma produced therein in at least some areas.",
"21.The plasma system as recited in claim 18, wherein: the precursor material produces a functional coating on a substrate using the plasma jet.",
"22.The plasma system as recited in claim 20, wherein: the enveloping gas separates the burner body from the plasma concentrically around the plasma.",
"23.The plasma system as recited in claim 18, further comprising: a chamber that communicates with the plasma jet source via the outlet orifice; a substrate that is exposed to the plasma jet and is placeable in the chamber; a substrate generator; and a substrate electrode that is electrically connected to the substrate generator and on which the substrate is placed.",
"24.The plasma system as recited in claim 23, further comprising: a feeder device provided in the chamber for supplying at least one of a reactive gas and the precursor material to the plasma jet.",
"25.The plasma system as recited in claim 24, wherein: the feeder device includes one of an injector and a gas spray.",
"26.A method of producing a functional coating on a substrate placed in a chamber, comprising: causing a high-frequency inductively coupled plasma jet source to produce a plasma having reactive particles; causing the plasma entering through an outlet orifice as a plasma jet from the plasma jet source into the chamber connected thereto to act on the substrate so that the functional coating is one of produced and deposited on the substrate; and periodically varying an intensity of the plasma jet on the substrate over time.",
"27.The method as recited in claim 26, wherein: the intensity of the plasma jet is varied at a frequency of 1 Hz to 10 kHz.",
"28.The method as recited in claim 26, wherein: the intensity of the plasma jet is varied at a frequency of 50 Hz to 1 kHz.",
"29.The method as recited in claim 26, wherein: the intensity of the plasma jet is varied between an adjustable upper limit and an adjustable lower limit.",
"30.The method as recited in claim 26, wherein: the intensity of the plasma jet is periodically extinguished for an adjustable period of time.",
"31.The method as recited in claim 26, further comprising: injecting an electric power of 500 watt to 50 kW into the plasma via a coil at a high frequency of 0.5 MHz to 20 MHz.",
"32.The method as recited in claim 26, further comprising: injecting an electric power of 1 kW to 10 kW into the plasma via a coil at a high frequency of 0.5 MHz to 20 MHz.",
"33.The method as recited in claim 26, further comprising: discharging the plasma as a jet out of the plasma jet source; and introducing the plasma into the chamber by supplying a gas at a gas flow rate of 5,000 sccm to 100,000 sccm to the plasma jet source through the outlet orifice.",
"34.The method as recited in claim 33, wherein: the gas includes argon, and the gas flow rate is 20,000 sccm to 70,000 sccm.",
"35.The method as recited in claim 26, further comprising: supplying one of at least one precursor material, a suspension of the at least one precursor material, and a reactive gas to at least one of the plasma through an inlet in the plasma jet source and the plasma jet through a feeder device located in the chamber.",
"36.The method as recited in claim 35, wherein: the at least precursor material includes one of a gaseous material, a microscale material, and a nanoscale material.",
"37.The method as recited in claim 35, wherein: the at least one precursor material forms the functional coating on the substrate after undergoing one of a chemical reaction and a chemical activation.",
"38.The method as recited in claim 35, wherein: the at least one precursor material is integrated into the substrate.",
"39.The method as recited in claim 26, further comprising: supplying to the plasma at least one of a carrier gas for a precursor material and a reactive gas for a chemical reaction with the precursor material.",
"40.The method according to claim 39, wherein: the carrier gas includes argon, and the reactive gas includes one of oxygen, nitrogen, ammonia, silane, acetylene, methane, and hydrogen.",
"41.The method as recited in claim 39, wherein: the precursor material includes one of an organic compound, an organosilicon compound, and an organometallic compound that is supplied to at least one of the plasma and the plasma jet in one of a gaseous form, a vapor form, and a liquid form as one of microscale powder particles, nanoscale powder particles, a liquid suspension in which is suspended one of microscale particles and nanoscale particles, and a mixture of one of gaseous and liquid substances with solids.",
"42.The method as recited in claim 26, wherein: a pressure gradient is produced at least intermittently between an interior of the chamber and a plasma generating space, causing acceleration of particles contained in the plasma jet onto the substrate.",
"43.The method as recited in claim 26, wherein: the plasma jet source is operated at a pressure of 1 mbar to 2 bar in an interior thereof, and a pressure in an interior of the chamber is kept below 50 mbar.",
"44.The method as recited in claim 26, wherein: the plasma jet source is operated at a pressure of 50 mbar to 1 bar in an interior thereof, and a pressure in an interior of the chamber is kept between 1 mbar and 10 mbar.",
"45.The method as recited in claim 26, wherein: the substrate is arranged on a substrate electrode that is acted upon by an electric voltage of 10 V to 5 kV at a frequency of 0 to 50 MHz.",
"46.The method according to claim 26, wherein: the substrate is arranged on a substrate electrode which is acted upon by an electric voltage of 50 V to 300 V at a frequency of 1 kHz to 100 kHz.",
"47.The method as recited in claim 45, wherein: the voltage is one of pulsed in phase opposition and varied over time in correlation with a change in the intensity of the plasma jet."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>Applying functional coatings to substrates is a widely used method of imparting desired properties to the surfaces of workpieces and/or components.",
"A conventional method of producing such functional layers is by plasma coating in a medium-high or high vacuum, which requires complex evacuation techniques and yields relatively low coating rates.",
"Therefore, this method is time-intensive and expensive.",
"Thermal plasmas in particular which allow high coating rates in the range of mm/h to be achieved are suitable for coating substrates in the atmospheric and subatmospheric pressure range.",
"Of the thermal plasma sources, the high-frequency inductively coupled plasma jet source (HF-ICP jet source) is especially promising, such as that known from E. Pfender and C. H. Chang “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6 th Workshop on Plasma Technology, Technical University of Illmenau, 1998.Furthermore, German Published Patent Application No.",
"199 58 474 has proposed a method of producing functional layers by using such a plasma jet source.",
"The advantages of the HF-ICP jet source include the range of operating pressures in the source, usually extending from 50 mbar to 1 bar or more, and also the great variety of materials that may be used and deposited with such a plasma jet source.",
"In particular, due to the fact that the starting materials are introduced axially into the very hot plasma jet, hard substances having a very high melting point may also be used.",
"Another advantage of the HF-ICP jet source is that it works without electrodes, i.e., contamination of the layers produced by the jet source electrode material are prevented.",
"One disadvantage of the known HF-ICP jet sources and plasma systems using such plasma jet sources is the high temperatures in the plasma jet of several thousand degrees Celsius to which the substrate that is to be coated is also exposed.",
"To this extent, the choice of usable substrates is considerably restricted."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a plasma system having an HF inductively coupled plasma jet source and a method implementable therewith for producing a functional coating on a substrate, so that the thermal load on the substrate in producing the functional coating is greatly reduced in comparison with the related art.",
"The plasma system according to the present invention and the method according to the present invention for producing a functional coating on a substrate by varying the plasma intensity over time have the advantage over the related art that the temperature to which the substrate is exposed may be reduced to less than half in comparison with the related art.",
"It is also advantageous that using the plasma system according to the present invention, the advantages of a high-rate deposition method taking place in the atmospheric or near-atmospheric pressure range are combined with a reduction in substrate temperature and a change in the chemical processes in the plasma thus produced.",
"It is advantageous in particular that the method according to the present invention is not a high-vacuum method, so that complex equipment for producing such a high vacuum is not necessary.",
"It is also advantageous that the method according to the present invention may also be used with virtually all industrially relevant substrate materials such as steel and, as the case may be, also polymers, and at the same time a wide selection of materials and/or compositions of the coating to be produced, e.g., including insulating materials such as ceramics or sintered metals, is also available.",
"In addition, due to the periodic change in intensity of the plasma jet, preferably to such an extent that the plasma jet is extinguished between intensity peaks, there is regularly a chemical and/or physical disequilibrium state in the plasma jet, which permits promising approaches for production of previously unknown layer systems, e.g., ceramic layers or layer systems.",
"In particular, the aforementioned disequilibrium states, which occur mainly on igniting and extinguishing the plasma, constitute a considerable portion of the total time during which the plasma jet acts on the substrate, given suitable pulsation of the plasma jet over time, so that chemical processes taking place in these disequilibrium states become a dominant factor for the entire deposition of functional coatings using such a plasma system and/or plasma jet source.",
"It is thus particularly advantageous if, in addition to a plasma jet whose intensity varies periodically, the substrate being coated is situated on a substrate electrode which receives a voltage which is in phase opposition or is varied, preferably pulsed, over time in correlation with the change in intensity of the plasma jet.",
"Another advantageous embodiment of the present invention provides for the supply of gas and/or precursor material to the plasma, i.e., the plasma jet, to be correlated in time, in particular synchronized, with the varying intensity of the plasma jet.",
"Finally, it is advantageous if, at least temporarily during the production of the functional layer, the greatest possible pressure gradient is produced between the inside of the chamber and the plasma generating space, causing an acceleration of particles contained in the plasma jet onto the substrate.",
"In this way, even deeper cavities in the surface of the substrate are better reached by the plasma and there is improved adhesion of the functional layer to the substrate."
],
[
"FIELD OF THE INVENTION The present invention relates to a plasma system having a high-frequency inductively coupled plasma jet source and a method of producing a functional coating on a substrate.",
"BACKGROUND INFORMATION Applying functional coatings to substrates is a widely used method of imparting desired properties to the surfaces of workpieces and/or components.",
"A conventional method of producing such functional layers is by plasma coating in a medium-high or high vacuum, which requires complex evacuation techniques and yields relatively low coating rates.",
"Therefore, this method is time-intensive and expensive.",
"Thermal plasmas in particular which allow high coating rates in the range of mm/h to be achieved are suitable for coating substrates in the atmospheric and subatmospheric pressure range.",
"Of the thermal plasma sources, the high-frequency inductively coupled plasma jet source (HF-ICP jet source) is especially promising, such as that known from E. Pfender and C. H. Chang “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6th Workshop on Plasma Technology, Technical University of Illmenau, 1998.Furthermore, German Published Patent Application No.",
"199 58 474 has proposed a method of producing functional layers by using such a plasma jet source.",
"The advantages of the HF-ICP jet source include the range of operating pressures in the source, usually extending from 50 mbar to 1 bar or more, and also the great variety of materials that may be used and deposited with such a plasma jet source.",
"In particular, due to the fact that the starting materials are introduced axially into the very hot plasma jet, hard substances having a very high melting point may also be used.",
"Another advantage of the HF-ICP jet source is that it works without electrodes, i.e., contamination of the layers produced by the jet source electrode material are prevented.",
"One disadvantage of the known HF-ICP jet sources and plasma systems using such plasma jet sources is the high temperatures in the plasma jet of several thousand degrees Celsius to which the substrate that is to be coated is also exposed.",
"To this extent, the choice of usable substrates is considerably restricted.",
"SUMMARY OF THE INVENTION An object of the present invention is to provide a plasma system having an HF inductively coupled plasma jet source and a method implementable therewith for producing a functional coating on a substrate, so that the thermal load on the substrate in producing the functional coating is greatly reduced in comparison with the related art.",
"The plasma system according to the present invention and the method according to the present invention for producing a functional coating on a substrate by varying the plasma intensity over time have the advantage over the related art that the temperature to which the substrate is exposed may be reduced to less than half in comparison with the related art.",
"It is also advantageous that using the plasma system according to the present invention, the advantages of a high-rate deposition method taking place in the atmospheric or near-atmospheric pressure range are combined with a reduction in substrate temperature and a change in the chemical processes in the plasma thus produced.",
"It is advantageous in particular that the method according to the present invention is not a high-vacuum method, so that complex equipment for producing such a high vacuum is not necessary.",
"It is also advantageous that the method according to the present invention may also be used with virtually all industrially relevant substrate materials such as steel and, as the case may be, also polymers, and at the same time a wide selection of materials and/or compositions of the coating to be produced, e.g., including insulating materials such as ceramics or sintered metals, is also available.",
"In addition, due to the periodic change in intensity of the plasma jet, preferably to such an extent that the plasma jet is extinguished between intensity peaks, there is regularly a chemical and/or physical disequilibrium state in the plasma jet, which permits promising approaches for production of previously unknown layer systems, e.g., ceramic layers or layer systems.",
"In particular, the aforementioned disequilibrium states, which occur mainly on igniting and extinguishing the plasma, constitute a considerable portion of the total time during which the plasma jet acts on the substrate, given suitable pulsation of the plasma jet over time, so that chemical processes taking place in these disequilibrium states become a dominant factor for the entire deposition of functional coatings using such a plasma system and/or plasma jet source.",
"It is thus particularly advantageous if, in addition to a plasma jet whose intensity varies periodically, the substrate being coated is situated on a substrate electrode which receives a voltage which is in phase opposition or is varied, preferably pulsed, over time in correlation with the change in intensity of the plasma jet.",
"Another advantageous embodiment of the present invention provides for the supply of gas and/or precursor material to the plasma, i.e., the plasma jet, to be correlated in time, in particular synchronized, with the varying intensity of the plasma jet.",
"Finally, it is advantageous if, at least temporarily during the production of the functional layer, the greatest possible pressure gradient is produced between the inside of the chamber and the plasma generating space, causing an acceleration of particles contained in the plasma jet onto the substrate.",
"In this way, even deeper cavities in the surface of the substrate are better reached by the plasma and there is improved adhesion of the functional layer to the substrate.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows a first embodiment of a plasma jet source in a sectional view.",
"FIG.",
"2 shows the periodic characteristic of the voltage across the plasma jet source over time.",
"FIGS.",
"3a through 3h show the plasma jet, whose intensity varies as a function of time.",
"FIG.",
"4 shows an exemplary embodiment of a plasma system having a plasma jet source.",
"FIG.",
"5 shows a second exemplary embodiment of a plasma system having a plasma jet source.",
"FIG.",
"6 shows a plasma jet exiting from the plasma jet source according to FIG.",
"4.DETAILED DESCRIPTION The present invention is based first on a plasma jet source 5, which is known fundamentally from E. Pfender and C. H. Chang, “Plasma Spray Jets and Plasma Particulate Interaction: Modeling and Experiments,” Convention Volume of the 6th Workshop on Plasma Technology, Technical University of Illmenau, 1998, or German Published Patent Application No.",
"199 58 474.This plasma jet source 5 has a pot-shaped burner body 25 having a rear injector as an inlet 10 for supplying an injector gas 11.In addition, a first cylindrical sleeve 14 and a second cylindrical sleeve 15 are provided, a central gas 12 being supplied to the interior of first sleeve 14 through a suitable first inlet (not shown) and an enveloping gas 13 being supplied to the interior of second sleeve 15 through a suitable second inlet (not shown).",
"Burner body 25 also has an outlet orifice 26 in the form of a circle, for example, having a diameter of 1 cm to 10 cm, for example, in particular 3 cm on its side facing away from inlet 10, this opening being provided with an orifice restrictor 22 shaped according to the shape of plasma jet 21 to be produced.",
"In addition, a water-cooled copper coil 17 is integrated into burner body 25 in the vicinity of outlet orifice 26 and is electrically connected to an HF generator 16.When injector gas 11, central gas 12 and enveloping gas 13 are supplied, an electric power of 500 W to 50 kW, in particular 1 kW to 10 kW, is injected into the interior of burner body 25 at a high frequency of 0.5 MHz to 20 MHz, in particular 0.5 to 4 MHz, via coil 17 and HF generator 16, so that a plasma 21 of reactive particles emerging from outlet orifice 26 of burner body 25 in the form of a plasma jet 20 may be ignited and sustained in a plasma generating space 27.This plasma jet 20 then continues to act on a substrate 19, e.g., a piece of steel situated on a substrate carrier or a substrate electrode 18, situated opposite outlet orifice 26, e.g., at a distance of 5 cm to 50 cm.",
"FIG.",
"1 also shows that, additionally in comparison with the related art, an electric component 28 is integrated into HF generator 16, for periodically varying the electric power delivered by HF generator 16 to coil 17, so that the intensity of the plasma jet thus produced is also varied periodically in this way.",
"Injector gas 11 introduced into burner body 25 through inlet 10, i.e., the injector is, for example, a precursor material for producing a functional coating on substrate 19.For example, a gas which reacts with injector gas 11 is suitable as central gas 12, which is optionally added.",
"Enveloping gas 13, preferably argon, protects the walls of burner body 25 and also causes plasma 21 which is produced to be blown as a jet out of plasma jet source 5 through outlet orifice 26, so that it acts as a bundled or guided plasma jet 20 on substrate 19.To do so, enveloping gas 13 is introduced at a gas flow rate of 5000 sccm to 100,000 sccm (standard cubic centimeters per minute), preferably 20,000 sccm to 70,000 sccm.",
"The periodic variation in intensity of plasma jet 20 using electronic component 28, which may also be connected as a separate component between coil 17 and HF generator 16, takes place at a frequency of 1 Hz to 10 kHz, in particular 50 Hz to 1 kHz, between an adjustable upper limit and an adjustable lower limit of intensity.",
"The lower limit is preferably set at zero, so that plasma jet 20 is periodically extinguished for a predefinable period of time.",
"As an alternative, however, it is likewise possible to provide for the intensity of plasma jet 20 to be varied between the two limits given above in virtually any desired form, e.g., without plasma 21 being extinguished in the meantime.",
"In particular, the intensity of plasma jet 20 may be varied in a rectangular, sinusoidal, sawtooth, rectangular or triangular form, optionally with a suitable offset, with respect to the resulting envelope.",
"For additional known details regarding the design of plasma jet source 5, as well as the methods performed with it for producing functional layers, reference is made to German Published Patent Application No.",
"199 58 474.FIG.",
"2 illustrates how the intensity of plasma jet 20 varies as a function of time when electric component 28 controls the HF generator, i.e., suitably varies the supply of electric power to coil 17.HF voltage U applied to coil 17 is plotted on the ordinate in FIG.",
"2, its absolute value and the shape of the envelope being approximately proportional to the intensity of plasma jet 20.The intensity of plasma jet 20 from plasma jet source 5 and emerging from outlet orifice 26 of burner body 25 is explained with the help of FIGS.",
"3a through 3h for various times t between t=0.3 ms and t=13.3 ms.",
"Plasma jet 20 emerges from outlet orifice 26 initially with a high intensity at time t=0 according to FIG.",
"3a; then this intensity diminishes significantly according to FIG.",
"3b, so that plasma jet 20 is completely extinguished shortly thereafter.",
"Next, plasma jet 20 is reignited according to FIGS.",
"3c through 3e, swinging back shortly before expanding continuously according to FIGS.",
"3f through 3h, so that after approx.",
"13.3 ms it has almost reached the starting state according to FIG.",
"3a again.",
"The pulsing of plasma jet 20 according to FIGS.",
"3a through 3h is induced by a change in the HF electric power injected into coil 17.FIGS.",
"3a through 3h show in particular that plasma jet 20 emerges from plasma jet source 5 with little divergence as a free and largely bundled plasma jet 20.FIG.",
"4 illustrates a plasma system having a conventional chamber 40 in which substrate 19 is situated on a substrate carrier 18 opposite outlet orifice 26 of plasma jet source 5, so that plasma jet 20 passes through outlet orifice 26 and enters into chamber 40, where it is able to act on substrate 19.In particular, FIG.",
"4 shows that substrate carrier 18 is secured in chamber 40 with the help of a mount 32 and is coolable with cooling water 39 through a cooling water inlet 31.According to FIG.",
"4, a first pressure p1 between 10 mbar and 2 bar, in particular between 50 mbar and 1 bar, prevails in the interior of plasma jet source 5, i.e., in a first pressure area 30, and a second pressure p2, which is a function of the size of outlet orifice 26 and the amount of enveloping gas 13 or injector gas 10 as well as the efficiency of the pumps connected to chamber 40, prevails in the interior of chamber 40, i.e., in a second pressure area 33.This pressure p2 is preferably much lower than pressure p1 due to a appropriately high pumping power, i.e., it is less than 100 mbar, for example, in particular less than 10 mbar.",
"In addition, argon is used as enveloping gas 13 in FIG.",
"4 and is introduced into plasma jet source 5 at a gas flow rate of 40,000 sccm td 60,000 sccm.",
"In particular, due to the fact that according to FIG.",
"4, plasma jet source 5, i.e., the production of plasma 21 is spatially separated from the production of the functional coating on substrate 19, it is possible to use plasma jet 20 in chamber 40 at a pressure of 1 mbar to 10 mbar, for example, as a result of which plasma jet 20 is greatly accelerated and expands at the same time on emerging from plasma jet source 5, in the interior of which a much higher pressure of 500 mbar, for example, prevails.",
"This is indicated schematically in FIG.",
"4 by plasma jet 20, which widens on emerging from outlet orifice 26.Such an expanded and accelerated plasma jet 20 in which the reactive particles present in the plasma jet may easily reach the velocity of sound or even supersonic velocity is capable of penetrating into deep cavities present on substrate 19.In addition, such an expansion of plasma jet 20 results in sudden cooling of plasma 21, which in turn further lowers the thermal load on substrate 19 and also yields chemical advantages with regard to an increase in plasma coating rate and an increase in the quality of the coating thus produced on the substrate.",
"In particular, the spatial separation of the processes in chamber 40 from plasma jet source 5 guarantees that plasma jet 20 may also be used in chamber 40 in a medium-high vacuum of 1 mbar without any change in the plasma mode, which is determined by plasma jet source 5.The acceleration and expansion of plasma jet 20 in the operating mode according to FIG.",
"4 is explained in greater detail with the help of FIG.",
"6, which illustrates the discharge of such an accelerated plasma jet 20 out of outlet orifice 26 into chamber 40.In particular, compression nodes 23 (Mach nodes) are clearly discernible there, indicating that plasma jet 20 is emerging from outlet orifice 26 at the velocity of sound, and thus the particles contained in plasma jet 20 at substrate 19 are at least partially accelerated to a velocity comparable to or even greater than the velocity of sound in plasma jet 20.The marked pressure gradient between plasma jet source 5 and chamber 40, which aspirates the ionized gas present in plasma 21, i.e., plasma jet 20, into chamber 40 at a high velocity, also achieves the result that the two regions 30, 33 are largely separated with respect to the pressures prevailing there via outlet orifice 26.The respective pressures are preferably selected so that the ratio of the pressure in first pressure range 30 to the pressure in second pressure area 33 is greater than 1.5, in particular greater than 3.For example, a pressure difference of more than 100 mbar between plasma generating space 27 in the interior of plasma jet source 5 and the interior of chamber 40 is maintained via a pumping device (not shown) which is connected to chamber 40.On the whole, the acceleration and expansion of plasma jet 20 according to FIG.",
"4 have the advantage that even complex geometries of substrate 19 may be provided with coatings with no problem, and the larger cross-sectional area of plasma jet 20 at substrate 19 results in a shortened coating time and at the same time an improved homogeneity in the coating of substrate 19.Mount 32 according to FIG.",
"4 is also used to introduce substrate 19 into plasma jet 20, so that plasma flows around it and works the surfaces of substrate 19, which are provided with or coated with the desired functional layer.",
"Due to the high velocity of the reactive particles in plasma jet 20, not only do deeper cavities in the substrate 19 come in contact with plasma 21 but also the diffusion boundary layer between substrate 19 and plasma 21 is reduced, which facilitates diffusion of reactive plasma constituents onto the surface of substrate 19 and thus shortens the duration of the treatment of substrate 19 with plasma jet 20.FIG.",
"5 illustrates another embodiment of a plasma system having a plasma jet source 5.In addition to FIG.",
"4, substrate 19 here is placed on a substrate electrode 18 which is connected to a substrate generator 37 by a generator feeder line 36 so that substrate 19 may be acted upon by an electric voltage.",
"Due to the electric power, i.e., voltage thus injected into substrate electrode 18, ions in plasma 21, i.e., plasma jet 20 are accelerated toward substrate 19, where they impinge with an increased energy.",
"Moreover FIG.",
"5 shows a conventional insulation 34 for electric separation of mount 32 and cooling water inlet 31 from substrate electrode 18.For effective movement of substrate 19 with respect to plasma jet 20 in particular during production of the functional layer, mount 32 of substrate 19 is also preferably designed to be rotatable and movable in all three directions in space.",
"In particular, substrate generator 37 applies an electric voltage of typically 10 V to 5 kV, in particular 5 V to 300 V, at a frequency of 0 Hz to 500 MHz, in particular 1 kHz to 50 kHz to substrate electrode 18.In a preferred variant of the exemplary embodiment according to FIG.",
"5, the voltage generated by substrate generator 37 is also varied, preferably pulsed, with plasma jet source 5 in a manner that correlates in time with the variation in intensity of plasma jet 21, in particular in phase opposition.",
"Variants of the exemplary embodiment according to FIG.",
"5 provide for expedient variations in the form of the electric voltage injected into substrate electrode 18, these variations being adapted to the individual case.",
"To do so, their amplitude, frequency and/or edge steepness may be varied, an offset of a positive or negative direct voltage may be used or the voltage may be pulsed.",
"In addition, it is not obligatory but merely advantageous if the electric voltage is varied periodically.",
"With regard to the pressures in first pressure area 30 and second pressure area 33 according to FIG.",
"5, it is advantageous if a pressure of more than 1 mbar, in particular 50 mbar to 1 bar, prevails inside plasma jet source 5, whereas a much lower pressure of less than 50 mbar, in particular 1 mbar to 10 mbar is maintained in chamber 40.This pressure ensures that an adequate mean free path length of the ions from plasma 21 prevails in chamber 40, so that the electric voltage applied to substrate electrode 18 does not result in a perceptible effect, i.e., an acceleration of the ions present in plasma jet 20 toward substrate 19.To this extent, this exemplary embodiment according to FIG.",
"5 operates in chamber 40 with a much lower pressure than the pressure generally used in producing coatings with the help of inductively coupled HF plasma jet sources.",
"In this way, by using the plasma system according to FIG.",
"5, it is readily possible to produce coatings on substrate 19 which may otherwise be produced only by CVD processes, in particular DLC (“diamond-like carbon”) layers.",
"On the whole, a great variety of coatings may be produced on substrate materials which are of industrial relevance with the help of the exemplary embodiments described above, and substrates 19 may be either electrically conducting or electrically insulating.",
"In particular, hard carbon layers may be produced in a low vacuum with the help of the above-mentioned plasma system and the method described here.",
"In addition, the plasma system described here may also be used for treating the surface of substrate 19, e.g., for carbonizing, nitriding or heating it.",
"With regard to materials that may be introduced into plasma jet source 5 for deposition of a coating on substrate 19 within the context of the preceding examples, reference is first made to German Published Patent Application No.",
"199 58 474.In particular, at least one gaseous or microscale or nanoscale precursor material, a suspension of such a precursor material, or a reactive gas is supplied to plasma 21 in chamber 40 through inlet 10, which is designed as an injector, in plasma jet source 5 and/or plasma jet 20 through a feeder device (not shown here), so that it forms the functional coating in a modified form on substrate 19 or is integrated into it, in particular after undergoing a chemical reaction or a chemical activation.",
"In addition, a carrier gas for the precursor material, in particular argon and/or a reactive gas for a chemical reaction with the precursor material, in particular oxygen, nitrogen, ammonia, a silane, acetylene, methane or hydrogen may be supplied to plasma 21 in plasma jet source 5, i.e., through the feeder device also located in chamber 40.The precursor material is preferably an organic, organosilicon or organometallic compound which is supplied to plasma 21 and/or plasma jet 20 in a gaseous or liquid form, as microscale or nanoscale powder particles, as a liquid suspension, in particular having microscale or nanoscale particles suspended in it, or as a mixture of gaseous or liquid substances containing solids.",
"In this way, a layer or a sequence of layers containing a metal silicide, a metal carbide, a silicon carbide, a metal oxide, a silicon oxide, a metal nitride, a silicon nitride, a metal boride, a metal sulfide, amorphous carbon, diamond-like carbon or a mixture of these materials may be produced as a functional coating on substrate 19 by using the plasma system explained here and the method explained here.",
"In conclusion, it should also be pointed out that HF generator 16 is preferably a tetrode generator, which makes is possible to generate plasma jet 20 with intensity modulation in a particularly simple manner as described here, so that the resulting temperature of substrate 19 is determined essentially by the average power of plasma jet 20 due to this intensity modulation.",
"Thus, the method according to the present invention also makes it possible to use very high powers of plasma jet 20 for short periods of time without creating a thermal overload on substrate 19.Furthermore, it is also possible for the regulation of the gases supplied to plasma jet source 5, e.g., central gas 12, injector gas 11 or enveloping gas 13 to correlate with the modulation of intensity of plasma jet 20 over time and/or the variation in the electric voltage applied to substrate electrode 18 over time."
]
] | Patent_10240477 |
[
[
"System and method for establishing eletronic business systems for supporting communications servuces commerce",
"A comprehensive electronic business support system comprises three layers: (1) the business layer, including various smart components which unify data and business processes across all customer interactions; (2) the integration layer, including various communications messaging interfaces and enterprise application integration adpaters, which provide a flexible, automated, and process driven solution for integrating across business applications and operations support systems; and (3) the presentation layer, including various customer views, which are presented via particular business portals.",
"A smart component server provides the core services and comprehensive business process logic required to successfully conduct business online.",
"The communications messaging interfaces integrate with back-office systems for functions such as billing, provisioning, and interconnection."
],
[
"1.An electronic business support system, comprising a smart component server, wherein said smart component server comprises: a plurality of activity smart components, each of which performs a specific business logic; a plurality of service smart components which provide common infrastructure capabilities required by said activity smart components; a plurality of communications messaging interfaces that transport messages from said support system to external systems; a plurality of transport adapters, which facilitate connectivity with external systems; an administrator console, which is a Java-based tool that uses XML files to map object attributes to a database which is associated with said electronic business support system; wherein said smart components are implemented as stateless session enterprise Java beans.",
"2.The electronic business support system of claim 1, wherein each of said activity smart components performs a communications-specific function.",
"3.The electronic business support system of claim 1, wherein said activity smart components comprise: an offer management server, which provides services for creating and presenting a collection of offer instances; a quote management server, which provides services for creating a quote upon placing said collection of offer instances into a shopping cart; and an order management server, which provides services for creating an order upon quoting all quote items.",
"4.The electronic business support system of claim 3, further comprising: a business rule management server, which provides services for defining and managing business rules that maintain valid relationships between objects.",
"5.The electronic business support system of claim 4, wherein said business rules are created in said administrator console using templates.",
"6.The electronic business support system of claim 4, wherein said business rules are invoked during a quote process.",
"7.The electronic business support system of claim 3, further comprising: a shop management server, which provides services for directing users in on-line shopping process.",
"8.The electronic business support system of claim 3, further comprising: a customer management server, which provides services for managing a customer, their accounts, and their communications-specific products in a hierarchical structure.",
"9.The electronic business support system of claim 3, further comprising: a sales management server, which provides services for sales management and promotion on-line.",
"10.The electronic business support system of claim 3, further comprising: an interaction management server, which provides services in support of interactions on-line.",
"11.The electronic business support system of claim 3, further comprising: a profile management server, which provides services for creating and managing profiles on-line.",
"12.The electronic business support system of claim 3, further comprising: a bill presentment management server, which provides services for presenting and managing bill points on-line.",
"13.The electronic business support system of claim 3, further comprising: a payment management server, which provides services for making payments on-line.",
"14.The electronic business support system of claim 3, further comprising: a trouble ticket server, which provides services for collecting and dispatching trouble tickets.",
"15.The electronic business support system of claim 3, further comprising: a notice management server, which provides services for posting notices and targeting notices to specific customers.",
"16.The electronic business support system of claim 1, wherein said service smart components comprise: a database access service, which includes a plurality of value objects and a data cursor service; an interconnect service, which uses said communications messaging interfaces and said transport adapters connecting said activity smart components with external systems; a security service, which uses standard Internet security protocols to ensure browser to server security and server to server security; a logging service, which creates files that report on specific actions within the electronic business support system; and a sequence service, which establishes default sequences for page flows; wherein said value objects include domain objects, which represent a specific row in a database table, and display objects, which are containers that deliver attribute values to JSP pages.",
"17.The electronic business support system of claim 16, wherein said data cursor service is implemented as a stateful enterprise Java bean; wherein said data cursor service creates a scrollable, read-only cursor; and wherein said read-only cursor is used when a large result set from a query is expected.",
"18.The electronic business system of claim 16, wherein said logging service comprises: a file logger, which sends information to a text file; and a console logger, which sends information to a screen.",
"19.The electronic business system of claim 18, wherein said logging service allows adding loggers to meet individual business needs.",
"20.The electronic business system of claim 16, wherein said logging service maintains an XML file which specifies a list of values that are used to determine whether to log a message.",
"21.The electronic business system of claim 20, wherein said list of values includes a severity level of an event, an architectural area wherein said event occurs, and a functional stage wherein said event occurs.",
"22.The electronic business support system of claim 1, wherein each of said communications messaging interfaces is associated with a routing policy that dispatches said communications messaging interface to a particular transport adapter which is used to handle a particular message.",
"23.The electronic business support system of claim 22, wherein each of said communications messaging interfaces is mapped to a plurality of strategies.",
"24.The electronic business support system of claim 23, wherein said strategies include: at least one source strategy that is used by said interconnect server to build up a payload message that is associated with one of said communications messaging interfaces before being dispatched to said transport adapter; and at least one target strategy that is used to update said support system with data from inbound messages.",
"25.The electronic business support system of claim 1, wherein said communications messaging interfaces are associated with multiple transport adapters to send different messages.",
"26.The electronic business support system of claim 1, wherein said transport adapters comprise: a file adapter, which prints out all information that it receives from a corresponding communications messaging interface; a business rules adapter, which determines and displays valid attachment points in a quote process; an electronic mail adapter, which sends electronic mail messages based on a corresponding communications messaging interface; an enterprise application integration adapter, which facilitates connection between said electronic business support system with an external system; and an open adapter, which is to be configured by the user according to a particular communications messaging interface.",
"27.The electronic business support system of claim 26, wherein said open adapter's handlers are stubs that are used by a plurality of communications messaging interfaces.",
"28.The electronic business support system of claim 26, wherein said electronic mail adapter's handlers determine who receives a particular electronic mail message and what template is used to create said message.",
"29.The electronic business support system of claim 26, wherein said enterprise application integration adapter is a stateless enterprise bean that runs within the electronic business support system's process space; and wherein said enterprise application integration adapter uses source and target connection models that run within said external system's process space to transmit data between the electronic business support system and said external system.",
"30.The electronic business support system of claim 29, wherein when a routing policy dispatches a communications messaging interface to said enterprise application integration adapter, a handler for said communications messaging interface creates an event; and wherein said event retains an association to the data referenced by said communications messaging interface.",
"31.The electronic business support system of claim 30, wherein said communications messaging interface comprises a plurality of objects that contain references to data which is to be sent as events into said external system.",
"32.The electronic business support system of claim 30, wherein said handler invokes a source connection model that populates an object with actual data and sends said object to said enterprise application integration adapter's automator model.",
"33.The electronic business support system of claim 26, wherein said external system is Vitria BusinessWare.",
"34.A method for publishing events from an electronic business support system to an enterprise application integration adapter, comprising the steps of: creating an automator model to handle an event that is associated with a particular communications messaging interface; setting up a routing policy for said communications messaging interface; creating a source connection model; populating a source strategy and a publisher channel for said communications messaging interface; and restarting the electronic business support system.",
"35.A method for updating an electronic business support system by subscribing events from an enterprise application integration adapter, comprising the steps of; creating an automator model to handle an event that is associated with a particular communications messaging interface; setting up a routing policy for said communications messaging interface; creating a target connection model; populating a target strategy and a subscriber channel for said communications messaging interface; retrieving, by said automator model, the published event data; publishing, by said automator model, a corresponding event to said subscriber channel; and updating, by said target connection model, the electronic business support system.",
"36.A process for sending a message from an electronic business support system to external systems, said process comprises the steps of: (a) calling a first method on an interconnect service with a communications messaging interface's code value; (b) returning, by said first method, an empty object for said communications messaging interface; (c) calling a second method on said object with an object identifier and a fully qualified class name of a domain object that contains event information to be sent; wherein said source strategy uses said event information to retrieve data for said message; (d) calling a third method on said interconnect service with a reference to said object that is created in step (b); (e) performing, by said interconnect service, a database lookup to determine a routing policy to be used for said communications messaging interface; (f) if a default routing policy is used or if no policy is specified, sending said communications messaging interface to a file adapter with a default payload; (g) if a generic routing policy is used, performing a database lookup in a table for generic policies to determine which adapter is used; (h) if a vendor product policy is used, performing a database lookup in a table for vendor product policies to determine which adapter is used; (i) performing a database lookup in a table for strategy policies to determine a source strategy to be used for said communications messaging interface and said payload; (j) sending said message, by said file adapter, out to the external systems; and (k) updating, by said interconnect service, said database.",
"37.The process of claim 36, wherein if a communications messaging interface has multiple entries, steps (e) through (k) are repeated.",
"38.A method for sending a communications messaging interface from the electronic business support system to an external system, comprising the steps of: obtaining a reference to an enterprise Java bean of said interconnect service; identifying a code that is associated with said communications messaging interface; creating an object for said communications messaging interface; adding event messages which are specific to said communications messaging interface; and sending said communications messaging interface to said interconnect service.",
"39.A method for adding a new communications messaging interface for the electronic business support system, comprising the steps of: confirming said communications messaging interface has not been defined in the electronic business support system; giving a code which assigns a unique identification to said communications messaging interface; adding said code and its corresponding routing policy class name to the database; creating source and target strategies; and making an entry for said communications messaging interface in the database.",
"40.A process for an electronic business support system to receive a message from external systems, said process comprises the steps of: receiving, by a transport adapter, a message from an external system; wherein said message includes a communications messaging interface code value, a communications messaging interface identification, and a payload type code; calling, by said transport adapter, a first method on said interconnect service to build a communications messaging interface based on said code value; returning, by said first method, an empty object for said communications messaging interface; wherein said adapter populates said communications messaging interface with the needed data before proceeding to the next step; calling, by said transport adapter, a second method on said interconnect service; performing, by said interconnect service, a database lookup in a table for strategy policies to determine a target strategy to be used for said communications messaging interface and a given payload; and calling, by said target strategy, a smart component server to update said support system.",
"41.An electronic business portal for the electronic business support system of claim 1, comprising: a plurality of JSP pages for dynamic content in a Web site; a plurality of display policy interfaces that access and manipulate data for display; a Web session controller which maintains user session state; a plurality of page transition policies which determine navigation logic through said Web site; and a plurality of resource bundles which display values or messages corresponding to codes in constant classes.",
"42.The electronic business portal of claim 41, wherein said Web session controller is a servlet that provides load balancing through a session-level algorithm that weighs server load information and routes requests accordingly.",
"43.The electronic business portal of claim 42, wherein said Web session controller provides fail-over by replacing HTTP session information across nodes in a cluster and maintains session state via a cookie-based session identification.",
"44.The electronic business portal of claim 41, wherein said Web session controller describes the lifecycle of data, using a page scope which passes data back and forth between a page and a display policy; and wherein said page scope begins when a JSP starts rendering and expires when the JSP finishes rendering.",
"45.The electronic business portal of claim 41, wherein said Web session controller describes the lifecycle of data, using a request scope which passes data from transition policies to JSPs and display policies or passes data between parent and sub JSPs; and wherein said request scope begins with a browser request that passes in HTML form data, lives throughout the life of said request from a client, until the destination JSP is rendered and sent back to the browser.",
"46.The electronic business portal of claim 41, wherein said Web session controller describes the lifecycle of data, using a session scope which stores data between requests; and wherein said session scope is created when a user first makes a request to said Web session controller and is destroyed when the session times out.",
"47.The electronic business portal of claim 41, wherein said Web session controller describes the lifecycle of data, using an application scope which begins with a server startup and ends when the server is shut down.",
"48.The electronic business portal of claim 41, wherein said transition policies are Java files that contain navigation and validation logic.",
"49.The electronic business portal of claim 41, wherein said transition policies access the electronic business support system's API to perform business logic.",
"50.A process for inserting a nested flow into a page flow at a transition point, comprising the steps of: invoking, by a transition policy for an originating page, a method which passes the identification for the first page of said nested flow, the identification of said originating page of said page flow, and the identification for the destination page of said page flow to a Web session controller; storing, by said Web session controller, said identifications; directing, by said Web session controller, the user into said nested flow; informing, by a transition directive on the last page of said nested flow, said Web session controller to re-execute the transition policy for said originating page; and when the transition policy for said originating page is re-executed, directing, by said Web session controller, the user to said destination page of said page flow; wherein said nested flow is a set of JSP pages representing a process that can be entered from multiple places within a portal.",
"51.The process of claim 50, further comprising the step of: performing at least one business process on the last page of said nested flow before re-executing the transition policy for said originating page.",
"52.A method for preventing a user from repeating a transition in a page flow, comprising the steps of: flagging a non-repeatable transition policy associated with a page; and directing the user to a destination page; wherein said destination page was determined during a prior execution of said non-repeatable transition policy.",
"53.A tag library that is used to introduce dynamic content into a Web page, comprising: a first source tag, which retrieves a display object; a second source tag which retrieves a list of display objects; a third source tag which creates a list of bundle item objects; a fourth source tag which creates any value object; a fifth source tag which creates any list of value objects; a first presentation tag which iterates through a list of objects and makes each object available through a scripting variable; a second presentation tag which checks a user's permissions; a third presentation tag which displays JSP content when said first presentation tag determines the user has permission; a fourth presentation tag which displays JSP content when said first presentation tag determines the user does not have permission; and a fifth presentation tag which provides access to validation information created by a transition policy.",
"54.The tag library of claim 53, wherein said first presentation tag also provides a counter variable.",
"55.The tag library of claim 53, wherein each of said source tags invokes a corresponding display policy interface which is used to modify said source tag's behavior.",
"56.The tag library of claim 53, wherein said first presentation tag invokes a corresponding display policy interface which is used to modify said presentation tag's behavior.",
"57.A process for electronically dealing a client request, comprising the steps of: receiving, by a Web session controller in an electronic business portal, a request; performing, by said Web session controller, a database lookup to invoke a transition policy; calling, by said transition policy, an activity smart component; calling, by said activity smart component, a data access service to create and persist value objects; passing control back to said transition policy; returning, by said transition policy, control to said Web session controller; resolving, by said Web session controller, page IID in the request to a URL; invoking a corresponding JSP page; invoking corresponding display policies; and returning a dynamic page to the client via HTTP.",
"58.An electronic business support system, comprising a smart component server, wherein said smart component server comprises: a plurality of activity smart components which encapsulate communications-specific functionality and business logic; a plurality of service smart components which provide common infrastructure capabilities required by said activity smart components; a plurality of communications messaging interfaces that transport messages from said support system to external systems; a plurality of transport adapters, which facilitate connectivity with external systems; an administrator console, which is a Java-based tool that uses XML files to map object attributes to a database which is associated with said electronic business support system; wherein said activity smart components include an offer management server which enables a user to present an offer collection; wherein said offer collection includes a set of determinants; wherein each of said determinants is represented by a page which includes a plurality of determinant items; and wherein each of said determinant items represents an offer on a determinant.",
"59.The electronic business support system of claim 58, wherein each offer instance is created by a user's each selection of a determinant item to a temporary collection object; wherein the user's determination of all selections constitutes a collection of offer instances to be added in a shopping cart; and wherein said offer collection has an associated sequence that establishes a default order in which said determinants are displayed within offer collection.",
"60.The electronic business support system of claim 58, wherein each of said determinants is selected from a group of determinant types comprising: fixed type, which is used when a collection is fixed; choose one type, which is used when the user must select one item from a list of items; choose multiple type, which is used when the user can select more than one item; and information request type, which is used to collect specific information form the user.",
"61.The electronic business support system of claim 58, wherein each of said determinant types corresponds to a specific JSP page whose transition policies provide functionality that is needed to create said offer collection.",
"62.The electronic business support system of claim 61, wherein said sequence may be overridden by an individual determinant item in a determinant if a next determinant to be displayed depends upon the selection of said individual determinant item.",
"63.A method for providing an offer collection over the Internet, comprising the steps of: presenting a set of determinants by pages, wherein each of said determinants is represented by a page which includes a plurality of determinant items, and wherein each of said determinant items represents an offer on a determinant; adding each offer instance which is created by a user's each selection of a determinant item to a temporary collection object; and upon the user's determination of all selections, placing a collection of offer instances in a shopping cart; wherein said offer collection has an associated sequence that establishes a default order in which said determinants are displayed within offer collection.",
"64.The method of claim 63, wherein only one determinant is needed for said offer collection; and wherein a composite of determinant items in said determinant is fixed.",
"65.The method of claim 63, wherein at least one of said determinant items includes a predetermined list of alternative items from which the user can make choices.",
"66.The method of claim 65, wherein said offer instances are associated with each other.",
"67.The method of claim 65, wherein said offer instances are not associated with each other.",
"68.The method of claim 63, wherein each of said determinants is selected from a group of determinant types comprising: fixed type, which is used when a collection is fixed; choose one type, which is used when the user must select one item from a list of items; choose multiple type, which is used when the user can select more than one item; and information request type, which is used to collect specific information from the user.",
"69.The method of claim 68, wherein each of said determinant types corresponds to a specific JSP page whose transition policies provide functionality that is needed to create said offer collection.",
"70.The method of claim 60, wherein said sequence may be overridden by an individual determinant item in a determinant if a next determinant to be displayed depends upon the selection of said individual determinant item.",
"71.The method of claim 64, wherein all offer instances associated with said composite of determinant items are transitioned together.",
"72.The method of claim 66, wherein an association between two offer instances is established by a transition which allows the user to upgrade from one offer instance associated with an assigned product to another predetermined offer.",
"73.An electronic business support system, comprising a smart component server, wherein said smart component server comprises: a plurality of activity smart components which encapsulate communications-specific functionality and business logic; a plurality of service smart components which provide common infrastructure capabilities required by said activity smart components; a plurality of communications messaging interfaces that transport messages from said support system to external systems; a plurality of transport adapters, which facilitate connectivity with external systems; an administrator console, which is a Java-based tool that uses XML files to map object attributes to a database which is associated with said electronic business support system; and wherein said smart component server organizes billing point objects and assigned product objects on an object hierarchy, said object hierarchy comprising: a root object, which is created during registration; a plurality of billing point objects, each of which is associated with said root object; and a plurality of assigned product objects, each of which is associated with one of said billing point objects; wherein each of said billing point object stores all information that is needed for billing purposes; wherein each of said assigned product objects is associated with an offer instance that is created by the user's selection of said product from a determinant; and wherein each of said objects has a specific object state that is maintained by said smart component server through API calls to a particular smart component.",
"74.The electronic business support system of claim 73, wherein said root object may be either in a pending state, which indicates that a request to add a new root or modifying an existing root is a process, or in an active state, which indicates that a request to add a new root or modifying an existing root has been approved.",
"75.The electronic business support system of claim 73, wherein said root object holds a set of information comprising: price group identity, which determines what products are available to a customer or a partner and at what price; logo URL, which is a link to where a logo for a customer or a partner can be stored and then displayed on a portal page; document URL, which is a link to where external documents regarding a customer or partner can be kept; and unique authorization identity of a customer or a partner.",
"76.The electronic business support system of claim 73, wherein each of said assigned product objects has an object state that is selected from a group of state types comprising: not ordered, which indicates a product is in a shop process but has not yet been ordered; pending, which indicates that an action on a product is not complete; rejected, which indicates that a product is not provisioned due to an error or unresolved issue; provisioned, which indicates that a process for provisioning a product is complete; unprovisioned, which indicates that a process for unprovisioning a product is complete; suspended, which indicates that a service on a product is still active and its charges can be invoiced, but no modifications can be made to said product; and canceled, which indicates that any pending action on a product is canceled.",
"77.The electronic business support system of claim 73, wherein each of said assigned product objects is either a provisioned product object or a composite product object; wherein said provisioned product object has a subscriber relationship upon which an account that said provisioned product is assigned to is charged on a periodic basis; and wherein said composite product object represents a group of products that are ordered as a bundle.",
"78.The electronic business support system of claim 73, wherein said object hierarchy can be displayed by category of summary point type.",
"79.An electronic business support system, comprising a smart component server, wherein said smart component server comprises: a plurality of activity smart components which encapsulate communications-specific functionality and business logic; a plurality of service smart components which provide common infrastructure capabilities required by said activity smart components; a plurality of communications messaging interfaces that transport messages from said support system to external systems; a plurality of transport adapters, which facilitate connectivity with external systems; an administrator console, which is a Java-based tool that uses XML files to map object attributes to a database which is associated with said electronic business support system; wherein said activity smart components include a quote management server which enables a user to create a quote upon placing a collection of offer instances into a shopping cart; wherein each offer instance is associated with a quote item; wherein each quote item is associated with the root object in a current object hierarchy in a session; wherein said quote management server displays charges for all items in said quote; and wherein said quote has a specific quote state that is maintained by said smart component server via API calls to said quote management server.",
"80.The electronic business support system of claim 79, wherein said object state for a quote is selected from a group of state types comprising: new, which indicates that a quote is created; pending configuration, which indicates that at least one quote item has not yet been completely configured; configured, which indicates that all quote items are completely configured; quoted, which indicates that all quote items are completely configured and contain all the information that is needed to order an entire quote; expired, which indicates that a quote has expired and can no longer be ordered; pending approval, which indicates that a quote cannot be completed until al least one requirement is met; canceled, which indicates that the user canceled a quote; rejected, which indicates that requirements for approval were not met; and completed, which indicates that a quote has been ordered.",
"81.The electronic business support system of claim 79, wherein each quote item has a specific object state that is maintained by said smart component server via API calls to said quote management server; and wherein said object state for a quote item is selected from a group of state types comprising: removed, which indicates that a quote item has been removed from said quote; pending configuration, which indicates that a quote item is not completely configured; configured, which indicates that a quote item is completely configured; expired, which indicates that either the offer associated with an quote item has expired or the quote that said quote item is associated with has expired; canceled, which indicates that the quote that a quote item is associated with has been canceled; completed, which indicates that a quote item has been ordered; replaced, which indicates that a quote item has been replaced by another quote item through a supplemental order; supplemental cancel, which indicates that a quote item was canceled through a supplemental order; and supplemental complete, which indicates that a quote item cannot be changed through a supplemental order.",
"82.The electronic business support system of claim 79, wherein said quote can be held for a predetermined amount of time before it expires.",
"83.The electronic business support system of claim 82, wherein if a price for the determinant item that is represented by a quote item changes while a quote is held, the offer instance that is associated with said quote item retains an association with the price at the time that said offer instance was created.",
"84.The electronic business support system of claim 83, wherein if an offer itself expires while a quote is held, the offer instance that is associated with said offer remains valid for said quote.",
"85.The electronic business support system of claim 79, wherein a number of values for each offer instance may be automatically populated during the transition from shopping cart to quote by using a plug-in; and wherein said plug-in is a dynamically deployed algorithm that can be implemented to perform a specific task.",
"86.The electronic business support system of claim 85, wherein an attachment point plug-in is used to attach assigned products; and wherein said plug-in, by default, attaches all assigned products to an account.",
"87.The electronic business support system of claim 85, wherein a parameter plug-in is used to set values for a parameter; and wherein said plug-in, by default, accepts values set as default for said parameter.",
"88.The electronic business support system of claim 85, wherein a service identifier plug-in is used; and wherein said plug-in, by default, obtains a next available service identifier.",
"89.The electronic business support system of claim 85, wherein a service address plug-in is used to set an address at which an item will be provisioned; and wherein said plug-in, by default, adopts the address associated with the root object with which said quote is also associated.",
"90.The electronic business support system of claim 79, wherein when several of the same determinant item are purchased, a number of values may be automatically populated from one quote item to all quote items that are associated with the same determinant item by using a plug-in.",
"91.The electronic business support system of claim 90, wherein an attachment point plug-in is used to attach assigned products.",
"92.The electronic business support system of claim 90, wherein a parameters plug-in is used to set all parameter values to match the parameter values for the original quote item.",
"93.The electronic business support system of claim 90, wherein a service identifier plug-in is used to obtain a next available service identifier.",
"94.The electronic business support system of claim 90, wherein a service address plug-in is used to copy the service address that is associated with the original quote item.",
"95.The electronic business support system of claim 90, wherein a shipping address plug-in is used to copy the shipping address that is associated with the original quote item.",
"96.The electronic business support system of claim 90, wherein a requested date plug-in is used to copy the requested provisioning date of the original quote item.",
"97.An electronic business support system, comprising a smart component server, wherein said smart component server comprises: a plurality of activity smart components which encapsulate communications-specific functionality and business logic; a plurality of service smart components which provide common infrastructure capabilities required by said activity smart components; a plurality of communications messaging interfaces that transport messages from said support system to external systems; a plurality of transport adapters, which facilitate connectivity with external systems; an administrator console, which is a Java-based tool that uses XML files to map object attributes to a database which is associated with said electronic business support system; wherein said activity smart components include an order management server which enables a user to create an order upon quoting all quote items; wherein each of said quote items is associated with an order item; wherein said order is associated with the same hierarchy root object in a session as the user is associated with; and wherein said order has a specific object state that is maintained by said smart component server via API calls to said order management server.",
"98.The electronic business support system of claim 97, wherein said object state for an order is selected from a group of state types consisting of: new, which indicates that a quote is ordered; pending dispatch, which indicates that all order items are created; dispatched, which indicates that an order is dispatched to a provisioning system; completed, which indicates that an order is provisioned; rejected, which indicates that an order is rejected; pending cancellation, which indicates that a request is submitted to cancel an order; and supplemented, which indicates that a user changed an order by creating a supplemental order.",
"99.The electronic business support system of claim 97, wherein each order item has a specific object state that is maintained by said smart component server via API calls to said order management server; and wherein said object state for an order item is selected from a group of state types consisting of: new, which indicates that a quote item is ordered and is now an order item; dispatched, which indicates that an order item is dispatched to a provisioning system; completed, which indicates that an order item is provisioned; rejected, which indicates that an order item is rejected; pending cancellation, which indicates that a request is submitted to cancel an order; canceled, which indicates that an order item is canceled; and supplemented, which indicates that an order has been replaced by an order which is created through a supplemental quote.",
"100.A process for submitting a supplemental order according to claim 99, comprising the steps of: creating a new quote which is populated with copies of each order item from the original order; and determining, by a supplemental order plug-in, which order items are eligible for supplement; wherein said plug-in, by default, only allows the order items that are in state dispatched to be supplemented; changing determined items; and submitting the supplemental order.",
"101.The process of claim 100, wherein the step of changing determined items comprises a plurality of actions, each of which is selected from a group of action types comprising: reconfiguring, which allows certain values to be changed; replacing, which allows for substitution of an existing order item with a new item; and canceling, which moves an item to state canceled.",
"102.The process of claim 100, further comprising the steps of: adding the items that were not eligible for supplement to the supplemental order; adding any replacement items to the supplemental order; adding any reconfigured items to the supplemental order; moving the quote for the supplemental order to state complete, whereby said quote becomes inactive; and moving the original order and all its order items to the state supplemented, whereby the original order becomes in active and the supplemental order becomes active.",
"103.The process of claim 102, further comprising the step of: before adding any reconfigured items to the supplemental order, running said supplemental order plug-in again to ensure that the original items that were reconfigured are still eligible for supplement; wherein if any item fails this check, an exception is thrown, an error displayed, and the entire supplemental order is rolled back.",
"104.The process of claim 100, further comprising the step of: validating, by a business rule adapter, all quote items that are associated with said new quote against all business rules; and wherein if any quote item relationship violates a business rule, a warning is displayed.",
"105.A method for creating a supplemental order according to claim 99, comprising the steps of: creating a new quote which is populated with copies of each order item from the original order; and determining, by a supplemental order plug-in, which order items are eligible for supplement; wherein said plug-in, by default, only allows the order items that are in state dispatched to be supplemented; changing determined items; and submitting the supplemental order.",
"106.The method of claim 105, wherein the step of changing determined items comprises a plurality of actions, each of which is selected from a group of action types comprising: reconfiguring, which allows certain values to be changed; replacing, which allows for substitution of an existing order item with a new item; and canceling, which moves an item to state canceled.",
"107.The method of claim 106, further comprising the steps of: adding the items that were not eligible for supplement to the supplemental order, adding any replacement items to the supplemental order; adding any reconfigured items to the supplemental order; moving the quote for the supplemental order to state complete, whereby said quote becomes inactive; and moving the original order and all its order items to the state supplemented, whereby the original order becomes in active and the supplemental order becomes active.",
"108.The method of claim 107, further comprising the step of: running said supplemental order plug-in again to ensure that the original items that were reconfigured are still eligible for supplement before adding any reconfigured items to the supplemental order; and wherein if any item fails this check, an exception is thrown, an error displayed, and the entire supplemental order is rolled back."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Bringing businesses online is challenging in any industry.",
"In the communications industry, complex operational infrastructures and intricate partner relationships pose an even bigger challenge in extending businesses to the Internet.",
"In addition, today's climate of active consolidation means all providers will eventually face the challenge of integrating existing operational systems with those of a newly-formed subsidiary, parent, or partner.",
"To remain competitive, providers must evolve into new organizations based on scalable infrastructures that incorporate dynamic and automated business processes to support customer-centric activities.",
"New systems must also be designed for flexibility and efficiency to ensure continuous and responsive customer support.",
"Retooling an existing infrastructure, however, is a costly and time-consuming option, even for new entrants burdened by few legacy systems.",
"What is desired is to develop a comprehensive, communications-specific electronic business (eBusiness) solution that communications service providers can easily deploy."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>This invention provides a comprehensive, modular solution that brings eBusiness to communications service providers.",
"The solution according to the invention is based on a deep understanding of communications business processes, operations support systems (OSS) complexity, and Internet technology.",
"It integrates marketing, sales, ordering, billing and service into a single, personalized Internet portal and brings communications service providers directly to their customers.",
"The Internet portal fulfills the promise of “one-stop shopping”, delivering targeted, convergent product bundles and round-the-clock service to valued customers.",
"The completely open, modular architecture unites functionally segmented processes and systems across all customer touch-points.",
"Together, these elements form a comprehensive solution that delivers speedy implementation, low maintenance cost, and rapid return on investment.",
"The solution is unique in that it allows customers to view their communications profile and requirements on their terms—in a format and flow consistently presented through a trusted and familiar Web browser interface.",
"The information hub translates customer management processes into myriad discrete service order transactions, each one tracked, coordinated and reported on via the browser interface.",
"This solution empowers customers and business partners with self-service capabilities, creating a revolutionary end-to-end customer experience that increases revenue and customer loyalty while dramatically reducing costs.",
"It allows customers to interact with communications service providers in a whole new way with simplicity, intimacy, transparency and immediacy.",
"Simplicity is possible because interfaces are clear, convenient and easy-to-use.",
"Intimacy is created through expressed preferences and personalized profiles that are remembered, and service presentations tailored to customer preferences.",
"Transparency is achieved through the presentation of meaningful status—customers understand exactly where their orders stand, reducing human intervention.",
"Immediacy is attainable because customer requests are handled in real time.",
"The system according to the invention is built on a Java platform using open Internet standards, proven enterprise technology and leading workflow and middleware applications.",
"It enables communications service providers to rapidly deploy a complete eBusiness solution all at once or in stages that fit their strategic and technical needs.",
"The eBusiness portals are the Internet interfaces designed for a particular sales channel and audience.",
"They provide a complete customer experience—one-stop-shopping and round-the-clock service.",
"The invention's eBusiness platform provides the foundation for a communications-specific business model that enables a single view of the customer across functional silos.",
"Its integration components automate business processes and leverage complex and heterogeneous OSS.",
"The eBusiness platform according to the invention provides a consolidated customer view and experience through a communications-specific business model spanning all customer touch-points.",
"This end-to-end experience allows more than self-service purchasing of product bundles, by allowing customers to administer accounts, view bills, open trouble tickets, and more.",
"The eBusiness platform contains Smart Components and an Administrator.",
"The Smart Components represent communications-specific data and business logic that is customer-focused, convergent, and cross-functional.",
"They provide a single unified customer view across functional silos from marketing, sales, ordering, billing, to service and are built to Sun's Enterprise JavaBeans specifications.",
"The Smart Components are grouped by logical functionality, providing a flexible design that makes it easy to add capabilities as the communications service provider's business evolves.",
"Each Smart Component is designed to manage the unique application logic, data, and business rules for the communications industry and can be easily extended to implement the specific business policies and processes of a communications provider.",
"The Administrator is a graphical user interface tool used by the communications service provider's internal staff to define and maintain the eBusiness site data.",
"The solution according to this invention allows for integration with operations support systems (OSS).",
"Specifically, the Interconnect Framework provides predefined interfaces and connectivity to infrastructures.",
"Through these components, the solution ensures that the customer experience remains simple, intimate, transparent and immediate.",
"The Interconnect Framework provides a flexible solution for integrating across business applications and OSSs.",
"The solution utilizes CMIs to communicate with a communications provider's OSS.",
"The communications messaging interfaces (CMIs) are predefined interfaces to any system, such as a communications provider's existing marketing, sales, ordering, billing, and service systems.",
"The eBusiness Portals, as embodiments of the solution, are the Internet interfaces that allow communications service providers to redefine the complete customer experience—integrating touch points across marketing, sales, ordering, billing and service into a personalized Web-based interface.",
"Each eBusiness portal provides the capabilities for a particular customer segment or sales channel.",
"They are designed to quickly deploy a branded Internet presence.",
"The eBusiness Portals present a communications provider communications-specific functionality and data dynamically and are easily configurable to evolve as its business changes and grows.",
"Each of the eBusiness Portals utilizes a set of unique interfaces to meet the specific needs of the channel and audience.",
"So whether the target customer is an IT administrator who is managing a communications service for 3,000 employees, a small business owner with 50 employees who needs Internet, phone, voice mail and other new products, or a residential customer who wants to add a new phone line or install DSL, the eBusiness Portals can be customized to support these specific customer needs.",
"Each of the eBusiness Portals consists of a set of JavaServer Pages (JSPs) that allow for quick and easy changes to the presentation of the communications provider's portal by using standard Web development tools."
],
[
"TECHNICAL FIELD The invention relates to electronic commerce technology.",
"More particularly, the invention relates to a comprehensive electronic business support system for communications service providers.",
"BACKGROUND OF THE INVENTION Bringing businesses online is challenging in any industry.",
"In the communications industry, complex operational infrastructures and intricate partner relationships pose an even bigger challenge in extending businesses to the Internet.",
"In addition, today's climate of active consolidation means all providers will eventually face the challenge of integrating existing operational systems with those of a newly-formed subsidiary, parent, or partner.",
"To remain competitive, providers must evolve into new organizations based on scalable infrastructures that incorporate dynamic and automated business processes to support customer-centric activities.",
"New systems must also be designed for flexibility and efficiency to ensure continuous and responsive customer support.",
"Retooling an existing infrastructure, however, is a costly and time-consuming option, even for new entrants burdened by few legacy systems.",
"What is desired is to develop a comprehensive, communications-specific electronic business (eBusiness) solution that communications service providers can easily deploy.",
"SUMMARY OF THE INVENTION This invention provides a comprehensive, modular solution that brings eBusiness to communications service providers.",
"The solution according to the invention is based on a deep understanding of communications business processes, operations support systems (OSS) complexity, and Internet technology.",
"It integrates marketing, sales, ordering, billing and service into a single, personalized Internet portal and brings communications service providers directly to their customers.",
"The Internet portal fulfills the promise of “one-stop shopping”, delivering targeted, convergent product bundles and round-the-clock service to valued customers.",
"The completely open, modular architecture unites functionally segmented processes and systems across all customer touch-points.",
"Together, these elements form a comprehensive solution that delivers speedy implementation, low maintenance cost, and rapid return on investment.",
"The solution is unique in that it allows customers to view their communications profile and requirements on their terms—in a format and flow consistently presented through a trusted and familiar Web browser interface.",
"The information hub translates customer management processes into myriad discrete service order transactions, each one tracked, coordinated and reported on via the browser interface.",
"This solution empowers customers and business partners with self-service capabilities, creating a revolutionary end-to-end customer experience that increases revenue and customer loyalty while dramatically reducing costs.",
"It allows customers to interact with communications service providers in a whole new way with simplicity, intimacy, transparency and immediacy.",
"Simplicity is possible because interfaces are clear, convenient and easy-to-use.",
"Intimacy is created through expressed preferences and personalized profiles that are remembered, and service presentations tailored to customer preferences.",
"Transparency is achieved through the presentation of meaningful status—customers understand exactly where their orders stand, reducing human intervention.",
"Immediacy is attainable because customer requests are handled in real time.",
"The system according to the invention is built on a Java platform using open Internet standards, proven enterprise technology and leading workflow and middleware applications.",
"It enables communications service providers to rapidly deploy a complete eBusiness solution all at once or in stages that fit their strategic and technical needs.",
"The eBusiness portals are the Internet interfaces designed for a particular sales channel and audience.",
"They provide a complete customer experience—one-stop-shopping and round-the-clock service.",
"The invention's eBusiness platform provides the foundation for a communications-specific business model that enables a single view of the customer across functional silos.",
"Its integration components automate business processes and leverage complex and heterogeneous OSS.",
"The eBusiness platform according to the invention provides a consolidated customer view and experience through a communications-specific business model spanning all customer touch-points.",
"This end-to-end experience allows more than self-service purchasing of product bundles, by allowing customers to administer accounts, view bills, open trouble tickets, and more.",
"The eBusiness platform contains Smart Components and an Administrator.",
"The Smart Components represent communications-specific data and business logic that is customer-focused, convergent, and cross-functional.",
"They provide a single unified customer view across functional silos from marketing, sales, ordering, billing, to service and are built to Sun's Enterprise JavaBeans specifications.",
"The Smart Components are grouped by logical functionality, providing a flexible design that makes it easy to add capabilities as the communications service provider's business evolves.",
"Each Smart Component is designed to manage the unique application logic, data, and business rules for the communications industry and can be easily extended to implement the specific business policies and processes of a communications provider.",
"The Administrator is a graphical user interface tool used by the communications service provider's internal staff to define and maintain the eBusiness site data.",
"The solution according to this invention allows for integration with operations support systems (OSS).",
"Specifically, the Interconnect Framework provides predefined interfaces and connectivity to infrastructures.",
"Through these components, the solution ensures that the customer experience remains simple, intimate, transparent and immediate.",
"The Interconnect Framework provides a flexible solution for integrating across business applications and OSSs.",
"The solution utilizes CMIs to communicate with a communications provider's OSS.",
"The communications messaging interfaces (CMIs) are predefined interfaces to any system, such as a communications provider's existing marketing, sales, ordering, billing, and service systems.",
"The eBusiness Portals, as embodiments of the solution, are the Internet interfaces that allow communications service providers to redefine the complete customer experience—integrating touch points across marketing, sales, ordering, billing and service into a personalized Web-based interface.",
"Each eBusiness portal provides the capabilities for a particular customer segment or sales channel.",
"They are designed to quickly deploy a branded Internet presence.",
"The eBusiness Portals present a communications provider communications-specific functionality and data dynamically and are easily configurable to evolve as its business changes and grows.",
"Each of the eBusiness Portals utilizes a set of unique interfaces to meet the specific needs of the channel and audience.",
"So whether the target customer is an IT administrator who is managing a communications service for 3,000 employees, a small business owner with 50 employees who needs Internet, phone, voice mail and other new products, or a residential customer who wants to add a new phone line or install DSL, the eBusiness Portals can be customized to support these specific customer needs.",
"Each of the eBusiness Portals consists of a set of JavaServer Pages (JSPs) that allow for quick and easy changes to the presentation of the communications provider's portal by using standard Web development tools.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a block diagram showing the architecture of the eBusiness support system; FIG.",
"2 is a block diagram showing the structure of the Smart Components; FIG.",
"3 is a block diagram showing a data access structure used in the system; FIG.",
"4 is a block diagram showing a structure of connectivity between the eBusiness support system and external systems; FIG.",
"5 is a block diagram illustrating a process for sending a message to an external system; FIG.",
"6 is a block diagram illustrating a process for receiving a-message from an external system; FIG.",
"7 is a page flow diagram illustrating a section of page flow used in the Small Business Portal that includes a registration process as a nested flow; FIG.",
"8 is block diagram illustrating an example of a non-repeatable transition; FIG.",
"9 is block diagram illustrating a process for submitting a request; FIG.",
"10 is a pictorial diagram showing a sample page wherein a user enters information to open a trouble ticket against a product in the hierarchy; FIG.",
"11 is a pictorial diagram showing a sample page returned to the client via HTTP; FIG.",
"12 is a block diagram showing a pricing model for one offer with two price groups; FIG.",
"13 is a page flow diagram showing a process to make a dynamic offer collection; FIG.",
"14 is a page flow diagram showing an offer determinant branch; FIG.",
"15 is a schematic page showing a fixed of bundle of offers; FIG.",
"16 is a page flow diagram showing a dynamic bundle of offers; FIG.",
"17 is a block diagram showing that a customer or partner views a menu associated with the same price group; FIG.",
"18 is a block diagram that illustrates a hierarchy object model of the billing points and products; FIG.",
"19 is a hierarchical graphic that illustrates how a product suspension is handled when the product is associated with other products; FIG.",
"20 is block diagram that illustrates a process for creating a supplemental order in the eBusiness support system; and FIG.",
"21 is a block diagram that illustrates the relationship between the smart component server hierarchy and the universal agent portal hierarchy.",
"DETAILED DESCRIPTION OF THE INVENTION Section 1.Architecture This section describes the architecture of the eBusiness support system according to the invention.",
"1.1.Overview Referring to FIG.",
"1, the architecture of the eBusiness support system 100 according to this invention includes three distinct layers: (1) Business Layer 101, which is the core of the system 100.Smart Components 104 employ Enterprise JavaBeans (EJB) technology, which allows them to encapsulate all the core functionality needed to complete communications specific business transactions.",
"By encapsulating this functionality, Smart Components 104 provide a unified view that seamlessly presents information obtained from disparate systems.",
"(2) Integration Layer 102, through which Smart Components 104 access external systems 110.The interconnect service within this layer uses communications messaging interfaces (CMIs) 105 and adapters 106/107 to transport messages.",
"The CMIs are pre-defined interfaces to common services (e.g.",
"rating, address validation, service reservation, etc.)",
"needed to complete client requests for customer, pre-order, order, and post order transactions.",
"The EAI Adapters 106 provide the flexibility to integrate with an EAI package 109.These adapters are configurable software interfaces for different EAI packages, such as Vitria BusinessWare or BEA eLink.",
"Other adapters are also available.",
"(3) Presentation Layer 103, in which eBusiness portals provide communications specific functionality tailored to particular types of users, such as small businesses or resellers.",
"These portals are eBusiness sites with interfaces and process flows dedicated to particular customer group.",
"They use JavaServer Pages (JSP) technology—standard HTML files that contain blocks of simple Java code—to dynamically generate customer views 108.The JSP-based portal architecture enables the separation of the user interface from application logic, enabling the design of the page to change without altering the underlying content.",
"Additional components of the portal architecture include a Web session controller, which maintains user session state, and page transition policies—Java classes that enforce page transition rules by accessing business functionality contained in the Smart Components 104.Administrator 111 is a Java-based tool that uses XML files to map object attributes to the database.",
"It enables users in product marketing to create and configure new products and services, without the need for software code changes.",
"It provides access to system maintenance functions for setting up secure user access.",
"The layered and component-based architecture provides a carrier-class performance, transactional integrity, and communications expertise.",
"For example, the Smart Component Server has the ability to cluster Smart Components for increased performance; and maintains transactional integrity and security; provides powerful, communications-specific capabilities.",
"The architecture provides fast and easy integration capabilities.",
"For example, the interconnect service facilitates interoperability with external systems; can be middleware-based of communicate directly with an external system; and provides the ability to add new message types and adapters to external systems.",
"The architecture also provides a flexible front-end user experience.",
"For example, the eBusiness portals are completely independent from the core Smart Components 104.Each portal can be redesigned without rebuilding or altering core functionality; can be one of many portals simultaneously accessing the same Smart Component Server; and is adaptable to new presentation vehicles.",
"The communications industry demands a carrier-class eBusiness solution that provides high performance, scalability and resiliency.",
"The Smart Components 104 in this invention are implemented as stateless session beans in order to meet these requirements.",
"The Smart Components 104 are clustered to increase performance.",
"Their stateless nature obviates the need to maintain instance-specific information over invocations.",
"This means that even though bean instances are pooled, no complex state activation logic is required.",
"Furthermore, in case of failure, instance data need not be replicated across nodes in a cluster.",
"New Smart Component instances can be dynamically generated as needed to meet increasing user demand.",
"The request load is then distributed among the Smart Components 104 to fully utilize all resources.",
"Because the Smart Components 104 do not maintain any state information, a client need not maintain contact with the same bean instance throughout the life of a session.",
"The Smart Components 104 can be replicated across servers.",
"In the event of an outage, clients are switched to another bean instance on a working server.",
"1.2.Smart Component Server Referring to FIG.",
"2, the business layer 101 contains two types of Smart Components: activity Smart Components 120 and service Smart Components 121.Activity Smart Components 120 encapsulate communications-specific functionality and business logic.",
"Service Smart Components 121 provide common infrastructure capabilities.",
"Both types of the Smart Components constitute the system's API.",
"1.2.1.Activity Smart Components The activity Smart Components 104 includes a plurality of managers.",
"For examples: (1) Shop Manager 122, which provides the ability to navigate through a catalog and place offers in a shopping cart; (2) Quote Manager 124, which provides the ability to dynamically quote product charges to the customer.",
"Possibly in conjunction with an integrated billing system, the Quoting Manager calculates a product charge based on an action (e.g.",
"add, remove, upgrade, recurring, etc.",
"), time period, customer pricing group and rate method.",
"It also manages the configuration of products and creates the quote; (3) Order Manager 125, which governs the creation of an order and sending it to an external provisioning system; (4) Customer Manager 126, which delivers a consolidated view of an enterprise vision representing the customer, accounts, users and services.",
"Customers manage their accounts, products, services, and users from their own perspective, which could be from an organizational, location or financial reporting structure; (5) Business Rule Manager 128, which defines valid business relationships between customers, accounts, products, and processes.",
"Works with business rules to define allowable configurations during the ordering process (e.g.",
"a customer must have an access line to order voice-mail).",
"Customers are now able to self-manage the complex communications-specific configurations during the ordering process and account management; (6) Interaction Manager 129, which allows customers to track an order or service request from start to finish.",
"Customers can immediately view provisioning steps that have been completed during the flow-through provisioning process.",
"A high-level status is provided to the customer while a detailed status is used by the provider to track potential performance enhancements; (7) Bill Presentation Manager, which presents consolidated billing views online using information from customer invoices.",
"The bill presentation manager interfaces with external billing systems to obtain billing information for display, dispute and adjustment; (8) Trouble Ticket Manager 133, which provides a self-service mechanism for the customer to request service assistance such as reporting trouble for a product.",
"When used with Interaction Manager 129, real-time trouble ticket status can be viewed.",
"The trouble ticket manager interfaces with an external trouble ticket application to handle the dispatch and resolution; (9) Sales Manager 127, which provides basic functionality to support a sales force in relation to a customer hierarchy; (10) Profile Manager 130, which provides a mechanism to collect and organize information about a customer; (11) Payment manager 132, which manages payments towards non-recurring charges either by adding the charges to the customer's next invoice or by collecting a payment method at the time of ordering; (12) Notice Manager 134, which provides a mechanism to store messages for display to targeted customers.",
"For situations where the processing logic is likely to change for each licensee, the addition of plug-ins is supported.",
"The plug-ins are small, dynamically deployed algorithms that a communications service provider (CSP) can implement to perform specific tasks.",
"For example, the CSP could create an algorithm that determines which news and information item to select based on where a customer lives.",
"To do this, the CSP would create a plug-in that selects the news and information item based on the customer's address.",
"The CSP could later override this behavior by creating and registering a new plug-in that selects the news and information item based on different criteria-for example, the customer's area code.",
"1.2.2.Service Smart Components Referring to FIG.",
"2 again, Service Smart Components 121 provide common infrastructure capabilities required by activity Smart Components 120, including: a data access service (DAS) 135, which includes a plurality of value objects and a data cursor service; an interconnect service 136, which uses the communications messaging interfaces and the adapters to connect the activity smart components to external systems; a security service 137, which uses standard Internet security protocols such as SSL to ensure browser-to-server security (via HTTPs) and server-to-server security; a logging service 138, which creates files that report on specific actions within the system; and a sequence service 139, which establishes default sequence for page flows.",
"(1) Data Access FIG.",
"3 illustrates a data access structure 140.DAS 135 provides an API that performs all create, read, update, and delete (CRUD) operations on the value objects 141 which represent data in the system.",
"There are two types of value objects: domain objects, which represent a specific row in a database table; and display objects, which are containers that deliver attribute values to the JSP pages in the presentation layer 103.DAS 135 also includes a value object builder 143.This factory instantiates value objects 141 and sets initial attribute values.",
"(2) Domain Objects The domain objects are created by DAS 135 by invoking a create method, which looks in the BUILDER table to determine the builder class to use to create the new object.",
"DAS 135 then passes the logical class name to the buildObject method in the builder class, which creates, initializes, and returns the new object.",
"The system employs TopLink for Java, an object relational mapping tool, to map attributes from a domain object or display object to the database.",
"TopLink accesses the database 145 via Java Database Connectivity (JDBC) 144.",
"(3) Data Cursor The data cursor service is implemented as a stateful enterprise bean.",
"It creates a scrollable, read-only cursor used when a large result set from a query is expected.",
"However, because the resulting list is read-only, the data cursor service cannot be used to retrieve attribute values that need to be updated.",
"(4) Logging Service The logging service 138 creates files that report on specific actions within the system.",
"A CSP can configure the logging service to meet individual business needs.",
"Log messages contain identifiers that report on the severity of an event (e.g.",
"fatal, critical, warning, etc.",
"), the architectural component where the event occurred (e.g.",
"DAS, interconnect service, activity object, etc.",
"), and the functionality that the application was attempting (e.g.",
"shopping cart, customer, menu, etc.).",
"Two types of loggers are used: a file logger that sends information to a text file, and a console logger that sends information to the screen.",
"(5) Authentication and Authorization An authentication service is used to verify that a user name and password is valid for the system.",
"An authorization service determines what a given user is allowed to do within a portal.",
"The authentication service is used by the Customer Manager 126 to register new user IDs and passwords, as well as authenticate users logging in to the system.",
"It provides an abstraction to a security provider that contains principal information used for authentication.",
"Principals are specific users of the system, and can be either individual users or defined groups.",
"In implementation, an RDBMS-based authentication security provider is used to assigns new users to specific groups.",
"This assignment happens when a user is created in either a Universal Agent Portal or a Small Business Portal.",
"The CSP can also use the LDAPRcalm class, which authenticates principals using a directory service such as the Netscape Directory Server.",
"New users and groups are also entered in this directory server.",
"Note that LDAP can only be used for authentication (verifying a user and password are valid for the system).",
"All authorization within the application must be done using the RDBMSRealm service.",
"The framework accesses the CYG-ACL table before rendering a page or executing a transition policy.",
"This table associates permissions (either allowing or restricting execution) for a JSP or transition policy with specific groups.",
"The groups include: (1) UA_LICENSEE_USER, which can use the Universal Agent Portal to shop for and purchase products, manage hierarchies on behalf of customers and partners, and create price overrides; (2) UA_LICENSEEADMINISTRATOR, which can use the Universal Agent Portal to create other users of that portal, create price overrides, and also use the Administrator Console and the licensee-level features in the Small Business Portal; (3) CP_ADMIN, which can shop for and purchase products and manage hierarchies in the Channel Partner Portal and create other users of the Channel Partner Portal; (4) CP_USR, which is a read-only user of the Channel Partner Portal; (5) SB_ADMIN, which can shop for and purchase products and manage hierarchies in the Small Business Portal and create other users of the Small Business Portal; and (6) SB_USR, which is a read-only user of the Small Business Portal.",
"For example, the users of a Small Business portal are assigned to one of two groups: (1) sb_customer_user, which has read-only access to the portal; and (2) sb_customer_admin, which can shop for products and manage products.",
"If a user assigned to one of these groups attempts to log into a Universal Agent Portal, an error message displays, and they are prevented from logging into that portal.",
"Similarly, within a portal, access to specific pages can be restricted, and through the use of the tags in a JSP, links and content can be displayed to and executed by certain user groups.",
"1.2.3.Constant Classes Constant classes are used throughout the application to store static data such as state names, street type codes, types of contacts, and so on.",
"When a CSP needs to add new codes and the corresponding values to the application, the CSP can either create its own constant class or subclass an existing constant class.",
"For example, the constant class StreetTypeCode defines codes that can be used by the application: public class StreetTypeCode { // FIELDS ------------------------------------------ /** Avenue */ public static final int AVENUE 1; /** Drive */ public static final int DRIVE 2; /** Place */ public static final int PLACE 3; /** Street */ public static final int STREET 4; /** Way */ public static final int WAY = 5; } // end StreetTypeCode class 1.3.Integration with External Systems The interconnect service EJB provides an interface that the activity smart components use to access external systems 110.Messages that contain information for external systems 110 are sent from the eBusiness support system through a particular type of communications messaging interface (CMI) 105.Examples of these CMI types include credit card validation, address validation, and service reservation, etc.",
"The CMIs are designed to work via EAI facilities.",
"They provide APIs built on best practices established by industry groups such as the TeleManagement Forum (TMF) and the Ordering and Billing Forum (OBF), and through participation with emerging standards bodies such as IPDR Working Group, and vendor programs such as SunConnect Framework for Communications.",
"Open architecture and a standards-based technology platform ensures that messages exchanged between the eBusiness platform and existing systems leverage standard interconnect techniologies such as XML CORBA Java Messaging Service, and TUXEDO.",
"Referring to FIG.",
"4, the activity Smart Components 104 or transition policies call the interconnect service 136 to create CMIs 105 whenever the eBusiness support system needs to communicate with an external system 110.Each CMI type is associated with a CMI routing policy 152 which determines the transport adapter 153 that handles that message (CMIs can be associated with multiple adapters in the case where they may send different messages via different transport methods).",
"Transport adapters 153 facilitate connectivity with the external system; they use strategies to retrieve data and update the system and use handlers to translate the data into a format that the external system expects.",
"Various transport adapters are available with the Smart Component Server.",
"For example: (1) Default adapter—this is a file adapter 156 that prints out all CMI information sent to it.",
"By default, all CMIs 105 deal with provisioning or trouble ticketing writing to the default adapter.",
"A CSP can change the routing data associated with the CMI so that these use a different adapter; (2) Open adapter 154—by default, this adapter's handlers are stubs used by CMIs 105 such as address validation or credit card authorization.",
"A CSP can configure these handlers to send the type of message appropriate to the transport.",
"The CSP can also create additional handlers for new CMIs, or configure existing CMIs to use different (or new) adapters or handlers.",
"However, if the CSP needs to send many messages via a particular transport type (for example IIOP or DCOM), and if that transport type requires specific adapter properties (configured in the adapter's XML file), the CSP should create a new transport-specific adapter; (3) SMTP email adapter 155—this adapter sends email messages based on CMIs.",
"Handlers for the email adapter 155 determine who receives the email and the template used to create the message; (4) EAI Adapter 106—this adapter is integrated with Vitria's BusinessWare, an enterprise application integration system that allows disparate systems to communicate via messages.",
"Each CMI 105 is also mapped to source and target strategy classes 151/152.The interconnect service 136 uses the source strategy to build up the payload message associated with the CMI before being dispatched to the transport adapter 153.Payloads are of a specific message type, such as name/value pairs or an XML, message.",
"Target strategies are used to update the system with data from inbound messages.",
"Various strategies are available.",
"For examples: (1) OidSourceStrategy, which builds a payload of OIDs.",
"This is the default strategy used by all CMIs 105.It is the only one that can be used by the EAI Adapter 106; (2) DummySourceStrategy, which used when no source strategy is needed; (3) DummyTargetStrategy, which used when no target strategy is needed.",
"A CSP needs to create source strategies for each CMI that the CSP wants to actually be sent to external systems.",
"When creating CMIs, the CSP can set the CMI_LOG flag so data about the dispatching of CMIs is persisted to the CMI_LOG table.",
"To receive inbound messages, inbound transport adapters 153 need to be created.",
"The inbound transport adapter 153 creates a CMI, invokes the remote method receiveCMI(cmi) on the interconnect EJB.",
"The interconnect service receives the inbound CMT and uses the target strategy to update the data model or to call the API.",
"1.3.1.How the Interconnect Service Sends CMIs FIG.",
"5 illustrates a process 160 that a transition policy 163 or an activity uses the interconnect service 136 to send a message to an external system.",
"The process includes the steps of: (1) The createCmi method on the interconnect service 136 is called, passing in the code value of the CMI.",
"(2) The createCmi method returns an empty CMI object.",
"(3) The putCmiDataItem method on the CMI object is called, passing in the Object Identifier (OID) and the fully qualified class name of the domain object that contains the event information the CSP wants to send.",
"The source strategy 150 uses this information to retrieve data for the message.",
"(4) The sendCmi method on the interconnect service 136 is called, passing in a reference to the CMI object created in step (2).",
"The sendCmi method returns information about the CMI response.",
"(5) The interconnect service 136 performs a database lookup in the CMI_CMI_POLICY table to determine which routing policy to use for the CMI.",
"This table maps each CMI code to the fully-qualified Java class of its routing policy.",
"The routing policy determines the adapter to invoke and if the CMI is to be logged.",
"The eBusiness support system provides three routing policy classes: (a) a default policy that sends CMIs to the default file adapter 156; (b) a generic policy that uses the GENERIC_CMI_POLICY table to determine the adapter to which the CMI is delivered; and (c) a vendor product policy that uses the VENDOR_PROD_CMI_POLICY table to determine the adapter to which the CMI is delivered based on a product's action.",
"For more advanced policies, a CSP needs to write its own policy class.",
"(6) If the default routing policy is used (or if no policy is specified) the routing policy sends the CMI to the file adapter 156 with a default payload (OIDs).",
"(7) If a generic routing policy is used, the routing policy class performs a database lookup in the GENERIC_CMI_POLICY table to determine which adapter to use.",
"This table associates the CMI with: (a) Adapter home, which is the JNDI-bound name of the adapter; (b) Context, which is used to determine how to translate the data into a format the external system expects.",
"Examples of context include the handler class name, an email address, a fax number, or any information the adapter needs to pass on the message; (c) Payload, which determines the type of message being sent, such as a class name and OID, a name/value pair, or an XMI, document.",
"There is a payload constant for each payload type.",
"(8) If a vendor product policy is used, the routing policy class performs a database lookup in the VENDOR_PROD_CMI_POLICY table to determine which adapter to use.",
"This table associates the CMI with: (a) an action, (b) vendor product OID, (c) adapter home, (d) context, and (e) payload.",
"Vendor product routing policies are useful when the adapter to use varies depending on the action performed on a product.",
"For example, to “Add” an access fine, the eBusiness support system might send a CMI to an external provisioning system.",
"However, to “Remove” the same access line, the system might send an email.",
"In this case, the VENDOR_PROD_CMI_POLICY table would contain one row for the “Add” action that sends the CMI to an adapter which uses handlers to deliver to the external system 110.The table would contain another row for the “Remove” action that calls an email adapter 155 to send an email to the CSP.",
"(9) The interconnect service 136 performs a database lookup in the SOURCE_TARGET_STRATEGY_POLICY table to determine the source strategy 150 to use for the CMI and the given payload.",
"The strategy is invoked and it calls the Smart Component Server (either through DAS or a direct API call) to collect the data needed to build up the payload.",
"If the default file adapter 156 was associated with the CMI, then the default source strategy 150 (OidSourceStrategy) is used.",
"(10) The adapter sends the message out to the external system 110.",
"(11) If in the CMI_CMI_POLICY table, the CMI_LOG_FLAG is set to 1, the interconnect service 136 populates the CMI_LOG table with dispatch information.",
"The interconnect service also updates the CMI_DOMAINOBJECT_LOG.",
"If a CMI has multiple entries in the CMI_CMI_POLICY table, then the steps (5) through (11) are repeated again for the CMI.",
"The order in which the process is repeated depends on the order in the database.",
"1.3.2.How the Interconnect Service Receives Messages FIG.",
"6 illustrate a process 170 that an external system 110 calls a transport adapter 153 to receive a message.",
"Note that a CSP must build inbound transport adapters specific to the messages the CSP expects to receive.",
"The process includes the steps of: (1) The external system 110 sends a message to a transport adapter 153.This adapter acts similar to a daemon process in that it listens to a specific port number or socket, or in the case of RMI, an RMI call.",
"This message must include a CMI code value, CMI ID, and a payload type code.",
"(2) The transport adapter 153 calls createCmi (cmi) on the interconnect service 136 to build the CMI based on the code value.",
"(3) The createCmi method returns an empty CMI object.",
"The adapter must be written such that it populates the CMI with the needed data before proceeding to the next step.",
"(4) The transport adapter 153 calls receiveCmi (cmi) on the interconnect service 136.",
"(5) The interconnect service 136 performs a database lookup in the SOURCE_TARGET_STRATEGY_POLICY table to determine the target strategy 151 to use for the CMI and the given payload.",
"(6) The target strategy 151 is invoked and it calls a smart component 120 (either through DAS 135 or a direct APT call) to update the system accordingly.",
"1.3.3.How the Interconnect Service Recovers from Errors It is possible that a CMI 105 may fail correctly to reach its intended destination, or that it may fail processing at that system.",
"In this event, the external system 110 should provide information describing the error in the response message.",
"The eBusiness support system provides an Error Correction Facility (ECF) to review, correct, and re-send CMIs which may have failed in this way.",
"The ECF can also be used to re-send successful CMIs, should this be desired.",
"The ECF relies on information written to CMI_LOG and CMI_DOMAIN_OBJECT_LOG when reviewing and correcting CMIs.",
"For this reason, if the CSP wants to recover and correct an erred CMI, the CSP must populate the CMI_LOG_FLG flag in the CMI_CMI_POLICY table with ‘1’ to insure that CMIs of that type are logged.",
"Only CMIs whose payload is composed of the OIDs can be reviewed, corrected and re-sent by the ECF.",
"The payload of a CMI 105 is determined by the source strategy 150.When reviewing a CMI 105, the ECF relies on data in the CMI_DOMAIN_OBJECT_LOG table to re-construct the CMI and allow users to change and correct domain object data which may have caused the CMI to fail.",
"Once corrected, the CMI and its payload can be re-sent to the external system 110.Any error information provided by the external system 110 is captured in three fields in the CMI_LOG table: (a) ERROR-CDE, which is a numeric code for the error encountered; (b) ERROR-CNTXT, which is the context in which the error occurred; and (c) ERROR-MSG, which is a text string describing the nature of the error encountered.",
"It is the job of the target policy 151 for the CMI 105 to receive this error information from the remote system's response and populate the eBusiness support system's database 145.Then, when the CMI 105 is reviewed in the Error Correction Facility, this error information is displayed.",
"1.4.eBusiness Portals The eBusiness support system provides portals that are specific to a market segment of the communications industry.",
"The Universal Agent Portal, Channel Partner Portal, and Small Business Portals all use: (1) JavaServer Pages (JSP) technology—these HTML pages with embedded Java source code allow for dynamic Web content; (2) Display policies—embedded within the JSP pages, these policies access and manipulate data for display; (3) A Web session controller 162—this servlet mediates the creation and display of JSP pages; (4) Transition policies 163—these Java files determine navigation logic through the site; and (5) Resource Bundles—the classes store display values or messages corresponding to codes in constant classes.",
"1.4.1.JavaServer Pages JSP pages are standard HTML files containing blocks of simple Java code that display dynamic content.",
"A CSP can use a standard HTML editor to create and edit these pages.",
"The eBusiness support system leverages JSP auto-compilation capabilities but defers all navigation and application logic to other components within the portal.",
"This approach clearly separates presentation responsibilities from application logic so a licensee can change events that occur during a page transition without having to alter the JSP page.",
"A JSP page uses display policies that handle any process logic needed to display a page.",
"To create or edit JSP pages for the portal, a CSP needs to understand various aspects of JAVA or HTML such as Page directives, Transition directives, Scriptlets, Expressions, Tags, and JavaScript libraries.",
"(1) JSP Page Directives.",
"The directives are used to set JSP page directives.",
"For example: <jsp:directive.page language=“java” /> <jsp:directive.page buffer=“[ n] K” /> <jsp:directive.page autoFlush=“faise” /> <jsp:directive.page errorPage=“/DefaultError.jsp” /> <jsp:directive.page extends=“cygent.portal.frame.jsp.DisplayJsp” /> The directives are also used to import needed classes.",
"For example: <jsp:directive.page import=“cygent.portal.frame.jsp.TypeConversion”/> <jsp:directive.page import=“cygent.portal.frame.jsp.CygentContext”/> <jsp:directive.page import=“cygent.portal.sb.display.logon.MyHomeUser ”/> (2) Transition Directives.",
"The transition directives are special HTML hidden form fields that appear on a JSP page.",
"They determine what navigation logic, if any, the server should execute.",
"Transition directives also determine the destination JSP page.",
"The CSP is required to set the following three transition directives on the JSP page: to—the intended destination page.",
"from—the originating page (the page the link is on).",
"Task—the task to accomplish during the transition from the originating page to the destination page.",
"The names that the CSP sets for “to” and “from” directives are logical names that uniquely represent pages on the server.",
"The framework uses the URL_MAP table to obtain a fully-qualified path for the page corresponding to the logical page name.",
"The following code might appear on a JSP page: <FORM NAME=IlmainForinll METHOD=“POST” ACTION=“/go” target=11_top11 onSubmit=11return (false);”> <INPUT TYPE=hidden NAME=from VALUE=“ApproveAccountRequest”> <INPUT TYPE=hidden NAME=to VALUE=’-> <INPUT TYPE=hidden NAME=task VALUE=’-> At submit time, the values for the “to” and “task” directives are populated by the subFormjavaScript function, which is defined in js_common.js, a library provided for input and form submit manipulation.",
"The script notifies the browser to submit all form data, including transition directives, to the server.",
"The server then parses through the data and looks in the TRANS_MAP table to determine if there is a transition policy associated with the “from” and “task” directives.",
"If a transition policy does not exist, the user is directed to the page set in the “to” directive.",
"If a transition policy exists, the policy is invoked and the server executes the navigation logic defined in the policy.",
"(3) Scriptlets.",
"Java code within the page is placed inside a scriptlet tag.",
"For example: <jsp:scriptlet> .java code </jsp:scriptlet> (4) Expressions.",
"Java code that evaluates a string is placed inside an expression tag.",
"For example: ‘<%=bundleItem.getCode( ) %>’ (5) Tags.",
"The Web framework provides a library of the tags that allow JSP page writers to retrieve value objects and display dynamic content using a simple tag syntax.",
"These tags are action tags used to instantiate object(s) and make them available through scripting variables.",
"They are specifically designed to simplify the process of retrieving or displaying information from the database.",
"The tags used in the eBusiness support system allow a CSP to introduce dynamic content into a page from a variety of sources, including databases, resource bundles, CMIS, and file systems.",
"Table 1.1 illustrates various tags, including source tags for retrieving and displaying dynamic content, and presentation tags for display purposes.",
"TABLE 1.1 Tag Name Tag Type Description queryObject source Retrieves a display object.",
"queryList source Retrieves a list of display objects.",
"Bundle source Creates a list of BundleItem objects.",
"GenericObject source Creates any value object.",
"GenericList source Creates any list of value objects.",
"Loop presentation Iterates through a list of objects and makes each object available through a scripting variable; also provides a counter variable.",
"Authorize presentation Checks a user's permissions.",
"Used in conjunction with either the pass or fail tag.",
"Pass presentation Displays JSP content when the authorize tag determines a user has permission.",
"Used in conjunction with the authorize tag.",
"Fail presentation Displays JSP content when the authorize tag determines a user does not have permission.",
"Used in conjunction with the authorize tag.",
"validation presentation Provides access to validation information created by a transition policy.",
"For each of the source tags, the CSP can implement a corresponding display policy.",
"Display policies can be used to modify the behavior of a custom tag.",
"To perform a simple data retrieval, such as retrieving an object by its primary key, a display policy is not necessary.",
"However, if the CSP had compound criteria for its data retrieval, or if the CSP wanted to do some post-query data manipulation (e.g.",
"calculate someone's age from their birth date), then the CSP would do those activities from a display policy.",
"At request time, the tag invokes methods defined in the display policy implementation.",
"In the following example, the queryObject tag invokes a display policy that constructs a query used to retrieve the display object for the <cygent:queryObject id=”customerDetail” obiectRef=”customerDetail” policy=“cygent.portal.sb.display.customerEdit.CustomerDetailPolicy” To access the tag library, add the following line of code below the page directives in the source file: <% @ taglib uri=“/cygent.tld” prefix=“cygent” %> (6) JavaScript Libraries.",
"The JavaScript libraries are used for client-side validation.",
"js_validation.",
"js provides JavaScript methods for validating data to submit to the server.",
"For example, it is used to verify that the required fields are filled in by the user.",
"Client-side validation should be used in conjunction with server-side validation.",
"The former is not a replacement for the later.",
"Although client-side validation may enable a better user experience by performing validation on the fly, it exposes the application to security risks from users who could bypass the JavaScript and submit erroneous data to the database.",
"The JavaScript libraries are also used for input and form submit manipulation.",
"js_common.js provides javaScript methods for setting request parameters and passing variables and values to the server.",
"All pages that submit form data should access the librares.",
"To access the libraries, the JavaScript codes must be included in the HEAD tag of the HTML page.",
"For example: <HTML> <HEAD> <TITLE>Order History</TITLE> <SCRIPT SRC=“/common/script/js_common.js” language = “javascript”> </SCRIPT> <SCRIPT SRC=“/common/script/js_validation.js” language = javascript”> </SCRIPT> </HEAD> (7) Implementing a Back Button.",
"Within JSP navigation there are two different types of contracts between JSPs.",
"One is explicit and the other is dynamic.",
"In an explicit contract, a button or link on JSPA always takes the user to JSPB.",
"The button or link on JSP_A has JSP_B coded into the navigation logic.",
"In the same fashion, the “back” or “cancel” button on JSP_B will always return the user to JSPA.",
"In a dynamic contract, one screen can be accessed from multiple JSPs.",
"For example, JSP_C can be accessed from either JSP_A or JSP_B.",
"In this case, the “back” or “cancel” button on JSP_C cannot be tied to a physical JSP.",
"The back functionality must rely on the history or stack that the browser stores to know where the user came from and what the previous JSP is.",
"Two methods that access the browser stack are history.go( ) and history.back( ).",
"history.back( ) always returns the user to the previous page.",
"If a user navigates from JSP_A to JSP_B, and then executes the “back” button on JSP_B, the application returns the user to JSP_A.",
"When JSP_B will return to JSP_A, implement history.back( ).",
"For more complex situations, history.go( ) can be implemented.",
"history.go ()is similar to history.back( ), but it accepts a parameter (Integer) that communicates how many pages the CSP wants the navigation to take the user forward (positive Integer value) or backwards (negative integer value).",
"A counter is maintained on the JSP that is then used as the input parameter to history.go( ).",
"There are various cases that warrant the use of history.go( ).",
"For examples: (a) When a JSP includes both validation and a “back” button.",
"If a user navigates to JSP_B, from JSP_A, and then experiences a validation error, JSP_B will be re-rendered with a validation message.",
"Because technically the previous page from JSP_B with validation errors is JSP_B, history.go( ) needs to be implemented.",
"The following code sample shows how the counter can be implemented: [ in the hidden fields of the JSP] <INPUT TYPE=hidden NAME=“timesValidationFailed” VALUE=“<jsp:expression>((val.getFieldValue(“timesValidation- Failed”) == null) ?",
"1 : 1 + Integer.parseInt(val.getFieldValue(“times- ValidationFailed”) )) </jsp:expression>II> [ in the back, or ‘cancel’ link] <A HREF= “javascript: history.",
"go (−(document.",
"forms[ 0 ] times- ValidationFailed.value))”> </A> (b) When a JSP includes cursoring and a “back” button.",
"If a user navigates to JSP_B, from JSP_A, then clicks “next” to see more items on a list, JSP_B is re-rendered with the next set of list items.",
"Because the last JSP from the second set of list items on JSP_B, if there is a “back” button, history.go must be implemented to return to JSP_A instead.",
"The following code sample shows how the counter can be implemented: [ in the hidden fields of the JSP] <INPUT TYPE=hidden NAME=“timesPageCursored” VALUE =“<%=((request.getParameter(“timesPageCursored”)== null) ?1 :1 + Integer.parseInt(request.getParameter(“timesPageCursored” )))%> ”> [ in the back’ or ‘cancel’ link) <A HREF=“javascript:history.go(−(document.forms[ O] .",
"timesPageCursored.value))”> </A> In the case where a JSP has cursoring logic, as well as validation, the above counters would have to be added together.",
"(c) When there is a sequence of screens that all have “back” buttons.",
"(Note that the JSPs in this sequence may also have validation and cursoring, so the above rules may also apply.)",
"An example might be JSP_A, JSP_B, JSP_C, and JSP_D, all of which have back functionality on them.",
"A user can navigate to JSP_A, then execute a link off JSP_A that begins the screen flow JSP_B, JSP_C, JSP_D, then back to JSP_A.",
"A concrete example is an AccountDetail JSP that allows a user to navigate to OpenTroubleTicket then TroubleTicketDetail, which then returns the user to AccountDetail.",
"In this scenario, a similar counter must be maintained by all JSPs in this sequence, in order that all “back” buttons work correctly.",
"In the trouble ticket example, the following counter would be implemented on all screens in the flow: [ in the hidden fields of the JSP] <INPUT TYPE=hidden NAME=“timesTroubleTicketPageAccessed” VALUE=“<jsp:expression>((request.getParameter(“timesTrouble Ticket-PageAccessed”) == null) ?",
"1 :1 + Integer.parseInt(request.getParameter- (“timesTroubleTicketPageAccessed”) )) </jsp:expression>” > [ in the ‘back’ or ‘cancel’ link] <A HREF=“javascript:history.go(−(document.forms[ O].times- TroubleTicketPage−Accessed.value))”> </A> (d) When a JSP requires multiple counters.",
"For example, a JSP may have validation and be part of a JSP sequence.",
"In this case, two counters are needed.",
"The counter for validation is added to the common counter, kept by all JSPs in a sequence.",
"For example: [ in the hidden fields of the JSP] <INPUT TYPE=hidden NAME=“timesScreenScreenRendered” VALUE=“<jsp:expression>((request.",
"getParameter(“timesScreenScreenRendered”) == null) ?",
"1 + Integer.parseInt(val.getFieldValue(“timesValidation-Failed”)) : 1 + Integer.parseInt(request.getParameter(“timesScreen- ScreenRendered”) )) </jsp:expression>” > <INPUT TYPE=hidden NAME=“timesValidationFailed” VALUE=“<jsp:expression>((val.getFieldvalue(“timesValidation Failed”) == null) ?1 : 1 + Integer.parseInt(val.getField− Value(“timesValidation-Failed”) )) </jsp:expression>” > [ in the ‘back’ or ‘cancel, link] <A HREF=“javascript:history.go(−(document.forms[ O].timesValidationFai- led.value)) ”></A> When adding screens into existingjSP sequences, the CSP must evaluate whether a counter needs to be added to the JSP.",
"This can be done by analyzing any counters that are on the screen before the new JSP and after the new JSP to see if a counter is being maintained.",
"(8) Display Objects.",
"Display objects are value objects in the eBusiness support system that hold attributes displayed on a page.",
"They are used as an alternative to domain objects when (a) the attributes to display span more than one domain object and (b) the domain object contains more attributes than what is needed to display.",
"For example, if a domain object called Foo contains four attributes, the CSP may want to display only one attribute.",
"The CSP could choose to carry the extra three attributes or create a display object that contains only the attribute that the CSP wants to display 1.4.2.Display Policies If the destination page in a transition requires more information than the parameters passed via the HTTP request from the Web session controller, or if a tag alone is not sufficient to retrieve and manipulate needed data, then a display policy is used.",
"Query and generic tags on the resulting page access these policies, which are then used to retrieve and optionally manipulate data.",
"Display policies are interfaces that a CSP can implement to extend the functionality of the tags.",
"Each tag of the tags has a corresponding display policy interface that the tag can invoke.",
"Table 1.2 illustrates various display policies and methods to implement.",
"TABLE 1.2 Display Policy Description Methods to Implement BundlePolicy Invoked by the bundle Must implement the following method: tag.",
"DoAfterLoadBundle Modifies the list of BundleItem Objects.",
"QueryObject Invoked by the query0bject Must implement the following method: Policy tag.",
"createExpression Creates the CygentExpression object; DoAfterQuery Modifies the object.",
"QueryListPolicy Invoked by the Must implement the following methods: queryList tag.",
"createExpression Creates the CygentExpression object; doAfterQuery Modifies the created list.",
"Genericobject Invoked by the Must implement the following method: Policy genericObject tag.",
"loadObject Creates any object to be available in the JSP page.",
"GenericList Invoked by the Must implement the following methods: Policy genericList tag.",
"loadList Creates a list of any object to be available in the JSP page.",
"LoopPolicy Invoked by the loop Must implement the following methods: tag.",
"doInitLoop Modifies values in CygentContext; doBeforeLoopBody Modifies the item in the list before it is displayed; DoAfterLoopBody Modifies the item in the list after it is displayed; doEndLoop Modifies values in CygentContext.",
"The CSP can also use JSPs to upload files to a server.",
"The properties file determines the directory to which any uploaded file will be saved, and the maximum allowable file size.",
"To allow an upload, the <FORM> tag must include the following attribute setting: ENCTYPE=“mulitpart/form-data” This upload request is handled slightly differently than any other page request from a JSP.",
"Normally, all data submitted from a JSP is available to a portal as parameters.",
"However, parameters are only allowed to be of type String.",
"In this case, since a file is created on the server, an attribute in Request scope is created.",
"It is accessible using the same form input name as indicated in the <INPUT TYPE=“FILE”>field on the JSP.",
"The getAttribute method in this case, returns an object of type File.",
"All other input fields are available, as usual, as parameters.",
"Additionally, to prevent overwriting of files on the server, the name of the file is prepended with the session ID, which is unique.",
"The original file name (without the session id) is available as a Parameter using the name as indicated in the <INPUT TYPE=“FILE”>field on a JSP.",
"Since the Web framework is unable to determine when the portal is done using the file, the framework is unable to know when the file should be deleted from the server.",
"It is left to the portal developers to delete this uploaded file, when no longer needed.",
"This can be done using the method delete on the Java File object.",
"1.4.3.Web Session Controller The Web session controller is a Java servlet that provides load balancing through a session-level, round-robin algorithm that weighs server load information and routes requests accordingly.",
"This servlet also provides failover by replicating HTTP session information across nodes in a cluster, and maintains session state via a cookie-based session ID.",
"Every link or form submission on a JSP page points to this controller, called Go.",
"When a transition starts, the following information is passed: to directive—the intended destination page.",
"from directive—the originating page (the page that link is on).",
"task directive—the task to accomplish during the transition from the originating page to the destination page.",
"any parameters required by the transition policy or destination page The Web session controller also resolves the page ID submitted with the request to an actual URL, in order to display the page.",
"The Web session controller uses scope as a way of describing the lifecycle of data as it travels from a client, through the framework, and back to the client.",
"There are four types of scope and they range from a very short lifecycle to a long running lifecycle.",
"These scopes are: (1) Page.",
"Page scope begins when a JSP starts rendering, and is available only to that page.",
"Page scope is primarily used to pass data back and forth between a page and a display policy.",
"Page scope expires when a JSP is finished rendering.",
"Sub JSPs each have their own scope, therefore the Request scope must be used to pass something from a parent to sub JSP.",
"(2) Request.",
"Request scope is analogous to parameter and attribute data in an HTTP request.",
"It begins with a browser request that passes in HTML form data, which is stored in the scope of parameters.",
"Request scope lives throughout the life of the request from a client, until the destination JSP is rendered and sent back to the browser.",
"Request scope is most often used to pass data from transition policies to JSPs and display policies.",
"It is also used to pass data between parent and sub JSPs.",
"(3) Session.",
"Session scope is analogous to attribute data in an HTTP session.",
"It is created when a user first makes a request to the Web session controller and is destroyed when the session times out.",
"Session scope is generally used to store data between requests.",
"Keep in mind that information placed in session scope lives until it is explicitly removed or the session is terminated.",
"Therefore, putting many objects into session scope can have performance impacts.",
"(4) Application.",
"Application scope spans all sessions on the server.",
"It begins with server startup and ends when the server is shut down.",
"It is not clustered; each node has its own application scope.",
"1.4.4.Transition Policies When a transition off a page requires data to be submitted or requires complex business processes, then a transition policy is invoked.",
"When a user clicks a button or link, the Web session controller accesses the TRANS_MAP database table to determine if a transition policy is needed, using the from and task directives.",
"It then routes the request either to that policy or if no policy exists, directly to the subsequent page.",
"Transition policies are Java files that contain navigation and validation logic.",
"They access the Smart Components' API to perform specific business logic through the process and parse methods.",
"These methods may also provide server-side validation against any submitted data.",
"Once this method is executed, control returns to Go, which then determines the URL of the destination page using the URL_MAP table in the database.",
"Transition policies may also use the parseAddOn method, which enables the CSP to handle any new form fields that are added to a JSP in addition to the fields handled by the parse method.",
"The parse method in the transition policy takes user input (HTML form data) and returns ParameterData, which contains at least one hash map of attributes and values, or at least one new (or updated) domain objects.",
"To add a new form field, the CSP must first subclass the existing transition policy for the task that needs to pass in the new field value.",
"In the subclass, the CSP implements the parseAddOn method, which receives as a parameter the ParameterData returned by parse.",
"To accommodate the new form field, the CSP should alter the hash map or object to include data that corresponds to the new fields.",
"The updated ParameterData is then used by the process method when executing the necessary navigation logic and calls to activity Smart Components.",
"For example: public void parseAdd0n(CygentContext context, ParameterData paramData) throws CygentException { ParameterDataEntry contactEntry; MyContact contact; // get entry from data structure contactEntry = paramData.getEntry(1); // get domain object from entry contact = (MyContact)contactEntry.getDomainobject( ); // update attributes contact.setSecondPhoneNumber (context.getParameter(“Contact.SecondTN”)); ... } To add validation for a new form field, it is necessary to call the add method on the validation context object from within the parseAddon method.",
"The process method then executes validation and handles validation errors for the new field.",
"(1) Nested Flows FIG.",
"7 illustrates a section of page flow 180 used in the Small Business Portal that includes a registration process 185 as a nested flow.",
"There are two places in the flow where a user can access the registration process: the Shopping Cart page 181 and the Splash page 182.Since there is more than one page that can access that page flow, there is more than one page that the user can end up on at the end of the registration process.",
"In the case of registration, there are two different pages: (a) Quote Summary 183, if the user enters the flow from a shopping cart; or (b) MyHome 184, if the user enters the flow from the Splash page 182.Whenever a page flow has an undetermined exit, that process is considered a nested flow 185.In these cases the transition policy for the transition into the nested flow invokes a method that passes in (a) the ID for the first page in the nested flow; (b) the origin page ID; and (c) the destination page ID.",
"The Web session controller then stores that information and directs the user into the nested flow 185.A transition directive on the last page in the nested flow informs the controller to re-execute the transition policy for the originating page.",
"However if needed, the last page in the flow may use a transition policy to first perform some business process, such as persisting data, before re-executing the originating page's transition policy.",
"If the DEST keyword is present, the server detects the keyword and re-executes the initial transition policy.",
"In some cases, the CSP may also want to execute a transition policy that performs some business process, such as persisting data, before re-executing the transition policy that initiated entry into the nested flow.",
"To do this, create an entry in the TRANS_MAP table that maps the logical page and task to the transition policy class.",
"Then, the new task can be passed to the subForm method to execute the transition policy.",
"After the original transition policy executes, the user exits the nested flow and forwards to the initial destination.",
"If the ORIG keyword is present, the server redirects the user to the page specified by the origPageld parameter without re-executing the transition that initiated entry into the nested flow.",
"In the previous registration example, when the transition policy for checkout on shopping cart 181 is re-executed, the user is now registered; therefore the destination page (either Quote Summary 183 or MyHome 184) is displayed.",
"If there is a need to reroute to another page, for example, to send the user to an error page, the nested flow 185 needs to be cleared.",
"To do this, the clearNestedState method on the CygentContext object in the transition policy should be called.",
"(2) Non-Repeatable Transitions The portal architecture also allows for the restriction of transition policies to be repeated.",
"For example, when an end user has placed an order, the user should not be allowed to use his browser back button and resubmit the same order.",
"To prevent this, transition policies can be flagged so that they cannot be repeated in the scope of a page flow.",
"When this flag is set, instead of re-executing the transition policy, the server redirects to the destination page determined during the first execution of the transition policy.",
"In some cases, the CSP may not want a user to execute the same transition twice during a critical flow.",
"For example, a user ordering an access line should not be able to submit the same order twice by using the back button to return to the order page after the order has already been submitted.",
"To avoid this, the CSP can create a clearly defined set of related logical pages that constitute a flow, and specify pages within the flow that have non-repeatable transition policies.",
"These transitions are non-repeatable within the scope of the flow.",
"Now referring to FIG.",
"8, an example of a non-repeatable transition 190 is illustrated.",
"Page C is associated with a transition policy (Policy3) that the user should not be allowed to execute more than once.",
"Also referring to Table 1.3, for the first execution of the submit link on page C, the server looks in the TRANS_MAP table and executes the transition policy-associated with the originating page ID (C) and task (submit).",
"TABLE 1.3 ORIG-PAGE-ID TASK CLASS_NAME NO_REPEAT_FLG A cancel Policy1 0 B replace Policy2 0 C submit Policy3 1 After clicking the “submit” button and transitioning to page D, the user may use the “back” button to return to page C. However, if the user (now on page C) tries to click the “submit” button again, the server does not re-execute the transition policy because the NO_RFPEAT_FLG is set to 1.Instead, it bypasses the transition policy and directs the user to the destination page determined during the first execution of the policy (when the FLOW table was accessed).",
"In this example, the next page would be page D. See Table 1.4.TABLE 1.4 Flow_ID Page_ID Page_Order Flow1 A 1 Flow1 B 2 Flow1 C 3 The user can now only execute Policy3 again by navigating through the application to page A (without using the browser button to go back).",
"For all pages with the same FLOW_ID, the data in the PAGE_ORDER column must consist of consecutive numbers that start with 1.",
"(3) Server-Side Validation Whenever the CSP collects HTML form data that will be persisted to the database, the server-side validation within the transition policies should be used.",
"Data that does not meet the validation criteria is placed in a validation exception (along with an error message retrieved from a resource bundle) and thrown.",
"The Web framework redirects the user to the previous screen and makes information about the exception, including the input data, available to the screen.",
"Table 1.5 illustrates the server-side validation types supported by the current system.",
"TABLE 1.5 TYPES DESCRIPTION credit card Returns true if the credit card number is valid based on the LUHN validation.",
"Otherwise, returns false.",
"The credit card number must have 13 to 17 digits in one of the following formats: 5555555555555555 5555 5555 5555 5555 5555 555555 55555 5555555555 date Validates a date string in the format MM/dd/yyyy.",
"(for example, Jan. 1, 2000) but will not except a shortened year format of M/d/yy (for example, Jan. 1, 2000).",
"code value in a Validates that a code value is valid.",
"resource bundle code value not Validates that the user made a selection from a drop-down menu zero populated using code values from a resource bundle.",
"email Validates that the email address includes an period, and ends in a three letter domain or two letter country.",
"Valid formats include: extension-greg@host.com quoted User-“Greg Jones”@host.com ipDomain-greg@[123.123.123.1] forCountry-Greg@host.co.uk maximum length Validates that a “form value” is less than or trim the value prior to validating.",
"non-numeric Returns true if the entire “input string” contains non-digits.",
"Returns false if the “input string” contains any digits.",
"non-zero Returns true if the “input string” contains one or more numeric numeric characters with at least one non-zero character.",
"Returns false if the “input string” contains non-numeric characters or only‘O’ characters.",
"not blank Returns false if the given “text field” is empty.",
"Returns true if the given “text field” contains a value.",
"numeric Returns true if the “input string” contains a numeric string.",
"Returns false if the “input string” contains any non-numeric characters.",
"positive non- Returns true if the “input string” contains a numeric string which is zero numeric greater than zero.",
"Returns false if the “input string” contains a negative numeric string or any non-numeric characters.",
"positive numeric Returns true if the “input string” contains a numeric string which is greater than or equal to zero.",
"Returns false if the “input string” contains a negative numeric string or any non-numeric characters.",
"telephone Verifies that the phone number is numeric with any combination of number dashes, parenthesis, spaces, and periods.",
"zip code Verifies that the zip code is numeric, dash allowed, and exactly 5 or 9 digits.",
"Valid formats include: 55555 55555-5555 555555555 The CSP can change the error messages that these validation types return by editting the resource bundle cygent.common.resource.ValidationMessageResourceBundle, or creating a new resource bundle and change the cygent.httpd.validation bundle property in the cygent properties file to use a different resource bundle class name.",
"1.4.5.Resource Bundles The eBusiness support system stores all static data displayed at the front end (except data coded directly into JSP pages) in Java classes called resource bundles.",
"Resource bundles associate text strings that appear in the portal with unique codes used internally by the application.",
"For example, static data for street types is stored in a bundle class called StreetTypeCodeResourceBundle: public class StreetTypeResourceBundle extends CygentResourceBundle { static final Object[ ][ ] contents = { //LOCALIZE THIS { “1”, “AVENUE”}, { “2”, “DRIVE”}, { “3”, “PLACE”}, { “4”, STREET”}, { “5”, “WAY”), //END OF MATERIAL TO LOCALIZE }; Each resource bundle must have a corresponding constant class that identifies the unique codes used by the application.",
"For this example, the constant class StreetTypeCode defines the codes that are used in the bundle class StreetTypeResourceBundle.",
"public class StreetTypeCode { //FIELDS --------------------------------- /**Avenue */ public static final int AVENUE = 1; /** Drive */ public static final int DRIVE= 2; /** Place */ public static final int PLACE = 3; /** Street */ public static final int STREET = 4; /** Way */ public static final int WAY = 5; } // end StreetTypeCode class 1.5.Client Request Process FIG.",
"9-11 illustrate a process for submitting a request.",
"The process includes the steps of: (1) A user clicks a link on a page.",
"Referring to FIG.",
"10 which illustrates a sample page 210, a user enters information to open a trouble ticket against a product in the hierarchy, and clicks submit button 211.Referring to FIG.",
"9, the entered data, along with the originating page, destination page, and task (CreateTrouble-Ticket, Trouble-TicketRecap, and submit) are sent via an HTTP request 161 to the Web session controller 162.",
"(2) The Web session controller 162 accesses the TRANS_MAP table in the database 145 to determine whether to invoke a transition policy 163.For example, the database row that contains CreateTroubleTicket as the origin page and submit as the task also contains the required transition policy, TroubleTicket-SubmitPolicy.",
"This class is invoked.",
"(3) The transition policy 163 calls an activity Smart Component 120.For example, the TroubleTicket transition policy calls the factory 143 to obtain the TroubleTicketManager Smart Component 133.",
"(4) The activity Smart Component 120 calls DAS 135 to create and persist value objects 141.The transition policy 163 passes in values for the object.",
"For example, the TroubleTicketManager Smart Component 133 calls DAS 135 to create new trouble ticket and trouble ticket contact domain objects.",
"It then updates the objects with form data and persists the updated objects.",
"(5) Control is passed back to the transition policy 163.For example: the transition policy populates the CMI 105 with needed form data, and passes the CMI 105 to the interconnect service 136.",
"(6) The transition policy 163 returns control to the Web session controller 162.",
"(7) The Web session controller 162 resolves the page ID passed in the HTTP request 161 to a URL, and invokes the corresponding JSP page 201.For example, the TroubleTicketRecapJSP page is invoked.",
"This file contains query tags to access the TroubleTicketRecapPolicy display policy.",
"(8) Corresponding display policies 202 are invoked and, if needed, a display object is created.",
"For example, the TroubleTicketRecapPolicy gets all the information needed to display the new trouble ticket.",
"(9) The dynamic HTML page is returned to the client via HTTP.",
"Referring to FIG.",
"11 for example, the user sees a recap 220 of the opened dispute.",
"Section 2.Data Structure This section describes the data structure for the eBusiness support system which includes various database tables.",
"2.1.Area Table Descriptions All tables of the database are grouped according to their area.",
"Each table lists the name of each field attribute, whether or not the field is required, the type and length, and a description for each field.",
"(1) Populating Tables Manually Every row in most database tables uses an Object Identifier (OID) as the primary key.",
"To manually populate a row in a database, the entry for the row's OID must be determined for that area, using the SEQ table.",
"The SEQ table contains an entry for each area of the application (the SEQ_NAME attribute) and a VALUE.",
"The value is the next number to be used as an OID for that area, and is the number to be used as the OID for the new row.",
"However, once this number is used, it must also be manually incremented, so that the next entry made will use a unique number.",
"(2) Type Some tables have a TYPE field, a class indicator field used by the persistence layer to determine the subclass for a particular object.",
"If the database is manually populated for an object that has subclasses, the correct type value must be entered.",
"If the database is populated using the Administrator console, these values are automatically entered.",
"To determine the correct value, the TopLink Builder console is used to view the inheritance properties for the superclass object.",
"(3) Write Lock Most tables have a WRITE_LOCK field.",
"This field works with the persistence layer to provide an optimistic lock that prevents access to a field if It is in the process of updating.",
"(4) Primary Keys The field name OID denotes the primary key for a table.",
"Any other field that uses “_OID” in its name is a foreign key.",
"(5) Dates: All unset dates are treated as Y.",
"2.2.Agent An agent is a user of the Universal Agent Portal.",
"TABLE 2.1 AGENT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the agent OVRRDE_THRSHD_VALUE N NUMBER (9) Threshold value for this agent OVRRDE_THRSHD_CD N NUMBER (9) Code indicating the override threshold type AGENT_TYPE_CD N NUMBER (9) Code indicating the agent type STATUS_CD N NUMBER (9) Code indicating the status for an agent type EMP_ID Y VARCHAR2 (50) Employee ID for this agent SINCE_DT N DATE The date this agent was created CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.2 maps an agent to an agent group.",
"TABLE 2.2 AGENT_GROUP_MEMBER_MAP Allows Attribute Name Nulls?",
"Type Description AGENT_OID N NUMBER (18) Object identifier for the agent AGENT_GROUP_OID N NUMBER (18) Object identifier for the agent group Table 2.3 maps an agent to an agent group to which they have visibility.",
"TABLE 2.3 AGENT_GROUP_VISBLTY_MAP Allows Attribute Name Nulls?",
"Type Description AGENT_OID N NUMBER (18) Object identifier for the agent AGENT_GROUP_OID N NUMBER (18) Object identifier for the agent group Table 2.4 maps an agent to a root (either a customer or a partner).",
"TABLE 2.4 AGENT_ROOT_MAP Allows Attribute Name Nulls?",
"Type Description AGENT_OID N NUMBER (18) Object identifier for the agent ROOT_OID N NUMBER (18) Object identifier for the root Table 2.5 creates a parent/child association between agent groups.",
"TABLE 2.5 AGENT_GROUP_GROUP_MAP Allows Attribute Name Nulls?",
"Type Description PARENT_OID N NUMBER (18) Object identifier for the parent CHILD_OID N NUMBER (18) Object identifier for the child TABLE 2.6 AGENT_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the agent group NAME N VARCHAR2 (50) Name of agent group CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.7 maps the agent to the root request with the root associated.",
"TABLE 2.7 AGENT_ROOT_RQST_MAP Allows Attribute Name Nulls?",
"Type Description AGENT_OID N NUMBER (18) Object identifier for the agent ROOT_RQST_OID N NUMBER (18) Object identifier for the root request 2.3.Bill Presentment Table 2.8 is a container used to pass billing point information to the portal.",
"TABLE 2.8 ACCT_RECVBL Allows Attribute Names Nulls?",
"Type Description OID N NUMBER (18) Object identifier for accounts receivable BLNG_POINT_OID N NUMBER (18) Object identifier for the billing point ENTERED_DT N DATE The date time for the date entered CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.9 represents an entry to an account.",
"TABLE 2.9 ACCT_RECVBL_ENTRY Allows Attribute Names Null?",
"Type Descriptions OID N NUMBER (18) Object identifier for accounts receivable entry ACCT_RECVBL_OID N NUMBER (18) Object identifier for accounts receivable ACCT_RECVBL_TYPE_CD N NUMBER (9) Code indicating the accounts receivable type AMT N NUMBER ( ) Amount of the accounts receivable entry CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.10 displays header information regarding charge amounts for a specific account imported from an external billing system.",
"TABLE 2.10 INVOICE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice INVOICE_ID Y VARCHAR2 (50) Invoice identifier used by an external system.",
"The default is the OID.",
"ACCT_OID N NUMBER (18) Object identifier for the associated account INVOICE_START_DT N DATE Start date of the billing cycle INVOICE_END_DT N DATE End date of the billing cycle TOTAL_AMT_DUE N NUMBER ( ) Total net price of all transactions for the given account during the billing cycle STATE_CD N NUMBER (9) Code indicating the state of the invoice after confirmation STATUS_CD N NUMBER (9) Code indicating the status for an invoice DSPLBL_INVOICE_PARTS_CD N NUMBER (9) Determines what to display for the invoice (i.e., use an external URL, use an internal invoice summary, or both) EXT_URL Y VARCHAR2 (240) External URL to link to an invoice CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.11 represents a dollar amount by which a charge is adjusted.",
"TABLE 2.11 INVOICE_ADJMNT_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the adjustment ADJMNT_TYPE_CD N NUMBER (9) Code indicating the type of adjustment ADJMNT_DT Y DATE Date the adjustment was created Table 2.12 represents the amount charged for a transaction against an object.",
"These values are imported from an external billing system.",
"TABLE 2.12 INVOICE_CHARGE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice charge ACTION_CD N NUMBER (9) Code indicating the type of action that created this charge UOM_CD N NUMBER (9) Code indicating the unit of measure QTY Y NUMBER (18) Quantity SVC_ID Y VARCHAR2 (240) Identifier for the service domain (i.e., a phone number or email address) SVC_DOMAIN_CD N NUMBER (9) Code indicating how the associated product will be tracked for usage or billing (e.g., a phone number or email address) OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer Table 2.13 represents an invoice charge item that represents a discount imported from an external billing system.",
"TABLE 2.13 INVOICE_DISC_ITEM Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the discount START_DT Y DATE Date the discount starts END_DT Y DATE Date the discount ends QTY Y NUMBER ( ) Number of units PRORTN Y NUMBER ( ) Amount that the discount is prorated UOM_CD N NUMBER (9) Code indicating the unit of measure Table 2.14 Represents information regarding a single line item imported from an external billing system.",
"TABLE 2.14 INVOICE_LINE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice line item INVOICE_OID N NUMBER (18) Object identifier for the associated invoice DSPL_ORDER N NUMBER (18) Order in which the line item is displayed EXT_SYS_REF Y VARCHAR2 (240) Reference to the external billing system DSCR Y VARCHAR2 (50) Description of the invoice line item AMT Y NUMBER ( ) Amount of the invoice line item CREATE_USR Y VARCHAR2 (40) Internal identifier of the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) Internal identifier of the user who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.15 represents an invoice charge that represents a non-recurring charge.",
"This is imported from an external billing system.",
"TABLE 2.15 INVOICE_NON_RCURRNG_CHARGE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the non-recurring charge CHARGE_DT N DATE Date of the charge Table 2.16 allows addition of external data in the form of new attributes to any classes within the bill presentment domain.",
"TABLE 2.16 INVOICE_LINE_ITEM_EXT_DATA Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice line item external data PARENT_OID N NUMBER (18) Object identifier for the parent object ATTRIB_NAME N VARCHAR2 (40) Name of the attribute ATTRIB_VALUE N VARCHAR2 (40) Value of the attribute WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.17 represents an invoice charge that represents a recurring charge.",
"This is imported from an external billing system.",
"TABLE 2.17 INVOICE_RCURRNG_CHARGE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Internal identifier for the recurring charge PRDCTY_CD N NUMBER (9) Code indicating the billing periodicity (i.e., weekly, monthly) START_DT N DATE Billing start date (exclusive) END_DT Y DATE Billing end date (inclusive) Table 2.18 represents invoice summary information, which is imported from an external billing system.",
"TABLE 2.18 INVOICE_SUMM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice summary INVOICE_OID N NUMBER (18) Object identifier for the associated invoice TOTAL_EXTENDED_PRICE_AMT N NUMBER ( ) Total extended price TAX_AMT N NUMBER ( ) Total tax amount ADJMNT_AMT Y NUMBER ( ) Total adjustment amount PREV_BAL_AMT Y NUMBER ( ) Previous balance DISC_AMT Y NUMBER ( ) Total amount of all discounts ONE_TIME_CHARGES_AMT Y NUMBER ( ) Total amount of any non- recurring charges MONTHLY_CHARGES_AMT Y NUMBER ( ) Total amount of recurring charges USAGE_CHARGES_AMT Y NUMBER ( ) Total amount of usage charges CREATE_USR Y VARCHAR2 (40) Internal identifier of the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) Internal identifier of the user who last modified the entry MOD_DT Y DATE Date the entry was last modified WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.19 represents an invoice charge item that represents a tax charge imported from an external billing system.",
"TABLE 2.19 INVOICE_TAX_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Internal identifier for the invoice tax item Table 2.20 represents an invoice charge item that represents usage charges imported from an external billing system.",
"TABLE 2.20 INVOICE_USAGE_CHARGE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the invoice usage charge START_DT N DATE Date the usage charge began END_DT Y DATE Date the usage charge ended TO_SVC_ID Y VARCHAR2 (240) The “to” service identifier FROM_SVC_ID Y VARCHAR2 (240) The “from” service identifier TO_SVC_DOMAIN_CD N NUMBER (9) Code indicating the service domain type for the “to” service identifier FROM_SVC_DOMAIN_CD N NUMBER (9) Code indicating the service domain type for the “from” service identifier Table 2.21.is a collection of attributes representing Adjustment Request to a Customer Bill.",
"TABLE 2.21 BILL_ADJMNT_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the bill adjustment request BLNG_POINT_OID N NUMBER (18) Object identifier for the billing point TARGET_OID N NUMBER (18) Object identifier for the target TARGET_CLASS N VARCHAR2 (80) The class of the target object (Invoice for e.g.)",
"associated with this bill adjustment request TYPE_CD N NUMBER (9) Code indicating the type of the bill adjustment request STATE_CD N NUMBER (9) Code indicating the state of the bill adjustment request STATUS_CD N NUMBER (9) Code indicating the status of the bill adjustment request REASON_CD N NUMBER (9) Code indicating the reason DSCR Y VARCHAR2 (240) Description of the bill adjustment request AMT N NUMBER ( ) Amount of the bill adjustment request CREATED_USR N VARCHAR2 (40) User name that created this bill adjustment request CREATED_DT N DATE The date this bill adjustment request is requested (created in system).",
"CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.4.Business Rules Table 2.22 contains information regarding rules used to maintain valid relationships between objects.",
"These rules are based on a finite set of atomic rules both simple (e.g., mutual exclusion) and complex (e.g., mutual exclusion plus a numerical limit).",
"TABLE 2.22 BUS_RULE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the business rule TEMPLT_CODE N NUMBER (9) Template code NAME Y VARCHAR2 (50) Name for the business rule VIOLTN_MSG Y VARCHAR2 (240) Violation message to be displayed in the portal DSCR Y VARCHAR2 (240) Description of the business rule CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER2 Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.23 contains parameter information which is used to describe a business rule.",
"TABLE 2.23 BUS_RULE_PARM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the business rule parameter BUS_RULE_OID N NUMBER (18) Object identifier for the associated business rule NAME N VARCHAR2 (50) Name of the parameter VALUE Y VARCHAR2 (50) Value of the parameter START_DT Y DATE Date the parameter becomes effective END_DT Y DATE Date the parameter is no longer effective CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.24 contains information regarding descriptor groups.",
"Descriptors are used to create groupings of objects or groupings of other descriptors that are used by business rules.",
"TABLE 2.24 DATA_OBJ_DSCPTR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the data object descriptor DSCR Y VARCHAR2 (240) Description of the data object descriptor CREATE_USR Y VARCHAR2 (40) User who created the entry TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.25 Contains information regarding individual items within descriptor groups.",
"Descriptors are used to create groupings of objects or groupings of other descriptors that are used by business rules.",
"TABLE 2.25 SIMPLE_DATA_OBJ_DSCPTR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the simple data object descriptor KEY_NAME Y VARCHAR2 (100) Name for the simple object descriptor VALUE Y VARCHAR2 (50) Value for the simple object descriptor Table 2.26 combines two simple object descriptors with a boolean operator to create grouping of objects used by business rules.",
"TABLE 2.26 CMPND_DATA_OBJ_DSCPTR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the compound descriptor OPERTN Y VARCHAR2 (10) Boolean operator (e.g., and/or) DATA_OBJ_DSCPTR1_OID N NUMBER (18) Object identifier for the first data object descriptor DATA_OBJ_DSCPTR2_OID N NUMBER (18) Object identifier for the second data object descriptor 2.5.Common Table 2.27 represents a group of principals that share a role for authorization purposes.",
"TABLE 2.27 CYG_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the Cygent group NAME Y VARCHAR2 (240) Name for the Cygent group value must match constant value in the CygGroupTypeCode.java constant class CREATE_USR Y VARCHAR2 (40) Date the entry was created CREATE_DT Y DATE User who created the entry MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.28 maps individual Cygent users to Cygent groups TABLE 2.28 CYG_GROUP_CYG_USR_MAP Allows Attribute Name Nulls?",
"Type Description CYG_GROUP_OID N NUMBER (18) Object identifier for the Cygent group CYG_USR_OID N NUMBER (18) Object identifier for the Cygent user CREATE_USR Y VARCHAR2 (40) Date the entry was created CREATE_DT Y DATE User who created the entry MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.29 contains an encrypted password for a user of the Cygent system.",
"TABLE 2.29 CYG_USR Allows Attribute Name Nulls?",
"Type Description PSWD N VARCHAR2 (240) Encrypted password OID N NUMBER (18) Object identifier for the Cygent user NAME N VARCHAR2 (40) Login name for the Cygent user ACTIVE_FLG N NUMBER (1) Indicates whether the Cygent user is active CREATE_USR Y VARCHAR2 (40) Date the entry was created CREATE_DT Y DATE User who created the entry MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.30 maps a CMI to a CMI policy.",
"TABLE 2.30 CMI_CMI_POLICY Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map CD_VALUE Y NUMBER (18) Code for the CMI POLICY_CLASS Y VARCHAR2 (240) Fully qualified path for the CMI policy CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.31 contains information used to grant a permission on a resource or class of resources to a list of Users and Groups.",
"TABLE 2.31 CYG_ACL Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the Cygent ACL ACL_NAME N VARCHAR2 (240) Name for the ACL ACL_PRNCPL_NAME N VARCHAR2 (240) Name of the user/group that has a specific permission for the ACL ACL_PRMSSN N VARCHAR2 (40) Permission assigned to the principal on this ACL CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.32 is used to group JSP pages into logical “flows” or sequences of pages which together perform a certain function.",
"TABLE 2.32 FLOW Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the flow FLOW_ID N VARCHAR2 (40) Identifier for the flow PAGE_ID N VARCHAR2 (40) Identifier for the JSP page PAGE_ORDER N NUMBER (18) Order in which the page should appear CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.33 determines which adapter to use if the CMI is associated with the generic routing policy.",
"TABLE 2.33 GENERIC_CMI_POLICY Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the generic CMI policy CD_VALUE N NUMBER (18) Code indicating the CMI policy to be called ADAPTER_HOME N VARCHAR2 (240) Home name for the CMI adapter CNTXT N VARCHAR2 (240) Implementation method for the adapter CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock TABLE 2.34 SOURCE_TARGET_STRATEGY_POLICY Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the source target strategy policy CD_VALUE N NUMBER (9) Code indicating the value PAYLOAD_CD N NUMBER (9) Code indicating he payload SOURCE_STRATEGY_CLASS N VARCHAR2 (240) The fully qualified source strategy class name TARGET_STRATEGY_CLASS N VARCHAR2 (240) The fully qualified target strategy class name CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock TABLE 2.35 CMI_LOG Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the CMI log ACTION_CD Y NUMBER (9) Code indicating the action for this CMI VENDOR_PROD_OID Y NUMBER (18) Object identifier for the vendor product associated with this CMI, if any.",
"CMI_ID N NUMBER (18) Object identifier for the CMI CD_VALUE N NUMBER (18) Code indicating the value CMI_DSCR Y VARCHAR2 (240) The CMI description STATUS_MSG Y VARCHAR2 (240) The CMI status, dispatched, completed or failed ADAPTER_HOME Y VARCHAR2 (240) The adapter home name that the CMI was delivered to ERROR_CD Y NUMBER (9) Code indicating the error ERROR_MSG Y VARCHAR2 (240) The CMI error message ERROR_CNTXT Y VARCHAR2 (500) The CMI error context DISPATCHED_CNT Y NUMBER (18) The number of times the CMI has been dispatched PAYLOAD_CD Y NUMBER (9) Code indicating the payload CORRECTED_CMI_ID Y NUMBER (18) The identifier of the corrected CMI CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.36 maps a domain class with a domain OID for a CMI.",
"TABLE 2.36 CMI_DOMAIN_OBJECT_LOG Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the CMI domain object log CD_LOG_OID N NUMBER (18) Object identifier for the CD log DOMAIN_OID N NUMBER (18) Object identifier for the domain DOMAIN_CLASS N VARCHAR2 (240) The domain class name CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.37 maps a plug-in constant to the appropriate plug-in class.",
"TABLE 2.37 PLUGIN_MAP Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the plug-in map PLUGIN_CD N NUMBER (9) Code indicating the plug-in PLUGIN_CLASS Y VARCHAR2 Fully qualified path for the plug-in class (240) CREATE_USR Y VARCHAR2 User who created the entry (40) CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 User who last modified the entry (40) MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.38 determines whether a transition policy is associated with the “from” and “Task” directives during a page transition at the front end.",
"TABLE 2.38 TRANS_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the transition map ORIG_PAGE_ID N VARCHAR2 Identifier of the origin page (80) TASK N VARCHAR2 Task to be executed (40) CLASS_NAME N VARCHAR2 Fully qualified class name for the association (240) transition policy NO_REPEAT_FLG N NUMBER(1) Flag indicating whether the transaction can be repeated CREATE_USR Y VARCHAR2 User who created the entry (40) CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 User who last modified the entry (40) MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.39 maps a page identifier to an external URL.",
"TABLE 2.39 URL_MAP Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the URL map PAGE_ID N VARCHAR2 Identifier for the page (80) URL N VARCHAR2 Relative path for the external URL (240) SECURE_FLG N NUMBER (1) Indicates whether the external URL uses a secure protocol CREATE_USR Y VARCHAR2 User who created the entry (40) CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 User who last modified the entry (40) MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.40 maps a validation constant to the appropriate validation policy class.",
"TABLE 2.40 VALDTN_MAP Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the validation map CD_VALUE Y NUMBER (18) Code for the associated constant POLICY_CLASS Y VARCHAR2 Associated policy class (240) CREATE_USR Y VARCHAR2 User who created the entry (40) CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 User who last modified the entry (40) MOD_DT Y MOD_DT Date the entry was last modified DATE TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.6.Customer Table 2.41 is default implementation of a billing point.",
"TABLE 2.41 ACCT Attribute Allows Name Nulls?",
"Type Description OID N NUMBER Object identifier for the account (18) ACCT_ID N NUMBER Identifier for the account used by external systems TABLE 2.42 ACCT_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the account request object ACCT_OID N NUMBER (18) Object identifier for the account STATE_CD N NUMBER (9) Code indicating the state ACTION_CD N NUMBER (9) Code indicating the action TOTAL_PRICE_AMT N NUMBER The amount of the total price of the account request TOTAL_TAX_AMT N NUMBER The amount of the total tax of the account request TOTAL_DISC_AMT N NUMBER The amount of the total discount of the account request SUBMTD_DT Y DATE The date the account request was submitted LABEL Y VARCHAR2 Label (50) BILLED_THRU_DT Y DATE The date the account request is billed through CREDIT_RATING_CD N NUMBER (9) Code indicating the credit rating BLNG_PRDCTY_CD N NUMBER (9) Code indicating the billing BILL_ROUND Y NUMBER TAX_GROUP_CD N NUMBER (9) Code indicating the tax group CREATE_USR Y VARCHAR2 User who created the entry (40) CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 User who last modified the entry (40) MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE LOCK N NUMBER (18) Optimistic lock Table 2.43 is a collection of attributes representing an address.",
"TABLE 2.43 ADDR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Internal identifier for an address object STREET_NBR Y VARCHAR2 (50) Street number STREET_NAME Y VARCHAR2 (50) Street name ADDL_NAME_LINE Y VARCHAR2 (240) Additional name line STREET_TYPE_CD N NUMBER (9) Code indicating the street type ADDL_ADDR_LINE Y VARCHAR2 (240) Second street address line DDL_ADDR_LINE2 Y VARCHAR2 (240) Third street address line STREET_DIRCTN_PRE_CD N NUMBER (9) Code indicating the street direction prefix STREET_DIRCTN_POST_CD N NUMBER (9) Code indicating the street direction postfix UNIT_NBR Y VARCHAR2 (50) Unit number UNIT_TYPE_CD N NUMBER (9) Code indicating the unit type ZIP_CD N VARCHAR2 (50) Zip code CITY N VARCHAR2 (50) City STATE_CD N NUMBER (9) Code indicating the state COUNTRY_CD N NUMBER (9) Code indicating the country HOUSE_NBR_SUFFIX Y VARCHAR2 (10) The house number suffix FLOOR Y VARCHAR2 (9) The floor for this address ROOM Y VARCHAR2 (10) The room number for this address COMMUNITY Y VARCHAR2 (32) The community name for this address CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.44 represents an offer instance that is either a provisioned product or a composite product, and has been assigned to a customer hierarchy.",
"TABLE 2.44 ASSGND_PROD Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the assigned product OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance BLNG_PRDCTY_CD N NUMBER (9) Code indicating the billing periodicity (e.g., monthly, weekly) BLNG_METHOD_CD N NUMBER (9) Code indicating the billing method (e.g., mail or electronic) PARTNER_CUST_OID Y NUMBER (18) Object identifier for the partner customer ORDRD_FOR_PROVSNG_DT Y DATE Date the product was ordered for provisioning ORDRD_FOR_UNPROVSNG_DT Y DATE Date the product was ordered to be unprovisioned SCHED_PROVSND_DT Y DATE Date the assigned product is scheduled to be provisioned SCHED_UNPROVSND_DT Y DATE Date the assigned product is scheduled to be unprovisioned PROVSND_DT Y DATE Date the assigned product was provisioned UNPROVSND_DT Y DATE Date the assigned product was unprovisioned RQSTD_PROVSND_DT Y DATE Date the assigned product was requested to be provisioned RQSTD_UNPROVSND_DT Y DATE Date the assigned product was requested to be unprovisioned TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.45 contains the information required for billing purposes.",
"To order billable products, a customer or partner must have at least one billing point.",
"A customer or partner can have multiple billing points.",
"TABLE 2.45 BLNG_POINT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the billing point PRICE_GROUP_OID Y NUMBER (18) Object identifier for the associated price group ACTIVE_DT Y DATE Date the billing point became active INACTIVE_DT Y DATE Date the billing point became inactive BILLED_THRU_DT Y DATE Last bill round date the account was billed BLNG_PRDCTY_CD N NUMBER (9) Code indicating the billing periodicity (e.g., weekly, monthly, etc.)",
"BLNG_METHOD_CD N NUMBER (9) Code indicating the billing method (i.e., mail, electronic, etc.)",
"TAX_GROUP_CD N NUMBER (9) Code indicating the tax group BILL_ROUND Y NUMBER ( ) Bill round CREDIT_RATING_CD N NUMBER (9) Code indicating the credit rating RUNNING_BAL N NUMBER ( ) Up-to-date balance of a customer's account TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.46 represents the group of products ordered using an offer collection.",
"TABLE 2.46 COMPOSITE_PROD Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the composite product TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.47 is a collection of attributes representing a person responsible for an entity in the Cygent system.",
"An entity can have one to many contacts.",
"TABLE 2.47 CNTCT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the contact NAME_OID N NUMBER (18) Object identifier for the name of the contact PHONE_NBR Y VARCHAR2 (50) Contact phone number CELL_NBR Y VARCHAR2 (50) Contact cell number PGR_NBR Y VARCHAR2 (80) Contact pager number DSCR Y VARCHAR2 (240) Description TITLE Y VARCHAR2 (40) Title CNTCT_TYPE_CD N NUMBER (9) Code indicating the contact type (e.g., technical, business) EMAIL_ADDR Y VARCHAR2 (240) Email address of the contact TARGET_OID N NUMBER (18) Object identifier for the entity instance that the contact is responsible for TARGET_CLASS N VARCHAR2 (80) Fully-qualified path for the target class CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.48 represents credit information for a hierarchy object TABLE 2.48 CREDIT_REF Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the credit reference CREDIT_TYPE_CD N NUMBER (9) Code indicating the type of credit reference (e.g., FID, SSN, VISA) CREDIT_ID N VARCHAR2 (50) Credit identifier associated with this credit reference for use by an external system.",
"The default is the OID.",
"TARGET_OID N NUMBER (18) Object identifier for the object with which the credit reference is associated TARGET_CLASS N VARCHAR2 (80) Fully-qualified path for the target class CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.49 is an implementation of the root hierarchy object.",
"TABLE 2.49 CUST Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the customer TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass CUST_ID N NUMBER The ID of this customer TABLE 2.50 CUST_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the customer request CUST_OID N NUMBER (18) Object identifier for the customer STATE_CD N NUMBER (9) Code indicating the state SUBMTD_DT Y DATE Date the customer request was submitted ACTION_CD N NUMBER (9) Code indicating the action TOTAL_PRICE_AMT Y NUMBER Amount of the total price of the customer request TOTAL_TAX_AMT Y NUMBER Amount of the total tax of the customer request TOTAL_DISC_AMT Y NUMBER Amount of the total discount of the customer request LABEL Y VARCHAR2 (50) Label CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.51 represents a partner as an implementation of a root.",
"TABLE 2.51 PARTNER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the partner TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.52 captures changes made to a Partner.",
"TABLE 2.52 PARTNER_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the partner request TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.53 represents Partner-defined ID for a partner's customer.",
"TABLE 2.53 PARTNER_CUST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the partner customer PARTNER_CUST_ID Y VARCHAR2 (50) Partner-defined ID for a partner's customer LABEL Y VARCHAR2 (50) Partner/customer label PARTNER_OID N NUMBER (18) Object identifier for the partner CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (18) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.54 contains information about geographical area where Partner is authorized to resale.",
"TABLE 2.54 RESALE_REGION Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the resale region REGION_CD N NUMBER (9) Code indicating the region PARTNER_OID N NUMBER (18) Object identifier for the partner CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.55 represents delivered products which are owned by an account and require no subscriber-type relationship.",
"TABLE 2.55 DELVRD_PROD Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the delivered product ORDRD_DT Y DATE Date the product was ordered PARTNER_CUST_OID Y NUMBER (18) Object identifier for the partner and customer RQSTD_DELVRD_DT Y DATE Date the product was requested to be delivered SCHED_DELVRD_DT Y DATE Date the product is scheduled to be delivered SHIPPED_DT Y DATE Date product was shipped RETURNED_DT Y DATE Date the product was returned DSPL_EXPRTN_DT Y DATE Last date the delivered product is displayed OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance INVENTORY_HIER_OBJ_OID Y NUMBER (18) Object identifier for the billing point or account the delivered product is associated with LABEL Y VARCHAR2 (50) Label for the delivered product DSCR Y VARCHAR2 (240) Description of the delivered product STATE_CD N NUMBER (9) Code that indicating state of the delivered product (e.g., pending, cancelled) STATUS_CD N NUMBER (9) Code indicating the status of the delivered product (e.g., shipped) CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.56 represents a point on the hierarchy.",
"A hierarchy objects can be a root, a billing point, or an assigned product.",
"TABLE 2.56 HIER_OBJECT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the hierarchy object LABEL Y VARCHAR2 (50) Label for the hierarchy object STATUS_CD N NUMBER (9) Code indicating the hierarchy object status STATE_CD N NUMBER (9) Code indicating the hierarchy object state PARENT_OID Y NUMBER (18) Object identifier for the parent hierarchy object HIER_OBJECT_TYPE_CD N NUMBER (9) Code indicating the object type (i.e., root, billing point, assigned product) ROOT_OID Y NUMBER (18) Object identifier for the hierarchy object that is the root of this hierarchy DSPL_EXPRTN_DT Y DATE Last date that the hierarchy object can be displayed DSCR Y VARCHAR2 (240) Description of the hierarchy object CREATE_USR Y VARCHAR2 (40) Internal identifier of the user who created the entry CREATE_DT Y DATE Date the entry was last modified MOD_USR Y VARCHAR2 (40) Internal identifier of the user who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.57 allows external information to be added to hierarchy classes in the form of attributes.",
"TABLE 2.57 HIER_OBJECT_EXT_DATA Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the hierarchy object external data PARENT_OID N NUMBER (18) Object identifier for the parent object ATTRIB_NAME N VARCHAR2 (40) Name for the attribute ATTRIB_VALUE N VARCHAR2 (40) Value for the attribute WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.58 maps a summary point to a hierarchy object.",
"TABLE 2.58 HIER_OBJECT_SUMM_POINT_MAP Allows Attribute Name Nulls?",
"Type Description HIER_OBJECT_OID N NUMBER (18) Object identifier for the hierarchy object OID N NUMBER (18) Object identifier for the map SUMM_POINT_OID N NUMBER (18) Object identifier for the summary point object CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.59 maps a delivered product to an associated hierarchy object.",
"TABLE 2.59 HIER_OBJ_DELVRD_PROD_MAP Allows Attribute Name Nulls?",
"Type Description HIER_OBJECT_OID N NUMBER (18) Object identifier for the hierarchy object DELVRD_PROD_OID N NUMBER (18) Object identifier for the delivered product Table 2.60 contains information regarding the customer's name.",
"TABLE 2.60 NAME Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the name SURNAME N VARCHAR2 (50) Last name GIVEN_NAME1 Y VARCHAR2 (50) First name GIVEN_NAME2 Y VARCHAR2 (50) Middle or second name GIVEN_NAME3 Y VARCHAR2 (50) Third or middle name PREFIX_CD N NUMBER (9) Code indicating the name prefix SUFFIX_CD N NUMBER (9) Code indicating a suffix to the name CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.61 maps an object to a specific address and address type.",
"This table is populated when an object can be associated with multiple addresses (each of a different type).",
"If an object is associated with only one address, the ADDR_OID is stored on the object itself and the OBJ_ADDR_MAP table is not used.",
"TABLE 2.61 OBJ_ADDR_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the object address map TARGET_OID N NUMBER (18) Object identifier for the associated target object TARGET_CLASS N VARCHAR2 (80) Fully-qualified path to the associated target class.",
"Target classes include classes in the customer domain, such as customer, account, contact, account request, and customer request.",
"ADDR_OID N NUMBER (18) Object identifier for the address ADDR_TYPE_CD N NUMBER (9) Code indicating the type of address ROOT_OID Y NUMBER (18) Object identifier for the root object CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER ( ) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.62 is used to apply rates specific to a group of customers or partners, such as residential or business price group.",
"Every root must be assigned to a price group.",
"TABLE 2.62 PRICE_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the price group NAME N VARCHAR2 (50) Name of the price group DSCR N VARCHAR2 (50) Description of the price group ROOT_MENU_OID N NUMBER (18) Object identifier for the associated root menu CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.63 represents products that have been provisioned.",
"TABLE 2.63 PROVSND_PROD Attribute Allows Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the provisioned product TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.64 represents the highest point in a hierarchy.",
"A root has no parent.",
"TABLE 2.64 ROOT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the root ROOT_ID Y VARCHAR2 (50) The root ID PRICE_GROUP_OID Y NUMBER (18) Object identifier for the associated price group ATHZTN_INFO Y VARCHAR2 (40) The authorization info for root LOGO_URL Y VARCHAR2 (240) The URL to this root's logo DOC_URL Y VARCHAR2 (240) The URL to a file SINCE_DT Y DATE Date the root was created TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.65 encapsulates a change instance of the Customer hierarchy object TABLE 2.65 ROOT_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the root request ROOT_OID N NUMBER (18) Object identifier for the root STATE_CD N NUMBER (9) Code indicating the state of the root request SUBMTD_DT N DATE The date when root request was submitted ACTION_CD N NUMBER (9) Code indicating the action type ROOT_ID Y VARCHAR2 (50) The root ID TOTAL_PRICE_AMT N NUMBER ( ) The total price for this root request TOTAL_TAX_AMT N NUMBER ( ) The total tax for this root request TOTAL_DISC_AMT N NUMBER ( ) The total discounts for this root request LABEL Y VARCHAR2 (50) The HierObjectImpl label PRICE_GROUP_OID Y NUMBER (18) The price group OID for this root request DOC_URL Y VARCHAR2 (240) The URL link to any documents/contracts associated with root ATHZTN_INFO Y VARCHAR2 (40) Authorization information CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.66 represents that a billing point request captures changes made to a billing point.",
"TABLE 2.66 BLNG_POINT_RQST Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the billing point request BLNG_POINT_OID N NUMBER (18) Object identifier for the billing point STATE_CD N NUMBER (9) Code indicating the state type ACTION_CD N NUMBER (9) Code indicating the action type TOTAL_PRICE_AMT N NUMBER ( ) The total price for this billing point request TOTAL_TAX_AMT N NUMBER ( ) The total tax for this billing point request TOTAL_DISC_AMT N NUMBER ( ) The total discounts for this billing point request SUBMTD_DT N DATE The date when the billing point request was submitted LABEL Y VARCHAR2 (50) The billing point label BILLED_THRU_DT Y DATE The billed through date of this billing point CREDIT_RATING_CD N NUMBER (9) Code indicating the credit rating BLNG_PRDCTY_CD N NUMBER (9) The billing periodicity of this billing point BLNG_METHOD_CD N NUMBER (9) Code indicating the billing method BILL_ROUND Y NUMBER ( ) The bill round associated with this Billing Point TAX_GROUP_CD N NUMBER (9) Code indicating the tax group CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic Lock Table 2.67 represents a specific category associated with a hierarchy point for creating different views of the hierarchy.",
"TABLE 2.67 SUMM_POINT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the summary point SUMM_POINT_TYPE_OID N NUMBER (18) Summary point type DSCR N VARCHAR2 (240) Description of the summary point NAME N VARCHAR2 (50) Summary point name CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.68 represents a general category from which specific summary points are created.",
"TABLE 2.68 SUMM_POINT_TYPE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the summary point type NAME N VARCHAR2 (50) Name of the summary point type ICON Y VARCHAR2 (240) Path to the icon that represents the summary point type DSCR N VARCHAR2 (240) Description of the summary point type ROOT_OID N NUMBER (18) Object identifier for the associated root hierarchy object CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.69 represents that a person at the provider site is responsible for a particular aspect of a customer or partners business.",
"TABLE 2.69 SPPRT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the support NAME_OID N NUMBER (18) Object identifier for the name PHONE_NBR Y VARCHAR2 (50) Phone number CELL_NBR Y VARCHAR2 (50) Cell number PGR_NBR Y VARCHAR2 (80) Pager number TITLE Y VARCHAR2 (40) Title EMAIL_ADDR Y VARCHAR2 (240) Email address for support DSCR Y VARCHAR2 (240) Description of support SPPRT_TYPE_CD Y NUMBER (9) Code indicating the support type CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.70 maps a support to a root.",
"TABLE 2.70 SPPRT_ROOT_MAP Allows Attribute Name Nulls?",
"Type Description ROOT_OID N NUMBER (18) Object identifier for the root SPPRT_OID N NUMBER (18) Object identifier for the support Table 2.71 contains temporary information necessary to hold descriptions of products that are being considered for removal.",
"TABLE 2.71 TEMP_REMOVE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the temp remove item USR_OID N NUMBER (18) Object identifier for the associated user HIER_OBJECT_OID N NUMBER (18) Object identifier for the associated hierarchy object TO_OFFER_OID Y NUMBER (18) Object identifier for the “to” offer ACTION_CD N NUMBER (9) Action code CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.72 represnets the user object that contains information pertaining to a registered users login name and password.",
"TABLE 2.72 USR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the user object ATHRTY Y NUMBER (18) Associated authority role PSWD_RMNDR Y VARCHAR2 (50) Password reminder CYG_USR_OID N NUMBER (18) Object identifier for the associated Cygent user object USR_TYPE_CD N NUMBER (9) User type which determines where to take the user upon login.",
"TARGET_CLASS Y VARCHAR2 (80) Class path for the associated target.",
"TARGET_OID Y NUMBER (18) Object with which the user is be associated (usually customer) CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.73 holds information pertaining to a user interaction with the system.",
"For all portals it is used to log that a user has registered or signed in.",
"For the Universal Agent Portal, a log is also created when an agent puts a user or customer into session.",
"TABLE 2.73 USR_INTRCTN_LOG Allow Attribute Name Nulls?",
"Type Descriptions OID N NUMBER (18) Object identifier for the user interaction log USR_OID N NUMBER (18) Object identifier for the user ROOT_OID Y NUMBER (18) Object identifier for the root INTRCTN_TYPE_CD N NUMBER (9) Code indicating the interaction type PORTAL_TYPE_CD N NUMBER (9) Code indicating the portal type CREATED_DT N DATE Date the user interaction log was generated CREATE_USER Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by Toplink to identity subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.7.Frame Table 2.74 is used with TopLink to create an aggregation of object attributes for OID, amount, and date.",
"TABLE 2.74 AGG_OBJ_MAP Attribute Allows Name Nulls?",
"Type Description AMOUNT Y NUMBER ( ) Holds an aggregation of attributes for a currency amount DT Y DATE Holds an aggregation of attributes for a date Table 2.75 is used to instantiate domain objects TABLE 2.75 BUILDER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the builder class DOM_NAME N VARCHAR2 (80) Domain name BLDR_CLASS N VARCHAR2 (80) Builder class BLK_SIZE N NUMBER (18) Block size used to indicate the number of OIDs CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.76 is for a sequence of numbers used to determine OIDs.",
"Every row in a database table uses the Object Identifier (OID) as the primary key.",
"To manually populate a row in a database, the entry for the row's OID for that area must be determined.",
"The SEQ table is used to determine this number.",
"Each area of the application should have its own entry in this table.",
"The value is the next number to be used as an OID for that area, and is the number to be used as the OID for the new row.",
"However, this number is used, it must also be manually incremented, so that the next entry made (either by an administrator or by the application) will use a unique number.",
"TABLE 2.76 SEQ Allows Attribute Name Nulls?",
"Type Description SEQ_NAME N VARCHAR2 (255) Name of the area of the application for which you are creating a new OID.",
"VALUE Y NUMBER (18) Next number to be used as an OID for the area.",
"This is the number you should use as the OID for the new row.",
"CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.8.Interaction Table 2.77 provides a portal view of an instance of an interaction model.",
"This object contains information that allows customers to view the past, current, and future states of an instance of a business process model managed by an external workflow tool.",
"Different portals may display different end-user views.",
"TABLE 2.77 END_USR_VIEW Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the end-user view NAME N VARCHAR2 (50) Name of the end-user view DSCR Y VARCHAR2 (240) Description of the end-user view INTRCTN_MODEL_OID N NUMBER (18) Object identifier for the associated interaction model CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.78 displays the end-user view of an instance state.",
"The past, current, and future states of an instance are displayed as part of the end-user view of that business process model.",
"TABLE 2.78 END_USR_VIEW_STATE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the end-user view state END_USR_VIEW_OID N NUMBER (18) Object identifier for the associated end-user view state NAME N VARCHAR2 (50) Name of the end-user view state DSCR Y VARCHAR2 (240) Description of the end- user view state ESTMTD_DURTN Y NUMBER (18) Expected duration of this end-user view state DSPL_ORDER N NUMBER (18) Order in which the state is displayed INTRCTN_MODEL_STATE_OID N NUMBER (18) Object identifier for the associated interaction model state CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.79 maintains state values of objects based on information from an external workflow tool.",
"TABLE 2.79 INTRCTN_MODEL Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the interaction model NAME N VARCHAR2 (50) Name of the interaction model DSCR Y VARCHAR2 (240) Description of interaction model CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.80 maps interaction model objects to end-user view objects, enabling the end user to view the interaction model information in the portal.",
"TABLE 2.80 INTRCTN_MODEL_END_USR_VIEW_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the interaction model PORTAL_TYPE_CD Y NUMBER (9) Code indicating the type of business portal to use END_USR_VIEW_OID N NUMBER (18) Object identifier for the end-user view START_DT Y DATE First date the end-user view object can be displayed END_DT Y DATE Last date the end-user view can be displayed CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.81 references an interaction model associated with states for a particular object instance.",
"TABLE 2.81 INTRCTN_MODEL_INSTNC Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the interaction model instance INTRCTN_MODEL_OID N NUMBER (18) Object identifier for the associated interaction model PENDING_TIME Y NUMBER ( ) Period of time before the object begins the first step of the model instance STATUS_CD N NUMBER (9) Code indicating the status of the interaction model instance INSTNC_START_DT Y DATE Starting date and time of the interaction model instance TARGET_CLASS N VARCHAR2 (80) Fully-qualified path of the target class for the interaction model instance TARGET_BUS_OBJ_OID N NUMBER (18) Object identifier for the associated target object CREATE_USR Y VARCHAR2 (40) Internal identifier of the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.82 references the state of a step within an interaction model instance.",
"TABLE 2.82 INTRCTN_MODEL_INSTNC_STATE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the instance state INTRCTN_MODEL_INSTNC_OID N NUMBER (18) Object identifier for the associated interaction model instance CMPLTD_DT Y DATE Date and time when the interaction model state was completed INTRCTN_MODEL_STATE_OID N NUMBER (18) Object identifier for the associated interaction model state EXTERNAL_STEP_MSG Y VARCHAR2 (240) The external step message associated with this interaction model instance state.",
"IS_DSPLD_FLG N NUMBER (1) Flag indicating whether the state is displayed STATUS_CD N NUMBER (9) Code indicating the status of the state INTRCTN_STATE_TYPE_CD N NUMBER (9) Code indicating the state type DSPL_ORDER N NUMBER (18) Order in which the state should be displayed CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.83 contains information regarding the state of a step within the interaction model.",
"TABLE 2.83 INTRCTN_MODEL_STATE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the interaction model state INTRCTN_MODEL_OID N NUMBER (18) Object identifier for the associated interaction model NAME N VARCHAR2 (50) Name of the interaction model state DSCR Y VARCHAR2 (240) Description of the interaction model state ESTMTD_DURTN Y NUMBER (18) Expected duration of the interaction model state INTRCTN_STATE_TYPE_CD N NUMBER (9) Code indicating type of interaction model state DSPL_ORDER N NUMBER (18) Order in which the model states should be displayed CREATE_USR Y VARCHAR2 (40) Internal identifier for the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic Lock Table 2.84 contains information used to select an order item for an interaction model.",
"TABLE 2.84 ORDR_ITEM_INTRCTN_MODEL_SELCTN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for this selection object INTRCTN_MODEL_OID N NUMBER (18) Object identifier for the interaction model ACTION_CD N NUMBER (9) Code indicating the action to be used VENDOR_PROD_OID Y NUMBER (18) Obejct identifier for the associated vendor product CREATE_USR Y VARCHAR2 (40) Internal identifier for the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic Lock 2.9.Note Table 2.85 represents notes that can be associated with a Customer, Quote or Order.",
"TABLE 2.85 NOTE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the note TARGET_OID N NUMBER (18) Object identifier for the target TARGET_CLASS N VARCHAR2 (80) The target class for this Note ROOT_OID N NUMBER (18) Object identifier for the root NOTE_TYPE_CD N NUMBER (9) Code indicating the note type SUBJECT N VARCHAR2 (50) A text description of the subject this Note USR_OID N NUMBER (18) Object identifier for the user CREATED_DT N DATE Date the note was generated CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.86 represents a note comment associated with a given note.",
"TABLE 2.86 NOTE_CMT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the note comment NOTE_OID N NUMBER (18) Object identifier for the note DSCR N VARCHAR2 Description (2000) USR_OID N NUMBER (18) Object identifier for the user CREATED_DT N DATE Date the note comment was generated CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.10.Notice Table 2.87 displays news and information to a user at the front end.",
"TABLE 2.87 CONTENT_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the content item OFFER_OID Y NUMBER (18) Object identifier for the associated offer NAME N VARCHAR2 (50) Name of the content item SHORT_DSCR Y VARCHAR2 (240) Short description of the content item LONG_DSCR Y VARCHAR2 Long description of the content item (2000) SMALL_ICON Y VARCHAR2 (240) Small icon displayed with the content item LARGE_ICON Y VARCHAR2 (240) Large icon displayed with the content item START_DT Y DATE Earliest date the content item can be displayed END_DT Y DATE Last date that the content item can be displayed DSPL_MAX_COUNT Y NUMBER ( ) Maximum number of times the content item can be displayed PLUGIN_CD N NUMBER (9) Code indicating the plug-in used to determine the content items to display CATGRY_CD N NUMBER (9) Code indicating the content item category TARGET_CLASS Y VARCHAR2 (80) Fully-qualified path of the target class such as a display attribute object used to display the content item TARGET_OID Y NUMBER (18) Object identifier for the target class CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.88 associates a content item with the specific object to which it is displayed.",
"TABLE 2.88 TARGET_CONTENT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the target content CLASS_NAME N VARCHAR2 (80) Fully-qualified path to which a content item will be displayed CLASS_OID N NUMBER (18) OID for the specific class instance for which a content item will be displayed CONTENT_ITEM_OID N NUMBER (18) Object identifier for the associated content item DSPL_COUNT Y NUMBER ( ) Number of times item is displayed to the target class CLICK_COUNT Y NUMBER ( ) Number of times item is clicked on by the target class PRIORITY_CD N NUMBER (9) Code indicating the priority of the associated content item CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.11.Offer Table 2.89 maps the disclosure that the user selected with the offer instance.",
"TABLE 2.89 ACCPTD_DISCL Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the accepted disclosure DISCL_OID N NUMBER (18) Object identifier for the associated disclosure OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.90 determines what happens to offers within an offer collection when the collection is broken, usually by the removal of an offer within the collection.",
"TABLE 2.90 BREAK_OFFER_COLLCTN_RULE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the rule OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the associated offer collection FROM_OFFER_OID N NUMBER (18) Object identifier for the “from” offer TO_OFFER_OID Y NUMBER (18) Object identifier for the “to” offer TRANSTN_ACTION_CD N NUMBER (9) Code indicating the action used CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock TABLE 2.91 COMPARISON_DEFN Represents the attributes by which products can be compared.",
"Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the comparison definition DSCR N VARCHAR2 (240) Text description of the definition for comparison CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.92 represents a group that contains offers to be compared against each other.",
"TABLE 2.92 COMPARISON_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the comparison group GROUP_NAME N VARCHAR2 (50) Name of the comparison group CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.93 maps a comparison group to a comparison definition.",
"TABLE 2.93 COMPARISON_GROUP_DEFN_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map L-Linkparatext_OID N NUMBER (18) Object identifier for the comparison group L-Linkparatext_OID N NUMBER (18) Object identifier for the comparison definition DSPL_ORDER N NUMBER (18) Determines the order in which the definition appears in a comparison group.",
"CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.94 maps a comparison group to a specific determinant.",
"TABLE 2.94 COMPARISON_GROUP_DTRMNT_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map L-Linkparatext_OID N NUMBER (18) Object identifier for the comparison group L-Linkparatext_OID N NUMBER (18) Object identifier for the determinant CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.95 stores values for comparison definitions for specific offers.",
"TABLE 2.95 COMPARISON_VALUE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the comparison definition value L-Linkparatext_OID N NUMBER (18) Object identifier for the associated comparison definition L-Linkparatext_OID N NUMBER (18) Object identifier for the associated offer COMPARISON_VALUE N VARCHAR2 (240) Value for the definition in string format.",
"For multiple values (i.e.",
"if the definition is “color” and there are multiple available colors), the VALUE must be a concatenated string.",
"CREATE_USR Y VARCHAR2 (40) User who created the rule CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who modified the entry MOD_DT Y DATE Date the entry was last modifier TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.96 represents the individual screens within an offer collection.",
"TABLE 2.96 DTRMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer collection determinant DTRMNT_TYPE_CD N NUMBER (9) Code indicating the type of determinant VALDTN_PLUG_CD N NUMBER (9) Code for the plug-in used to validate the determinant DSPL_ATTRIBS_OID N NUMBER (18) Object identifier for the associated display attributes MIN_RANGE N NUMBER (9) Specifies the minimum range of the determinant.",
"Used only when the determinant is of type choose multiple.",
"MAX_RANGE N NUMBER (9) Specifies the maximum range of the determinant.",
"Used only when the determinant is of type choose multiple DFLT_SELCTN N NUMBER (9) Specifies the selection that appears as the default.",
"Used only when the determinant is of type choose multiple.",
"CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE N NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.97 represents the offers in an offer collection from which the user can make a selection.",
"TABLE 2.97 DTRMNT_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the determinant item OVRRIDE_DTRMNT_SEQ_OID Y NUMBER (18) Object identifier for the determinant sequence table designating the next offer collection determinant.",
"Overrides the next determinant OID on the determinant item's offer determinant DTRMNT_OID N NUMBER (18) Object identifier for the associated offer collection determinant OFFER_OID N NUMBER (18) Object identifier for the associated offer DSPL_ORDER N NUMBER (18) Indicates the order in which to display the determinant item in the offer collection determinant INSTNC_DSPL_ORDER N NUMBER (18) Indicates the order in which to display the offer instances in collection groups in the subsequent containers, such as a cart or an offer CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.98 maps one offer collection determinant to another.",
"TABLE 2.98 DTRMNT_SEQ Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer collection determinant map OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the associated offer collection TO_DTRMNT_OID Y NUMBER (18) Object identifier for the determinant that is being mapped to FROM_DTRMNT_OID Y NUMBER (18) Object identifier for the determinant being mapped from OVRRDE_FLG N NUMBER (1) Flag indicating whether the determinant sequence overrides the order created in the determinant item DESTINATION_DTRMNT_TYPE_CD N NUMBER (9) Code indicating the type of the destination LOGICAL_DSPL_ORDER N NUMBER (18) Order in which the determinants appear at the front end CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK Y NUMBER (18) Optimistic lock Table 2.99 represents legal information that a user must agree to before ordering a quote.",
"TABLE 2.99 DISCL Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the disclosure SHORT_DSCR Y VARCHAR2 (240) Short description for the disclosure LONG_DSCR Y VARCHAR2 Long description for the disclosure (2000) ACTION_CD N NUMBER (9) Code indicating the action the disclosure is associated with CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT T DATE Date the entry was created MOD_USR Y VARCHAR2 (40) Internal identifier of the user who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.100 represents a single marketable entity offered by a licensee to its customers.",
"An offer contains all the data necessary for displaying the offer to a customer, which is then related to one or more vendor products.",
"TABLE 2.100 OFFER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer ORDERABLE_DT Y DATE Date that the offer can be ordered EXPRTN_DT Y DATE Date that the offer expires and can no longer be ordered EXPRTN_OID Y NUMBER (18) Object identifier for the offer that can replace this offer when it expires DSPL_ATTRIBS_OID N NUMBER (18) Object identifier for the corresponding display attributes OFFER_TYPE_CD N NUMBER (9) Code indicating the offer type PLUGIN_CD N NUMBER (9) OID for the plug-in used to select the vendor product TRANSTN_DTRMNT_OID Y NUMBER (18) Object identifier for the determinant used to transition the offer if it is part of an offer collection TRANSTN_DTRMNT_TYPE_CD N NUMBER (9) Code indicating the determinant type used to transition the offer if it is part of an offer collection RPLCMNT_DTRMNT_OID Y NUMBER (18) Object identifier for the replacement determinant RPLCMNT_DTRMNT_TYPE_CD N NUMBER (9) Code indicating the replacement determinant type UPDATABLE_QTY_FLG N NUMBER (1) Flag indicating whether the offer quantity is updatable ADDR_MAINTENANCE_CD N NUMBER (9) Code to indicating whether address maintenance is required CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.101 represents a set of screens used to display and select offers purchased as a group.",
"TABLE 2.101 OFFER_COLLCTN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer collection OFFER_COLLCTN_TYPE_CD N NUMBER (9) Code indicating the offer collection type COMPOSITE_OFFER_OID Y NUMBER (18) Object identifier for the composite offer associated with the offer collection TRANSTN_DTRMNT_OID Y NUMBER (18) Object identifier for the determinant to use when transitioning this collection TRANSTN_DTRMNT_TYPE_CD N NUMBER (9) Code indicating the type of determinant to use for a transition of this collection CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.102 contains information used for mapping source offer collection objects with target offer collection objects during a transition.",
"TABLE 2.102 OFFER_COLLCTN_OFFER_TRANSTN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for this object OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the associated offer collection object FROM_OFFER_OID N NUMBER (18) Object identifier for the offer the transition is being mapped from TO_OFFER_OID N NUMBER (18) Object identifier for the offer the transition is mapping to FIXED_COMPOSITE_OID Y NUMBER (18) Object identifier for the associated fixed composite offer SHORT_DSCR Y VARCHAR2 (240) Short description of the transition LONG_DSCR Y VARCHAR2 Long description of the transition (2000) CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.103 represents licensee-entered price that overrides an associated price TABLE 2.103 OVRRDE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the override TARGET_CLASS N VARCHAR2 (80) Fully-qualified path of the target class for the override TARGET_OID N NUMBER (18) Object identifier for the override UNIT_PRICE_OID Y NUMBER (18) Object identifier for the unit price OVRRDE_TYPE_CD N NUMBER (9) Object identifier for the override type AMT N NUMBER ( ) Amount of the override CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.104 represents associated reason for an override.",
"TABLE 2.104 OVRRDE_REASON Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the override reason REASON_CD N NUMBER (9) Code indicating the override reason REASON_DSCR Y VARCHAR2 (240) Reason description CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic Lock Table 2.105 represents a specific instance of an offer as it moves through cart, quote, and order, and is assigned to the customer hierarchy.",
"Each offer instance object contains a reference to the offer being ordered, the vendor product that needs to be provisioned, the non-recurring prices associated with the item, and the configured parameters.",
"TABLE 2.105 OFFER_INSTNC Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer instance OFFER_OID N NUMBER (18) Object identifier for the associated offer DTRMNT_ITEM_OID Y NUMBER (18) Object identifier for the associated determinant item VENDOR_PROD_OID Y NUMBER (18) Object identifier for the associated vendor product SVC_DOMAIN_CD N NUMBER (9) Code indicating how the associated product will be tracked for usage for billing (e.g., a phone number or IP address) OVRRDE_REASON_OID Y NUMBER (18) Object identifier for the override reason TOTAL_PRICE_AMT N NUMBER ( ) Total non-recurring charges TOTAL_TAX_AMT N NUMBER ( ) Total taxes TOTAL_DISC_AMT N NUMBER ( ) Total discount EXTENDED_PRICE_AMT N NUMBER ( ) Extended price (price × quantity) MONTHLY_RCURRNG_DSPL_AMT Y NUMBER ( ) Monthly rate displayed in the portal CREATED_DT N DATE Date the offer instance was generated OFFER_COLLCTN_INSTNC_ID Y NUMBER (18) Identifier of the associated offer collection instance OFEER_COLLCTN_DSPL_ORDER Y NUMBER (18) Order in which to display the offer instance within the offer collection OFFER_COLLCTN_COMPOSITE_FLG N NUMBER (1) Flag indicating whether this offer instance is a composite product OFFER_COLLECTION_OID Y NUMBER (18) Object identifier for the offer collection determinant from which the offer instance was collected PRE_CONFIGD_CD N NUMBER (9) Code indicating whether the offer instance has already been configured REPRICE_RATES_FLG N NUMBER (1) Flag indicating whether the rates can be repriced ADDR_OID Y NUMBER (18) Object identifier for the associated service address.",
"CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.106 contains the discount line items that are attributed to a particular offer instance.",
"TABLE 2.106 OFFER_INSTNC_DISC Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the discount DSCR Y VARCHAR2 (240) Description of the discount line item QTY Y NUMBER ( ) Quantity AMT N NUMBER ( ) Discount amount OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance FORMAT_CD Y NUMBER (9) Code indicating how the discount is formatted in an external system EXT_SYS_REF Y VARCHAR2 (240) Reference to external system CREATE_USR Y VARCHAR2 (40) Internal identifier of the user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.107 contains information regarding a non-recurring charge for an offer instance.",
"TABLE 2.107 OFFER_INSTNC_NON_RCURRNG_CHARG Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for this object OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance PRICE_ARRGMNT_OID N NUMBER (18) Object identifier for the associated price arrangement AMT N NUMBER ( ) Amount of the charge DSCR Y VARCHAR2 (240) Description of the charge QTY Y NUMBER ( ) Number of items FORMAT_CD N NUMBER (9) Code indicating how the discount is formatted in an external system UOM_CD N NUMBER (9) Code indicating the unit of measure CHARGE_DT N DATE Date the charge transaction is calculated CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18 Optimistic lock Table 2.108 contains information regarding a non-recurring charge for an offer instance TABLE 2.108 OFFER_INSTNC_RCURRNG_RATE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer instance recurring rate OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance PRICE_ARRGMNT_OID N NUMBER (18) Object identifier for the associated price arrangement CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.109 represents information regarding any tax rate associated with an offer instance.",
"TABLE 2.109 OFFER_INSTNC_TAX Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the tax DSCR N VARCHAR2 (240) Description of the tax AMT N NUMBER ( ) Amount of the tax OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance EXT_SYS_REF Y VARCHAR2 (240) Reference to the external billing system FORMAT_CD Y NUMBER (9) Code indicating how the discount is formatted in an external system CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.110 references information regarding a usage rate for an offer instance.",
"TABLE 2.110 OFFER_INSTNC_USAGE_RATE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the usage object OFFER_INSTNC_OID N NUMBER (18) Object identifier for the offer instance PRICE_ARRGMNT_OID N NUMBER (18) Object identifier for the price arrangement CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.111 maps source offer objects to target offer objects.",
"TABLE 2.111 OFFER_TRANSTN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer transition FROM_OFFER_OID N NUMBER (18) Object identifier for the from offer TO_OFFER_OID N NUMBER (18) Object identifier for the “to” offer SHORT_DSCR Y VARCHAR2 (240) Short description of the offer transition LONG_DSCR Y VARCHAR2 Long description of the offer transition (2000) CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.112 maps offers to vendor products.",
"TABLE 2.112 OFFER_VENDOR_PROD_MAP Allows Attribute Name Nulls?",
"Type Description VENDOR_PROD_OID N NUMBER (18) Object identifier for the vendor product OFFER_OID N NUMBER (18) Object identifier for the offer Table 2.113 maps source Offer's with target Offers during a Replacement TABLE 2.113 OFFER_RPLCMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer replacement FROM_OFFER_OID N NUMBER (18) Object identifier for the from offer TO_OFFER_OID N NUMBER (18) Object identifier for the to offer SHORT_DSCR Y VARCHAR2 (240) Short description of the offer replacement LONG_DSCR Y VARCHAR2 Long description of the offer (2000) replacement CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.114 maps source Offer Collection's with target Offer Collections during a transition TABLE 2.114 OFFER_COLLCTN_OFFER_RPLCMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the offer collection offer replacement OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the offer collection FROM_OFFER_OID N NUMBER (18) Object identifier for the from offer TO_OFFER_OID N NUMBER (18) Object identifier for the to offer FIXED_COMPOSITE_OID Y NUMBER (18) Object identifier for the fixed composite SHORT_DSCR Y VARCHAR2 (240) Short description for the offer collection offer replacement LONG_DSCR Y VARCHAR2 Long description for the offer (2000) collection offer replacement CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y VARCHAR2 (40) Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.115 determines an offer that is an upsell of another offer.",
"TABLE 2.115 UPSELL_OFFER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the upsell offer OFFER_OID N NUMBER (18) Object identifier for the offer TARGET_OID N NUMBER (18) Object identifier for the target upsell offer TARGET_CLASS N VARCHAR2 (80) The class of the upsell object (either Offer or Offer Collection) CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.116 is used by determinant items to create parent/child relationships.",
"TABLE 2.116 PARENT_ASSIGNMENT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the parent assignment OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the offer collection CHILD_DTRMNT_ITEM_OID N NUMBER (18) Object identifier for the determinant that is the child PARENT_DTRMNT_ITEM_OID N NUMBER (18) Object identifier for the offer collection determinant that is the parent CARDINALITY_CD N NUMBER (9) Code indicating whether the relationship is one-to- one or one-to-many CREATE_USR Y VARCHAR2 (40) Date the entry was created CREATE_DT Y DATE User who created the entry MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.117 represents information necessary to display, validate, and collect parameters for products.",
"TABLE 2.117 PARM_DEFN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the parameter definition DSPL_LABEL Y VARCHAR2 (50) Label displayed at the front end CONFIGD_LABEL Y VARCHAR2 (50) Label displayed at the front end once the parameter is configured READ_ONLY_FLG N NUMBER (1) Flag indicating whether the parameter value can be modified USR_VSBL_FLG N NUMBER (1) Flag indicating whether the parameter is visible at the front end for configuration VALDTN_CD N NUMBER (9) Validation code for the parameter PLUGIN_CD N NUMBER (9) Plug-in used to validate the parameter GUI_WIDGT_CD N NUMBER (9) Code indicating the type of GUI widget to display at the front-end when collecting the parameter information LOW_VALUE Y VARCHAR2 (50) Minimum value for the parameter HIGH_VALUE Y VARCHAR2 (50) Maximum value for the parameter DFLT_VALUE Y VARCHAR2 (50) Default value that is displayed for the parameter PARM_DEFN_TYPE_CD N NUMBER (9) Code indicating the parameter type PARM_SIZE N NUMBER (18) Size of the parameter REQUIRED_FLG N NUMBER (1) Flag indicating whether or not the parameter is required CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.118 represents default parameter value for a specific vendor product.",
"TABLE 2.118 PARM_DEFN_DFLT_VALUE_OVRRDE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map PARM_DEFN_OID N NUMBER (18) Object identifier for the parameter definition VENDOR_PROD_OID N NUMBER (18) Object identifier for the vendor product DFLT_VALUE N VARCHAR2 (50) Value of the default CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.119 maps enumerations to parameter definitions TABLE 2.119 PARM_DEFN_PARM_ENUM_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map PARM_DEFN_OID N NUMBER (18) Object identifier for the parameter definition PARM_ENUM_OID N NUMBER (18) Object identifier for the parameter enumeration definition object DSPL_ORDER N NUMBER (18) Display order of the enumerations within the definition CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.120 contains the list of values for an associated parameter.",
"TABLE 2.120 PARM_ENUM_DEFN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Ojbect identifier for the parameter enumeration definition EXTERNAL_NAME Y VARCHAR2 (50) Name displayed to the user (e.g., “Three”) INTERNAL_VALUE Y VARCHAR2 (50) Internal value used by the system (e.g., “3”) PARM_ENUM_TYPE_CD N NUMBER (9) Code indicating the parameter enumeration type CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.121 allows the user to validate against a group of parameters as if they were one entity (e.g., address validation).",
"Parameters must belong to a parameter group TABLE 2.121 PARM_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the parameter group GROUP_NAME N VARCHAR2 (50) Name of the parameter group PLUGIN_CD N NUMBER (9) Plug-in to call to validate the group VALDTN_CD N NUMBER (9) Code used to validate the group USR_VSBL_FLG N NUMBER (1) Determines whether the group is visible to the end user CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.122 maps the parameter group object to the parameter definition object.",
"TABLE 2.122 PARM_GROUP_PARM_DEFN_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map PARM_GROUP_OID N NUMBER (18) Object identifier for the parameter group PARM_DEFN_OID N NUMBER (18) Object identifier for the parameter definition DSPL_ORDER N NUMBER (18) Order in which the parameter definition appears in the group DSPL_NEXT N NUMBER (1) Indicates the next parameter displayed in the group CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.123 contains the configured parameter value selected by the user for a given offer instance.",
"TABLE 2.123 PARM_VALUE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the parameter value PARM_DEFN_OID N NUMBER (18) Object identifier for the associated parameter definition PARM_GROUP_OID N NUMBER (18) Object identifier for the associated parameter group VALUE Y VARCHAR2 (50) Value of the parameter OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance PARM_GROUP_DSPL_ORDER Y NUMBER (18) Order in which the value is displayed in the group STATUS_CD N NUMBER (9) Code indicating the parameter status EXTERNAL_NAME Y VARCHAR2 (50) Name for the parameter value used by external systems CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.124 maps a Cygent offer to an external provider's.offer.",
"TABLE 2.124 PRVDR_SVC_OFFER_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map L-Linkparatext_OID N NUMBER (18) Object identifier for the associated determinant item L-Linkparatext_OID N NUMBER (18) Object identifier for the associated determinant PRVDR_SVC_ID Y NUMBER (18) External provider's ID for the returned service PRVDR_SVC_NAME Y VARCHAR2 (50) External provider's name for the returned service CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.125 represents displayed link from one offer to another related offer.",
"TABLE 2.125 RLTD_OFFER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the related offer link OFFER_OID N NUMBER (18) Object identifier for the associated offer RLTD_OFFER_OID N NUMBER (18) Object identifier for the related offer CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.126 represents a single line item indicating what the offer instance was created from.",
"An offer instance can be created in many different ways.",
"(e.g., from the menu, an existing service, etc.)",
"The offer instance can handle zero to many instances of source line items.",
"TABLE 2.126 SOURCE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the source line item DSCR N VARCHAR2 (240) Description of the source line item OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance SOURCE_FROM_CD Y NUMBER (9) Code indicating the type of source CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.127 represnets the lead time required to complete an action on an offer.",
"TABLE 2.127 SVC_LEVEL_AGRMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the service level agreement TARGET_CLASS N VARCHAR2 (80) Fully-qualified path of the class to which you are mapping the service level agreement, usually vendor product TARGET_OID N NUMBER (18) Object identifier for the object specified by the target class ACTION_CD N NUMBER (9) Code indicating the action VALUE N NUMBER (18) Time needed to perform the action UOM_CD N NUMBER (9) Code indicating the unit of measure CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.128 temporarily stores offer instances during the selection of an offer collection.",
"TABLE 2.128 TEMP_OFFER_COLLCTN_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the temporary offer collection item TEMP_OFFER_COLLCTN_ID N NUMBER (18) Identifier of the temp offer collection instance OFFER_COLLCTN_OID N NUMBER (18) Object identifier for the associated offer collection USR_OID Y NUMBER (18) Object identifier for the current user SESSION_ID Y VARCHAR (240) Identifier for the session in which the cart item was created TARGET_OID N NUMBER (18) Object identifier for the object to which the offer instance will be associated TARGET_CLASS N VARCHAR2 (80) Fully-qualified class path for the target object DTRMNT_OID Y NUMBER (18) Object identifier for the associated offer collection determinant OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance LOGICAL_DSPL_ORDER Y NUMBER (18) Location of this item within the logical sequence of this item DTRMNT_ITEM_OID Y NUMBER (18) Object identifier for the associated determinant item DSPL_ORDER N NUMBER (18) Location of this item within the display QTY Y NUMBER (25) Quantity of the offer instance CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.129 represnets a vendor who supplies a product to the licensee.",
"TABLE 2.129 VENDOR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the vendor NAME N VARCHAR2 (50) Vendor's name CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.130 represents an actual product that can be shipped or provisioned.",
"TABLE 2.130 VENDOR_PROD Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier of the vendor product NAME N VARCHAR2 (50) Name of the vendor product DSCR N VARCHAR2 (50) Description of the vendor product PROVSNG_CD Y VARCHAR2 (240) Code indicating the provisioning type SVC_DOMAIN_CD N NUMBER (9) Code indicating the type of service domain (e.g., phone number, email address) PROVSNG_START_DT Y DATE First date that the vendor product can be provisioned PROVSNG_END_DT Y DATE Last date that the vendor product can be provisioned VENDOR_OID N NUMBER (18) Object identifier for the associated vendor IS_SVC_ID_REQUIRED_CD N NUMBER (9) Indicates whether a service identifier is required CLASS_OF_SVC_CD N NUMBER (9) Code indicating the class of service CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.131 maps a vendor product to a CMI policy.",
"TABLE 2.131 VENDOR_PROD_CMI_POLICY Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the vendor product to a CMI policy ACTION_CD N NUMBER (9) Action code VENDOR_PROD_OID N NUMBER (18) Object identifier fort the associated Vendor product ADAPTER_HOME N VARCHAR2 (240) The associated adapter CNTXT N VARCHAR2 (240) Associated contact object CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Optimistic lock WRITE_LOCK N NUMBER (18) User who created the entry Table 2.132 maps a disclosure to a vendor product.",
"TABLE 2.132 VENDOR_PROD_DISCL_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map DISCL_OID N NUMBER (18) Object identifier for the disclosure VENDOR_PROD_OID N NUMBER (18) Object identifier for the vendor product DSCR Y VARCHAR2 (240) Description of the map CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.133 maps vendor products to parameter groups.",
"TABLE 2.133 VENDOR_PROD_PARM_GROUP_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the map PARM_GROUP_OID N NUMBER (18) Object identifier for the parameter group VENDOR_PROD_OID N NUMBER (18) Object identifier for the vendor product DSPL_ORDER N NUMBER (18) The display order for the parameter group.",
"STATUS_CD N NUMBER (9) Code indicating the status of the map CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.134 represents a charge related to the creation of a supplemental order.",
"TABLE 2.134 SPLMNTL_CHARGE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the supplemental charge TARGET_OID N NUMBER (18) Object identifier for the target TARGET_CLASS N VARCHAR2 (80) Fully qualified class name of the target object OVRRDE_REASON_OID Y NUMBER (18) Object identifier for the override reason AMT N NUMBER ( ) Amount of the supplemental charge DSCR Y VARCHAR2 (240) Description of the supplemental charge QTY Y NUMBER ( ) The quantity of the items that the charge was calculated on FORMAT_CD N NUMBER (9) Code indicating the format UOM_CD N NUMBER (9) Code indicating the UOM CHARGE_DT N DATE Date the charge was generated CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.12.Order Table 2.135 represents a customer commitment to purchase a set of offers.",
"TABLE 2.135 ORDR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the order ORDR_ID Y NUMBER (18) Identifier for the order used by an external system ID Y VARCHAR2 (50) Identifier for the order used by an external system STATE_CD N NUMBER (9) Code indicating the state of the order.",
"The order state attribute is populated from the interaction log.",
"STATUS_CD N NUMBER (9) Code indicating the status of the order ESCLTN_FLG N NUMBER (1) Escalation flag SPLMNTD_ORDR_OID Y NUMBER (18) Object identifier for the supplemental order ORDER_TYPE_CD N NUMBER (9) Code indicating the order type ROOT_OID N NUMBER (18) Object identifier for the root hierarchy object QUOTE_OID Y NUMBER (18) Object identifier for the associated quote TOTAL_AMT N NUMBER ( ) Total amount of the order DSCR Y VARCHAR2 (50) User-entered description of the order SHPNG_ADDR_OID Y NUMBER (18) Object identifier for the shipping address for delivered products PAYMNT_OID Y NUMBER (18) Object identifier for the associated payment MONTHLY_RCURRNG— Y NUMBER ( ) Monthly rate for an order DSPL_SUMM_AMT displayed to the customer TOTAL_SPLMNTL_AMT N NUMBER ( ) Total supplemental charge amount SUBMTD_DT Y DATE Date order was submitted PURCH_ORDR_NBR Y VARCHAR2 (50) Purchase order number AGENT_OID Y NUMBER (18) Object identifier for the agent LAST_MODIFIED_USR N VARCHAR2 (40) Name of last modified user CREATED_USR N VARCHAR2 (40) Name of created user EMAIL_ADDR Y VARCHAR2 (240) Email address PARTNER_CUST_OID Y NUMBER (18) Object identifier for the partner customer CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify Subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.136 represents an external request for an offer.",
"The scheduled due date and start work flow date are set when the preceding Quote Item is configured.",
"TABLE 2.136 ORDR_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the order item ORDR_OID N NUMBER (18) Object identifier for the order OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance TARGET_OID N NUMBER (18) Object identifier for the hierarchy object to which the order item is associated TARGET_CLASS N VARCHAR2 (80) Fully-qualified path of the associated target object SPLMNTD_ORDR_ITEM_OID Y NUMBER (18) Object identified for the supplemental order item SPLMNTL_TYPE_CD N NUMBER (9) Code indicating the supplemental type ESCLTN_FLG N NUMBER (1) Escalation flag PRVDR_ORDER_ID Y VARCHAR (50) Identifier for the associated order provider CAN_REMOVE_FROM— N NUMBER (1) Flag indicating whether the ORDR_FLG order can be removed from cart PURCH_ORDR_NBR Y VARCHAR2 (50) Purchase order number RQST_DUE_DT Y DATE Date the order item is requested for provisioning SCHED_DUE_DT Y DATE Scheduled due date of service, otherwise known as the Service Level SUBMTD_DT Y DATE Date order was submitted EXECUTED_DT Y DATE Date the action on the order item was resolved LABEL Y VARCHAR2 (50) Label for the order item PAYMNT_OID Y NUMBER (18) Object identifier for the associated payment STATUS_CD N NUMBER (9) Code indicating the status of the order item STATE_CD N NUMBER (9) Code indicating the state of the order item.",
"This is populated from the interaction log DSPL_ORDER N NUMBER (18) Number indicating the order in which the item is displayed in relation to other order items QTY Y NUMBER ( ) Order item quantity ACTION_CD N NUMBER (9) Code indicating the action required to create the order item SHPNG_ADDR_OID Y NUMBER (18) Object identifier for the shipping address object.",
"This column is not updated by the Cygent application, but is included for customization purposes only.",
"Shipping addresses are stored at the order level.",
"DSCR Y VARCHAR2 (50) Description for order item CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.13.Payment Table 2.137 represents a subclass of payment.",
"The credit card payment contains credit card information regarding a specific payment.",
"TABLE 2.137 CREDIT_CARD_PAYMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the credit card payment object CARD_TYPE_CD N NUMBER (9) Code that indicates the credit type (e.g., Visa, American Express) CARD_HOLDER_FIRST_NAME N VARCHAR2 (50) First name of the cardholder CARD_HOLDER_MIDDLE_NAME Y VARCHAR2 (50) Middle name of the cardholder CARD_HOLDER_LAST_NAME N VARCHAR2 (50) Last name of the cardholder BLNG_ADDR_OID N NUMBER (18) Object identifier for the associated billing address ENCRYPTED_CARD_NBR N VARCHAR2 (50) Encrypted credit card number ENCRYPTED_PIN_NBR Y VARCHAR2 (50) Encrypted PIN number EXPRTN_DT N DATE Credit card expiration date ATHZTN_CD Y VARCHAR2 (50) Authorization code for this payment ATHZTN_SUBMIT_DT Y DATE Date the payment was submitted for authorization ATHZTN_EXPRTN_DT Y DATE Date the authorization expires for this payment SETTLMNT_MSG Y VARCHAR2 (240) Message returned from the settlement request SETTLMNT_SUBMIT_DT Y DATE Date this payment was submitted for settlement ATHZTN_SOURCE_CD Y VARCHAR2 (50) Code indicating the source of the authorization code APPROVAL_CD Y VARCHAR2 (50) Code indicating payment approval AVS_CD Y VARCHAR2 (50) Code indicating the address verification system SETTLMNT_CD Y VARCHAR2 (50) Code for the settlement transaction SETTLMNT_BATCH_NBR Y VARCHAR2 (50) Batch number in which the settlement request was made PSP_STATUS Y VARCHAR2 (50) Status of the payment as defined by the payment service provider PSP_TXN_NBR Y VARCHAR2 (50) Transaction number associated with this payment SETTLMNT_AMT Y NUMBER ( ) Dollar amount paid by the issuing credit card company PSP_RETURN_CD Y VARCHAR2 (50) Return code for the payment as defined by the payment service provider PSP_ACTION Y VARCHAR2 (50) Action for this payment as defined by the payment service provider PSP_ERROR_MSG Y VARCHAR2 (50) Error message received from the payment service provider PSP_NAME Y VARCHAR2 (50) Name of the payment service provider TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass Table 2.138 contains information regarding payment transactions made by users.",
"TABLE 2.138 PAYMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the payment CREATED_DT N DATE Date this payment was created STATUS_CD N NUMBER (9) Code indicating the payment status AMT N NUMBER ( ) Payment amount TARGET_OID N NUMBER (18) Object identifier for the object to which the payment is associated TARGET_CLASS N VARCHAR2 (80) Fully-qualified path for the target class USR_OID N NUMBER (18) Object identifier for the user CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.139 allows addition of new attributes to any classes within the Payment domain.",
"TABLE 2.139 PAYMNT_EXT_DATA Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the payment external data PARENT_OID N NUMBER (18) Object identifier for the parent class ATTRIB_NAME N VARCHAR2 (40) Name for the attribute ATTRIB_VALUE N VARCHAR2 (40) Value for the attribute WRITE_LOCK N NUMBER (18) Optimistic lock 2.14.Pricing Table 2.140 represents the unique key for an action used for a price TABLE 2.140 CNTXT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the context NAME N VARCHAR2 (50) Name of the context CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.141 is used to indicate which unit price is valid for different levels in a multi-level price arrangement TABLE 2.141 CRTRIA Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Internal identifier of the criteria object LOW_VALUE N VARCHAR2 (50) Low value of this level.",
"This is number is inclusive HIGH_VALUE Y VARCHAR2 (50) High value of this level.",
"This number is exclusive.",
"OPER_CD N NUMBER (9) Code that describes the relationships between the levels (e.g., between, greater than) UOM_CD N NUMBER (9) Unit of measure code CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.142 determines the active price arrangement for a given price group supported.",
"TABLE 2.142 PRICE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the price CNTXT_OID Y NUMBER (18) Object identifier for the associated context ACTIVE_PRICE_ARRGMNT_OID Y NUMBER (18) Object identifier for the currently active price arrangement DSCR Y VARCHAR2 (50) Description of the price LONG_DSCR Y VARCHAR2 (240) Long description of the price PRICE_GROUP_SPPRTD_OID N NUMBER (18) Object identifier for the price group supported EXT_SYS_REF Y VARCHAR2 (240) Reference to the external billing system DO_NOT_SUMMARY_PRICE_FLG N NUMBER (1) Indicates whether the price should not be included in the monthly recurring summary on the associated offer instance FORMAT_CD N NUMBER (9) Code indicating how the invoice is formatted in an external system CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.143 determines how a price is calculated (e.g., flat, tier).",
"TABLE 2.143 PRICE_ARRGMNT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the price arrangement PRICE_OID N NUMBER (18) Object identifier for the associated price PRICE_ARRGMNT_TYPE_CD N NUMBER (9) Code indicating the type of the price arrangement DSCR Y VARCHAR2 (50) Description of the price arrangement FORMAT_CD N NUMBER (9) Code indicating how the discount is formatted in an external system CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.144 represents the junction of a price group and an action supported for a given offer.",
"TABLE 2.144 PRICE_GROUP_SPPRTD Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the price group supported PRICE_GROUP_OID N NUMBER (18) Object identifier for the price group OFFER_OID N NUMBER (18) Object identifier for the offer ACTION_CD N NUMBER (9) Code indicating the action NO_PRICE_FLG N NUMBER (1) Indicates whether the price group does not have a price for this action on this offer CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.145 provides the actual monetary amount to be charged per unit for a given price arrangement.",
"TABLE 2.145 UNIT_PRICE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the unit price EXT_SYS_REF Y VARCHAR2 (240) Reference to the external billing system UOM_CD N NUMBER (9) Code indicating the unit of measure for the price UNIT_AMT Y NUMBER ( ) Unit dollar amount PRICE_ARRGMNT_OID N NUMBER (18) Object identifier for the associated price arrangement CRTRIA_OID Y NUMBER (18) Object identifier for the criteria that indicates when this price is used in a multi-level price agreement FORMAT_CD N NUMBER (9) Code indicating how the invoice is formatted in an external system SEQ_NBR N NUMBER (18) Number indicating the sequence of this unit price CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.15.Profile Table 2.146 defines a profile that can be associated with any Cygent class.",
"TABLE 2.146 PROF_DEFN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile definition DSPL_LABEL Y VARCHAR2 (50) The label displayed before profile is configured CONFIGD_LABEL Y VARCHAR2 (50) The label displayed after profile is configured PROF_DEFN_TYPE_CD N NUMBER (9) Code indicating the profile definition type STATUS_CD N NUMBER (9) Code indicating status GUI_WIDGET_CD N NUMBER (9) Code indicating the GUI widget GUI_WIDGET_SIZE Y NUMBER (18) The size of the widget that is displayed when collecting the profile LOW_VALUE Y VARCHAR2 (50) The min value for the profile HIGH_VALUE Y VARCHAR2 (50) The max value for the profile DFLT_VALUE Y VARCHAR2 (50) The default value for the profile REQUIRED_FLG N NUMBER (1) Flag indicating whether or not the profile is required or optional CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.147 defines an element of a list from which a user can select a value for a profile.",
"A set of ProfileEnumerations are associated with a Profile Definition through Profile Definition.",
"Profile Enumeration Maps.",
"TABLE 2.147 PROF_ENUM_DEFN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile enumeration PROF_ENUM_TYPE_CD N NUMBER (9) Code indicating the profile enumeration type EXT_NAME Y VARCHAR2 (50) The name that is displayed to the user INTERNAL_VALUE Y VARCHAR2 (50) The internal value used by the system CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.148 is used to group profile definitions.",
"All profile definitions must be assigned to a ProfileGroup.",
"TABLE 2.148 PROF_GROUP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile group GROUP_NAME Y VARCHAR2 (50) The name of the profile group STATUS_CD N NUMBER (9) Code indicating the status of the profile group CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.149 associates profile definitions with ProfileGroups.",
"Each ProfileDefinition must be assigned with one or more ProfileGroups TABLE 2.149 PROF_GROUP_PROF_DEFN_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile group profile definition map PROF_GROUP_OID N NUMBER (18) Object identifier for the profile group PROF_DEFN_OID N NUMBER (18) Object identifier for the profile definition DSPL_ORDER N NUMBER (18) The display order for profile defintion within the group DSPL_NEXT_FLG N NUMBER (1) Indicates whether the profile definition is displayed next to the previous definition CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.150 associates individual Profile Enumerations with Profile Definitions.",
"TABLE 2.150 PROF_DEFN_PROF_ENUM_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile definition profile enumeration map PROF_DEFN_OID N NUMBER (18) Object identifier for the profile definition PROF_ENUM_DEFN_OID N NUMBER (18) Object identifier for the profile enumeration definition DSPL_ORDER Y NUMBER (18) The display order for the enumeration CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.151 associates Profile Groups with Cygent objects.",
"TABLE 2.151 PROF_KEY_PROF_GROUP_MAP Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile key profile group map PROF_KEY N VARCHAR2 (80) The profile key PROF_GROUP_OID N NUMBER (18) Object identifier for the profile group PROF_KEY_STATUS_CD N NUMBER (9) Code indicating the profile key status DSPL_ORDER N NUMBER (18) The display order for the profile group CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.152 contains the value of profile definitions selected by the user.",
"TABLE 2.152 PROF_VALUE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the profile value PROF_GROUP_OID N NUMBER (18) Object identifier for the profile group PROF_DEFN_OID N NUMBER (18) Object identifier for the profile definition TARGET_OID N NUMBER (18) Object identifier for the target TARGET_CLASS N VARCHAR2 (80) The object class name this profile value belongs to STATUS_CD N NUMBER (9) Code indicating the status of the profile value VALUE Y VARCHAR2 (50) The profile value CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.16.Quote Table 2.153 represents the point in the ordering process flow where a limited-time price is offered for items a user wants to purchase.",
"TABLE 2.153 QUOTE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the quote STATE_CD N NUMBER (9) Code indicating the state of the quote STATUS_CD N NUMBER (9) Status code QUOTE_TYPE_CD N NUMBER (9) Code indicating the quote type SPLMNTD_ORDR_OID Y NUMBER (18) Object identifier for the supplemented order ROOT_OID N NUMBER (18) Object identifier for the associated hierarchy root PAYMNT_OID Y NUMBER (18) Object identifier for the payment SHPNG_ADDR_OID Y NUMBER (18) Object identifier for the shipping address for delivered products MONTHLY_RCURRNG— Y NUMBER ( ) Total recurring monthly charge for DSPL_SUMM_AMT the displayed quote TOTAL_SPLMNTL_AMT N NUMBER ( ) Total supplemental charge amount QUOTE_ID Y VARCHAR2 (50) Identifier used by an external system AGENT_OID Y NUMBER (18) Object identifier for the agent PARTNER_CUST_OID Y NUMBER (18) Object identifier for the partner customer LAST_MODIFIED_USR N VARCHAR2 (40) The name of the user that last modified this quote * Note that this is different from lastModUser field used for auditing EMAIL_ADDR Y VARCHAR2 (240) Email address associated with this quote CREATED_USR N VARCHAR2 (40) Name of created user CREATED_DT N DATE Date the quote was created SUBMTD_DT Y DATE Date the quote was submitted QUOTED_DT Y DATE Date the quote was locked VALID_UNTIL_DT Y DATE Last date that the quote is valid AMT Y NUMBER ( ) Total amount of the quote DSCR Y VARCHAR2 (50) Description of the quote PURCH_ORDR_NBR Y VARCHAR2 (50) Purchase order number for quote CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.154 represents an offer instance in a quote TABLE 2.154 QUOTE_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the quote item STATE_CD N NUMBER (9) Code indicating the state of the quote item STATUS_CD N NUMBER (9) Code indicating the status OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance QUOTE_OID N NUMBER (18) Object identifier for the associated quote TARGET_OID N NUMBER (18) Obejct identifier for the object to which the offer instance that is associated after provisioning TARGET_CLASS N VARCHAR2 (80) Fully-qualified path to the target class FROM_OFFER_OID Y NUMBER (18) Object identifier for the source offer SPLMNTD_ORDR_ITEM_OID Y NUMBER (18) Object identifier for the supplemental order item SPLMNTL_TYPE_CD N NUMBER (9) Code indicating the supplemental type ESCLTN_FLG N NUMBER (1) Boolean indicating if the quote item is escalated PAYMNT_OID Y NUMBER (18) Object identifier for the associated payment SHPNG_ADDR_OID Y NUMBER (18) Object identifier for the address object.",
"This column is not updated by the Cygent application, but is included for customization purposes only.",
"Shipping addresses are stored at the quote level.",
"CREATED_DT N DATE Date the quote item was created RQSTD_DT Y DATE Date the quote item was requested to be active SCHED_DT Y DATE Date the quote item is scheduled to be active CAN_REMOVE_FROM— Y NUMBER (1) Boolean flag indicating QUOTE_FLG whether the quote item can be removed from the quote LABEL Y VARCHAR2 (50) Label for the quote item ACTION_CD N NUMBER (9) Code indicating the action to use PURCH_ORDR_NBR Y VARCHAR2 (50) Purchase order number for the quote item (stored as string) DSCR Y VARCHAR2 (50) Description of the quote item QTY Y NUMBER ( ) Quote item quantity DSPL_ORDER Y NUMBER (18) Indicates the display order of the quote items CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last mod TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.155 identifies groups of attributes on a quote item that will be populated either by preconfiguration or propagation.",
"Groups include the product parameters, attachment date, requested date, and others.",
"TABLE 2.155 QUOTE_ITEM_POPULATN_DSCPTR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the quote item population descriptor OFFER_OID N NUMBER (18) Object identifier for the offer POPULATN_OPERTN— N NUMBER (9) Code indicating the population TYPE_CD operation type TARGET_ATTRIBUTES_CD N NUMBER (9) Code indicating the target attributes PLUGIN_CD N NUMBER (9) Code indicating the plug-in CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.17.Shop Table 2.156 contains information regarding offers placed in a customer or partners shopping cart.",
"TABLE 2.156 CART_ITEM Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the cart item object TARGET_OID Y NUMBER (18) Object identifier for the instance that is in the cart TARGET_CLASS Y VARCHAR2 (80) Fully-qualified class path of the target class CART_ID Y NUMBER (18) Identifier for the cart used by external systems.",
"Default is the OID.",
"CAN_REMOVE— N NUMBER (1) Flag indicating whether the item can FROM_CART_FLG be removed from the cart.",
"QTY Y NUMBER ( ) Number of items in the cart OFFER_INSTNC_OID N NUMBER (18) Object identifier for the associated offer instance USR_OID Y NUMBER (18) Object identifier for the associated user SESSION_ID Y VARCHAR (240) Identifier for the session in which the cart item was created ACTION_CD N NUMBER (9) Code indicating the action being performed on this cart item DSPL_ORDER Y NUMBER (18) Number designating priority with which the cart item is displayed in the cart CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.157 contains the information needed to display a menu, offer, or offer collection at the front end.",
"TABLE 2.157 DSPL_ATTRIBS Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the display attribute ICON Y VARCHAR2 (240) Illustration or photograph that appears with the object when it displays at the front end.",
"LABEL Y VARCHAR2 (240) Name for the object that appears at the front end LABEL_HEX_COLOR Y VARCHAR2 (50) Color used to display the offer text SHORT_DSCR Y VARCHAR2 (240) Brief description of the associated object LONG_DSCR Y VARCHAR2 Detailed description of the associated (2000) object SIDEBAR_GRAPHIC Y VARCHAR2 (240) Clickable image or symbol link that appears next to the link to the object in the portal HIGHLIGHTED— Y VARCHAR2 (240) Rollover version of the clickable SIDEBAR_GRAPHIC image SALES_MSG Y VARCHAR2 (240) The sales message to be displayed with this shop item CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.158 is a menu of other menus or offers displayed to the user.",
"Users see root menus corresponding to their price groups.",
"Each user has a price group and each price group has a root menu.",
"TABLE 2.158 MENU Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the menu DSPL_ORDER N NUMBER (18) Indicate the display order of the group TARGET_OID Y NUMBER (18) Object identifier for the associated offer TARGET_CLASS Y VARCHAR2 (80) Fully-qualified path for the target class DSPL_ATTRIBS_OID N NUMBER (18) Object identifier for the associated display attributes PARENT_OID Y NUMBER (18) Object identifier for the parent menu ROOT_MENU_OID N NUMBER (18) Object identifier for the root menu CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_DT Y DATE Date the entry was last modified MOD_USR Y VARCHAR2 (40) User who last modified the entry TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.18.Task Manager These tables are specifically for the task manager, which is an optional product.",
"These tables are populated only if you have purchased the cygent task manager.",
"Table 2.159 represents an object to which a task can be assigned.",
"TABLE 2.159 TM_PERFRMR Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the performer TARGET_OID N NUMBER (18) Object identifier for the target object associated with the performer TARGET_CLASS N VARCHAR2 (80) Fully-qualified class name of the target object associated with the performer CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.160 represents a specific initiated instance of a process flow.",
"TABLE 2.160 TM_PROCESS_FLOW_INSTNC Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the process flow instance DEF_ID N NUMBER (18) External identifier for the process flow definition NAME Y VARCHAR2 (50) Name of the process flow instance DSCR Y VARCHAR2 (240) Description of the process flow STATE_CD N NUMBER (9) Code indicating the process instance state STATUS_CD N NUMBER (9) Code indicating the process instance status TARGET_OID Y NUMBER (18) Object identifier for the target object that invoked the process instance TARGET_CLASS Y VARCHAR2 (80) Fully-qualified class name of the target object that invoked the process instance ATTACHMENT Y VARCHAR2 (80) Extra information included with the process flow instance START_DT N DATE Date that the process instance was invoked END_DT Y DATE Date the process instance completed CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.161 represents a group of objects qualified to perform a certain type of task.",
"TABLE 2.161 TM_ROLE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the role NAME N VARCHAR2 (50) Name of the role STATUS_CD N NUMBER (9) Code indicating the role′s current status ESCLTN_RECPT— Y NUMBER (18) Object identifier for the target object TARGET_OID notified when an associated role has an escalated task ESCLTN_RECPT— Y VARCHAR2 (80) Fully-qualified class name of the target TARGET_CLASS object CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.162 maps a role to a specific performer.",
"TABLE 2.162 TM_ROLE_TM_PERFRMR_MAP Allows Attribute Name Nulls?",
"Type Description L-Linkparatext_OID N NUMBER (18) Object identifier for the role L-Linkparatext_OID N NUMBER (18) Object identifier for the performer Table 2.163 represents a specific instance of an invoked task.",
"TABLE 2.163 TM_TASK_INSTNC Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the task instance DEF_ID N NUMBER (18) External identifier for the task instance NAME Y VARCHAR2 (50) Name of the task instance DSCR Y VARCHAR2 (240) Description of the task instance TASK_TYPE_CD N NUMBER (9) Code indicating the task type STATE_CD N NUMBER (9) Code indicating the task instance state STATUS_CD N NUMBER (9) Code indicating the task instance status CMI_CD_VALUE Y NUMBER (18) Code indicating a CMI if used for the task DURTN Y VARCHAR2 (10) Length of the tasks duration ESCALATION— Y NUMBER (18) Object identifier for the role notified if L-Linkparatext_OID the task reaches an escalation point (exceeds the duration without completing) ESCLTN_FLG N NUMBER (1) Boolean indicates if the task is escalated (exceeded the duration with completing) ATTACHMENT Y VARCHAR2 (80) Extra information included with the task instance START_DT N DATE Date that the task instance was invoked END_DT Y DATE Date the task instance completed PAGE_ID Y VARCHAR2 (80) Identifier for the JSP page used to begin any JSP page flow created specifically for this task L-Linkparatext_OID N NUMBER (18) Object identifier for the role associated with the task L-Linkparatext_OID N NUMBER (18) Object identifier for the process flow instance to which this task instance belongs L-Linkparatext_OID Y NUMBER (18) Object identifier for the performer to which this task is assigned CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.164 represents the value of a task variable for a specific task.",
"TABLE 2.164 TM_TASK_VALUE Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the task variable value NAME N VARCHAR2 (50) Name of the task variable TASK_VALUE N VARCHAR2 (50) Value of the task variable TYPE_CD N NUMBER (9) Code indicating the task variable type L-Linkparatext_OID N NUMBER (18) Object identifier for the corresponding task instance CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.19.Trouble Ticket Table 2.165 represents a trouble ticket which provides a way for users to report issues regarding objects in their hierarchy or for an invoice.",
"TABLE 2.165 TRBL_TKT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the trouble ticket CATGRY_CD N NUMBER (9) Code indicating the type of the trouble ticket TRBL_TKT_ID N VARCHAR2 (50) Trouble ticket identifier used by external systems TYPE_CD N NUMBER(9) Code indicating the trouble ticket type NAME Y VARCHAR2 (240) Name for the trouble ticket USR_OID N NUMBER (18) Object identifier for the associated user ROOT_OID N NUMBER (18) Object identifier for the associated root object PARTNER_CUST_OID Y NUMBER (18) Object identifier for the partner customer PRIORITY_LEVEL Y NUMBER () Numerical value indicating the trouble ticket's priority or severity RSOLTN Y VARCHAR2 (240) Indicates the trouble ticket resolution STATE_CD N NUMBER (9) Code indicating the trouble ticket state STATUS_CD N NUMBER (9) Code indicating the trouble ticket status CREATED_DT N DATE Date that the trouble ticket was created RSOLTN_DT Y DATE Date the trouble ticket was resolved TARGET_OID Y NUMBER (18) Object identifier for the target object that the trouble ticket was raised against TARGET_CLASS Y VARCHAR2 (80) Fully-qualified class name of the target object CNTCT_NAME Y VARCHAR2 (240) Name of the contact for the trouble ticket CNTCT_EMAIL_ADDR Y VARCHAR2 (240) Contact's email address CNTCT_PHONE_NBR Y VARCHAR2 (50) Contact's phone number PREFERRED_CONTACT_CD N NUMBER (9) Customer-selected code indicating the preferred method of notification NOTIFY_ON_EVENT_CD N NUMBER (9) Customer-selected code indicating the frequency of notification SVC_REP_ID Y VARCHAR2 (240) Licensee defined ID for an associated service representative CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.166 contains comments for a specific trouble ticket object.",
"A trouble ticket can have zero to many associated comments.",
"TABLE 2.166 TRBL_TKT_CMT Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the trouble ticket comment DSCR Y VARCHAR2 (240) Comment text DSPL_CD N NUMBER (9) Code indicating how the trouble ticket is displayed TRBL_TKT_OID N NUMBER (18) Object identifier for the associated trouble ticket CREATED_DT N DATE Date the trouble ticket comment was generated CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry USR_OID N NUMBER (18) Object identifier for the user MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock Table 2.167 contains information used to map a trouble ticket to an interaction model selection.",
"TABLE 2.167 TRBL_TKT_INTRCTN_MODEL_SELCTN Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the trouble ticket interaction model selection INTRCTN_MODEL_OID N NUMBER (18) Object identifier for the associated interaction model CREATE_USR Y VARCHAR2 (40) The user who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.20.Vitria Adapter Table 2.168 is specifically for the EAI adapter to Vitria BusinessWare, which is an optional product.",
"This table determines the source and target strategies for vendor products that use the Vitria adapter.",
"It is populated only if you have purchased that product.",
"TABLE 2.168 VITRIA_ADAPTER Allows Attribute Name Nulls?",
"Type Description OID N NUMBER (18) Object identifier for the adapter CD_VALUE Y NUMBER (18) Code indicating the CMI policy to be called PUBLISH_CHANNEL Y VARCHAR2 (240) Fully-qualified class name of the target object SOURCE_STRATEGY— Y VARCHAR2 (240) Fully-qualified class name of the CLASS source strategy class TARGET_STRATEGY— Y VARCHAR2 (240) Fully-qualified class name of the CLASS target strategy class L-Linkparatext_OID N NUMBER (18) Object identifier for the vendor product cmi policy CREATE_USR Y VARCHAR2 (40) User who created the entry CREATE_DT Y DATE Date the entry was created MOD_USR Y VARCHAR2 (40) User who last modified the entry MOD_DT Y DATE Date the entry was last modified TYPE Y NUMBER (2) Used by TopLink to identify subclass tables related to a superclass WRITE_LOCK N NUMBER (18) Optimistic lock 2.21.View Tables Views are logical representations of physical tables; they retrieve data based on operations on their underlying base tables.",
"Table 2.169 retrieves offer instance, address and partner customer information for an assigned product, when applicable.",
"Base tables include: ASSGND_PROD, OFFER_INSTNC, HIER_OBJECT, ADDR, and PARTNER_CUST.",
"TABLE 2.169 ASSGND_PROD_VIEW Attribute Name Attribute Name Attribute Name L-Linkparatext_LABEL L-Linkparatext_STATE_CD L-Linkparatext_ADDR_OID L-Linkparatext_ROOT_OID L- OFFER_INSTNC_OID Linkparatext_COUNTRY_CD L-Linkparatext_STATUS_CD L- L- Linkparatext_CREATE_USR Linkparatext_OFFER_COLLCTN— COMPOSITE_FLAG ADDR_OID L- L- Linkparatext_HOUSE_NBR— Linkparatext_OFFER_COLLCTN— SUFFIX INSTANCE_ID L- L-Linkparatext_FLOOR L-Linkparatext_OFFER_OID Linkparatext_STREET_NBR L-Linkparatext_STREET— L-Linkparatext_ROOM L-Linkparatext_SVC_ID NAME L- L- PARTNER_CUST_OID Linkparatext_ADDL_NAME— Linkparatext_COMMUNITY LINE L-Linkparatext_STREET— L-Linkparatext_ZIP_CD L-Linkparatext_LABEL TYPE_CD L- ASSGND_PROD_OID L-Linkparatext_CUST_ID Linkparatext_ADDL_ADDR— LINE L- L- Linkparatext_STREET_DIRCTN— Linkparatext_OFFER_INSTNC— PRE_CD OID L- L- Linkparatext_STREET_DIRCTN— Linkparatext_PARTNER_CUST— POST_CD OID L-Linkparatext_UNIT_NBR L- Linkparatext_UNPROVSND— DT L- L- Linkparatext_UNIT_TYPE_CD Linkparatext_PROVSND_DT L-Linkparatext_CITY L- Linkparatext_RQSTD_PROVSND— DT Table 2.170 retrieves offer instance, address and partner customer information for an assigned product, when applicable.",
"Base tables include: ASSGND_PROD, HIER_OBJECT, and ADDR.",
"TABLE 2.170 ASSGND_PROD_ADDR_VIEW Attribute Name Attribute Name L-Linkparatext_ADDL_ADDR_LINE L-Linkparatext_UNIT_NBR L-Linkparatext_ADDL_NAME_LINE L-Linkparatext_UNIT_TYPE_CD L-Linkparatext_CITY L-Linkparatext_ZIP_CD L-Linkparatext_COMMUNITY L-Linkparatext_PARTNER_CUST_OID L-Linkparatext_COUNTRY_CD L-Linkparatext_ROOT_OID L-Linkparatext_FLOOR L-Linkparatext_HOUSE_NBR_SUFFIX L-Linkparatext_ROOM L-Linkparatext_STATE_CD L- Linkparatext_STREET_DIRCTN_PRE_CD L- Linkparatext_STREET_DIRCTN_POST_CD L-Linkparatext_STREET_NBR L-Linkparatext_STREET_NAME L-Linkparatext_STREET_TYPE_CD Table 2.171 retrieves offer instance, address and partner customer information for an assigned product, when applicable.",
"Base tables include: ASSGND_PROD, HIER_OBJECT, and ADDR.",
"TABLE 2.171 ASSGND_PROD_ADDR_2_VIEW Attribute Name Attribute Name L-Linkparatext_ADDL_ADDR_LINE L-Linkparatext_UNIT_NBR L-Linkparatext_ADDL_NAME_LINE L-Linkparatext_UNIT_TYPE_CD L-Linkparatext_CITY L-Linkparatext_ZIP_CD L-Linkparatext_COMMUNITY L-Linkparatext_PARTNER_CUST_OID L-Linkparatext_COUNTRY_CD L-Linkparatext_ROOT_OID L-Linkparatext_FLOOR L-Linkparatext_HOUSE_NBR_SUFFIX L-Linkparatext_ROOM L-Linkparatext_STATE_CD L- Linkparatext_STREET_DIRCTN_PRE_CD L- Linkparatext_STREET_DIRCTN_POST_CD L-Linkparatext_STREET_NBR L-Linkparatext_STREET_NAME L-Linkparatext_STREET_TYPE_CD Table 2.172 retrieves data for actions taken when an offer collection is broken, when applicable.",
"Base tables include: DSPL_ATTRIBS, BREAK_OFFER_COLLCTN_RULE, OFFER, and OFFER_COLLCTN.",
"TABLE 2.172 BREAK_OFFER_COLLCTN_RULE_VIEW Attribute Name BREAK_OFFER_COLLCTN_RULE_OID OFFER_COLLCTN_OID L-Linkparatext_LABEL FROM_L-Linkparatext_OID FROM_L-Linkparatext_LABEL TO_L-Linkparatext_OID TO_L-Linkparatext_LABEL TRANSTN_ACTION_CD Table 2.173 retrieves data object descriptors and compound data object descriptors combined with a boolean operator to create grouping of objects used by business rules, when applicable.",
"Base tables include: CMPND_DATA_OBJ_DSCPTR and DATA_OBJ_DSCPTR.",
"TABLE 2.173 CMPND_DATA_OBJ_DSCPTR_VIEW Attribute Name CMPND_DATA_OBJ_DSCPTR_OID L-Linkparatext_DSCR L-Linkparatext_OPERTN DATA_OBJ_DSCPTR1_OID L-Linkparatext1_DSCR DATA_OBJ_DSCPTR2_OID L-Linkparatext2_DSCR Table 2.174 retrieves content items.",
"TABLE 2.174 CONTENT_ITEM_VIEW Attribute Name L-Linkparatext_SHORT_DSCR L-Linkparatext_SMALL_ICON L-Linkparatext_START_DT L-Linkparatext_TARGET_CLASS L-Linkparatext_TARGET_OID L-Linkparatext_CATGRY_CD L-Linkparatext_DSPL_MAX_COUNT L-Linkparatext_END_DT L-Linkparatext_LARGE_ICON L-Linkparatext_LONG_DSCR L-Linkparatext_NAME L-Linkparatext_OFFER_OID L-Linkparatext_OID L-Linkparatext_PLUGIN_CD L-Linkparatext_LABEL Table 2.175 retrieves offer instance, address and partner customer information for a delivered product, when applicable.",
"Base tables include: DELVRD_PROD, PARTNER_CUST, ADDR, OFFER_INSTNC.",
"TABLE 2.175 DELVRD_PROD_ADDR_VIEW Attribute Name DELVRD_PROD_OID L- Linkparatext_STREET_DIRCTN_PRE_CD L-Linkparatext_INV_HIER_OBJ_OID L- Linkparatext_STREET_DIRCTN_POST_CD L-Linkparatext_SHIPPED_DT L-Linkparatext_UNIT_NBR L-Linkparatext_RQSTD_DELVRD_DT L-Linkparatext_UNIT_TYPE_CD L-Linkparatext_LABEL L-Linkparatext_ZIP_CD L-Linkparatext_STATUS_CD L-Linkparatext_CITY PARTNER_CUST_OID L-Linkparatext_STATE_CD L-Linkparatext_CUST_ID L-Linkparatext_COUNTRY_CD L-Linkparatext_LABEL L-Linkparatext_ROOT_OID OFFER_INSTNC_OID L-Linkparatext_OFFER_OID L-Linkparatext_SVC_ID L- Linkparatext_OFFER_COLLECTION_INSTNC— ID L- Linkparatext_OFFER_COLLECTION_COMPOSITE— FLG ADDR_OID L-Linkparatext_STREET_NBR L-Linkparatext_STREET_NAME L-Linkparatext_ADDL_NAME_LINE L-Linkparatext_STREET_TYPE_CD L-Linkparatext_ADDL_ADDR_LINE Table 2.176 retrieves partner customer information for a delivered product, when applicable.",
"Base tables include: DELVRD_PROD, PARTNER_CUST.",
"TABLE 2.176 DELVRD_PROD_VIEW Attribute Name DELVRD_PROD_OID L-Linkparatext_PARTNER_CUST_OID L-Linkparatext_SHIPPED_DT L-Linkparatext_STATUS_CD L-Linkparatext_LABEL L-Linkparatext_OFFER_INSTNC_OID PARTNER_CUST_OID L-Linkparatext_LABEL Table 2.177 retrieves data for determinant items and their associated offers when applicable.",
"Base tables include: DSPL_ATTRIBS, DTRMNT, OFFER, and DTRMNT_ITEM.",
"TABLE 2.177 DTRMNT_ITEM_VIEW Attribute Name DTRMNT_OID L-Linkparatext_LABEL DTRMNT_ITEM_OID L-Linkparatext_DSPL_ORDER L-Linkparatext_INSTNC_DSPL_ORDER L-Linkparatext_OVRRDE_DTRMNT_SEQ_OID OFFER_OID L-Linkparatext_LABEL Table 2.178 retrieves data for mappings from one offer collection determinant to another, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, OFFER_COLLCTN, DTRMNT_SEQ, and DTRMNT.",
"TABLE 2.178 DTRMNT_SEQ_VIEW Attribute Name DTRMNT_SEQ_OID L-Linkparatext_FROM_DTRMNT_OID L-Linkparatext_TO_DTRMNT_OID L-Linkparatext_LOGICAL_DSPL_ORDER L-Linkparatext_OVRRDE_FLG TO_L-Linkparatext_LABEL FROM_L-Linkparatext_LABEL DESTINATION_L-Linkparatext_TYPE_CD OFFER_COLLCTN_OID L-Linkparatext_LABEL Table 2.179 retrieves data for offer collections and their determinants, when applicable.",
"Base tables include: OFFER_COLLCTN, DSPL_ATTRIBS, DTRMNT, and OFFER.",
"TABLE 2.179 OFFER_COLLCTN_VIEW Attribute Name OFFER_COLLCTN_OID L-Linkparatext_TYPE_CD COMPOSITE_L-Linkparatext_OID COMPOSITE_L-Linkparatext_LABEL L-Linkparatext_TRANSTN_DTRMNT_OID L-Linkparatext_TRANSTN_DTRMNT_LABEL L-Linkparatext_TRANSTN_DTRMNT_TYPE_CD Table 2.180 retrieves data for mappings of source offer collection's with target offer collections during a replacement, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, OFFER_COLLCTN, and OFFER_COLLCTN_OFFER _RPLCMNT.",
"TABLE 2.180 OFFER_COLLCTN_OFFER_RPLCT_VIEW Attribute Name OID OFFER_COLLCTN_OID OFFER_COLLCTN_LABEL (L-Linkparatext) FROM_OFFER_OID FROM_OFFER_LABEL TO_OFFER_OID TO_OFFER_LABEL FIXED_COMPOSITE_OID FIXED_COMPOSITE_LABEL SHORT_DSCR LONG_DSCR Table 2.181 retrieves data for mappings of source offer collection's with target offer collections during a transition, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, OFFER_COLLCTN, and OFFER_COLLCTN_OFFER _RPLCMNT.",
"TABLE 2.181 OFFER_COLLCTN_OFFER_TRANS_VIEW Attribute Name OID OFFER_COLLCTN_OID OFFER_COLLCTN_LABEL FROM_OFFER_OID FROM_OFFER_LABEL TO_OFFER_OID TO_OFFER_LABEL FIXED_COMPOSITE_OID FIXED_COMPOSITE_LABEL SHORT_DSCR LONG_DSCR Table 2.182 retrieves data for offer transitions, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, and OFFER_TRANSTN.",
"TABLE 2.182 OFFER_TRANSTN_VIEW Attribute Name FROM_OFFER_LABEL OFFER_TRANSTN_FROM_OFFER_OID OFFER_TRANSTN_OID OFFER_TRANSTN_TO_OFFER_OID TO_OFFER_LABEL OFFER_TRANSTN_SHORT_DSCR OFFER_TRANSTN_LONG_DSCR Table 2.183 retrieves data for replacement offers, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, and OFFER_RPLCMNT.",
"TABLE 2.183 OFFER_RPLCMNT_VIEW Attribute Name FROM_OFFER_LABEL OFFER_RPLCMNT_FROM_OFFER_OID OFFER_RPLCMNT_OID OFFER_RPLCMNT_TO_OFFER_OID TO_OFFER_LABEL OFFER_RPLCMNT_SHORT_DSCR OFFER_RPLCMNT_LONG_DSCR Table 2.184 retrieves information for an offer, when applicable.",
"Base tables include: DISPL_ATTRIBS, DTRMNT, OFFER.",
"TABLE 2.184 OFFER_VIEW Attribute Name L-Linkparatext1_LABEL EXPRTN_LABEL L-Linkparatext_ADDR_MAINTENANCE_CD L-Linkparatext_DSPL_ATTRIBS_OID L-Linkparatext_EXPRTN_DT L-Linkparatext_EXPRTN_OID L-Linkparatext_OFFER_TYPE_CD L-Linkparatext_OID L-Linkparatext_ORDERABLE_DT L-Linkparatext_PLUGIN_CD L-Linkparatext_DTRMNT_OID L-Linkparatext_DTRMNT_TYPE_CD L-Linkparatext_DTRMNT_OID L-Linkparatext_DTRMNT_TYPE_CD L-Linkparatext_UPDATABLE_QTY_FLG RPLCMNT_DTRMNT_LABEL TRANSTN_DTRMNT_LABEL Table 2.185 retrieves information for an order item interaction model, when applicable.",
"Base tables include: ORDR_ITEM_INTRCTN_MODEL_SELCTN, INTRCTN_MODEL, and VENDOR_PROD.",
"TABLE 2.185 ORDR_ITEM_INTRCTN_MODLSEL_VIEW Attribute Name OID INTRCTN_MODEL_OID INTRCTN_MODEL_NAME ACTION_CD VENDOR_PROD_OID L-Linkparatext_NAME TYPE Table 2.186 retrieves partner customer, agent and supplemented order information for an order, when applicable.",
"Base tables include: ORDR, CNTCT, NAME, ORDR, HIER_OBJECT and PARTNER_CUST.",
"TABLE 2.186 ORDR_VIEW Attribute Name L-Linkparatext1_OID L-Linkparatext1_ROOT_OID L-Linkparatext1_SUBMTD_DT L-Linkparatext1_PARTNER_CUST_OID L-Linkparatext1_CREATED_USR L-Linkparatext1_LAST-MODIFIED_USER L-Linkparatext1_STATUS_CD L-Linkparatext1_ESCLTN_FLG L-Linkparatext1_EMAIL_ADDR L-Linkparatext1_AGENT_OID L-Linkparatext1_SPLMNTD_ORDR_ID L-Linkparatext1_ORDR_ID L-Linkparatext1_ID L-Linkparatext1_PURCH_ORDR_NBR L-Linkparatext1_DSCR L-Linkparatext1_MONTHLY_RCURRNG_DSPL_SUM L-Linkparatext1_TOTAL_SPLMNTL_AMT L-Linkparatext1_TOTAL_AMT PARTNER_CUST_OID L-Linkparatext_LABEL L-Linkparatext_GIVEN_NAME1 L-Linkparatext_SURNAME L-Linkparatext_LABEL Table 2.187 retrieves data for determinant items to create parent/child relationships, when applicable.",
"Base tables include: OFFER, OFFER_COLLCTN, DSPL_ATTRIBS, DTRMNT_ITEM, and PARENT_ASSIGNMENT.",
"TABLE 2.187 PARENT_ASSIGNMENT_VIEW Attribute Name PARENT_ASSIGNMENT_OID OFFER_COLLCTN_OID L-Linkparatext_LABEL CHILD_DTRMNT_ITEM_OID CHILD_DTRMNT_ITEM_LABEL PARENT_DTRMNT_ITEM_OID PARENT_DTRMNT_ITEM_LABEL PARENT_ASSIGNMENT_CARDINALITY— Table 2.188 retrieves information for prices, contexts, and price arrangements, when applicable.",
"Base tables include: PRICE, PRICE_ARRGMNT, and CNTXT TABLE 2.188 PRICE_VIEW Attribute Name PRICE_OID CNTXT_OID L-Linkparatext_NAME L-Linkparatext_ACTIVE_PRICE_ARRGMNT_OID L-Linkparatext_DSCR L-Linkparatext_DSCR L-Linkparatext_LONG_DSCR L-Linkparatext_PRICE_GROUP_SPPRTD_OID L-Linkparatext_EXT_SYS_REF L-Linkparatext_DO_NOT_SUMMARY_PRICE_FLG L-Linkparatext_FORMAT_CD L-Linkparatext_TYPE Table 2.189 retrieves information to display returned provider offers for the service availability pre-qualification function.",
"Base tables include: DTRMNT, DTRMNT_ITEM, PRVDR_SVC_OFFER_MAP.",
"TABLE 2.189 PRVDR_SVC_OFFER_MAP_VIEW Attribute Name L-Linkparatext_LABEL L-Linkparatext_OID L-Linkparatext_LABEL L-Linkparatext_OID L-Linkparatext_PRVDR_SVC_ID L-Linkparatext_PRVDR_SVC_NAME L-Linkparatext_OID Table 2.190 retrieves partner customer, agent and supplemented order information for a quote, when applicable.",
"Base tables include: QUOTE, CNTCT, NAME, ORDR and PARTNER_CUST.",
"TABLE 2.190 QUOTE_VIEW Attribute Name QUOTE_OID L-Linkparatext_ROOT_OID L-Linkparatext_QUOTE_TYPE_CD L-Linkparatext_CREATED_DT L-Linkparatext_PARTNER_CUST_OID L-Linkparatext_STATUS_CD L-Linkparatext_LAST_MODIFIED_USR L-Linkparatext_CREATED_USR L-Linkparatext_EMAIL_ADDR L-Linkparatext_AGENT_OID L-Linkparatext_ID L-Linkparatext_PURCH_ORDR_NBR L-Linkparatext_DSCR L-Linkparatext_VALID_UNTIL_DT L-Linkparatext_STATE_CD L-Linkparatext_SPLMNTD_ORDR_OID L-Linkparatext_MONTHLY RCURRNG_DISPL_SUM L-Linkparatext_TOTAL_SPLMNTL_AMT L-Linkparatext_AMT L-Linkparatext_SUBMTD_DT L-Linkparatext_QUOTED_DT L-Linkparatext_QUOTE_ID L-Linkparatext_ORDR_ID L-Linkparatext_ID L-Linkparatext_GIVEN_NAME1 L-Linkparatext_SURNAME PARTNER_CUST_OID L-Linkparatext_LABEL Table 2.191 retrieves data for links between related offers, when applicable.",
"Base tables include: DSPL_ATTRIBS, OFFER, and RLTD_OFFER.",
"TABLE 2.191 RLTD_OFFER_VIEW Attribute Name L-Linkparatext_OID L-Linkparatext_OFFER_OID L-Linkparatext_RLTD_OFFER_OID RLTD_OFFER_LABEL (L-Linkparatext) OFFER_LABEL (L-Linkparatext) Table 2.192 retrieves agent and contact information for a given root (partner or customer), when applicable.",
"Base tables include: HIER_OBJECT, NAME, ROOT, AGENT_ROOT_MAP, CNTCT.",
"TABLE 2.192 ROOT_AGENT_VIEW Attribute Name ROOT_OID L-Linkparatext_LABEL L-Linkparatext_ID L-Linkparatext_CNTCT_GIVEN_NAME1 L-Linkparatext_CNTCT_SURNAME L-Linkparatext_CNTCT_PHONE_NBR L-Linkparatext_OID AGENT_L-Linkparatext_TARGET_CLASS L-Linkparatext_GIVEN_NAME1 L-Linkparatext_SURNAME Table 2.193 retrieves data needed to display a root request for a given agent.",
"Base tables include: AGENT and ROOT_RQST.",
"TABLE 2.193 ROOT_RQST_AGENT_VIEW Attribute Name L-Linkparatext_CNTCT_OID L-Linkparatext_CNTCT_TARGET_CLASS L-Linkparatext_GIVEN_NAME1 L-Linkparatext_NAME_OID L-Linkparatext_OID L-Linkparatext_SURNAME L-Linkparatext_ACTION_CD L-Linkparatext_LABEL L-Linkparatext_OID L-Linkparatext_ROOT_OID L-Linkparatext_STATE_CD L-Linkparatext_SUBMTD_DT Table 2.194 retrieves data needed to display items in the news and information (notice) area.",
"Base tables include: TARGET_CONTENT, CONTENT_ITEM, MENU, DSPL_ATTRIBS.",
"TABLE 2.194 TARGET_CONTENT_VIEW Attribute Name L-Linkparatext_OID L-Linkparatext_PRIORITY_CD L-Linkparatext_CLASS_NAME L-Linkparatext_CLASS_OID L-Linkparatext_CONTENT_ITEM_OID L-Linkparatext_DSPL_COUNT L-Linkparatext_CLICK_COUNT L-Linkparatext_OID L-Linkparatext_TARGET_OID L-Linkparatext_TARGET_CLASS L-Linkparatext_CATGRY_CD L-Linkparatext_DSPL_MAX_COUNT L-Linkparatext_NAME L-Linkparatext_SHORT_DSCR L-Linkparatext_SMALL_ICON L-Linkparatext_START_DT L-Linkparatext_END_DT L-Linkparatext_OID L-Linkparatext_DSPL_ATTRIBS_OID L-Linkparatext_TARGET_CLASS L-Linkparatext_TARGET_OID L-Linkparatext_ROOT_MENU_OID L-Linkparatext_LABEL L-Linkparatext_LONG_DSCR Table 2.195 retrieves partner customer information for a given trouble ticket, when applicable.",
"Base tables include: TRBL_TKT, PARTNER_CUST.",
"TABLE 2.195 TBL_TKT_VIEW Attribute Name TRBL_TKT_OID L-Linkparatext_TARGET_OID L-Linkparatext_TARGET_CLASS L-Linkparatext_CATGRY_CD L-Linkparatext_ROOT_OID L-Linkparatext_USR_OID L-Linkparatext_ID L-Linkparatext_PREFERRED_CONTACT_CD L-Linkparatext_NAME L-Linkparatext_STATUS_CD L-Linkparatext_TRBL_TKT_ID L-Linkparatext_CNTCT_NAME L-Linkparatext_CNTCT_EMAIL_ADDR L-Linkparatext_CONTCT_PHONE_NBR L-Linkparatext_RSOLTN L-Linkparatext_RSOLTN_DT L-Linkparatext_CREATED_DT L-Linkparatext_MODIFIED_DT L-Linkparatext_MODIFIED_USR PARTNER_CUST_OID L-Linkparatext_PARTNER_CUST_ID L-Linkparatext_LABEL Section 3.Operations This section describes the basic operations of the Smart Component Server (SCS), including the core services and comprehensive business process logic required to successfully conduct business online.",
"3.1.Users, Groups, and Agents 3.1.1.Users Users are those who access the Smart Component Server through an eBusiness portal, or through the Administrator Console.",
"Each user has a unique user name/password combination that provides authentication within the system.",
"The user name and password are stored in an encrypted table (USR).",
"The USR table contains all other user information, such as the hierarchy object or agent that a user is associated with.",
"3.1.2.Groups Once a password is established, a user is assigned to one of the groups for the system.",
"These groups are used by the ACL table to determine which JSPs and transition policies can be executed.",
"Various groups are available.",
"For example: (1) UA_LICENSEE _USER.",
"This group can use the Universal Agent Portal to shop for and purchase products, manage hierarchies on behalf of customers and partners, and create price overrides.",
"(2) UA_LICENSEE_ADMINISTRATOR.",
"This group can use the Universal Agent Portal to create other users of that portal, create price overrides, and use the Administrator Console, as well as the licensee-level features in the Small Business Portal.",
"It can also use the Error Correction Facility.",
"(3) CP_ADMIN.",
"The group can shop for and purchase products and manage hierarchies in the Channel Partner Portal.",
"It can also create other users of the Channel Partner Portal.",
"(4) CP_USR.",
"This group is a read-only user of the Channel Partner Portal.",
"(5).",
"SB_ADMIN.",
"This group can shop for and purchase products and manage hierarchies in the Small Business Portal.",
"It can also create other users of the Small Business Portal.",
"(6) SB_USR.",
"This group is a read-only user of the Small Business Portal.",
"3.1.3.Agents Users, who use the Universal Agent Portal and who belong to the UA_LICENSEE_USER and UA_LICENSEE_ADMINISTRATOR groups, are agents with associated agent types.",
"While the existing groups in the system determine application-level security, additional groups are needed to determine the actual data displayed to agents in the Universal Agent Portal.",
"Customers and partners are associated with agents during the registration process.",
"The associated agent will always be able to see that root's associated data.",
"However, a communications services provider (CSP) may want certain agents to be able to view data associated with other agents.",
"For example, the CSP may want sales agent managers to view data for all customers and partners associated with the sales agents they are responsible for.",
"This ability is handled through the use of agent groups and agent group visibilities.",
"(1) Agent Groups.",
"A CSP may create agent groups to fit its business needs.",
"For example, the CSP might create agent groups named California, Illinois, and New York, to which certain sales agents are members.",
"The CSP then might create agent groups named West Coast, Midwest, and East Coast to which both sales agents and the state groups belong.",
"(2) Agent Group Visibility.",
"An agent that is a member of a group does not automatically have access to that group's data.",
"Agents can always view data associated with the customers or partners they are assigned to.",
"However, any other viewable customer or partner data must be explicitly designated when the agent is created.",
"For example, both Bob and John belong to the New Jersey group.",
"They can each view their own customers' data.",
"However, only Bob has visibility into the New Jersey group.",
"This means he can view data associated with any other member of that group, in this case John's customer data, but because John does not have visibility into the group, he cannot view any data associated with Bobs customers.",
"The CSP can use agent groups and agent group visibility to create complex viewing scenarios.",
"Group assignment and visibility work together to create data-level security.",
"Although the New Jersey and Florida groups both belong to the East Coast group, they cannot see each other's data as they do not have visibility into that group.",
"Sally belongs to the East Coast group.",
"Because Sally has visibility into the East Coast group, she can see all data associated with any member of the East Coast group, which includes all members of the New Jersey group and all members of the Florida group-whether or not those members have visibility into the groups.",
"Sally also belongs the Regional Managers group, as does Marla.",
"But because neither of them have visibility into that group, Maria cannot view anything associated with the East Coast group, and Sally cannot view anything associated with the West Coast group.",
"Christine does have visibility into the Regional Managers group, even though she is not a member.",
"This means she has visibility to everything Sally and Maria also have visibility to.",
"When any customer or partner data is requested by a portal, the Agent Visibility plug-in first determines whether the user associated with the request is an administrator or a CSR agent.",
"If they are, all customer and partner data matching the request is returned.",
"If not, then only the data for which the agent has visibility rights is returned.",
"3.2.Offers Offers can either be single offers or can be associated and sold with other offers, such as bundles.",
"Once offers are created, a CSP can create associations for upsell and related products, as well as upgrade and replacement paths for a particular offer.",
"The CSP uses the Administrator Console to create offers and all associated objects such as vendor products, prices, and transitions.",
"3.2.1.Vendor Products Vendor products contain information needed to provision products such as provisioning codes and provisioning start and end dates.",
"Product parameters, disclosures, and service level agreements (the time it takes to complete a specific action such as “add” or “remove”) are also associated with vendor products.",
"Users are never shown vendor products.",
"Instead, they see the offers associated with vendor products.",
"More than one vendor product can be associated with a single offer.",
"For example, an offer item of “Access Line” might be associated with two vendor products: one from Company ABC and one from Company XYZ.",
"The ChooseVendorProduct plug-in in the system simply chooses the first associated vendor product found in the database.",
"The CSP can develop a new plug-in that determines the valid vendor product for a given circumstance using different logic.",
"Or the CSP could elect to determine the vendor product manually after the order is placed.",
"It should be noted that the ChooseVendorProduct plug-in is only called if an offer is associated with more than one vendor product.",
"(1) Parameters Product parameters provide information for the installation and operation of products; for example, the number of rings before voice mail activates.",
"During the configuration process, form fields such as dropdown lists, radio buttons, and text entry fields collect the selections the user makes.",
"These values are then persisted in the database.",
"Every parameter is associated with a parameter group.",
"This allows the CSP to group similar parameters together.",
"Vendor products are associated with parameter groups and not with individual parameters.",
"To have the user select from a list of valid values for a parameter, the CSP create a parameter enumeration.",
"For example, if the CSP creates a parameter named “Number of rings before voice mail activates,” then the CSP might create a list containing the values “2,” “4,” and “6.” If more than one vendor product is associated with an offer, and a plug-in is not used to determine the correct vendor product for a given customer, the user configures parameters for all associated products.",
"The CSP may have a situation where vendor products share parameter definitions, but each vendor product should have a different default value.",
"For example, the CSP might have a parameter definition called speed.",
"This definition might be used by both a dial-up access product and a DSL product.",
"The CSP might want the default for the dial-up access to be 58K, and the default for the DSL to be 512K.",
"The CSP can set a default value override for any vendor product.",
"This ensures that the specified vendor product always has a certain parameter value set by default.",
"Parameters (groups, definitions, and enumerations), as well as value overrides are all created in the Administrator Console.",
"(2) Disclosures Disclosures are sets of terms and conditions that the CSP requires a customer or partner to read and agree to before a product can be provisioned.",
"A single disclosure can be associated with many vendor products, and a single vendor product can be associated with many disclosures.",
"Users are shown all disclosures for all vendor products associated with an offer.",
"3.2.2.Offers Offers contain all the information needed to make purchasing decisions, such as price and descriptive text.",
"Offers have an availability date and an expiration date.",
"When an offer reaches its expiration date, it can no longer be offered for purchase.",
"However, an offer can be associated with another offer to which the original offer transitions when it expires.",
"This is the expiration offer.",
"If no expiration offer is associated, then the user might see a message stating that they can no longer order the original offer.",
"All offers to be displayed in an eBusiness portal menu are associated with a DisplayAttributes object.",
"The display attribute contains all formatting information needed to display an object in an eBusiness portal; for example, label, icon, font color, and description.",
"A sales message can also be held in a display attribute.",
"This message can be targeted towards an internal user such as a sales agent, to prompt them with information they should impart to their customer when selling the offer.",
"3.2.3.Offer Instances When a user elects to buy an offer, an offer instance is created.",
"An offer instance represents the single, unique instance of an offer purchased by a particular user.",
"For example, when users shop the menu, they are looking at offers-representations of products they might want to buy.",
"When they elect to place the product in their cart, they receive their own offer instance that they can configure as needed.",
"Offer instances are created whenever a cart item is created in the case of shopping the menu, or are created when a quote item is created, as in the case of reordering an existing product.",
"Additionally, when an offer instance is created because a user is adding a product to the hierarchy, an associated assigned product hierarchy object or an associated delivered product is also created.",
"An offer instance retains its association with the offer and vendor product through the shopping, quoting, and ordering processes and when it is assigned to the customer hierarchy.",
"An instance also retains an association with the parameter values selected and any active rates.",
"Users can change parameter values and can remove products from their hierarchy.",
"Both these actions can have a related price.",
"Offers may have different relationships with other offers.",
"For examples: (1) Related Offers.",
"The CSP can associate offers so that when users view an offer, they also see links to any related offers.",
"For example, voice mail might be a related offer for an access line.",
"(2) Upsell Offers.",
"The CSP can associate offers so that when users view an offer, they see links to offers that are of higher quality or provide more features.",
"For example, voice mail with three mailboxes might be an upsell from voice mail with a single mailbox.",
"(3) Transitionable Offers.",
"The CSP can also set up offers so that once provisioned and assigned to a hierarchy, customers and partners can upgrade to another offer.",
"(4) Replacement Offers.",
"The CSP can set up offers so that after an order is placed, but before the resulting order item is provisioned, that order item can be replaced by a different offer instance.",
"3.2.4.Pricing When a user views an offer in a menu or cart, a pricing CMI prices that offer based on the price group for the root associated with the current customer or partner.",
"Once a quote reaches the “quoted” stage, the price for non-recurring charges is calculated and assigned to that offer instance.",
"Additionally, committed prices for recurring and usage charges are also associated with the offer instance.",
"At this point, any future changes to the active price for recurring or usage rates on the associated offer are not reflected on the offer instance—the rates originally associated with the offer instance remain valid.",
"(1) Price Groups Price groups allow the CSP to market and provide specific products and prices to specific sets of customers.",
"For example, an access line could have one set of prices for a residential price group and a different set for a business price group.",
"Price groups are associated with root menus.",
"Additionally, every root of a customer hierarchy (customer) must be assigned to a price group, and every price must be associated with a price group.",
"This way, users only view prices associated with the root of their customer hierarchy.",
"Because Universal Agent Portal users are not themselves associated with a root object, they are shown the defaultInternalUserRootMenu as defined in the properties file.",
"They are shown prices from the default price group.",
"To set a price group as the default, the CSP must edit the properties file.",
"A price group adapter is available, which automatically assigns all customers to the default price group specified in the properties file.",
"The CSP can create its adapters to call an external system to determine a price group.",
"A price is derived from the following objects: (a) Price group supported.",
"This is the intersection of a specific offer, price group, and action (e.g., add, remove, usage).",
"(b) Price.",
"This determines the active price arrangement for a given price group supported.",
"(c) Context.",
"This provides a differentiator for each action.",
"For example, an offer may have two prices that use the “add” action: one for labor and one for a one-time fee.",
"Context is also used for transition pricing.",
"If a CSP wish to incur a charge when a customer upgrades a product, the context for that price must be the offer the customer is transitioning from.",
"(d) Price arrangement.",
"This determines how the price is calculated; for example, flat, tier, or threshold.",
"(e) Unit price.",
"This specifies the monetary amount charged per unit for a given price arrangement.",
"(f) Criteria.",
"For a multi-level price arrangement (i.e., tier or threshold), criteria determine each of the levels within the arrangement.",
"Referring to FIG.",
"12, a pricing model is illustrated.",
"This example shows one offer that two price groups can order.",
"For price group A, the product has two prices—one for usage for long distance and one for usage for local toll calls (the context provides the differentiator).",
"For each of these prices, there is both an inactive price arrangement and an active price arrangement.",
"The long distance price uses a tier arrangement whose criteria determine the tier levels.",
"The local toll price uses a flat unit arrangement.",
"For price group B, there is only a price for usage for local toll.",
"Because there is no price created for long distance, any user associated with price group B will be shown the long distance usage price associated with the default price group.",
"(2) Price Group Supported A price group supported (PGS) determines the price based on a given price group and the action performed on a specific offer.",
"The default actions that come with the eBusiness support system include actions that create non-recurring charges and actions that create recurring and usage rates.",
"(a) Actions that create non-recurring charges include: Add—The account is charged this price when a product is added to the account's hierarchy.",
"Change—The account is charged this price when a user changes any configuration parameters for an existing product in the accounts hierarchy.",
"Remove—The account is charged this price when a user removes the product from an accounts hierarchy.",
"Transition—The account is charged this price when a user upgrades an existing product, or replaces a pending product using a supplemental order.",
"Suspend—The account is charged this price when the product is placed under suspension.",
"Resume—The account is charged this price to resume a previously suspended product.",
"(b) Actions that create recurring rates include: Recur—The account is charged this price at an interval determined by the unit of measure attribute in the unit price.",
"(c) Actions that create usage rates include: Usage—The account is charged this price based on usage of the product.",
"It should be noted that by default, the system only calculates charges for non-recurring actions.",
"Unregistered users, or internal agents who shop for customers or partners before registering them, are shown prices associated with the default price group.",
"Therefore, it is important to post notification that the final price may be different from the price displayed in the menu or cart.",
"Because customers and partners must be registered before a quote can be created, prices shown in a quote reflect the price group assigned during registration.",
"Additionally, if a PGS does not exist for an action on an offer given a price group, then the user is shown the price associated with the default price group.",
"If instead, CSP do not want members of a price group to be charged for a particular action, CSP must set the NO_PRICE_INDICATOR flag for that price group supported (unless there is also no PGS for the default price group for that action, in which case no price is shown).",
"For example, the Call Waiting offer has a PGS for the add action for Price Group 1 (the default), but does not have a PGS for Price Group 2.Therefore, any customer or partner associated with Price Group 2 is shown the price for Price Group 1.",
"(If there were also no price for the add action for the default price group, then no price would be shown.)",
"However, if a PGS is created for Price Group 2 with the NO_PRICE_INDICATOR flag activated, then an customer or partner associated with Price Group 2 is not shown any price for the add action on the Call Waiting offer.",
"(3) Price The price is the intersection of a PGS, a context, and the active price arrangement.",
"Context differentiates actions of the same type.",
"Referring to FIG.",
"12, the offer has two prices with a PGS action of “usage.” However, because each price has a different context (long distance and local toll), two separate prices are created.",
"When users drill down through an invoice, they can view that breakdown.",
"For prices charged for the action “transition” the context must contain the OID of the offer that the user is transitioning from.",
"The price can display a short and long description.",
"Additionally, the format code determines where the price appears on the bill.",
"For example, the CSP may want to group all rates of the same action together.",
"The CSP create format codes in constant classes to work in conjunction with external systems.",
"(4) Price Arrangement The price arrangement determines how to calculate the price.",
"For example, a price may be multi-level such as tier or threshold, or it may be flat.",
"The price arrangement code specifies the type of price arrangement.",
"(5) Unit Price The unit price contains the monetary amount per unit, such as the dollar amount and the unit of measure (e.g., minute, hour).",
"If a unit price is one of many prices in a price arrangement, the CSP need to create criteria to indicate the valid unit price for each level of the arrangement.",
"(6) Criteria Criteria determine the valid price for a unit of measure in a multi-level price arrangement.",
"The operand code represents text that describes the levels, such as “equals” or “less than.” 3.3.Offer Collections The offer collection feature of the eBusiness support system allows the CSP to market and provision complex collections of products.",
"FIG.",
"13 illustrates a an offer collection process 400.The CSP may create fixed bundles of products or allow a user to create a bundle dynamically by having them select from “menus” of individual offers.",
"For example, the CSP might sell a fixed bundle consisting of a specific access line offer, a specific call forwarding offer, and a specific voice mail offer.",
"Or, if the CSP are selling a Web access bundle that consists of Internet access, Web site hosting, and email boxes, the CSP might create an offer collection that allows users to select from a list of access speeds, disk space sizes, and quantities of mailboxes.",
"The CSP can also use offer collections to ask questions that help determine the appropriate group of products to present during the shopping process.",
"During the shopping process, an offer collection is presented as a set of screens that gathers the information needed to create a grouping of products.",
"As the user selects an item from a screen, the corresponding offer instance is placed in a temporary collection object 403.Once all selections are determined, the user verifies the grouping, and the individual offer instances are placed in their shopping cart.",
"3.3.1.Creating an Offer Collection To allow users to order groups of products, the CSP needs to create: (a) offer collection 403, which is a representation of the groups of screens that displays offers to users; (b) determinant 401, which provides more functionality than that of a simple menu.",
"For example, a determinant allows a user to select from a list of offers to create a bundle; (c) determinant item 402, which is an individual item that represents an offer on a determinant.",
"(1) Offer Collections An offer collection represents the set of screens that displays and captures information needed to create a group of offers.",
"There are three types of offer collections: Fixed Collection.",
"The collection is pre-determined; the user cannot change the offers associated with the collection.",
"Dynamic Collection.",
"The user can make choices from a pre-determined list for each item in the collection.",
"Independent Collection.",
"When a collection is of this type, the created offer instances are not associated with each other when ordered.",
"When offers purchased through a collection need to retain an association with each other, the CSP must also create a composite offer that represents the collection as a whole.",
"In the example shown in FIG.",
"13 the composite offer may be “Business Dial-Up Service Bundle.” All offer instances created using the collection, including the composite offer instance, retain an association with an offer collection ID.",
"Business rules dictate that once provisioned, the composite product is attached to the account, while the associated offer instances can be placed throughout the hierarchy.",
"The composite and assigned products retain an association through a common offer collection ID attribute.",
"Fixed and dynamic collections require composite offers.",
"Products created from independent collections retain no association with each other.",
"Therefore once provisioned, individual items can be deleted or transitioned without affecting the others.",
"(2) Determinants Determinants, rather than menus, are used to display offers within an offer collection.",
"Each determinant type corresponds to a specific JSP page whose transition policies use smart component functionality to create the collection.",
"New determinants may be created by CSP.",
"The solution provides the following determinant types: Fixed Determinant.",
"The user cannot select the individual offers that make up the collection.",
"This type of determinants is used when the collection is fixed.",
"Choose One Determinant.",
"This type of determinant is used when the user must select one item from a list of items.",
"Choose Multiple Determinant.",
"This type of determinant is used when the user can select more than one item.",
"If a determinant is of this type, predetermined minimum and maximum values validate the users selections.",
"Service Address Request.",
"This type of determinant is used to collect an address.",
"(3) Determinant Items Determinant items represent the offers from which the user can make a selection.",
"They are associated with both a determinant and the actual offer they represent.",
"When a user selects a determinant item, an offer instance is created and placed in the temporary collection.",
"The CSP must be sure that associated offers have not expired.",
"If expired offers are associated with determinant items, the user is still shown the determinant items, and the offer and collection expire when the user elects to purchase the associated quote.",
"Each offer collection has an associated sequence that establishes the default order in which determinants are displayed within the collection.",
"However, individual determinant items can override that sequence if the next determinant displayed depends on the determinant item selected.",
"For example, in the Web bundle shown in FIG.",
"13, the CSP might decide that users can select from larger disk spaces when they select higher connection speeds.",
"Referring to FIG.",
"14, to create a branching navigation, the CSP must override the default determinant sequence by entering an overriding determinant OID for each of the determinant items on the first page.",
"In this example, the CSP would create three different determinants for the second page.",
"3.3.2.Ordering a Fixed Bundle of Offers FIG.",
"15 illustrate an example of how the CSP might use an offer 420 collection to sell a fixed bundle with three determinant items 423 consisting of an access line, call forwarding, and voice mail.",
"Only one determinant 421 is needed for fixed collections, as the user cannot select the individual offers.",
"When the user adds the collection to a cart, each individual offer instance is added, as well as an offer instance for the “Essential Bundle” composite product 422.3.3.3.Ordering a Dynamic Bundle of Offers FIG.",
"16 illustrates an example of how the CSP might use offer collections 430 to present the ordering of T1 access 431.In this example, the CSP would use the determinant type ChooseOne for the first two determinants 432-433.Note that the determinant items displayed on the third page (and therefore the determinant 433) depend on the determinant item 432 the user selected on the second page.",
"For the third determinant 434, the CSP might create a new determinant type that automatically displays parameters and collects values for a specific parameter group.",
"The fourth determinant 435 would use a custom determinant type that creates a quantity of associated offer instances based on the quantity entered.",
"3.4.Creating Offer Transitions Transitions allow users to upgrade from an offer instance associated with an assigned product to another pre-determined offer.",
"When a user chooses to upgrade a product, the assigned product becomes associated with the new offer instance and a quote item associated with the new offer is created.",
"The CSP create offer transitions in the Administrator console.",
"For any given offer, the CSP can create one to many transitions.",
"However, the CSP cannot create transitions for delivered products.",
"3.4.1.Transitioning Offer Collections The CSP also creates transitions for each offer within an offer collection.",
"Additionally, the CSP can create transitions for when a user breaks an offer collection by attempting to remove an associated product.",
"For dynamic collections, users can transition the individual offers.",
"Therefore the CSP creates transitions for each offer associated with determinant items in the collection.",
"The composite itself cannot be transitioned—the transitioned products retain an association to the original composite.",
"For fixed offer collections, the user can only transition the composite product, and at this time, all offer instances associated with that composite are automatically transitioned.",
"Therefore, the CSP must also create transitions for each of those offers, along with a transition for the composite itself.",
"3.5.Catalogs and Menus Menus comprise the catalog from which users select product offers.",
"The CSP creates menus using the Administrator Console.",
"A menu can contain a list of links to other menus or it can contain information regarding an offer.",
"When a menu has no associated parent menu, it is a root menu.",
"The root menu displayed to a user (and therefore all subsequent menus associated with the root menu) depends on the root object currently in session.",
"Users are either associated directly with a root object (as is the case with Small Business Portal and Channel Partner Portal users), or they are agents (as in the Universal Agent Portal).",
"Referring to FIG.",
"17, when customers or partners 443 shop, they are only shown the menu 442 and prices associated with the price group 441 with which their root object is also associated.",
"When agents shop the menu, and they do not have a particular customer in session (that is they have not selected a particular customer to shop for), they are shown the default internal root menu.",
"The prices displayed are prices associated with the default price group.",
"Both the default menu and the default price group are determined in the properties file.",
"However, once agents have a customer 443 in session, they are shown menus and prices associated with that customer or partner's price group 441.The CSP use the Administrator Console to create menus.",
"Each menu is associated with a DisplayAttributes object that determines display information such as icons, text color, and description.",
"When creating a menu that will be associated with the default menu, be sure to include only offers or offer collections that everyone who will shop on the CSP's Web site can order.",
"3.6.Hierarchy In the eBusiness support system, billing points and products that a user procures are organized on a hierarchy.",
"Referring to FIG.",
"18, there are three types of objects on the hierarchy 450: root 452, billing point 453, and assigned product 454.The root 452 is the top level of the hierarchy.",
"Each root can have one or more billing points 453—the point at which all charges are collected.",
"Assigned products 454 are either provisioned products 455 or composite products 456.Products such as CPEs that are shipped directly to a customer are considered delivered products, and are not hierarchy objects, but instead retain an association with a hierarchy object, such as the billing point to which they are assigned.",
"To allow for different objects in the eBusiness support system to have the same hierarchy properties, hierarchy objects 451 may be subclassed.",
"For example, in the Channel Partner Portal, the partner object subclasses the root object and is therefore the root in that portal's hierarchy.",
"In the Small Business Portal, the customer object subclasses the root object.",
"Each hierarchy object has a valid state at any given time.",
"States determine the current stage in the lifecycle and the actions that can be performed on the object.",
"3.6.1.Creating a Hierarchy Root and billing point hierarchy objects are created during registration.",
"At this point, other objects can also be associated with these hierarchy objects, such as contacts, names, and addresses.",
"Assigned product hierarchy objects are created during the shop process when the cart items and associated offer instances are created.",
"(1) Request Objects Whenever a hierarchy object is created or modified, before the new or changed information is persisted to the actual hierarchy object, a request specific to that object is created (for assigned and delivered products, the request object is a quote item, then an order item).",
"For example, before an actual customer is created, a customer request is created.",
"This request object contains all information that might be needed by any external system that also needs to create a corresponding object.",
"(2) Root The root of a hierarchy is at the top level; that is, it has no parent.",
"Products cannot be assigned to a root.",
"The act of registration creates a root hierarchy object to which billing points and products can be associated.",
"The root object holds the following information: Price group, which determines what products are available to the customer or partner and at what price.",
"Logo URL, which is a link to where a logo for customers or partners can be stored and then displayed on portal pages.",
"Doc URL, which is a link to where external documents regarding the customer or partner can be kept.",
"Authorization, which is unique information regarding a customer or partner, such as a social security number or mother's maiden name.",
"The root's associated hierarchy object stores state information.",
"A root can have the following states: Pending, which is a request to add a new root or modify an existing root is in the process of being approved.",
"Active, which is the root request is approved, and the root is created or updated.",
"A root can be either a customer or a partner.",
"If the root is a customer, it can have an associated customer ID.",
"If the root is a partner, it has an associated partner ID and can also be associated with a resale region.",
"(3) Billing Point A root hierarchy object has one or more billing point objects as a child.",
"This is the point at which billing for all associated products occurs.",
"All information needed for billing purposes is stored on the billing point, such as active/inactive dates, billing periodicity; bill round, and running balance.",
"The billing point's hierarchy object stores state information.",
"A billing point has the following valid states: Pending, which indicates that a billing point request (either the addition of a new billing point, or a modification to the existing billing point) is in the process of being approved.",
"Active, which indicates that the billing point request is approved, and the billing point is created or updated.",
"(4) Products A product is a representation of an item that a user has purchased and placed on a hierarchy (this is done during the quote process).",
"Products retain an association with an instance of an offer—the entity that the user shopped and elected to purchase.",
"Products must be attached to either a billing point or another product.",
"There are two types of products: assigned products and delivered products.",
"An assigned product is a hierarchy object, and can be either a provisioned product or a composite product.",
"A delivered product is not a hierarchy object, but can be associated with a hierarchy object.",
"(a) Provisioned Product.",
"Provisioned products have a subscriber relationship.",
"That is, the account to which the product is assigned is charged on a periodic basis during the life of the product.",
"These charges can be a one-time fee, a recurring fee, or a fee for usage of the product.",
"An example of a provisioned product is voice mail.",
"Provisioned products can have the following states: Not ordered, which indicates that a product is in the “shop” process, but has not yet been ordered.",
"Pending, which indicates that an action on a product (e.g., add/remove/cancel/modify) is not complete.",
"Rejected, which indicates that the product is not provisioned due to an error or unresolved issue.",
"Provisioned, which indicates that the process of provisioning the product is complete.",
"Unprovisioned, which indicates that the process of unprovisioning the product is complete.",
"Suspended, which indicates that the service is still active and its charges can be invoiced, but no modifications can be made to the product.",
"Cancelled, which indicates that any pending action on the product is cancelled.",
"This state is only valid while the product is associated with an order item.",
"(b) Composite product.",
"Composite products represent a group of products ordered as a bundle.",
"An example of a composite product might be “The Essential Bundle,” which maintains an association with an access line, voice mail, and call forwarding provisioned products (these associated products can be placed anywhere on the hierarchy).",
"Composite products always have a billing point as a parent, and do not have children.",
"Composite products can have the following states: Not ordered, which indicates that a product is in the “shop” process, but has not yet been ordered.",
"Pending, which indicates that an action on a product (e.g., add/remove/cancel/modify) is not complete.",
"Rejected, which indicates that the product is not provisioned due to an error or unresolved issue.",
"Provisioned, which indicates that the process of provisioning the product is complete.",
"Unprovisioned, which indicates that the process of unprovisioning the product is complete.",
"Suspended, which indicates that the service is still active and its charges can be invoiced, but no modifications can be made to the product.",
"Cancelled, which indicates that any pending action on the product is cancelled.",
"This state is only valid while the product is associated with an order item.",
"(c) Delivered Products.",
"Delivered products are normally shipped to and owned outright by the customer and require no subscriber relationship.",
"That is, the billing point is charged for the product only once.",
"They are not hierarchy objects themselves, but instead, may be associated with a hierarchy object.",
"An example of a delivered product is a cell phone associated with a wireless service program.",
"Delivered products can have the following states: Not ordered, which indicates that a product is in the “shop” process, but has not yet been ordered.",
"Pending, which indicates that the product has been ordered, but not shipped.",
"Rejected, which indicates that the product is not provisioned due to an error or issue.",
"Delivered, which indicates that the product has been shipped.",
"Returned, which indicates that the product has been returned.",
"Canceled, which indicates that any pending action on the product is canceled.",
"This state is only valid while the product is associated with a quote item.",
"3.6.2.Managing Hierarchies Once a hierarchy is created, it can be managed.",
"The Small Business Portal and the Channel Partner Portal users can self-manage their hierarchies.",
"The Universal Agent Portal users manage hierarchies on behalf of customers and partners.",
"The actions that a user can initiate against a hierarchy include: (1) Add an account or modify an existing billing point; (2) Modify customer or partner information, including addresses and contacts; (3) Upgrade assigned products.",
"When a user upgrades an assigned product, the association the assigned product had to an offer instance changes to the offer instance the user upgraded to; (4) Re-order products and attach the new products to other points in the hierarchy; (5) Modify the configuration of an vendor product associated with an assigned product; (6) Suspend and resume an offer instance associated with an assigned product (Universal Agent Portal users only); and (7) Report trouble on any hierarchy object.",
"3.6.3.Hierarchy Related Objects The following listed are objects that can also be created and then associated with hierarchy objects.",
"(1) Name.",
"This object holds all name information (e.g.",
"surname, given name, suffix).",
"(2) Address.",
"This object holds all address information.",
"The CSP can create an association between an address and any other object in the eBusiness support system using the OBJ_ADDR_MAP table.",
"(3) Contacts.",
"Users can create contacts, which represent a person responsible for a specific entity in the eBusiness support system.",
"They can be either contacts at the customer/partner site, or at the provider site.",
"Contacts at the customer/partner site can be associated with any point in the hierarchy.",
"(4) Support Personnel.",
"Contacts at the provider site are called “supports” in the eBusiness support system, and by default are associated with the root hierarchy object.",
"The CSP can create support personnel that represent people internal to the CSP's organization who are responsible for specific areas of customer or partner support.",
"For example, the CSP may create a billing support and a sales support that are then assigned to individual customers or partners upon registration.",
"(5) Profiles.",
"The CSP may add profile creation to the registration process.",
"Customers and partners can be associated with selected information, such as their industry or their region.",
"By creating profiles, the CSP can gain better insight into customer and partner demographics.",
"For example, during the registration process, form fields such as dropdown lists, radio buttons, and text entry fields collect the selections the user makes in response to predetermined questions (called profile definitions).",
"These values are then persisted in the database and are associated with a target class and target OID (for example, the customer who answered the questions).",
"To have the user select from a list of valid values for a profile definition, the CSP create a profile enumeration.",
"For example, if the CSP creates a profile definition “Choose your industry,” the CSP might create a list containing the values “Software,” “Consulting,” and “Hardware.” By mapping profile definitions to profile groups, the CSP can display sets of parameter definitions together.",
"By further associating these groups to profile keys, the CSP can determine which profile groups are displayed under what circumstances.",
"For example, the CSP might create a profile key of customer and a profile key of partner.",
"The CSP would then create its JSPs such that those that display the profiles to customers only display groups associated with the customer key.",
"(6) Billing and Accounts Receivable.",
"Users can view invoices, open a dispute against an invoice, and create an adjustment against an invoice for a specific billing point.",
"(7) History.",
"A history of creation of, and changes to, associated objects during the life of the customer hierarchy is maintained.",
"For example, a user can view a history of orders they have placed, or trouble tickets they have raised.",
"The objects for which a user can view history depend on the portal.",
"3.7.Creating Hierarchy Views A user can create associations among hierarchy objects to display them by category.",
"To do this, a user creates summary point types and specific summary points.",
"For example, a summary point type might be “Location” and the summary points might be “East,” “West,” and “Midwest.” Once the summary point types and summary points are created, a user can go through the hierarchy and assign summary points to specific billing points or assigned products.",
"A user can assign many summary points to a hierarchy object.",
"For example, in addition to “Location,” a user might also create a summary point type of “State” and individual summary points for specific states, and then assign both summary points to hierarchy objects.",
"Once summary points are assigned, users can change the hierarchy view by sorting according to summary point type.",
"The user can select up to three different summary point types to determine sorting priorities; for example, first by location, then by state.",
"Items that are not assigned a summary point type used in the sort order are not displayed in the view Views do not retain any hierarchical information.",
"All objects are shown as a list.",
"These views cannot be saved.",
"Users must determine a sorting order each time they wish to view the hierarchy in an order other than the default.",
"A user can rename and remove summary point types and summary points.",
"If a user removes a summary point type, all summary points associated with it are also removed.",
"When a summary point is removed, any billing point or product that was associated with the summary point loses that association.",
"3.8.Shopping Cart When a user elects to purchase an offer from a menu, the following happens: (1) An offer instance is created.",
"This offer instance retains an association to the offer chosen in the menu.",
"(2) A cart item is created.",
"The cart item retains an association to the offer instance, user, and a target class (such as an assigned product or delivered product-these objects are also created at this time).",
"(3) The action “add” is set on the cart item.",
"The cart acts as a “sandbox,” where offers can be added and deleted, and quantities changed, before moving on the quote process where the offer instances are configured.",
"An user can remove a cart item if it does not belong to a fixed or dynamic offer collection.",
"If the removed item is a composite product, then all other products associated with the same offer collection are also removed.",
"3.9.Quotes Users create a quote by selecting to purchase the items in their cart.",
"Each offer instance associated with a cart item becomes associated with a quote item, and the quote becomes associated with the root of the current hierarchy in session.",
"This allows the user to view that hierarchy during the configuration process to determine where to assign or associate the items in the quote.",
"Quotes display the total monthly recurring charges as well as the total non-recurring (one-time) charges for all items in the quote.",
"Users can enter a description for the quote.",
"Quotes are associated with the customer or partner's root object.",
"They also maintain an association with an agent if the quote was created using the Universal Agent portal.",
"Quotes may have various states including: (1) New, which indicates that the quote is created; (2) Pending Configuration, which indicates that not all quote items are completely configured; (3) Configured, which indicates that all quote items are completely configured; (4) Quoted, which indicates that all quote items are configured and contain all the information needed to order the entire quote.",
"The user can no longer alter the quote; (5) Expired, which indicates that the quote has expired and can no longer be ordered; (6) Pending Approval, which indicates that the quote cannot be completed until one or more requirements are met.",
"For example, at this point you might have an external system run a credit check; (7) Canceled, which indicates that the user canceled the quote; (8) Rejected, which indicates that requirements in the approval state were not met; and (9) Completed, which indicates that the quote has been ordered.",
"3.9.1.Held Quotes Once a quote reaches the negotiated quote stage, it can be held for a predetermined amount of time before it expires and can no longer be ordered.",
"Once a quote expires, the user must create a new one.",
"When a quote expires depends on when the user requested provisioning during the configuration process.",
"If the user accepted the earliest possible start date for all items in the quote, then the quote expires 30 days later.",
"If the user selected a later date for provisioning, then the quote expires on the day that is the earliest of all provisioning dates.",
"However, no quote is valid more than 30 days.",
"If the date selected for provisioning is more than 30 days, the quote still expires after 30 days.",
"The CSP can change the 30-day period for maximum valid days to any length of time by editing the properties file.",
"If a price for an offer represented by a negotiated quote item changes while a quote is held, the associated offer instance retains the association to the original price and not the new price.",
"And if the offer itself expires, the associated offer instance remains valid for the quote.",
"3.9.2.Quote Items Quote items, which are the individual items that make up the quote, are associated with offer instances.",
"During the quoting process, users configure parameters for each quote item.",
"The parameters they configure, and the values they can choose from, are determined by the vendor product associated with the offer instance for the quote item.",
"Once the parameter values are set, those values are directly associated with the offer instance.",
"It is also during the configuration that a user determines where the quote items should be attached: either to an existing point in their hierarchy or to another item in the current quote.",
"The eBusiness support system uses business rules to determine and display only valid attachment points.",
"Quote items can have the following states: Removed, which indicates the quote item has been removed from the quote.",
"However, the quote item is not deleted and still retains an association with the quote.",
"Pending Configuration, which indicates the quote item is not completely configured.",
"Configured, which indicates the quote item is completely configured.",
"Expired, which indicates either the offer associated with the quote item has expired and can no longer be ordered, or the quote that the quote item is associated with has expired.",
"Canceled, which indicates the user has canceled the quote.",
"Completed, which indicates the quote item has been ordered.",
"Replaced, which indicates the quote item has been replaced by another quote item through a supplemental order.",
"Supplemental Cancel, which indicates the quote item was canceled through a supplemental order.",
"Supplemental Complete, which indicates the quote item cannot be changed through a supplemental order.",
"If the quote is created because the user is transitioning from an existing product in their hierarchy, then the quote item retains an association with the original offer for transition pricing purposes.",
"When a user elects to purchase a quote, all disclosures for the offer items associated with the quote items are displayed.",
"If the disclosure is not accepted, the user can return to the quote and remove the associated quote item.",
"Once all disclosures are accepted, a CMI sends information regarding the relationships of all quote items to the business rule adapter.",
"If any quote item violates a business rule, a warning is displayed.",
"(1) Pre-populating Default Configuration Values The eBusiness support system can be configured such that certain values for an offer instance are automatically populated during the transition from the cart to the quote through the use of plug-ins.",
"The plug-ins default the values for the following attributes: Attachment point.",
"This is the point at which the assigned product will be attached, By default, this plug-in attaches all products to the account.",
"Parameters.",
"This plug-in sets parameter values.",
"By default, the plug-in accepts the values set as the default for that parameter.",
"Service identifier.",
"This plug-in sets the service identifier.",
"By default, the plug-in obtains the next available service identifier.",
"Service address.",
"This plug-in sets the address at which the item will be provisioned.",
"By default, the plug-in uses the address associated with the foot with which the quote is also associated.",
"When configuration values are pre-populated, the status for each item is set to “configured” during the transition from cart to quote.",
"If needed, the user can then change any of the defaulted values.",
"(2) Propagating Configuration Values When a user purchases several of the same item, instead of having to configure each item, the user can elect to propagate values from one quote item to all quote items associated with the same offer item, so that the values are the same for all items.",
"As with setting default configuration values, this is done with one of the plug-ins that can propagate the following configuration values: Attachment point.",
"This is the point at which the assigned products will be attached.",
"Parameters.",
"All parameter values are set to match the original quote item parameter values.",
"Service identifier.",
"Instead of copying the service identifier on the original quote item, the plug in uses the next available service identifier obtained from the ServiceId adapter.",
"Service address.",
"This plug-in copies the address associated with the original quote item.",
"Shipping address.",
"For delivered products, the plug-in copies the shipping address of the original quote item.",
"Requested date.",
"The plug-in copies the original quote items requested provisioning date.",
"If needed, the user can then change any of the defaulted values.",
"(3) Price Overrides The prices displayed for a quote item are those associated with the corresponding offer item.",
"However users of the Universal Agent Portal can override these prices.",
"A price override can be either a percentage off an existing price, or it can be a new dollar amount that represents the final price.",
"This is determined by the price override type.",
"Overrides reasons are pre-configured using constant class codes.",
"Users pick a reason from this pre-configured list.",
"Each agent has a threshold that determines the amount they can reduce a charge or rate (overrides can only be used to reduce a rate or charge).",
"This override amount cannot be exceeded when the quote is submitted to be ordered.",
"If a price is overridden in the Universal Agent portal, this overridden price is the price displayed in the Small Business and Channel Partner portals.",
"3.9.3.Modifying Quotes Once quotes reach the negotiated state, they can be modified.",
"However, instead of changing the current quote, all information is copied onto a new quote and the original quote remains the same.",
"This allows the user to compare quotes.",
"When these quotes contain the same offer instance(s), only one of the quotes can actually be ordered.",
"3.9.4.Suspend/Resume Services Quotes are also created when a user wishes to suspend or resume an existing assigned product.",
"It may be desirable to suspend rather than remove or unprovision products for a number of reasons.",
"For example, a subscriber may be a large customer who has allowed one or more accounts to fall into arrears.",
"Rather than terminate service (which may damage the customer relationship), services can be suspended while the subscriber is given a chance to bring their account current.",
"Only assigned products that are in state Provisioned may be suspended.",
"Delivered products may not be suspended or resumed.",
"(1) Suspension While an assigned product is suspended, it is put into state Suspended and is no longer a valid attachment point for new products.",
"A quote that contains the products to be suspended is priced as usual, showing charges on the associated offer for the action “Suspend.” The assigned product is then moved to a pending state and once the suspend order is submitted and complete, the assigned product moves to state Suspended.",
"(2) Suspension of Products in Offer Collections The process described above is true for single, a la carte products.",
"The situation is slightly more complicated in hierarchies with multiple child products or products which were provisioned as part of an Offer Collection.",
"FIG.",
"19 is a hierarchy 460 illustrating how a product suspension is handled when the product is associated with other products.",
"In this example, there is one customer, ACME 461 which has two accounts, WEST 462 and EAST 463.An offer collection 464, called “Essential Bundle” has been provisioned to ACME-EAST and its constituent products distributed throughout the hierarchy, some to the account ACME-WEST.",
"Those provisioned products which are constituents of the offer collection are marked with “*”.",
"One provisioned product, “Caller ID” 465 is an a-la-carte product attached to “Access Line 2” 466.There are also two delivered products, both “Switch Equipment” 467-468.When an agent attempts to suspend a product, the DetermineSuspendProductList plug-in checks to determine if the product can be suspended.",
"The default plug-in logic is as follows: If the agent is attempting to suspend an a-la-carte product that has no children, the product is suspended.",
"If the agent is attempting to suspend an a-la-carte product, the product and all of its child products are suspended.",
"If the agent is attempting to suspend a product that is part of an offer collection, but is not the offer collection composite, that product and all of its child products are suspended.",
"If the agent is attempting to suspend the composite of an offer collection, the entire offer collection is suspended.",
"This will suspend all products that are part of the collection, and their children, even if the children were not part of the original collection.",
"As stated above, only products in state Provisioned may be suspended.",
"If a child product is in a state other than provisioned, the plug-in will throw an exception, an error message will be presented, and the entire transaction will be rolled back.",
"(3) Resuming Resuming products functions similarly.",
"Resuming a product will resume all of its child products, and only products that are in a state Suspended can be resumed.",
"However, if there is a need to resume a product that is in a Provisioned state, it will not throw an error.",
"For example, in the hierarchy in FIG.",
"19, assume that the entire “Essential Bundle” offer collection 464 is suspended, and that an agent resumes “Access Line 3” 469.This also resumes “Call Forward 3” 470 and “Voicemail 3.” If, at a later date, the agent then attempts to resume the entire offer collection by resuming the “Essential Bundle” composite product, the system will traverse the collection and recursively resume all products and their children.",
"But, unlike in the case of suspend, when the system encounters “Access Line 3” and sees that it is not in state Suspended, it will silently skip it (and its children) without raising an exception.",
"3.10.Orders When the user purchases all items on an existing quote that has not expired, an order is created.",
"All items in a state of Configured on the quote become order items.",
"Like a quote, an order is associated with the same hierarchy root as the user.",
"In a default implementation, orders can have the following states: (1) New, which indicates that the quote is ordered; (2) Pending Dispatch, which indicates that all order items are created; (3) Dispatched, which indicates that the order is dispatched to the provisioning system; (4) Completed, which indicates that the order is provisioned; (5) Rejected, which indicates that the order is rejected; (6) Pending Cancellation, which indicates that a request is submitted to cancel the order; (7) Canceled, which indicates that the order is canceled; and (8) Supplemented, which indicates that a user changed the order by creating a supplemental order.",
"An order retains an association with any shipping address associated with the quote, as well as the total of the order, any payment made, and the date the order was submitted.",
"3.10.1.Order Items Order items derive much of their information from the associated quote item, except for their order, their requested, scheduled, and submitted dates, and their state.",
"The valid states for an order item include: (1) New, which indicates that the quote item is ordered and is now an order item; (2) Dispatched, which indicates that the order item is dispatched to the provisioning system; (3) Completed, which indicates that the order item is provisioned; (4) Rejected, which indicates that the order item is rejected; (5) Pending Cancellation, which indicates that a request is submitted to cancel the order item; (6) Canceled, which indicates that the order item is canceled; and (7) Supplemented, which indicates that the order has been replaced by an order created through a supplemental quote.",
"3.10.2.Supplemental Orders Users can change an order once it has been submitted as long as the item they wish to change has not completed the provisioning process.",
"This is done through the creation of a supplemental order.",
"This process is similar, but not identical to, the standard quote-to-order process.",
"When a user elects to create a supplemental order, the system creates a new quote which is populated with copies of each of the order items from the original order.",
"During this transition, the SupplementalOrder plug-in determines which items in the original order are eligible for supplement.",
"By default, this plug-in only allows supplemental orders for items that are in a Dispatched state.",
"Items in any other state still appear in the new supplemental order quote, but have the Supplemental Complete state, indicating that they are complete and no further action need (or may) be taken.",
"The CSP can modify this plug-in to include any other logic, the only requirement is that it return a “True” or “False” value for each order item from the original order indicating if that item is eligible for supplement.",
"For the quote items that can be changed, users can elect to do one of the following: Reconfigure.",
"Reconfiguring allows certain values to be changed, similar to the configuration process on a regular quote.",
"The Service ID can be changed during a reconfigure order, as can any product parameters.",
"Certain reconfigurations are not allowed, however.",
"An item cannot be attached to a new hierarchy point.",
"Also, if the item has a service level agreement, the requested date of the item can be moved, as long as it is not prior to the earliest start date for that product's SLA.",
"Replace.",
"Replacing an order item allows for substitution of an existing order item with a similar item.",
"The CSP determines which replacements are allowed by creating replacement transitions.",
"When a replacement is executed, the supplemental quote item copied from the original order item moves to Replaced state and the new item is added to the supplemental quote in the Pending Configuration state.",
"Cancel.",
"Canceling an item in a supplemental quote moves the item to Supplemental Cancel state.",
"The original order item is moved to state Pending Cancel.",
"(1) Submitting a Supplemental Order The following actions are taken when an agent submits a supplemental quote: Items that were not eligible for supplement, which were added to the supplemental quote in the state SUPP_COMPLETE are added to the supplemental order.",
"Their state from the original order is copied (Complete to Complete, Rejected to Rejected, etc.).",
"The system skips any order items that were replaced by new order items (they have a status of Replaced).",
"They are not added to the new supplemental order, no new order item is created.",
"Any replacement items are added to the new supplemental order in state New.",
"Items that were supplementable in the original order and were reconfigured in the supplemental quote are added to the supplemental order, but first the plug-in runs again on the order items in the original order.",
"This is to ensure that the original order item is still eligible for supplement.",
"For example, if the supplemental quote was held for a period of time, it is possible that items in the original order to be supplemented are now in the state of Completed, thus making them ineligible for supplementation.",
"If any item fails this check, an exception is thrown, an error displayed and the entire supplemental order is rolled back.",
"5.The supplemental quote, and all its quote items move to state Complete and the quote ceases to be active.",
"6.The original order and all its order items move to the state Supplemented and the original order ceases to be active.",
"The new supplemental order becomes the active order.",
"Once this process is complete, the proper CMI is dispatched to the external system.",
"This CMI is the ServiceRequest CMI in the case of add or change, and the CancelServiceRequest CMI in the case of cancellations.",
"FIG.",
"23 illustrates a process 480 about how a supplemental order is created in the system.",
"First, an agent chooses to supplement the original order 481.The Access Line in the order is in a Complete state.",
"It is ineligible for supplement (according to the default plug-in behavior) and arrives in the supplemental quote in state Supplemental Complete.",
"All other items in the original order are eligible, and are placed in the supplemental quote in state Pending Configuration.",
"Second, the agent cancels the Call Waiting product in the supplemental quote, moving the quote item to Supplemental Cancel 482.Third, the agent replaces the Voicemail product with the Super V-Mail product 483.The original Voicemail quote item moves to state Replaced, and a new quote item for Super V-mail is added to the supplemental quote in state Pending Configuration.",
"Forth, the agent must configure those products in state Pending Configuration; Super V-Mail (which was just added via the replacement) and Call Forward 484.Finally, the agent submits the order 485.The plug-in runs again on the items in the original order and finds that none of them have become ineligible for supplement (which might occur if one of the items in the original order had subsequently completed provisioning and changed state from Dispatched to Complete), and so allows the supplemental order to be created.",
"Access Line, since it was not eligible for supplement, enters the Supplemental Order in the same state (Complete) that it was in the original order.",
"New or reconfigured items (Super V-Mail, Call Forward) are added in state Dispatched.",
"Call Waiting, which was canceled, shows up in the supplemental order in a Pending Cancel state.",
"Finally, Voicemail does not appear in the order since it was replaced by Super V-Mail.",
"After all items have been added, the proper CMIs are sent to the external system.",
"Note that this includes a CMI for Access Line, even though it is expected that the external system will do nothing (since no change was made to the order item), (2) Charges from Supplemental Orders The action of creating a supplemental order can create a charge.",
"However, unlike charges or rates associated with a particular offer, the system uses the CalculateSupplementalCharge plug-in to create the charge.",
"By default, this plug-in creates a supplemental charge object, which is a flat fee, and then associates it to a hierarchy object (by default, the billing point).",
"The logic to create the charge can be modified.",
"(3) Escalations Users can escalate an supplemental order.",
"This sets the escalation_flag attribute on the supplemental quote and order.",
"3.11.Business Rules The CSP can define rules that maintain valid relationships between objects.",
"These rules are based on a finite set of atomic rules both simple (e.g., mutual exclusion) and complex (e.g., mutual exclusion plus a numerical limit).",
"For example, when defining a voice mail product, a rule might be assigned that does not allow a customer to order voice mail unless they already have phone service.",
"Rules are invoked during the quote process.",
"When users determine where to attach a quote item (either to an existing point in their customer hierarchy, or to another quote item), the system uses the business rule adapter to determine and display only valid attachment points.",
"Later in the quote process, once all disclosures are accepted, a CMI sends information regarding all quote items to the business rule adapter.",
"At both points, the adapter validates all given items against all existing rules.",
"If any quote item relationship violates a business rule, a warning is displayed.",
"3.11.1.Rule Templates The CSP creates rules in the Administrator Console using templates.",
"Within each template are parameters that are defined by specific data-for example, the vendor product the rule is created for.",
"(1) Template Descriptions Following are descriptions of each of the business rule templates.",
"Parameters whose values must be determined to create a rule are placed between the brackets [].",
"Template 8 Rule Statement: Any hierarchy item described as an [anchorDescriptor] can have between [min] and [max] relatives of [correspondentRelationship] which are described as a [correspondentDescriptor|.",
"Rule Example #1: A business access line must be attached to an account.",
"Rule Example #2: Nothing can be attached to a custom calling feature.",
"Template 9 Rule Statement: At any time when a hierarchy item described as [anchorDescriptor] is in the state [anchorState] it must have at least one relative given by [correspondentRelationship] which is described as [correspondentDescriptor] and is in the state [correspondentState].",
"Rule Example: Whenever a voice mail product is effective, it must have a sibling call forwarding product that is also effective (i.e., voice mail requires call forwarding).",
"Template 10 Rule Statement: At any time when a hierarchy item described as [anchorDescriptor] is in the state [anchorState] it cannot have any relative given by [correspondentRelationship] which is described as [correspondentDescriptor] and is in the state [correspondentState].",
"Rule Example: Two voice mail products cannot be siblings and be effective at the same time.",
"Template 11 Rule Statement: At any time when a product described by [anchorDescriptor] is in the state [anchorState] it cannot have any relative given by [correspondentRelationship] which is described by [correspondentDescriptor], has the same vendor product OID, has the same [parameterToMatch], and is in the state [correspondentState].",
"Rule Example #1: Two voice mailboxes with the same mailbox number cannot be assigned to the same parent and be effective at the same time.",
"Rule Example #2: Only one of each type of custom calling feature can be assigned to an object and be effective at a time.",
"(2) Descriptors Often a rule does not apply to one object but to a group of similar objects.",
"For example, in the following rule: Voice mail must be attached to an access line there may be many vendor products that are defined as voice mail and many other vendor products that are defined as access lines.",
"Descriptors are used to create groupings of objects or groupings of other descriptors.",
"3.12.Paying Non-recurring Charges The system allows users to pay for any non-recurring charges (i.e.",
"one-time charges) by credit card.",
"Once all credit card information is entered, a CMI sends that information, along with the total dollar amount to an external system, for authorization.",
"The external system then returns authorization messages that can be displayed to the user.",
"The CSP may need to create new CMIs specific to the CSP's external payment system.",
"3.13.Notes Notes are text-based messages that the user creates for future reference.",
"Notes are associated with certain objects such as a quote or order which are created in different portals.",
"No matter in which portal the note was created, it retains an association with a specific root object.",
"However, users can only view a note from the portal in which that note was created.",
"For example, if a Universal Agent portal user creates a note regarding a partner, a Channel Partner Portal user also associated with that customer cannot view it.",
"However, any other Universal Agent user that can view that customer's information can view that note.",
"Notes retain an association with the user who created the note, the date and time the note was created, and the comment (this is the actual note text).",
"Notes cannot be appended; that is, if a user wishes to add a comment to an existing note, they must create a new note comment.",
"However, multiple notes can be associated with the same object.",
"3.14.Viewing Invoices To display invoices, the CSP uses the API to import billing information from an external billing system into the invoice tables.",
"Or instead of importing information for all billing points, the CSP can create a link to existing external invoices.",
"The INVOICE table holds imported header-type information, such as a reference to the account, date information, total amount due, and state information.",
"The INVOICE_SUMMARY table contains the total charges for recurring, usage, and non-recurring charges, as well as tax, adjustment, and discount totals.",
"Individual charge data is stored in the INVOICE_LINE_ITEM table.",
"Invoice line items retain associations with the tables that contain the detailed information for each charge imported from the external billing system.",
"Once invoice information is imported, a user can view individual invoices (i.e.",
"each month's invoice), past due balances aged at current, 30, 60, 90, and greater than 90 days, and payment history.",
"3.14.1.Disputes When a customer or partner questions an invoice line item, an agent can raise a dispute.",
"Disputes are handled in exactly the same way as trouble tickets.",
"3.14.2.Adjustments Agent users can create billing adjustments when an error on an existing invoice has been found.",
"Instead of actually creating an adjustment, the user creates a request for adjustment, which an external billing system then accesses, calculates, and returns to the system in the INVOICE_ADJMNT_ITEM table.",
"Adjustment reasons are pre-defined using constant classes, so that users can select the appropriate reason from a drop-down menu.",
"The CSP can add reasons to this class as needed.",
"3.15.Reporting Trouble The system allows users to report trouble for any object in the system by creating a trouble ticket (or in the case of an issue with an invoice, a dispute).",
"Users enter a description of the issue, including a detailed comment.",
"After the information is submitted, the system's server dispatches a CMI to an external trouble ticket system.",
"As the external trouble ticket system resolves the issue, it notifies the server of each step taken towards resolution.",
"A user can then view those steps.",
"During this time, users can create additional comments associated with the trouble ticket.",
"Agent users can resolve trouble tickets.",
"Users can also request cancellation of a trouble ticket.",
"When this occurs, a request is sent via a CMI to the trouble ticket system.",
"The external trouble ticket system must be configured to notify the system whether or not the trouble ticket is canceled.",
"3.16.Viewing Status The system's server includes interaction models that allow users to view the past, present, and future steps related to business processes managed by external systems.",
"For example, users can view the status of steps for the provisioning of order items and the handling of trouble tickets.",
"3.16.1.Interaction Models Interaction models reflect the workflow that external systems use to complete a given task.",
"Interaction model states reflect the individual steps within that workflow.",
"When a user kicks off an event associated with a model, an instance of the model and an instance for each of its states are created, specific to that user's event.",
"As the event travels through the external workflow, the status is passed back to the system.",
"(The configuration of the external workflow determines how the status is passed back.)",
"At this point, the status for the current model instance state is updated.",
"Once a state reaches a status of complete, the completion date is populated for that state, and the next state (specified by the display order), becomes active.",
"For example, an external provisioning workflow for an access line might have the following steps: (1) Provision access line; (2) Line set up; (3) Establish external service; (4) Establish internal service; (5) Test tine; and (6) Provisioning complete The CSP would create an interaction model named PROVISION_ACCESS_LINE and then create states for each of these steps.",
"3.16.2.End-User Views For users to view the status of each state, the CSP creates end-user views that correlate to interaction models, and end-user view states that correlate to interaction model states.",
"Together, these allow users to see pending, in progress, and completed steps for an interaction model instance in language they understand.",
"The CSP can create multiple views for one model to reflect the different portals that can access the data.",
"Referring to Table 3.1, for example, the interaction model for provisioning an access line might have two views—one for the Small Business Portal viewed by customers, and one for the Universal Agent portal.",
"TABLE 3.1 Interaction Model States CSR End User View State Customer View State Provision access Provisioning of the access Access line is in the line line has begun process of being provisioned Line set up Access line being set up in central office Establish external Running a new line from service the central office to the external termination point Establish internal Running a new line from service the external termination point to the jack Test line Testing line Testing line Provisioning Provisioning complete Provisioning complete complete Dates can be associated with end user views to determine when they are valid.",
"This way, if the CSP needs to change an interaction model, any user associated with a model instance that has not completed all steps is shown the view for the model as it was before the CSP changed the model.",
"However, any new instances of the model are shown the new end-user view.",
"3.17.Viewing Notices The CSP can create an information queue to display pertinent news and product information to-a specific object in the customer hierarchy.",
"For example, the CSP could create a notice about an outage and only show it to affected accounts.",
"Content items are the actual items that the CSP creates for display.",
"They can contain a reference to a specific offer.",
"The CSP can also set the maximum amount of times the content item is shown.",
"For each content item that CSP creates, the CSP must also create a target.",
"The target determines who sees the notice.",
"The system's server keeps track of each time the content item is shown to a specific target, and also each time that target clicks through to view the associated offer (if applicable).",
"3.18.Reports The CSP can generate reports using the system's definition files pulled from the database.",
"The definition files include: (1) Financial Bookings (Universal Agent portal only), which shows monthly recurring and non-recurring charges for a provider's customer and partner base; (2) Financial Bookings for a partner, which shows monthly recurring and non-recurring charges for one Partner; (3) Financial Forecast (Universal Agent Portal only), which shows forecast recurring and non-recurring charges based on orders placed but not yet completed, for a provider's customer and partner base; (4) Financial Forecast for a partner, which shows forecast recurring and non-recurring charges for a partner's products; (5) Orders Past Due, which shows all past due orders for a partner.",
"From Crystal Reports, the CSP can edit these definition files or create its own to suit its needs.",
"When the CSP generates these reports, it needs to save them using a standardized report name.",
"Section 4.Small Business Portal One embodiment of the invention is the Small Business Portal (SBP) that allows customers, either business or residential, to manage their accounts with a provider.",
"SBP users can use the portal to shop for products and services, manage these services, and report and track trouble tickets and disputes.",
"In this section, “CSP” refers to a communications service provider from whom the customers purchase products and services to resell.",
"CSP deploys the SBP to enable its customers to manage their accounts.",
"The “customer” refers to other organizations and individuals who purchase products and services from the CSP.",
"The “user” refers to any user of the SBP.",
"These users will typically be either residential or small business users or personnel at the customer organization.",
"There are two kinds of users: Users and Administrator Users.",
"These two kinds of users have different authority, which is discussed in more detail in the following subsections.",
"For the purposes of this document, the term user will refer to a user with all authority.",
"3.1.The Small Business Portal 3.1.1.Logging In The SBP user logs in using a user name and password.",
"By default, two security levels are supported: Customer Administrator, which has authority to do everything in the portal except manual approval.",
"Customer User, which has authority to browse the catalog and create quotes, but cannot order quotes or create new objects in the system.",
"The CSP creates the first user (typically an Administrator) for a customer via the Universal Agent portal, or customers can register themselves.",
"Once at least one administrator user is created for a customer, either by the provider or the customer, that user may then in turn create other users.",
"3.1.2.Self-Registering A new customer may self-register by clicking the “New User Register Here” link from the splash page.",
"Clicking this link takes the new customer to a page that prompts them to the Create User page, where they enter a user name and password.",
"By default, new users have customer administrator authority.",
"Once they have created this information, they can create customer information by clicking on the “Accounts” link in the My Menu navigation bar.",
"A new customer may shop (they will be shown the menu for the default price group) without creating customer information, but will be prompted to enter the information when they attempt to create an order from their cart.",
"3.2.Small Business Portal Home After a SBP user logs in to the portal, he sees the customer homepage, called My Home.",
"This page contains the following areas: (1) The main navigation bar, which is visible throughout the SBP and allows the user to navigate to the following areas: Home: Takes the CSP to the SBP splash page.",
"My Home: Brings the user to the customer homepage.",
"Search: Currently not implemented, this link can be customized to point to whatever search engine the CSP may design and deploy with the SBP.",
"Browse Catalog: Brings the user to the main catalog menu where they can shop for products and services.",
"Help: Currently not implemented, this link can be customized to point to whatever online help system the CSP may design and deploy with the SBP.",
"(2) The My Menu Navigation Bar, which sits below the main navigation bar and contains the following: Cart: Takes the customer to the shopping cart to view any products previously placed there.",
"Accounts: Takes the customer to the Account Detail page.",
"Products: Takes the customer to the My Products page.",
"The My Home page also contains the following: Company Name and Logo: The name and logo of the provider are displayed at the top of the My Home page, as well as the name of the user currently logged in.",
"News and Information: This area displays any news (such as reports of outages, new service offerings, etc.)",
"or other promotional messages targeted at the SBP user.",
"The contents of this area are configurable via the Administrator Console.",
"My History: The activity history area allows users to view past activity in the following areas: orders, quotes, payments, disputes, and trouble tickets.",
"My Accounts: This area contains links to view and manage customer information, accounts, ordered services, and bills.",
"This section also contains functionality to manage users.",
"3.3.Shopping A customer can use the SBP to shop, select, configure and order products.",
"Ordering products in the SBP consists of the three stages: (1) shopping; (2) creating and configuring items in a quote; and (3) Ordering the items in a quote.",
"The following subsections detail how the SBP implements this life cycle, and the options available to SBP users.",
"The SBP user begins shopping by clicking the “Browse Catalog” link in the main navigation bar on the My Home page.",
"3.3.1.Menus Menus are navigable lists of products offered for ordering.",
"In the SBP, the user shops product menus, then chooses and configures offered products.",
"Menus typically break product offerings down into categories desired to be presented by the licensee such as “Business Services” and “Residential Services.” When the user is browsing the product menu he will see a set of product offerings determined by their assigned price group.",
"The price group determines the root menu and prices for each offer.",
"If a user browses the menu before registering, he is shown the root menu and price associated with the default price group.",
"The SBP shows a “breadcrumb” displaying the path thus far taken through the hierarchy.",
"The user can click back at any link in the chain to return to the menu at that level.",
"3.3.2.Offers The offers in menus are listed with a brief description and an “Add to Cart” button.",
"(1) Offer Detail.",
"When the user shops and finds an offer they wish to purchase, they can choose to drill in to the offer detail page by clicking on the product name link.",
"The detail page displays the name of the offer, itemized prices for recurring, non-recurring and usage charges, and any related offers.",
"The user can then add that product to the cart then continue shopping.",
"(2) Related Offers.",
"When viewing an individual offer via the detail page, the user is shown related offers if any exist.",
"Related offers are offers that have some connection or marketing synergy with the offer being viewed.",
"Both related offers are displayed on the detail page as links.",
"These related offers can themselves be drilled into from the first offer's detail page.",
"3.3.3.Offer Collections Since-simple, one-product offers may not be sufficient to meet a providers product offering requirements, the system allows for individual offers to be bundled together into offer collections.",
"Offer collections (and also simple offers) are configured in the Administrator Console.",
"To select an offer collection for purchase the user must click the “Add Bundle” link for that offer and then select which options for that collection they wish to purchase.",
"If the offer collection is fixed, then all offers in the collection will be automatically selected; no choices can be made.",
"If the collection is dynamic, the user is guided through the collection's determinants where the user selects the individual offers that will make up the collection.",
"At any point in this process, the user can go back to a previous determinant to select a different offer.",
"The navigation path through the collection, as well as the determinants used and the offers included are all configured through the Administrator Console.",
"3.3.4.Shopping Cart As the user selects offers for purchase, they are placed in the shopping cart.",
"At any point in the shopping process, the user can view their shopping cart by selecting the “Cart” link from the My Menu navigation bar in the My Home page.",
"This is a view of all products that have been selected for ordering, their total recurring and total non-recurring charges, the quantity currently selected for quote and a text field to input an updated quantity.",
"The user may also remove items from a quote by checking the remove button and clicking the “Update” button.",
"3.4.Quotes 3.4.1.Creating Quotes When the user has shopped and selected all the desired products, clicking “Checkout” takes the customer to the Choose Quote page.",
"The customer can then select an existing quote or click the “Create Quote” button to add the items to the cart to create a quote from the current shopping cart.",
"All of the items that were in the cart now appear as quote items in the Quote Summary page.",
"3.4.2.Managing Quote Summaries The Quote Summary page shows all the items in the user's quote.",
"The top of the summary page shows the quote number, creation date, and status.",
"The user can also enter a master purchase order number, a brief description, and an e-mail address.",
"Items in a quote display the item number (ordinal number of item in quote) product offer name, the action for this item (i.e., add, remove, change, etc.",
"), the purchase order number (if one was provided), the status (configured, pending configuration, etc.",
"), the price for recurring and non-recurring charges, and a checkbox with which the user can remove items from the quote.",
"If the user removes any items from a quote, they must click “Update” to commit those changes.",
"If there are more items than will fit on one page, a pair of previous/next links allows users to navigate between pages.",
"The user can hold or cancel the quote, shop more, continue the quote—to-order process.",
"Holding a quote allows a quote to be held in its current state and then re-opened to order at a later time.",
"Once held, it is persisted.",
"Within this time frame, the user can re-open a non-expired held quote at any time and continue working on it by shopping more, configuring items, ordering it or canceling it.",
"After a quote expires, it is no longer available for ordering, but will still appear in and be viewable via the quote history as an “expired quote.” Held quotes are displayed in the current quote history, available from the activity history area on the My Home page.",
"Canceling a quote causes the quote to become inactive, but it will continue to be viewable as a “Canceled quote” in the quote history.",
"Canceling a quote stops the ordering process and moves the current quote to state “Canceled.” Canceled quotes can always be viewed from the quote history available from the My History menu.",
"3.4.3.Configuring Quote Items Before quote items can be purchased and moved to order items, they must be configured.",
"Configuration is reached by selecting the “Configure” link which is next to every quote item requiring configuration.",
"If the user attempts to click “Continue” without configuring all quote items, the SBP will prompt for configuration information for each unconfigured item in the quote.",
"The following attributes are configured in a quote: (1) Service Start Date.",
"Users can choose to accept the earliest possible start date for a quote item, or select another date (as long as the date they select is not earlier than the earliest possible date).",
"The earliest possible start date is calculated from the Service Level Agreement between the CSP and the User.",
"A constraint on service start dates arises when a quote item is attached to an existing quote item.",
"Users cannot select a start date for a dependent product that is earlier than the start date selected for its parent product.",
"(2) Service Address.",
"For some offers, Users must input information for the service address.",
"This associates the product to a physical location, typically a customer address.",
"(3) Selecting Service Identifier.",
"Users must select a service identifier (a telephone number for access line, for example).",
"The system uses a plug-in which can be interfaced with an external system to retrieve a set of service identifiers from which to choose.",
"(4) Product Association.",
"The user must also attach each quote item either to a place in the customer's hierarchy or to another item in the quote.",
"The determination of which hierarchy objects or quote items are valid attachment points is handled by the business rules engine.",
"(5) Product Parameters.",
"Finally, the user must provide values for product parameters (such as number of rings before voicemail picks up, guaranteed level of service, etc.).",
"What parameters are required is an attribute of the offer's associated vendor product.",
"These parameters are defined in the Administrator Console.",
"3.4.4.Automating Quote Item Configuration In addition to manually configuring quote items, users can take advantage of features of the Smart Component Server to streamline the quote configuration process, pre-population.",
"Certain offered products can be set up to have values for their parameters pre-populated when they are added to a quote.",
"By default, when an offer item moves from a cart item to a quote item, a plug-in can then give pre-populated values to certain product parameters.",
"By default, various parameters can be pre-populated: For examples: attachment point, service identifier, and start date.",
"The parameters to be pre-populated are determined by the PreConfigure*.",
"java plug-ins.",
"By default, the pre-populated values for product parameters are taken from the default values specified when the offer was configured in the Administrator Console.",
"Other parameters can be pre- populated as follows: Attachment point.",
"The plug-in picks the first account associated with the customer.",
"Service identifier.",
"The plug-in picks an identifier at random from the pool.",
"Start date.",
"The plug-in uses the default earliest-available start date determined by the vendor product associated with this quote item's associated offer.",
"Once the user accepts the pre-populated configuration information, they can at any time before ordering the quote go back and modify the configuration of individual quote items which were pre-configured.",
"3.4.5.Disclosures Offer items in the quote have disclosures associated with them which communicate legal or other information the customer must agree to before purchasing.",
"All associated disclosures for quote items are displayed and the user must accept them before the quote can proceed.",
"3.4.6 Negotiated Quotes Once all the configuration described has been completed and disclosures have been accepted, a quote reaches the “quoted” stage, and the display updates to reflect this by bringing the user to the Negotiated Quote page.",
"Once a user elects to order a negotiated quote, all quote items become order items.",
"(1) Prices of Held Negotiated Quotes.",
"Once a quote reaches the negotiated stage, the price for that quote is fixed.",
"If the user holds the quote, and prices for offers ordered in that quote and for that customer's price group should change, the quote will remain at the price at which it was held.",
"The held negotiated quote information is persisted the same way as a held quote summary, except that a held quote summary will be repriced every time it is accessed.",
"(2) Modifying a Negotiated Quote.",
"Selecting “Modify Quote” brings the user back to the quote summary page.",
"All of the user's previous configuration information is preserved, and they can make any changes they wish.",
"They then can proceed with ordering the quote.",
"3.4.7.Orders Once in the Negotiated Quote page, the user can create an order.",
"Offers which represent equipment (cell phones, pagers) which are physically delivered to a customer are called delivered products in the SBP.",
"When purchasing a quote containing one or more delivered products, users are required to single shipping address into the Shipping Address page to which all delivered products in the quote will be shipped.",
"If there are no offers corresponding to delivered products in the quote, then no shipping address is required.",
"Clicking the “Order Now” button in the Negotiated Quote or Shipping Address page takes the user to the Payment Option page.",
"At this point the customer can select to either add the charges for the order to a bill, or to pay immediately via credit card.",
"If the user chooses to pay by credit card, they are taken to the Make Payment page where they provide credit card and billing address information.",
"When they have entered the credit card information and clicked the “Order Now” button a CMI sends all credit infor to an external system for approval and the user is taken to a confirmation page.",
"3.5.My History Users can view history information from the links in the activity history section of the My Home page.",
"The user can choose from orders, quotes, or trouble tickets.",
"Choosing one of these categories brings up the history page for that item.",
"Depending on the type of item whose history the user is viewing, they may take different actions.",
"3.5.1.Order History SBP users can view a history of service orders from the My Home page.",
"The following information is returned for each item on the order history page: Order number; Date Created; Description; One-time Charges; Recurring Charges; and Status.",
"(1) Order Detail.",
"By clicking on the order number, the user can then bring up a detail of the order from which they can also request to cancel or reorder the quote.",
"This page lists: Order number; Date Ordered; Status; Master PO number (if any); Description; and each of the constituent order items.",
"Each order item in the list displays: Item number; Action (Add, Remove, etc.",
"); Description, which is a link which allows the CSP to drill down and view an charges for the item; Status.",
"The status that is visible from the order item line-item in the order detail page is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that order item; A link to view the detail for that item.",
"The detail page for an order item displays: product name, purchase order number, totals for one-time and recurring/monthly charges, and all the configuration information which was determined when that item was configured in a quote.",
"(2) Request to Cancel an Order.",
"Canceling an order is not guaranteed to completely cancel all products ordered.",
"Since the system relies on external systems to handle provisioning, it is possible that an ordered item can already be provisioned at the time the user is requesting cancellation, but that the new state information has not been communicated to the system.",
"Therefore, canceling an order from the order detail page simply requests a cancellation and sends the appropriate CMI to the external system.",
"If the external system is successful, the order will be canceled and the state reflected in the order history.",
"(3) Reordering.",
"When the user selects “Reorder” from the Order Detail page, they are brought to the Quote Summary page for a new quote, populated with any items which were in the original order having an action of “Add”.",
"This prevents reordering any items which were in the original order as upgrades, or which were removed, changed suspended or resumed.",
"The items in the re-ordered quote are in a pre-configured state, but the user can then change the configuration of the quote items.",
"They then can resubmit them as a new order, leaving the original order unchanged.",
"3.5.2.Quote History Users can view quotes from the quote history available from the My Home page in the activity history.",
"For each current quote, the quote history page displays: Quote number; Date Created; Description; Status; and Detail.",
"A user can also view ordered, expired and canceled quotes.",
"The user can switch between these categories via links on the main quote history page.",
"(1) Quote Detail.",
"The user can view a detail page for a quote by clicking the “View Detail” link in the history list.",
"The detail page summarizes the data displayed in the history entry, and also shows a list of every item in that quote.",
"As in quote summaries, the user can also request to hold, cancel, modify, or order the quote from this page.",
"For each item, the quote detail displays: Item number; Product Name (Clicking this link shows associated charges for the product); The action (add, remove, etc.)",
"for that item which caused it to appear in the quote; The PO number (if any); Item Status; One Time Charge; and Recurring Charge.",
"3.5.3.Payment History Users can view payments in the payment history page.",
"This page is accessed via the “Payments” link in the “My History” menu in the My Home page.",
"For each current quote, the payment history page displays: Date; Amount; Status; and Details.",
"The user can view a detail page for a payment by clicking the “View Detail” link in the history fist.",
"For each payment, the Payment Detail page displays: Payment Date; Payment Amount; Order Number (the Order associated with the payment); Credit Card Holder Name; Credit Card Billing Address; and Credit Card Information.",
"3.5.4.Dispute History Past trouble tickets are viewed via the trouble ticket history link in the My Home page.",
"The following information is returned for each item on the Dispute History page: Dispute number (Clicking the dispute number in the history list or search result set opens the detail page for that ticket showing a summary of the ticket); Date Created; Category and Name of the dispute; Dispute Status (The user can view a detail for a dispute by clicking on it from the history list.",
"The dispute detail page lists: Dispute Number; Dispute Name; Status (The dispute status displayed is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that dispute); Created By; Created Date; Last Modified By; Last Modified Date; Preferred Contact Method; Contact to Notify; Phone Number; Email Address; The entity (customer, account, product) for which the ticket was raised; The category; The ticket description text; and The resolution text (if any).",
"3.5.5.Trouble Ticket History Past trouble tickets are viewed via the trouble ticket history in the My Home page.",
"The following information is returned for each item on the trouble ticket history page: Ticket number (Clicking the trouble ticket number in the history list or search result set opens the detail page for that ticket showing a summary of the ticket); Date; Category and Name of trouble ticket; Ticket Status (The user can view a detail for a trouble ticket by clicking on it from the history list.",
"The ticket detail page lists: Trouble Ticket Number; Trouble Ticket Name; Status (The ticket status displayed is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that trouble ticket); Created By; Created Date; Last Modified By; Last Modified Date; Preferred Contact Method; Contact to Notify; Phone Number; Email Address; The entity (customer, account, product) for which the ticket was raised); The category; The ticket description text; and The resolution text.",
"3.6.My Accounts 3.6.1.Managing Accounts Accounts allow the CSP's customers to segment their business into logical units such as geographic location or department.",
"The My Home page provides the SBP user with a list of all of their accounts in the My Accounts section.",
"The list gives the name of the account, its status, and a link to view products for that account.",
"Clicking on the account name brings the user to the account detail page, where they can view all information pertaining to the selected account such as billing address, credit information, and the names of any contact personnel assigned to the account.",
"A drop-down menu at the top allows the user quickly to switch to other accounts, which then displays that account's information.",
"From the account detail page, the user can: view the account's history; modify any of the account's information; report trouble for this account, which will raise a trouble ticket; and add, modify and remove contacts by clicking on the appropriate links.",
"3.6.2.Creating Accounts Users of the SBP can create accounts from the My Home page by clicking the “Add Account” link.",
"The user must provide an account name, billing address and credit information for every account they create.",
"Once created, the account will be displayed in the list of accounts on the customer accounts page.",
"3.6.3.Managing Products in an Account From the account management page the user can view products and services provisioned to a particular account by clicking the “view the products” link next to that account.",
"This leads to the My Products page, which displays a hierarchical view of all products provisioned to that account.",
"Arrows next to products allows the user to click to expand or collapse that level of the product hierarchy.",
"Clicking on the name of a product brings the user to the detail page for that product.",
"(1) Product Detail Page.",
"For assigned products, the detail page shows the name of the product, a brief description, the date the product was provisioned, and a summary of the configuration information.",
"Any contacts assigned to this product are also visible, as is a link to add new contact information.",
"The user can then perform the following operations to manage the product hierarchy, for assigned products only: (a) upgrade; (b) remove; (c) report trouble; and (d) reorder.",
"For delivered products, users may only report trouble and add/remove contacts.",
"(2) Modifying Products.",
"The user modifies an assigned product by clicking “Modify Product” from the assigned product detail page.",
"Since an external provisioning system must be used to make the desired modifications to a provisioned product or service, the SBP implements product modification as an order.",
"Modifying an assigned product takes the user to the Quote Summary page for a new quote, consisting only of the product to be modified.",
"From this new quote detail, users can then configure any parameters including a “start” date when the modification will take effect as well as any other product parameters, service identifier, etc.",
"The user can then submit the quote.",
"(3) Removing Products.",
"The user can request to discontinue an assigned product by clicking “Remove Product” from the assigned product detail page.",
"Once again, since an external provisioning system must be used to remove a product, the SBP implements product removal as an order.",
"Removing a product brings up the quote summary page of a new quote, consisting only of the product to be removed.",
"The only configuration possible for this removal quote is the termination date.",
"Analogous to the service start date selected when ordering the product, the user can choose the earliest possible date or a specific date, as long as the date chosen is not earlier than the earliest possible date.",
"(4) Upgrading Products.",
"Product upgrades are initiated from the detail page for the provisioned product to be upgraded.",
"Which product offers are on a given product's upgrade path is configurable through the Administrator Con-sole.",
"Once an upgrade product is chosen, that new product is quoted and ordered like a normal product offer.",
"Once the upgrade order is complete, the new offer instance replaces the original.",
"(5) Reporting Trouble.",
"The user can raise a trouble ticket on a product.",
"(6) Reordering.",
"When the user selects “Reorder” from the product detail page, they are brought to the Quote Summary page for a new quote, populated with an order item corresponding to the product being reordered.",
"The item in the quote is in a configured state, but the user can then change the configuration.",
"They then can re-submit them as a new order.",
"3.6.4.Customer Details The customer detail page presents the SBP user with a summary of their customer information.",
"The page displays customer information, resale authority, address, and contacts.",
"The user can also view their customer history, modify their address information, raise a trouble ticket, or add, modify, and remove contacts.",
"Customer history consists of any changes made to customer information such as mailing address.",
"(1) Modifying customer Information.",
"The SBP user can click the “Modify Customer Information” link on the customer detail page to provide new or updated address information.",
"(2) Reporting Trouble.",
"The user can raise a trouble ticket on an account.",
"(3) Adding and Removing Contacts.",
"The process for adding, modifying, and removing contacts for a customer is identical to the process for adding contacts to an account.",
"3.6.5.About Contacts Contacts are individuals in the customer's organization who are assigned responsibility for various aspects of the customer's relationship with the provider.",
"Users of the SBP may assign contacts to customers and accounts.",
"When adding a contact, the user must provide a name, address and telephone number.",
"Contacts are of a particular type such as “primary” or “technical.” Users of the SBP can assign as many contacts as they desire.",
"There can also be multiple contacts of a given type.",
"What contact types are defined is determined by a constant class which may be changed.",
"Customer contacts are displayed on the customer, account, and product detail pages, where the user can add new contacts, modify an existing contact, or remove an existing contact.",
"3.6.6.Managing Bills The Invoice Summary page presents the SBP user with a summary of their billing information.",
"Clicking on the “View Bills” link next to the account name in the My Home page brings the user to the Invoice Summary page, where they can view billing information pertaining to the selected account.",
"Drop-down menus at the top allow the user quickly to switch to other accounts and other billing cycles, which will then display in this page.",
"From the Invoice Summary page, the user can view detail pages about: One-time Charges; Monthly Charges; Usage Charges; Adjustments; Discounts; and Taxes.",
"The user can create a billing dispute by clicking on the “Open Dispute” button.",
"3.6.7.Managing Users SBP users with administrator authority can create other users for their customer account from the manage users area of the customer My Home page.",
"The user must provide a user name, password, and password hint.",
"They must also select the authority level.",
"The two authority levels and their default permissions are: Customer Administrator, which has authority to do everything in the portal except manual approval; and Customer User, which has authority to browse the catalog and create quotes, but cannot order quotes or create new objects in the system.",
"An SBP user of sufficient authority can also remove existing users by checking the Remove radio button and clicking the “Update” button.",
"The selected user is then made inactive and will be unable to log in to the SBP.",
"3.7.Creating Trouble Tickets and Disputes 3.7.1.Issuing Trouble Tickets Users of the SBP can issue trouble tickets against the following items by clicking the “Report Trouble” link on the following detail pages: (a) customer; (b) account; (c) provisioned product; (d) delivered products; (e) composite product.",
"The trouble ticket is date-stamped and marked with the ID of the SBP user submitting it.",
"The user must supply contact information for the customer making the trouble report and specify a category or reason for the report.",
"The user must also include a detailed text description of the problem, and any recommended action.",
"Once the user has provided all the information required for the ticket and submitted it, the Trouble Ticket Detail page displays a summary of the ticket.",
"3.7.2.Creating Billing Disputes Users of the SBP can create billing disputes against invoices by clicking on the “Open Dispute” button in the Invoice Summary page.",
"The new dispute is date-stamped and marked with the ID of the SBP user submitting it.",
"The user must supply contact information for the customer making the trouble report and specify a category or reason for the report.",
"The user must also include a detailed text description of the problem, and any recommended action.",
"Once the user has provided all the information required for the dispute and submitted it, The Dispute Detail page displays a summary of the dispute.",
"3.8.Manual Approval The SBP allows administrators at the provider site to manually set the status of order items, account requests, and customer requests.",
"This is useful in testing or in deployments which do not use external systems for provisioning or trouble ticketing.",
"By default, the SBP allows a licensee to set the “pending,” “rejected,” and “complete” states, plus the “canceled” state for an order item.",
"The CSP must launch the approval screens from a new browser.",
"The following URL will bring up the approval sign-in page: http://<hostname>:<port_number>/ go?to=UA_ApprovalSignin&from=nafrom&task=naTask In this URL, replace <localhost> and <port_number> with the appropriate values for the CSP's Smart Component Server deployment.",
"At the sign-in page, enter the following login information, entering each token exactly as it appears after the colon: username: userB0022: This is a user that belongs to the UA-Liscensee-Admin Cyg-User groups; password: “password”.",
"This user has Licensee_Admin level authority.",
"Do not give this user name to anyone who should not have this authority.",
"The CSP will then see the approval menu which contains the following: Approve Partner Requests; Approve Partner Requests; Approve Account Requests; Approve Order Item Requests.",
"Each of these menus presents a list of items (appropriate to the menu chosen), their item number, the date the item was created, the current status, and radio buttons allowing the CSP to alter the state of the item.",
"Once the CSP have made the desired modifications, clicking “Update” will commit those changes and change the state of the items within the system.",
"Section 5.Universal Agent Portal One embodiment of the invention is the Universal Agent Portal (UAP), which allows users (customer service representatives, sales agents, or other administrative users) at a communications service provider (CSP) site quick access to customer and partner data, enable them to create and modify customers and partners, shop for products, report trouble, and perform many other functions offered in the eBusiness support system on behalf of customers and partners.",
"5.1.Navigating the UAP The functionality available in the UAP can be broken down between the user home page and customer home page.",
"The user home page is displayed after a UAP user have logged in to the portal.",
"Much of the functionality in the UAP is only available once the UAP user has selected a customer or partner with whom to work.",
"This is referred to in this document as making the customer “active.” Different navigation controls are available at different points in the portal.",
"5.1.1.Starting the UAP First start the Smart Component Server; then open a browser and navigate to the following URL http://<webserver>:<listen_port>/go?to=UA-Signin&from=nafrom&task=naTask When the splash Page appears, enter the user name and password and click “SIGN IN” to log in to the portal.",
"5.1.2.Main Navigation Bar The Main navigation bar appears at all times, regardless of whether a customer or partner is active and allows the UAP user to navigate to the following areas: User Homepage: Takes the UAP user to the Internal User Homepage, which allows the UAP user to search for customers, orders, product instances, and trouble tickets.",
"Clicking the “USER HOME” link also makes the user navigation bar visible if it is not already visible.",
"Customer/Partner Home: This link takes the UAP user to the customer/partner Summary page and makes the customer/partner navigation bar visible if it is not already visible.",
"If there is no active customer, this link is inactive.",
"Help: Opens online help in a separate window.",
"Shop: Takes the UAP user to the first page of the catalog.",
"Release Customer/ Partner Releases the customer/partner currently active.",
"Logout: Logs the UAP user out of the portal.",
"5.1.3.Customer/Partner Navigation bar The customer/partner navigation bar is active throughout the portal whenever a customer or partner is active.",
"This navigation bar allows the UAP user to navigate the following locations: orders: Takes the UAP user to the order history page for the active customer/partner.",
"quotes: Takes the UAP user to the quote history page for the active customer/partner.",
"payments: Takes the UAP user to the payment history page for the active customer/partner.",
"adjustments: Takes the UAP user to the adjustment history page for the active customer/partner.",
"trouble tickets: Takes the UAP user to the trouble ticket history page for the active customer/partner.",
"disputes: Takes the UAP user to the dispute history page for the active customer/partner.",
"products: Takes the UAP user to the product history page for the active customer/partner.",
"notes: Takes the UAP user to the note history page for the active customer/partner.",
"bills: Takes the UAP user to the billing history page for the active customer/partner.",
"a/r: Takes the UAP user to the accounts receivable page for the active customer/Partner.",
"Support: Takes the UAP user to the support page for the active customer/partner.",
"approval menu: Brings up the manual approval menu.",
"The approval menus may only be viewed by users belonging to the ua-licensee-admin group.",
"quote preview: Allows the UAP user to view a quote-in-progress (a shopping cart) before the UAP user order a quote.",
"This is the only link on the customer/partner navigation bar which is active when there is no active customer.",
"5.1.4.User Navigation Bar This bar is visible throughout the portal and contains the following links: new customer: Takes the UAP user to the create new customer page.",
"new partner: Takes the UAP user to the create new partner page.",
"bulk pre-qualification: Takes the UAP user to the bulk pre-qualification input page.",
"manage agents: Takes the UAP user to the manage agents page (Administrators only).",
"manage agent groups: Takes the UAP user to the manage agent groups page (Administrators only).",
"manage support: takes the UAP user to the manage support page.",
"change my password: Takes the UAP user to the change user password page.",
"5.2.Agents, Groups, and Users in the UAP Universal Agent Portal users are those who access the eBusiness support system through the Universal Agent Portal.",
"Each UAP user has a unique user name/password combination that provides authentication within the system.",
"5.2.1.Agents It is necessary not only to restrict the functionality available but also the data visible to the different types of UAP users.",
"The primary-motivation is to protect customer/partner data from inappropriate or inconsistent modification.",
"The system implements this data-level security via agents.",
"Agents are distinct from users, but every UAP user is also of a particular agent type.",
"The following three agent types are defined, by default: (a) CSR; (b) Sales Agent; and (c) Administrator.",
"Sales Agents are subject to visibility restrictions.",
"CSRs and Administrators are exempt from these restrictions.",
"New agent types can be created by modifying the appropriate constant class.",
"A user's agent type affects what data they can see when executing customer searches.",
"When searching for customers or partners, a plug-in runs and examines the agent type of the user executing the search.",
"The default behavior of the plug-in is as follows: If the agent executing the search is of agent type CSR, or Administrator no check is made to agent group visibility table.",
"Otherwise, the table is consulted, and the data returned is restricted according to the visibility rights granted to the agent.",
"5.2.2.Agent Groups Agents are further associated into groups.",
"An agent group may be a geographical.",
"division (East Coast office, West Coast office, etc.)",
"or by agent's last name, or any other division.",
"Agent groups, and group visibility are what implement the data-level security.",
"UAP administrator users can create new agent groups.",
"5.2.3.Creating Users and Agents By default, only administrators can create new UAP agents.",
"The “manage agents” link from the user navigation bar brings the UAP user to the manage agents page.",
"Here, the UAP user is presented with a list of existing agents, which the UAP user can view or modify.",
"The “CREATE” link allows the UAP user to create a new user and the associated agent.",
"The UAP user must provide a name and password for this user.",
"This name and password is stored with the user.",
"The UAP user must also select an agent type and a security type (one of Administrator or User) and must enter contact information (name, phone, email, pager, etc.)",
"for this agent.",
"From the Agent Group Assignment page, the UAP user must assign the agent to one or more groups.",
"Finally, the UAP user must make visibility assignments for this agent.",
"Giving an agent visibility into a group allows that agent to see all customer data associated with members of that group.",
"5.2.4.Managing Agent Groups The “manage agent groups” link from the user navigation bar brings the UAP user to the manage group page where the UAP user can create, modify or remove groups.",
"The Manage Groups page shows the UAP user a list of all agent groups, and allows the UAP user to remove, view or modify them, or to create a new group.",
"Note that the UAP user can assign entire groups as members of a group, so that the UAP user could, for example, create a group called “North America” and assign the groups “East Coast;” “West Coast,” and “Midwest” to it.",
"An agent group may be removed from the system.",
"Agents or agent groups that were members of, or had visibility into the removed group continue to exist.",
"When a group is removed, all agents who had visibility into that group and could see its associated data will no longer have that visibility.",
"This can cause agents to be unable to see the customer data they require.",
"5.3.The User Homepage Once logged in, a UAP user are presented with the User Homepage.",
"The UAP user see any news and information targeted to the UAP user, the user navigation bar, and the search pane.",
"5.3.1.Agent Visibility and Customer Data What data is visible to the UAP user depends on the following criteria: (1) Certain agent types are exempt from these visibility rules.",
"The choice of which agents are subject to/exempt from visibility rules is made by a plug-in whose default behavior exempts agents of type CSR or Administrator.",
"All other agent types (that is, Sales Agent) are subject to the visibility restrictions.",
"(2) The UAP user's agent group and agent group visibility assignments affect what customer data is visible to the UAP user.",
"When executing a search for a customer/partner or a related item, the set of data against which the UAP user search is executed is determined by the UAP user's agent group visibility.",
"5.3.2.Selecting an Existing Customer Much of the functionality offered in the UAP is only available when the UAP user has made a customer (or partner) “active.” In order to select a customer or partner, the UAP user must perform a search.",
"By selecting a customer or partner from the search result set, the UAP user activates that customer or partner active.",
"The UAP allows users to search for customers and partners using the attributes of: (a) Customer Name; (b) Customer ID; (c) Customer Contact Phone Number; (d) Customer Contact First Name; (e) Customer Contact Last Name; and (f) Assigned Sales Agent (sort by first and last name individually).",
"Searches may also be made ascending or descending.",
"Once a customer is selected and made active, the agent is brought to the Customer Homepage.",
"5.3.3.Searching for Related Items The UAP user can execute searches for objects other than customers.",
"A dropdown menu on the search pane controls which type of object the UAP user are searching for.",
"Changing this drop-down causes the search pane to redraw with input fields for search criteria appropriate to that object.",
"Once the UAP user specify values for the search criteria, executing the search returns a set of matching items (if any).",
"Selecting an item from the result set causes the associated customer to become the active customer, and replaces the current active customer/partner.",
"The search then brings the UAP user to the appropriate detail page for that item (product detail page for a provisioned product, trouble ticket detail for a trouble ticket, etc.).",
"With a customer or partner active, the UAP user can also navigate to pages for trouble ticket, product, and service order from the customer/partner navigation bar.",
"These searches will only be against data specific to the active customer or Partner.",
"The UAP user can execute searches for the following types of items from the user home page: (1) Product Instance.",
"Search on and Sort on: (a) Product Name; (b) Service Identifier (e.g., telephone number); (c) Customer Name; (d) Customer ID; (e) Partner-Customer ID; and (f) Requested Due Date.",
"Search on only: (a) Service Street Number; (b) Service Street Name; (d) Service City; (e) Service State/Province; and (f) ZIP/Postal Code.",
"(2) Service Order.",
"Search and Sort on: (a) Order Number; (b) Purchase Order Number; (c) Order Status; (d) Assigned Sales Agent; (e) Customer Name; (f) Customer ID; (h) Partner-Customer ID; (i) Expedite Flag; and (j) Date Created.",
"Sort on only: (a) Agent First Name; (b) Agent Last Name (3) Trouble Ticket.",
"Search on and Sort By: (a) Ticket Number; (b) Ticket Status; (c) Customer Name; (d) Customer ID; (e) Partner-Customer ID; (f) Ticket Contact Name; and (g) Date Created.",
"When the UAP user finishes working with a customer or partner, the UAP user can choose to release the customer or partner, by selecting the “RELEASE CUSTOMER” link on the main navigation bar.",
"Doing so will remove everything related to that customer/partner from the current session and return the UAP user to the user home page.",
"The UAP user need not explicitly release a customer or partner from being active.",
"Selecting a new active customer or partner from a customer search result set will automatically make the newly selected customer active in lieu of the original one.",
"5.3.4.Bulk Pre-Qualification Some services may require pre-qualification before they can be provisioned.",
"The UAP allows the UAP user to create and submit requests for bulk pre-qualification of customers to an external system.",
"From the user navigation bar, the UAP user can select “bulk pre-qualification.” The bulk pre-qualification page allows the UAP user to browse for and select a file containing information needed to qualify a group of customers for a product.",
"The UAP user must give a name to the request and an email address that will be notified when the request has been processed.",
"The UAP user can then submit the request.",
"5.4.Registering New Customers/Partners 5.4.1.Creating Customers or Partners Any UAP user can create customers and partners.",
"The process is slightly different for the two entities.",
"(1) Creating Customers When creating a customer, the UAP user enter relevant information such as mailing address and company name.",
"Also, if there are any profiles defined the data required for the profile is captured.",
"The UAP user can also specify: (a) a document URL that points to contracts or other documentation relevant to this customer; (b) a price group for the customer; and (c) an assigned sales agent.",
"(2) Creating Partners For partners, additional information is captured during creation.",
"Most notably, partners are often only allowed to resell services in certain geographical areas.",
"Hence, the UAP user must select from a list of (configurable) geographical regions and indicate explicitly in which regions the new partner is authorized to resell services.",
"For partners, the particular partner program in which this new partner is enrolled is called “partner level” This is simply the implementation of price group for partners.",
"In addition to a document URL (which for partners might contain a link to legal or other contractual documents for the partner), the UAP user specifies a URL pointing to the Partner Logo image.",
"This logo is used in the Channel Partner Portal to re-brand the interface for the partner user.",
"(3) Profiles Profiles provide a way to capture various demographic or marketing information for a customer or partner at the time that customer is created.",
"What information is gathered is entirely configurable through the Administrator Console.",
"(4) Assigning Contacts During the registration process, the UAP user is prompted for information for a contact.",
"(5) Creating a User Finally, the UAP user create a user for the customer or partner just created.",
"The UAP user specifies a user name and password, a password hint, and selects a user type.",
"It is this name and password that allows customers to log in to the Small Business Portal and partners to log in to the Channel Partner Portal to manage their partner relationship on their own behalf.",
"(6) Assigning Support The UAP user can also assign support resources from the UAP user's organization to a customer.",
"5.4.2.Creating Accounts Customers and partners have associated accounts.",
"Once the customer or partner is created, the UAP user can then proceed to create one or more accounts, capturing the required name, address and credit/payment information.",
"Referring to FIG.",
"21, customer data is organized in a hierarchy 490 which consists of root 452, billing point 453 and assigned product 454.In the UAP, Root 452 is implemented as either a customer or a partner 491; Billing point 453 is implemented as an account 492 (all charges accrue at the account level).",
"The implementation of Assigned products is same as that in the Smart Component Server.",
"When the UAP user makes a customer or partner 491 active, the UAP user can view data belonging to the associated hierarchy 490.The eBusiness support system relies on external systems to do the actual network provisioning of customers and their accounts and products.",
"If there is no external system, all objects (customers/partners, accounts, orders) will persist in a pending state until manually changed via the approval pages.",
"5.5.Customer/Partner Homepage Once a customer/partner has been selected and made active, the UAP user is shown that customer or partner's home page.",
"This page displays the following: a customer summary section, showing each of the customer/partner's accounts and a link to add new accounts; a news and information pane showing news and promotional messages appropriate to that customer/partner; a history pane of the last five customer activity items (trouble tickets, orders, quotes, disputes, etc.)",
"at the bottom of the page.",
"5.5.1.Customer/Partner Detail The name of the customer/partner on the home page is a link to the customer/partner detail page.",
"This page summarizes the information for that customer/partner (including resale authority for partners and customer address for customers) and allows the UAP user to modify the customer/partner information, report trouble, and manage contacts.",
"5.5.2.Accounts On the customer summary page, the UAP user is presented with a list of every account associated with that customer.",
"For each account in the list are the following links: Account Name: Clicking the account name itself brings up the detail page for that account.",
"View Services: Clicking this link allows the UAP user to view all provisioned services (i.e.",
"products) for that account.",
"Manage Bills: Bills for this account are available through the Manage Bills link.",
"A/R: A summary of Accounts Receivable information is available through the A/R link.",
"Once the UAP user have selected one of these links, each of the subsequent account-related pages has a drop-down menu containing all other accounts associated with this customer, allowing the UAP user to move from viewing information for one account to viewing information for a different account without needing to return to the customer detail page.",
"(1) Account Detail Clicking the name of an account in the listing on the customer summary page brings the UAP user to the account detail page for that account.",
"This detail page contains a summary of the account's information such as name, billing information, billing address, and credit information.",
"Also listed are the contacts (if any) for that account.",
"The account detail page includes links which allow the UAP user to view history items for this account, modify account information, raise a trouble ticket on this account and add, modify or remove contacts for that account.",
"(2) Bills Next to each account in the account listing on the customer summary page is the “Manage Bills” link which allows the UAP user to view bills for that account.",
"This page presents a list of bills for the selected account, a drop-down list to select which billing cycle to view, and a drop-down menu from which the UAP user can select a different account whose bills are then displayed in place of the original account selected.",
"Selecting a bill from the list brings up the bill detail page, which gives a breakdown with subtotals for various types of invoice items, including: (a) Monthly, or Recurring Charges; (b) One-time, or Non-Recurring Charges; (c) Usage charges; (d) Taxes; (e) Adjustments; and (f) Discounts.",
"The UAP user can then drill in to each type of charge and see a list of charge instances for that type and for that invoice.",
"(3) Accounts Receivable In addition to viewing invoices, an “A/R” link next to each account in the customer summary list allows the UAP user to view past balances for a customer account, broken down into intervals of 30 days, 60 days, 90 days, and 90 days and above.",
"The UAP user can then select a different account for the current customer and view that account's A/R balances as well.",
"(4) Creating an Account Accounts can be created at any time from the customer summary page by clicking the “ADD NEW ACCOUNT” link.",
"The UAP user must provide an account name, billing address and credit information for every account the UAP user create.",
"Once created, the account will be displayed in the list of accounts on the customer summary page.",
"5.5.3.History From the customer/partner home page, the UAP user can view history, showing events in the life-cycle of the customer to date.",
"By default, a list of the last five history items is shown.",
"If the agent wishes to view history information for a particular category, those histories are available from the navigation bar.",
"These history items include the categories of: (a) Orders; (b) Quotes; (c) Payments; (d) Adjustments; (e) Disputes; and (f) Trouble Tickets.",
"From a history item link, the UAP user can then drill in to the specific item to view its detail page.",
"5.6.Shopping The UAP user can use the UAP to shop, select, configure and order products on behalf of customers or partners.",
"Ordering products in the UAP consists of the stages of: (a) Shopping; (b) Creating and configuring items in a quote; and (c) Ordering the items.",
"5.6.1.Price Group Price groups are a mechanism by which the UAP user can segment the UAP users customers, perhaps into “VIP,” “Executive” and “Standard” groups, for example.",
"Price groups determine what products and what prices for those products are available to a given customer.",
"The prices displayed throughout the shop process are determined by the active price group, but can later be negotiated or overridden.",
"What price groups exist and their associated prices are both configured in the Administrator Console.",
"What price group is active when using the UAP is dependent on whether or not the UAP user have an active customer.",
"If there is no active customer or partner, the default price group determines the associated product prices.",
"If a customer or partner is active, then the price group for that customer (which was assigned when the customer or partner was created) determines which associated prices are displayed.",
"5.6.2.Menus Menus are navigable lists of products offered for ordering.",
"In the UAP, the UAP user shop product catalogs, and choose and configure offered products for the customers or partners on whose behalf they are acting.",
"Menus typically break product offerings down into categories such as “Business Services” and “Residential Services.” What categories exist and what offers are available through them is configured through the Administrator Console.",
"(1) Default Menu When the UAP user are browsing the product catalogue with no active customer or partner, the UAP user will see a set of product offerings determined by the parameter customer.defaultInternalUserRootMenu0id in the properties file.",
"Once a partner or customer is active, and that partner's price group becomes active and in turn determines the root menu the UAP user see when shopping the catalog.",
"The contents of all menus, including the default menu is configurable through the Administrator Console.",
"(2) Alphabetical Product Index In addition to the hierarchical menus, the UAP user can also locate products by name.",
"Selecting a letter from the alphabetical index will bring the UAP user td a menu of products beginning with that letter.",
"The names on which the UAP user search are taken from the menu item label, in the DISPLATTRIBS table entry for that menu item.",
"(3) Breadcrumbs While browsing the hierarchical menus (but not while viewing an alphabetical list), the UAP shows a .“breadcrumb” displaying the path thus far taken through the catalog.",
"The UAP user can click back at any fink in the chain to return to the menu at that level.",
"5.6.3.Offers The offers in menus are listed with a brief description (from the DISPL_ATTRIBS table entry for that menu item), the total price for recurring rates and for non-recurring charges, a field displaying both the current quantity in a quote and a field for entering a different quantity.",
"When the UAP user shops and finds an offer the customer/partner wishes to purchase for a customer, the UAP user can choose to drill down to the offer detail page.",
"The page displays the name of the offer, a list of which price groups can purchase this offer, itemized prices for recurring, non-recurring and usage charges, and any upsell or related offers.",
"The UAP user can then specify a quantity of the product to be ordered, just as in the order's line-item in the menu page.",
"Whenever the UAP user changes the quantity of an item to be purchased, either from the menu page or the offer detail page, the UAP user must click “UPDATE” to save the new quantity before moving to a new page, This commits the items the UAP user has selected to the shopping cart, which can be viewed at any time by selecting “quote preview”.",
"The UAP user can then continue shopping, perhaps in a different category of product offerings.",
"When viewing an individual offer via the detail page, the UAP user are shown related offers or upsell offers, if any exist.",
"Related offers are offers which have some connection or marketing synergy with the offer being viewed.",
"Upsell offers are related offers that might be interesting to the user.",
"Both related and upsell offers are displayed on the detail page in a one-line format similar to the offers on a menu page.",
"These related offers can themselves be drilled into from the first offer's detail page, or the UAP user can specify quantities immediately for purchase.",
"For example, when the UAP user clicks on the Business Access Line product offer, the UAP user is taken to the offer detail page for Business Access Line.",
"There, in addition to the Business Access Line product, the UAP user see various Upsell Products including: Business Voice Mail w/Pager Notification; Essential Bundle; and Business Messaging Bundle.",
"The UAP user can enter a quantity for purchase for any of these upsell offers and click “UPDATE” to add those offers to the customer/partner cart.",
"Which offers are related to one another and which are on an upsell path is configurable through the Administrator Console.",
"5.6.4.Offer Collections Since simple, one-product offers may not be sufficient to meet a providers product offering requirements, the system allows for individual offers to be bundled together into offer collections.",
"Offer collections (as well as simple offers) are configured in the Administrator Console.",
"Instead of selecting a quantity to purchase, as the UAP user does with simple offers, to select an offer collection for purchase the UAP user must click the “add Bundle” link for that offer and then select which options for the collection the UAP user wish to purchase.",
"If the offer collection is fixed, then the UAP user will automatically select all of the offers in the collection; no choices can be made.",
"If the collection is dynamic, the UAP user are guided through the collection's determinants where the UAP user select the individual offers that will make up the collection.",
"At any point in this process, the UAP user can go back to a previous determinant to select a different offer.",
"The navigation path through the collection, as well as the determinants used and the offers included are all configured through the Administrator Console.",
"5.6.5.Quote Preview At any point in the shopping process, the UAP user can view a quote preview by selecting that link from the customer/partner navigation bar.",
"This is a view of all products that have been selected for ordering, their total recurring and total non-recurring charges, the quantity currently selected for quote and a text field to input an updated quantity.",
"As the UAP user select offers for purchase, they are placed in the quote preview.",
"The quote preview is the UAP implementation of the shopping cart.",
"The UAP user can adjust the quantity of remove items from the UAP user“s cart.",
"5.7.Quotes 5.7.1.Creating Quotes When the UAP user has shopped and selected all the products desired, clicking “CHECKOUT” creates a quote.",
"All of the items that were in the UAP user's quote preview when the UAP user selected checkout now appear as quote items.",
"5.7.2.Quote Summary The quote summary page shows all the items in the UAP user's current quote.",
"The top of the summary page shows the quote number, creation date, status, last-modified user, and create user.",
"The UAP user can also enter a master purchase order number, a brief description, e-mail address, select a sales agent, and either select an existing partner-customer ID or enter a new one (only if there is a partner active).",
"Partner-Customer ID is a field where the UAP user can store a partner's internal ID for their end-customers.",
"When the UAP user is shopping on behalf of a partner, the partner can provide the UAP user with its end-customer's ID which the UAP user can then associate with the quote via this field.",
"Items in a quote display the item number (ordinal number of item in quote) product offer name, the action for this item, the purchase order number (if one was provided), the status (configured, pending configuration, etc.",
"), the price for recurring usage, and non-recurring charges, and a checkbox with which the UAP user can remove items from the quote.",
"If the UAP user remove any items from a quote, the UAP user must click “UPDATE” to commit those changes.",
"If there are more items than will fit on one page, a pair of previous/next links allow the UAP user to navigate between pages.",
"The UAP user can hold or cancel the quote, add notes, shop more, or continue.",
"Canceling a quote causes the quote to become inactive, but it will continue to be available as a “Canceled quote” in the customer's quote history.",
"Adding a note to a quote allows the UAP user to attach an annotation to the quote that will then be visible to anyone else working with that quote in the UAP.",
"Any notes created in the UAP can only be viewed in the UAP.",
"Users of other e-Business portals cannot view or modify them.",
"If the UAP user chooses to “shop more” from the quote summary, the UAP user is returned to the product catalog.",
"Any products the UAP user purchased now are added to the current quote.",
"5.7.3.Holding a Quote Holding a negotiated quote causes the quote to persist for a period of time.",
"The UAP user can re-open a held quote at any time and continue working on it by shopping more, configuring items, ordering it or canceling it.",
"After a quote expires, it is no longer available for ordering, but will still appear in and be viewable via the quote history as an “expired quote.” 5.7.4.Canceling a Quote Canceling a quote stops the ordering process and moves the current quote to a “Canceled” state.",
"Canceled quotes can always be viewed from the quote history available from the customer/partner navigation bar.",
"5.7.5.Configuring Quote Items Before quote items can be purchased and moved to order items, they must be configured.",
"Configuration is reached by selecting the “Configure” link which is next to every quote item requiring configuration.",
"If the UAP user attempt to click “CONTINUE” without configuring all quote items, the UAP will prompt the UAP user for configuration information for each item in the quote.",
"(1) Service Start Date The UAP user can choose to accept the earliest possible start date for a quote item, or select another date (as long as the date the UAP user selects is not earlier than the earliest possible date).",
"The Service Level Agreement (SLA) is an attribute of the offer's associated vendor product, configured via the Administrator Console when product offerings are created.",
"The SLA is added to the current date resulting in the earliest possible start date.",
"This represents, for example, the amount of time it would take a technician to perform all the necessary network provisioning (perhaps including a customer premises visit) before the offered product is available for use.",
"A constraint on service start dates arises when the UAP user attaches a quote item to an existing quote item.",
"The UAP user may not select a start date earlier than the start date for a dependent product that is earlier than the start date selected for its “parent” product.",
"(2) Service Address The UAP user must input information for the service address.",
"This associates the product to a physical location, typically a customer address.",
"(3) Selecting Service Identifier The UAP user must select a service identifier (a telephone number for access line, for example).",
"The system uses a plug-in to interface with an external provisioning or telephone-number-inventory system to retrieve a set of service identifiers from which to choose.",
"(4) Product Association The UAP user must also attach each quote item either to a place in the customer's hierarchy, or to another item in the quote.",
"The determination of which hierarchy objects or products are valid attachment points is handled by the business rules manager.",
"(5) Product Parameters Finally, the UAP user must provide values for product parameters (such as number of rings before voice mail picks up, guaranteed level of service, etc.).",
"What parameters are required is an attribute of the offer's associated vendor product.",
"These parameters are defined in the Administrator Console.",
"5.7.6.Automating Quote Item Configuration In addition to manually configuring quote items, the UAP user can take advantage of two features of the UAP to streamline the quote configuration process, pre-population and propagation.",
"(1) Pre-population of Parameters Certain offered products can be set up to have values for their parameters pre-populated when they are purchased in a quote.",
"By default, when an offer item moves from a cart item to a quote item, a plug-in can then give pre-populated values to certain product parameters.",
"By default, the following parameters can be pre-populated: (a) attachment point; (b) service address (customers only); (c) service identifier; (d) start date; and (e) other product parameters.",
"The parameters to be pre-populated are determined by the PreConfigure*, java plug-ins.",
"By default, the pre-populated values for product parameters are taken from the default values specified when the offer was configured in the Administrator Console.",
"Other parameters are populated as follows: attachment point—The plug-in picks the first account it finds associated with the partner.",
"service address—For a customer, defaults to customer's address.",
"For a partner, the plug-in does nothing.",
"service identifier—The plug-in picks an identifier at from the pool.",
"In the default implementation this is a static list; in an actual deployment this identifier would likely come from some external system.",
"start date—The plug-in uses the default earliest available start date determined by the vendor product associated with this quote item's associated offer.",
"Once the UAP user accepts the pre-populated configuration information, the UAP user can at any time before ordering the quote go back and modify the configuration of individual quote items which were pre-configured, until the quote is submitted as an order.",
"(2) Propagation of Parameters If there is more than one of the same quote items to be ordered, the UAP allows configuration parameters to propagate to subsequent items in the quote.",
"Once the UAP user has configured a quote item, the UAP user can check the box marked “Propagate to all items (same product)”.",
"Then, when accepting the configuration for that item, the UAP user will see a confirmation page indicating which items in this quote (if any) have been selected by the plug-in to receive the propagated configuration information.",
"The UAP user can then accept or cancel the propagation.",
"If the UAP user accepts the propagation, the indicated quote items receive the configuration information from the “primary” quote item from which the UAP user elected to propagate.",
"Certain configuration parameters for a quote item can be propagated.",
"By default, they are: (a) attachment point; (b) service address; (c) service identifier: (d) start date; and (e) other product parameters.",
"The determination of which quote items are to be configured by propagation is also determined by the plug-in.",
"By default, the plug-in propagates configuration to the following class of quote items: (a) quote items that are of the same product type (determined by vendor product type) as the “root” quote item; and (b) of those quote items, only quote items in state “pending configuration” are eligible for propagation.",
"Once the UAP user propagates configuration information, the UAP user can at any time go back and modify the configuration of individual quote items which were propagated.",
"5.7.7.Disclosures Offer items in the quote can have disclosures associated with them which communicate legal or other information the customer must agree to before purchasing.",
"All disclosures for each item in the quote which has them are displayed and the UAP user must accept them before the quote can proceed.",
"5.7.8.Negotiated Quote Once all the configuration described has been completed for a quote, it reaches the “quoted” stage, and the display updates to reflect this by bringing the UAP user to the Negotiated Quote page.",
"Once a user elects to order a negotiated quote, A quote items become order items.",
"(1) Price Overrides UAP agents can issue price overrides to a quote item once the containing quote has reached the negotiated stage.",
"Every item in a negotiated quote has a link labeled “Modify” which brings up the price override page.",
"The UAP user must specify a reason for the override, a description of the particular reason (i.e.",
"reason is “promotion” and the description of the particular promotion is “Summer DSL Blowout.”) The UAP user can then specify a percentage discount for each item in the One-time Charges, Monthly Rates or Usage Rates categories.",
"The system calculates and displays the new final charge.",
"The UAP user can either reset or click “APPLY” and commit the override.",
"The total quote price is recalculated based on the overridden items, and the quote summary updates to reflect this new price.",
"(2) Prices of Held Quotes Once a quote reaches the negotiated stage, the price for that quote is fixed.",
"If the UAP user holds the quote, and prices for items in that quote and for that customer's price group should change, the quote will remain at the price at which it was held.",
"5.7.9.Shipping Address for Delivered Products Offers which represent equipment (cell phones, pagers) which are physically delivered to a customer or partner are called delivered products in the UAP.",
"When purchasing a quote containing one or more delivered products, the UAP user is required to give a single shipping address to which all delivered products in the quote will be shipped.",
"If there are no offers corresponding to delivered products in the quote, then no shipping address is required.",
"5.8.Orders Once products have been selected for ordering, configured in a quote and submitted by clicking the “ORDER NOW” link, an order is created.",
"5.8.1.Order History The UAP user can search on service orders from the user home page, or orders can be tracked from the Order History off of the customer/partner navigation bar.",
"The order history page displays: (a) Order number; (b) Date Created; (c) Partner-Customer ID; (e) Description; (f) Status; and (g) Last Modified By.",
"(1) Order Detail By clicking on the order number, the user can then bring up a detail of the order.",
"This page lists: (a) Order number; (b) Date Created; (c) Status; (d) Master PO number (if any); (e) Description; (f) Partner-Customer ID; and (g) each of the constituent order items Each order item in the list displays: Item number; Action (Add, Remove, etc.",
"); Description.",
"The description is a link which allows the UAP user to drill down and view all charges for the item); Status.",
"The status that is visible from the order item line-item in the order detail page is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that order item.",
"a link to view a detail for that item.",
"The detail page for an order item displays the product name, the purchase order number, and all the configuration information which was determined when that item was configured in a quote.",
"From the order detail page, the user can request cancellation of the order, or re-order the entire order.",
"The user can also view any notes attached to this order.",
"totals for one-time and recurring/monthly charges (2) Item Status and Interaction Models The status that is visible from the order item line-item in the order detail page is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that order item.",
"5.8.2.Canceling an Order Canceling an order is not guaranteed to completely cancel all products ordered.",
"Since the system relies on external systems to handle provisioning, it is possible that an ordered item can already be provisioned at the time the UAP user are requesting cancellation, but that the new state information has not been communicated to the system.",
"Therefore, canceling an order from the order detail page simply requests a cancellation and sends the appropriate CMI to the external system.",
"If the external system is successful, the order will be canceled and the state reflected in the order history.",
"5.8.3.Reordering When the UAP user select “REORDER” from the order detail page, the UAP user are brought to the quote summary page for a new quote, populated with any items which were in the original order with an action of “Add”.",
"This excludes from reordering any items which were in the original order as upgrades, or which were removed, changed suspended or resumed.",
"The items in the re-ordered quote are in a configured state, but the UAP user can then change the configuration of the quote items.",
"The UAP user then can re-submit them as a new order, leaving the original order unchanged.",
"5.8.4.Supplemental Orders From the Order Detail page, the UAP user can elect to supplement the order.",
"Supplemental orders are a way for UAP agents to add or change aspects of an order before it has been fully provisioned.",
"What items in an order are eligible for supplement is controlled by a plug-in, but by default only products who have not reached state “complete” can be supplemented.",
"The supplemental quote contains those order items which the plug-in determined were eligible for supplement.",
"All other items which were ineligible also appear in the supplemental order quote, but are un-modifiable and retain their state from the original order.",
"The UAP user can make whatever supplemental changes are required, such as replace items in the quote for supplemental order, reconfigure them or cancel them outright.",
"Once the UAP user make these supplemental changes, the UAP user submit the supplemental quote and creates a supplemental order.",
"(1) Charges for Supplemental Orders A plug-in calculates charges for a supplemental order.",
"By default, this charge is a flat-fee which is then assessed to the account and associated with the quote.",
"This behavior can be customized.",
"The supplemental charge is displayed on the Negotiated Quote page in the totals area.",
"The UAP user can also elect to waive this supplemental charge from the Negotiate Quote page by clicking the “Waive Charge” link next to the supplemental charge amount.",
"The charge is then removed and no longer displays.",
"(2) Escalations While submitting a supplemental order, the UAP user can flag individual items as “escalation” items, indicating that the item is in response to an issue raised by a customer.",
"5.9.Service Management 5.9.1.Products in the UAP Products such as access lines, voice mail, and call forwarding for which there is an ongoing subscriber relationship are referred to as assigned products in the system.",
"Assigned products can be modified, suspended, resumed, or removed.",
"Delivered products represent actual end-user equipment such as a cell phone or other customer premise equipment.",
"These delivered products maintain an association to an account within the system, but are not eligible for the same actions as provisioned products; they cannot be modified, upgraded, suspended or resumed, or removed.",
"5.9.2.Viewing Services From the Customer Summary page, the UAP user can view products and services provisioned to particular account by clicking the “view services” link next to that account.",
"The UAP user is then shown a hierarchical view of all products provisioned to that account.",
"Arrows next to products indicate that the UAP user can click to expand or collapse that level of the product hierarchy.",
"Clicking on the name of a product brings the UAP user to the detail page for that product.",
"The UAP user can also bring up the products page by clicking the “products” link from the customer/partner navigation bar.",
"(1) Product Detail Page For assigned products, the detail page shows the name of the product, a brief description, the date the product was provisioned, and a summary of the configuration information.",
"Any contacts assigned to this product are also visible, as is a link to add new contact information.",
"To manage the product hierarchy, the UAP user can modify, remove, suspend/resume, upgrade, report trouble, and contacts for the assigned products.",
"For delivered products, the UAP user may only report trouble and add/remove contacts.",
"5.9.3.Modifying Products The UAP user modifies an assigned product by clicking “Modify Product” from the assigned product detail page.",
"Since an external provisioning system must be used to make the desired modifications to a provisioned product or service, the UAP implements product modification as an order.",
"Modifying an assigned product takes the UAP user to the quote summary page for a new quote, consisting only of the product to be modified.",
"From this new quote detail, the UAP user can then configure the active parameters on the offer including a “start” date when the customer wishes the modification to take effect as well as any other product parameters, service address, service identifier, etc.",
"The UAP user can then submit the quote.",
"5.9.4.Removing Products The UAP user can request an assigned product to be discontinued by clicking “Remove Product” from the assigned product detail page.",
"Removing a product brings the UAP user to the quote summary page of a new quote, consisting only of the product to be removed.",
"The only configuration possible for this removal quote is the termination date.",
"Analogous to the service start date the UAP user selected when ordering the product, the UAP user can choose either the earliest possible date, or a specific date, as long as the date the UAP user choose is not earlier than the earliest possible date.",
"5.9.5.Suspend/Resume Product From the product detail page, the UAP user can suspend products that are provisioned and active.",
"When the UAP user selects a product to be suspended, the system will check to see if the product is an Offer Composite.",
"If it is, the entire offer collection and any of its constituents' child products are suspended, even if they reside on other accounts associated with the active customer.",
"Otherwise, the selected product and all of its children are suspended.",
"When the UAP user wish to resume a product, the UAP user first locate it in the product hierarchy and select “RESUME”.",
"The system traverses that product's children and resumes them as well.",
"Finally, if the UAP user is resuming a previously suspended composite offer, the entire collection and any of its constituents' children are resumed.",
"Since an external provisioning system must be used to make the desired suspensions/resumptions to a provisioned product or service, the UAP implements product suspend/resume as an order.",
"Suspending or resuming an assigned product takes the UAP user to the quote summary page for a new quote, consisting only of the products to be suspended or resumed.",
"5.9.6.Upgrading Products Product upgrades are initiated from the detail page for the provisioned product to be upgraded.",
"Which product offers are on a given product's upgrade path is configurable through the Administrator Console.",
"Once an upgrade product is chosen, that new product is quoted and ordered like a normal product offer.",
"Once the upgrade order is complete, the new offer instance replaces the original.",
"5.9.7.Contacts The UAP user may also view or remove existing contacts for this product, and add new contacts.",
"5.9.8.Reporting Trouble The UAP user may also raise trouble tickets against specific products.",
"5.10.Support Personnel Support Personnel are individuals within the UAP user's organization with assigned areas of responsibility and whom the UAP user can then associate with customers.",
"By default, the UAP has three categories for support: (a) business; (b) primary; and (c) technical.",
"These categories reside in a constant class and can be modified.",
"5.10.1.Assigning Support The UAP user can assign support personnel to the active customer by clicking “support” on the customer navigation bar.",
"The assign support page presents the UAP user with a list of all available support personnel.",
"Clicking on “ASSIGN” associates the selected support person to the account, and adds them to the list of assigned supports displayed at the bottom of the page.",
"The UAP user can also un-assign support personnel from this page.",
"Note that the UAP user can assign multiple support resources from a single category; there is no limit to the number of business, technical or primary supports for a customer.",
"5.10.2.Managing Support Personnel Administrative users can create new support personnel and can modify or remove existing ones.",
"If the UAP user is an administrative user, the “manage support” link appears on the user navigation bar and brings up the manage support page.",
"Here, the UAP user is presented with a list of existing support personnel and can view or modify a support person by clicking the corresponding link.",
"Creating a new support person requires the UAP user to select a category of support from the drop-down menu (these categories can be customized via a constant class).",
"Then, the UAP user must enter name and contact information for this support person, and can enter any comments.",
"From the manage support page, the UAP user can remove an existing support person outright by selecting them from the list and clicking “REMOVE.” 5.12.Contacts Contacts are individuals in the customer's or partners organization who are assigned responsibility for various aspects of the relationship with the provider.",
"The UAP user may assign contacts to (a) the customer/partner; (b) an account; and (c) a product instance.",
"The UAP user also must provide at least one contact for the customer/partner during registration.",
"When adding a contact, the UAP user must provide a name, address and telephone number.",
"Contacts are of a particular type such as “primary” or “technical.” The UAP user can assign as many contacts as needed.",
"There can also be multiple contacts of a given type.",
"What contact types are defined is determined by a constant class which may be changed.",
"5.11.1.Managing Contacts Customer contacts are displayed on the customer/partner, account, and product detail pages.",
"From there, the UAP user can add new contacts, modify an existing contact, or remove an existing contact.",
"5.12.Reporting Trouble 5.12.1.Issuing Trouble Tickets By clicking the “Report Trouble” link on the detail pages, the UAP user can issue trouble tickets against customer, account, and provisioned product The trouble ticket is date-stamped and marked with the ID of the UAP agent submitting it.",
"The UAP user must supply contact information for the customer or partner claimant making the trouble report and specify a category or reason for the trouble report.",
"The UAP user should also provide a detailed text description of the problem, and any recommended action.",
"If the UAP user specifies an e-mail address and indicate that this is the preferred contact method, a notification is automatically sent to that e-mail address as the ticket changes state.",
"Once the UAP user has provided all the information required for the ticket and submitted it, a summary page is displayed.",
"5.12.2.Tracking Trouble Tickets Past trouble tickets against a customer are viewed either by executing a search from the user home page or via the history list from the customer/partner navigation bar.",
"Clicking the trouble ticket number in the history list opens the detail page for that ticket showing a summary of the ticket.",
"The status link brings the UAP user to the interaction status for the ticket, which displays the state of the ticket as reported from the external ticketing system through an end-user view of an interaction model.",
"As issues are worked, the UAP user can open trouble tickets and click the modify link to add updated information.",
"The systems always records and displays the last-modified date and the user ID of the UAP agent who last modified the trouble ticket.",
"5.12.3.Resolving a Trouble Ticket When an issue has been resolved, the UAP user can open the associated trouble ticket to modify it and mark it resolved.",
"The UAP user must put in a description of how the issue was resolved when resolving a ticket.",
"If an external system is in use for tracking trouble tickets, the system sends a CMI to notify the external system of the ticket's resolution.",
"5.13.Adjustments and Disputes 5.13.1.Adjustments The UAP user can view the adjustment history for a customer by selecting the “adjustments” link from the customer navigation bar.",
"A drop-down list with all accounts associated with that customer allows the agent to view adjustment histories for each account.",
"From the adjustment history, the UAP user can issue a new adjustment to the customer.",
"The UAP user selects an account, an invoice against which to issue the adjustment (optional), whether the adjustment is a credit (reduces the amount a customer owes) or debit, (increases the amount the customer owes), an adjustment reason (allowable adjustment reasons are defined in a constant class and are configurable), and a detailed description of the problem or issue that led to the adjustment.",
"The UAP user may also issue an adjustment when resolving a dispute.",
"5.13.2.Disputes When viewing the invoice summary page, or a detail page for a specific invoice, the UAP user can open a dispute on behalf of a customer or partner.",
"The UAP user must specify the partner or customer ID, a name, contact information for the claimant, select a category for the dispute (these categories reside in a constant class and are configurable), and write a detailed text description of the problem.",
"Once submitted, the UAP user can view the dispute, along with any other for that customer at any time by clicking the ‘disputes’ link in the customer navigation bar.",
"The dispute history gives the dispute ID, the date created, a category and brief description, and the state of the dispute.",
"The UAP user can only create disputes from an invoice summary page, not from the dispute history page.",
"If the UAP user needs to add or change information in a dispute record, the UAP user can do so from the dispute history by opening that dispute and modifying the description.",
"When the issue which occasioned the dispute has been resolved, the UAP user can open and modify the dispute record, add a resolution description, and then either save the record, or save and issue an adjustment to the account.",
"If the UAP user chooses to save and issue adjustment the UAP user is then brought to the adjustment page and can issue the adjustment.",
"5.14.Notes The UAP users can create notes to record activity taken on various entities in the system.",
"Notes capture the date and time they were created, the UAP agent who created them, the customer with whom they are associated, and the entity the note is attached to.",
"Notes can be attached to: (a) Customers or partners, from their respective detail pages; (b) Orders, from the Order Detail page; and (c) Quotes, from the Quote Detail page.",
"Also, a note history can be viewed for each of these entities from their detail pages.",
"5.15.Reporting The UAP user can view five different reports from the UAP.",
"Two are visible from the user home page: (a) Financial Forecast; and (b) Financial Bookings.",
"These two reports summarize revenue forecast or booked, respectively broken out by product line and charge type (recurring vs. non-recurring) for the current month.",
"Three reports are specific to partners and are visible from the partner home page for a particular partner in session.",
"They are: (a) Financial Forecast; (b) Financial Bookings; and (c) Orders Past Due.",
"The first two reports are analogous to the two reports on the user home page, except that they cover revenue for the active partner alone.",
"The last, Orders Past Due summarizes any outstanding orders which are not complete and are past their requested due date.",
"The actual data in these reports comes from the database, but the reports themselves are provided by an external system; the portal merely supplies links to those reports.",
"5.16.Manual Approval The Universal Agent Portal provides pages that allow the UAP user to manually set the status of order items, account requests, and customer requests.",
"This is useful in testing or in deployments which do not use external systems for provisioning or trouble ticketing.",
"By default, the UAP allows a licensee to set the “pending,” “rejected,” and “complete” states, plus the “canceled” state for an order item.",
"Click the “approval menu” link off of the user navigation bar to bring up the approval menu.",
"The UAP user will then see the approval menu which contains: (a) Approve Customer Requests; (b) Approve Partner Requests; (c) Approve Account Requests; and (d) Approve Order Item Requests.",
"Each of these menus presents a list of items (appropriate to the menu chosen), their item number, the date the item was created, the current status, and radio buttons allowing the UAP user to alter the state of the item.",
"Once the UAP user has made the desired modifications, clicking “UPDATE” will commit those changes and change the state of the items within system.",
"Section 6.The Channel Partner Portal One embodiment of the invention is the Channel Partner Portal (CPP), which allows users at communications service provider sites to manage their accounts with a provider.",
"CPP users can use the portal to shop for products and services (typically on behalf of their own end-customers), manage these services, view reports, and report and track trouble tickets.",
"6.1.CPP Logging In The CPP user logs in using a user name and password set up for their organization by a communications service provider (CSP).",
"The CSP can set up multiple users for a partner, with differing levels of security or access to data.",
"By default, two security levels are supported: administrator and user.",
"The CSP creates the first user (typically an administrator) for a partner via the Universal Agent Portal.",
"Once the CSP has created at least one administrator user for a partner, that user may then in turn create other users for the partner.",
"A CPP user's security level determines what transition policies they may execute and what JSP's they may view.",
"The two security levels, administrator and user have the following permissions and restrictions for navigating the CPP: (1) CP_partner_user.",
"User may not add, remove, or modify accounts; may not access bulk pre-qualification; may not add, remove or modify support resources; may not add, remove or modify CPP users; may not reorder products or existing orders.",
"(2) Partner_admin.",
"Administrator may access the entire portal except the manual approval screens.",
"6.2.CPP Home After a CPP user logs in to the portal, the partner home page comes to screen.",
"This page contains the following areas: (1) Navigation.",
"The main navigation bar is visible throughout the CPP and allows the user to navigate to the following areas: Home: brings the user to the partner home page.",
"Search: brings the user to the search page.",
"Shop: brings the user to the main catalog menu where they can shop for products and services.",
"Support: brings the user to the support page where they can view support resources assigned to them.",
"Help: This link can be customized to point to whatever online help system the CSP may design and deploy with the CPP.",
"(2) Partner Name and Logo.",
"The partner's name and logo are displayed at the top of the partner home page, as well as the name of the user currently logged in.",
"(3) News and information.",
"This area displays any news (such as reports of outages, new service offerings, etc.)",
"or other promotional messages targeted at the CPP user.",
"The contents of this area are configurable via the Administrator Console.",
"(3) Search.",
"The search area of the partner home page, as well as the “SEARCH” link on the navigation bar, brings the CPP user to the search page from where they can execute three different types of searches.",
"(4) Information Management.",
"The information management area contains links to three types of partner-specific reports.",
"It also provides a link where CPP users can create bulk pre-qualification requests.",
"CPP users can also manage accounts and view their partner detail page.",
"(5) Activity History.",
"The activity history area allows CPP users to view past activity in three areas: quotes, orders and trouble tickets.",
"(6) Administering Users.",
"Functions in this area allow CPP users to change their password, and (if they have sufficient authority) to create other partner users.",
"6.3.Searching From the search area on the partner home page, the user can execute searches for three types of objects: orders, trouble tickets, and products.",
"Clicking any one of these links brings the user to the search page for that type of object.",
"6.3.1.Search Types On the search page, a drop-down menu controls which type of object the user is searching for.",
"Changing the selection in this drop-down reloads the search page with input fields for search criteria appropriate to that object.",
"The user can execute searches for the following types of items: Service Order.",
"Search on and sort by: (a) Order Number; (b) Purchase Order Number; (c) Order Status; (d) Partner-Customer ID; (e) Escalation Flag; and (f) Date Ordered.",
"Trouble Ticket.",
"Search on and sort by: (a) Ticket Number; (b) Ticket Status; (d) Partner-Customer ID; and (e) Date Created.",
"Product Instance.",
"Search on and sort by: (a) Product Name; and (b) Service Identifier (e.g., telephone number).",
"Partner Customer ID.",
"Search on only: (a) Service Street Number; (b) Service Street Name; (c) Service City; (d) Service State/Province, and (e) ZIP/Postal Code.",
"In addition, the user can specify whether any sort should be ascending or descending.",
"6.3.2.Search Results Once the user specifies values for the search criteria, executing the search returns a set of matching items (if any).",
"By clicking on a column header, the user can cause the result set to be sorted on that columns parameter.",
"Clicking again toggles the sort from ascending to descending.",
"Clicking on an item in a result set brings the user to the appropriate detail page for that item (product detail page for a provisioned product, trouble ticket detail for a trouble ticket, etc.)",
"6.4.Shopping A partner can use the CPP to shop, select, configure and order products.",
"Ordering products in the CPP consists of the stages of: (a) shopping; (b) creating and configuring items in a quote; and (c) Ordering the items in a quote.",
"6.4.1.Menus Menus are navigable lists of products offered for ordering.",
"In the CPP, the user shops product menus, and selects offered products for purchasing.",
"Menus typically break product offerings down into categories such as “Business Services” and “Residential Services.” (1) Default Menu and Price Groups When the user is browsing the product menu they will see a set of product offerings determined by their assigned price group.",
"The price group determines the root menu and prices for each offer.",
"(2) Alphabetical Product Index In addition to the hierarchical menus, the user can also locate products by name.",
"Selecting a letter from the alphabetical index will bring the user to a menu of products beginning with that letter.",
"(3) Breadcrumbs While browsing the hierarchical menus (but not while viewing an alphabetical list), the CPP shows a “breadcrumb” displaying the path thus far taken through the menu hierarchy.",
"The user can click back at any link in the chain to return to the menu at that level.",
"6.4.2.Offers The offers in menus are listed with a brief description, the total price for recurring charges and for non-recurring charges, a field displaying both the current quantity in the quote and a field for entering a different quantity.",
"(1) Offer Detail When the user shops and finds an offer they wish to purchase, they can choose to drill in to the offer detail page.",
"The detail page displays the name of the offer, itemized prices for recurring, non-recurring and usage charges, and any upsell or related offers.",
"The user can then specify a quantity of the product to be ordered, just as in the order's line-item in the menu page.",
"Whenever the user changes the quantity of an item to be purchased, either from the menu page or the offer detail page, they must click “UPDATE” to save the new quantity before moving to a new page.",
"This commits the items the user has selected to their shopping cart, which can be viewed at any time by selecting “Quote Preview”.",
"The user can then continue shopping, perhaps in a different category of product offerings.",
"(2) Related Offers and Upsell Offers When viewing an individual offer via the detail page, the user is shown related offers or upsell offers, if any exist.",
"Related offers are offers which have some connection or marketing synergy with the offer being viewed.",
"Both related and upsell offers are displayed on the detail page in a one-line format similar to the offers on a menu page.",
"These related offers can themselves be drilled into from the first offer's detail page.",
"For example, when the user clicks on the Business Access Line product offer, they are taken to the offer detail page for Business Access Line.",
"There, in addition to the Business Access Line product, the user sees: Business Voice Mail w/Pager Notification, Essential Bundle, and Business Messaging Bundle.",
"The user can enter a quantity for purchase for any of these upsell offers and click “UPDATE” to add those offers to their cart.",
"Which offers are related to one another and which are on an upsell path is configurable through the Administrator Console.",
"(3) Offer Collections Since simple, one-product offers may not be sufficient to meet a providers' product offering requirements, the system allows for individual offers to be bundled together into offer collections.",
"Offer collections (and also simple offers) are configured in the Administrator Console.",
"Instead of selecting a quantity to purchase, as with simple offers, to select an offer collection for purchase the user must click the “add Bundle, link for that offer and then select which options for that collection they wish to purchase.",
"If the offer collection is fixed, then the user will automatically receive all of the offers in the collection; no choices can be made.",
"If the collection is dynamic, the user is guided through the collection's determinants where the user selects the individual offers that will make up the collection.",
"At any point in this process, the user can go back to a previous determinant to select a different offer.",
"The navigation path through the collection, as well as the determinants used, and the offers included are all configured through the Administrator Console.",
"6.4.4.Quote Preview At any point in the shopping process, the user can view a quote preview by selecting that link from the top of any menu page.",
"This is a view of all products that have been selected for ordering, their total recurring and total non-recurring charges, the quantity currently selected for quote and a text field to input an updated quantity.",
"The user may also remove items from a quote.",
"The quote preview is the CPP implementation of the shopping cart.",
"As the user selects offers for purchase, they are placed in the quote preview.",
"6.5.Quotes 6.5.1.Creating Quote When the user has shopped and selected all the products desired, clicking “CHECKOUT” creates a quote.",
"All of the items that were in their quote preview when they selected checkout now appear as quote items.",
"6.5.2.Quote Summary The quote summary page shows all the items in the users current quote.",
"The top of the summary page shows the quote number, creation date, status, the name of the user who created the quote, and the last-modified user.",
"The user can also enter a master purchase order number, a brief description, select an existing partner-customer ID (or enter a new one), and enter an e-mail address.",
"Partner-Customer ID is a convenience field where the user can store an internal ID for their end-customers.",
"Items in a quote display the item number (ordinal number of item in quote) product offer name, the action for this item (i.e., add, remove, change, etc.",
"), the purchase order number (if one was provided), the status (configured, pending configuration, etc.",
"), the price for recurring and non-recurring charges, and a checkbox with which the user can remove items from the quote.",
"If the user removes any items from a quote, they must click “UPDATE” to commit those changes.",
"If there are more items than will fit on one page, a pair of previous/next links allows users to navigate between pages.",
"(1) Working With a Quote The user can hold or cancel the quote, add notes, shop more, or continue.",
"Holding a quote allows a quote to be held in its current state and then re-opened at a later time.",
"Held quotes are displayed in the current quote history, available from the activity history area on the partner home page.",
"Canceling a quote causes the quote to become inactive, but it will continue to be available as a “Canceled quote” in the quote history.",
"If the user chooses to “shop more” from the quote summary, they are returned to the product catalog.",
"Any products they purchase now are added to the current quote.",
"Adding a note to a quote allows the user to attach an annotation to the quote that will then be visible to anyone else working with that quote in the CPP.",
"Any notes created in the CPP can only be viewed in the CPP.",
"Users of other e-Business portals cannot view or modify them.",
"6.5.3.Holding a Quote Holding a non-negotiated quote causes the quote to persist for a period of time.",
"The user can re-open a non-expired held quote at any time and continue working on it by shopping more, configuring items, ordering it or canceling it.",
"6.5.4.Canceling a Quote Canceling a quote stops the ordering process and moves the current quote to state “Canceled.” Canceled quotes can always be viewed from the quote history available from the main navigation bar.",
"6.5.5.Configuring Quote Items Before quote items can be purchased and moved to order items, they must be configured.",
"Configuration is reached by selecting the “Configure” link which is next to every quote item requiring configuration.",
"If the user attempts to click “CONTINUE” without configuring all quote items, the CPP will prompt for configuration information for each item in the quote.",
"The following attributes are configured in a quote: (1) Service Start Date.",
"Users can choose to accept the earliest possible start date for a quote item, or select another date (as long as the date they select is not earlier than the earliest possible date.",
"The earliest possible start date is an attribute of the offer's associated vendor product configured via the Administrator Console when product offerings are created.",
"A constraint on service start dates arises when a quote item is attached to an existing quote item.",
"Users cannot select a start date for a dependent product that is earlier than the start date selected for its parent product.",
"(2) Service Address.",
"Users must input information for the service address.",
"This associates the product to a physical location, typically a customer address.",
"(3) Selecting Service Identifier.",
"Users must select a service identifier (a telephone number for access line, for example).",
"The system uses a plug-in to interface with an external system to retrieve a set of service identifiers from which to choose.",
"(4) Product Association.",
"The user must also attach each quote item either to a place in the partner's hierarchy or to another item in the quote.",
"The determination of which hierarchy objects or quote items are valid attachment points is handled by the business rules manager.",
"(5) Product Parameters.",
"Finally, the user must provide values for product parameters (such as number of rings before voice mail picks up, guaranteed level of service, etc.).",
"What parameters are required is an attribute of the offer's associated vendor product.",
"These parameters are defined in the Administrator Console.",
"6.5.6.Automating Quote Item Configuration In addition to manually configuring quote items, users can take advantage of two features of the CPP to streamline the quote configuration process, pre-population and propagation.",
"(1) Pre-population of Parameters Certain offered products can be set up to have values for their parameters pre-populated when they are added to a quote.",
"By default, when an offer item moves from a cart item to a quote item, a plug-in can then give pre-populated values to certain product parameters.",
"By default, the following parameters can be pre-populated: (a) attachment point; (b) service identifier; (c) start date; and (d) Other product parameters.",
"The parameters to be pre-populated are determined by the PreConfigure*.Java plug-ins.",
"By default, the pre-populated values for product parameters are taken from the default values specified when the offer was configured in the Administrator Console.",
"Other parameters are populated as follows: attachment point.",
"The plug-in picks the first account found under the partner.",
"service identifier.",
"The plug-in picks an identifier at random from the pool.",
"start date.",
"The plug-in uses the default earliest-available start date determined by the vendor product associated with this quote item's associated offer.",
"Once the user accepts the pre-populated configuration information, they can at any time before ordering the quote go back and modify the configuration of individual quote items which were pre-configured.",
"(2) Propagation of Parameters If there is more than one of the same quote item to be ordered, the CPP allows configuration parameters to propagate to subsequent items in the quote.",
"Once the user has configured a quote item, they can check the box marked “Propagate to all items (same product)”.",
"Then, when accepting the configuration for that item, they will see a confirmation page indicating which items in this quote (if any) have been selected by the plug-in to receive the propagated configuration information.",
"They can then accept or cancel the propagation.",
"If they accept, the indicated quote items receive the configuration information from the “primary” quote item from which they elected to propagate.",
"Certain configuration parameters for a quote item can be propagated.",
"By default, they are: (a) attachment point; (b) service address; (c) service identifier; (d) start date; and (e) other product parameters.",
"The determination of which quote items are to be configured by propagation is also determined by the plug-in.",
"By default, the plug-in propagates configuration to the following class of quote items: (a) quote items that are of the same product type (determined by vendor product type) as the ‘root’ quote item; and (b) of those quote items, only quote items in state “pending configuration” are eligible for propagation Once the user propagates configuration information, they can at any time before purchasing the quote go back and modify the configuration of individual quote items which were propagated, until the quote is submitted as an order.",
"After configuration is complete, the user is returned to the quote detail page.",
"Clicking “Continue” from here, after having configured the quote, will bring up the disclosures page.",
"6.5.7.Disclosures Offer items in the quote can have disclosures associated with them which communicate legal or other information the customer must agree to before purchasing.",
"All disclosures for each item in the quote which has them are displayed and the user must accept them before the quote can proceed.",
"6.5.8.Negotiated Quote Once all the configuration described has been completed for a quote, and disclosures have been accepted, it reaches the “quoted” stage, and the display updates to reflect this by bringing the user to the Negotiated Quote page.",
"Once a user elects to order a negotiated quote, all quote items become order items.",
"(1) Prices of Held Quotes Once a quote reaches the negotiated stage, the price for that quote is fixed.",
"If the user holds the quote, and prices for items in that quote and for that customer's price group should change, the quote will remain at the price at which it was held.",
"A held negotiated quote will expire after a predetermined period of time has elapsed, measured from when the quote was first created.",
"This time limit is specified in the properties file by the parameter quote.defaultquote-validtimeduration.",
"This parameter has a value of 30 days, by default.",
"After a quote expires, it is no longer available for ordering, but will still appear in and be viewable via the quote history as an “expired quote.” (2) Modifying a Quote Selecting “Modify Quote” brings the user back to the quote summary page.",
"All of the user's previous configuration information is presented, and they can make changes.",
"The new quote will be repriced.",
"They can proceed with ordering the modified quote.",
"6.5.9.Shipping Address for Delivered Products Offers which represent equipment (cell phones, pagers) which are physically delivered to a partner are called delivered products in the CPP.",
"When purchasing a quote containing one or more delivered products, users are required to give a single shipping address to which all delivered products in the quote will be shipped.",
"If there are no offers corresponding to delivered products in the quote, then no shipping address is required.",
"6.6.Managing Support Support Personnel are individuals within the CSP's organization who are assigned areas of responsibility and whom the CSP can then associate with partners via the Universal Agent Portal.",
"CPP users can then view support information to determine the correct contact for a given situation.",
"Users of the CPP can view the support resources assigned to them via the “SUPPORT” link on the main navigation bar.",
"This displays the support list, from which users can select a support resource and view their detailed information.",
"By default, the CPP has three categories for support: (a) business; (b) primary; (c) technical.",
"6.7.Viewing Activity History Users can view history information from the links in the activity history section of the partner home page.",
"The user can choose from orders, quotes or trouble tickets.",
"Choosing one of these categories brings up the history page for that item.",
"Depending on the type of item whose history the user is viewing, they may take different actions.",
"6.7.1.Order History CPP users can search on service orders from the home page.",
"The following information is returned for each item on the order history page: (a) Order number; (b) Date Created; (c) Partner-Customer ID; (d) Description; (e) Status; and (e) Last Modified By.",
"(1) Order Detail By clicking on the order number, the user can then bring up a detail of the order.",
"This page lists: (a) Order number; (b) Date Created; (c) Status; (d) Master PO number (if any); (e) Description; (e Partner-Customer ID; and (g) each of the constituent order items.",
"Each order item in the list displays: Item number; Action (Add, Remove, etc.",
"); Description.",
"The description is a link which allows the CSP to drill down and view all charges for the item; Status.",
"The status that is visible from the order item line-item in the order detail page is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that order item; a link to view a detail for that item.",
"The detail page for an order item displays the following data: (a) product name; and (b) purchase order all the configuration information which was determined when that item was configured in a quote.",
"From the order detail page, the user can request cancellation of the order, or re-order the entire order.",
"The user can also view any notes attached to this order; totals for one-time and recurring/monthly charges.",
"No actions, apart from returning to the order detail page, are available from the order item detail page.",
"(2) Canceling an Order Canceling an order is not guaranteed to completely cancel all products ordered.",
"Since the system relies on external systems to handle provisioning, it is possible that an ordered item can already be provisioned at the time the user is requesting cancellation, but that the new state information has not been communicated to the system.",
"Therefore, canceling an order from the order detail page simply requests a cancellation and sends the appropriate CMI to the external system.",
"If the external system is successful, the order will be canceled and the state reflected in the order history.",
"(3) Reordering When the user selects “REORDER” from the order detail page, they are brought to the quote summary page for a new quote, populated with any items which were in the original order with an action of “Add”.",
"This excludes from reordering any items which were in the original order as upgrades, or which were removed, changed suspended or resumed.",
"The items in the re-ordered quote are in a pre-configured state, but the user can then change the configuration of the quote items.",
"They then can resubmit them as a new order, leaving the original order unchanged.",
"(4) Notes Users may attach notes to an order.",
"6.7.2.Quote History Users can view quotes from the quote history available from the partner home page in the activity history.",
"The quote history page displays the following information for each current quote: (a) Quote number; (b) Date Created; (c) Partner-Customer ID; (d) Description; (e) Status; and (e Last Modified By.",
"A user can also view ordered, expired and canceled quotes.",
"The user can switch between these three categories via links on the main quote history page.",
"(1) Quote Detail The user can view a detail page for a quote by clicking the quote in the history list.",
"The detail page summarizes the data displayed in the history entry, and also shows a list of every item in that quote.",
"For each item, the quote detail displays: (a) Item number; (b) Product Name (Clicking this link shows associated charges for the product); (e) The action (add, remove, etc.)",
"for that item which caused it to appear in the quote; (f The PO number (if any); (g) Item Status; (h) One Time Charge; and (i) Recurring Charge.",
"(2) Quote Item Detail Each quote item also has a detail link which shows the user a detail page for that quote item, including the configuration parameters which were chosen when the quote item was purchased.",
"6.7.3.Trouble Ticket History Past trouble tickets are viewed either by executing a search, or by viewing the trouble ticket history, both from the partner home page.",
"The following information is returned for each item on the trouble ticket history page: (a) Ticket number (Clicking the trouble ticket number in the history list or search result set opens the detail page for that ticket showing a summary of the ticket); (b) Date Created; (c) Category; (d) Name of trouble ticket (a headline); and (e) Ticket Status.",
"The user can view a detail for a trouble ticket by clicking on it from the history list.",
"The ticket detail page lists: (a) Trouble Ticket Name; (b) Trouble Ticket Number; (c) Partner-Customer ID; (d) Status (The ticket status displayed is obtained from an interaction model; it is a view into the external provisioning process.",
"Clicking on this status will bring up the interaction history for that trouble ticket); (e) Created By; (f) Created Date; (g) Last Modified By; (h) Last Modified Date; (i) Contact to Notify; (j) Phone Number; (k) Email Address; (I) The entity (partner, account, product) for which the ticket was raised; (m) The category; (n) The ticket description text; and (o) The resolution text (if any).",
"6.8.Information Management 6.8.1.Reports The CPP allows users to view three different reports from the partner home page.",
"The default reports are: (a) Financial Forecast; (b) Financial Bookings; and (c) Orders Past Due.",
"The first two reports summarize revenue forecast or revenue booked, respectively broken out by product line, charge type (recurring vs. non-recurring), and by month.",
"The last report is a summary of all open orders which are past their current due date.",
"The actual data in these reports comes from the database, but the reports themselves are provided by an external system; the portal merely supplies links to those reports.",
"6.8.2.Bulk Pre-Qualification Some services may require pre-qualification before they can be provisioned.",
"The CPP allows partners to create and submit requests for bulk pre-qualification of a group of customers to an external system.",
"From the partner home page, CPP users can select “bulk pre-qualification.” The bulk pre-qualification screen allows users to browse for and select a file containing information on customers to be qualified.",
"They must give a name to the request, and an email address that will be notified when the request has been processed.",
"They then submit the request.",
"The file is then sent to a system which can handle the request.",
"6.8.3.Account Management Accounts allow the CSP's partners to segment their business into logical units such as geographic location or department.",
"The partner accounts page provides the CPP user with a list of all of their accounts.",
"The list gives the name of the account, its status, and a link to view products for that account.",
"Clicking on the account name brings the user to the account detail page, where they can view all information pertaining to the selected account such as billing address, credit information, and the names of any contact personnel assigned to the account.",
"A drop-down menu at the top allows the user quickly to switch to other accounts, which will then display their information.",
"From the account detail page, the user can view the account's history, modify any of the account's information, and report trouble for this account, which will raise a trouble ticket, as, well as add, modify or remove contacts.",
"Users of the CPP can create accounts from the partner accounts page by clicking the “Add Account” link.",
"The user must provide an account name, billing address and credit information for every account they create.",
"Once created, the account will be displayed in the list of accounts on the partner accounts page.",
"6.8.4.Managing Products From the account management page the user can view products and services provisioned to a particular account by clicking the “view the products” link next to that account.",
"They are then shown a hierarchical view of all products provisioned to that account.",
"Arrows next to products allow the user to click to expand or collapse that level of the product hierarchy.",
"Clicking on the name of a product brings the user to the detail page for that product.",
"(1) Product Detail Page For assigned products, the detail page shows the name of the product, a brief description, the date the product was provisioned, and a summary of the configuration information.",
"Any contacts assigned to this product are also visible, as is a link to add new contact information.",
"To manage the product hierarchy, the user can modify, upgrade, remove, report trouble, and reorder the assigned products.",
"For delivered products, users may only report trouble and add/remove contacts.",
"(2) Modifying Products The user modifies an assigned product by clicking “Modify Product” from the assigned product detail page.",
"Modifying an assigned product takes the user to the quote summary page for a new quote, consisting only of the product to be modified.",
"From this new quote detail, users can then configure any parameters which were available when the product was first ordered, including a “start” date when the modification will take effect as well as any other product parameters, service address, service identifier, etc.",
"The user can then submit the quote.",
"(3) Removing Products The user can request an assigned product to be removed from the hierarchy by clicking “Remove Product” from the assigned product detail page.",
"Removing a product brings up the quote summary page of a new quote, consisting only of the product to be removed.",
"The only configuration possible for this removal quote is the termination date.",
"Analogous to the service start date selected when ordering the product, the user can choose either the earliest possible date, or a specific date, as long as the date chosen is not earlier than the earliest possible date.",
"(4) Upgrading Products Product upgrades are initiated from the detail page for the provisioned product to be upgraded.",
"Which product offers are on a given product's upgrade path is configurable through the Administrator Console.",
"Once an upgrade product is chosen, that new product is quoted and ordered like a normal product offer.",
"(4) Reporting Trouble The user can raise a trouble ticket on a product.",
"(5) Reordering When the user selects “REORDER” from the product detail page, they are brought to the quote summary page for a new quote, populated with a quote item corresponding to the order items in the order which had an action of ‘Add’.",
"The item in the re-ordered quote is in a configured state, but the user can then change the configuration of the quote items.",
"They then can re-submit them as a new order.",
"6.8.5.Partner Detail The partner detail page presents the CPP user with a summary of their partner information.",
"The page displays partner information, resale authority, address, and contacts.",
"The user can also view their partner history, modify their address information, raise a trouble ticket, and add, modify, or remove contacts.",
"Partner history consists of any changes made to partner information such as mailing address.",
"(1) Modifying Partner Information The CPP user can click the “Modify Partner Address” link on the partner detail page to provide new or updated address information.",
"Once they click modify, the new information is displayed on the partner detail page.",
"(2) Reporting Trouble The user can raise a trouble ticket on an account.",
"(3) Adding, Modifying and Removing Contacts The process for adding contacts to a partner is identical to the process for adding contacts to an account.",
"6.9.User Administration 6.9.1.Changing Passwords CPP users may change their passwords from the user administration area of the partner home page.",
"They must provide their current password, type their new password twice, and give a password hint which can be shown to a user if they forget their password.",
"6.9.2.Managing Users (1) Adding New Users.",
"CPP users with sufficient authority can create other users for their partner account from the manage users area of the partner home page.",
"The user must provide a user name, password, and password hint.",
"They must also select the authority level.",
"The two authority levels and their default permissions are: (a) User: read-only user who may not create orders of perform maintenance activities; and (b) Administrator: user who has full access to all of the features.",
"(2) Removing Existing Users.",
"From the Manage Users page, a CPP user of sufficient authority can remove existing users by selecting them from the drop-down menu and clicking “update.” The selected user is then made inactive and will be unable to log in to the CPP.",
"6.10.Issuing Trouble Tickets Users of the CPP can issue trouble tickets against the following items by clicking the “Report Trouble” link on the following detail pages: (a) partner, (b) account; (c) provisioned product; (d) composite product; and (e) delivered product The trouble ticket is date-stamped and marked with the ID of the CPP user submitting it.",
"The user must supply contact information for the partner making the trouble report and specify a category or reason for the report.",
"The user must also include a detailed text description of the problem, and any recommended action.",
"If the user specifies an e-mail address and indicates that this is the preferred contact method a notification is automatically sent to that e-mail address as the ticket changes state.",
"Once the user has provided all the information required for the ticket and submitted it, summary page is displayed.",
"6.11.Contacts Contacts are individuals in the partner's organization who are assigned responsibility for various aspects of the partner's relationship with the provider.",
"Users of the CPP may assign contacts to the following entities: (a) the partner; (b) an account; and (c) a product instance.",
"When adding a contact, the user must provide a name, address and telephone number.",
"Contacts are of a particular type such as “primary” or “technical.” Users of the CPP can assign as many contacts as they desire.",
"There can also be multiple contacts of a given type.",
"What contact types are defined is determined by a constant class which may be changed.",
"Customer contacts are displayed on the customer, account, and product detail pages, where the user can add new contacts, modify an existing contact, or remove an existing contact.",
"6.12.Notes CPP users can create notes to record activity taken on various entities in the system.",
"Notes capture the date and time they were created, the CPP agent who created them, the customer with whom they are associated, and the entity the note is attached to.",
"Notes can be attached to: (a) Customers or partners, from their respective detail pages; (b) Orders, from the order detail page; and (c) Quotes, from the quote detail page Also, a note history can be viewed for each of these entities from their detail pages.",
"6.13.Manual Approval The CPP allows administrators to manually set the status of order items, account requests, and customer requests.",
"This is useful in testing or in deployments which do not use external systems for provisioning or trouble ticketing.",
"By default, the CPP allows a licensee to set the “pending,” “rejected” and “complete” states, plus the “canceled” state for an order item.",
"Upon navigating to the Manual Approval page, the CPP user will then see the following options: (a) Approve Customer Requests; (b) Approve Partner Requests; (c) Approve Account Requests; and (d) Approve Order Item Requests.",
"Each of these menus presents a list of items (appropriate to the menu chosen), their item number, the date the item was created, the current status, and radio buttons allowing the CSP to alter the state of the interim.",
"Once the CSP have made the desired modifications, clicking “UPDATE” will commit those changes and change the state of the items within system.",
"Section 7.EAI Adapter The EAI adapter runs within the eBusiness support system's process space specifically as part of the Interconnect Service.",
"It uses source and target strategies that run within an external system's process space to transmit data between the Smart Component Server (SCS) and the external system.",
"Once integrated with the external system, the eBusiness support system can publish events into the external system, and objects within the eBusiness support system can be updated based on events within the external system.",
"7.1.Creating Events The eBusiness support system employs communications messaging interfaces (CMIs), objects within the system that contain references to data needed to be sent as events into an external system such as BusinessWare.",
"Each CMI is associated with a routing policy that dispatches it to the correct adapter.",
"When a routing policy dispatches the CMI to the BusinessWare adapter, the appropriate handler for that CMI creates an event.",
"This event retains an association to the data referenced by the CMI.",
"There are six generic handlers for the Vitria's BusinessWare adapter, which correspond to six generic events that the system publishes: ObjectAdd, which is used when an object needs to be added, for example to provision a new product.",
"ObjectAddChange, which is used when it is not known if an object currently exists.",
"If it doesn't, the object is added.",
"If it does, the passed in data is used to update the object.",
"ObjectChange, which is used when an object changes, for example, to change parameters on a provisioned product.",
"ObjectDelete, which is used when an object is deleted, for example to remove an order.",
"ObjectNotify, which is used when a system needs to be notified of an event.",
"ObjectRequest, which is used when a synchronous request is needed.",
"When this type of event is used, the adapter cannot process any other events until it receives the returned data.",
"7.4.Using the BusinessWare Adapter To use the BusinessWare adapter to publish events to an automator model in BusinessWare, the CSP must complete the following steps for each CMI that will use the adapter: (1) Create a BusinessWare automator model to handle the event associated with the CMI.",
"(2) Populate the CMI_CMI_POLICY table so that the CMI uses the generic routing policy.",
"(3) If the CMI data is for an object other than a vendor product, populate the GENERIC_CMI_POLICY table so that the ADAPTER_HOME attribute is the Java Naming Directory (JNDI) deployed name of the BusinessWare adapter EJB and the CNTXT attribute is the fully qualified class name for the appropriate handler.",
"(5) If the CMI transports data for a vendor product, populate the VENDOR_PROD_CMI_POLICY.",
"This table associates a handler with each specific action on a vendor product.",
"The ADAPTER_HOME and CNTXT attributes are populated as described in step (3).",
"(5) Create a source connection model, and if the system is to be updated with data returned from the automator model, create a target connection model as well.",
"(6) Populate the VITRIA_ADAPTER table.",
"This table contains the source and target strategies, as well as the publish channel, for a given CMI.",
"(7) Stop and restart the service.",
"Because the system reads the VITRIA_ADAPTER table at startup and loads it into JNDI, changes are not recognized until the service is restarted.",
"7.5.Creating a Source Connection Model The system provides a source connection template that the BusinessWare console uses to create a new connection model.",
"The process for creating a source connection model using the console includes the steps of: (1) Create a source strategy specific to the CMI.",
"The RetrieveCygentData flow uses the source strategy to access the data for the associated CMI.",
"Each CMI associated with the BusinessWare adapter should have its own source strategy; (2) Create a new model.",
"In the BusinessWare console, navigate to the directory where the instance to be saved.",
"From the File menu, select: New>Connection Model>Cygent Source Connection Model.",
"Then, rename the connection model appropriately; (3) Configure the subscriber channel.",
"This is the channel that the handler uses to publish events.",
"This channel must be the same as the value for the corresponding PUBLISH_CHANNEL in the VITRIA_ADAPTER table; (4) Configure the RetrieveCygentData flow.",
"This includes setting the Server URL, the user name, and the appropriate password; and (5) Configure the publisher channel.",
"This is the channel that publishes the event to the automater model.",
"7.6.Creating a Target Connection Model The system provides a target connection template that the BusinessWare console uses to create a new connection model.",
"The process for creating a target connection model using the console includes the steps of: (1) Create a target strategy specific to the CMI.",
"The update flow uses the target strategy to update data within the system; (2) Create a new model.",
"In the BusinessWare console, navigate to the directory where the instance to be saved.",
"From the File menu, select: New>Connection Model>Cygent Target Connection Model.",
"Then, rename the connection model appropriately; (3) Configure the subscriber channel.",
"This is the channel that the automator model uses to publish the event; and (4) Configure the Update flow.",
"This includes setting the Server URL, the user name, and the appropriate password.",
"Section 8.Application in DSL Commerce One embodiment of the invention is the application of the Smart Component Server (SCS) in DSL commerce.",
"The application is based on the integration of the DSLAdvantage, SCS, and the adapters for Vitria BusinessWare via an HTTP connection to provide flow-through provisioning for DSL products.",
"8.1.Connection Model Communication Message Interfaces (CMIs) transport references to data collected in the portal to the BusinessWare adapter.",
"A DSLAdvantage source connection model translates the CMI into specific event data based on the request, then sends it to the appropriate automator model.",
"This model translates the message to XML and sends the message via an HTTP connection directly to the DSL provider.",
"When the BusinessWare adapter receives a return XML message, it uses the same automator model to translate the message before sending it to the DSLAdvantage target connection model, which updates the appropriate objects.",
"DSLAdvantage uses the BusinessWare adapter to interact with a DSL service provider at three key points: (a) Service availability; (b) Order entry; and (c) Order status 8.2.Service Availability The service availability function enables the eBusiness support system to present available services to an end user by sending pre-qualification information to the DSL service provider via a synchronous request, so that the provider immediately returns a list of services the customer qualifies for.",
"Then a determinant allows a user to select from a list of offers to create a dynamic bundle.",
"Offer collections for DSL services use two determinants to capture and send pre-qualification information to the DSL provider: the SERVICE_ADDRESS_REQUEST determinant, which collects address and site information, and the SERVICE_AVAILABILITY_CHOOSE_ONE determinant, which sends that site information to the DSL provider and then displays the returned products.",
"The following steps illustrate how these determinants work in a DSL offer collection: (1) The end user enters, then submits, pre-qualification information.",
"This page uses the address determinant to collect the information.",
"The service availability determinant (the next determinant in the offer collection sequence) sends the address and site information to the BusinessWare adapter via a CMI.",
"(2) This information is sent to the DSL provider.",
"The BusinessWare adapter uses the service availability source connection model to translate this information into service availability-specific data, and then sends it to the service availability automater model.",
"This model converts the data into an XML form, and sends it to the DSL service provider via an HTTP connection.",
"This event is synchronous—the adapter waits for a response from the provider.",
"(3) The provider returns a list of DSL services.",
"The DSL service provider immediately returns a list of all products the customer qualifies for.",
"(4) The provider's products are mapped to the offers.",
"The system uses the DetermineDeterminantItems plug-in and the PRVDR_SVC_OFFER_MAP table to map the returned product IDs with corresponding the offers.",
"The system then updates the service availability agreements (SMs) based on the returned install date for the corresponding vendor product.",
"(5) The service availability determinant displays the offers.",
"An offer collection is set up so that once a user selects a DSL product, the following determinant displays the corresponding customer premises equipment (CPE) needed to complete the DSL offer collection.",
"8.3.Order Entry Once the end user completes the parameter configuration of all items in a quote and accepts the associated disclosures, an order is created.",
"At this point, all order items associated with DSL services, along with the end user pre-qualification information, are sent to the provider via the BusinessWare adapter.",
"This event is asynchronous—the adapter does not wait for a response from the provider.",
"Once the provider accepts the order, it assigns an order ID to each item and sends that information back to the system via the BusinessWare adapter.",
"The order entry target connection model translates the information and populates the PRVDR_ORDR_ID attribute for each order item.",
"8.4.Order Status Once the DSL service provider has returned the order ID, the provisioning status of a DSL item can be requested.",
"When an end user checks the status of a DSL item, the BusinessWare adapter uses the provider order ID saved to the corresponding order item to send a status request to the provider via the BusinessWare adapter.",
"This event is synchronous—the adapter waits for a response from the provider.",
"Once the provider sends status information back, the system posts this information to the DSL service provider status page.",
"The system neither translates nor saves the status information—it is posted to this page exactly as it is sent by the DSL service provider.",
"If the provider does not yet have status on the order, the system displays a message stating that provisioning status is not yet available.",
"If the provider's site is not active when the order is submitted, the provider might not receive the order.",
"For this reason, it is recommended that the user check the status of new orders daily to ensure the provider receive all orders sent.",
"8.5.Creating DSL Vendor Products and Offers Vendor products and offers for DSL services and CPF are created in the same way as any other products and offers in the Cygent system.",
"The user must associate a vendor with a vendor product, associate any disclosures, service level agreements, and parameter groups, and then create corresponding offers and prices.",
"The user can also create new business rules specific to the relationship between the DSL services and CPE.",
"The user may also want to create a placeholder CPE vendor product and offer with a price of $0, for use when a customer already owns the specified CPE.",
"This allows a customer to select this placeholder product when they don't need to order a CPE, thereby not breaking any business rules.",
"Additionally, a representation in their account services for the CPE shows the delivered product was not provisioned by the DSL service provider.",
"BusinessWare is configured to send specific provisioning information to the DSL service provider when the order is submitted.",
"To get this information to BusinessWare, the use needs to create vendor product parameters to associate with the DSL service vendor products.",
"8.6.Creating a DSL Offer Collection To create offer collections for DSL services, the user must first design the flow of the collection, create the determinants, create corresponding determinant items for the DSL offers, then populate the determinant sequence table.",
"When creating a DSL offer collection, adhere to the following: (1) Use the SERVICE_ADDRESS_REQUEST determinant.",
"This determinant collects address information.",
"(2) The determinant after SERVICE_ADDRESS_REQUEST must be the SERVICE_AVAILABILITY_CHOOSE_ONE determinant.",
"The corresponding display policy for this determinant sends the pre-qualification information to the interconnect service, and then displays the returned products as determinant items.",
"(3) Populate the PRVDR_SVC_OFFER_MAP table.",
"This creates the association between the DSL provider's products and the determinant items.",
"The DSL service provider must provide a list of valid product IDs."
]
] | Patent_10240614 |
[
[
"Electronic device, communication system and method, information processing terminal and method, and information processing apparatus and method",
"An electronic device, a communication system and method, an information processing terminal and method, and an information processing apparatus and method capable of easily and rapidly performing communication.",
"When a link pin (21A) is inserted into a VCR (3), the VCR (3) reads a pin ID stored in an RF tag of the link pin (21A) and reports the pin ID and the address to a management server (1).",
"When the management server (1) has already acquired a pin ID of the link pin (21B) read out by a television receiver (5), the management server (1) determines whether a group ID included in the pin ID reported from the VCR (3) is identical a group ID included in the pin ID reported from the television receiver (5).",
"If the group IDs are determined to be identical, the VCR (3) is connected to the television receiver (5).",
"The present invention can be applied to various information processing apparatuses connected to the network."
],
[
"1.An electronic unit for associating information processing terminals to be connected through a network with each other, characterized by comprising: identification means identifiable by the sense of sight; storage means for storing identification information which includes at least information that associates the information processing terminals to be connected through the network with each other; and providing means for providing, when inserted into or placed on a predetermined information processing terminal, the information processing terminal with the identification information stored by the storage means.",
"2.A communication system comprising an information processing terminal and an information processing apparatus, characterized in that: the information processing terminal comprises: reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, first identification information stored in the electronic unit; transmission means for transmitting the first identification information read by the reading means and first positional information indicating the own position on a network to the information processing apparatus for managing a connection to another information processing terminal; and connection means for connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the first identification information and the first positional information performed by the transmission means, to the another information processing terminal according to the second positional information, and the information processing apparatus comprises: receiving means for receiving the first identification information and the first positional information when they are transmitted; management means for managing a plurality of pieces of identification information and positional information which include the first identification information and the first positional information received by the receiving means; and reporting means for reporting, when among the identification information managed by the management means the first identification information sent from the information processing terminal and the second identification information sent from the another information processing terminal are the same, the first positional information of the information processing terminal to the another information processing terminal, and for reporting the second positional information of the another information processing terminal to the information processing terminal.",
"3.A communication method for a communication system comprising an information processing terminal and an information processing apparatus, characterized in that: an information processing method for the information processing terminal comprises: a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, first identification information stored in the electronic unit; a transmission step of transmitting the first identification information read by the process of the reading step and first positional information indicating the own position on a network to the information processing apparatus for managing a connection with another information processing terminal; and a connection step of connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the first identification information and the first positional information performed by the process of the transmission step, to the another information processing terminal according to the second positional information, and an information processing method for the information processing apparatus comprises: a receiving step of receiving the first identification information and the first positional information when they are transmitted; a management step of managing a plurality of pieces of identification information and positional information which include the first identification information and the first positional information received by the process of the receiving step; and a reporting step of reporting, when among the identification information managed by the process of the management step the first identification information sent from the information processing terminal and the second identification information sent from the another information processing terminal are the same, the first positional information of the information processing terminal to the another information processing terminal, and for reporting the second positional information of the another information processing terminal to the information processing terminal.",
"4.An information processing terminal characterized by comprising: reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; transmission means for transmitting the identification information read by the reading means and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and connection means for connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the transmission means, to the another information processing terminal according to the second positional information.",
"5.An information processing terminal according to claim 4, characterized by further comprising reporting means for reporting, when the reading means cannot read the identification information, to the information processing apparatus that the connection is to be terminated.",
"6.An information processing method characterized by comprising: a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; a transmission step of transmitting the identification information read by the process of the reading step and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and a connection step of connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the process of the transmission step, to the another information processing terminal according to the second positional information.",
"7.A program for making a computer execute: a reading control step of controlling reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the electronic unit; a transmission control step of controlling the transmission of the identification information read by the process of the reading control step and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and a connection control step of controlling the connection, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the process of the transmission control step, to the another information processing terminal according to the second positional information.",
"8.An information processing apparatus for managing a connection of information processing terminals, characterized by comprising: receiving means for receiving identification information of electronic units inserted into or placed on the information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; management means for managing the identification information and the positional information received by the receiving means; and reporting means for reporting, when among the identification information managed by the management means first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, the positional information of the first information processing terminal to the second information processing terminal, and for reporting the positional information of the second information processing terminal to the first information processing terminal.",
"9.An information processing method for an information processing apparatus which manages a connection of information processing terminals, characterized by comprising: a receiving step of receiving identification information of electronic units inserted into or placed on the information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; a management step of managing the identification information and the positional information received by the process of the receiving step; and a reporting step for reporting, when among the identification information managed by the process of the management step first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, the positional information of the first information processing terminal to the second information processing terminal, and for reporting the positional information of the second information processing terminal to the first information processing terminal.",
"10.A program for making a computer of an information processing apparatus for managing a connection of information processing terminals execute: a receiving control step of controlling receiving of identification information of electronic units inserted into or placed on the information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; a management control step of controlling the management of the identification information and the positional information received by the process of the receiving control step; and a reporting control step for controlling reporting, when among the identification information managed by the process of the management control step first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, of the positional information of the first information processing terminal to the second information processing terminal, and reporting of the positional information of the second information processing terminal to the first information processing terminal.",
"11.An information processing terminal characterized by comprising: reading means for reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the first electronic unit; transmission means for transmitting the identification information read by the reading means and first positional information indicating the own position on a network; receiving means for receiving second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the transmission means; and connection means for connecting to the another information processing terminal according to the second positional information received by the receiving means.",
"12.An information processing method characterized by comprising: a reading step of reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the first electronic unit; a transmission step of transmitting the identification information read by the process of the reading step and first positional information indicating the own position on a network; a receiving step of receiving second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the process of the transmission step; and a connection step of connecting to the another information processing terminal according to the second positional information received by the process of the receiving step.",
"13.A program for making a computer execute: a reading control step of controlling reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the first electronic unit; a transmission control step of controlling the transmission of the identification information read by the process of the reading control step and first positional information indicating the own position on a network; a receiving control step of controlling receiving of second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the process of the transmission control step; and a connection control step of controlling the connection to the another information processing terminal according to the second positional information received by the process of the receiving control step.",
"14.An information processing terminal characterized by comprising: reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; synchronization establishment means for establishing synchronization with another information processing terminal located in a vicinity; requesting means for requesting the another information processing terminal with which synchronization has been established by the synchronization establishment means to report terminal-name information specified in the another information processing terminal; and connection means for connecting, when the terminal-name information has been reported in response to the request made by the requesting means, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the reading means.",
"15.An information processing terminal according to claim 14, characterized by further comprising generation means for generating the terminal-name information which includes at least the identification information read by the reading means.",
"16.An information processing method characterized by comprising: a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; a synchronization establishment step of establishing synchronization with another information processing terminal located in a vicinity; a requesting step of requesting the another information processing terminal with which synchronization has been established by the process of the synchronization establishment step to report terminal-name information specified in the another information processing terminal; and a connection step of connecting, when the terminal-name information has been reported in response to the request made by the process of the requesting step, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the process of the reading step.",
"17.A program for making a computer execute: a reading control step of controlling reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the electronic unit; a synchronization-establishment control step of controlling the establishment of synchronization with another information processing terminal disposed in a vicinity; a requesting control step of controlling requesting the another information processing terminal with which synchronization has been established by the process of the synchronization-establishment control step to report terminal-name information specified in the another information processing terminal; and a connection control step of controlling the connection, when the terminal-name information has been reported in response to the request made by the process of the requesting control step, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the process of the reading control step."
],
[
"<SOH> BACKGROUND ART <EOH>As communication technologies have been advanced, various types of units, including not only personal computers but also PDAs (personal digital assistants) and television receivers, have been able to be connected to a network these days.",
"Various systems have been proposed such as that in which images reproduced by a personal computer are sent to a television receiver through a home network and viewed by using the television receiver.",
"Although connecting each unit to a network has been gradually made possible with a simple setting, however, it is necessary to set the address of a mating unit and others to execute communication with the unit in each case, and this setting is troublesome.",
"To display an image reproduced by a personal computer on a television receiver, for example, the user needs to operate the personal computer to specify the address of the television receiver and others.",
"When many units are connected to a network, it is difficult to check that which unit is connected to which unit.",
"In consideration of the current situation, in which it is expected that structuring networks by radio communication, such as a radio LAN (local area network) and Bluetooth™, has been further spread, there is the possibility that the issue will become further serious in which units connected to each other are not known."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a view showing an example structure of a communication system to which the present invention is applied.",
"FIG.",
"2 is a perspective view showing example appearances of link pins shown in FIG.",
"1 .",
"FIG.",
"3A is a perspective view showing an example appearance of a VCR shown in FIG.",
"1 .",
"FIG.",
"3B is a perspective view showing an example appearance of a television receiver shown in FIG.",
"1 .",
"FIG.",
"4 is a block diagram showing an example structure provided for each client shown in FIG.",
"1 .",
"FIG.",
"5 is a block diagram showing an example structure of a management server shown in FIG.",
"1 .",
"FIG.",
"6 is a view showing an example list managed by the management server.",
"FIG.",
"7 is a flowchart describing processing of a client shown in FIG.",
"1 .",
"FIG.",
"8 is a flowchart describing processing of the management server shown in FIG.",
"1 .",
"FIG.",
"9 is a view showing another example structure of the communication system to which the present invention is applied.",
"FIG.",
"10 is a flowchart describing processing of a unit shown in FIG.",
"9 .",
"FIG.",
"11 is a flowchart describing another processing of a unit shown in FIG.",
"9 .",
"FIG.",
"12A is a perspective view showing another example appearances of link pins.",
"FIG.",
"12B is a perspective view showing still another example appearances of link pins.",
"FIG.",
"12C is a perspective view showing example appearances of link pins.",
"FIG.",
"13 is a perspective view showing an example appearance of a VCR on which a link card is placed.",
"FIG.",
"14A is a view showing another example appearances of link cards.",
"FIG.",
"14B is a view showing still another example appearances of link cards.",
"FIG.",
"14C is a view showing example appearances of link cards.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD The present invention relates to electronic units, communication systems and methods, information processing terminals and methods, and information processing apparatuses and methods, and more particularly, to electronic units, communication systems and methods, information processing terminals and methods, and information processing apparatuses and methods which allow easy and quick communications.",
"BACKGROUND ART As communication technologies have been advanced, various types of units, including not only personal computers but also PDAs (personal digital assistants) and television receivers, have been able to be connected to a network these days.",
"Various systems have been proposed such as that in which images reproduced by a personal computer are sent to a television receiver through a home network and viewed by using the television receiver.",
"Although connecting each unit to a network has been gradually made possible with a simple setting, however, it is necessary to set the address of a mating unit and others to execute communication with the unit in each case, and this setting is troublesome.",
"To display an image reproduced by a personal computer on a television receiver, for example, the user needs to operate the personal computer to specify the address of the television receiver and others.",
"When many units are connected to a network, it is difficult to check that which unit is connected to which unit.",
"In consideration of the current situation, in which it is expected that structuring networks by radio communication, such as a radio LAN (local area network) and Bluetooth™, has been further spread, there is the possibility that the issue will become further serious in which units connected to each other are not known.",
"DISCLOSURE OF INVENTION The present invention has been made in consideration of such a condition, and allows easy and quick communications and also easy recognition of connected units.",
"An electronic unit according to the present invention is characterized by including identification means identifiable by the sense of sight; storage means for storing identification information; and providing means for providing, when inserted into or placed on a predetermined information processing terminal, the information processing terminal with the identification information stored by the storage means.",
"An information processing terminal of a communication system according to the present invention is characterized by including reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, first identification information stored in the electronic unit; transmission means for transmitting the first identification information read by the reading means and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and connection means for connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the first identification information and the first positional information performed by the transmission means, to the another information processing terminal according to the second positional information.",
"The information processing apparatus is characterized by including receiving means for receiving the first identification information and the first positional information when they are transmitted; management means for managing a plurality of pieces of identification information and positional information which include the first identification information and the first positional information received by the receiving means; and reporting means for reporting, when among the identification information managed by the management means the first identification information sent from the information processing terminal and the second identification information sent from the another information processing terminal are the same, the first positional information of the information processing terminal to the another information processing terminal, and for reporting the second positional information of the another information processing terminal to the information processing terminal.",
"A communication method for a communication system according to the present invention is characterized by including a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, first identification information stored in the electronic unit; a transmission step of transmitting the first identification information read by the process of the reading step and first positional information indicating the own position on a network to an information processing apparatus for managing a connection with another information processing terminal; a connection step of connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the first identification information and the first positional information performed by the process of the transmission step, to the another information processing terminal according to the second positional information; a receiving step of receiving the first identification information and the first positional information when they are transmitted; a management step of managing a plurality of pieces of identification information and positional information which include the first identification information and the first positional information received by the process of the receiving step; and a reporting step of reporting, when among the identification information managed by the process of the management step the first identification information sent from the information processing terminal and the second identification information sent from the another information processing terminal are the same, the first positional information of the information processing terminal to the another information processing terminal, and for reporting the second positional information of the another information processing terminal to the information processing terminal.",
"A first information processing terminal according to the present invention is characterized by including reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; transmission means for transmitting the identification information read by the reading means and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and connection means for connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the transmission means, to the another information processing terminal according to the second positional information.",
"The information processing terminal may further include reporting means for reporting, when the reading means cannot read the identification information, to the information processing apparatus that the connection is to be terminated.",
"An information processing method for the first information processing terminal according to the present invention is characterized by including a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; a transmission step of transmitting the identification information read by the process of the reading step and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and a connection step of connecting, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the process of the transmission step, to the another information processing terminal according to the second positional information.",
"A program according to the present invention is characterized by making a, computer execute a reading control step of controlling reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the electronic unit; a transmission control step of controlling the transmission of the identification information read by the process of the reading control step and first positional information indicating the own position on a network to an information processing apparatus for managing a connection to another information processing terminal; and a connection control step of controlling the connection, when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the process of the transmission control step, to the another information processing terminal according to the second positional information.",
"An information processing apparatus according to the present invention is characterized by including receiving means for receiving identification information of electronic units inserted into or placed on information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; management means for managing the identification information and the positional information received by the receiving means; and reporting means for reporting, when among the identification information managed by the management means first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, the positional information of the first information processing terminal to the second information processing terminal, and for reporting the positional information of the second information processing terminal to the first information processing terminal.",
"An information processing method for an information processing apparatus according to the present invention is characterized by including a receiving step of receiving identification information of electronic units inserted into or placed on information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; a management step of managing the identification information and the positional information received by the process of the receiving step; and a reporting step for reporting, when among the identification information managed by the process of the management step first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, the positional information of the first information processing terminal to the second information processing terminal, and for reporting the positional information of the second information processing terminal to the first information processing terminal.",
"A program according to the present invention is characterized by making a computer execute a receiving control step of controlling receiving of identification information of electronic units inserted into or placed on information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network, when they are transmitted; a management control step of controlling the management of the identification information and the positional information received by the process of the receiving control step; and a reporting control step for controlling reporting, when among the identification information managed by the process of the management control step first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, of the positional information of the first information processing terminal to the second information processing terminal, and reporting of the positional information of the second information processing terminal to the first information processing terminal.",
"A second information processing terminal according to the present invention is characterized by including reading means for reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the first electronic unit; transmission means for transmitting the identification information read by the reading means and first positional information indicating the own position on a network; receiving means for receiving second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the transmission means; and connection means for connecting to the another information processing terminal according to the second positional information received by the receiving means.",
"An information processing method for the second information processing terminal according to the present invention is characterized by including a reading step of reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the first electronic unit; a transmission step of transmitting the identification information read by the process of the reading step and first positional information indicating the own position on a network; a receiving step of receiving second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the process of the transmission step; and a connection step of connecting to the another information processing terminal according to the second positional information received by the process of the receiving step.",
"A program according to the present invention is characterized by making a computer execute a reading control step of controlling reading, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the first electronic unit; a transmission control step of controlling the transmission of the identification information read by the process of the reading control step and first positional information indicating the own position on a network; a receiving control step of controlling receiving of second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information performed by the process of the transmission control step; and a connection control step of controlling the connection to the another information processing terminal according to the second positional information received by the process of the receiving control step.",
"A third information processing terminal according to the present invention is characterized by including reading means for reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; synchronization establishment means for establishing synchronization with another information processing terminal located in a vicinity; requesting means for requesting the another information processing terminal with which synchronization has been established by the synchronization establishment means to report terminal-name information specified in the another information processing terminal; and connection means for connecting, when the terminal-name information has been reported in response to the request made by the requesting means, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the reading means.",
"The information processing terminal may further include generation means for generating the terminal-name information which includes at least the identification information read by the reading means.",
"An information processing method for the third information processing terminal according to the present invention is characterized by including a reading step of reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit; a synchronization establishment step of establishing synchronization with another information processing terminal located in a vicinity; a requesting step of requesting the another information processing terminal with which synchronization has been established by the process of the synchronization establishment step to report terminal-name information specified in the another information processing terminal; and a connection step of connecting, when the terminal-name information has been reported in response to the request made by the process of the requesting step, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the process of the reading step.",
"A program according to the present invention is characterized by making a computer execute a reading control step of controlling reading, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, of identification information stored in the electronic unit; a synchronization-establishment control step of controlling the establishment of synchronization with another information processing terminal located in a vicinity; a requesting control step of controlling requesting the another information processing terminal with which synchronization has been established by the process of the synchronization-establishment control step to report terminal-name information specified in the another information processing terminal; and a connection control step of controlling the connection, when the terminal-name information has been reported in response to the request made by the process of the requesting control step, to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the identification information read by the process of the reading control step.",
"In an electronic unit according to the present invention, it is identifiable by the sense of sight; identification information is stored; when inserted into or placed on a predetermined information processing terminal, the stored identification information is sent to the information processing terminal.",
"In a communication system and a communication method according to the present invention, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, an information processing terminal reads first identification information stored in the electronic unit; the read first identification information and first positional information indicating the own position on a network are sent to an information processing apparatus for managing a connection to another information processing terminal; and when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the first identification information and the first positional information, a connection is made to the another information processing terminal according to the second positional information.",
"Further, the information processing apparatus receives the first identification information and the first positional information when they are transmitted; a plurality of pieces of identification information and positional information which includes the first identification information and the first positional information received are managed; and when among the managed identification information the first identification information sent from the information processing terminal and the second identification information sent from the another information processing terminal are the same, the first positional information of the information processing terminal is reported to the another information processing terminal, and the second positional information of the another information processing terminal is reported to the information processing terminal.",
"In a first information processing terminal, an information processing method, and a program according to the present invention, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit is read; the read identification information and first positional information indicating the own position on a network are sent to an information processing apparatus for managing a connection to another information processing terminal; and when second positional information of the another information processing terminal is reported from the information processing apparatus in response to the transmission of the identification information and the first positional information performed by the transmission means, a connection is made to the another information processing terminal according to the second positional information.",
"In an information processing apparatus, an information processing method, and a program according to the present invention, identification information of electronic units inserted into or placed on information processing terminals, the identification information being read by the information processing terminals, and positional information of the information processing terminals on a network are received when they are transmitted; the identification information and the positional information received are managed; and when among the managed identification information first identification information sent from a first information processing terminal and second identification information sent from a second information processing terminal are the same, the positional information of the first information processing terminal is reported to the second information processing terminal, and the positional information of the second information processing terminal is reported to the first information processing terminal.",
"In a second information processing terminal, an information processing method, and a program according to the present invention, when a first electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the first electronic unit is read; the read identification information and first positional information indicating the own position on a network are sent; second positional information indicating the position of another information processing terminal which has read the same identification information as the identification information from a second electronic unit, on the network, sent from the another information processing terminal in response to the transmission of the identification information and the first positional information is received; and a connection is made to the another information processing terminal according to the received second positional information.",
"In a third information processing terminal, an information processing method, and a program according to the present invention, when an electronic unit for associating information processing terminals to be connected, with each other is inserted or placed, identification information stored in the electronic unit is read; synchronization with another information processing terminal located in a vicinity is established; the another information processing terminal with which synchronization has been established is requested to report terminal-name information specified in the another information processing terminal; and when the terminal-name information has been reported in response to the request, a connection is made to the another information processing terminal which has reported the terminal-name information that includes the same identification information as the read identification information.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a view showing an example structure of a communication system to which the present invention is applied.",
"FIG.",
"2 is a perspective view showing example appearances of link pins shown in FIG.",
"1.FIG.",
"3A is a perspective view showing an example appearance of a VCR shown in FIG.",
"1.FIG.",
"3B is a perspective view showing an example appearance of a television receiver shown in FIG.",
"1.FIG.",
"4 is a block diagram showing an example structure provided for each client shown in FIG.",
"1.FIG.",
"5 is a block diagram showing an example structure of a management server shown in FIG.",
"1.FIG.",
"6 is a view showing an example list managed by the management server.",
"FIG.",
"7 is a flowchart describing processing of a client shown in FIG.",
"1.FIG.",
"8 is a flowchart describing processing of the management server shown in FIG.",
"1.FIG.",
"9 is a view showing another example structure of the communication system to which the present invention is applied.",
"FIG.",
"10 is a flowchart describing processing of a unit shown in FIG.",
"9.FIG.",
"11 is a flowchart describing another processing of a unit shown in FIG.",
"9.FIG.",
"12A is a perspective view showing another example appearances of link pins.",
"FIG.",
"12B is a perspective view showing still another example appearances of link pins.",
"FIG.",
"12C is a perspective view showing example appearances of link pins.",
"FIG.",
"13 is a perspective view showing an example appearance of a VCR on which a link card is placed.",
"FIG.",
"14A is a view showing another example appearances of link cards.",
"FIG.",
"14B is a view showing still another example appearances of link cards.",
"FIG.",
"14C is a view showing example appearances of link cards.",
"BEST MODE FOR CARRYING OUT THE INVENTION FIG.",
"1 is a view showing the concept of a communication system to which the present invention is applied.",
"A management server 1 manages the connections of units connected to a network 2.For example, when a link pin 21A and a link pin 21B are inserted to a VCR (video cassette recorder) 3 and a television receiver 5, respectively, the management server 1 connects the VCR 3 and the television receiver 5, as indicated by a one-dot chain line.",
"The network 2 is formed, for example, of an Ethernet or a radio LAN (local area network) such as IEEE (Institute of Electrical and Electronics engineers) 802.11a or 802.11b.",
"With this, for example, an image reproduced by the VCR 3 is sent to the television receiver 5 through the network 2, and displayed on the television receiver 5.When a link pin 22A is inserted to a game machine 6, and a link pin 22B is inserted to a projector 7, the management server 1 establishes the connection between the units, as indicated by a one-dot chain line, to display an image reproduced by the game machine 6 on the projector 7.In this example, the link pin 21A and the line pin 21B form a pair and the link pin 22A and the link pin 22B form a pair.",
"The user can check the head (circular part in the figure) of each pin to identify the pair.",
"Details of the link pin 21A and the link pin 21B will be described later.",
"A personal computer 4 is also connected to a network 2.The user can change units to be connected to each other without setting addresses and other, just by inserting the link pin 21A and the link pin 21B, and the link pin 22A and the link pin 22B to the units.",
"(Hereinafter, when it is not necessary to distinguish the units connected to the network 2, from the VCR 3 to the projector 7, they are called clients accordingly.)",
"FIG.",
"2 is a view showing example appearances of the link pin 21A and link pin 21B.",
"As shown in the figure, the link pin 21A is formed basically of an identification part 31A and a shaft 32A, and an RF tag 33A is provided at a predetermined position of the shaft 32A.",
"In the same way, the link pin 21B is also formed of an identification part 31B and a shaft 32B, and an RF tag 33B is provided at a predetermined position of the shaft 32B.",
"The identification part 31A of the link pin 21A and the identification part 31B of the link pin 21B have a sphere shape, for example, and are colored by the same color.",
"Therefore, the user can recognize at a glance that the link pin 21A and the link pin 21B form a pair, that is, units to which the link pin 21A and the link pin 21B are inserted are connected through the network 2.Although not shown in a figure, the identification parts of the above-described link pin 22A and the link pin 22B are colored by a different color from that of the identification parts 31A of the link pin 21A and the identification part 31B of the link pin 21B.",
"When the shaft 32A and the shaft 32B are inserted into the insertion sections provided at predetermined positions of clients (the VCR 3 to the projector 7), the RF tag 33A which is built in the shaft 32A, and the RF tag 33B which is built in the shaft 32B, provide stored identification information for the clients.",
"More specifically, the insertion sections of the clients are provided with readers for reading the identification information stored in the RF tag 33A and the RF tag 33B.",
"The identification information (hereinafter called a pin ID, if necessary) stored in the RF tag 33A or the RF tag 33B is formed, for example, of a group ID and an individual ID.",
"The group ID is set such that the RF tag 33A and the RF tag 33B has the same group ID, and the individual ID is set such that each RF tag has a different individual ID.",
"For example, a pin ID of “pin00001-01” formed of a group ID of “pin00001” and an individual ID of “01” is assigned to the RF tag 33A, and a pin ID of “pin00001-02” formed of a group ID of “pin00001” and an individual ID of “02” is assigned to the RF tag 33B.",
"Therefore, when the management server 1 receives pin IDs read by clients, the management server 1 can check the group ID included in the pin IDs to connect the clients to which the link pin 21A and the link pin 21B have been inserted.",
"FIG.",
"3A is a perspective view showing an example appearance of the VCR 3, and FIG.",
"3B is a perspective view of an example appearance of the television receiver 5.As shown in FIG.",
"3A, a link-pin insertion section 42 is provided at the lower right of a front face 3A at which a cassette insertion section 41 is provided, and the shaft 32A of the link pin 21A, for example, can be inserted into and pulled out of the link-pin insertion section 42.In a vicinity of the link-pin insertion section 42, a reader for reading the pin ID stored in the RF tag 33A of the inserted link pin 21A is provided.",
"As shown in FIG.",
"3B, a link-pin insertion section 52 is provided, in the same way as in the VCR 3, at the lower right of a face 5A of the television receiver 5, at which a display section 51 is provided, and the shaft 32B of the link pin 21B, for example, can be inserted into and pulled out of the link-pin insertion section 52.Also in a vicinity of the link-pin insertion section 52, a reader for reading the pin ID stored in the RF tag 33B of the inserted link pin 21B is provided.",
"To change the connection destination of a client, the user pulls out the link pin inserted into the link-pin insertion section provided at a predetermined position in this way, and inserts the link pin into the link-pin insertion section of a client to which a new connection is made.",
"FIG.",
"4 is a view showing an example structure of a client.",
"Such a structure is provided for each client shown in FIG.",
"1.When the link pin 21A, for example, is inserted into the link-pin insertion section provided in a vicinity of a reader 61, the reader 61 reads the pin ID stored in the RF tag 33A, and outputs it to a communication control section 62.When the inserted link pin 21A is pulled out, the reader 61 reports the state to the communication control section 62.The communication control section 62 sends, for example, the pin ID sent from the reader 61 to the management server 1 through a communication section 64 and the network 2, according to an instruction sent from a control section 63 which controls the operation of the whole of the client.",
"The communication control section 62 also sends data sent from the control section 63 to another client through the communication section 64 and others, and if necessary, outputs data sent from another client to the control section 63.When the structure shown in FIG.",
"4 is provided for the VCR 3, and the VCR 3 and the television receiver 5 are connected (link pins having the same group ID have been inserted thereinto), for example, the communication control section 62 sends video data and audio data sent from the control section 63 to the television receiver 5.When the structure shown in FIG.",
"4 is provided for the projector 7, and the projector 7 and the game machine 6 are connected, the communication control section 62 sends video data and audio data of a game, sent from the communication section 64 to the control section 63.The control section 63 controls the operation of the whole of the client.",
"When the structure shown in FIG.",
"4 is provided for the VCR 3, for example, the control section 63 controls the operation of a head not shown, and others, reads video data and audio data recorded in a mounted cassette, and outputs them to the communication control section 62.When the structure shown in FIG.",
"4 is provided for the projector 7, the control section 63 outputs video data sent from the communication control section 62 to a reproduction section not shown to display the image, and also outputs received audio data to the reproduction section not shown to outputs the sound from a speaker.",
"The communication section 64 is connected to the network 2, and sends data sent from the communication control section 62, to another client and to the management server 1.The communication section 64 also receives data sent from another client and others, and outputs it to the communication control section 62.FIG.",
"5 is a block diagram showing an example structure of the management server 1.A CPU (central processing unit) 71 executes various types of processing according to a program stored in a ROM (read only memory) 72 or a program loaded from a storage section 78 to a RAM 73 (random access memory).",
"The RAM 73 also stores, if necessary, data required by the CPU 71 to execute the various types of processing.",
"The CPU 71, the ROM 72, and the RAM 73 are connected to each other through a bus 74.An input-and-output interface 75 is also connected to the bus 74.An input section 76 formed of a keyboard, a mouse, and others, an output section 77 formed of a display, such as a CRT (cathode ray tube) or an LCD (liquid-crystal display), a speaker, and others, the storage section 78 formed of a hard disk and others, and a communication section 79 are connected to the input-and-output interface 75.The storage section 78 stores various types of application software used by the CPU 71, and sends application software to the CPU 71, if necessary.",
"The storage section 78 also generates a list for managing clients connected through the network 2, according to an instruction sent from the CPU 71, and stores the list.",
"FIG.",
"6 is a view showing an example list stored in the storage section 78.The list shown in the figure stores clients to which link pins are inserted to which a group ID of “pin00001” has been assigned.",
"As described above, When the link pin 21A has a pin ID of “pin00001-01”, the link pin 21B has a pin ID of “pin00001-02”, the link pin 21A is inserted into the VCR 3, and the link pin 21B is inserted into the television receiver 5, the VCR 3 and the television receiver (TV) 5 are registered on the list as shown in FIG.",
"6.Both of the link pin 21A and the link pin 21B have a group ID of “pin 00001”.",
"In the example shown in FIG.",
"6, the address (address on the network 2) of the VCR 3 registered as a client 1 is set to “255:255:255:254” and the address of the television receiver 5 registered as a client 2 is set to “255:255:255:255”.",
"These addresses are managed by the corresponding clients, and sent from the clients together with the pin IDs when the link pins are inserted.",
"These addresses may be registered on the management server 1 in advance.",
"In this case, a client to which a link pin is inserted sends the pin ID and information (information which includes the address) identifying the client.",
"The storage section 78 manages such a list according to an instruction sent from the CPU 71, and, when the VCR 3 reports that the inserted link pin 21A was pulled out, for example, deletes the entry of the VCR 3 from the list.",
"Back to the description made by referring to FIG.",
"5, the communication 79 performs communications through the network 2.When the VCR 3 reports that the link pin 21A has been inserted, for example, the communication section 79 outputs the report to the storage section 78 and others.",
"A drive 80 is also connected to the input-and-output interface 75, if necessary.",
"A magnetic disk 81, an optical disk 82, a magneto-optical disk 83, or a semiconductor memory 84 is mounted to the drive 80, if necessary, and a computer program read therefrom is installed in the storage section 78, as required.",
"The operation of the communication system shown in FIG.",
"1 will be described next.",
"Processing of a predetermined client, for example, the VCR 3, for connecting to another client will be described first by referring to a flowchart shown in FIG.",
"7.In step S1, the reader 61 of the VCR 3 determines whether a link pin has been inserted into the link-pin insertion section 42 (whether a pin ID has been read), and waits for until it determines that a link pin has been inserted.",
"When the user inserts the link pin 21A into the link-pin inserting section 42, for example, the processing proceeds to step S2, and the reader 61 reads the pin ID stored in the RF tag 33A.",
"The pin ID read by the reader 61 is sent to the communication control section 62.The pin ID may be encrypted by a predetermined method and provided by the RF tag 33.In step S3, the communication control section 62 sends the pin ID and the address assigned to the VCR 3 to the management server 1.In step S4, the communication control section 62 determines whether the address of another client (any of the personal computer 4 to the projector 7) has been received.",
"When a client sends the pin ID and others, the management server 1 sends the address registered on the list corresponding to the group ID included in the pin ID, to the client which has sent the pin ID and others, as will be described later by referring to a flowchart shown in FIG.",
"8.When the communication control section 62 determines in step S4 that the address of another client has not been received, the processing skips the following steps S5 to S7, and proceeds to step S8.When the communication control section 62 determines in step S4 that the address of another client has been received, the processing proceeds to step S5, and the received address is managed as the address of a client to be connected through the network 2, and a connection to the client is made.",
"When the address of the television receiver 5 has been entered into a list such as that shown in FIG.",
"6, for example, since the address is sent from the management server 1, the communication control section 62 makes a connection to the television receiver 5 according to the received address.",
"In step S6, the communication control section 62 determines whether the address of a client which requests the end of connection has been sent from the management server 1.When it is determined that the address has been sent, the processing proceeds to step S7, and the connection to the client is terminated.",
"When the link pin 21B is removed from the television receiver 5, and this removal is reported to the management server 1, for example, the information of the television receiver 5 is deleted from the list and the end of connection is reported to the VCR 3.With this, the connection between the VCR 3 and the television receiver 5 is terminated.",
"When the communication control section 62 determines in step S6 that the address of a client with which a connection is terminated has been sent from the management server 1, the process of step S7 is skipped and the processing proceeds to step S8.In step S8, the reader 61 determines whether the link pin 21A has been removed from the link-pin insertion section 42.When the reader 61 determines that the link pin has not been removed, and a so-called time out, in which a connection is terminated after a predetermined time elapses, has been set, the processing returns to step S3, and the subsequent processes are repeatedly executed.",
"With this, in formation such as a pin ID read by the reader 61 is sent to the management server 1 at a predetermined interval.",
"When a time out is set, if a cause such as a physical disconnection of the network occurs, other than “the removal of the link pin 21A”, the connection is terminated because of the time out.",
"When a time out is not set, the processes of step S4 and subsequent steps are executed.",
"When the user makes a change the output of the television receiver 5 to one which is reproduced by the personal computer 4, the user removes the link pin 21A inserted into the VCR 3, and inserts it into the insertion section of the personal computer 4.When the reader 61 determines in step S8 that the link pin 21A has been removed, the reader 61 reports the removal to the communication control section 62.When the reader 61 reports the removal of the link pin 21A, the communication control section 62 reports to the management server 1 in step S9 that the link pin 21A has been removed.",
"When a connection to another client exists, the communication control section 62 terminates the connection to the client in step S10.The above-described processing is repeatedly executed thereafter.",
"Such processing is also executed in clients other than the VCR 3 to connect to each other clients to which link pins having the same group IDs are inserted.",
"Processing of the management server 1, executed correspondingly to the processing shown in FIG.",
"7 will be described next by referring to the flowchart shown in FIG.",
"8.In step S21, the CPU 71 of the management server 1 checks information received by the communication section 79 to determine whether any client has reported the pin ID and the address.",
"When the CPU 71 determines in step S21 that the pin ID and the address have not been reported, the processing skips the processes of step S22 to step S26, described later.",
"When the CPU 71 determines in step S21 that the pin ID and the address have been reported, the processing proceeds to step S22.In step S22, the CPU 71 determines whether the address of another client has already been registered on the list (list of clients to which link pins having the same group ID are inserted) corresponding to the group ID included in the reported pin ID.",
"For example, as shown in FIG.",
"6, when the television receiver has already been registered on the list corresponding to a group ID of “pin00001”, if a pin ID of “pin00001-01” and an address of “255:255:255:254” are reported by the VCR 3 in that state, the CPU 71 determines in step S22 that the address of another client (television receiver 5) has been registered on the list corresponding to the group ID included in the reported ID, and the processing proceeds to step S23.The list also stores, if necessary, the time when the pin ID and others were reported, and others.",
"In step S23, the CPU 71 reports the addresses of all clients entered in the list, that is, the address of the television receiver 5, to the VCR 3.If link pins to which a group ID of “pin00001” has been assigned are inserted into clients other than the television receiver 5, the addresses of those clients are also reported.",
"In step S24, the CPU 71 reports the address of the VCR 3 to the television receiver 5 already registered on the list.",
"The processing proceeds to step S25, and the CPU 71 registers the address of the VCR 3 and the current time on the list, if necessary, and stores it, which is similar to that shown in FIG.",
"6, in the storage section 78.With this, the VCR 3 and the television receiver 5 obtains the addresses of the mating units, and communication is established.",
"The management server 1 may control the switching of the selector connected to each client to physically connect the clients registered on the list to each other.",
"When the CPU 71 determines in step S22 that the address of another client has not been registered on the list corresponding to the group ID, the processing proceeds to step S26, and the CPU 71 registers the address of the client which reported the pin ID and the time on the list corresponding to the group ID.",
"In other words, when the reported address is sent from a client already registered (one client has been registered on the list), the entry time is updated.",
"In step S27, the CPU 71 determines whether a client has reported the removal of the link pin.",
"When it is determined that a client has reported the removal of the link pin, the processing proceeds to step S28, and the CPU 71 determines whether, in the list in which the address of the client has been registered, the address of another client has been registered.",
"When the CPU 71 determines in step S28 that the address of another client has not been registered, the processing proceeds to step S29, and the CPU 71 deletes the address of the client from which the link pin has been removed, from the list.",
"Then, the processing returns to step S21, and the subsequent processes are repeatedly executed.",
"When the CPU 71 determines in step S28 that, in the list in which the address of the client which reported the removal of the link pin has been registered, the address of another client has been registered, the processing proceeds to step S30, and the CPU 71 reports the address of the client (client which reported the removal of the link pin) for which a connection is terminated, to the registered another client.",
"With this, the connection between the clients into which the link pins having the same group ID were inserted is released.",
"The processing proceeds to step S31, and the CPU 71 deletes the address of the client which reported the removal of the link pin, from the list.",
"When the CPU 71 determines in step S27 that a client has not reported the removal of the link pin, the processing proceeds to step S32, and the CPU 71 determines next whether time out occurs in a unit.",
"As described above, the management server 1 also manages the time when the pin ID and others were reported, and the connection to a predetermined unit is terminated (time-out occurs in the unit), if necessary, according to the time.",
"When the CPU 71 determines in step S32 that time out has not occurred in any unit, the processing returns to step S21, and the processes of the subsequent steps are repeatedly executed.",
"When the CPU 71 determines in step S32 that time out has occurred in a unit, the processing proceeds to step S30, and the above-described processes are executed.",
"More specifically, the address of the client for which the connection is terminated is reported to the other clients, and then, is deleted from the list.",
"For example, as described above, when the VCR 3 and the television receiver 5 are connected, and the VCR 3 has reported the removal of the link pin 21A, the CPU 21 reports the address of the VCR 3 to the television receiver 5, and deletes the address of the VCR 3 from the list.",
"Then, the processing proceeds to step S21, and the above processes are repeatedly executed.",
"With this, the user can perform communications easily and quickly just by inserting link pins to desired clients.",
"In addition, the user can easily understand connected clients just by checking the identification sections of the inserted link pins.",
"For example, when the user inserts the link pin 21A into the personal computer 4, and the link pin 21B into a printer not shown, the personal computer 4 recognizes according to the address of the printer, reported from the management server 1 that the connected client is the printer, and sends data to be printed out to the printer, due to the above-described processing.",
"Then, the address reported from the management server 1 may be entered into the personal computer 4 as the address of the default printer.",
"A case in which the management server 1 is connected to the network 2 has been described so far.",
"As shown in FIG.",
"9, even if the management server 1 does not exist, the user can connect desired clients just by inserting link pins to the clients.",
"In a case in which the management server 1 does not exist, as shown in FIG.",
"9, when the link pin 21A is inserted into the personal computer 4, for example, the personal computer 4 reads the pin ID, broadcasts the read pin ID and the address to all units (clients), and detects a unit into which a link pin having the same group ID.",
"For example, when the link pin 21B, to which the same group ID is assigned as to the link pin 21A, is inserted into the television receiver 5, the television receiver 5 responds to the detection from the personal computer, and reports its address to the personal computer 4.Communications are established between units which have exchanged the addresses with each other.",
"Processing of each unit constituting the communication system shown in FIG.",
"9 will be described next by referring to a flowchart shown in FIG.",
"10.In this example, processing of the personal computer 4 will be described.",
"In step S41, the reader 61 of the personal computer 4 determines whether a link pin has been inserted into the link-pin insertion section of the personal computer 4, and waits for until it determines that a link pin has been inserted.",
"When the reader 61 determines, for example, that the link pin 21A is inserted, the processing proceeds to step S42, and the reader 61 reads a pin ID formed of a group ID and an individual ID, such as those described above.",
"When the reader 61 sends the pin ID to the communication control section 62, the communication control section 62 broadcasts its own address and the pin ID of the link pin 21A, and detects a unit to which a link pin having the same group ID is inserted.",
"In step S44, the communication control section determines whether a unit to which a link pin having the same group ID is inserted has been detected.",
"When it is determined that such a unit has not been detected, the processing proceeds to step S45, and it is determined whether the link pin 21A has been removed.",
"When the communication control section 62 determines in step S45 that the link pin 21A has been removed, the processing proceeds to step S41, and the above-described processes are repeatedly executed.",
"Contrarily, when it is determined the link pin 21A has not been removed, the processing returns to step S43, and the subsequent processes are repeatedly performed.",
"When the communication control section determines in step S44 that a unit to which a link pin having the same group ID is inserted has been detected, the processing proceeds to step S46, and the communication control section 62 receives the address sent from the unit.",
"In step S47, the communication control section 62 makes a connection to the detected unit, to which a link pin having the same group ID is inserted, according to the received address.",
"When link pins having the same group ID are inserted into a plurality of units and those units are detected, all the detected units may be shown to the user and selected.",
"For example, when the television receiver 5 to which the link pin 21B having the same group ID as the link pin 21A is inserted receives the address of the personal computer 4 and others sent for unit detection, the television receiver 5 responds thereto, and reports its own address to the personal computer 4.In step S48, the communication control section 62 determines whether a connected unit, such as the television receiver 5, has requested the release of the connection (whether the link pin 21B has been removed from the television receiver 5).",
"When it is determined that the release of the connection has been requested, the processing proceeds to step S49.In step S49, the communication control section 49 releases the connection to the television receiver 5.The processing returns to step S43, and the subsequent processes are repeatedly executed.",
"Contrarily, when the communication control section 62 determines that the release of the connection has not been requested, the processing proceeds to step S50, and the communication control section 62 determines whether the link pin 21A has been removed from the link-pin insertion section.",
"When the communication control section 62 determines in step S50 that the link pin 21A has not been removed, the processing returns to step S47, and the connection to the television receiver 5 continues.",
"When the communication control section 62 determines that the link pin 21A has been removed, the processing proceeds to step S51.In step S51, the communication control section 62 requests the television receiver 5 to release the connection.",
"The processing proceeds to step S52, and the connection is terminated.",
"Then, the processing returns to step S41, and the above processes are executed.",
"Processing of each unit in a case in which the network 2 shown in FIG.",
"9 uses Bluetooth will be described next by referring to FIG.",
"11.Also in FIG.",
"11, processing of the personal computer 4 will be described.",
"The processes of step S61 and step S62 are the same as those of step S41 and step S42 shown in FIG.",
"10.More specifically, the reader 61 of the personal computer 4 determines in step S61 whether a link pin has been inserted.",
"When the reader 61 determines that the link pin 21A has been inserted, the processing proceeds to step S62, and the reader 61 reads the pin ID thereof.",
"In step S63, the communication control section 62 generates a Bluetooth device name by using the pin ID.",
"The Bluetooth device name indicates the unit name of a device (Bluetooth device) provided with a Bluetooth module, and can be changed as desired by each Bluetooth device.",
"More specifically, the communication control section 62 generates a Bluetooth device name according to the pin ID and information, such as a Bluetooth address, specified in advance unique to each Bluetooth device.",
"For example, when the reader 61 reads a pin ID of “pin00001-01” and the personal computer 4 has a Bluetooth address of “pc1”, the communication control section 62 combines them to generate a Bluetooth device name of “pc1-pin00001-01” formed of the Bluetooth address, the group ID, and the individual ID.",
"Also in each unit to which a link pin has been inserted, a Bluetooth device name is generated in the same way.",
"The processing proceeds to step S64, and the communication control section 62 establishes synchronization with a Bluetooth device disposed in its vicinity.",
"More specifically, in step S64, the communication control section 62 performs “inquiry” and “page” specified in Bluetooth to establish synchronization with a Bluetooth device located in the vicinity in a frequency axis (frequency hopping pattern) and a time axis (time slot).",
"In step S65, the communication control section 62 requests the Bluetooth device with which synchronization has been established, to send the Bluetooth device name.",
"In step S66, the communication control section 62 determines whether the reported Bluetooth device name includes the same group ID as that of the link pin 21A inserted into the personal computer 4.For example, as shown in FIG.",
"9, when the link pin 21B having a pin ID of “pin00001-02” is inserted into the television receiver 5, the television receiver 5 also generates a Bluetooth device name as described above, and sends a Bluetooth device name of, for example, “tv1-pin00001-02”.",
"When the communication control section 62 determines in step S66 that the reported Bluetooth device name does not include the same group ID as that of the link pin 21A, the processing proceeds to step S67, and the communication control section 62 determines whether the link pin 21A has been removed.",
"When the communication control section 62 determines in step S67 that the link pin 21A has been removed, the processing returns to step S61, and the above-described processes are repeatedly performed.",
"Contrarily, when the communication control section 62 determines that the link pin 21A has not been removed, the processing returns to step S64, and the subsequent processes are repeatedly performed.",
"When the communication control section 62 determines in step S66 that the reported Bluetooth device name includes the same group ID as that of the link pin 21A, the communication control section 62 sets the unit which reported the Bluetooth device name as a unit to be connected, and the processing proceeds to step S68.In step S68, the communication control section 62 determines whether the individual ID of the link pin 21A inserted into the personal computer 4 is larger than the individual ID of the unit to be connected.",
"When it is determined that the individual ID of its own (the link pin 21A) is larger, the processing proceeds to step S69, and a connection is made to the television receiver 5 as a Bluetooth master.",
"For example, when the link pin 21A is inserted into the personal computer 4, and the Bluetooth device name such as that described above has been reported from the television receiver 5, the communication control section 62 recognizes that the individual ID of the personal computer 4 is “01” and the individual ID of the television receiver 5 is “02”.",
"Therefore, in this case, the communication control section 62 determines in step S68 that the individual ID of its own smaller than the individual ID of the television receiver 5, and the processing proceeds to step S70.The communication control section 62 connects to the television receiver 5 as a slave, and performs communications by Bluetooth.",
"The television receiver 5 performs communications with the personal computer 1 by Bluetooth as the master.",
"The processes of step S71 to step S75 are basically the same as the processes of step S47 to step S51 shown in FIG.",
"10.More specifically, in step S71, the communication control section 62 determines whether the television receiver 5 has requested the release of the connection.",
"When it is determined that the release of the connection has been requested, the processing proceeds to step S72, the connection is released, then, the processing returns to step S64, and the subsequent processes are repeatedly executed.",
"Contrarily, when the communication control section 62 determines in step S71 that the release of the connection has not been requested, the processing proceeds to step S73, and the communication control section 62 determines whether the link pin 21A has been removed.",
"When the communication control section 62 determines that the link pin 21A has not been removed, the processing returns to step S71, and the subsequent processes are executed.",
"When the communication control section 62 determines in step S73 that the link pin 21A has been removed, the processing proceeds to step S74.The communication control section 62 requests the television receiver 5 to release the connection in step S74, the processing proceeds to step S75, and the connection is released.",
"Then, the processing proceeds to step S61, and the subsequent processes are repeatedly executed.",
"As described above, even when the management server 1 is not connected, units to which link pins having the same group ID are inserted can be connected to each other.",
"In addition, even when the network 2 is a radio network such as a radio LAN and a Bluetooth, the user can easily understand connected units by referring to the colors of the inserted link pins.",
"The communication systems described above can be applied to various units.",
"For example, when a server for distributing musical contents is located at a predetermined position in a house, and a speaker is provided for each room, the user can continue to listen to the music being distributed from the server even in a room to which the user has moved by carrying a link pin and inserting it into the speaker.",
"In this case, a link pin is always inserted into the server; when the user goes out of the room, the user pulls out the link pin inserted into the speaker of the room; and the user inserts the link pin into the speaker in the room where the user moves.",
"The above described processing is executed every time when the link pin is removed and inserted.",
"The output destination of the music being distributed from the server is changed to the speaker to which the link pin is newly inserted.",
"The user having no link pin cannot access the server.",
"This condition can also be used as an access right.",
"In other words, the server administrator distributes link pins having the same group ID as that of the link pin inserted into the server to only the users to which a connection to the server is allowed.",
"In the above description, to identify the pair of the link pin 21A and the link pin 21B, and the pair of the link pin 22A and the link pin 22B, for example, the colors of the identification sections are used.",
"As shown in FIG.",
"12A to FIG.",
"12C, they can also be identified by the shapes of the identification sections or characters printed on the identification sections.",
"The identification section 31A of the link pin 21A and the identification section 31B of the link pin 21B shown in FIG.",
"12A have an almost cubic shape.",
"Connected units may be identified by the shape of the identification sections in this way.",
"On the identification section 31A of the link pin 21A and the identification section 31B of the link pin 21B shown in FIG.",
"12B, a character of “A” is printed.",
"On the identification section 31A of the link pin 21A and the identification section 31B of the link pin 21B shown in FIG.",
"12C, a character of “B” is printed.",
"Therefore, when the four link pins shown in FIG.",
"12B and FIG.",
"12C are inserted into units, the user can easily recognize connected units by checking the characters printed on the identification sections.",
"A link pin itself may be made to have a card shape, as shown in FIG.",
"13.FIG.",
"13 is a perspective view showing a part of the appearance of the VCR 3.To control a unit connection by using a link card 101A, a reader 61 such as that described above is provided, for example, at a corner of the upper surface 3B of the VCR 3.FIG.",
"14A is a view showing example appearances of the link card 101A and a link card 101B to which the same group ID as to the link card 101A has been assigned.",
"As shown in the figure, for example, circles are drawn on the surfaces of the link card 101A and the link card 101B as an identification section 111A and an identification section 111B, respectively.",
"An RF tag 112A and an RF tag 112B are disposed at the upper right of the link card 101A and at the upper right of the link card 101B, respectively, to manage the same group ID.",
"When the user wants to connect the VCR 3 and the television receiver 5, for example, the user places the link card 101A on the reader 61 of the VCR 3, and places the link card 101B on a reader provided at a predetermined position on the upper surface of the television receiver 5.With this, the units read the card IDs (group IDs and individual IDs) stored in the RF tags of the link cards to establish a connection between the VCR 3 and the television receiver 5, as described above.",
"Link cards shown in FIG.",
"14B and FIG.",
"14C have different patterns on their identification sections from those of the link card 101A and the link card 101B shown in FIG.",
"14A.",
"On the identification section 111A of the link card 101A and the identification section 111B of the link card 101B shown in FIG.",
"14B, “squares” are drawn.",
"On the identification section 111A of the link card 101A and the identification section 111B of the link card 101B shown in FIG.",
"14C, “triangles” are drawn.",
"In this way, the user can recognize connected units by checking the patterns drawn on the surfaces of link cards.",
"Not only pin-shaped and card-shaped objects, such as those described above, but also block-shaped objects can control connections.",
"An RF tag may be built in a doll to control a connection.",
"Further, to allow connected units to be more easily recognized, LEDs (light emitting diodes), which emit predetermined-color light, may be provided for the identification sections of link pins or link cards.",
"In the above description, when a link pin is inserted to a unit, the unit connects to a mating unit immediately.",
"The unit may connect to the mating unit according to an instruction of the user.",
"In the above description, when a link pin is removed from the unit, a connection to the unit is released immediately.",
"The connection to the unit may be released at predetermined timing, such as after predetermined data is sent or after an instruction is issued by the user.",
"In the above description, an RF tag provides identification information to the unit.",
"Information corresponding to a group ID such as that described above may be provided by using a memory card such as a Memory Stick (registered trademark) or by using capacitance or resistance managed by a link pin.",
"The above-described series of processing can be executed not only by hardware but also by software.",
"When the series of processing is executed by software, a program constituting the software is installed from a network or a recording medium into a computer which is built in special hardware, or into a machine, such as a general-purpose personal computer, which can execute various functions by installing various programs.",
"The recording medium is formed not only of a package medium, such as the magnetic disk 81 (including a floppy disk), the optical disk 82 (including a CD-ROM (compact disk read only memory) and a DVD (digital versatile disk)), the magneto-optical disk 83 (including an MD (registered trademark, Mini Disk)), or the semiconductor memory 84, into which the program is recorded and which is distributed to provide the user with the program separately from the apparatus body, as shown in FIG.",
"5, but also of the ROM 72, or a hard disk included in the storage section 78 which records the program, which has been embedded in advance in"
]
] | Patent_10250341 |
[
[
"Soft restraining system",
"A device for restraining a miscreant includes a bell shaped tubular body adapted to envelop the torso of a miscreant.",
"Openings at each end allow the body to be passed over the head to surround the torso of the miscreant.",
"Restraining straps are each adapted to encircle one half of the tubular body.",
"Each end of each of the straps may be locked at two diametrically opposed locations on the body.",
"To deploy the device, it is held generally above the head of the miscreant and pulled downwardly over his head and torso.",
"When the upper opening has passed the head of the miscreant to encircle his neck, one free end of each strap is pulled manually to cause lockable tightening thereof around the body of the device, thereby restraining the miscreant."
],
[
"1.A device for restraining a miscreant, said device comprising a substantially tubular body adapted to envelop the torso of a miscreant and having an upper, in use, opening adapted to be passed over the head to surround the neck of the miscreant, and a lower, in use, opening, characterised in that said body is provided with a restraint strap system which comprises at least one pair of straps each adapted to encircle substantially one half of the tubular body and locking means for each end of each of said straps disposed at two substantially diametrically opposed locations on the tubular body.",
"2.A device according to claim 1, characterised in that the straps so pass through the locking means as to be manually graspable at either free end, whereby manual traction of either free end of a strap will cause lockable tightening of that strap around part of the body.",
"3.A device according to claim 1, characterised in that the restraint strap system comprises two pairs of straps, one disposed above, in use, the other.",
"4.A device according to claim 3, characterised in that the upper, in use, pair of straps is adapted to encircle the miscreant in the region of his elbows and the lower, in use, pair of straps is adapted to encircle the miscreant in the region of his knees.",
"5.A device according to claim 1, characterised in that the body has a substantially frustoconical shape extending from a comparatively narrow upper, in use, end to a wider lower, in use, end.",
"6.A device according to claim 1, characterised in that the material of the body comprises a foraminous woven material.",
"7.A device according to claim 1, characterised in that the locking means comprises a pair of D-rings at each end of the strap.",
"8.A method of restraining a miscreant comprising the steps of providing a device according to claim 1, said device generally above the head of said miscreant, pulling the device downwardly over the head and torso of the miscreant until the opening at the upper end of the tubular body has passed the head of the miscreant, characterised by the steps of manually pulling one free end of each strap to cause lockable tightening thereof around the body of the device, thereby restraining the miscreant at at least one location.",
"9.A method according to claim 8, wherein the restraint strap system comprises two pairs of straps, one pair disposed above in use the other pair, and characterised by the further step of tightening the upper, in use, strap pair prior to tightening the lower, in use, strap pair."
],
[
"The present invention relates to a soft restraint system.",
"More particularly, but not exclusively, it relates to a soft restraint system enabling people upholding the law and other responsible personnel to restrain a troublemaker or miscreant engaged in disorderly or criminal behaviour.",
"In one example it may be used on board aircraft.",
"The term ‘air rage’ has been used in recent times to describe airline passengers who, for whatever reason, become disruptive or violent whilst an aircraft is in flight.",
"In so doing, they may annoy fellow passengers, or may even disrupt or endanger the flight.",
"This has caused airline companies considerable concern.",
"Indeed, on occasions, aircraft have been diverted to unload the miscreant.",
"The soft restraint system may of course be used physically to restrain miscreants or other troublesome individuals in many other situations, such as by police, prison officers, nurses or other personnel engaged in law enforcement operations, or in any other situation where people are likely to be faced with a person out of control or likely to do violence either to himself herself or those around him or her, such as in psychiatric hospitals, etc.",
"One increasing problem these days is that often, the staff most immediately available to deal with a miscreant are female or people less strong than the person who must be restrained.",
"At present, it is known to use a restraining set of handcuffs, but these are not easy to apply and a certain amount of force is needed to bring the miscreant's hands together before the handcuffs can be applied.",
"It is also known from our co-pending UK Patent Application No.",
"9927364.1 to provide a restraint system which includes a tubular member adapted to be placed loosely over the head and around the torso of the miscreant.",
"However, this restraint system then needs to be held in place by means of padlocks, firstly to narrow the neck opening of the tubular member and secondly to lock the lower end of the tubular member between the legs of the miscreant.",
"By use of this device, the miscreant can be rendered harmless since the hands and any weapons carried thereby will be enclosed within the tubular member.",
"However, the member is not always easy to apply, particularly if the miscreant does not wish it to be applied.",
"Finding and applying the padlocks can take valuable seconds.",
"Furthermore, the shape of the restraint system sometimes makes it difficult or awkward to apply the system over the head and shoulders of a miscreant.",
"It is an object of the invention to provide a system which overcomes the above disadvantages and which enables application of a first degree of restraint to the miscreant, i.e.",
"to make it difficult but not impossible for him to react.",
"Furthermore, it should enable application of a second degree of restraint to be applied immediately or soon thereafter so that the miscreant is restrained from further damage to himself, herself or those around him or her.",
"According to a first aspect of the present invention, there is provided a device for restraining a miscreant, said device comprising a substantially tubular body adapted to envelop the torso of a miscreant and having an upper, in use, opening adapted to be passed over the head to surround the neck of the miscreant, and a lower, in use, opening, said body being provided with a restraint strap system which comprises a pair of straps each adapted to encircle substantially one half of the tubular body and locking means for each end of each of said straps disposed at two substantially diametrically opposed locations on the tubular body.",
"Preferably, said straps so pass through the locking means as to be manually graspable at either free end, whereby manual traction of either free end of a strap will cause lockable tightening of that strap around part of the body.",
"Advantageously, the restraint strap system comprises two pairs of straps, one disposed above, in use, the other.",
"In this case, the upper, in use, pair of straps is adapted to encircle the miscreant in the region of his elbows and the lower, in use, pair of straps is adapted to encircle the miscreant in the region of his knees.",
"The body preferably has a substantially frustoconical shape extending from a comparatively narrow upper, in use, end to a wider lower, in use, end.",
"The substantially frustoconical shape may be described as a bell-shape, in which case a wide, lowermost end is adapted more easily to envelop the head and shoulders of the miscreant.",
"Also narrowing of the bell-shape towards an upper end of the body is adapted to constrict the shoulders and/or upper arms of the miscreant as the body of the device is pulled downwardly over the torso.",
"The material of the body is preferably of a foraminous woven material.",
"The locking means may comprise a pair of D-rings at each end of each strap.",
"According to a second aspect of the present invention, there is provided a method of restraining a miscreant comprising the steps of providing a device as described above, deploying said device generally above the head of said miscreant, pulling the device downwardly over the head and torso of the miscreant until the opening at the upper end of the tubular body has passed the head of the miscreant, and manually pulling one free end of each strap means to cause lockable tightening thereof around the body of the device, thereby restraining the miscreant at at least one location.",
"In the case where there are two strap systems, it is preferred that the upper, in use, strap system be tightened prior to tightening the lower, in use, strap system.",
"An embodiment of the present invention will now be more particularly described by way of example and with reference to the accompanying drawings in which: FIG.",
"1 is a front elevation of a device embodying the invention; FIG.",
"2 is a rear elevation of the device shown in FIG.",
"1; FIG.",
"3 is a scrap view of locking means for the device when in a relaxed condition to enable unlocking; FIG.",
"4 is a scrap view of the locking means when the straps are locked; and FIG.",
"5 shows an alleged miscreant suitably restrained by the device.",
"Referring now to the drawings, more particularly to FIGS.",
"1 and 2, there is shown a restraint device which comprises a substantially bell-shaped body 1 of foraminous woven material generally dimensioned to encircle the torso of a miscreant of at least average size.",
"The device may be produced in a range of sizes to suit the problem.",
"The body 1 is formed of two pieces of material joined at side seams 6a and 6b.",
"At its upper, in use, end is an opening 2, defined by a substantially inextendible seam 3, dimensioned to fit over the head of the miscreant and encircle his neck.",
"The lower, in use, end of the body 1 is of much greater circumference so as more easily to encapsulate the head and shoulders of the miscreant, in a manner which will be described below.",
"The lower opening 5 is encircled by a seam 4 of substantially inextendible material and is provided with two pairs of handles, 7a and 7b.",
"These handles 7a and 7b extend to the side seams 6a and 6b respectively and are affixed to the body and to the lower and the side seams by stitching or the like along their length.",
"This prevents tearing of the foraminous material when force is applied to the handles.",
"In order to carry out a first phase of restraint of a miscreant, an empowered operative, who may be a police officer, a prison warder, a member of airline personnel, a nurse or the like should have the device to hand.",
"It may possibly be so folded into a belt-mounted bag that it may be deployed at a moment's notice.",
"Preferably, the handles 7a and 7b protrude from the bag or are easily available for use by a pair of operatives.",
"The handles may be so colour coded or otherwise identifiable that one operative may take the pair of handles 7a and the other may take the pair of handles 7b.",
"The two operatives then place the bell mouth lower opening 5 above the head of the miscreant and bring the device 1 down sharply so that it envelops first the head and then the torso of the miscreant, with the handles 7a and 7b generally finishing in the calf region of the miscreant.",
"The upper opening 2 will at that point surround the neck of the miscreant.",
"The upper part of the bell-shape of the device 1 will tend, as it is pulled downwardly, to constrict the shoulders and upper arms of the miscreant so that it is more difficult, but not yet impossible, for the miscreant to deploy his arms aggressively.",
"At this point, it is important to move on to the second stage of restraint as soon as possible.",
"For this, there are provided two strap systems:— upper, in use, straps 8a and 8b, are located to encircle the miscreant in the region just above the elbows; and lower, in use, straps 18a and 18b, are located to encircle the miscreant in the region just above the knees.",
"The upper strap system comprises two straps 8a and 8b, each passing around a respective side of the body 1 from substantially the middle of the front to substantially the middle of the back face of the body 1.The straps 8a and 8b pass through loops 10, each fixed to a reinforcement patch 11, themselves attached to the material of the body 1.As shown, there are at least two loops 10 for each strap 8a,8b (one at the front and one at the rear), although more may be provided if so desired.",
"At the centre of both front and rear panels of the body 1 there is provided a reinforcement patch 12 to which are mounted pairs of locking D-rings 14a and 15a, 14b and 15b.",
"As shown in FIGS.",
"3 and 4, each end of each strap 8 passes through an upper D-ring 14 and then through and around a lower D-ring 15, passing back through the upper D-ring 14 and ending in a grip 9a or 9b.",
"The end of each grip 9 is folded over on itself and sewn at 16a,16b.",
"FIG.",
"3 shows this arrangement in relaxed condition where a strap may be released by passing the strap 8 through the upper D-ring 14, toward the centre of the reinforcement patch 12.FIG.",
"4 shows the arrangement when the grips 9a and 9b have been pulled outwardly to tighten the straps 8a and 8b.",
"The D-ring 15 is flipped over to trap the strap 8 between it and D-ring 14, thereby causing the strap to become locked.",
"It can be further tightened by pulling the grip 9a, but cannot be released without moving D-ring 15 back to its position as shown in FIG.",
"3.To this end, each D-ring 15 may optionally be provided with a tag or pull-string to facilitate the release process.",
"As can be seen, there are two pairs of grips 9a,9b—one pair to the front of the body 1 and one pair to the rear.",
"Pulling on either grip will cause tightening of the corresponding strap 8 around the respective side of the miscreant.",
"The other end of that strap will be or become locked by the D-rings at the other side of the body 1.Either hand of each operative may be used to pull the grip at either the front or rear of the respective side of the miscreant.",
"Ideally, both operatives will pull the grips on the same side of the body 1, since this gives a more controlled action with regard to the miscreant.",
"However, when urgent action is required, it may be necessary for one operative to pull his rear grip and the other to pull his front grip.",
"Indeed, it may be possible for a single operative to pull both grips 9a and 9b of one pair, one away from the other.",
"The lower strap system includes two straps 18a, 18b, each passing around a respective side of the body, from the middle of the front to the middle of its back.",
"The straps pass through loops 20 stitched to reinforcement patches 21 and again pass through pairs of D-rings 24a, 25a and 24b and 25b, all stitched or attached to reinforcement patch 22.The loose ends of the straps 18a and 18b form respectively pulling grips 19a and 19b.",
"Again, if each operative pulls one of the grips 19a or 19b, the straps will become tightened and locked so that the knees of the miscreant will be restrained and he will be finally unable to make nuisance of himself.",
"This situation is shown in FIG.",
"5.As has been described, the system is especially suitable for use by operatives less strong than the miscreant since they can pull downwardly against the individual's struggles upwardly.",
"In such a contest, the downward force usually wins.",
"The device of the invention is generally safe to use as it avoids restraint techniques associated with medical problems such as positional asphyxia and it is made of foraminous breathable material.",
"It is also lightweight and therefore the miscreant should not suffer from overheating or be forced to carry a heavy burden.",
"Hence, it can be retained in position for lengthy periods of time until the miscreant can be handed over to the police or be dealt with by more permanent forms of restraint, if that is appropriate.",
"The apparatus embodying the invention is very easy to use, and does not involve intensive training.",
"It can be applied quickly from a stowage container which can be carried by the person of an operative or may be located at convenient points within any space where trouble could be expected.",
"The device may be used in aeroplanes, in police cells, in prisons, in hospitals and in arrest situations and indeed anywhere where a miscreant may be breaking the law or causing civil unrest."
]
] | Patent_10250409 |
[
[
"Recombinant viral switches for the control of gene expression in plants",
"The invention describes a method of controlling a biochemical process or a biochemical cascade in plants utilizing a process of interaction between a heterologous DNA sequence in a transgenic plant, on one side, and a heterologous DNA sequence in a plant viral transfection vector, on the other.",
"Optionally, the process of interaction further involves a low molecular weight component.",
"The process of interaction makes the infection with a viral transfection vector a gene-“switch” which switches on a biochemical process or cascade of interest via various reactions such as nucleic acid recombination, replication, transcription, restriction, translation, protein folding, assembly, targeting, posttranslational processing, or enzymatic reaction.",
"Further a process for producing a product in a transgenic plant and kit of parts for such a process is provided."
],
[
"1.A process of controlling a biochemical process (II) or biochemical cascade (III) of interest in a plant, said process comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous DNA sequences; and (b) infecting the plant with at least one viral transfection vector containing in its genome one or more second heterologous DNA or RNA sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and (ii) one or more second heterologous DNA or RNA sequences of the transfection vector and/or expression products of the second heterologous DNA or RNA sequences, and (iii) optionally one or more externally added low molecular weight components, thus switching on the biochemical process (II) or biochemical cascade (II) of interest that was not operable prior to said interaction.",
"2.The process according to claim 1, wherein the process of interaction requires an expression product of a first heterologous DNA sequence stably integrated in the nuclear genome of the plant.",
"3.The process according to claim 1, wherein said interaction requires an expression product of a second heterologous DNA or RNA sequence of said transfer vector.",
"4.The process according to claim 1, wherein the infection of the plant in step (b) is achieved by an assembled virus particle or infectious viral nucleic acids, or by activating a transfection process by release of viral nucleic acids that were previously incorporated into the plant genome.",
"5.The process of claim 4, wherein said assembled virus particle or said infectious viral nucleic acid is or comprises RNA.",
"6.The process according to claim 1, wherein the infection of the plant in step (b) comprises Agrobacterium-mediated transfer of nucleic acid sequences into cells of said plant.",
"7.The process according to claim 1, wherein a further vector is introduced in step (b) and wherein a sequence and/or an expression product of said further vector is involved in said process of interaction.",
"8.The process according to claim 1, wherein the infection of the plant in step (b) is achieved by introducing one or more vectors into cells of said plant, whereby said vector(s) are adapted to undergo processing to generate said viral transfection vector in cells of said plant.",
"9.The process according to claim 1, wherein said process of interaction is a viral transfection vector-generated process.",
"10.The process according to claim 1, wherein the process of interaction involves DNA transposition.",
"11.The process according to claim 1, wherein the process of interaction involves DNA recombination.",
"12.The process according to claim 11, wherein the biochemical process or cascade of interest comprises expression of a first or second DNA or RNA sequence comprising a promoterless gene in anti-sense orientation which is placed into sense orientation towards a constitutive promoter in said process of interaction.",
"13.The process according to claim 1, wherein the process of interaction involves recognition of a heterologous promoter by a heterologous RNA polymerase.",
"14.The process according to claim 13, wherein said first and said second DNA or RNA sequence comprises a heterologous sequence to be expressed under the control of a heterologous promoter not recognized by a plant RNA polymerase, and transcription of said sequence to be expressed is switched on by interaction of said promoter with an RNA polymerase functional therewith and being encoded by said second or said first DNA sequence, respectively.",
"15.The process according to claim 14, wherein said RNA polymerase is a bacteriophage RNA polymerase and said heterologous promoter is a bacteriophage promoter.",
"16.The process according to claim 1, wherein the process of interaction involves a DNA reaction such as DNA replication, ligation, hybridisation, transcription, or DNA restriction.",
"17.The process according to claim 1, wherein the process of interaction involves an RNA reaction such as replication, processing, splicing, reverse transcription, hybridization or translation, or activiation, inhibition or modification thereof.",
"18.The process according to claim 1, wherein the process of interaction involves a protein reaction such as protein folding, assembly, activation, posttranslational modification, targeting, binding, enzymatic activity or signal transduction, or activation, inhibition or modification thereof.",
"19.The process according to claim 1, wherein (i) the biochemical process or cascade of interest comprises expression of a first or second DNA sequence separated from its promoter by a DNA insert capable of preventing transcription of the first or second DNA sequence, and (ii) the process of interaction triggered in step (b) results in the excision of the DNA insert whereby the first or second DNA sequence is expressed.",
"20.The process according to claim 19, wherein the DNA insert is a non-autonomous transposable element which is excised by a transposase (i) encoded by a second DNA sequence on the viral vector for an insert in the nuclear genome, or (ii) encoded by a first DNA sequence in the nuclear genome for an insert in the viral vector.",
"21.The process according to claim 19, wherein the DNA insert is flanked by unidirectional sites recognizable by a site-specific DNA recombinase (i) encoded by a second DNA sequence on the viral vector for an insert in the nuclear genome, or (ii) encoded by a first DNA sequence in the nuclear genome for an insert in the viral vector.",
"22.The process according to claim 1, wherein transcription of a first or a second DNA sequence is switched on by a heterologous or engineered transcription factor capable of recognizing a heterologous or engineered or chimaeric promoter operably linked to a heterologous gene of interest of said first or second DNA sequence, whereby said promoter is not recognizable by any natural plant transcription factor and said heterologous or engineered transcription factor is encoded by a second or a first DNA sequence, respectively.",
"23.The process according to claim 22, wherein the transcription factor is inducible by an externally applied low molecular weight component.",
"24.The process according claim 1, wherein said first heterologous DNA sequence of step (a) is not of plant viral origin.",
"25.A process of controlling a biochemical process (II) or biochemical cascade (III) of interest in a plant, said process comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous nucleic acid sequences; and (b) infecting the plant with at least one vector containing in its genome one or more second heterologous nucleic acid sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous nucleic acid sequences on the nuclear genome and/or expression products of the first heterologous nucleic acid sequences, and (ii) one or more second heterologous nucleic acid sequences of the trarsfection vector and/or expression products of the second heterologous nucleic acid sequences, and (iii) optionally one or more externally added low molecular weight components, whereby a viral transfection vector is generated in cells of said plant, thus switching on the biochemical process (II) or biochemical cascade (III) of interest that was not operable prior to said interaction.",
"26.The process of claim 5, wherein the process of interaction requires an expression product of a first heterologous DNA sequence stably integrated in the nuclear genome of the plant.",
"27.A process of producing a product in a transgenic plant comprising: (a) introducing into the nuclear genome of the plant one or more first heterologous DNA sequences: and (b) infecting the plant with at least one viral transfection vector containing in its genome one or more second heterologous DNA or RNA sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and (ii) one or more second heterologous DNA or RNA sequences of the transfection vector and/or expression products of the second heterologous DNA or RNA sequences, and (iii) optionally one or more externally added low molecular weight components, thus switching on the biochemical process (II) or biochemical cascade (III) of interest that was not operable prior to said interaction, thereby producing the product in the transgenic plant.",
"28.The process of claim 25 further comprising: (a) growing the transgenic plant to a desired stage; (b) infecting the plant with one or more viral transfection vectors, and optionally contacting the plant with one or more low molecular weight components, this switching on the biochemical process or cascade necessary to produce the product, said process or cascade not being operable prior to said interaction; and (c) producing the product in the plant.",
"29.Kit-of-parts for performing the process of claim 1, comprising (i) a transgenic plant or seeds thereof, and (ii) a vector, notably a viral transfection vector.",
"30.The vector for performing step (b) claim 1.31.The plant used in the process of claim 27.32.The plant used in the process of claim 28."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Controllable Transgene Expression Systems in Plants One of the major problems in plant biotechnology is the achievement of reliable control over transgene expression.",
"Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc.",
"Natl.",
"Acad.",
"Sci, 91,12760-12764; John & Keller, 1996, Proc.",
"Natl.",
"Acad.",
"Sci., 93, 12768-12773; U.S. Pat.",
"No.",
"6,103,956; U.S. Pat.",
"No.",
"5,650,555) or protein toxins (U.S. Pat.",
"No.",
"6,140,075).",
"Existing technologies for controlling gene expression in plants are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e.",
"they are “leaky”.",
"Tissue-specific promoters (U.S. Pat.",
"No.",
"5,955,361;.WO09828431) present a powerful tool but their use is restricted to very specific areas of applications, e.g.",
"for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat.",
"No.",
"5,608,152).",
"Inducible promoters can be divided into two categories according to their induction conditions—those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack.",
"Examples of the first category are heat-inducible (U.S. Pat.",
"No.",
"5,187,287) and cold-inducible (U.S. Pat.",
"No.",
"5,847,102) promoters, a copper-inducible system (Mett et al., 1993, Proc.",
"Natl.",
"Acad.",
"Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat.",
"No.",
"6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569).",
"One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95).",
"For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin.",
"Biotechnol., 11, 146-151).",
"Other examples of inducible promoters are promoters which control the expression of patogenesis-related (PR) genes in plants.",
"These promoters can be induced by treatment of the plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat.",
"No.",
"5,942,662).",
"There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat.",
"No.",
"6,093,554; U.S. Pat.",
"No.",
"5,919,705).",
"In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase.",
"The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification.",
"The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression.",
"Additionally, the use of antibiotics and steroids as chemical inducers is not desirable for the large-scale applications.",
"When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression.",
"The tissue or organ-specific promoters are restricted to very narrow areas of applications, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will.",
"Plant Viral Vectors and Their Use in the Field of Applied Plant Virology Presently, there are three distinct major fields in the area of applied plant virology: a) use of viruses as vectors for transgene overexpression; b) use of viruses as vectors for plant functional genomics, and c) use of viral components in the field of phytopathology for generating virus-resistant transgenic plants.",
"Plant viruses can serve as efficient tools for high level expression of transgenes in host plant species.",
"The use of transgenic plant virus in field does not seem to compromise any biosafety issues.",
"For example, Animal and Plant Health Inspection Service, USDA, did not find any significant impact after field trials with genetically modified TMV (tobacco mosaic virus) and tobacco etch viruses containing heterologous genes of pharmaceutical interest.",
"As a result, two permissions were issued in 1996 and 1998.Work has been conducted in the area of developing viral vectors for transferring foreign genetic material into plant hosts for the purposes of expression (U.S. Pat.",
"No.",
"4,885,248; U.S. Pat.",
"No.",
"5,173,410).",
"There are several patents which describe the first viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat.",
"No.",
"5,316,931; U.S. Pat.",
"No.",
"5,589,367; U.S. Pat.",
"No.",
"5,866,785).",
"In general, these vectors can express foreign genes from an additional subgenomic promoter (U.S. Pat.",
"No.",
"5,466,788; U.S. Pat.",
"No.",
"5,670,353; U.S. Pat.",
"No.",
"5,866,785), as translational fusions with viral proteins (U.S. Pat.",
"No.",
"5,491,076; U.S. Pat.",
"No.",
"5,977,438) or from polycistronic viral RNA using IRES elements for independent protein translation, also used herein, according to ANNEX A corresponding to German Patent Application No 10049587.7.Carrington et al., (U.S. Pat.",
"No.",
"5,491,076) describe the use of an endogenous viral protease to cleave heterologous proteins from viral polyproteins.",
"Another area of application for viral vectors is plant functional genomics.",
"Della-Cioppa et al., (WO993651) describe the use of TMV-based viral vectors for expression of plant cDNA libraries with the purpose of silencing endogenous genes.",
"Angell & Baulcombe (1997, EMBO J, 16, 3675-3684; WO9836083) describe a PVX-based system called “Amplicon™” designed for down-regulating the targeted genes in plants.",
"The same system in combination with Hc-Pro that suppresses transgene silencing in plants (Pruss et al., 1997, Plant Cell, 9, 859-868; U.S. Pat.",
"No.",
"5,939,541) is used for overexpression of transgenes.",
"U.S. Pat.",
"No.",
"5,939,541 describes an approach based on using the 5′proximal region (booster sequence, including the Hc-Pro gene) of the potyvirus to enhance expression of any gene in plants.",
"This sequence can be stably integrated into the plant genome or delivered by a virus.",
"It is worth mentioning that Hc-Pro has a pronounced pleiotropic effect and enhances the expression of both transgenes and endogenous plant genes.",
"Thus, these systems provide at best a quantitative improvement of total protein expression over existing processes.",
"They do so by influencing many components of the protein production machinery by an unknown mechanism and in a hardly controlled manner.",
"There is an abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g.",
"U.S. Pat.",
"No.",
"5,792,926; U.S. Pat.",
"No.",
"6,040,496).",
"It is also worth mentioning that an environmental risk is associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch.",
"Virol, 145, 1867-1883).",
"Therefore, it is an object of the present invention to provide an environmentally safe process of controlling a biochemical process or a biochemical cascade of interest in a plant whereby the process or cascade may be selectively switched on at any predetermined time.",
"It is another object of this invention to provide a process for producing a product in a transgenic plant wherein the production of the product may be selectively switched on after the plant has grown to a desired stage, whereby the process is environmentally safe and does not lead to the release of potentially hazardous functional transgenes in the environment.",
"Another object of this invention is to provide a kit of parts for performing such processes."
],
[
"<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG.",
"1A is a schematic representation of a process according to the invention.",
"FIG.",
"1B is a schematic representation of possible classes of processes of interaction in an infected plant cell.",
"FIG.",
"2 depicts crTMV-based vectors pIC1111 and pIC1123 containing IRES cp,148 CR -Ac transposase and and IRES mp,75 CR -Ac transposase, respectively.",
"Also shown is the T-DNA region of binary vector pSLJ744 containing p35S::Ds::GUS-3′ocs.",
"FIG.",
"3 depicts crTMV-based vectors pIC2541 and pIC2531 containing IRES cp,148 CR -Cre recombinase and and IRES mp,75 CR -Cre recombinase, respectively.",
"Also shown is the T-DNA region of the binary vector pIC2561 containing the GUS gene flanked by two loxP sites in direct orientation.",
"FIG.",
"4 depicts crTMV-based vectors pIC2541 and pIC2531 (see also FIG.",
"3 ) in combination with the T-DNA region of the binary vector pIC1641 containing the GUS gene flanked by two inverted loxP sites.",
"FIG.",
"5 depicts the T-DNA region of the binary vector pIC2691 carrying the GUS gene under control of T7 promoter and crTMV-based vector pIC2631 containing the T7 polymerase gene.",
"FIG.",
"6 shows X-gluc stained leaves of transgenic Arabidopsis plants transformed with pSLJ744.Transcription of the GUS gene is prevented by the insertion of Ds element.",
"A—leaves inoculated with the transcript from pIC1123.B—leaves inoculated with the transcript from pIC1111.C—leaves inoculated with water.",
"FIG.",
"7 depicts the TMV-based viral provectors pICH4371 and pICH4461 end of provector (RdRp: RNA dependent RNA polymerase; MP: movement protein; sGFP: synthetic green fluorescent protein; 3′NTR: 3′non-translated region of TMV; sgp: subgenomic promoter).",
"FIG.",
"8 depicts the T-DNA of binary vector pICH1754 providing a Cre recombinase expression cassette.",
"FIG.",
"9 depicts a scheme of formation of viral vectors from provectors in the presence of Cre recombinase.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION The present invention relates to a process of controlling a biochemical process or biochemical cascade of interest in a plant according to the preamble of claim 1.Moreover, the present invention relates to a process for producing a product in a transgenic plant by using the process of controlling a biochemical process or biochemical cascade of interest according to the invention.",
"Further, the present invention relates to a kit-of-parts for performing the processes of the invention.",
"The process of the invention allows for the selective control of transgene expression in a transgenic plant whereby a biochemical process or biochemical cascade of interest previously non-operable in the plant may be selectively switched on at any predetermined time.",
"BACKGROUND OF THE INVENTION Controllable Transgene Expression Systems in Plants One of the major problems in plant biotechnology is the achievement of reliable control over transgene expression.",
"Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc.",
"Natl.",
"Acad.",
"Sci, 91,12760-12764; John & Keller, 1996, Proc.",
"Natl.",
"Acad.",
"Sci., 93, 12768-12773; U.S. Pat.",
"No.",
"6,103,956; U.S. Pat.",
"No.",
"5,650,555) or protein toxins (U.S. Pat.",
"No.",
"6,140,075).",
"Existing technologies for controlling gene expression in plants are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e.",
"they are “leaky”.",
"Tissue-specific promoters (U.S. Pat.",
"No.",
"5,955,361;.WO09828431) present a powerful tool but their use is restricted to very specific areas of applications, e.g.",
"for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat.",
"No.",
"5,608,152).",
"Inducible promoters can be divided into two categories according to their induction conditions—those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack.",
"Examples of the first category are heat-inducible (U.S. Pat.",
"No.",
"5,187,287) and cold-inducible (U.S. Pat.",
"No.",
"5,847,102) promoters, a copper-inducible system (Mett et al., 1993, Proc.",
"Natl.",
"Acad.",
"Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat.",
"No.",
"6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569).",
"One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95).",
"For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin.",
"Biotechnol., 11, 146-151).",
"Other examples of inducible promoters are promoters which control the expression of patogenesis-related (PR) genes in plants.",
"These promoters can be induced by treatment of the plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat.",
"No.",
"5,942,662).",
"There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat.",
"No.",
"6,093,554; U.S. Pat.",
"No.",
"5,919,705).",
"In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase.",
"The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification.",
"The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression.",
"Additionally, the use of antibiotics and steroids as chemical inducers is not desirable for the large-scale applications.",
"When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression.",
"The tissue or organ-specific promoters are restricted to very narrow areas of applications, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will.",
"Plant Viral Vectors and Their Use in the Field of Applied Plant Virology Presently, there are three distinct major fields in the area of applied plant virology: a) use of viruses as vectors for transgene overexpression; b) use of viruses as vectors for plant functional genomics, and c) use of viral components in the field of phytopathology for generating virus-resistant transgenic plants.",
"Plant viruses can serve as efficient tools for high level expression of transgenes in host plant species.",
"The use of transgenic plant virus in field does not seem to compromise any biosafety issues.",
"For example, Animal and Plant Health Inspection Service, USDA, did not find any significant impact after field trials with genetically modified TMV (tobacco mosaic virus) and tobacco etch viruses containing heterologous genes of pharmaceutical interest.",
"As a result, two permissions were issued in 1996 and 1998.Work has been conducted in the area of developing viral vectors for transferring foreign genetic material into plant hosts for the purposes of expression (U.S. Pat.",
"No.",
"4,885,248; U.S. Pat.",
"No.",
"5,173,410).",
"There are several patents which describe the first viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat.",
"No.",
"5,316,931; U.S. Pat.",
"No.",
"5,589,367; U.S. Pat.",
"No.",
"5,866,785).",
"In general, these vectors can express foreign genes from an additional subgenomic promoter (U.S. Pat.",
"No.",
"5,466,788; U.S. Pat.",
"No.",
"5,670,353; U.S. Pat.",
"No.",
"5,866,785), as translational fusions with viral proteins (U.S. Pat.",
"No.",
"5,491,076; U.S. Pat.",
"No.",
"5,977,438) or from polycistronic viral RNA using IRES elements for independent protein translation, also used herein, according to ANNEX A corresponding to German Patent Application No 10049587.7.Carrington et al., (U.S. Pat.",
"No.",
"5,491,076) describe the use of an endogenous viral protease to cleave heterologous proteins from viral polyproteins.",
"Another area of application for viral vectors is plant functional genomics.",
"Della-Cioppa et al., (WO993651) describe the use of TMV-based viral vectors for expression of plant cDNA libraries with the purpose of silencing endogenous genes.",
"Angell & Baulcombe (1997, EMBO J, 16, 3675-3684; WO9836083) describe a PVX-based system called “Amplicon™” designed for down-regulating the targeted genes in plants.",
"The same system in combination with Hc-Pro that suppresses transgene silencing in plants (Pruss et al., 1997, Plant Cell, 9, 859-868; U.S. Pat.",
"No.",
"5,939,541) is used for overexpression of transgenes.",
"U.S. Pat.",
"No.",
"5,939,541 describes an approach based on using the 5′proximal region (booster sequence, including the Hc-Pro gene) of the potyvirus to enhance expression of any gene in plants.",
"This sequence can be stably integrated into the plant genome or delivered by a virus.",
"It is worth mentioning that Hc-Pro has a pronounced pleiotropic effect and enhances the expression of both transgenes and endogenous plant genes.",
"Thus, these systems provide at best a quantitative improvement of total protein expression over existing processes.",
"They do so by influencing many components of the protein production machinery by an unknown mechanism and in a hardly controlled manner.",
"There is an abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g.",
"U.S. Pat.",
"No.",
"5,792,926; U.S. Pat.",
"No.",
"6,040,496).",
"It is also worth mentioning that an environmental risk is associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch.",
"Virol, 145, 1867-1883).",
"Therefore, it is an object of the present invention to provide an environmentally safe process of controlling a biochemical process or a biochemical cascade of interest in a plant whereby the process or cascade may be selectively switched on at any predetermined time.",
"It is another object of this invention to provide a process for producing a product in a transgenic plant wherein the production of the product may be selectively switched on after the plant has grown to a desired stage, whereby the process is environmentally safe and does not lead to the release of potentially hazardous functional transgenes in the environment.",
"Another object of this invention is to provide a kit of parts for performing such processes.",
"GENERAL DESCRIPTION OF THE INVENTION These objects are achieved by a process according to claim 1.More specifically, these objects are achieved by a process of controlling a biochemical process or biochemical cascade of interest in a plant, said process being characterized by comprising the following steps: (a) introducing into the nuclear genome of the plant one or more first heterologous DNA sequences, (b) infecting the plant with at least one viral transfection vector containing in its genome one or more second heterologous DNA sequences, thus triggering a process of interaction in the plant between (i) one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and (ii) one or more second heterologous DNA sequences of the transfection vector and/or expression products of the second heterologous DNA sequences, and (iii) optionally one or more externally added low molecular weight components, thus switching on the biochemical process or cascade of interest that was not operable prior to said interaction.",
"Preferably, said first heterologous DNA sequence(s) in the above processes are of non-plant viral origin, i.e.",
"do not originate from a plant virus.",
"The present invention further provides a process of producing a product in a transgenic plant comprising the process of controlling a biochemical process or a biochemical cascade of interest in a plant according to the invention.",
"In particular, the process further comprises the following steps: (a) growing the transgenic plant to a desired stage, followed by (b) infecting the plant with one or more vectors, and optionally contacting the plant with one or more low molecular weight components, thus switching on the biochemical process or cascade necessary for the production of the product, said process or cascade not being operable prior to said interaction, and (c) producing the product in the plant, whereby said vector is preferably a viral transfection vector.",
"Further a kit of parts is provided for the above processes comprising a transgenic plant or seeds thereof and a virus-based vector.",
"Also, a kit of parts is provided comprising a transgenic plant and one or more vectors, whereby said vector(s) may give rise to one or more viral transfection vectors in a plant.",
"Said transgenic plant preferably contains a first heterologous DNA sequence according to step (a) of the process of the invention.",
"Further, a vector for performing step (b) of the process of the invention and a plant obtained or obtainable by the process of the invention is provided.",
"According to the invention it is possible to selectively switch on a biochemical process or biochemical cascade in a transgenic plant by infecting the transgenic plant with one or more viral transfection vectors.",
"The biochemical process or cascade is not operable in the transgenic plant prior to the infection with the viral vector for lack of essential elements or functions necessary to perform the biochemical process or cascade.",
"Essential elements may be e.g.",
"a promoter, an RNA polymerase, a transcription factor or the like.",
"Essential functions may be transcription, translation or enzymatic activity which is not operable e.g.",
"for lack of functional coupling of a promoter with a downstream sequence to be expressed.",
"The biochemical process or cascade becomes operable by a process of interaction triggered by the infection.",
"The process of interaction in the plant requires one or more first heterologous DNA sequences of the nuclear genome and/or expression products of the first heterologous DNA sequences, and one or more second heterologous DNA sequences of the transfection vector and/or expression products of the second heterologous DNA sequences, and optionally one or more externally added low molecular weight components.",
"Preferably the process of interaction switching on the biochemical process or cascade of interest requires one first heterologous DNA sequence of the nuclear genome and/or expression product of the first heterologous DNA sequence, and one second heterologous DNA sequence of the transfection vector and/or expression product of the second heterologous DNA sequence.",
"The DNA sequences used according to the invention may be obtained via the use of RNA sequences.",
"Specifically, the DNA sequences of steps (a) or (b) may be an expression product of RNA sequences, e.g.",
"of an RNA virus.",
"In the absence of any one of the first and second heterologous DNA sequences or expression products of the first and second heterologous DNA sequences required for the process of interaction, none of the present first and second heterologous DNA sequences, expression products of the first and second heterologous DNA sequences or the externally added low molecular weight components are able, alone or in combination, to switch on the biochemical process or cascade of interest.",
"Moreover, the biochemical process or cascade of interest is not a process which has been silenced by a mechanism such as post-transcriptional gene silencing which may be still operating at a low level.",
"The biochemical process or biochemical cascade of interest is not operable in the transgenic plant prior to the infection with a corresponding viral transfection vector and prior to the optional addition of a low molecular weight component.",
"Moreover, a viral transfection vector according to the invention is unable to switch on the biochemical process or biochemical cascade of interest in a plant not having the corresponding first heterologous DNA sequence required according to the invention.",
"Finally, the biochemical process or cascade of interest cannot be switched on in a plant by contacting the plant with a low molecular weight component in the absence of the first and second heterologous DNA sequences or expression products required for switching on the process or cascade of interest according to the invention.",
"The process of the invention provides control over a biochemical process or cascade of interest with a hitherto unattainable technical precision and environmental safety.",
"Thereby novel applications in plant biotechnology are available for solving problems which cannot be solved by conventional technologies involving basal transgene expression activity in the plant, particularly when producing toxic substances or biodegradable polymers.",
"Moreover, the precise control according to the invention allows to grow a transgenic plant to a desired stage where the plant is best suited for performing the biochemical process or cascade of interest without burdening the plant with a basal expression activity slowing down the growth of the plant.",
"Once the plant is ready for efficiently performing the biochemical process or cascade of interest, the process or cascade of interest may be switched on and performed with high efficiency.",
"Accordingly, the process of the invention allows to safely decouple the growth phase and the production phase of a transgenic plant.",
"Moreover, it is possible to design multi-component systems for multiple biochemical processes or cascades of interest, whereby one or more desired processes or cascades can be selectively switched on.",
"In a first embodiment, the system comprises a transgenic plant containing a heterologous DNA sequence providing an expression product which is necessary to control expression of a desired product encoded by a viral transfection vector.",
"The system further comprises different viral vectors each encoding a different product to be expressed in the transgenic plant.",
"Thereby, it is possible to safely use the same transgenic plant for the production of different products depending on the viral vector used.",
"The advantage of this system is clear in the light of the fact that it may take years to provide a stably transformed transgenic plant whereas the preparation of a viral vector may be accomplished in a few weeks.",
"In a second embodiment, the transgenic plant contains multiple heterologous DNA sequences which may encode for different desired gene products.",
"Each of the multiple heterologous DNA sequences may be controlled by a different viral vector.",
"Thereby, it is possible to selectively control the heterologous DNA sequences of the transgenic plant by the choice of the corresponding viral vector.",
"Moreover, in a third embodiment, it is possible to design a system wherein the process of interaction switching on the biochemical process or cascade of interest requires the infection with more than one viral vector and the optional application of one or more externally added low molecular weight components whereby present technology is even safer to operate.",
"A biochemical process or cascade to be controlled according to the invention may be any process or cascade which may take place in a living plant system.",
"Preferred biochemical processes or cascades lead to the production of a product in the plant.",
"Examples of products of interest which may be obtained by the process of this invention include polypeptides or proteins as primary products, (posttranslationally) modified or otherwise processed proteins which may be enzymes, proteins having a desired glycosylation pattern, non-proteinaceous low-molecular weight products and oligomerisation products thereof like carbohydrates or biodegradable plastics etc.",
"Most preferred are pharmaceutical polypeptides.",
"Said biochemical process or cascade, notably expression of a protein, may involve formation of sub-genomic RNA, notably from a viral transfection vector.",
"The process of controlling a biochemical process or cascade involves at least the following two components: (1) a transgenic plant containing a first heterologous DNA sequence preferably not originating from a plant virus and (2) a viral transfection vector containing a second heterologous DNA sequence.",
"For the purposes of this invention, a heterologous DNA sequence is a sequence which neither occurs naturally in the plant species employed nor in the wild-type virus on which the viral vector is based on, respectively.",
"Nevertheless, such a sequence may comprise sequence portions native to the plant and/or the virus of interest besides heterologous portions.",
"Said heterologous DNA sequence may comprise more than one functional element.",
"Examples of such functional elements include promoter, enhancer, transcription termination region, coding region, non-translated spacer region, translation initiation region, IRES (internal ribosome entry site) region, stop codon etc.",
"or parts thereof.",
"Said first heterologous DNA sequence is preferably heterologous to said host plant.",
"Said first heterologous DNA sequence may be of viral origin.",
"Preferably, however, said first heterologous DNA sequence is of non-plant viral origin, i.e it is not of plant viral origin.",
"Said second heterologous DNA (or RNA) sequence is preferably heterologous to the virus on which the viral vector is based on.",
"Said second heterologous DNA sequence may be of plant origin.",
"Infection of the transgenic plant (1) with a viral vector (2) triggers a process of interaction between said at least first and second heterologous DNA sequence or expression product(s) thereof, thus switching on the biochemical process or cascade of interest.",
"The fact that the at least two components (1) and (2) are required means that interaction of said components is a necessary condition for switching on said biochemical process or cascade.",
"Prior to said interaction, said biochemical process is not operable whereby “leaky” expression of a transgene cannot occur.",
"In prior art systems, expression of a transgene can merely be induced by a quantitative increase or an enhancement of an already existing, albeit lower, expression level.",
"The present invention not only provides a quantitative increase but also a qualitative change in that a previously not operable process or cascade becomes operable.",
"This advantage of the present invention is of particular importance when a biochemical process or cascades of interest involves formation of a toxic or growth-retarding product.",
"According to the invention it is possible to entirely separate plant growth and production of said product whereby interference with or retardation of plant growth by the presence of the desired product in the growing plant is avoided.",
"Therefore, the stages of biomass accumulation and production of a product of interest may be decoupled.",
"The transgenic plant and the transgenic vector of the invention are not functional for controlling a biochemical process or biochemical cascade with viruses or plants not containing the corresponding heterologous DNA sequences, respectively.",
"Consequently, this invention represents a significant progress in terms of biological safety in plant biotechnology.",
"Said processes of interaction which are triggered by infecting the transgenic plant with a viral vector and which lead to switching on of a biochemical process include DNA recombination, DNA replication, transcription, restriction, ligation, hybridisation, RNA replication, reverse transcription, RNA processing, splicing, translation, protein folding, assembly, targeting, posttranslational processing, enzymatic activity.",
"Said expression products of said first or said second heterolgous DNA sequence include RNA, notably mRNA, and polypeptides or proteins.",
"Said process of interaction between said first and said second heterologous sequences (and optionally further sequences) does preferably not include complementation (genetic reassembly) of viral functions or of an infectious viral vector.",
"This invention preferably relates to multicellular plants.",
"Examples for plant species of interest are monocotyledonous plants like wheat, maize, rice, barley, oats, millet and the like or dicotyledonous plants like rape seed, canola, sugar beet, soybean, peas, alfalfa, cotton, sunflower, potato, tomato, tobacco and the like.",
"The fact that there are specific viruses for each of such plants, contributes to the broad versatility and applicability of this invention.",
"The viral transfection vector used in this invention may be derived from any such plant specific virus.",
"The viral vector may be based on an RNA or on a DNA double-stranded or single-stranded virus.",
"Specific examples of viral transfection vectors are given below and in ANNEX A and ANNEX B.",
"In step (a), the plant may be a natural plant or a genetically modified plant.",
"The genetic modification may be either in the nuclear genome of the plant or in an organelle genome such as plastid or mitochondria genome.",
"In step (a) a heterologous sequence is introduced in the nuclear genome, and preferably a stable genome modification is provided.",
"Step (a) may be carried out more than once in order to introduce more than one heterologous DNA sequence.",
"In this way several heterologous functions may be introduced in the target plant e.g.",
"for engineering a whole biochemical pathway.",
"In step (b), the transgenic plant obtained according to step (a) is infected with a viral transfection vector.",
"The infecting may be achieved by supplying the plant with an assembled virus particle, or with infectious viral nucleic acids, or by activating a transfection process by release of viral nucleic acids previously incorporated into the plant genome.",
"The assembled virus particle may contain RNA and the infectious viral nucleic acids may be RNA, notably if they are based on an RNA virus (cf.",
"examples 2 and 3).",
"More than one vector may be used to control the biochemical process or biochemical cascade of interest.",
"Preferably, only one vector containing the desired heterologous sequence(s) is used for reasons of reproducibility of the process.",
"Infection may be done by contacting the viral vector with said transgenic plant.",
"Preferably, mechanical stimulation like rubbing or scatching of leaves or other plant tissue may be used to initiate infection.",
"Infection may also be achieved by activating the viral vector previously integrated in the genome of the host plant.",
"Viral vectors capable of systemic infection of the plant are preferred.",
"The infection of the plant in step (b) may further comprise Agrobacterium-mediated transfer of nucleic acid sequences into cells of said plant.",
"Agrobacterium-mediated transfer may e.g.",
"be used to integrate sequences into the genome of the host plant.",
"A viral vector may be activated from such sequences integrated the genome of the plant.",
"An RNA virus-based vector may e.g.",
"be activated by transcribing a cDNA copy of said virus, notably by transcribing a cDNA copy integrated into the genome.",
"However, integration of sequences introduced into plant cells by Agrobacterium-mediated transfer do not have to lead to integration into the genome.",
"Agrobacterium-mediated transfer may provide transient expression of a gene flanked by T-DNA.",
"Notably, sequences on a Ti-plasmid may exert a function in the process of the invention without or before integration into the genome.",
"If more than one vector is introduced in step (b), the same or different methods may be used for these vectors.",
"Notably, more than one vector may be introduced by Agrobacterium-mediated transfer using different Agrobacterium strains simultaneously (e.g.",
"using an Agrobacterium mixture) or consecutively.",
"In one embodiment of the invention, a further vector in addition to said viral infection vector may be introduced in step (b) of the process of the invention.",
"Said further vector may be or may not be a viral transfection vector.",
"Said further vector may provide a further nucleic acid sequence as a necessary condition for switching on said biochemical process of the invention (cf.",
"example 6).",
"In another embodiment of the invention, infecting the plant in step (b) is achieved by introducing one or more vectors into cells of said plant, whereby said vector(s) are adapted to undergo processing to generate a viral transfection vector in cells of said plant.",
"Three, four or more vectors may be introduced in cells of said plant in this embodiment.",
"Preferably, two vectors are introduced.",
"Said vectors may or may not be viral transfection vectors.",
"Preferably, at least one of said vectors is a viral transfection vector (cf.",
"example 6).",
"However, according to this embodiment, a viral transfection vector may also be generated from introduced vectors none of which is a viral transfection vector.",
"Said biochemical process or pathway may be switched on by the assembly and appearance of said viral transfection vector in cells of the plant by said processing, triggered by measures (a) and (b) of the process of the invention.",
"Steps (a) and (b) of the process of this invention may be carried out on the same plant.",
"However, it is preferred that a stable plant line is obtained according to conventional processes based on a plant in which at least one first heterologous DNA sequence of interest was introduced according to step (a).",
"Transgenic plants may then be grown from seeds of a stably transformed plant, and infection according to step (b) may be performed when initiation of said biochemical process is desired.",
"Step (b) is preferably carried out in a greenhouse.",
"A viral transfection vector is a nucleic acid (RNA or DNA) or nucleoprotein which upon invading a wild type or genetically engineered host is capable of replication or amplification in cells of said host and of amplification and/or expression of heterologous sequence(s) of interest.",
"Preferably, said viral transfection vector is further capable of cell to cell movement.",
"More preferably, a viral vector retains additional viral capabilities such as long distance movement, assembly of viral particles or infectivity.",
"In the process of this invention, a viral vector might not have all the properties mentioned above, but such functions can be provided in trans in the context of host cell.",
"Preferred viral transfection vectors encode and express a movement protein.",
"Further, they may encode a virus-specific DNA or RNA polymerase (replicase); a RNA-dependent RNA polymerase (RdRp) is preferred.",
"In a first specific embodiment of this invention, the biochemical process of interest is expression of a heterologous DNA sequence of interest.",
"This process may be called primary biochemical process.",
"This primary process results in an RNA or polypeptide molecule.",
"In the simplest case, one of the RNA or polypeptide molecule is the product of interest.",
"In a biochemical cascade, the product of such a primary process may cause a secondary biochemical process e.g.",
"by way of its catalytic activity or by way of regulating the other biochemical process.",
"In a biochemical cascade, more than one biochemical process takes place, whereby each such process depends on a previous biochemical process.",
"Said controlling or switching is preferably directed to said primary biochemical process in this embodiment.",
"In the first specific embodiment, the heterologous DNA sequence of interest which is to be expressed, may either be a first heterologous DNA sequence of the plant nuclear genome or a second heterologous DNA (or RNA) sequence of said viral transfection vector.",
"In a second specific embodiment of this invention, the biochemical process of interest is the production of non-proteinaceous compound of interest by the plant.",
"In a further specific embodiment of the invention, a process of controlling a biochemical process (II) or biochemical cascade (III) of interest in a plant is provided, said process being characterized by comprising the following steps: (a) introducing into the nuclear genome of the plant one or more first heterologous nucleic acid sequences, (b) infecting the plant with at least one vector containing in its genome one or more second heterologous nucleic acid sequences, thus triggering a process of interaction (I) in the plant between (i) one or more first heterologous nucleic acid sequences of the nuclear genome and/or expression products of the first heterologous nucleic acid sequences, and (ii) one or more second heterologous nucleic acid sequences of the transfection vector and/or expression products of the second heterologous nucleic acid sequences, and (iii) optionally one or more externally added low molecular weight components, whereby a viral transfection vector is generated in cells of said plant, thus switching on the biochemical process (II) or biochemical cascade (III) of interest that was not operable prior to said interaction.",
"Further, specific embodiments of this process may be as described above, where applicable.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1A is a schematic representation of a process according to the invention.",
"FIG.",
"1B is a schematic representation of possible classes of processes of interaction in an infected plant cell.",
"FIG.",
"2 depicts crTMV-based vectors pIC1111 and pIC1123 containing IREScp,148CR-Ac transposase and and IRESmp,75CR-Ac transposase, respectively.",
"Also shown is the T-DNA region of binary vector pSLJ744 containing p35S::Ds::GUS-3′ocs.",
"FIG.",
"3 depicts crTMV-based vectors pIC2541 and pIC2531 containing IREScp,148CR-Cre recombinase and and IRESmp,75CR-Cre recombinase, respectively.",
"Also shown is the T-DNA region of the binary vector pIC2561 containing the GUS gene flanked by two loxP sites in direct orientation.",
"FIG.",
"4 depicts crTMV-based vectors pIC2541 and pIC2531 (see also FIG.",
"3) in combination with the T-DNA region of the binary vector pIC1641 containing the GUS gene flanked by two inverted loxP sites.",
"FIG.",
"5 depicts the T-DNA region of the binary vector pIC2691 carrying the GUS gene under control of T7 promoter and crTMV-based vector pIC2631 containing the T7 polymerase gene.",
"FIG.",
"6 shows X-gluc stained leaves of transgenic Arabidopsis plants transformed with pSLJ744.Transcription of the GUS gene is prevented by the insertion of Ds element.",
"A—leaves inoculated with the transcript from pIC1123.B—leaves inoculated with the transcript from pIC1111.C—leaves inoculated with water.",
"FIG.",
"7 depicts the TMV-based viral provectors pICH4371 and pICH4461 end of provector (RdRp: RNA dependent RNA polymerase; MP: movement protein; sGFP: synthetic green fluorescent protein; 3′NTR: 3′non-translated region of TMV; sgp: subgenomic promoter).",
"FIG.",
"8 depicts the T-DNA of binary vector pICH1754 providing a Cre recombinase expression cassette.",
"FIG.",
"9 depicts a scheme of formation of viral vectors from provectors in the presence of Cre recombinase.",
"Appendices 1 to 11 depict vectors and constructs used in example 6.DETAILED DESCRIPTION OF THE INVENTION As shown by FIG.",
"1A, the present invention provides a process of controlling a biochemical process or biochemical cascade of interest in a plant whereby the process comprises a process of interaction (I), switching on a biochemical process of interest (II), which in turn may be causal for a biochemical cascade of interest (III).",
"The process of interaction may involve any one of the following reactions or combinations of the reactions of DNA, RNA and Proteins.",
"DNA reactions contemplated in this invention are restriction, recombination, replication, transposition, amplification, and transcription.",
"RNA reactions contemplated in this invention are RNA processing, replication, reverse transcription, hybridisation, and translation.",
"Protein reactions contemplated in this invention are protein processing, folding, assembly, post-translational modifications, activation, targeting, binding activity modification, signal transduction.",
"Process (III) may be present or absent.",
"The production of a product is the preferred result of the process of the invention.",
"As shown by FIG.",
"1B, possible processes of interaction may belong to one or more of the classes of interaction shown by the figure.",
"In the figure, a transgenic plant cell infected with a viral transfection vector is shown schematically.",
"The recombinant plant genome contains one or more heterologous DNA sequences which may lead to one or more expression products.",
"The genome of the viral transfection vector contains one or more heterologous DNA sequences which may lead to one or more expression products.",
"The transgenic plant cell may be contacted with one or more low molecular weight components capable of entering the cell.",
"In the process of interaction switching on the biochemical process or biochemical cascade of interest in the plant cell, the following interactions may occur which are indicated by arrows in FIG.",
"1B.",
"One or more heterologous DNA sequences of the recombinant plant genome may interact with one or more heterologous DNA sequences of the viral transfection vector.",
"One or more heterologous DNA sequences of the recombinant plant genome may interact with one or more expression products of the heterologous DNA sequences of the viral transfection vector.",
"One or more expression products of the heterologous DNA sequences of the recombinant plant genome may interact with one or more expression products of the heterologous DNA sequences of the viral transfection vector.",
"The expression product may be an RNA or a polypeptide.",
"Any of these interactions may also involve or require the presence of one or more low molecular weight components added externally to the infected transgenic plant cell.",
"The low molecular weight components may be necessary or desired for switching on or for promoting the biochemical process or biochemical cascade of interest.",
"The low molecular weight components are unable to switch on the processes or cascades of interest in the absence of the viral transfection vector or the heterologous DNA sequence in the plant nuclear genome.",
"According to the first specific embodiment of this invention, a novel process to achieve transfection-based reliable control over either the expression of a transgene stably integrated into a plant, or over expression of a heterologous gene of a viral vector inside a transgenic plant host is provided.",
"This process makes use of an interaction of at least two heterologous DNA sequences or expression products thereof, which is triggered only when the virus vector infection process is initiated.",
"One of these sequences may be stably incorporated in the plant nuclear genome and the other one is provided by said viral vector.",
"Such a switchable two-component expression system can be used to control a biochemical process or cascade that may be controlled at various levels, e.g.",
"by triggering interaction reactions such as, but not limited to: DNA recombination, replication, transcription, restriction, RNA replication, reverse transcription, processing, translation, protein folding, assembly, targeting, posttranslational processing, enzymatic activity, etc.",
"This process requires at least a heterologous DNA in a transgenic plant and a recombinant virus-based vector comprising a heterologous DNA or RNA sequence.",
"The general scheme of this process is shown in FIG.",
"1.A transgenic plant containing in its nuclear genome one or more stably integrated heterologous DNA sequence(s) of interest can be engineered using standard transcriptional or translational vectors and standard transformation protocols.",
"Construction of transcriptional vectors for stable plant transformation has been described by numerous authors (for review, see Hansen & Wright, 1999, Trends in Plant Science, 4, 226-231; Gelvin, S. B., 1998, Curr.",
"Opin.",
"Biotech., 9, 227-232).",
"The basic principle of all these constructs is identical: a fully functional transcription unit consisting of, in 5′ to 3′ direction, a plant-specific promoter, the structural part of a gene of interest and a transcriptional terminator has to be introduced into the plant cell and stably integrated into the genome in order to achieve expression of the gene of interest.",
"Construction of translational plant vectors is described in German Patent Application Nos.",
"100 49 587.7 and 10061150.8 (ANNEX A and ANNEX B).",
"The principal difference to transcriptional vectors is that translational vectors do not require a transcriptional promoter for expression of the gene of interest but rely on the plant transcription machinery following their integration into plant genome.",
"Different methods may be used for the delivery of an expression vector into plant cells such as direct introduction of said vector into the cells by the means of microprojectile bombardment, electroporation or PEG-mediated transformation of protoplasts.",
"Agrobacterium-mediated plant transformation also represents an efficient way of vector delivery.",
"Thus, DNA may be transformed into plant cells by various suitable technologies such as by a Ti-plasmid vector carried by Agrobacterium (U.S. Pat.",
"No.",
"5,591,616; U.S. Pat.",
"No.",
"4,940,838; U.S. Pat.",
"No.",
"5,464,763), particle or microprojectile bombardment (U.S. Pat.",
"No.",
"5,100,792; EP 00444882B1; EP 00434616B1).",
"Agrobacterium can serve not only for stable nuclear transformation, but also for an efficient delivery of T-DNA for transient expression of gene(s) of interest.",
"This so called agroinfiltration protocol was first developed to analyze foreign genes expression and gene silencing in plants (Kaplia et al., 1997, Plant Science, 122, 101-108; Schöb et al., 1997, Mol.",
"Gen. Genet, 256, 581-588).",
"In principle, other plant transformation methods can also be used e.g.",
"microinjection (WO 09209696; WO 09400583A1; EP 175966B1), electroporation (EP00564595B1; EP00290395B1; WO 08706614A1) etc.",
"The choice of the transformation method depends on the plant species to be transformed.",
"For example, microprojectile bombardment may be preferred for monocots transformation, while for dicots, Agrobacterium-mediated transformation gives generally better results.",
"Construction of plant viruses for the expression of non-viral genes in plants has been described in several papers (Dawson et al., 1989, Virology, 172, 285-293; Brisson et al., 1986, Methods in Enzymology, 118, 659; MacFarlane & Popovich, 2000, Virology, 267, 29-35; Gopinath et al., 2000, Virology, 267, 159-173; Voinnet et al., 1999, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 96, 14147-14152) and can be easily performed by those skilled in the art.",
"In one specific embodiment of our invention, the transgene in a plant genome is separated from its promoter by a DNA insert sufficiently long to prevent the transcription of said transgene (FIGS.",
"2, 3).",
"Said DNA insert may be, for example, a non-autonomous transposable element or any DNA fragment flanked by unidirected sites recognizable by a site-specific DNA recombinase.",
"The appropriate transposase or site-specific DNA recombinase may be delivered by a viral vector which functions as a vector switch (FIGS.",
"2, 3).",
"After expression of said vector-encoded transposase or recombinase, the catalytic activity of such an enzyme leads to excision of the DNA insert or fragment that was separating the promoter from the transgene switching on expression of the transgene (FIG.",
"6).",
"Site-specific recombinases/integrases from bacteriophages and yeasts are widely used for manipulating DNA in vitro and in plants.",
"Preferred recombinases-recombination sites for the use in this invention are the following: Cre recombinase-LoxP recombination site, FLP recombinase-FRT recombination sites, R recombinase-RS recombination sites, etc.",
"Transposons are widely used for the discovery of gene function in plants.",
"Preferred transposon systems for use in the present invention include Ac/Ds, En/Spm, transposons belonging to “mariner” family, etc.",
"In another embodiment of this invention, the transgenic plant may carry a promoterless transgenic sequence flanked by two inverted loxP sites (FIG.",
"4).",
"Such an orientation of recombination sites may lead to the inversion of the flanked DNA sequence when exposed to Cre recombinase.",
"As a consequence of such an inversion, a promoterless gene withnos terminator will be placed from anti-sense in sense orientation towards the constitutive promoter.",
"Example 4 exemplifies this approach using a promoterless GUS gene with a nos terminator.",
"Another embodiment of this invention describes the possibility to assemble a functional viral vector construct in vivo in an engineered plant cell.",
"This means required elements of a viral vector (precursors) are delivered separately with two (FIG.",
"7) or more constructs into the plant cell.",
"After e.g.",
"Agrobacterium tumefaciens mediated delivery of such precursors into a plant cell expressing the site-specific DNA recombinase (FIG.",
"8), site specific recombination can lead to the assembly of functional viral vector expressing transgene of interest (example 6, FIG.",
"9).",
"Heterologous transcription factors and RNA polymerases may also be used as transgene switches.",
"This approach is demonstrated in Example 5 wherein a transgenic plant carries the GUS gene under control of a bacteriophage T7 promoter (see FIG.",
"5).",
"No GUS expression can be detected in transgenic Arabidopsis containing such construct as the plant RNA polymerases do not recognize prokaryotic promoters.",
"Viral delivery of the bacteriophage T7 RNA polymerase triggers expression of the GUS gene (FIG.",
"5).",
"The expression of a plant transgene that is under control of a bacteriophage promoter (e.g.",
"T3, T7, SP6, K11) with the corresponding DNA/RNA polymerase delivered by a viral vector may be another efficient approach for the development of transgene switches contemplated in this invention.",
"Another useful approach may be the use of heterologous or chimaeric or other artificial promoters which require heterologous or engineered transcription factors for their activation.",
"In some cases, the existing inducible systems for transgene expression may be used.",
"Examples are the copper-controllable (Mett et al., 1993, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA., 90, 4567-4571) and the ethanol-inducible gene expression systems (Caddick et al., 1998, Nature Biotech., 16, 177-180) which may be modified such that the transcription factors (ACE1 for copper-inducible or ALCR for the ethanol-inducible system) are provided in trans by viral delivery, thus further reducing the leakiness of the expression systems.",
"Alternatively, heterologous transcription factors may be modified in such that no activating ligand-inducer will be required to drive the transcription factor into the active state.",
"Other embodiments contemplated in this invention include triggering reactions such as DNA restriction and/or DNA replication.",
"An example of a biochemical cascade that can be triggered by restriction is a two-component system wherein a DNA sequence containing an origin of replication and being integrated into a nuclear genome is specifically excised and converted into an autosomally replicating plasmid by a rare-cutting restriction enzyme delivered by viral vector, thus triggering the cascade.",
"Alternatively, a DNA viral vector with a modified system of replication initiation may be made operable only in the presence of a factor in a transgenic host that allows for efficient replication of the modified viral vector in question.",
"There are numerous reactions affecting RNA molecules that may be used as efficient triggering devices of a cascade according to the present invention.",
"These include, inter alia, reactions such as RNA replication, reverse transcription, editing, silencing, or translation.",
"For example, a DNA derived from an viral RNA vector may be reverse transcribed by a transgenic host into a DNA which in turn could participate in processes such as DNA integration into a nuclear genome or DNA-mediated mutagenesis.",
"Another recombinant viral switch contemplated under the invention is a process that relies on posttranslational modification of one or more transgene expression products.",
"There are many possible implementations of such switches that could operate by controlling steps such as polypeptide folding, oligomer formation, removal of targeting signals, conversion of a pro-enzyme into an enzyme, blocking enzymatic activity, etc.",
"For example, expression of a polypeptide from a viral expression vector may trigger a biochemical process of interest only if a genetically engineered host specifically cleaves a pro-enzyme thus converting it into an active enzyme, if a product is targeted to a particular cellular compartment because of the host's ability to cleave or modify targeting motif, or if a product is specifically mobilized due to the removal of a specific binding sequence.",
"The process of this invention relies on the interaction of at least two components, but multi-component systems based on interactions between more than one heterologous DNA in host nuclear genome or more than one viral transfection vectors are also contemplated.",
"The same is true with regard to multi-component systems that involve, in addition to the above named two components (heterologous DNA or its product in a host plant and a heterologous DNA or its product in a viral vector), additional elements such as low molecular weight effectors or nucleic acids or proteins that are not integrated into a plant chromosome.",
"Such a low molecular weight component is defined as a non-proteinaceous molecule or ion having a molecular weight of less than 5 kD.",
"The ultimate purpose of a recombinant switch system contemplated herein is an operational control of a process in a plant production system, such as biochemical pathway or a cascade of biochemical reactions of interest.",
"A pathway or a biochemical cascade is a chain of biochemical reactions in a host production system that upon completion, yields a specific product, effect or trait.",
"The approaches described herein, in addition to being versatile and leakage-proof gene switches, provide an efficient production control method.",
"The two-component process described above is in essence a “key-lock” system, whereby a company can efficiently control access to production by selling the transfection switch component.",
"EXAMPLES With regard to additional disclosure of specific vectors and constructs used in the following examples, reference is made to ANNEX A and ANNEX B.",
"Example 1 Construction of Viral Vectors for Plant Infection, Carrying the Genes Involved in DNA Recombination: Ac Transposase and Cre Recombinase Series of crTMV-based viral expression vectors carrying the genes involved in DNA recombination, were constructed according to a standard molecular biology protocols (Maniatis et al., 1982, Molecular cloning: a Laboratory Manual.",
"Cold Spring Harbor Laboratory, New York).",
"Detailed information concerning commonly used vectors, genes and gene fragments used in this and the following examples can be found in public domain databases.",
"Two-step cloning strategy was used for all constructs.",
"First, an intermediate construct was made to fuse the gene of interest (GUS) with the appropriate IRES-sequence and the 3′-nontranslated region (NTR) of the crTMV (pseudoknots and t-RNA-like structure).",
"For the IRESmp75CR and IREScp148CR-fusions (Skulachev et al.",
"1999, Virology 263, 139-154) the gene of interest (GUS) was subcloned into the plasmid pIC766 (IRESmp75CR-GUS-3′-NTR in pBS(SK+) and into the plasmid pIC751 (IREScp148CR-GUS-3′-NTR in pBS(SK+), respectively.",
"Convenient restriction sites for sub-cloning, like Nco I at the 5′-end and BamH I- or Xba I at the 3′-end of the gene of interest were introduced by PCR if necessary.",
"DNA sequencing analysis was used to confirm the sequences of all PCR-amplified parts of the construct.",
"In the final step of cloning, the IRES/GUS/3′-NTR-fragment was sub-cloned further into the viral expression vector pIC797 (T7 promoter—crTMV cDNA with the GUS gene following the viral CP gene (RdRp-MP-CP-HindIII-IRESmp228CR-GUS-3′NTR)-NotI-Xbal-Spel-BamHI in pUC19) as a HindIII/NotI fragment.",
"For this purpose, the plasmid pIC797 was digested with SacII and NotI, the large fragment was gel purified and ligated with the 1.3 kb SacII/HindIII fragment of the same plasmid and the HindIII/NotI-fragment of the intermediate construct (pIC2251 for Cre recombinase).",
"In case of the Ac-transposase a four-fragments ligation was necessary due to the presence of a HindIII-restriction site in the coding part of the Ac gene.",
"The final constructs (pIC1111 and pIC1123 for Ac transposase; pIC2541 and pIC2531 for Cre recombinase) are shown in FIGS.",
"2 and 4 respectively.",
"Example 2 In Vitro Transcription of Viral Vector Constructs The plasmids pIC1111, pIC1123, pIC2541 and pIC2531 (FIGS.",
"2 and 4, respectively) were linearized by digestion with Not I restriction endonuclease.",
"The linearized plasmids were transcribed in vitro as described by Dawson et al.",
"(1986, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA., 83, 1832-1836).",
"Quality and quantity of full-length RNA transcripts were determined by agarose gel electrophoresis (Maniatis et al., 1982, Molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, New York).",
"Example 3 Activation of a Transgene Stably Integrated in a Plant Genome by Virus-Delivered Ac Transposase The T-DNA of plasmid pSLJ744 (obtained from J. Jones, Sainsbury Laboratory, JIC, Norwich, UK) (FIG.",
"2) was introduced in Arabidopsis thaliana (Col-0) plants as descried by Bent et al., (1994, Science, 285, 1856-1860).",
"Seeds were harvested three weeks after vacuum-infiltration, sterilised and screened for transformants on GM+1% glucose medium (Valvekens et al., 1988, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 85, 5536-5540.)",
"containing 50 mg/L kanamycin.",
"Rosette leaves of five weeks old Arabidopsis transformants were inoculated with full-length transcript-RNA as obtained in example 2 by mechanical wounding.",
"For this purpose, the RNA was mixed with 3× GKP-buffer (50 mM glycine, 30 mM K2HPO4, 3% celite, 3% benthonite) and scratched gently on the upper side of the leaves.",
"The T-DNA of plasmid SLJ744 contained a non-autonomous Ds element inserted between the CaMV 35S promoter and the GUS gene (FIG.",
"2).",
"Excision of the Ds element caused by action of virus-delivered Ac transposase leads to the expression of the GUS-gene, which can be easily monitored by histochemical staining of inoculated leaves (Jefferson, 1987, Plant Mol.",
"Biol.",
"Reporter, 5, 387-405).",
"Inoculated leaves were collected 9-14 days after the transfection with full-length transcript RNA.",
"Samples were infiltrated using X-gluc solution (Jefferson, 1987, Plant Mol.",
"Biol.",
"Reporter, 5, 387-405).",
"After incubation overnight at 37° C., the leaves were destained in 70% ethanol and examined by light microscopy.",
"Large sectors of GUS-stained tissues were observed in primarily inoculated leaves.",
"No GUS staining was detected in the control transgenic plants inoculated by distilled H2O.",
"The results are shown in FIG.",
"6.The sectors of GUS staining are consistent with the sectors of viral infection in primarily inoculated leaves.",
"This is evidence for the high efficiency of this approach: Ds excision and, as a consequence, GUS expression took place in all infected cells.",
"In comparison, constant presence of Ac transposase in plants carrying a copy of the Ac transgene stably integrated in the genome leads to Ds excision sectors only in a minor fraction of the plant tissue (results not shown).",
"Example 4 Activation of a Transgene Stably Integrated in the Plant Genome by Virus-Delivered Cre Recombinase Two different constructs pIC2561 and pIC1641 (FIGS.",
"3 and 4, respectively) with loxP-recombination sites were designed as targets for Cre-mediated recombination.",
"In construct pIC2561, the GUS gene with the 3′NOS transcription termination signal is flanked by two direct loxP-sites.",
"This fragment was inserted between the CaMV 35S promoter and a synthetic GFP gene (sGFP).",
"The recombination between the two loxP sites, once exposed to virus-delivered Cre recombinase, leads to excision of the GUS gene.",
"This event can be easily monitored by GFP expression and absence of GUS-acitivity in the inoculated leaves.",
"For the construction of plasmid pIC2561, the SLJ4K1 (Jones et al.",
"1992, Transgenic Research 1, 285-292) the GUS gene was amplified with primers carrying loxP sites and Cla1 (5′CCG ATC GAT ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAT GTT ACG TCC TGT AGA AAC CC3′) and Nco1 (5′GGC CAT GGA TAA CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT TGC ATG CCT GCA GGT CGA TCT AGT AAC3′) restriction sites were introduced at the 5′ and 3′ ends of the gene, respectively.",
"Said PCR-product was digested with Cla1 and Nco1 restriction enzymes and subcloned into the Nco1-Cla1 sites of the plasmid pIC591 (pHBT:ClaI/NcoI-sGFP-3′NOS).",
"The HBT promoter (Sheen, J.",
"1995, EMBO J., 12, 3497-3505) (not functional in Arabidopsis) of this intermediate construct was replaced by the CaMV 35S promoter by ligating together its gel-purified large HindIII/Klenow—Cla1 fragment with the 1.4 kb EcoR1/Klenow—Cla1 fragment of (35S promoter) of SLJ4K1.Functional clones were determined in microprojectile co-bombardment experiments with DNA of pIC1422 (cre recombinase under control of HBT promoter).",
"For further subcloning into the binary vector pICBV1 (proprietary development of Icon Genetics AG, Munich, Germany, however, any other binary vector is suitable as well), the pICBV1-DNA was digested with EcoRI and Ecl13611 restriction enzymes, gel-purified and ligated with the large Xho1/Klenow—EcoR1 fragment (p35S:-loxP-GUS-3′OCS-loxP-sGFP-3′NOS) of said functional intermediate clone.",
"The T-DNA region of the final construct pIC2561 is shown in FIG.",
"3.The second construct carrying the GUS gene flanked by two inverted loxP sites is shown in FIG.",
"4.To make this construct, two PCR primers (5′CTG AAG CTT ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAC CAT GG CTG CAG ATA ACT TCG TAT3′ and 5′GCC TCG AGA TAA CTT CGT ATA ATG TAT GCT ATA CGA AG TT ATC TGC AGC CAT GGT ATA ACT TCG TA3′) with 18 bb of complementary 3′ ends were designed, annealed and filled in with the Klenow fragment of DNA polymerase I.",
"The final DNA fragment contained two inverted loxP sites separated by Pst1, Nco1 and flanked by Xho1 (from the Pst1 side) and Hind1111 restriction sites.",
"After a Xho1-Hind111 digestion, the fragment was ligated with large Xho1-Hind111 fragment of pSLJ4D4 (Jones et al., 1992, Transgenic Research, 1, 285-292).",
"The resulting plasmid was digested with Pst1-Nco1, gel-purified and ligated with the 2.6 kb Nco1-Pst1 fragment of pSLJ4D4.As the final step of cloning, the whole cassette (CaMV p35S-loxP-3′nos-GUS-loxP) was subcloned into the binary vector pBIN19 (Bevan, M. 1984, Nucl.",
"Acid Research, 12, 8711-8721) as HindIII/EcoRI-fragment.",
"The T-DNA of this construct (pIC1641) is shown in FIG.",
"4.Transgenic Arabidopsis lines were obtained by Agrobacterium tumefaciens mediated transformation according to the modified vacuum infiltration protocol of Bent et al.",
"(1994, Science, 265, 1856-1859).",
"The presence of the transgene in segregating T1-population was confirmed by PCR-analysis.",
"Transcription of viral a cDNA clone (pIC2531) and inoculation of transgenic Arabidopsis lines with viral RNA was performed as described in examples 2 and 3, respectively.",
"GFP and GUS Detection A LEICA stereo fluorescent microscope system was used to monitor GFP expression (excitation at 450-490 nm, emission at 500-550 nm).",
"The sGFP used in our experiments can be excited by blue and UV-light.",
"GUS detection was performed as described in example 3.Example 5 Activation of a Transgene Stably Integrated in a Plant Genome by Virus-Delivered T7 RNA Polymerase Construction of the Vectors The binary vector for plant transformation with the GUS gene reporter under control of the T7 promoter was made as follows.",
"The gel-purified 2 kb BssH11/T4 polymerase—Sal 1 fragment of pIC057 carrying the T7 promoter-GUS gene construct was ligated with Sma1-Sal1 digested expression vector pIC056, adding the 35S transcription termination signal to the 3′ end of the GUS gene.",
"The resulting construct pIC2641 was digested with Sac1 and Xho1, gel-purified from the vector backbone and ligated with Sac1/Sal1 digested pBIN19.The final construct pIC2651 (FIG.",
"5) was used for the Arabidopsis transformation as described above.",
"The viral vector expressing T7 polymerase was made as follows.",
"The plasmid pIC2603 was digested with Sph1 and Sal1 and the gel purified 2.8 kb fragment carrying the T7 polymerase gene was ligated with the large Nco1-Sal1 fragment of pIC1018.Resulting plasmid pIC2621 has the T7 gene flanked by the IRESmp75CR at its 5′ end and by the 3′ nontranslated region (NTR) of crTMV at its 3′ end.",
"The final cloning step included the ligation of the small Hind111-Not1 fragment of pIC2621 with the large Sac11-Not1 and small Sac11-Hind111 fragments of pIC1087.The final construct pIC 2631 (FIG.",
"5) containing the T7 polymerase gene in a crTMV viral vector was used for transcription and plant transfection as described in examples 2 and 3, respectively.",
"Example 6 Activation of a Transgene from Viral Amplicon Precursors Construction of the Vectors In order to introduce LoxP-sites recognized by Cre recombinase into a basic construct, IPCR was performed with primers containing LoxP-sites in opposite orientation flanked by convenient restriction sites (primer 1: 5′-TATCTGCAGG AGCTCATAAC TTCGTATAAT GTATGCTATA CGAAGTTATA AGCTTCTGGC CGTCGTTTTA C-3′; primer 2: 5′-CTCCTGCAGA TAACTTCGTA TAATGTATGC TATACGAAGT TATCTCGAGG AATTCGGCGT AATCATGGTC A-3′).",
"These primers were annealed to the multi-cloning site of the pUC119 vector in order to amplify the whole plasmid in an IPCR-reaction.",
"Overlapping sequences of the primers contained a Pst I-restriction site.",
"After restriction of the IPCR-product with Pst I and religation, the intermediate construct pICH1212 (Appendix 1) was obtained.",
"The Xho1-EcoR1 fragment of MP-gene containing a translation stop codon 25 AA before the natural translation termination signal was ligated with Xho1-EcoR1 large fragment of PICH1212.In the resulting construct pICH3431 (Appendix 2) the 3′-MP-part is located next to a LoxP-site.",
"To fuse this MP-LoxP element to the 5′-part of a MP-gene in a vector, which contains also the Arabidopsis Actin 2-promoter and the RdRp-polymerase, the MP-LoxP element from pICH3431 was subcloned as EcoRI-Ecl136II fragment into the plasmid pICH3301 (Appendix 3) cut with EcoRI and NotI, resulting in the plasmid pICH3461 (Appendix 4).",
"The NotI restriction site was treated with Klenow fragment of DNA polymerase 1 before subcloning.",
"The KpnI-XhoI and Xho-HindIII fragments at 5′-end of the resulting vector were further used for cloning into the Kpn1 and HindIII-treated binary vector pICBV10 (T-DNA region of pICBV10 is shown in Appendix 5) in a three fragment ligation reaction.",
"The final construct pICH4371 is depicted in FIG.",
"7.For making a 3′-end of the viral vector precursor, an XhoI-NcoI fragment containing a LoxP site next to an Ω-leader-sequence from construct pICH2744 (Appendix 6) was subcloned into plasmid pICH1721 (Appendix 7) to fuse the LoxP-site/Ω-leader-sequence-element to the 5′-end of the sGFP-gene which was flanked by a 3′NTR-sequence at the 3′-end (construct pICH3421, Appendix 8).",
"In order to add a nopaline synthase transcription termination signal to this ORF, plasmid pICH3421 was cut by KpnI and NotI and the resulting small fragment was cloned into the plasmid pICH3232 resulting in construct pICH3441 (Appendix 9).",
"For the Agrobacterium tumefaciens-mediated delivery this 3′-end of the viral precursor vector was further subcloned into the binary vector pICBV10 (Appendix 5) as KpnI/HindIII fragment.",
"The final construct pICH4461 is shown in FIG.",
"7.The Cre recombinase construct pICH529 (wheat histone H4 promoter-LoxP-Cre recombinase-NOS terminator, see Appendix 10) was modified to clone the Cre recombinase into the binary vector used for obtaining nuclear transformants of Nicotiana.",
"First the wheat H4 promoter was replaced by the Actin2 promoter from Arabidopsis by subcloning an Ecl136II/Pst l-fragment from construct pIC04 (Arabidopsis Actin2-promoter without intron, not shown) into pICH529 digested with HindIII (blunt) and Pstl.",
"This resulted in construct pICH1262 (Appendix 11).",
"In order to replace the NOS-terminator by the OCS-terminator flanked at its 5′-end by a LoxM recombination-site, the OCS-terminator was PCR-amplified from plasmid pICH495 (NOS promoter-BAR-gene-OCS terminator, not shown) and further subcloned as SphI/SacI-fragment into the plasmid pICH1262 , producing the construct pICH1321 (not shown).",
"The sequence of the forward primer (5′-CGGCATGCAT AACTTCGTAT AATCTATACT ATACGAAGTT AGGATCGATC CTAGAGTCCT GC-3′) used for this amplification, included the SphI-restriction site and the LoxM-recombination site.",
"The SacI-restriction site at the 3′-end of the PCR-product was introduced by the sequence of the reverse primer (5′-CGGAGCTCGT CAAGGTTTGA CCTGCACTTC-3′).",
"Finally, the resulting construct (Actin2 promoter-LoxP-Cre recombinase-LoxM-OCS terminator) was further subcloned into the binary vector pIC00015 as NotI/SacI-fragment, resulting inconstruct pICH1754 (FIG.",
"8).",
"To clone this fragment into the binary vector it was necessary to fill in the NotI-site of the fragment and the EcoRI-site in the polylinker of the binary vector.Transformation of tobacco leaf discs Transgenic Nicotiana lines (species tabacum and benthamiana), containing T-DNA of pICH1754, were obtained by Agrobacterium-mediated transformation of leaf discs as described by Horsch et al., (1985, Science, 227, 129-131).",
"Leaf discs were incubated for 30 min with Agrobacterium strain GV3101 transformed with the construct pICH1754.After three days of incubation on medium (MS-medium 0.1 mg/l NAA, 1 mg/l BAP) without selective agent, selection of transformants was performed on the same MS-medium supplemented with 100 mg/L Kanamycin.",
"In ordero reduce the growth of Agrobacterium, the medium was also supplemented with 300 mg/L carbenicilin and 300 mg/L cefataxime.",
"Regenerants were incubated on selective MS-medium without hormones supplemented with the same concentration of the selective agents to induce the rooting.",
"The presence of the transgene in segregating T2-populations was confirmed by PCR-analysis.",
"Delivery of Viral Vector Precursors by Agro-Infiltration The agroinfiltration of transgenic tobacco plants was performed according to a modified protocol described by Yang et al., 2000, Plant Journal, 22(6), 543-551.Agrobacterium tumefaciens strain GV3101 transformed with individual constructs (pICH4371 and pICH4461) was grown in LB-medium supplemented with Rifampicin 50 mg/l, carbencilin 50 mg/l and 100 μM acetosyringone at 28° C. Agrobacterium cells of an overnight culture (5 ml) were collected by centrifugation (10 min, 4500 g) and resuspended in 10 mM MES (pH 5.5) buffer supplemented with 10 mM MgSO4 and 100 μM acetosyringone.",
"Bacterial suspension was adjusted to a final OD600 of 0.8.In case of delivery of several constructs agrobacterial clones carrying different constructs were mixed before infiltration.",
"Agroinfiltration was conducted on near fully expanded leaves that were still attached to the intact plant.",
"Bacterial suspension was infiltrated with a 5 ml syringe.",
"By infiltrating 100 μl of bacterial suspension into each spot (typically 3-4 cm2 of infiltrated area) eight to 16 spots separated by veins could be placed in a single tobacco leaf.",
"After infiltration plants were further grown under greenhouse conditions at 22° C. and 16 h light.",
"Sixteen days after infiltration, leaves of transgenic tobacco plants (pICH1754, Nicotiana tabacum) infiltrated with construct pICH4371 and pICH4461 showed growing sectors of strong GFP-expression which could be observed under UV-light on intact plants.",
"No GFP-expression was visible on leaves of non-transformed tobacco infiltrated with the same Agrobacterium suspension mix.",
"Annex A Vector System for Plants FIELD OF INVENTION This invention relates to a vector capable of amplification and expression and/or suppression of a gene in a plant, as well as uses thereof, and a method and pro-vector for generating said vector.",
"BACKGROUND OF THE INVENTION Vectors for genetic engineering of plants are highly desirable for the production of proteins, for endowing a host plant with a new trait, for suppressing a gene of the host plant, or for determining the function of a gene, notably a gene determined by genomics.",
"Vectors, notably viral vectors, for the genetic engineering of plants are already known.",
"These must be capable of infection, amplification and movement (both cell-to-cell and long-distance) in a plant in addition to having at least one sequence for gene expression or suppression.",
"Prior art vectors rely on subgenomic promoters as transcriptional elements.",
"A subgenomic promoter has the effect that, in a transfected plant cell, transcription of a vector nucleic acid sequence starts in part at said subgenomic promoter to generate a shorter RNA so that translation of a gene downstream from said promoters by the plant translation machinery is enabled.",
"Translation may then proceed cap-dependent.",
"Such multiple transcriptions are kinetically disadvantageous because of waste of replicase capacity.",
"Such vectors have a number of further shortcomings.",
"The introduction of a virus subgenomic promoter into a vector sequence makes said sequence longer and thus less efficient.",
"Moreover, the presence of several identical or similar subgenomic promoters which are well adapted to transcription in the host gives rise to frequent recombination events and instability with loss of sequence portions.",
"On the other hand, if significantly different subgenomic promoters are used, recombination may be suppressed but such promoters may be too different to be effectively recognized by the transcription system, which means loss of efficiency.",
"Moreover, vectors are usually highly integrated entities with several interdependent functional elements or genes tightly packed into a sequence.",
"This is the reason why the operability of a vector for certain heterologous genes or the like is somewhat idiosyncratic and frequently gives unpredictable results, notably in terms of infectivity and expression.",
"Further, the available sequence space for promoters is usually constrained if sequence overlaps with upstream genes are present.",
"Therefore, it is an object of this invention to provide a novel vector for plant genetic engineering which is capable of efficient and stable operation in a host plant.",
"It is a further object to provide a vector which is capable of high-level expression of a gene in a plant.",
"It has been surprisingly found that these objects can be achieved with a vector capable of amplification and expression of a gene in a plant comprising a nucleic acid having a sequence for at least one non-viral gene to be expressed and having or coding for at least one IRES element necessary for translation of a gene downstream thereof.",
"It has been previously suggested (WO 98/54342) to use a plant IRES element in a recombinant DNA molecule that has merely the function of gene expression (after integration into the host genome).",
"However, the expression level is low.",
"The exact reasons for this low expression level are not known.",
"In any event, expression is limited to the very plant cells transformed, thus the overall efficiency in whole plants is extremely low.",
"It has been surprisingly found that it is possible to construct a plant vector which, when introduced into a plant cell, has not only the capability of gene expression but which has several additional functions which are all required for amplification and spreading throughout the plant so that the overall efficiency is extremely high.",
"These functions comprise infection, amplification, cell-to-cell movement and long-distance movement.",
"It is surprising that the required high degree of integration of functional and structural elements on a vector does not impair gene expression from said vector.",
"The IRES element of said vector can be located upstream of said non-viral gene to be expressed for directly supporting its translation.",
"Alternatively, said IRES element may indirectly support the translation of said gene to be expressed by directly supporting the translation of another gene essential for a function of said vector selected from the group of infection, amplification and cell-to-cell or long-distance movement of said vector.",
"It is a further object to provide a vector which is capable of the effective suppression of a gene in a plant.",
"This object has been achieved by a vector capable of amplification in a plant comprising a nucleic acid having or coding for at least one IRES element necessary for translation of a gene required for amplification of said vector and located downstream of said IRES element, said vector further comprising at least a portion of a sequence of the host plant genome in an anti-sense orientation for suppressing a gene of the host plant.",
"Further preferred embodiments are defined in the subclaims.",
"Here, the first plant expression and amplification vectors based on plant active translational (IRES) elements are described.",
"Existing IRES elements isolated from animal viruses do not support translation in plant cells.",
"Therefore, knowledge accumulated in animal expression systems is not applicable to plants.",
"Animal IRES elements have never been tested for other functional properties, such as residual promoter activity, so this invention discloses the first bona fide cases of gene expression in plants relying exclusively on translation rather than on transcription with a subgenomic promoter necessary for expression of a gene downstream thereof.",
"The vectors of this invention allows preferably for regulation and preferential expression of a gene of interest in a plant by suppressing cap-dependent translation.",
"In another preferred embodiment, very short homologous or artificial IRES elements are used, thus adding to the stability of the resulting vectors.",
"A preferred advantage of this strategy is that IRES sequences can be inserted upstream or downstream of viral gene(s) (e.g.",
"the coat protein gene of tobacco mosaic virus such that translation of downstream foreign gene(s) or the viral gene(s), respectively, may occur via cap-independent internal ribosome entry pathway.",
"Thus, said cap-independent translation of foreign gene(s) will occur from bicistronic or/and polycistronic RNAs.",
"General Problem Situation and Definitions Upon infection of a plant with a virus the early events of viral infection (entry and genome uncoating) occur.",
"Then the virus must engage in activities that enable its genome to be expressed and replicated.",
"The viral genome may consist of one (monopartite) or more (multipartite) RNA or DNA segments, and each of these segments may under certain conditions be capable of replicating in the infected cell.",
"A viral replicon has been defined as “a polynucleotide of viral sequences that is replicated in host cells during the virus multiplication cycle” (Huisman et al., 1992, “Genetic engineering with plant viruses”, T. M. A. Wilson and J. W. Davies eds., 1992, CRC Press, Inc.).",
"In this invention we use the term “amplification-based expression system” to designate either a full-length viral genome or any fragment of viral RNA or DNA that (i) contains and is able to express foreign sequences, non-native for the wild-type parental virus (ii) replicates either by itself or as a result of complementation by a helper virus or by a product of the transgenic plant host.",
"The terms “amplification-based expression system” and “recombinant viral vector” are closely similar.",
"These systems represent a recombinant nucleic acid containing additional sequences, homologous (native) or foreign, heterologous (non-native) with respect to the viral genome.",
"The term “non-native” means that this nucleic acid sequence does not occur naturally in the wild-type genome of the virus and originates from another virus or represents an artificial synthetic nucleotide sequence.",
"Such an amplification-based system derived from viral elements is capable of replicating and, in many cases, cell-to-cell as well as long-distance movement either in a normal or/and in a genetically modified transgenic host plant.",
"In the latter case the transgenic plant should complement the viral components of a vector which may be deficient in a certain function, i.e.",
"the product(s) of a transgene essential for vector replication and/or expression of its genes or long-distance transport should be provided by the transgenic plant.",
"Plant virus amplification-based vectors based on the monopartite (e.g.",
"tobacco mosaic virus, TMV) or multipartite (e.g.",
"members of Bromoviridae family) genome have been shown to express foreign genes in host plants (for review, see “Genetic engineering with plant viruses”, T. M. A. Wilson and J. W. Davies eds.,1992, CRC Press, Inc.).",
"The majority (about 80%) of known plant viruses contains plus-sense single-stranded RNA (ssRNA) genomes that are infectious when being isolated from the virions in a form of free RNA.",
"This means that at the first step of the virus replication cycle, genomic RNA must be translated in order to produce the virus-specific RNA-dependent RNA polymerase (replicase) that is absent from uninfected plant cells and, therefore, is essential for viral RNA replication (for review, see Y. Okada 1999, Philosoph.",
"Transact.",
"of Royal Soc., B, 354, 569-582).",
"It should be mentioned that plus-sense ssRNA viruses differ in translation strategies used for genome expression: the genomes of so called picoma-like viruses represent a single continuous open reading frame (ORF) translated by the ribosome into a large polyprotein which is then proteolytically processed into functionally active virus-coded proteins.",
"The virus-specific proteinase(s) are involved in polyprotein processing.",
"A second peculiar feature of picorna-like viruses is that their genomic RNA contains, instead of cap structure, a small viral protein covalently linked to the 5′-end of the genome.",
"In this invention we most preferably focus on viruses of the so-called Sindbis-like superfamily that comprises many plant viruses, in particular, more than a dozen of viruses belonging to the genus Tobamovirus (for review, see A. Gibbs, 1999, Philosoph.",
"Transact.",
"of Royal Soc., B, 354, 593-602).",
"The technology ensures cap-independent and viral promoter-independent expression of foreign genes.",
"The genome of tobamoviruses (TMV U1 is the type member) contains four large ORFs.",
"The two components of the replicase (the 130-kDa and its readthrough 183-kDa proteins) are encoded by the 5′-proximal region of the genomic RNA and are translated directly from genomic RNA.",
"The 3′-terminal 15 nucleotides of the 180-kDa protein gene of TMV U1 overlap with the ORF coding for the 30-kDa protein responsible for cell-to-cell movement of TMV infection (movement protein, MP).",
"In TMV U1 this gene terminates two nucleotides before the initiation codon of the last gene which encodes the 17-kDa coat protein (CP) located upstream of the 3-proximal nontranslated region (3′-NTR) consisting of 204 nucleotides (in TMV U1).",
"Translation of RNA of tobamoviruses occurs by a ribosome scanning mechanism common for the majority of eukaryotic mRNAs (for reviews, see Kozak, 1989, J. Mol.",
"Biol.",
"108, 229-241; Pain, 1996 ; Merrick and Hershey,1996, In “Translational control”, eds.",
"Hershey, Matthews and Sonenberg, Cold Spring Harbour Press, pp.",
"31-69; Sachs and Varani, 2000, Nature Structural Biology 7, 356-360).",
"In accordance with this mechanism, structurally polycistronic tobamovirus RNA is functionally monocistronic, i.e., only the 5′-proximal ORF encoding the replicative proteins (130-kDa protein and its readthrough product) can be translated from full-length genomic RNA (reviewed by Palukaitis and Zaitlin,1986, In “The Plant Viruses”, van Regermortel and Fraenkel-Conrat eds., vol.2, pp.105-131, Plenum Press, NY).",
"It should be emphasized that the 68-nucleotide 5′-terminal nontranslated leader sequence of TMV U1 termed omega (Ω) has been shown to play the role of an efficient translational enhancer stimulating the translation of the 5′-proximal ORF.",
"The 5-distal MP and CP genes are translationally silent in full-length TMV U1 RNA, however, they are translated from separate mRNAs referred to as subgenomic RNAs (sgRNA).",
"Apparently, the tobamovirus sgRNAs are transcribed from negative-sense genomic RNA and share a common 3′-terminus.",
"The expression of TMV genes that are translated from sgRNAs is regulated independently, both quantitatively and temporarily: the MP is produced transiently during early steps of infection and accumulates to relatively low levels (about 1% of total plant protein), whereas the CP constitutes up to 70% of total plant protein synthesis and the CP can accumulate up to 10% of total cellular protein (Fraser, 1987, In “Biochemistry of virus-infected plants”, pp.1-7, Research Studies Press Ltd., Letchworth, England).",
"It is clear that production of each sgRNA is controlled by different cis-acting sequences termed “subgenomic mRNA promoter” (sgPR).",
"Generally, this term indicates the region of the viral genome (presumably in a minus-sense RNA copy) that can be recognized by the replicase complex to initiate transcription from the internally located sgPR sequence to produce sgRNA.",
"However, for convenience, by the term “subgenomic promoter” we conventionally mean a nucleotide sequence in plus-sense viral RNA that is usually located upstream of the coding sequence and the start point of sgRNA and which is functionally involved in the initiation of the sgRNA synthesis.",
"However, it should be taken into consideration that some viral sgPRs are located not only upstream of the controlled viral gene, but can even overlap with this gene (Balmori et al., 1993, Biochimie (Paris) 75, 517-521).",
"Each sgPR occupies a different position in the TMV genome.",
"None of the sgPRs of TMV has been precisely mapped, but the 250 nucleotides upstream of the CP gene have been shown to promote synthesis of the CP sgRNA (Dawson et al., 1989, Virology 172, 285-292).",
"Lehto et al.",
"(1990, Virology 174, 145-157) inserted in the TMV genome (in front of the MP gene) sequences (253 and 49 nucleotides) preceding the CP gene in order to estimate the size of the CP sgPR.",
"The insertion did not remove the native MP sgPR, but separated it from the MP ORF.",
"The mutant (called KK6) with an inserted 253 nt promoter region replicated stably and moved systemically over the infected plant.",
"It is not unexpected that in the KK6 mutant the insertion changed the length of the MP sgRNA leader (Lehto et al., 1990, Virology 174, 145-157) (see FIG.",
"18).",
"The KK6 MP sgRNA leader was 24 nucleotides compared to 9 b.p.",
"for the CP sgRNA.",
"By contrast, the mutant with an inserted 49-nt fragment of the promoter region replicated only transiently before being overtaken by a progeny of wild-type virus with the insert deleted.",
"In addition, it has been shown (Meshi et al., 1987, EMBO J., 6, 2557-2563) that production of the CP sgRNA was reduced when the 96-nt region derived from CP sgPR was used.",
"It is concluded that the 49-96 nt sequences upstream of the CP gene did not contain the entire sgPR of the TMV U1 CP gene, whereas the 250-nt sequence included complete sgPR.There is little information about the structure and mapping of sgPR controlling the expression of the TMV MP gene.",
"Because the putative MP sgPR sequence overlaps with the 183-kDa replicase protein, the mutational analysis of the MP sgPR was complicated.",
"Preliminary results of W. Dawson and co-workers reported recently delineated the boundaries of the minimal and full MP sgPR of TMV U1 (Grdzelishvili et al., 2000, Virology 276, in press).",
"Computer folding of the region upstream of the MP gene reveals two stem-loop structures, located 5′-proximally to the 75-nt region preceding AUG codon of the MP gene.",
"It is assumed that in contrast to genomic RNA and the CP sgRNA, the sgRNA of the MP gene (so called I2 sgRNA) is uncapped (for review see: Okada, 1999, Philosoph.",
"Transact.",
"Of Royal Soc., B, 354, 569-582).",
"The present invention provides the results confirming the absence of the cap-structure in I2 sgRNAs of both TMV U1 and crTMV (FIG.",
"16).",
"It has been shown by W. Dawson with colleagues that an important factor affecting the expression of a foreign gene from the vector virus is the position of the foreign gene relative to the 3′-terminus of viral genome: the efficiency of expression increased dramatically when the gene was placed closer to the 3′-terminus (Culver et al., 1993, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 90, 2055-2059).",
"The highest expressed gene is that of the CP which is adjacent to the 3′-NTR that consists (in TMV U1 RNA) of three pseudoknots followed by a tRNA-like structure.",
"It was suggested (Shivprasad et al., 1999, Virology 355, 312-323) that the proximity of the gene to the pseudoknots rather than to the 3-terminus was the main factor increasing expression of the foreign gene.",
"Many important aspects of the TMV sg PRs structure were clarified due to the efforts of W. Dawson's group, however, the general conclusion of these authors was that “we are still in the empirical stage of vector building” (Shivprasad et al., 1999, Virology 355, 312-323).",
"The above shows that the synthesis of sgRNAs is essential for expression of the 5′-distal genes of TMV genome, since these genes are translationally silent in full-length RNA.",
"The mechanism of gene autonomization by subgenomization can be regarded as a strategy used by TMV in order to overcome the inability of eukaryotic ribosomes to initiate translation of the 5′-distal genes from polycistronic mRNA.",
"According to the traditional ribosome scanning model (Kozak, 1999, Gene 234, 187-208), the internal genes of a polycistronic eukaryotic mRNA are not accessible to ribosomes.",
"Recently, we have isolated a crucifer infecting tobamovirus (crTMV) from Oleracia officinalis L. plants.",
"A peculiar feature of crTMV was its ability to infect systemically members of Brassicaceae family.",
"In addition, this virus was able to systemically infect plants of the Solanaceae family and other plants susceptible to TMV U1.The genome of crTMV (6312 nucleotides) was sequenced (Dorokhov et al., 1994, FEBS Letters 350, 5-8) and was shown to contain four traditional ORFs encoding proteins of 122-kDa (ORF1), 178-kDa (ORF2), the readthrough product of 122-kDa protein, a 30-kDa MP (ORF3), and a 17-kDa CP (ORF4).",
"A unique structural feature of crTMV RNA was that, unlike other tobamoviruses, the coding regions of the MP and CP genes of crTMV are overlapped by 75 nucleotides, i.e.",
"the 5′-proximal part of the CP coding region also encodes the C-terminal part of the MP.",
"In order to provide a clear and consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided: Adjacent: A position in a nucleotide sequence immediately 5′ or 3′ to a defined sequence.",
"Amplification vector: A type of gene vector that, upon introduction into a host cell, is capable of replicating therein.",
"Anti-Sense Mechanism: A type of gene regulation based on controlling the rate of translation of mRNA to protein due to the presence in a cell of an RNA molecule complementary to at least a portion of the mRNA being translated.",
"Chimeric Sequence or Gene: A nucleotide sequence derived from at least two heterologous parts.",
"The sequence may comprise DNA or RNA.",
"Coding Sequence: A deoxyribonucleotide sequence which, when transcribed and translated, results in the formation of a cellular polypeptide or a ribonucleotide sequence which, when translated, results in the formation of a cellular polypeptide.",
"Compatible: The capability of operating with other components of a system.",
"A vector or plant viral nucleic acid which is compatible with a host is one which is capable of replicating in that host.",
"A coat protein which is compatible with a viral nucleotide sequence is one capable of encapsidating that viral sequence.",
"Gene: A discrete nucleic acid sequence responsible for a discrete cellular product.",
"Gene to be expressed: A gene of technological interest to be expressed.",
"Host: A cell, tissue or organism capable of replicating a vector or plant viral nucleic acid and which is capable of being infected by a virus containing the viral vector or plant viral nucleic acid.",
"This term is intended to include procaryotic and eukaryotic cells, organs, tissues or organisms, where appropriate.",
"Host Plant Genome: This term mean preferably the nuclear genome of a host plant cell, but may also include mitochondrial or chloroplast DNA.",
"Infection: The ability of a virus or amplification-based vector to transfer its nucleic acid to a host or introduce nucleic acid into a host, wherein the viral nucleic acid or a vector is replicated, viral proteins are synthesized, and new viral particles assembled.",
"In this context, the terms “transmissible” and “infective” are used interchangeably herein.",
"Internal Ribosome Entry Site (IRES) element, or IRES: a nucleotide sequence of viral, cellular or synthetic origin, which at the stage of translation is responsible for internal initiation.",
"IRES element necessary for translation of a gene downstream thereof: IRES element which is effective for translation of said gene in the sense that without such IRES element no technologically significant translation of this gene will occur.",
"Non-viral gene: A gene not functional for the life cycle of a virus.",
"Phenotypic Trait: An observable property resulting from the expression of a gene.",
"Plant Cell: The structural and physiological unit of plants, consisting of a protoplast and the cell wall.",
"Plant Organ: A distinct and visibly differentiated part of a plant, such as root, stem, leaf or embryo.",
"Plant Tissue: Any tissue of a plant in planta or in culture.",
"This term is intended to include a whole plant, plant cell, plant organ, protoplast, cell culture, or any group of plant cells organized into a structural and functional unit.",
"Production Cell: A cell of a tissue or organism capable of replicating a vector or a viral vector, but which is not necessarily a host to the virus.",
"This term is intended to include prokaryotic and eukaryotic cells, organs, tissues or organisms, such as bacteria, yeast, fungus and plant tissue.",
"Promoter: The 5′-non-coding sequence upstream to and operationally connected to a coding sequence which is involved in the initiation of transcription of the coding sequence.",
"Protoplast: An isolated plant cell without cell walls, having the potency of regeneration into cell culture or a whole plant.",
"Recombinant Plant Viral Nucleic Acid: Plant viral nucleic acid which has been modified to contain nonnative nucleic acid sequences.",
"Recombinant Plant Virus: A plant virus containing the recombinant plant viral nucleic acid.",
"Reporter Gene: A gene the gene product of which can be easily detected.",
"Subgenomic Promoter (sgPR): A promoter of a subgenomic mRNA of a vector or a viral nucleic acid.",
"Substantial Sequence Homology: Denotes nucleotide sequences that are homologous so as to be substantially functionally equivalent to one another.",
"Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting function of the gene products or an RNA coded for by such sequence.",
"Transcription: Production of an RNA molecule by RNA polymerase as a complementary copy of a DNA sequence.",
"Translation: Production of a polypeptide by a ribosome (frequently by means of scanning a messenger RNA).",
"Vector: A nucleic acid, which is capable of genetically modifying a host cell.",
"The vector may be single-stranded (ss) (+), ss (−) or double-stranded (ds).",
"Virus: An infectious agent composed of a nucleic acid encapsidated in a protein.",
"A virus may be a mono-, di-, tri- or multi-partite virus.",
"Advantages of the Invention This invention provides a novel strategy for constructing the amplification-based vectors for foreign (heterologous, non-native) gene expression such that translation of these genes can occur through an IRES-mediated internal ribosome entry mechanism from a polycistronic RNA and/or through IRES-mediated cap-independent internal ribosome entry mechanism from bi- and multicistronic sgRNA produced from the vector in the infected cell.",
"In either event, the IRES element is necessary for translation of a gene.",
"One of the advantages of this strategy is that it does not require any specific manipulation in terms of sgPRs: the only sequences that should be inserted into the vector are the IRES-sequence(s) (native or/and non-native) upstream of gene(s) to be translated.",
"As a result, translation of downstream gene(s) is promoted by the inserted IRES sequences, i.e.",
"is cap-independent.",
"The sequence segment harboring an IRES element preferably does not function as subgenomic promoter to a technically significant degree.",
"This means that this sequence segment either does not cause any detectable production of corresponding subgenomic RNA or that for the translation of any such subgenomic RNA, if formed by any residual subgenomic promoter activity of said sequence segment, this IRES element is still necessary for the translation of a downstream gene.",
"Consequently, in a special case, primary recombinant RNA produced by the vector comprises: one or more structural genes preferably of viral origin, said IRES sequence, the (foreign) gene of interest located downstream of the IRES and the 3′-NTR.",
"It is important that this strategy allows a simultaneous expression of more than one foreign gene by insertion of a tandem of two (or more) foreign genes, each being controlled by a separate IRES sequence.",
"The present invention is preferably directed to nucleic acids and recombinant viruses which are characterised by cap- independent expression of the viral genome or of its subgenomic RNAs or of non-native (foreign) nucleic acid sequences and which are capable of expressing systemically in a host plant such foreign sequences via additional plant-specific IRES element(s).",
"In a first preferred embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence and native CP subgenomic promoter have been deleted from a viral nucleic acid, and a non-native plant viral coat protein coding sequence with upstream located plant virus IRES element has been inserted that allows for cap-independent expression in a host plant, whereas packaging of the recombinant plant viral nucleic acid and subsequent systemic infection of the host by the recombinant plant viral nucleic acid are maintained.",
"The recombinant plant viral nucleic acid may contain one or more additional native or non-native IRES elements that function as translation elements and which have no transcriptional activity, i.e.",
"are effecticely unable to function as a subgenomic promoter.",
"Each native or non-native IRES element is capable of providing cap-independent expression of adjacent genes or nucleic acid sequences in the host plant.",
"In a second preferred embodiment, an amplification and expression vector is provided in which native or non-native plant virus IRES element(s) located upstream of foreign nucleic acid sequences are inserted downstream of a native coat protein gene.",
"The inserted plant virus IRES element may direct cap-independent expression of adjacent genes in a host plant.",
"Non-native nucleic acid sequences may be inserted adjacent to the IRES element such that said sequences are expressed in the host plant under translational control of the IRES element to synthesize the desired product.",
"In a third preferred embodiment, a recombinant vector nucleic acid is provided as in the second embodiment except that the native or non-native plant viral IRES element(s) with downstream located foreign nucleic acid sequences are inserted upstream of native coat protein subgenomic promoter and coat protein gene.",
"In a fourth preferred embodiment, a recombinant vector nucleic acid is provided in which native or non-native plant viral IRES element(s) is (are) used at the 5′ end of the viral genome or in the viral subgenomic RNAs so as to render translation of a downstream gene(s) cap-independent.",
"In a fifth preferred embodiment, inhibition of cap-dependent translation is being utilised to increase the level of cap-independent translation from said vectors.",
"The viral-based amplification vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus.",
"The recombinant plant viral nucleic acid is capable of replication in the host, systemic spreading in the host, and cap-independent expression of foreign gene(s) or cap-independent expression of the whole viral genome or of subgenomic RNAs in the host to produce the desired product.",
"Such products include therapeutic and other useful polypeptides or proteins such as, but not limited to, enzymes, complex biomolecules, or polypeptides or traits or products resulting from anti-sense RNA production.",
"Examples for desirable input traits are resistance to herbicides, resistance to insects, resistance to fungi, resistance to viruses, resistance to bacteria, resistance to abiotic stresses, and improved energy and material utilization.",
"Examples for desirable output traits are modified carbohydrates, modified polysaccharides, modified lipids, modified amino acid content and amount, modified secondary metabolites, and pharmaceutical proteins, including enzymes, antibodies, antigens and the like.",
"Examples for trait regulation components are gene switches, control of gene expression, control of hybrid seed production, and control of apomixis.",
"The present invention is also directed to methods for creation of artificial, non-natural IRES elements (as opposed to IRESs isolated from living organisms) providing cap-independent and promoter independent expression of a gene of interest in plant cells (and perhaps additionally in yeast or animal cells).",
"Examples for living organisms from which IRESs may be isolated are animal viruses and plant viruses.",
"Examples for animal viruses are hepatitis C virus, infectious bronchitis virus, picornaviruses such as poliovirus and encephalomiocarditis virus, and retroviruses such as moloney murine leukemia virus, and harvey murine sarcoma virus.",
"Examples for plant viruses are potato virus X, potyviruses such as potato virus Y and turnip mosaic virus, tobamoviruses such as crucifer-infecting tobamovirus, and comoviruses such as cowpea mosaic virus.",
"Alternatively, natural IRESs may be isolated from cellular messenger RNAs like those derived from antennapedia homeotic gene, human fibroblast growth factor 2, and translation initiation factor elF-4G.",
"In a sixth preferred embodiment, artificial, non-natural IRES elements are created on the basis of complementarity to 18S rRNA of eukaryotic cells, including yeast, animal and plant cells.",
"In a seventh preferred embodiment, artificial, non-natural IRES elements are created on the basis of repeated short stretches of adenosin/guanosin bases.",
"In an eighth preferred embodiment of this invention, a method of engineering and using viral-based amplification vectors is presented, wherein viral genome expression in plant cells occurs under the control of a plant-specific artificial transcription promoter.",
"In a ninth preferred embodiment of the present invention, a method of construction and using viral-based amplification vectors is presented, which vectors allow for expression from replicons being formed in plant cells as a result of primary nuclear transcript processing.",
"In a tenth preferred embodiment of this invention, a procedure is described for using circular single-stranded viral-based amplification vectors for cap-independent expression of foreign genes in plants.",
"In an eleventh preferred embodiment of the present invention, methods are presented that allow for expression of a gene of interest in cells under conditions favoring cap-independent translation.",
"In one example, cells infected with an amplification vector are treated with a compound inhibiting cap-dependent translation.",
"In another example, the vector itself contains a gene, the product of which has an inhibiting effect on cap-dependent translation in the host or an anti-sense sequence having said function.",
"In a twelvth preferred embodiment of this invention, a method is described that allows, by using in vivo genetic selection, to identify an IRES sequence that provides cap-independent expression of gene of interest or a reporter gene in an expression vector.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"10 depicts vectors T7/crTMV and SP6/crTMV.",
"FIG.",
"11 depicts vectors T7/crTMV/IRESMP,75CR-GUS, T7/crTMV/IRESMP,75UI-GUS, T7/crTMV/IRESMP,228CR-GUS, T7/crTMV/IRESCP,148CR-GUS, T7/crTMV/SPACERCP,148UI-GUS and T7/crTMV/PL-GUS.",
"FIG.",
"12.Mapping of the 5′end of the crTMV I2 sgRNA by primer extention (A) and putative secondary structure of 12 sgRNA 5′NTR.",
"FIG.",
"13.crTMV 12 sgRNA 5′NTR contains translation inhibiting hairpin structure.",
"(A)-depicts artificial transcripts used for in vitro translation in wheat germ extracts (WGE); (B)-shows translation products synthesized in WGE.",
"FIG.",
"14.Tobamoviruses contain a putative translation inhibiting hairpin structure upstream of the MP gene.",
"FIG.",
"15.Method of the specific detection of capped mRNAs.",
"A, B. RNA-tag with known sequence is ligated specifically to the cap of tested RNA.",
"C. Reverse transcription with 3′-specific primer and synthesis of first strand of cDNA.",
"Tag sequence is included to the sequence of cDNA.",
"D. PCR with tag-specific and 3′-specific primers.",
"The appearance of the respective PCR band indicates the presence of cap-structure in the tested RNA.",
"E. PCR with 5′-specific and 3′-specific primers.",
"The appearance of PCR band serves as a control for PCR reaction and indicates a presence of the specific tested RNA in the reaction.",
"F Relative comparison of the lengths of obtained PCR bands.",
"FIG.",
"16a and 16b.",
"Detection of the presence of a cap-structure at the 5′-terminus of viral RNAs in a 2% agarose gel.",
"Arrows indicate the respective PCR bands.",
"FIG.",
"17.depicts KK6-based TMV vectors.",
"FIG.",
"18.Nucleotide sequence of 5′NTR of KK6 and KK6-IRESMP,75CR I2sgRNA.",
"FIG.",
"19.Time-course of CP and MP accumulation in leaves inoculated with KK6-IRESMP,75CR (K86), KK6 and TMV UI.",
"FIG.",
"20.CP accumulation in tobacco infected with KK6, KK6-IRESMP,75CR, KK6-IRESMP,125CR, and KK6-H-PL and KK6-PL.",
"FIG.",
"21 depicts a crTMV IRESmp multimer structure and complementarity to 18S rRNA.",
"FIG.",
"22 depicts bicistronic transcripts containing IRESMP,75CR the tetramers of 18-nt segment of IRESCP,148CR, 19-nt segment of IRESMP,75CR, polylinker (PL) as intercistronic spacer and products of their translation in RRL.",
"FIG.",
"23 depicts the IRESCP,148CR structure.",
"FIG.",
"24 depicts constructs used for IRESCP,148CR sequence elements testing in vitro and in vivo.",
"FIG.",
"25.GUS activity testing in WGE after translation of transcripts depicted in FIG.",
"30.FIG.",
"26.GUS activity test in tobacco protoplasts transfected with 35S promoter-based constructs analogous to those depicted on FIG.",
"30.FIG.",
"27 depicts a scheme of cloning of two infectious TMV vectors containing IRESMP,75CR in 5′NTR.",
"FIG.",
"28 depicts vector Act2/crTMV.",
"FIG.",
"29 depicts pUC-based vector Act2/crTMV/IRESMP,75CR-GUS.",
"FIG.",
"30 depicts circular single-stranded vector KS/Act2/crTMV/IRESMP,75CR-GUS.",
"FIG.",
"31 depicts vector KS/Act2/crTMV/IRESMP,75CR-GUS FIG.",
"32 depicts construct 35S/CP/IRESMP,75CR/GUS.",
"FIG.",
"33 depicts construct 35S/GUS/IRESMP,75CR/CP.",
"FIG.",
"34 depicts construct 35S/CP-VPg/IRESMP,75CR/GUS.",
"FIG.",
"35 shows a construct for in vivo genetic selection to identify a viral subgenomic promoter or an IRES sequence that provides cap-independent expression of a gene of interest in an expression vector.",
"DETAILED DESCRIPTION OF THE INVENTION A primary objective of this invention is to provide a novel strategy for the construction of amplification-based vectors for foreign (heterologous, non-native) gene expression such that translation of these genes will occur by virtue of IRES-mediated cap-independent internal ribosome entry mechanism from polycistronic genomic viral RNAs and/or from bi- and multicistronic sgRNAs produced by an amplification vector, preferably a viral vector in a plant cell.",
"Construction of recombinant plant viral RNAs and creation of amplification-based vectors for the introduction and expression of foreign genes in plants has been demonstrated by numerous authors using the genomes of viruses belonging to different taxonomic groups (for review, see “Genetic Engineering With Plant Viruses”, 1992, eds.",
"Wilson and Davies, CRC Press, Inc.).",
"Tobamoviruses are considered to be convenient subjects for the construction of viral vectors.",
"Donson et al.",
"(U.S. Pat.",
"Nos.",
"5,316,931; 5,589,367 and 5,866,785) created TMV-based vectors capable of expressing different foreign genes in a host plant.",
"Thus, neomycin phosphotransferase, a-trichosantin and several other foreign genes were inserted adjacent to the subgenomic promoter (sgPR) of TMV CP.",
"Donson et al., (1993, PCT WO 93/03161) developed on the basis of a tobamovirus “a recombinant plant viral nucleic acid comprising a native plant viral subgenomic promoter, at least one non-native plant viral subgenomic promoter and a plant viral coat protein coding sequence, wherein said non-native plant viral subgenomic promoter is capable of initiating transcription of an adjacent nucleic acid sequence in a host plant and is incapable of recombination with the recombinant plant viral nucleic acid subgenomic promoters and said recombinant plant viral nucleic acid is capable of systemic infection in a host plant”.",
"Contrary to the technology of Donson et al., the present invention is not concerned with sgPRs in order to construct a viral replicon-based plant expression system.",
"Instead of sgPRs, our technology manipulates with IRES-sequences of different origin (native or non-native for the virus), the sequences of which effectively lack sgPR activity, i.e.",
"are effectively unable to promote sgRNA production.",
"Therefore, these IRES sequences should not be regarded as sgPRs even in the case they represent a nonfunctional segment of a sgPR.",
"It is generally believed that uncapped transcripts of full-length viral RNA obtained after in vitro transcription of cDNA clones are generally non-infectious for intact plants and isolated protoplasts.",
"Therefore, capping of a virus expression vector RNA transcript is generally considered as a prerequisite for in vitro transcript infectivity.",
"Capped RNA transcripts are commonly used for introducing a viral vector RNA into a plant.",
"It is important to note that in some cases viral RNA may be encapsidated by the coat protein using a simple procedure of in vitro assembly.",
"Thus, TMV virions as well as pseudovirions containing vector RNA can be readily produced from CP and in vitro transcripts or purified authentic viral RNA.",
"About fifteen years ago, it has been shown by Meshi et al.",
"(1986, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 85, 5043-5047) that (1) the uncapped transcripts of full-length TMV RNA produced in vitro are infectious in the absence of a cap analogue, although their specific infectivity is very low.",
"In the present invention, uncapped expression vector RNA reassembled with TMV CP can be used for plant inoculations in order to overcome its low infectivity.",
"At least one of the additional approaches described in this invention opens the technical possibilities for plant infection with a cap-independent plant viral vector.",
"This is the method of insertion of a full-length single-stranded (ss) DNA copy of a viral vector under control of an appropriate DNA promoter.",
"After inoculation of a host plant with the recombinant viral DNA, the infectious full-length RNA of a plant viral vector, which will be able to replicate and spread over the plant, will be produced.",
"In other words, these procedures, taken together with the fact of cap-independent expression of foreign gene(s) promoted by IRES sequences, make both processes, namely host plant inoculation and foreign gene expression, entirely cap-independent.",
"An important preferred object of the present invention is the creation of a series of crTMV genome-based viral vectors with the “IRES-foreign gene” block inserted between the CP gene and 3′-NTR.",
"Various IRES and control sequences were used (see FIG.",
"11) in combination with two different reporter genes (GUS and GFP).",
"A unique feature of this invention is that the foreign genes that were located outside of the viral sgPR sequences were expressed in the infected plant cap-independently from the 3′-proximal position of genomic and sgRNAs produced by the vector.",
"In particular, the IRESMP,75CR sequence representing the 3′-terminal part of the 5′-nontranslated leader sequence of crTMV sgRNA I2 was efficient in mediating cap-independent expression of the 3′-proximal foreign gene in plants infected with a viral vector.",
"It should be emphasized that said crTMV-based viral vectors produce three types of viral plus-sense ssRNAs in infected plants, including: i) full-length genomic RNA, ii) tricistronic I2 sgRNA (our data show that the latter sgRNA is uncapped, contrary to full-length RNA), and iii) bicistronic sgRNA containing the first CP gene and the second foreign gene.",
"Therefore, all these RNAs are 3′-coterminal and cap-independent translation of their 3′-proximal gene from either capped (full-length and bicistronic) or uncapped (tricistronic) RNAs is promoted by the preceding IRES sequence.",
"An important characteristic of virus-based vectors is their stability.",
"However, the TMV-based vectors with foreign genes usually do not move efficiently through phloem in plants that can be systemically infected with wild-type virus.",
"This may be due to increased length of the recombinant viral RNA and/or to the presence of the repeated sequences, which could lead to recombinations and deletions resulting in reversions to wild-type virus.",
"The conversion of the progeny population to wild-type virus occurs in systemically infected leaves.",
"An important characteristic for a virus-based vector is the level of foreign protein gene expression and the level of protein accumulation.",
"The vector is able to produce readily visible bands corresponding to GUS stained in SDS-PAGE.",
"The technologies suitable for construction of amplification-based vectors capable of expressing foreign sequences in host plants have been developed on the basis of different viral genomes (e.g., see G. Della-Cioppa et al., 1999, PCT WO 99/36516).",
"The central feature of those inventions was that the recombinant plant viral nucleic acid “contains one or more non-native subgenomic promoters which are capable of transcribing or expressing adjacent nucleic acid sequences in the host plant.",
"The recombinant plant viral nucleic acids may be further modified to delete all or part of the native coat protein coding sequence and to contain a non-native coat protein coding sequence under control of the native or one of the non-native plant viral subgenomic promoters, or put the native coat protein coding sequence under the control of a non-native plant viral subgenomic promoter”.",
"In other words, the most important element(s) of that invention is/are the native and non-native sgPR sequences used for artificial sgRNAs production by the viral vector.",
"An important feature that distinguishes the invention presented by our group from others is that according to WO 99/36516, the foreign gene must be inevitably located directly downstream of the sgPR sequence, i.e.",
"should be located at the 5′-proximal position of the chimeric sgRNA produced by the viral vector in the host plant.",
"By contrast, our invention proposes that the foreign gene is separated from a sgPR (if present) at least by one (or more) viral gene(s) such that said foreign gene is located 3′-proximally or internally within the functionally active chimeric sgRNA produced by the vector.",
"Thus, foreign gene expression is promoted by the IRES sequence, native or non-native of the wild-type virus.",
"The next preferred object of this invention is the construction of a novel type of non-native IRES sequences, namely artificial, non-natural synthetic IRESs capable of promoting cap-independent translation of 5′-distal genes from eukaryotic polycistronic mRNAs.",
"We show that intercistronic spacers complementary to 18S rRNA of varying length and composition are able to mediate cap-independent translation of the 3′-proximal GUS gene in bicistronic H-GFP-IRES-GUS mRNA (FIG.",
"22).",
"The last but not least advantage provided by the present invention is the possibility to combine repeats of two or more foreign genes each being preceded by the native or non-native IRES sequence in the amplification-based vector genome.",
"Expression of such a cassette of an “IRES-foreign gene” will allow the simultaneous production of two or more foreign proteins by the vector.",
"Viruses belonging to different taxonomic groups can be used for the construction of virus-based vectors according to the principles of the present invention.",
"This is right for both RNA- and DNA-containing viruses, examples for which are given in the following (throughout this document, each type species name is preceded by the name of the order, family and genus it belongs to.",
"Names of orders, families and genera are in italic script, if they are approved by the ICTV.",
"Taxa names in quotes (and not in italic script) indicate that this taxon does not have an ICTV international approved name.",
"Species (vernacular) names are given in regular script.",
"Viruses with no formal assignment to genus or family are indicated): DNA Viruses: Circular dsDNA Viruses: Family: Caulimoviridae, Genus: Badnavirus, Type species: commelina yellow mottle virus, Genus: Caulimovirus, Type species: cauliflower mosaic virus, Genus “SbCMV-like viruses”, Type species: Soybean chloroticmottle virus, Genus “CsVMV-like viruses”, Type species: Cassava vein mosaicvirus, Genus “RTBV-like viruses”, Type species: Rice tungro bacilliformvirus, Genus: “Petunia vein clearing-like viruses”, Type species: Petunia vein clearing virus; Circular ssDNA Viruses: Family: Geminiviridae, Genus: Mastrevirus (Subgroup I Geminivirus), Type species: maize streak virus, Genus: Curtovirus (Subgroup II Geminivirus), Type species: beet curly top virus, Genus: Begomovirus (Subgroup III Geminivirus), Type species: bean golden mosaic virus; RNA Viruses: ssRNA Viruses: Family: Bromoviridae, Genus: Alfamovirus, Type species: alfalfa mosaic virus, Genus: Ilarvirus, Type species: tobacco streak virus, Genus: Bromovirus, Type species: brome mosaic virus Genus: Cucumovirus, Type species: cucumber mosaic virus; Family: Closteroviridae Genus: Closterovirus, Type species: beet vellows virus, Genus: Crinivirus, Type species: Lettuce infectious yellows virus, Family: Comoviridae, Genus: Comovirus Type species: cowpea mosaic virus, Genus: Fabavirus, Type species: broad bean wilt virus 1, Genus: Nepovirus, Type species: tobacco ringspot virus: Family: Potyviridae, Genus: Potyvirus, Type species: potato virus Y, Genus: Rymovirus, Type species: ryegrass mosaic virus, Genus: Bymovirus, Type species: barley yellow mosaic virus; Family: Sequiviridae, Genus: Sequivirus, Type species: parsnip vellow fleck virus, Genus: Waikavirus, Type species: rice tungro spherical virus: Family: Tombusviridae, Genus: Carmovirus, Type species: carnation mottle virus, Genus: Dianthovirus, Type species: carnation ringspot virus, Genus: Machlomovirus, Type species: maize chlorotic mottle virus, Genus: Necrovirus, Type species: tobacco necrosis virus, Genus: Tombusvirus, Type species: tomato bushy stunt virus, Unassigned Genera of ssRNA viruses, Genus: Capillovirus, Type species: apple stem grooving virus; Genus: Carlavirus, Type species: carnation latent virus: Genus: Enamovirus, Type species: pea enation mosaic virus.",
"Genus: Furovirus, Type species: soil-bome wheat mosaic virus.",
"Genus: Hordeivirus, Type species: barley stripe mosaic virus, Genus: Idaeovirus, Type species: raspberry bushy dwarf virus; Genus: Luteovirus, Type species: barley yellow dwarf virus: Genus: Marafivirus, Type species: maize rayado fino virus: Genus: Potexvirus, Type species: Dotato virus X; Genus: Sobemovirus, Type species: Southern bean mosaic virus.",
"Genus: Tenuivirus, Type species: rice stripe virus, Genus: Tobamovirus, Type species: tobacco mosaic virus, Genus: Tobravirus, Type species: tobacco rattle virus, Genus: Trichovirus, Type species: apple chlorotic leaf spot virus, Genus: Tymovirus, Type species: turnip yellow mosaic virus, Genus: Umbravirus, Type species: carrot mottle virus, Negative ssRNA Viruses: Order: Mononegavirales, Family: Rhabdoviridae, Genus: Cytorhabdovirus, Type Species: lettuce necrotic vellows virus, Genus: Nucleorhabdovirus, Type species: potato yellow dwarf virus; Negative ssRNA Viruses: Family: Bunyaviridae, Genus: Tospovirus, Type species: tomato spotted wilt virus: dsRNA Viruses: Family: Partitiviridae, Genus: Alphacryptovirus, Type species: white clover cryptic virus 1, Genus: Betacryotovirus, Type species: white clover cryptic virus 2, Family: Reoviridae, Genus: Fijivirus, Type species: Fiji disease virus, Genus: Phytoreovirus, Type species: wound tumor virus, Genus: Oryzavirus, Type species: rice ragged stunt virus; Unassigned Viruses: Genome ssDNA: Species banana bunchy top virus, Species coconut foliar decay virus, Species subterranean clover stunt virus, Genome dsDNA, Species cucumber vein yellowing virus, Genome dsRNA Species tobacco stunt virus, Genome ssRNA Species Garlic viruses A,B,C,D, Species grapevine fleck virus, Species maize white line mosaic virus, Species olive latent virus 2, Species ourmia melon virus, Species Pelargonium zonate spot virus; Satellites and Viroids: Satellites: ssRNA Satellite Viruses: Subgroup 2 Satellite Viruses, Type species: tobacco necrosis satellite, Satellite RNA, Subgroup 2 B Type mRNA Satellites, Subgroup 3 C Type linear RNA Satellites, Subgroup 4 D Type circular RNA Satellites, Viroids, Type species: potato spindle tuber viroid.",
"In particular, the methods of the present invention can preferably be applied to the construction of virus replicon-based vectors using the recombinant genomes of plus-sense ssRNA viruses preferably belonging to the genus Tobamovirus or to the families Bromoviridae or Potyviridae as well as DNA-containing viruses.",
"In the latter case the foreign gene should preferably be located downstream of a viral gene and its expression can be mediated by the IRES sequence from bicistronic or polycistronic mRNA transcribed by a DNA-dependent RNA polymerase from a genomic transcription promoter.",
"A separate preferred aspect of this invention is concerned with the application of the methods of the invention to the construction of ssDNA-based vectors.",
"The geminivirus-based vectors expressing the foreign gene(s) under control of an IRES sequence can exemplify this aspect.",
"The geminiviruses represent a group of plant viruses with monopartite or bipartite circular ssDNA that have twinned quasiicosahedral particles (reviewed by Hull and Davies, 1983, Adv.",
"Virus Res.",
"28, 1-45; Mullineaux et al., 1992, “Genetic engineering with plant viruses”, Wilson and Davies, eds., 1992, CRC Press, Inc.).",
"The two ssDNA components of the bipartite geminiviruses referred to as A and B encode for 4 and 2 proteins, respectively.",
"The DNA A contains the CP gene and three genes involved in DNA replication, whereas the DNA B encodes for two proteins essential for the viral movement.",
"It has been demonstrated that the genomes of bipartite geminiviruses belonging to the genus Begomovirus, such as tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) can replicate and spread over a certain host plant despite the deletion of the CP gene (Gardiner et al., 1988, EMBO J.",
"7, 899-904; Jeffrey et al., 1996, Virology 223, 208-218; Azzam et al., 1994, Virology 204, 289-296).",
"It is noteworthy that some begomoviruses including BGMV exhibit phloem-limitation and are restricted to cells of the vascular system.",
"Thus, BGMV remains phloem-limited, while TGMV is capable of invading the mesophyll tissue in systemically infected leaves (Petty and Morra, 2000, Abstracts of 19th Annual meeting of American Society for Virology, p.127).",
"The present invention proposes to insert the foreign gene in a bipartite geminivirus genome by two ways: (i) downstream of one of its (e.g., BGMV) genes, in particularthe CP gene such that the CP ORF will be intact or 3′-truncated and the IRES sequence will be inserted upstream of the foreign gene.",
"Therefore, the mRNA transcription will proceed from the native DNA promoter resulting in production of bicistronic chimeric mRNA comprising the first viral gene (or a part thereof), the IRES sequence and the 3′-proximal foreign gene expression of which is mediated by the IRES.",
"Alternatively (ii), the full-length DNA copy of the the RNA genome of the viral vector can be inserted into a DNA of a CP-deficient bipartite geminivirus under control of the CP gene promoter.",
"The RNA genome of the RNA-vector-virus will be produced as a result of DNA A transcription in the plant cell inoculated with a mixture of recombinant DNA A and unmodified DNA B.",
"An advantage of this method is that the geminivirus-vector is needed as a vehicle used only for delivering the vector to primary-inoculated cells: all other steps will be performed by a tobamovirus vector itself including production of IRES-carrying vector RNA after geminivirus-vector DNA transcription by a cellular RNA polymerase, its replication, translation and systemic spread over the host plant and foreign gene(s) expression.",
"As an additional possibility for the creation of a ssDNA vector, cloning of the viral cDNA and the foreign gene into a phagemid vector and production of the ssDNA according to standard methods can be mentioned.",
"Taking into account that tobamovirus-derived IRES sequences are shown to be functionally active in animal cells (our previous patent application), the methods of the present invention can be used for constructing the recombinant viral RNAs and producing the viral vectors on the basis of animal viruses, e.g.",
"the viruses belonging to the families Togaviridae, Caliciviridae, Astroviridae, Picomaviridae, Flaviviridae in order to produce new vectors expressing the foreign genes under control of plant virus-derived IRES sequences.",
"Such animal virus-based vectors for plants and animals can be useful in the fields of vaccine production or for gene therapy.",
"It should be noted, however, that the rod-like virions of Tobamoviruses and, in particular, the flexible and long virions of filamentous Potexviruses, Carlaviruses, Potyviruses and Closteroviruses apparently provide the best models for realization of the methods of the present invention.",
"The next preferred objective of this invention is to use the IRES sequence in such a way that the virus-based amplification vector will contain the IRES-sequence within its 5′-NTR.",
"It is presumed that insertion of an IRES sequence does not prevent viral replication, but is able to ensure an efficient cap-independent translation of transcripts of genomic vector RNA.",
"Therefore, said construct may comprise: (i) An IRES element within or downstream of the 5′-untranslated leader sequence that is native or non-native for said viral vector and promotes cap-independent translation of the viral 5′-proximal gene (the RdRp), and (ii) at least one native or non-native IRES sequence located downstream of one or more viral structural genes and upstream of foreign gene(s) in order to promote their cap-independent translation.",
"According to this method, the specific infectivity of uncapped full-length vector transcripts will be increased due to efficient 5′-IRES-mediated translation of the parental RNA molecules in the primary inoculated cells.",
"Yet another preferred objective of the present invention is the method of producing one or several protein(s) of interest in plant cells based on the introduction and cap-independent expression of a foreign gene from a mono- or polycistronic mRNA sequence mediated by the plant specific IRES sequence located upstream of said foreign gene sequence.",
"A particular feature of this method is that the technology involves a procedure that allows to selectively switch off the cellular cap-dependent mRNA translation with the help of certain chemical compounds.",
"However, this procedure does not affect the cap-independent IRES-mediated translation of mRNAs artificially introduced in the plant cells, thus allowing to control and enhance cap-independent expression.",
"Alternatively, the means for inhibiting the translation of cellular capped mRNA can be applied to plants infected with said viral vector itself that expresses the foreign gene(s) in a cap-independent manner.",
"Under conditions when the translation of the cellular capped mRNAs is prevented, selective expression of the foreign gene(s) from said virus vector will occur.",
"The vector of the invention may be an RNA or DNA vector.",
"It may be ss(+), ss(−) or ds.",
"It may show any of the modes of amplification known from viruses.",
"This includes the multiplication of the vector nucleic acid and optionally the production of coat protein and optionally the production of proteins for cell-to-cell movement or long-distance movement.",
"The genes for the required replication and/or coat and/or movement may be wholly or partially encoded in an appropriately engineered host plant.",
"In this manner, a system is generated consisting of mutually adapted vector and host plant.",
"The vector may be derived form a virus by modification or it may be synthesized de novo.",
"It may have only IRES elements effectively devoid of any subgenomic promoter activity.",
"However, the vector may combine one or several subgenomic promoters with one or several IRES elements effectively devoid of subgenomic promoter function, so that the number of cistrons is greater than the number of promoters.",
"Considering the simplest case of one IRES element, said element may be located upstream of a (foreign) gene of interest to be expressed directly by said IRES element and optionally downstream of a (viral) gene for, say replication, to be expressed IRES-independent.",
"Alternatively, the gene of interest may be upstream of an IRES element and expressed IRES-independent and the IRES element serves for the expression of a downstream viral gene.",
"These simplest cases may of course be incorporated singly or multiply in a more complex vector.",
"The vector may contain a sequence in anti-sense orientation for suppressing a host gene.",
"This suppression function may exist alone or in combination with the expression of a (foreign) gene of interest.",
"A particularly preferred case involves the suppression of a gene essential for cap-dependent translation, e.g.",
"a gene for a translation initiation factor (e.g.",
"elF4) associated with cap-dependent translation, so that the translation machinery of the host plant is wholy in service of vector gene translation.",
"In this case, the vector must be wholy cap-independent.",
"Of course, the vector may be generated within a plant cell from a pro-vector by the plant nucleid acid processing machinery, e.g.",
"by intron splicing.",
"The IRES element may be of plant viral origin.",
"Alternatively, it may be of any other viral origin as long as it satisfies the requirement of operation in a plant cell.",
"Further, an IRES element operative in a plant cell may be a synthetic or an artificial element.",
"Synthesis may be guided by the sequence of the 18S rRNA of the host plant, namely the segment operative for IRES binding.",
"It should be sufficiently complementary thereto.",
"Sufficiency of complementarity can simply be monitored by testing for IRES functionality.",
"Complementarity in this sense comprises GC, AU and to some extent GU base pairing.",
"Further, such IRES element may be a multimer of such a complementary sequence to increase efficiency.",
"The multimer may consist of identical essentially complementary sequence units or of different essentially complementary sequence units.",
"Moreover, artificial IRES elements with high translation efficiency and effectively no subgenomic promoter activity may be generated by a process of directed evolution (as described e.g.",
"in U.S. Pat.",
"No.",
"6,096,548 or U.S. Pat.",
"No.",
"6,117,679).",
"This may be done in vitro in cell culture with a population of vectors with IRES element sequences that have been randomized as known per se.",
"The clones which express a reporter gene operably linked to the potential IRES element are selected by a method known per se.",
"Those clones which show subgenomic promoter activity are eliminated.",
"Further rounds of randomization and selection may follow.",
"The IRES element of the vector of the invention may be effectively devoid of promoter activity.",
"This means that that the expression of a gene operably linked to an IRES element would not occur by a residual subgenomic promoter activity.",
"This mode of action may be determined by standard molecular biology methods such as Northern blotting, primer extension analysis (Current Protocols in Molecular Biology, Ed.",
"By F. Ausubel et al., 1999, John Wiley & Sons), 5′ RACE technology (GibcoBRL, USA), and alike.",
"It should be added that IRES elements that show detectable subgenomic promoter activity but operate essentially as translational rather than transcriptional elements, are also subject of our invention.",
"Such discrimination could be derived, for example, by measuring quantitatively the relative amounts of two types of mRNAs on Northern blots, namely the short mRNA due to sgPR activity and the long mRNA not due to sgPR activity.",
"If the IRES element does not essentially operate as a residual viral subgenomic promoter, the relative amount of corresponding short mRNA should be lower than 20%, preferably lower than 10% and most preferably.",
"lower than 5% of the sum of the short and long mRNA.",
"Thus we provide as a preferred embodiment a vector capable of amplification of a gene in a plant comprising a nucleic acid having a sequence for at least one non-viral gene to be expressed and having or coding for at least one IRES element necessary for translation of said gene in said plant with the proviso that the expression of said gene is essentially derived from translational rather than transcriptional properties of said IRES element sequence when measured by standard procedures of molecular biology.",
"The novel vectors of the invention open new avenues for genetic modification of plants.",
"As a first possibility we suggest the use for determining the function of a structural gene of a plant.",
"This is notably of interest for genomics.",
"Therefore, a plant for which the genome has been sequenced is of particular interest.",
"This is a small scale (plant-by plant) application.",
"The vector of this invention is highly effective for this application, since it allows suppression of genes of interest and/or overexpression of genes to bring out the gene function to be discovered in an intensified manner.",
"In a large scale application the vector may be used to generate a trait or to produce a protein in a host plant.",
"Infection of plants with the vector may be done on a farm field previously planted with unmodified plants.",
"This allows for the first time a genetic modification of plants on a field, whereby the farmer has greatest freedom in terms of selection of seeds and vectors from a variety of sources for producing a desired protein or trait.",
"Examples for plant species of interest for the application of this invention are monocotyledonous plants like wheat, maize, rice, barley, oats, millet and the like or dicotyledonous plants like rape seed, canola, sugar beet, soybean, peas, alfalfa, cotton, sunflower, potato, tomato, tobacco and the like.",
"In the following, the invention will be further described using specific examples.",
"Standard molecular biological techniques were carried out according to Sambrook et al.",
"(1989, Molecular Cloning: a Laboratory Manual.",
"2nd edn.",
"Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).",
"All plasmids utilized in the invention can be prepared according to the directions of the specification by a person of ordinary skill in the art without undue experimentation employing materials readily available in the art.",
"EXAMPLE 1 Construction of a tobamovirus Vector Infecting cruciferous Plants Virions of a known tobamovirus called crucifer tobamovirus (crTMV) which is able to infect systemically crucifer plants were isolated from Olearacia officinalis L. with mosaic symptoms.",
"Results of crTMV host-range examination are presented in Table 1.Plasmid Constructions CrTMV cDNA was characterized by dideoxynucleotide sequencing (Dorokhov et al., 1994 FEBS Letters 350, 5-8).",
"Full length T7 RNA polymerase promoter-based infectious crTMV cDNA clones were obtained by RT-PCR from crTMV RNA using oligonucleotides crTMV1-Kpn 5′-gcatggtaccccttaatacgactcactataGTTTAGTTTTATTGCAACAACAACAA (upstream), wherein the italic bold letters are a sequence of a Kpn I site, the underlined lowercase letters are nucleotide sequence of the T7 RNA polymerase promoter, the uppercase letters are from the 5′-termini of crTMV cDNA; and crTMV2 5′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG (downstream), wherein the italic bold letters are sequence of NotI site, the uppercase letters are from 3′-termini of crTMV cDNA and cloning into pUC19 between KpnI and Bam HI restriction sites (FIG.",
"10).",
"Full length SP6 RNA polymerase promoter-based infectious crTMV cDNA clones were obtained by RT-PCR from crTMV RNA by using oligonucleotides crTMV1-SP6 5′-gcatggtaccatttaggtgacactatagaactcGTTTTAGTTTTATTGCAACAACAACAA (upstream), wherein the italic bold letters are a sequence of a Kpn I site, the underlined lowercase letters are a nucleotide sequence of the T7 RNA polymerase promoter, the uppercase letters are from the 5′-termini of crTMV cDNA; and crTMV2 5′-gcatgcggccgcTGGGCCCCTACCCGGGGTTAGGG (downstream), wherein the italic bold letters are a sequence of a Not I site, the uppercase letters are from 3′-termini of crTMV cDNA and cloning into pUC19 between KpnI and Bam HI restriction sites (FIG.",
"10).",
"The full-length crTMV cDNA clones were characterized by dideoxynucleotide sequencing.",
"The ability of crTMV infectious transcripts to infect systemically Nicotiana and crucifer species was confirmed by infection tests on respectively Nicotiana tabacum var.",
"Samsun and Arabidopsis thaliana.",
"TABLE 1 Virus detection and symptoms caused by crTMV in mechanically infected plants.",
"Non-inoculated Inoculated Leaves Upper Leaves Species Symptoms* Virus** Symptoms Virus Nicotiana tabacum L. cv.",
"Samsun C + M + cv.",
"Samsun NN.",
"L + s − Nicotiana clevelandii L. L + N + M + Nicotiana glutinosa L. L + N + s − Nicotiana sylvestris L. L + N + s + Nicotiana benthamiana L. L + N + M + Nicotiana rustica L. C + M + Lycopersicum esculentum L. L + N + s − Solanum tuberosum L. s − s − Capsicum frutescens L. L + N + M + Brassica chinensis L. C + M + Brassica rapa L. C + M + Brassica napus L C + M + Brassica oleracea L. L + s − Brassica compestris L. C + M + Brassica cauliflora L. C + s − Arabidopsis thaliana L. L + N + M + Chenopodium L + N + s + amaranticolor L. Coste and Reyn.",
"Chenopodium quinoa L + N + s − L. Willd.",
"Chenopodium murale L. L + N + s − Datura stramonium L. L + N + s − Plantago major L. L + N + M + Tetragonia expansa L. L + N + s − Beta vulgaris L. L + N + s − Petunia hybrida L. C + M + Cucumis sativus L. L + N + s − Phaseolus vulgaris L. s − s − Raphanus sativus L. s − s − Sinapis alba L. C + M 0 *C, chlorosis; L, local lesion; M, mosaic; N, necrosis; s, symptomless.",
"**Virus detected (+) or not (−) by ELISA.",
"EXAMPLE 2 Construction of tobamoviral Vectors for Expression of GUS Genes in Nicotiana and crucifer Plants via Viral IRESs Series of IRES-mediated expression vectors T7/crTMV/GUS were constructed as follows.",
"First, Hind III and Xba I sites were inserted in the end of the CP gene of Sac II/Not I fragment of T7/crTMV vector (FIG.",
"10) by a polymerase chain reaction (PCR) and two pairs of specific primers.",
"Second, IRESMP,75CR-GUS, IRESMP,76UI-GUS, IRESMP,228CR-GUS, IRESCP,148CR-GUS, IRESCP,148UI-GUS, PL-GUS cDNA described in Skulachev et al.",
"(1999, Virology 263, 139-154) were inserted into Hind III and Xba I containing Sac II/Not I fragment of T7/crTMV vector to obtain Sac I-IRESMP,75CR-GUS-Not I, Sac II-IRESMP,75UI-GUS-Not I, Sac II-I-RESMP,228CR-GUS-Not I, Sac I-IRESCP,148CR-GUS-Not I, Sac II-IRESCP,148UI-GUS-Not I, Sac II-PL-GUS-Not I cDNA, respectively.",
"Third, Sac II-Not I cDNA fragment of T7/crTMV vector was replaced by Sac I-IRESMP,75CR-GUS-Not I or Sac II-IRESMP,75UI-GUS-Not I or Sac II-IRESMP,228CR-GUS-Not I or Sac II-IRESCP,148CR-GUS-Not I or Sac I-IRESCP,148UI-GUS-Not I or Sac II-PL-GUS-Not I cDNA to obtain respectively, vector T7/crTMV/IRESMP,75CR-GUS (FIG.",
"11), vector T7crTMV/IRESMP,75UI-GUS (FIG.",
"11), vector T7/crTMV/IRESMMP,228CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148UI-GUS (FIG.",
"11 and vectorT7/crTMV/PL-GUS (FIG.",
"11).",
"EXAMPLE 3 Expression of GUS Gene in Transfected Nicotiana and crucifer Plants via Viral IRESs This example demonstrates the tobamovirus IRES-mediated expression of the GUS gene in Nicotiana benthamiana and Arabidopsis thaliana plants infected crTMV-based vectors: T7/crTMV/IRESMP,75CR-GUS (FIG.",
"11), vector T7/crTMV/IRESMP,75UI-GUS (FIG.",
"11), vector T7/crTMV/IRESMP,228CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148UI-GUS (FIG.",
"11) and vectorT7/crTMV/PL-GUS (FIG.",
"11).",
"In vitro Transcription The plasmids T7/crTMV/IRESMP,75CR-GUS (FIG.",
"11), vector T7/crTMV/IRESMP,75UI-GUS (FIG.",
"11), vector T7/crTMV/IRESMP,228CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148CR-GUS (FIG.",
"11), vector T7/crTMV/IRESCP,148UI-GUS (FIG.",
"11) and vectorT7/crTMV/PL-GUS (FIG.",
"11) were linearized by Not I.",
"The recombinant plasmids were transcribed in vitro as described by Dawson et al.",
"(1986 Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 83, 1832-1836).",
"Agarose gel electrophoresis of RNA transcripts confirmed that they were intact.",
"The RNA concentration was quantified by agarose gel electrophoresis and spectrophotometry.",
"GUS Detection Inoculated leaves were collected 10-14 days after transfection with capped full-length transcripts.",
"IRES activity was monitored by histochemical detection of GUS expression as described earlier (Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405).",
"Samples were infiltrated using the calorimetric GUS substrate, but the method (De Block and Debrouwer, 1992, Plant J.",
"2, 261-266) was modified to limit the diffusion of the intermediate products of the reaction: 0.115 M phosphate buffer, pH 7.0 containing 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) 600 μg/ml; 3 mM potassium ferricyanide; 10 mM EDTA.",
"After incubation overnight at 37° C., the leaves were destained in 70% ethanol and examined by light microscopy.",
"EXAMPLE 4 IRESMP,75CR does not Function as MP Subaenomic Promoter but Provides MP Gene Expression via Cap-Independent Internal Initiation of Translation in TMV-Infected Plants This example uses different approaches to confirm the possibility of IRESMP,75CR used in viral vectors for cap-independent expression of a gene of interest.",
"CrTMV MP Subgenomic RNA has a 125-nt Long 5′-Nontranslated Region (5′NTR) and Contains a Translation Inhibiting Stem-Loop Secondary Structure To determine the length and nucleotide sequence of TMV UI and crTMV MP subgenomic RNA (I2 sgRNA) 5′NTR, the protocol of primer extension experiments described by Lehto et al.",
"(1990, Virology 174, 145-157) was changed in the following way: (i) AMV reverse transcriptase (RT); (ii) RT reaction under 45° C.; (iii) the GC-rich primer; (iv) increased dNTP concentration; (v) dITP to avoid secondary structure.",
"It has been shown (FIG.",
"12) that the 5′UTR sequence of crTMV I2 sgRNAs consists of 125 nucleotides.",
"This result was confirmed by direct 5′UTR RT sequencing.",
"FIG.",
"12B shows that crTMV 5′NTR contains a stable hairpin-loop structure.",
"Being placed just upstream of the MP gene of artificial transcript, it is able to inhibit MP gene translation in vitro (FIG.",
"13).",
"This means that IRESMP,75CR located between 5′HI2CR and the MP gene can provide efficient cap-independent internal initiation of translation.",
"FIG.",
"14 shows that homologous to 5′HI2CR putative translation inhibiting hairpin-loop structure can be revealed in the 125-nt sequence upstream of the MP gene of other tobamoviruse.",
"CrTMV and TMV UI MP Subgenomic RNAs are not Capped To study the structure of the 5′-terminus of the subgenomic RNA coding for the 30K movement protein (MP) gene of crTMV, the “Jump-Start” method offered by Active Motif was used.",
"Jump-Start™ is the method of chemical ligation of an RNA tag specifically to the 5′-end of capped mRNAs.",
"During reverse transcription, the ribo-oligonucleotide tag of a known sequence becomes incorporated into the 3′-end of a first strand cDNA.",
"This creates a known priming site suitable for PCR.",
"Initially, the 5′-terminal 2′-3′-cis-glycol groups of capped RNA were converted to reactive di-aldehydes via sodium periodate oxidation.",
"1-2 μl of a tested RNA (1 μg/μl) were mixed with 14 μl of pure water and 1 μl of sodium acetate buffer (pH 5.5), then 4 μl of 0.1 M sodium periodate were added and the reaction mixture was incubated for 1 hour.",
"Then a 3′-aminoalkyl derivatized synthetic ribo-oligonucleotide tag was chemically ligated to the di-aldehyde ends of oxidized RNA via reductive amination in the presence of sodium cyanoborohydride.",
"5 μl of sodium hypophosphite were added and the reaction mixture was incubated for 10 minutes.",
"Then 23 μl of water, 1 μl of sodium acetate buffer (pH 4.5) and 2 μl of ribo-oligonucleotide tag 5′-CTAATACGACTCACTATAGGG (28.5 pmol/μl) were added to the reaction mixture and incubated for 15 minutes.",
"Then 10 μl of sodium cyanoborohydride were added and incubated for 2 hours.",
"Then 400 μl of 2% lithium perchlorate in acetone were added, incubated for 15 minutes at −20° C. and centrifugated for 5 minutes.",
"The pellet was washed with acetone twice, then dissolved in 20 μl of water.",
"To remove an abundance of the RNA tag, CTAB precipitation in the presence of 0.3 M NaCl was used.",
"CTAB is a strong cationic detergent that binds to nucleic acids to form an insoluble complex.",
"Complex formation is influenced by the salt concentration: when the salt concentration is above 1 M, no complex formation occurs; when it is below 0.2 M, all nucleic acids are efficiently included in the complex; and when between 0.3 M and 0.4 M, the incorporation of small single-stranded nucleic acids into the complex is very inefficient (Belyavsky et al., 1989, Nucleic Acids Res.",
"25, 2919-2932; Bertioli et al., 1994, BioTechniques 16, 1054-1058).",
"10 μl of 1.2 M NaCl (to a final concentration of 0.4 M) and 3 μl of 10% CTAB (to a final concentration of 1%) were added, the reaction mixture was incubated for 15 minutes at room temperature and then centrifugated for 5 minutes.",
"The pellet was resuspended in 10 μl of NaCl, 20 μl of water and 3 μl 10% CTAB were added and the reaction mixture was incubated for 15 minutes at room temperature and then centrifugated for 5 minutes.",
"The pellet was dissolved in 30 μl of 1.2 M NaCl, 80 μl of 96% ethanol was added, and the reaction mixture was incubated overnight at −20° C. Then it was centrifugated for 5 minutes and washed with 70% ethanol.",
"Then the pellet of tagged RNA was dissolved in 24 μl of water.",
"Finally, reverse transcription with 3′-gene specific primers resulted in incorporation of the 5′-tag sequence at the 3′-terminus of first-strand cDNA.",
"For reverse transcription, 12 μl of tagged RNA, 1 μl of specific 3′-end primers, 4 μl of 5× buffer for SuperScript™II (Gibco BRL Life Technologies) containing 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 were mixed and heated at 95° C. for 30 seconds, then cooled on ice.",
"Then to the reaction mixture 0.5 μl of DTT (to 1 mM final concentration), 2 μl of 10 mM dNTP, 0.5 μl of RNAsine, 0.5 μl of SuperScript™II were added and incubated for 1 hour at 42° C. Then 1 μl of 40 mM MnCl2 was added and the reaction mixture was incubated for 15 minutes at 42° C. The presence of MnCl2 in the reaction mixture allows SuperScript™ to overcome the cap structure during reverse transcription more efficiently: when using 3 mM MgCl2 and 2 mM MnCl2, the reverse transcriptase was shown to reveal an extraordinary high cap-dependent transferase activity, and typically the enzyme added preferentially three or four cytosine residues in the presence of 5′-capped mRNA templates (Chenchik et al., 1998, Gene cloning and analysis by RT-PCR, edited by Paul Siebert and James Larrick, BioTechniques Books, Natlck, Mass.",
"; Schmidt and Mueller, 1999, Nucleic Acids Res.",
"27, 331).",
"For the PCR reaction, two sets of primers were used for each tested RNA-3′-specific/5′-specific primers and 3′-specific/tag-specific primers (FIG.",
"15).",
"To determine the possibility of using the method of chemical ligation of RNA with tag known sequence specifically to the cap-structure of viral RNAs, the genomic RNA of tobacco mosaic virus (TMV) U1 strain which is known to be capped (Dunigan and Zaitlin, 1990, J. Biol.",
"Chem.",
"265, 7779-7786.)",
"was used as control.",
"The respective PCR bands were detected when specific primers, U1-Spn and corresponding to RNA-tag primer 779 were used in the PCR reaction (Table 2, FIG.",
"16).",
"TABLE 2 Templates and primers used for PCR.",
"Corresponding PCR Forward band and detection Template primer Reverse primer of cap-structrure Genomic TMV (U1) RNA U1-Spn + Genomic TMV (U1) RNA 779 U1-Spn + (cap) Non-capped RNA transcript of TMV U1-Spn + Non-capped RNA transcript of TMV 779 U1-Spn − (non-capped) Complete cDNA clone of TMV (U1) U1-Spn + Genomic crTMV RNA K5 2PM + Genomic crTMV RNA 779 2PM + (?",
"- cap?)",
"Non-capped RNA transcript of crTMV K5 2PM + Non-capped RNA transcript of crTMV 779 2PM − (non-capped) Complete cDNA clone of crTMV K5 2PM + Subgenomic TMV (U1) RNA for MP 2211 UM50-54 + Subgenomic TMV (U1) RNA for MP 779 UM50-54 − (non-capped) Complete cDNA clone of TMV (U1) 2211 UM50-54 + Subgenomic crTMV RNA for MP 1038 CPF25 + Subgenomic crTMV RNA for MP 779 CPF25 − (non-capped) Complete cDNA clone of crTMV 1038 CPF25 0 As a control, the non-capped RNA-transcript of the complete cDNA clone of TMV (U1) was used, and the cap structure was not found as expected (Table 2, FIG.",
"16).",
"Then the presence of a cap structure at the 5′-terminus of the genomic RNA of crTMV was tested.",
"For these experiments, the specific PCR primers K5, 2PM and primer 779 which corresponds to the RNA-tag were taken (Table 1, FIG.",
"16).",
"Interestingly, the mobility of the PCR band observed with the primers 779 and 2PM, was higher than expected (FIG.",
"16).",
"This could reflect the presence of a strong secondary structure at the 5′-terminus of the genomic RNA of crTMV (Dorokhov et al., 1994, FEBS Letters 350, 5-8).",
"This secondary structure is absent at the 5′-terminal part of related TMVs (Goelet et al., 1982, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 79, 5818-5822).",
"In control experiments with non-capped transcript of the complete cDNA clone of crTMV, no respective PCR band was observed, as expected.",
"For subgenomic RNA coding for the TMV (U1) MP gene, the absence of a cap-structure at the 5′-terminus was proposed.",
"We tested the respective sgRNA with the specific primers 2211, UM50-54 and primer 779 corresponding to the RNA-tag.",
"No cap structure was found (Table 2, FIG.",
"16).",
"The same results were obtained with the respective subgenomic RNA of crTMV (Table 2, FIG.",
"16) indicating that cap-structure is absent at the 5′-terminus of this subgenomic RNA of tobamoviruses.",
"Insertion of IRESMP,75CR into a TMV UI Based Vector that is Deficient of MP Gene Expression, KK6 Provides Efficient Cap-Independent MP Gene Expression The KK6 vector (Lehto et al., 1990, Virology 174, 145-157) contains two CP subgenomic promoters (sgPr).",
"The first CP sgPr-1 is in its proper place, upstream of the CP gene, whereas the second, CP sgPr-2 is placed upstream of the MP gene.",
"It was shown that the MP gene was expressed via CP sgPr-2 instead of native MP sgPr.",
"As a result of this insertion, KK6 lost the capability of efficient cell-to-cell movement.",
"Analysis showed that I2 sgRNA does not contain an IRESMP,75CR element in its 5′-nontranslated leader.",
"It has been proposed that IRESMP,75CR-lacking KK6 I2 sgRNA cannot express the MP gene efficiently.",
"In order to examine this suggestion, IRESMP,75CR was inserted into KK6 between the CP sgPr-2 and the MP gene and we were able to obtain KK6-IRESMP75 that was stable in progeny (FIG.",
"17).",
"It was shown that KK6-IRESMP75 provides synthesis of I2 sgRNA containing crTMV IRESMP75 (FIG.",
"18).",
"It can be seen that the start of KK6-IRESMP75 I2 sgRNA is not changed in comparison to KK6, which means that IRESMP75 does not serve as MP sgPr.",
"This insertion drastically improved cell-to-cell movement.",
"KK6 infected Samsun plants systemically but the first symptoms developed slowly (15-17 days) compared to those induced by wild-type TMV (TMV 304) (about 7 days).",
"Symptoms in the upper leaves of KK6-infected plants were distinct: yellow spots in contrast to mosaic symptoms were produced by wild-type TMV.",
"KK6 virus progeny produced numerous lesions in N. glutinosa that developed slower than lesions induced by wild-type TMV UI.",
"The average size of local lesions induced by KK6 was approximately 0.1 mm in comparison to those induced by TMV UI (1.1 mm).",
"Plants inoculated by KK6-IRESMP75 looked like KK6-infected Samsun plants but: (i) the first systemic symptoms were developed more rapidly (about 10 days) and (ii) they were much brighter including yellow spots and mosaic.",
"In contrast to KK6 the average size of local lesions induced by K86 in N. glutinosa was increased to 0.6-0.7 mm.",
"Examination of the time-course of MP accumulation showed that KK6-IRESMP75 MP is detected earlier than KK6 MP in inoculated leaves (FIG.",
"19).",
"These results allowed the conclusion that insertion of IRESMP75CR upstream of the KK6 MP gene partially restores the movement properties of KK6 defective in cell-to-cell and long-distance transport.",
"In order to obtain additional evidences of the essential role of IRES in cap-independent MP gene expression of TMV cDNA vectors and in the life cycle of tobamoviruses, series of additional KK6-based vectors was constructed (FIG.",
"17).",
"KK6-IRESMP125 contains a natural hairpin-loop structure which is able to inhibit translation of the MP gene in vitro in the presence of WT crTMV 5′leader of I2 sgRNA (FIG.",
"13) and IRESMP75.KK6-H-PL contains a natural hairpin-loop structure and a 72-nt artificial polylinker sequence.",
"KK6-PL contains the polylinker region only.",
"Results of tests for infectivity on Nicotiana tabacum cv.",
"Samsun plants (systemic host) are presented in Table.3.FIG.",
"20 shows the results of a Western test of CP accumulation in tobacco leaves infected with KK6-based vectors.",
"Replacement of IRESMP75CR by a nonfunctional PL-sequence drastically blocked vector multiplication.",
"TABLE 3 Virus accumulation in tobacco systemically infected by KK6-based vectors.",
"cDNA copies Virus accumulation TMV 304 (WT) +++ KK6 + KK6-IRESMP75 ++ KK6-IRESMP125 ++ KK6-H-PL +/− KK6-PL +/− EXAMPLE 5 Creation of Artificial, Non-Natural IRES Elements Without Subgenomic Promoter Activity Provides Cap-Independent Expression of Genes of Interest in Eukaryotic Cells The goal of this example is to demonstrate the approaches for creation of artificial, non-natural IRES elements free of subgenomic promoter activity, which provide cap-independent expression of a gene of interest in eukaryotic cells.",
"Construction of an Artificial, Non-Natural IRES Element on the Basis of 18-nt Segment of IRESMP,75CR Analysis of the IRESMP,75CR nucleotide sequence shows that it has a multimer structure and contains four nucleotide sequence segments being a variation of element (−72) GUUUGCUUUUUG(−61) and having high complementarity to A. thaliana 18S rRNA (FIG.",
"21).",
"In order to design an artificial, non-natural IRES, the 18-nt sequence CGUUUGCUUUUUGUAGUA was selected.",
"Four oligos were synthesized: MP1(+): 5′-CGCGCAAGCTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTACTGCAGGCGGG-3′ MP1(−): 5′-CCCGCCTGCAGTACTACAAAAAGCAAACGTACTACAAAAAGCAAAGCTTGCGCG-3′ MP2(+): 5′-GGCGGCTGCAGTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTAGAATTCGG-GC-3′ MP2(−): 5′-GCCCGAATTCTACTACAAAAAGCAAACGTACTACAAAAAGCAAACTGCAGCCG-CC-3′ Primers MP1(+) and MP1(−) were annealed to each other yelding dsDNA fragment A: CGCGCAAGCTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTACTGCAGGCGGG GCGCGTTCGAAACGAAAAACATCATGCAAACGAAAAACATCATGACGTCCGCCC HindIII PstI Primers MP2(+) and MP2(−) were annealed to each other yelding dsDNA fragment B: GGCGGCTGCAGTTTGCTTTTTGTAGTACGTTTGCTTTTTGTAGTAGAATTCGGGC CCGCCGACGTCAAACGAAAAACATCATGCAAACGAAAAACATCATCTTAAGCCCG PstI EcoRI Both fragments were digested with PstI and ligated to each other.",
"Then the ligation product A+B was extracted using agarose electrophoresis and digested with HindIII and EcoRI followed by ligation into the hGFP-GUS vector described by Skulachev et al.",
"(1999, Virology 263, 139-154) using HindIII and EcoRI cloning sites (FIG.",
"22).",
"Results The transcripts depicted in FIG.",
"22 were translated in rabbit reticulocyte lysate (RRL) as described by Skulachev et al.",
"(1999, Virology 263, 139-154) and synthesized products were analyzed by gel electrophoresis.",
"Results represented in FIG.",
"22 show that an artificial, non-natural sequence based on a 18-nt segment of IRESMP,75CR provides 3′-proximal-located GUS gene expression.",
"This means that two features, namely complementarity to 18S rRNA and multimer structure are essential for IRESMP,75CR function and effectiveness.",
"A tetramer of 18-nt segment does not reach the level of IRESMP,75CR activity but there is a way to improve the activity of artificial, non-natural IRES elements using the 12-nt segment GCUUGCUUUGAG which is complementary to 18S rRNA.",
"Construction of an Artificial, Non-Natural IRES using 19-nt Segment of IRESCP,148CR Analysis of structural elements essential for IRESCP,148CR activity (FIGS.",
"23-26) shows that a polypurine (PP) segment is crucial for IRESCP,148CR functioning.",
"As a prominent element of the PP tract, a 9-nt direct repeat in 19-nt sequence: AAAAGAAGGAAAAAGAAGG (called direct repeat (DR)) was used for the construction of an artificial IRES.",
"In order to obtain the tetramer of DR the following primers were used: CPI(+): 5′-CGCGCAAGCTTAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGCT- GCAGGCGGG-3′ CP1(−): 5′-CCCGCCTGCAGCCTTCTTTTTCCTTCTTTTCCTTCTTTTTCCTTCTTTTAAGCT-TGCGCG- 3′ CP2(+): 5′-GGCGGCTGCAGAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGAA- TTCGGGC-3′ CP2(−): 5′-GCCCGAATTCCTTCTTTTTCCTTCTTTTCCTTCTTTTTCCTTCTTTTCTGCAGC-CGCC-3′ According to the experimental procedure described above, the following IRES element was used as intercistronic spacer: 5′-CGCGCAAGCUUAAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGCU-GCAG AAAAGAAGGAAAAAGAAGGAAAAGAAGGAAAAAGAAGGAAUUCAUG-3′ Results The transcripts depicted in FIG.",
"22 were translated in rabbit reticulocyte lysate (RRL) as described by Skulachev et al.",
"(1999, Virology 263, 139-154) and synthesized products were analyzed by gel electrophoresis.",
"The results represented in FIG.",
"22 show that an artificial, non-natural sequence based on repeated 19-nt segment of IRESCP,148CR provides the efficient expression of a 3′-proximally located GUS gene.",
"EXAMPLE 6 Construction of a TMV cDNA Transcription Vector Expressing a Replicase Gene in Infected Cells in a Cap-Independent Manner The main goal of this example was to obtain two new TMV U1-based viruses with modified 5′UTR providing expression of the replicase gene in a cap-independent manner: 1) Omega-leader of TMV was completely substituted by IRESMP,75CR.",
"GUUCGUUUCGUUUUUGUAGUAUAAUUAAAUAUUUGUCAGAUAAGAGAUUGGUUAGAG AUUUGUUCUUUGUUUGACCAUGG.",
"2) Since it is believed that the first 8 nucleotides of the TMV 5′UTR are essential for virus replication (Watanabe et al., 1996, J. Gen. Virol.",
"77, 2353-2357), IRESMP,75CR was inserted into TMV leaving the first 8 nucleotides intact: GUAUUUUUGUAGUAUAAUUAAAUAUUUGUCAGAUAAGAGAUUGGUUAGAGAUUUGUU CUUUGUUUGACCAUGG.",
"The following primers were used: a) SP6-IRES-1 (in the case of the first variant) Xbal SP6 Promotor IRESMP,75CR GGGTCTAGATTTAGGTGACACTATAGTTCGTTTCGTTTTTGTAGTA b) SP6-IRES-2 (in the case of the second variant) Xbal SP6 Promotor IRESMP,75CR GGGTCTAGATTTAGGTGACACTATAGTATTTTTGTAGTATAATTAAATATTTGTC.",
"c) IRES-NcoI (reverse primer to obtain IRES with a NcoI site at 3′end): GGGCCATGGTCAAACAAAGAACAAATCTCTAAAC.",
"d) TMV-NcoI (direct primer to obtain TMV polymerase, starting from NcoI site): NcoI GGGCCATGGCATACACACAGACAGCTAC.",
"e) TMV-Xho (reverse primer to obtain 5′-part of replicase from AUG to SphI site) XhoI ATGTCTCGAGCGTCCAGGTTGGGC.",
"Cloning Strategy: PCR fragment A was obtained using oligos SP6-IRES1 and IRES-NcoI and crTMV clone as template.",
"PCR fragment B was obtained using oligos TMV-NcoI and TMV-XhoI and TMV-304L clone.",
"Fragments A and B were cloned simultaneously into the pBluscriptSK+vector using Xbal and XhoI sites (fragments were ligated together through NcoI site).",
"The same procedure was applied to obtain the second variant of the virus using SP6-IRES2 oligo.",
"At the next stage, the whole TMV cDNA was cloned into the obtained vector using SphI and KpnI sites to restore the viral genome (FIG.",
"27).",
"EXAMPLE 7 Construction of Tobamoviral Vectors Act2/crTMV and Act2/crTMV IRESMP,75CR-GUS Based on Actin 2 Transcription Promoters The main goal of this example is the demonstration of the construction strategy of a new crTMV-based vector with which viral genome expression in plant cells occurs under the control of an efficient Actin 2 transcription promoter.",
"It allows the use of the vector Act2/crTMV/IRESMP,75CR-GUS for gene expression in plants.",
"Cloning Act2 into pUC19 The Act2 transcription promoter (about 1 000 bp) was cut out of plasmid pACRS029 by digestion with KpnI and Pst and cloned into pUC19 digested with KpnI and PstI.",
"Creation of a PstI site in Plasmid T7/crTMV (see FIG.",
"10) Upstream of crTMV Genome Start 334-nt cDNA fragment of the 5′-terminal portion of the crTMV genome obtained by PCR using the direct primer ATGCTGCAGGTTTTAGTTTTATTGCAACAACAA (the PstI site is underlined) and the reverse primer ATGCGATCGAAGCCACCGGCCMGGAGTGCA (PvuI site is also underlined) was digested with PvuI and PstI and inserted into pUC19Act2 together with the part of crTMV genome (PvuI-SpeI fragment).",
"Cloning of the Rest of the Genome Together with the Last Construct The Act2 containing construct was inserted into plasmid T7/crTMV after digestion with KpnI/SpeI.",
"Fusion of 5′-Terminus of crTMV to Act2 Transcriptional Start without Additional Sequences This step was carried out by site-directed mutagenesis using oligonucleotide primer specific for both Act2 and crTMV to obtain the final construct Act2/crTMV (FIG.",
"28).",
"To get the vector Act2/crTMV/IRESMP,75CR-GUS (FIG.",
"29) the SpeI-NotI cDNA fragment of plasmid Act2/crTMV (FIG.",
"28) was replaced by the SpeI-NotI DNA fragment of T7/crTMV/IRESMP,75CR-GUS construct (FIG.",
"11) that contains the GUS gene under the control of IRESMP,75CR.",
"EXAMPLE 8 Construction of Circular Single-Stranded Tobamoviral Vector KS/Act2/crTMV/IRESMP,75CR-GUS (FIG.",
"30) The main goal of this example is to demonstrate the possibility of using circular single-stranded DNA vectors for foreign gene expression in plants.",
"In order to construct KS/crTMV/IRESMP,75CR-GUS (FIG.",
"30), 9.2 kb KpnI-NotI cDNA fragment of vector Act2/crTMV/IRESMP,75CR-GUS was inserted into plasmid pBluescript II KS+ (Stratagene) digested with KpnI-SalI and containing the phage f1 replication origin.",
"Single-stranded DNA of vector KS/Act2/crTMV/IRESMP,75CR-GUS was prepared according to Sambrook et al., 1989 (Molecular Cloning: a Laboratory Manual, 2ed edn.",
"Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).",
"EXAMPLE 9 Construction of Tobamoviral Vector KS/Act2/crTMV-Int/IRESMP,75CR-GUS Containing Oleosin Intron from Arabidonsis thaliana The main goal of this example is to create vector KS/Act2/crTMV/IRESMP,75CR-GUS containing Arabidopsis thaliana oleosin gene intron that should be removed after transcript processing (FIG.",
"31).",
"The cloning strategy comprised the following steps: ps 1.Cloning of A. thaliana Oleosin Gene Intron.",
"A. thaliana oleosin gene intron was obtained by PCR using A. thaliana genomic DNA and specific primers: A.th./Int (direct) ATGCTGCAGgttttagttCAGTAAGCACACATTTATCATC (PstI site is underlined, lowercase letters depict crTMV 5′terminal sequence) and A.th/Int (reverse) ATGAGGCCTGGTGCTCTCCCGTTGCGTACCTA (StuI is underlined).",
"2.Insertion of A. thaliana Oleosin Gene Intron into 334-nt 5′-Terminal Fragment of crTMV cDNA.",
"cDNA containing A. thaliana oleosin gene intron was digested with PstI/StuI and ligated with DNA fragment obtained by PCR using primers corresponding to positions 10-334 of crTMV genome: atgAGGCCTTTATTGCAACAACAACAACAAATTA (StuI site is underlined) and ATGCGATCGAAGCCACCGGCCAAGGAGTGCA (PvuI site is underlined).",
"The next steps were as described in EXAMPLE 7.EXAMPLE 10 Influence of Rapamycin as an Inhibitor of Cap-Dependent Initiation of Translation on GUS Gene Expression in Tobacco Protoplasts transfected with IRESMP,75CR Containing Bicistronic Transcription Vectors, 35S/CP/IRESMP,75CR/GUS (FIG.",
"32) and 35S/GUS/ IRESMP,75CR/CP (FIG.",
"33) The aim of this example is to demonstrate the principal possibility to use inhibitors of cap-dependent translation to increase efficiency of IRES-mediated cap-independent translation of a gene of interest.",
"Rapamycin as an inhibitor of cap-dependent initiation of translation was selected.",
"Recently, a novel repressor of cap-mediated translation, termed 4E-BPI (elF-4E binding protein-1) or PHAS-1 was characterized (Lin et al., 1994, Science 266, 653-656; Pause et al., Nature 371, 762-767).",
"4E-BP1 is a heat- and acid-stable protein and its activity is regulated by phosphorylation (Lin et al., 1994 Science 266, 653-656; Pause et al., Nature 371, 762-767).",
"Interaction of 4EBP1 with elF-4E results in specific inhibition of cap-dependent translation, both in vitro and in vivo (Pause et_al., Nature 371, 762-767).",
"It has been shown that rapamycin induces dephosphorylation and consequent activation of 4E-BP1 (Beretta et al., 1996, EMBO J.",
"15, 658-664).",
"Construction of IRES- and GUS gene-containing vectors 35S/CP/ IRESMP,75CR/GUS (FIG.",
"32), 35S/GUSI IRESMP,75CR/CP (FIG.",
"33) and a method of tobacco protoplast transfection with 35S-based cDNA were described by Skulachev et al.",
"(1999, Virology 263, 139-154).",
"Comparison of GUS gene expression in tobacco protoplats treated by rapamycin and transfected with bicistronic cDNA with GUS gene in 3′- and 5′-proximal location shows the possibility to increase IRES-mediated cap-independent translation of the GUS gene.",
"EXAMPLE 11 Influence of Potwirus VPg as a inhibitor of Cap-Dependent Initiation of Translation on GUS Gene in Tobacco Protoplasts Transfected with IRESPM,75CR Containing Bicistronic Transcription Vectors 35S/CP/IRESMP,75CR/GUS (FIG.",
"32) and 35S/CP-VPg/IRESMP,75CR/GUS This example demonstrates the principal possibility of using a gene product to inhibit cap-dependent translation (FIG.",
"34).",
"Recently, interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the eukaryotic translation initiation factor elF(iso)4E of Arabidopsis thaliana has been reported (Wittman et al., 1997, Virology 234, 84-92).",
"Interaction domain of VPg was mapped to a stretch of 35 amino acids and substitution of an aspartic acid residue within this region completely abolished the interaction.",
"The cap structure analogue m7GTP, but not GTP, inhibited VPg-elF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for elF(iso)4E binding (Leonard et al., 2000, J. Virology 74, 7730-7737).",
"The capability of VPg to bind elF(iso)4E could be used for inhibition of cap-dependent translation.",
"We propose to use the vector 35S/CP-VPg/IRESMP,75CR/GUS (FIG.",
"34) wherein CP is fused with VPg from potyvirus potato virus A.",
"Comparison of GUS gene expression in protoplasts transfected with 35S/CP-VPg/IRESMP,75CR/GUS or 35S/CP/IRESMP,75CR/GUS would allow to increase IRES-mediated and cap-independent GUS gene expression.",
"EXAMPLE 12 In vivo Genetic Selection of an IRES Sequence or a Subqenomic Promoter using TMV Vector This example demonstrates the possibility of using in vivo genetic selection or Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a subgenomic promoter or an IRES sequence providing cap-independent expression of a gene of interest in a viral vector.",
"This approach proposes using side-by-side selection from a large number of random sequences as well as sequence evolution (Ellington and Szostak, 1990, Nature 346, 818-822; Tuerk and Gold, 1990, Science 249, 505-510; Carpenter and Simon, 1998, Nucleic Acids Res.",
"26, 2426-2432).",
"The project encompasses: In vitro synthesis of crTMV-based defective-interfering (DI) transcript containing the following elements (5′-3′ direction): (i) a T7 transcription promoter, (ii) a 5′-terminal part of crTMV genome with a sequence responsible for viral genome complementary (minus chain) synthesis, (iii) a sequence coding for the N-terminal part of a viral replicase, (iv) a sequence containing 75-nt randomized bases, (v) a neomycin phosphotransferase 11 (NPT II) gene, (vi) a crTMV origin of assembly (Oa), and (vii) a 3′-terminal part of the crTMV genome with minus chain genome promoter sequence (FIG.",
"35).",
"Co-transfection of tobacco protoplasts by a transcript together with crTMV genomic RNA (FIG.",
"10).",
"Protoplasts will grow and regenerate in media containing kanamycin.",
"Selection and isolation of an IRES or a subgenomic promoter element providing protoplast survival and regeneration in the presence of kanamycin.",
"ANNEX B Processes and Vectors for Producing Transgencic Plants FIELD OF THE INVENTION The present invention relates to processes and vectors for producing transgenic plants as well as plant cells obtained thereby.",
"BACKGROUND OF THE INVENTION Achievement of a desirable and stably inheritable pattern of transgene expression remains one of the major problems in plant biotechnology.",
"The standard approach is to introduce a transgene as part of a fully independent transcription unit in a vector, where the transgene is under transcriptional control of a plant-specific heterologous or a homologous promoter and transcription termination sequences (for example, see U.S. Pat.",
"No.",
"05,591,605; U.S. Pat.",
"No.",
"05,977,441; WO 0053762 A2; U.S. Pat.",
"No.",
"05,352,605, etc).",
"However, after the integration into the genomic DNA, because of random insertion of exogenous DNA into plant genomic DNA, gene expression from such transcriptional vectors becomes affected by many different host factors.",
"These factors make transgene expression unstable, unpredictable and often lead to the transgene silencing in progeny (Matzke & Matzke, 2000, Plant Mol Biol., 43, 401-415; S. B. Gelvin, 1998, Curr.",
"Opin.",
"Biotechnol., 9, 227-232; Vaucheret et al., 1998, Plant J., 16, 651-659).",
"There are well-documented instances of transgene silencing in plants, which include the processes of transcriptional (TGS) and posttranscriptional gene silencing (PTGS).",
"Recent findings reveal a close relationship between methylation and chromatin structure in TGS and involvement of RNA-dependent RNA-polymerase and a nuclease in PTGS (Meyer, P., 2000, Plant Mol.",
"Biol., 43 221-234; Ding, S. W., 2000, Curr.",
"Opin.",
"Biotechnol., 11, 152-156; lyer et al., Plant Mol.",
"Biol., 2000, 43, 323-346).",
"For example, in TGS, the promoter of the transgene can often undergo methylation at many integration sites with chromatin structure not favorable for stable transgene expression.",
"As a result, practicing existing methods requires many independent transgenic plants to be produced and analyzed for several generations in order to find those with the desired stable expression pattern.",
"Moreover, even such plants displaying a stable transgene expression pattern through the generations can become subsequently silenced under naturally occurring conditions, such as a stress or pathogen attack.",
"Existing approaches aiming at improved expression control, such as use of scaffold attachment regions (Allen, G. C., 1996, Plant Cell, 8, 899-913; Clapham, D., 1995, J. Exp.",
"Bot., 46, 655-662; Allen, G. C., 1993, Plant Cell, 5, 603-613) flanking the transcription unit, could potentially increase the independency and stability of transgene expression by decreasing dependency from so-called “position effect variation” (Matzke & Matzke, 1998, Curr.Opin.",
"Plant Biol., 1, 142-148; S. B. Gelvin, 1998, Curr.",
"Opin.",
"Biotechnol., 9, 227-232; WO 9844 139 A1; WO 006757 A1; EP 1 005 560 A1; AU 00,018,331 A1).",
"However, they only provide a partial solution to the existing problem of designing plants with a required expression pattern of a transgene.",
"Gene silencing can be triggered as a plant defence mechanism by viruses infecting the plant (Ratcliff et al., 1997, Science, 276, 1558-1560; Al-Kaff et al., 1998, Science, 279 2113-2115).",
"In non-transgenic plants, such silencing is directed against the pathogen, but in transgenic plants it can also silence the transgene, especially when the transgene shares homology with a pathogen.",
"This is a problem, especially when many different elements of viral origin are used in designing transcriptional vectors.",
"An illustrative example is the recent publication by Al-Kaff and colleagues (Al-Kaff et al., 2000, Nature Biotech., 18, 995-999) who demonstrated that CaMV (cauliflower mosaic virus) infection of a transgenic plant can silence the BAR gene under the control of the CaMV-derived 35S promoter.",
"During the last years, the set of cis-regulatory elements has significantly increased and presently includes tools for sophisticated spatial and temporal control of transgene expression.",
"These include several transcriptional elements such as various promoters and transcription terminators as well as translational regulatory elements/enhancers of gene expression.",
"In general, translation enhancers can be defined as cis-acting elements which, together with cellular trans-acting factors, promote the translation of the mRNA.",
"Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5′ end of the capped mRNA.",
"However, initiation of translation may also occur by a mechanism which is independent of the cap structure.",
"In this case, the ribosomes are directed to the translation start codon by internal ribosome entry site (IRES) elements.",
"These elements, initially discovered in picomaviruses (Jackson & Kaminski, 1995, RNA, 1, 985-1000), have also been identified in other viral and cellular eucaryotic mRNAs.",
"IRESs are cis-acting elements that, together with other cellular trans-acting factors, promote assembly of the ribosomal complex at the internal start codon of the mRNA.",
"This feature of IRES elements has been exploited in vectors that allow for expression of two or more proteins from polycistronic transcription units in animal or insect cells.",
"At present, they are widely used in bicistronic expression vectors for animal systems, in which the first gene is translated in a cap-dependent manner and the second one is under the control of an IRES element (Mounfford & Smith, 1995, Trends Genet, 4, 179-184; Martines-Salas, 1999, Curr Opin Biotech., 19, 458-464).",
"Usually the expression of a gene under the control of an IRES varies significantly and is within a range of 6-100% compared to cap-dependent expression of the first one (Mizuguchi et al., 2000, Mol.",
"Ther., 1, 376-382).",
"These findings have important implications for the use of IRESs, for example for determining which gene shall be used as the first one in a bicistronic vector.",
"The presence of an IRES in an expression vector confers selective translation not only under normal conditions, but also under conditions when cap-dependent translation is inhibited.",
"This usually happens under stress conditions (viral infection, heat shock, growth arrest, etc.",
"), normally because of the absence of necessary trans-acting factors (Johannes & Sarnow, 1998, RNA, 4, 1500-1513; Sonenberg & Gingras, 1998, Cur.",
"Opin.",
"Cell Biol., 10, 268-275).",
"Translation-based vectors recently attracted attention of researchers working with animal cell systems.",
"There is one report connected with the use of an IRES-Cre recombinase cassette for obtaining tissue-specific expression of cre recombinase in mice (Michael et al., 1999, Mech.",
"Dev., 85, 35-47).",
"In this work, a novel IRES-Cre cassette was introduced into the exon sequence of the EphA2 gene, encoding an Eph receptor of protein tyrosine kinase expressed early in development.",
"This work is of specific interest as it is the first demonstration of the use of translational vectors for tissue-specific expression of a transgene in animal cells that relies on transcriptional control of the host DNA.",
"Another important application for IRES elements is their use in vectors for the insertional mutagenesis.",
"In such vectors, the reporter or selectable marker gene is under the control of an IRES element and can only be expressed if it inserts within the transcribed region of a transcriptionally active gene (Zambrowich et al., 1998, Nature, 392, 608-611; Araki et al., 1999, Cell Mol Biol., 45, 737-750).",
"However, despite the progress made in the application of IRESs in animal systems, IRES elements from these systems are not functional in plant cells.",
"Moreover, since site-directed or homologous recombination in plant cells is extremely rare and of no practical use, similar approaches with plant cells were not contemplated.",
"There are significantly less data about plant-specific IRES elements.",
"Recently, however, several IRESs that are also active in plants were discovered in tobamovirus crTMV (a TMV virus infecting Cruciferae plants) (Ivanov et al., 1997, Virology, 232, 32-43; Skulachev et al., 1999, Virology, 263, 139-154; WO 98/54342) and there are indications of IRES translation control in other plant viruses (Hefferon et al., 1997, J. Gen Virol., 78, 3051-3059; Niepel & Gallie, 1999, J.",
"Virol., 73, 9080-9088).",
"IRES technology has a great potential for the use in transgenic plants and plant viral vectors providing convenient alternative to existing vectors.",
"Up to date, the only known application of plant IRES elements for stable nuclear transformation is connected with the use of IRESs to express a gene of interest in bicistronic constructs (WO 98/54342).",
"The construct in question comprises, in 5′ to 3′ direction, a transcription promoter, the first gene linked to the said transcription promoter, an IRES element located 3′ to the first gene and the second gene located 3′ to the IRES element, i.e., it still contains a full set of transcription control elements.",
"Surprisingly, we have found that translational vectors that are devoid of their own transcription control elements and rely entirely on insertion into a transcriptionally active genomic DNA of a plant host, allow recovery of numerous transformants which express the gene of interest.",
"Even more surprisingly, such transformants could be easily detected even in host plants with a very low proportion of transcriptionally active DNA in their genome such as wheat.",
"This invention is the basis of the proposed process that allows for design of transgene expression that is entirely controlled by the host's transcriptional machinery, thus minimizing the amount of xenogenetic DNA elements known to trigger transgene silencing.",
"It also allows to control transgene expression in a novel way, by modulating the ratio of cap-dependent versus cap-independent translation.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"36 shows transgene expression from four of many possible translational vector variants.",
"A—the vector contains a translation enhancer and a translation stop codon; B—the vector contains an IRES as translation enhancer and a transcription termination region; C—as in B, except that the IRES is preceded by translation stop codons for all three reading frames; D—as in C, except that the vector is flanked by intron/exon boundary regions (3′I-5′E and 3′E-5′I) to provide the features of an exon and to facilitate its incorporation into the spliced mRNA.",
"FIG.",
"37 depicts vector pIC1301 containing IRESMP,75CR, BAR and the 35S terminator.",
"FIG.",
"38 depicts vector pIC1521 containing a “hairpin”, IRESCP,148CR, BAR and the 35S terminator.",
"The “hairpin” structure serves as an alternative to the translation stop codon, preventing the formation of the translational fusion products.",
"FIG.",
"39 depicts vector pIC1451 containing a promoterless BAR gene and the 35S terminator.",
"FIG.",
"40 depicts vector pIC052 containing loxP, HPT and nos terminator.",
"FIG.",
"41 depicts vector pIC06-IRES containing IRESMP,75CR, the AHAS gene, whereby AHAS is the mutated version of the Arabidopsis acetohydroxyacid synthase gene conferring resistance to imidazoline herbicides.",
"FIG.",
"42 depicts vectors pIC-DOG and pIC-CRE containing the coding sequence of the yeast 2-deoxyglucose-6-phosphate (2-DOG-6-P) phosphatase and cre recombinase under the control of the rice actin promoter, respectively.",
"FIG.",
"43 depicts the transposon-incorporated translational vector pIC-dSpm and vector pIC1491 containing a transposase.",
"PHBT is a chimaeric promoter consisting of p35S enhancers fused to the basal part of the wheat C4PPDK gene.",
"DETAILED DESCRIPTION OF THE INVENTION A primary objective of this invention is to provide a novel process or vector to produce transgenic plants for the stable expression of transgenic material integrated into a plant genome.",
"This object is achieved by a process for producing transgenic plants or plant cells capable of expressing a transgenic coding sequence of interest under transcriptional control of a host nuclear promoter by introducing into the nuclear genome a vector comprising in its transcript a sequence for binding a plant cytoplasmic ribosome in a form functional for initiation of translation and, downstream thereof, said transgenic coding sequence, and subsequently selecting plant cells or plants expressing said transgenic coding sequence.",
"The gene of interest is under control of a translation signal, such as but not limited to, an IRES element and it has no promoter operably linked to it.",
"Such vectors rely on transgene insertions into transcriptionally active DNA of the host genome.",
"Further a novel vector is provided for transforming plant cells, comprising, optionally after processing in the host cell, in its transcript a sequence for binding a plant cytoplasmic ribosome in a form functional for the initiation of translation and, downstream thereof, a coding sequence, said vector being devoid of a promoter functional for the transcription of said coding sequence.",
"Preferred embodiments are defined in the subclaims.",
"Construction of vectors for stable transformation of plants has been described by numerous authors (for review, see Hansen & Wright, 1999, Trends in Plant Science, 4, 226-231; Gelvin, S. B., 1998, Curr.",
"Opin.",
"Biotech., 9, 227-232).",
"The basic principle of all these constructs is identical—a fully functional transcription unit consisting of, in 5′ to 3′direction, a plant-specific promoter, a structural part of a gene of interest and a transcriptional terminator, has to be introduced into the plant cell and stably integrated into the genome in order to achieve expression of a gene of interest.",
"We have developed a different technology for obtaining stable nuclear transformants of plants.",
"Our invention relies on the surprising finding that the host plant transcription machinery is able to drive the formation of mRNA from a transgene of interest in a transformed plant cell.",
"The proposed process utilizes vectors having a gene of interest that is not operationally linked to a promoter in said vector.",
"Rather, they comprise the coding region of a gene of interest under the control of translation elements only.",
"Said translational element may be a sequence for binding, preferably after transcription, a plant cytoplasmic ribosome thus enabling translation of a coding sequence downstream thereof.",
"Preferably, said translational element is a ribosome entry site functional in plants and more preferably a plant-specific IRES element, notably an IRES element of plant viral origin, of plant origin, of non-plant origin or an artificially designed IRES element.",
"Such a vector DNA, after integration into the transcribed region of a resident plant gene, yields chimaeric mRNA and is subsequently translated into the protein of interest via initiation of translation from said sequence for binding a plant cytoplasmic ribosome (FIG.",
"36).",
"To the best of our knowledge, there is no prior art concerning this approach for generating stable nuclear plant transformants.",
"It was very surprising, that, given the low proportion of transcriptionally active DNA in most plant genomes, transformation experiments utilizing translation vectors described in the present invention, yielded numerous transformants expressing the gene of interest.",
"Our invention addresses imminent problems of reliable transgene expression.",
"The transgene integrated into host genome using our invented process, relies on the transcription machinery including all or most of the transcriptional regulatory elements of the host's resident gene, thus minimizing transgene silencing usually triggered by xenogenetic DNA elements.",
"The vectors for transgene delivery can be built in many different ways.",
"The simplest versions consist only of the coding region of a gene of interest or a portion thereof with a translation signal (basic translational vector).",
"In a preferred vector, a translational stop signal is provided upstream of said sequences for binding a plant cytoplasmic ribosome.",
"The stop signal may for example be at least one stop codon and/or an RNA hairpin secondary structure or the like.",
"This stop signal causes abortion of upstream translation.",
"More advanced versions may include a plant-specific IRES element followed by the coding region (of a gene) of interest.",
"Advanced versions of the translational vector may include sequences for site-specific recombination (for review, see Corman & Bullock, 2000, Curr Opin Biotechnol, 11, 455-460) allowing either the replacement of an existing transgene or integration of any additional gene of interest into the transcribed region of the host DNA.",
"Site-specific recombinases/integrases from bacteriophages and yeasts are widely used for manipulating DNA in vitro and in plants.",
"Examples for recombinases-recombination sites for the use in this invention include the following: cre recombinase-LoxP recombination site, FLP recombinase-FRT recombination sites, R recombinase-RS recombination sites, phiC31 integrase—attP/attB recombination sites etc.",
"The introduction of splicing sites into the translation vector may be used to increase the probability of transgene incorporation into the processed transcript.",
"The vector may further comprise a sequence coding for a targeting signal peptide between said sequence for binding a plant cytoplasmic ribosome and said coding sequence.",
"Preferable examples of such signal peptides include a plastid transit peptide, a mitochondrial transit peptide, a nuclear targeting signal peptide, a vacuole targeting peptide, and a secretion signal peptide.",
"Various methods can be used to deliver translational vectors into plant cells, including direct introduction of said vector into a plant cell by means of microprojectile bombardment, electroporation or PEG-mediated treatment of protoplasts.",
"Agrobacterium-mediated plant transformation also presents an efficient way of the translational vector delivery.",
"The T-DNA insertional mutagenesis in Arabidopsis and Nicotiana with the promoterless reporter APH(3′)II gene closely linked to the right T-DNA border showed that at least 30% of all inserts induced transcriptional and translational gene fusions (Koncz et al., 1989, Proc.",
"Natl.",
"Acad.",
"Sci., 86, 8467-8471).",
"A translational vector can also be cloned into transposable elements, facilitating the search for suitable transcribed regions and providing either a constitutive or tissue/organ-specific pattern of transgene expression.",
"Transposable elements are extensively used in plants with the purpose of inactivation-based gene tagging (Pereira & Aarts, 1998, Methods Mol Biol., 82, 329-338; Long & Coupland, 82, 315-328; Martin G B., 1998, Curr Opin Biotechnol., 9, 220-226).",
"Different versions of the transposon-tagging systems were developed.",
"In the simplest version, transposons are used for insertional mutagenesis without any modifications except, possibly, for deletions or frame-shift mutations in order to generate non-autonomous transposable elements.",
"In more sophisticated versions, additional genes are inserted into the transposable elements, e.g.",
"reinsertion markers, reporter genes, plasmid-rescue vectors (Carroll et al., 1995, Genetics, 13, 407-420; Tissier et al., 1999, Plant Cell, 11, 1841-1852).",
"There are so-called enhancer-trap and gene-trap systems (Sundaresan et al., 1995, Genes Dev., 9, 1797-810; Fedorov & Smith, 1993, Plant J., 3, 273-89).",
"Transposable elements in such systems are equipped either with a promoterless reporter gene or a reporter gene under the control of a minimal promoter.",
"In the first case, the reporter gene can be expressed following insertion into the transcribed region of host DNA just after the host promoter or insertion into the coding region of the host gene and creation of “in frame” fusion with the host gene transcript.",
"The chance for successful “in frame” fusion can be significantly increased by placing in front of the reporter gene a set of splicing donor and acceptor sites for all three reading frames (Nussaume et al., 1995, Mol Gen Genet., 249, 91-101).",
"In the second case, transcription of a reporter gene will be activated from the minimal promoter following insertion near the active host promoter (Klimyuk et al., 1995, Mol Gen Genet., 249, 357-65).",
"The success of such approaches for transposon tagging favors the use of a similar approach for the translational vectors with IRES elements in front of the gene of interest.",
"All approaches described above aim at designing a system that places a transgene under expression control of the resident gene in which the insertion occurred.",
"This might be advantageous for specific tasks and cases.",
"In many other cases, a modified pattern of transgene expression might be preferable.",
"For such purposes, the translational vector can be equipped with transcriptionally active elements, such as enhancers which can modulate the expression pattern of a transgene.",
"It is known that enhancer sequences can affect the strength of promoters located as far as several thousand base pairs away (Müller, J., 2000, Current Biology, 10, R241-R244).",
"The feasibility of such an approach was demonstrated in experiments with activation tagging in Arabidopsis (Weigel et al., 2000, Plant Physiol., 122, 1003-1013), where T-DNA-located 35S enhancer elements changed the expression pattern of resident genes, and in enhancer-trap transposon tagging described above.",
"In the latter example, resident gene enhancers determined the expression pattern of the reporter transgene.",
"This approach might be useful, for example, at the initial stages of plant transformation, or when modulation of the transgene expression pattern is required after the transformation.",
"The enhancer sequences can be easily manipulated by means of sequence-specific recombination systems (inserted, replaced or removed) depending on the needs of the application.",
"Our approach was to preferably make a set of constructs based on different IRES elements functional in plant cells.",
"The constructs contain IRES elements followed by a plant selectable marker gene and a transcription/translation termination signal.",
"These constructs can be used directly for plant cells transformation after being linearized from the 5′ end in front of the IRES sequences or can be cloned into the T-DNA for Agrobacterium-mediated DNA transfer.",
"Another set of constructs, serving as controls, contained either a promoterless selectable gene (a negative control) or a selectable gene under the control of a constitutive promoter functional in monocot and/or dicot cells (a positive control).",
"DNA was transformed into plant cells using different suitable technologies, such as Ti-plasmid vector carried by Agrobacterium (U.S. Pat.",
"No.",
"5,591,616; U.S. Pat.",
"No.",
"4,940,838; U.S. Pat.",
"No.",
"5,464,763), particle or microprojectile bombardment (U.S. Pat.",
"No.",
"05,100,792; EP 00444882 B1; EP 00434616 B1).",
"In principle, other plant transformation methods could be used, such as but not limited to, microinjection (WO 09209696; WO 09400583 A1; EP 175966 B1), electroporation (EP 00564595 B1; EP 00290395 B1; WO 08706614 A1).",
"The transformation method depends on the plant species to be transformed.",
"Our exemplification includes data on the transformation efficiency for representatives of monocot (e.g.",
"Triticum monococcum) and dicot (e.g.",
"Brassica napus, Orichophragmus violaceous) plant species, thus demonstrating the feasibility of our approach for plant species of different phylogenetic origin and with different densities of transcribed regions within a species genome.",
"The transgenic coding sequence in the vector may represent only part of a gene of interest, which gene is reconstructed to a functional length as a result of site-directed or homologous recombination.",
"The translation of the sequence of interest is preferably cap-independent.",
"The host may be modified for inhibiting (or enhancing) cap-dependent translation or for enhancing (or inhibiting) cap-independent translation.",
"This may be accomplished by treatment with exogenous agents or by including a sequence in the vector or said plant, which expression has the desired effect.",
"EXAMPLES Example 1 Construction of IRES Containing Vectors Series of IRES-mediated expression vectors were constructed using standard molecular biology techniques (Maniatis et al., 1982, Molecular cloning: a Laboratory Manual.",
"Cold Spring Harbor Laboratory, N.Y.).",
"Vector pIC1301 (FIG.",
"37) was made by digesting plasmid pIC501 (p35S-GFP-IRESMP,75CR-BAR-35S terminator in pUC120) with HindIII and religating large gel-purified fragment.",
"The IRESMP,75CR sequence represents the 3′ terminal 75 bases of the 5′-nontranslated leader sequence of the subgenomic RNA of the movement protein (MP) of a crucifer (CR)-infecting tombamovirus.",
"Vector pIC1521 (FIG.",
"38) was made following three steps of cloning.",
"In the first step pIC1311 was constructed by ligating the large HindIII-PstI fragment of pIC031 with the small HindIII-NcoI fragment of pIC032 and the small BspHI-PstI fragment of pIC018.The resulting construct pIC1311 (not shown) containing the BAR gene under the control of the 35S promoter was used as the comparative control in the transformation experiments.",
"Plasmid pIC1311 was digested with HindIII-NruI and blunt-ended by treatment with Klenow fragment of DNA polymerase I.",
"The large restriction fragment was gel-purified and religated producing pIC1451 (promoterless BAR-35S terminator; see FIG.",
"39).",
"Ligation of the large SacI-PstI fragment of pIC1451 with the small SacI-NcoI fragment of pIC033 and the small BspHI-PstI fragment of pIC018 produced pIC1521 (FIG.",
"38).",
"This construct contains a “hairpin” in front of the IREScp,148CR (CP stands for coat protein) element.",
"The “hairpin” structure is formed by the presence of an inverted tandem repeat formed by KpnI-EcoRI and ClaI-KpnI fragments from the Bluescript II SK+ polylinker sequence.",
"All vectors were linearized for use in the transformation experiments by digesting either with SacI (pIC1521; pIC1451) or HindIII (pIC1311; pIC1301) restriction enzyme and gel-purified to separate from undigested vectors.",
"Example 2 PEG-Mediated Protoplast Transformation of Brassica napus Isolation of Protoplasts The isolation of Brassica protoplasts was based on previously described protocols (Glimelius K., 1984, Physiol.Plant, 61, 38-44; Sundberg & Glimelius, 1986, Plant Science, 43, 155-162 and Sundberg et al., 1987, Theor.",
"Appl.",
"Genet., 75, 96-104).",
"Sterilized seeds (see Appendix) were germinated in 90 mm Petri dishes containing ½ MS medium with 0.3% Gelrite.",
"The seeds were placed in rows slightly separated from each other.",
"The Petri dishes were sealed, tilted at an angle of 45° and kept in the dark for 6 days at 28° C. The hypocotyls were cut into 1-3 mm long peaces with a sharp razor blade.",
"The blades were often replaced to avoid the maceration of the material.",
"The peaces of hypocotyls were placed into the TVL solution (see Appendix) to plasmolise the cells.",
"The material was treated for 1-3 hours at room temperature.",
"This pre-treatment significantly improves the yield of intact protoplasts.",
"The preplasmolysis solution was replaced with 8-10 ml of enzyme solution (see Appendix).",
"The enzyme solution should cover all the material but should not to be used in excess.",
"The material was incubated at 20-25° C. in dark for at least 15 hours.",
"The Petri dishes were kept on a rotary shaker with very gentle agitation.",
"The mixture of protoplasts and cellular debris was filtered through 70 mm mesh size filter.",
"The Petri dishes were rinsed with 5-10 ml of W5 solution (Menczel et al., 1981, Theor.",
"Appl.",
"Genet., 59, 191-195) (also see Appendix) that was also filtered and combined with the rest of the suspension.",
"The protoplast suspension was transferred to 40 ml sterile Falcon tubes and the protoplasts were pelleted by centrifugation at 120 g for 7 min.",
"The supernatant was removed and the pellet of protoplasts was re-suspended in 0.5 M sucrose.",
"The suspension was placed into 10 ml sterile centrifuge tubes (8 ml per tube) and loaded with 2 ml of W5 solution.",
"After 10 min of centrifugation at 190 g the intact protoplasts were collected from the interphase with a Pasteur pipette.",
"They were transferred to new centrifuge tubes, resuspended in 0.5 M mannitol with 10 mM CaCl2 and pelleted at 120 g for 5 min.",
"PEG Treatment The protoplasts were resuspended in the transformation buffer (see Appendix).",
"The protoplast concentration was determined using the counting chamber and than adjusted to 1-1.5×106 protoplasts/ml.",
"The 100 μl drop of this suspension was placed at the lower edge of the tilted 6-cm Petri dish and left for a few minutes allowing the protoplasts to settle.",
"The protoplasts were than gently mixed with 50-100 μl of DNA solution (Qiagen purified, dissolved in TE at the concentration 1 mg/ml).",
"Than 200 μl of PEG solution (see Appendix) was added dropwise to the protoplasts/DNA mixture.",
"After 15-30 min the transformation buffer (or W5 solution) was added in small aliquots (dropwise) until the dish was almost filled (˜6 ml).",
"The suspension was left to settle for 1-5 hours.",
"Then the protoplast were transferred to the centrifuge tubes, re-suspended in W5 solution and pelleted at 120 g for 5-7 min.",
"Protoplast Culture and Selection for Transformants The protoplasts were transferred to the culture media 8 pM (Kao & Michayluk, 1975, Planta, 126, 105-110; also see the Appendix) and incubated at 25° C., low light density, in 2.5 cm or 5 cm Petri dishes with 0.5 ml or 1.5 ml of media, respectively.",
"Protoplast density was 2.5×104 protoplasts/ml.",
"The three volumes of fresh 8 pM media without any hormones were added right after the first protoplasts division.",
"The cells were incubated at high light intensity, 16 hours per day.",
"After 10-14 days the cells were transferred to K3 media (Nagy & Maliga, 1976, Z.",
"Pflanzenphysiol., 78, 453-455) with 0.1 M sucrose, 0.13% agarose, 5-15 mg/L of PPT and the hormone concentration four times less than in 8 pM medium.",
"To facilitate the transfer to fresh media, the cells were placed on the top of sterile filter paper by carefully spreading them in a thin layer.",
"The cells were kept at high light intensity, 16 hours per day.",
"The cell colonies were transferred to Petri dishes with differentiation media K3 after their size had reached about 0.5 cm in diameter.",
"Example 3 Transformation of Triticum monococcum by Microproiectile Bombardment Plant Cell Culture Suspension cell line of T. monococcum L. was grown in MS2 (MS salts (Murashige & Skoog, 1962 Physiol.",
"Plant, 15, 473-497), 0.5 mg/L Thiamine HCl, 100 mg/L inosit, 30 g/L sucrose, 200 mg/L Bacto-Tryptone, 2 mg/L 2,4-D) medium in 250 ml flasks on a gyrotary shaker at 160 rpm at 25° C. and was subcultured weekly.",
"Four days after a subculture the cells were spread onto sterile 50 mm filter paper disks on a gelrite-solidified (4 g/L) MS2 with 0.5 M sucrose.",
"Microprojectile Bombardment Microprojectile bombardment was performed utilizing the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad).",
"The cells were bombarded at 900-1100 psi, with 15 mm distance from a macrocarrier launch point to the stopping screen and 60 mm distance from the stopping screen to a target tissue.",
"The distance between the rupture disk and a launch point of the macrocarrier was 12 mm.",
"The cells were bombarded after 4 hours of osmotic pretreatment.",
"A DNA-gold coating according to the original Bio-Rad's protocol (Sanford et al., 1993, In: Methods in Enzymology, ed.",
"R. Wu, 217, 483-509) was done as follows: 25 μl of gold powder (0.6, 1.0 mm) in 50% glycerol (60 mg/ml) was mixed with 5 μl of plasmid DNA at 0.2 μg/μl, 25 μl CaCl2 (2.5 M) and 10 μl of 0.1 M spermidine.",
"The mixture was vortexed for 2 min followed by incubation for 30 min at room temperature, centrifugation (2000 rpm, 1 min), washing by 70% and 99.5% ethanol.",
"Finally, the pellet was resuspended in 30 μl of 99.5% ethanol (6 μl/shot).",
"A new DNA-gold coating procedure (PEG/Mg) was performed as follows: 25 μl of gold suspension (60 mg/ml in 50% glycerol) was mixed with 5 μl of plasmid DNA in an Eppendorf tube and supplemented subsequently by 30 μl of 40% PEG in 1.0 M MgCl2.The mixture was vortexed for 2 min and than incubated for 30 min at room temperature without mixing.",
"After centrifugation (2000 rpm, 1 min) the pellet was washed twice with 1 ml of 70% ethanol, once by 1 ml of 99.5% ethanol and dispersed finally in 30 μl of 99.5% ethanol.",
"Aliquots (6 μl) of DNA-gold suspension in ethanol were loaded onto macrocarrier disks and allowed to dry up for 5-10 min.",
"Plasmid DNA Preparation Plasmids were transformed into E.coli strain DH10B, maxi preps were grown in LB medium and DNA was purified using the Qiagen kit.",
"Selection For stable transformation experiments, the filters with the treated cells were transferred onto the solid MS2 medium with the appropriate filter-sterilized selective agent (150 mg/L hygromycin B (Duchefa); 10 mg/L bialaphos (Duchefa).",
"The plates were incubated in the dark at 26° C. Example 4 Transformation of Orychophracimus violaceus by Microproiectile Bombardment Preparation of the Suspension Culture Plants of O. violaceus are grown in vitro on MS medium, 0.3% Gelrite (alternatively, ½ MS, 2% sucrose and 0.8% agar) at 24° C. and 16/8 hours day/night photoperiod for 3-4 weeks.",
"Four-six leaves (depending of size) were cut into small peaces and transferred to the Magenta box with 30 ml of Callus Inducing Medium (CIM) (see the Appendix).",
"The material was kept for 4-5 weeks at dim light (or in dark) at 24° C. and vigorous agitation.",
"During this period the fresh CIM media was added to keep the plant tissue in the Magenta box covered with liquid.",
"The cells sticking to the wall of the Magenta box were released into the media by vigorous inverting and shaking of the box.",
"Preparation of Plant Material for Microproiectile Bombardment The aliquote of cell suspension was carefully placed onto the sterile filter paper supported by solid CIM media in Petri dish.",
"The Petri dish with plant material was kept in the dark for 5-7 days.",
"Four hours before the procedure, the filter paper with cells was moved to fresh CIM with 10% sucrose.",
"Microprojectile bombardment was performed as described in Example 3.Fourteen hours after the bombardment the material was transferred to CIM with 3% sucrose and kept in the dark.",
"Selection for Transformants Two-four days after the bombardment, the filter paper with cells was transferred to the plate with CIM supplemented with the appropriate selection agent (10-15 μg/ml PPT).",
"Every seven days the material was transferred to fresh selection media.",
"The plates were kept in the dark and after approximately 6 weeks the plant material was transferred to the Petri plates with Morphogenesis Inducing Medium (MIM) (see the Appendix) supplemented with the appropriate selection agent (10-15 μug/ml PPT).",
"The plates were incubated at high light intensity, 16 hours day length.",
"Example 5 Transformation of Triticum monococcum with Promoterless loxP-HPT Gene The construct pIC052 (FIG.",
"41) was linearized by digestion with HindIII restriction enzyme, gel-purified to separate undigested material and used for the microprojectile bombardment as described above (see EXAMPLE 3).",
"The linearized vector contains pUC19 polylinker (57 bp) followed by a loxP site from the 5′ end of the HPT gene.",
"In general, approximately 100 bp is located at the 5′ end of translation start codon of HPT gene.",
"Thirty four plates were transformed and after 1.5 months of selection on hygromycin-containing media (EXAMPLE 3), three hygromycin resistant colonies were recovered.",
"The sequence of the integration sites recovered by IPCR, confirmed the independency of all three transformants.",
"Example 6 Transformation of Orychophragmus Leaves with Promoterless IRESMP,75CR-AHAS Plant acetohydroxyacid synthase (AHAS) is a nuclear encoded, chloroplast targeted protein which catalyses the first step in the biosynthesis of the branched chain amino acids.",
"It is under allosteric control by these amino acids and can be inhibited by several classes of herbicides.",
"The construct pIC06-IRES was made by replacing the promoter of the Arabidopsis AHAS(Ser653-Asn) gene (1.3 Kb PstI-NcoI fragment) in pIC06 with the IRESMP,75CR sequence.",
"The final construct (FIG.",
"41) contained the mutated version of the Arabidopsis acetohydroxyacid synthase (AHAS) gene with a single amino acid substitution (Ser653Asn) conferring resistance to the imidazoline herbicide family (Sathasivan, Haughn & Murai, 1991, Plant Physiol., 97, 1044-1050).",
"The plasmid was linearized by treatment with SalI restriction enzyme and used for microprojectile bombardment of freshly induced O. violaceous suspension culture.",
"Leaves of sterile O. violaceous plants were cut onto the small peaces and placed in the liquid High Auxin Medium (HAM) (see the Appendix) in Magenta boxes on a rotary shaker to induce suspension culture.",
"After 7-14 days the suspension culture was transferred to the Petri dishes with Greening Medium (GM) covered by sterile filter paper (see the Appendix).",
"After 3 days the filter paper with the cells was transferred on GM supplemented with 0.4 M sucrose.",
"After four hours the cells were used for microprojectile bombardment with linearized DNA of pIC06-IRES, as described in EXAMPLE 3.After 14 hours the filter paper with cells was transferred to GM, 3% sucrose.",
"Two days later the cells were transferred to GM with 0.7 μM imazethapyr (AC263, 499 or Pursuit, American Cyanamid).",
"The cells were subcultured every 7-10 days.",
"Putative events were identified after approximately four-six weeks and the transformants were selected under high light intensity, 16 hours per day, on the regeneration medium (RM) with 1-2 μM imazethapyr.",
"Example 7 Expression of 2-DOG-6-P Gene using Translational Vector The aim of this example is to demonstrate the possibility of manipulation with transgenic plant cells already containing translational vector sequences with the sequence-specific recombination sites.",
"The hygromycin-resistant T. monococcum cells transformed with vector pIC052 (EXAMPLE 5) were used for microprojectile co-bombardment with two plasmids, pIC-DOG and pIC-CRE (FIG.",
"42).",
"Plasmid pIC-DOG contains promoterless 2-deoxyglucose-6-phosphate (2-DOG-6-P) phosphatase cDNA (patent WO 98/45456) flanked by two loxP sites.",
"Cre-mediated integration of the 2-DOG-6-P gene into the loxP site of pIC052-containing transformants leads to the expression of 2-DOG-6-P from a resident promoter.",
"Such expression confers resistance to 2-deoxyglucose (2-DOG).",
"The resistant colonies were selected as described in EXAMPLE 3, but using 0.075-0.1% of 2-DOG as the selective agent.",
"Example 8 Transposon-Incorporated Translational Vector The aim of this example is to show an alternative way to the direct transformation of directing translational vector to a desired transcriptional site in a host genome.",
"Co-transformation of O. violaceous cells with the constructs shown in FIG.",
"43 and selection for transformants was performed as described in EXAMPLE 4.The non-autonomous transposable dSpm element contains a promoterless BAR gene preceeded from its 5′ end IRESMP,75CR.",
"The transposition induced by Spm transposase facilitates the search for transcriptionally active regions with a desired expression pattern (in this case—constitutive) in said host genome, thus increasing the number of recovered primary transformants.",
"Indeed, the number of transformants was 3-4 times higher than with the IRESMP75CR-BAR gene alone (pIC1301, FIG.",
"37).",
"Appendix Seed sterilization Soak the seeds in 1% PPM solution for at least 2 hours (overnight is preferable).",
"Wash the seeds in 70% EtOH for 1 minute than sterilize in 10% chlorine solution with 0.01% SDS or Tween 20) in 250 ml flask placed on the rotary shaker.",
"Wash the seeds in 0.5 L of sterile water.",
"TVL Enzyme solution 0.3 M sorbitol 1% cellulase R10 0.05 M CaCl2 × 2H2O 0.2% macerase R10 pH 5.6-5.8 0.1% dricelase dissolved in 8 pM macrosalt with 0.5 M pH 5.6-5.8 W5 PEG solution 18.4 g/L CaCl2 × 2H2O 40% (w/v) of PEG-2000 in H2O 9.0 g/L NaCl 1.0 g/L glucose 0.8 g/L KCl pH 5.6-5.8 CIM MIM Macro MS Macro MS Micro MS Micro MS Vitamin B5 Vitamin B5 MES 500 mg/L MES 500 mg/L PVP 500 mg/L PVP 500 mg/L Sucrose 30 g/L Sucrose 30 g/L 2.4-D 5 mg/L ABA 1 mg/L Kin 0.25 mg/L BA 0.5 mg/L Gelrite 3 g/L IAA 0.1 mg/L pH 5.6-5.8 Gelrite 3 g/L pH 5.6-5.8 Greening High Auxine Medium (GM) Medium (HAM) Macro MS Macro MS Micro MS Micro MS Vit B5 Vit B5 MES 500 mg/L MES 500 mg/L PVP 500 mg/L PVP 500 mg/L Sucrose 30 g/L Sucrose 30 g/L BA 2 mg/L NAA 5 mg/L Kin 0.5 mg/L Kin 0.25 mg/L NAA 0.1 mg/L BA 0.25 mg/L pH 5.6-5.8 pH 5.6-5.8 Regeneration Medium Macro MS Micro MS Vit B5 MES 500 mg/L PVP 500 mg/L Sucrose 30 g/L ABA 1 mg/L BA 0.5 mg/L IAA 0.1 mg/L pH 5.6-5.8 Hormone solutions were filter sterilized and added to the autoclaved media."
]
] | Patent_10250413 |
[
[
"Process and device for desulphurizing hydrocarbons containing thiophene derivatives",
"The invention relates to a selective desulphurisation method for thiophene derivatives contained in the hydrocarbons emitted from the distillation of crude oil, refined or not, consisting in oxidising the atoms of thiophene sulphur in sulphone in the presence of an oxidising agent and separating the sulphonated compounds from said hydrocarbons.",
"This inventive method comprises at least one first stage involving the oxidation/absorption by heterogeneous catalysis of the sulphurous compounds in an organic environment, at a temperature of at least 40?C, at atmospheric pressure in the presence of an organic oxidiser from the family of peroxides and peracids, in the presence of a catalyst having a specific surface area greater than 100 m2/g and a porosity varying from 0.2 to 4 ml/g, and a second stage wherein the used catalyst is regenerated."
],
[
"1.Process for selectively desulfurizing the thiophenic derivatives contained in the hydrocarbons produced from the distillation of crude oil, refined or not, consisting of oxidizing the thiophenic sulfur atoms into sulfones in the presence of an oxidizing agent, and of separating the sulfonated compounds from said hydrocarbons, this process being characterized in that it includes at least a first step of oxidation/adsorption by heterogeneous catalysis of the sulfurous compounds, in organic medium, at a temperature of at least 40° C., in the presence of an organic oxidizer from the family of peroxides and peracids and in the presence of a catalyst having a specific surface area greater than 100 m2/g and a porosity varying from 0.2 to 4 ml/g, and a second step of regeneration of the used catalyst, the regeneration step always following the oxidation/adsorption step.",
"2.Process according to claim 1, characterized in that the oxidation/adsorption and regeneration steps are carried out successively in the same reactor on the same catalyst.",
"3.Process according to claim 1, characterized in that the oxidation/adsorption and regeneration steps are carried out simultaneously in reactors (1, 2) arranged in parallel and operating alternately for one and other steps.",
"4.Process according to claim 1, characterized in that the oxidation/adsorption and regeneration steps are carried out in two moving-bed reactors (20a, 20b) connected to each other by the catalytic bed, one being used for the oxidation, the other for the regeneration.",
"5.Process according to claim 1, characterized in that the oxidizing agent is selected from the group consisting of organic peroxides, organic hydroperoxides, and peracids.",
"6.Process according to claim 1, characterized in that the catalyst includes a support selected from the group consisting of silicas, aluminas, zirconias, amorphous or crystalline aluminosilicates, aluminophosphates, mesoporous solids, activated carbon, clays, and their mixtures.",
"7.Process according to claim 6, characterized in that the catalyst contains at least a metal selected from the group consisting of titanium, zirconium, vanadium, chromium, molybdenum, iron, manganese and tungsten, this metal being introduced into the matrix of the support or deposited in oxide form on the support.",
"8.Process according to claim 1, characterized in that the catalyst contains from 0 to 30% by weight of metal in oxide form.",
"9.Process according to claim 1, characterized in that the catalyst is comprised of at least a support selected from gamma-alumina, silica and silicic mesoporous solids and silicoaluminates.",
"10.Process according to claim 9, characterized in that the supported catalyst is selected from catalysts containing tungsten on a support selected from silicas and aluminum oxides, alone or in mixture.",
"11.Process according to claim 1, characterized in that the oxidizer/total sulfur mol ratio in the hydrocarbons varies from 2 to 20.12.Process according to claim 1, characterized in that the oxidizer is a compound with the general formula R1OOR2, in which R1 and R2 are selected identical or different from the group consisting of the hydrogen atom and the alkyl groups, linear or branched, having from 1 to 30 carbon atoms, while R1 and R2 cannot be hydrogen simultaneously.",
"13.Process according to claim 12, characterized in that the oxidizer is selected from the group consisting of tert-butyl hydroperoxide and di-tert butyl peroxide.",
"14.Process according to claim 1, characterized in that the oxidizer is a peracid of formula R3COOOH, in which R3 is hydrogen or a linear or branched alkyl group having from 1 to 30 carbon atoms.",
"15.Process according to claim 14, characterized in that the oxidizer is selected from the group consisting of peracetic acid, performic acid, and perbenzoic acid.",
"16.Process according to claim 1, characterized in that the catalyst regeneration step consists of eliminating the formed deposits by washing or combustion.",
"17.Process according to claim 1, wherein the hydrocarbons produced from the distillation of crude oil are selected from the group consisting of hydrotreated gasoils, kerosenes, and gasolines.",
"18.Process according to claim 8, characterized in that the catalyst contains from 0 to 20% by weight of metal in oxide form.",
"19.Process according to claim 11, characterized in that the oxidizer/total sulfur mol ratio in the hydrocarbons varies from 2 to 6.20.Process according to claim 17, wherein the hydrocarbons produced from the distillation of crude oil are gasolines produced from catalytic cracking."
],
[
"The present invention concerns a process and a device for desulfurizing hydrocarbons, and particularly, for desulfurizing fuel bases for gasoils, kerosenes, and gasolines.",
"In particular, it concerns the desulfurization of fuel bases containing dibenzothiophenic compounds.",
"The presence of sulfur in fuels constitutes what is considered today to be a major problem for the environment.",
"Indeed, through combustion, sulfur is converted to various sulfur oxides that can be transformed into acids, thus contributing to the formation of acid rain.",
"In general, refineries use catalytic hydrosulfurization processes to reduce the sulfur content in fuels.",
"Thus, gasoils derived directly from distillation are hydrotreated between 300° and 400° C. under pressure of hydrogen varying between 30 and 100 bars (30 to 100·105 Pa), in the presence of a catalyst on a fixed bed and composed of sulfides of metals of groups VIb and VIII deposited on aluminum oxide, for example cobalt and molybdenum sulfides or nickel and molybdenum sulfides.",
"Because of the operating conditions and the consumption of hydrogen, these processes can be costly both in investment and in operation, particularly if fuels with very low sulfur content are to be produced.",
"Consequently, in order to desulfurize a fuel initially containing 1% sulfur by weight until it has a concentration of sulfur of between 0.05 and 0.005% by weight, the size of the reactor can be multiplied by four and the quantity of hydrogen needed for the reaction must be increased by about 20%.",
"It is particularly difficult to eliminate traces of sulfur by such processes, especially if the sulfur belongs to refractory molecules such as alkyl dibenzothiophene in position 4, or 4 and 6.In some countries like Sweden, the United States, particularly in California, and others, the total sulfur content of gasoils is already limited to 0.005% by weight.",
"This limitation could become generalized in time within the OECD countries.",
"For Europe, this goal of 0.005% by weight of total sulfur could be achieved in 2005.Unlike gasoils, gasolines are not only distilled directly from crude oil, these gasolines being then slightly sulfurous, but can also be obtained by several processes such as reforming of naphthas, isomerization of light naphthas, alkylation of butane or propane producing isooctane, methoxylation of isobutene, and the catalytic cracking of distillates under vacuum or atmospheric residue.",
"In particular, catalytic cracking provides between 20% and 60% by weight of final gasoline.",
"However, these gasolines contain up to 0.1% by weight of sulfur.",
"It is common, therefore, to desulfurize gasolines produced by catalytic cracking using processes similar to those described for the hydrodesulfurization of gasoils, for which the operating conditions of hydrogen pressure, space velocity, and temperature are more stringent.",
"These processes, although costly, do not enable total sulfur content in cracked gasoline of between 0.005% and 0.03% by weight to be obtained by conventional means.",
"Although refiners, in order to reduce this sulfur content, have thought that including additives in the cracking catalyst would break down the sulfurous compounds formed during the process, particularly mercaptans and sulfides, these additives have only a limited or even no effect on the benzothiophenic derivatives, even when the mercaptans and the sulfides have been eliminated before cracking.",
"In the case of gasolines from catalytic cracking that generate sulfur in gasolines, hydrodesulfurization is not only ineffective with respect to thiophenic compounds, but it is also destructive with respect to the octane index of the gasoline.",
"Indeed, during the hydrodesulfurization reaction, there is a partial hydrogenation of the olefins contained in these cracked gasolines, their disappearance resulting in a decrease in the octane index of the gasoline and thus a deterioration in the quality of the gasoline.",
"To compensate for this loss, it is possible to introduce other components to improve this index or to reprocess the gasoline itself to increase this index.",
"The inclusion of an additive or the reprocessing to improve the quality of the gasoline affects the production cost that much more, and it is therefore advantageous to have a processing method that enables the direct elimination of the refractory sulfurous compounds, such as benzothiophenic derivatives, by limiting the use of hydrogen.",
"Processes for selective oxidation of sulfurous compounds are among treatment processes that can achieve this end.",
"Among the methods and processes developed to reduce the quantity of sulfur in fuels in the form of derivatives of thiophene, oxidation by organic peroxides, organic hydroperoxides, hydrogen peroxide, and organic peracids, has been considered either without catalyst, or by homogenous catalysis in the presence of catalysts based on organometallic compounds or metallic oxides in aqueous phase (see U.S. Pat.",
"No.",
"3,668,117, U.S. Pat.",
"No.",
"3,565,793, EP 0,565,324, and publications by T. A. KOCH, K. R. KRAUSE, L. EMANZER, H. MEHDIZADEH, J. M. ODOM, S. K. SENGUPTA, New J.",
"Chem., 1996, 20, 163-173, and by F. M. COLLINS, A. R. LUCY, C. SHARP, J. of Molecular Catalysis A: Chemical 117 (1997), 397-403).",
"Processes using molybdenum- and tungsten-based metallic catalysts in the presence of hydrogen peroxide in aqueous solution (heterogeneous catalysis) take place at temperatures of more than 60° C., and there is excessive consumption of hydrogen peroxide, a part of this oxidant being broken down by the catalyst used.",
"The peracids used, very powerful oxidants, obtained by reaction of hydrogen peroxide and a carboxylic acid such as formic acid or acetic acid, are generally less effective than hydrogen peroxide and less selective with respect to sulfurous compounds and in particular can oxidize olefins.",
"Other oxide sulfurization processes in organic medium have been proposed.",
"They consist of placing in contact powdered metallic oxides, or of forming metallic compounds having peroxo groups in aqueous or organic solutions with the hydrocarbons containing these refractory sulfurous compounds, whether or not in the presence of organic or aqueous peroxides which are introduced with an alcohol type solvent or in the water (see U.S. Pat.",
"No.",
"3,816,301, U.S. Pat.",
"No.",
"4,956,578, U.S. Pat.",
"No.",
"5,958,224).",
"Another process, described in U.S. Pat.",
"No.",
"3,945,914, consists of producing a desulfurized hydrocarbonated material in three processing steps.",
"The first step consists of at least partially oxidizing the sulfurous compounds by placing them in contact with peroxides in the presence of metallic catalysts containing metals from the group including titanium, zirconium, molybdenum, tungsten, vanadium, tantalum, chromium, and their mixtures, in liquid or solid form possibly supported, although the supports are not essential for the reaction.",
"The second step consists of placing the hydrocarbonated material containing the oxidized compounds in contact with another metallic component, metallic oxide or peroxide (metals from the group including nickel, molybdenum, cobalt, tungsten, iron, zinc, vanadium, copper, manganese, mercury, and their mixtures), at a temperature varying from 250° C. to 730° C., under hydrogen pressure.",
"The third step consists of recovering the desulfurized hydrocarbonated material.",
"In all these methods and processes, the derivatives of thiophene in their sulfonated and/or sulfonic form are transformed.",
"However, for some of these compounds, even when working at high temperature, the reaction is relatively slow and total conversion is not achieved in less than one hour, except by using very strong concentrations of oxidant, often far greater than the quantities necessary for the oxidation of the sulfurous derivatives.",
"In other cases, it is possible to work in several steps, but they are costly in time and in monitoring the unit.",
"The present invention therefore proposes a process for desulfurizing hydrocarbons, particularly those used as bases for fuels containing thiophenic derivatives, without reducing the index of the octane number or of the cetane number, sometimes even increasing these indices.",
"In particular, it concerns the finish treatment of hydrotreated gasoils, kerosenes, and catalytic cracked gasolines with high concentrations of refractory thiophenic derivatives in hydrogenations.",
"Furthermore, the invention proposes such a process that makes it possible to reach oxidation levels that are identical if not greater than the known processes, while limiting the reaction and separation times of the oxidized sulfurous compounds from the desulfurized hydrocarbons.",
"An object of the present invention is therefore a process for selectively desulfurizing the thiophenic compounds contained in the hydrocarbons produced from the distillation of crude oil, refined or not, consisting of oxidizing the thiophenic sulfur atoms into sulfones in the presence of an oxidizing agent and a catalyst, and of separating the obtained sulfonated compounds from said hydrocarbons, this process being characterized in that it comprises at least a first step of oxidation/adsorption by heterogeneous catalysis of the sulfurous compounds, in an organic medium, at a temperature of at least 40° C., in the presence of an organic oxidizer from the family of peroxides and peracids and in the presence of a catalyst having a specific surface area greater than 100 m2/g and a porosity varying from 0.2 to 4 ml/g, and a second step of regeneration of the used catalyst, the regeneration step always following the oxidation/adsorption step.",
"Within the scope of the present invention, derivatives of thiophene are understood as being benzothiophenic, polybenzothiophenic compounds and their alkyl derivatives, among which are the alkyldibenzothiophenes, particularly refractory to the conversion processes usually used by refiners.",
"The process of the invention has the advantage, on the one hand, of ensuring the oxidation at atmospheric pressure of all of the sulfur contained in the hydrocarbons, and more selectively a conversion of the thiophenic derivatives into sulfones, this by means of a simple industrial process, and on the other hand, of simultaneously adsorbing these sulfoxide compounds on the catalyst.",
"In fact, the separation of the hydrocarbons from most of the formed sulfones and sulfoxides is immediate, with the latter ending up in solid form deposited on the catalyst or deposited in a form that can be filtered by known means, in the treated hydrocarbons.",
"This catalyst, on which these sulfoxide compounds have been absorbed, constitutes the “used catalyst.” The sulfones that may have been dissolved in the treated hydrocarbons can be extracted.",
"Moreover, this oxidation/adsorption has no effect on the olefins, which in catalytic cracked gasolines does not change the octane index, or the concentration of unsulfurous aromatic compounds.",
"In addition, the oxidation process according to the invention improves the cetane number of the gasoils.",
"Without being limited by a theory, it has become clear that the greater the specific surface area of the catalyst, the longer it remains active.",
"Furthermore, because compounds of the sulfone and sulfoxide type have a strongly polar nature, they are kept on the surface of the catalyst, probably at the catalyst's Lewis acid sites.",
"In addition, the larger the size of the pores, the less the catalyst's pores risk becoming quickly clogged and the greater the longevity of the catalyst during the oxidation cycle.",
"For the present invention, one has to select the catalyst that has the best compromise of specific surface area and pore size to obtain sufficient activity, for as long as possible, to be the most effective for oxidation/adsorption.",
"When the process is continuously implemented intermittently, the oxidation/adsorption and regeneration steps can be performed in the same reactor or simultaneously in reactors arranged in parallel and operating alternatively for one or the other of the fixed bed steps, or in at least two moving-bed reactors connected to each other by the catalytic bed, one being used for oxidation/adsorption and the other for regeneration.",
"With a fixed bed, the first reactor containing a fixed catalyst bed receives the flow of hydrocarbons and oxidizing agent and the second receives, for the regeneration of the catalyst, liquid effluents, such as a washing solvent, or oxidizing gaseous effluents like air or an air/N2 mixture, the temperature of the catalytic bed being increased.",
"These reactors change function when the effectiveness of the catalyst in the oxidation/adsorption reactor is no longer sufficient in oxidation and/or adsorption.",
"With a moving-bed, the hydrocarbons are brought into the first reactor where the oxidation takes place, the catalyst being pushed progressively toward the second reactor where it is regenerated before being returned to the oxidation/adsorption reactor.",
"The moving-bed reactors, well known particularly in the area of reforming, can be used in this device.",
"In this form of embodiment, a third reactor is used, placed between the first two reactors and making it possible to eliminate the hydrocarbons from the used catalyst before washing it or burning off the trapped sulfone and sulfoxide compounds.",
"The catalysts used according to the present invention are selected among the supports from the group consisting of silicas, aluminum oxides, zirconias, amorphous or crystalline aluminosilicates, aluminophosphates, mesoporous silicic and silicoaluminate solids, activated carbon and clays, these supports being used alone or in mixture.",
"In the catalysts of the invention, these supports can be used advantageously as supports of metals of the group consisting of titanium, zirconium, vanadium, chromium, molybdenum, iron, manganese, cerium, and tungsten; these metals in oxide form can be introduced into the matrix of the support or deposited on the surface of the support.",
"In fact, a synergistic effect has been noted of the metal with the support, that is, an unexpected increase of activity of the catalyst with respect to the oxidation of the thiopenic compounds, and at the same time, an increased trapping of the sulfone and sulfoxide compounds in the pores of the catalyst, without any of them being subsequently desorbed.",
"In the process according to the invention, the catalyst contains from 0 to 30% by weight of metal in oxide form on at least one support.",
"Preferably, the catalyst contains from 0 to 20% metal in oxide form.",
"Among the supports composed of refractory oxides, gamma-aluminum oxides, silicon oxides, silicic mesoporous solids, and silicoaluminates are preferred.",
"Among the supported catalysts, catalysts containing tungsten or titanium in oxide form are preferred, deposited on a support or introduced into the matrix, this support being selected from among the silicon oxides, aluminum oxides and aluminosilicates, alone or in mixture.",
"In a preferred form of implementation of the process, the total oxidizer/sulfur mol ratio contained in the hydrocarbons is between 2 and 20, and preferably between 2 and 6.According to the invention, the oxidizers are selected from among the compounds with the general formula R1OOR2, in which R1 and R2 are identical or different, selected from among hydrogen, linear or branched alkyl groups having from 1 to 30 carbon atoms and aryl or alkylaryl groups the aryl motif of which can be replaced by alkyl groups, while R1 and R2 cannot be hydrogen simultaneously.",
"In a preferred embodiment, the oxidizer of the formula R1OOR2 is selected from the group consisting of tert-butyl hydroperoxide and di-tert butyl peroxide.",
"Other oxidizers of the invention, the peracids of formula R3COOOH, are selected so that R3 is hydrogen or a linear or branched alkyl group having from 1 to 30 carbon atoms.",
"They are preferably selected from the group consisting of peracetic acid, performic acid, and perbenzoic acid.",
"In the process of the invention, the catalyst regeneration step consists of eliminating the formed deposits by washing or combustion.",
"For the washing, a solvent is used, preferably polar, from the group consisting of water, linear or branched alcanols having from 1 to 30 carbon atoms, alone or in mixture with water, alkylnitriles having from 1 to 6 carbon atoms.",
"Water, acetonitrile, methanol, and their mixtures are preferred.",
"By combustion, the catalyst is brought up to a temperature of no more than 800° C., preferably a temperature equal to or less than 650° C., under a pressure varying from 105 Pa to 106 Pa, preferably from 105 Pa to 2×105 Pa, in the presence of an oxidizing gas.",
"Oxidizing gas is understood as being pure oxygen and all mixtures of gas containing oxygen, particularly mixtures of oxygen and nitrogen and air itself.",
"The quantity of oxygen in the nitrogen is adjusted in order to limit the formation of water vapor, since too great a quantity of water vapor has the side effect of modifying the structure of the pores of the catalyst by decreasing their volume, specifically when it contains crystalline alumunosilicates such as zeolites or alumuninophosphates as support.",
"Moreover, this adjustment makes it possible to control the temperature variations related to the exothermicity of the combustion.",
"A second object of the invention is a device for implementing the process defined above, this device comprising at least a first reactor containing an oxidation catalyst and having feed pipes for the hydrocarbons and the oxidizer and an outlet pipe for the desulfurized hydrocarbons, and possibly a second reactor having feed pipes for solvent or oxidizing gas of the catalyst, in order to regenerate it, and an outlet pipe for the combustion gases.",
"Oxidizing gas is understood here as oxygen/air, air/nitrogen, and oxygen/nitrogen mixtures.",
"When the device includes two reactors, the reactors can operate with a fixed bed or a moving bed.",
"A third object of the invention is the application of the process defined above to the specific finish treatment of gasolines produced from catalytic cracking, or the treatment of gasoils having been previously hydrotreated and kerosenes, for better economy of the process.",
"The invention will now be described in more detail, with reference to the appended drawings in which: FIG.",
"1 is a diagram of a device with two reactors operating alternatively for oxidizing and for regenerating the catalyst; FIG.",
"2 is a diagram of a device having two moving-bed reactors, the first corresponding to the oxidation step, the second to the catalyst regeneration step, a pipe for return of the regenerated catalyst being added to the system; FIGS.",
"3-1 and 3-2 show graphs illustrating the total sulfur content, as a function of time, of the hydrocarbons treated according to the invention in the Example III below.",
"The device of FIG.",
"1 has two reactors 1 and 2 charged with a catalyst arranged as fixed-bed.",
"When the reactor 1 is operating in oxidation and the reactor 2 operates in regeneration, the pipe 3 takes the sulfurous hydrocarbon load, into which the oxidizer has been introduced by the pipe 4, the three-way valve 6a and the pipe 8a.",
"The flow of desulfurized hydrocarbons leaves the reactor 1 by the pipe 9a and reaches the desulfurized hydrocarbon outlet pipe 10a via the three-way valve 7a.",
"At the same time, the pipe 5 takes to the reactor 2 either an appropriate solvent or an oxidizing gas, via the three-way valve 6b and the pipe 8b.",
"When the reactor operates in combustion, the temperature of the catalytic bed is held at 500° C. The solvent containing the sulfones recovered on the catalyst or the combustion gases, primarily SO2, CO, and CO2, are evacuated via the pipe 9b, the three-way valve 7b and the pipe 11b in the pipe 11a.",
"When the regeneration of the catalyst is done and the activity of the catalyst of reactor 1 becomes insufficient, the function of the two reactors is exchanged.",
"Thus, the hydrocarbons/oxidizer mixture passes through the pipe 3a and the valve 6b to enter the reactor 2.The desulfurized hydrocarbons are removed by the pipe 9b and are taken to the outlet pipe 10a via the valve 7b and the pipe 10b.",
"At the same time, the solvent or the oxidizing gas arriving by the pipe 5 is sent to the reactor 1 by the pipe 3a, the valve 6a and the pipe 8a.",
"The solvent or the oxidation gases are taken back in the outlet pipe 11a via the pipe 9a and the valve 7a.",
"The valves 6a, 6b, 7a, and 7b can be exchanged according to a common procedure, in order to allow the circulation of the proposed flows.",
"A filter can be placed advantageously on one of the pipes 9a or 9b, or on 10a or 10b, to recover the solid sulfones formed during oxidation and remaining in suspension in the hydrocarbons.",
"Sulfur traps equipped with absorbents such as silica or activated alumina can be advantageously added to these same pipes, downstream from these filters, to trap sulfones that are still dissolved in the treated hydrocarbons.",
"The device of FIG.",
"2 includes two reactors 20a and 20b, arranged in series, each containing a moving-bed catalyst, the reactor 20a operating in oxidation mode and the reactor 20b operating in regenerative mode, and a propulsion device 30 to allow the catalyst from the reactor 20b to return to the reactor 20a.",
"The hydrocarbons are taken by the pipe 40 into the reactor 20a, after having been doped by the oxidizer via the pipe 50.For example, the reactor 20a can be selected from among funnel-shaped reactors, the moving bed of the catalyst being moved by gravity toward the lower part of the reactor.",
"In this way, while the desulfurized hydrocarbons are removed by the pipe 60, the catalyst is forced by gravity into the reactor 20b through the pipe 70.The solvent or combustion gas is introduced via the channel 80 in the reactor 20b.",
"In order to effect regeneration by combustion, the temperature is increased to and held at 500° C. The sulfones-containing solvent or the combustion gases are removed by the pipe 100.Since these moving-beds generally operate intermittently, the catalyst not being moved continuously, it is beneficial to place on the reactor 20b a solvent or nitrogen purge to eliminate the hydrocarbons prior to washing, and/or eliminate the combustion gases by stripping the nitrogen.",
"When it leaves the reactor 20b, the regenerated catalyst is taken via the pipe 110 to the device 30.This device can be a pressurized gas propulsion device or a worm gear.",
"It takes the regenerated catalyst back to the reactor 20a via the pipe 120.In some special forms of embodiment of these moving reactors, the reactors 20a and 20b can be part of the same unit having two separate stages.",
"The following examples illustrate the efficiency of the process of the invention without limitation thereto.",
"EXAMPLE 1 The present example describes the effectiveness of the process, according to the invention, with respect to the elimination of the derivatives of the dibenzothiophene present in the partially desulfurized bases for fuels.",
"The samples of catalyst used are of two types, the catalysts formed from a single support and those to which are combined one or more metals deposited by impregnation.",
"Table 1 below provides the specific surface area and porosity characteristics of each of them.",
"TABLE I Catalyst Type Specific surface Pore size sample of support area (m2/g) (Angströms) Metal oxides C1 SiO2 160 252 C2 SiO2 140 300 WO3 C3 Al2O3 gamma 245 104 WO3 C4 Beta zeolite 470 30 TiO2 C5 Mesoporous 1000 85 C6 Mesoporous 830 70 MoO3 C7 Al2O3 γ 210 95 WO3 The catalysts C2, C3, and C6 were obtained by wet impregnation with a metallic salt, respectively ammonium metatungstate and ammonium hexamolybdate, in a concentration of 140 mg of metal per gram of support, then dried and finally calcinated at a temperature of 500° C. The catalyst C4 was obtained by treating a beta zeolite with commercially available titanium according to the procedure described in patent EP 0,842,114.To test the oxidation activity of these catalysts as a function of time, 20 ml of catalyst is introduced into a 150 ml micropilot.",
"A charge of average distillates after hydrotreatment, containing 212 ppm of residual sulfur remaining from hydrotreatment, doped with 1,800 ppm of tert-butyl hydroperoxide (tBHP), is circulated over the catalyst at an hourly space velocity (HSV) of 1 h−1, under atmospheric pressure at a temperature of 70° C. Samples are taken regularly during oxidation to measure the activity of the catalyst over time.",
"A comparative sample called T1, corresponding to the use of a catalyst alone without peroxide, is also monitored.",
"In Table II below, the results are given of the effectiveness of these catalysts over time.",
"TABLE II Total sulfur (ppm) after different periods of operation Sample Catalyst 2 hrs 4 hrs 5 hrs 6 hrs T1 C1 121 185 196 202 X1 C1 49 46 48 49 X2 C2 28 14 9 16 X3 C3 30 28 23 27 X4 C4 18 16 11 11 X5 C5 35 32 38 35 X6 C6 23 20 17 22 X7 C7 34 31 25 31 After two hours of operation, these results confirm that, apart from the effect due to the nature of the catalyst, the greater the catalyst pore size and specific surface area, the lower the sulfur content of the treated hydrocarbons.",
"Moreover, it is verified that the activity of the catalyst increases when it is comprised of a metal oxide with support.",
"However, after 24 hours, irrespective of the catalyst, a slight increase is observed of the sulfur content of the desulfurized hydrocarbons, which may correspond to the beginning of clogging of the catalyst's pores, the sulfones and sulfoxides being attached thereto.",
"By this process, it is clear that the choice of a catalyst for the process of the invention is the result of a compromise between the nature of the catalyst, its specific surface area, and the size of the pores thereof.",
"EXAMPLE II In this Example, the effectiveness of the catalyst is measured as a function of the oxidation of the compounds.",
"The same process as in Example I is used, with catalysts C1-C6, and the formation is monitored of sulfones and sulfoxides with reference to the dibenzothiophene compounds, particularly the benzothiophene (BT), dibenzothiophene (DBT) and the 4,6 dimethyldibenzothiophene (DMBT), by gas chromatography equipped with a sulfur specific detector (SIEVERS process).",
"Table III below shows the results obtained.",
"TABLE III Oxidizer % Sulfone oxidation Catalyst concentration (eqS) BT DBT DMBT C1 3 80 78 81 C2 3 90 95 93 C3 3 88 92 89 C4 3 96 99 95 C5 3 85 87 88 C6 3 92 94 93 These results show that there is a conversion of at least 80% of the refractory thiophenic derivatives into sulfones, with catalysts composed of a single support, and of more than 90% with catalysts composed of supports and at least a metal in the form of metal oxide inserted into the matrix of the support or deposited on the support.",
"EXAMPLE III The present example seeks to show, at the same time as the oxidation, the effect as a function of time of the adsorption of the sulfone and sulfoxide compounds on the oxidation/adsorption and regeneration sequences, and the effectiveness with reference to the oxidation/adsorption.",
"This was done with the C3 catalyst under the operating conditions described in Example I on a middle distillate containing 44 ppm of sulfur after hydrotreatment, and in the presence of 600 ppm of tBHP.",
"The results of the oxidation/adsorption are given in FIG.",
"3-1, when the catalyst is fresh.",
"After two days, the total concentration of sulfur in the hydrocarbons increases substantially again to the initial value, in the absence of the treatment, according to the invention.",
"The results of FIG.",
"3-2 correspond to monitoring the sulfur content of these same hydrocarbons when this same catalyst C3, regenerated by combustion, is used.",
"The results obtained on a fresh catalyst are nearly identical to those obtained on the same regenerated catalyst.",
"These two graphs show the importance of the process of the invention that proposes an alternative operation of the same catalyst for oxidation/adsorption or for regeneration, the oxidation/adsorption time naturally being adapted to the content in sulfur."
]
] | Patent_10250431 |
[
[
"Orbital implant",
"An orbital implant includes a body of bioactive material having macropores of at least 400 μm, and a cap of bioactive material having substantially no pores or only micropores smaller than 50 μm.",
"The cap covers a portion of the body."
],
[
"1.An orbital implant which includes a body of bioactive material having macropores of at least 400 μm, and a cap of bioactive material having substantially no pores or only micropores smaller than 50 μm, with the cap covering a portion of the body.",
"2.An implant according to claim 1, which is substantially spherical.",
"3.An implant according to claim 2, which has a diameter of about 20 mm.",
"4.An implant according to claim 1, wherein the macropores in the body are substantially spherical so that they have diameters of at least 400 μm, and, optionally, wherein the diameters of the macropores do not exceed 1000 μm.",
"5.An implant according to claim 1, wherein some macropores are in communication with the outer surface of the body and wherein adjacent macropores in the body are interconnected by openings and/or passageways, so that open paths to the outer surface of the body are thereby provided in the body.",
"6.An implant according to claim 5, wherein substantially no isolated or closed macropores are present in the body.",
"7.An implant according to claim 5, wherein the openings and/or passageways which interconnect adjacent macorpores have diameters greater than 50 μm.",
"8.An implant according to claim 5, wherein the macropores in the body occupy from 40% to 85% by volume of the body.",
"9.An implant according to claim 5, wherein the body has micropores smaller than 50 μm.",
"10.An implant according to claim 9, wherein at least some of the micropores are of irregular shape, and have a maximum dimension smaller than 50 μm.",
"11.An implant according to claim 9, wherein at least some of the micropores are substantially spherical so that their diameters are thus smaller than 50 μm.",
"12.An implant according to claim 9, wherein adjacent micropores in the body are interconnected by openings and/or passageways and wherein some micropores are also interconnected to the macropores by openings and/or passageways so that the micropores, by means of those openings and/or passages, provide open paths to the macropores.",
"13.An implant according to claim 12, wherein substantially no isolated or closed micropores are present in the body.",
"14.An implant according to claim 9, wherein some of the micropores are of irregular shape and are in the form of interstitial spaces between incompletely sintered bioactive material particles, and wherein some of the micropores are of substantially spherical shape, with irregular micropores interconnecting adjacent spherical micropores, and also connecting spherical micropores to macropores.",
"15.An implant according to claim 14, wherein all the spherical micropores are of substantially the same size while all the irregular micropores are of substantially the same size, with the irregular micropores being smaller than the spherical micropores.",
"16.An implant according to claim 14, wherein substantially no isolated or closed micropores are present in the body.",
"17.An implant according to claim 9, wherein the micropores occupy from 3% to 70% by volume of the macropore-free bioactive material.",
"18.An implant according to claim 1, wherein the cap is in the form of a circular concave disc integrated with the body of bioactive material.",
"19.An implant according to claim 1, which is substantially spherical, and wherein the cap has a thickness which is no more than half the diameter of the implant.",
"20.An implant according to claim 1, wherein the bioactive material of the body and that of the cap are the same, and is hydroxyapatite."
],
[
"THIS INVENTION relates to an orbital implant.",
"According to the invention, there is provided an orbital implant which includes a body of bioactive material having macropores of at least 400 μm, and a cap of bioactive material having substantially no pores or only micropores smaller than 50 μm, with the cap covering a portion of the body.",
"The term ‘bioactive material’ used in this specification has its usual generally accepted meaning or definition, namely that it is ‘a material that elicits a specific biological response at the interface of the material which results in the formation of a bond between the tissues and the material’, as provided by L. L. Hench and J. Wilson in “An Introduction to Bioceramics”, Advanced Series in Ceramics—Vol.",
"1, Ed.",
"L. L. Hench and J. Wilson, World Scientific, Singapore, N.J., London, Hong Kong (1993) p. 7.The bioactive material may be a calcium phosphate material or compound such as a hydroxyapatite; a bioactive glass, which can typically be based on SiO2, Na2O, CaO and/or P2O5; a bioactive glass ceramic, which can be similar in composition to bioactive glass but which incorporates additionally MgO, CaF2 and/or metal oxides; or a composite material CONFIRMATION COPY comprising a polymer containing bioactive material particles, such as particles of a calcium phosphate compound, a bioactive glass and/or a bioactive glass ceramic.",
"The orbital implant may preferably be spherical.",
"It will thus be of a size so that it can be inserted into, and fit into, the orbit of a mammal, either to replace the contents of an eye following evisceration or to replace the eyeball following enucleation.",
"Thus, when it is to be implanted into the orbit of an adult human, it may have a diameter of about 20 mm.",
"The macropores of the body may be substantially spherical so that they have diameters of said at least 400 μm.",
"Preferably, the diameters of the macropores do not exceed 1000 μm.",
"Some macropores may be in communication with the outer surface of the body.",
"In other words, when such macropores are present, the body will have irregularly spaced surface indentations or dimples.",
"Adjacent macropores in the body may be interconnected by openings and/or passageways.",
"Thus, by means of the macropores which are in communication with the body outer surface and the openings and/or passageways between adjacent macropores, open paths to the body outer surface, defined by the macropores, are provided in the implant body.",
"The interconnecting openings or passageways between adjacent macropores may have diameters greater than 50 μm, preferably greater than 100 μm.",
"In other words, the body may contain substantially no isolated or closed macropores.",
"The macropores in the body may occupy from 40% to 85% by volume, preferably about 60% by volume, of the body.",
"The body may also have micropores smaller than 50 μm.",
"At least some of these micropores may be of irregular shape.",
"Thus, they may be in the form of interstitial spaces, for example, interstitial spaces between particles of bioactive material, resulting from incomplete sintering of the particles during formation of the body.",
"The sizes of these micropores are then dependent on the sizes of the bioactive material particles from which the body is sintered.",
"However, these micropores will have a maximum dimension smaller than 50 μm, and their maximum dimension may typically be of the order of 1 μm, or even smaller.",
"Instead, or additionally, at least some of the micropores may be of regular shape, eg substantially spherical so that their diameters are thus smaller than 50 μm.",
"The micropores, when present, may occupy from 3% to 70% by volume, preferably about 40% by volume, of the macropore-free bioactive material, ie the residual bioactive material around the macropores.",
"All the spherical micropores present in the body may be of substantially the same size, while all the irregularly shaped micropores may be of substantially the same size.",
"The irregularly shaped micropores may be smaller than the spherical micropores.",
"For example, when the irregularly shaped micropores are of the order of 1 μm or smaller, the spherical micropores may have diameters of at least 10 μm, and may typically have diameters of 10-45 μm.",
"Adjacent micropores in the body are then preferably interconnected by openings and/or passageways.",
"Some micropores may also be interconnected to the macropores by openings and/or passageways.",
"The micropores will thus, by means of these openings and/or passageways, provide open paths to the macropores, as well as, together with the macropores, open paths to the outer surface of the body.",
"In other words, there may thus be substantially no isolated or closed micropores in the body.",
"More specifically, both interstitial micropores and spherical micropores may be present in the body, with adjacent spherical micropores being interconnected by interstitial micropores which thus constitute the interconnecting openings or passageways.",
"The interstitial micropores will then also interconnect the spherical micropores to the macropores.",
"The body may thus have a trimodal pore size distribution, comprising macropores, which may be in the size range 400-1000 μm; larger micropores which may be in the size range smaller than 50 μm but at least 10 μm; and smaller micropores which are 1 μm or smaller.",
"The cap may, in one embodiment of the invention, be of bioactive material containing substantially no pores.",
"However, in another embodiment of the invention, the cap may contain pores; however, the pores will then be micropores smaller than 50 μm, ie the pores will then be irregular micropores and/or spherical micropores, as hereinbefore described.",
"In other words, the cap is then characterized thereby that it contains no pores larger than 50 μm.",
"Thus, it will contain no macropores as hereinbefore described.",
"The cap, which is thus an anterior cap, may be in the form of a circular concave or dish-shaped disc integrated with or embedded in the body of bioactive material.",
"The diameter of the rim of the cap may be the same as the diameter of the implant; however, preferably, it has a smaller diameter than that of the implant.",
"Preferably, the diameter of the rim of the cap may be about three-quarters that of the implant.",
"The cap will thus be thin relative to the diameter of the implant.",
"Thus, its thickness may be no more than half the diameter of the implant, and preferably about one-fortieth of the diameter of the implant.",
"While the bioactive material of the cap can, at least in principle, be different to that of the body, it is envisaged that the body and the cap will normally be of the same bioactive material.",
"The bioactive material may, in particular, be synthetic hydroxyapatite.",
"The orbital implant of the invention is thus, in use, placed into an orbit of a mammal.",
"The placing of an orbital implant of the integrated type, ie an orbital implant which, in use, becomes integrated through tissue ingrowth and vascularization, such as that of the invention, following evisceration or enucleation, is known.",
"The mammal will thus be one who has had an ocular enucleation or evisceration, or who needs an implant replacement.",
"Use of the orbital implant according to the invention will, it is believed, result in fibrovascular tissue ingrowth into the entire body of the implant, with the comparatively smooth cap resulting in little or no erosion of anterior tissue, including the conjunctiva, taking place.",
"After the implant has been placed into the orbit, eye muscles are typically attached to the implant, whereafter the implant is covered with tissue including conjunctiva, and a period of healing allowed during which fibrovascular tissue ingrowth into the implant occurs.",
"Thereafter, an artificial eye or prosthesis can be fitted over the conjunctiva, adjacent the cap of the implant.",
"It follows thus that when the implant is placed into the orbit, it is orientated such that the cap faces anterior tissue including the conjunctiva.",
"The invention will now be described in more detail by way of example and with reference to the accompanying diagrammatic drawings.",
"In the drawings, FIG.",
"1 shows a front view of an orbital implant according to one embodiment of the invention; FIG.",
"2 shows a side view of the orbital implant of FIG.",
"1; FIG.",
"3 shows an enlarged cross-sectional view of part of the orbital implant of FIG.",
"1; FIG.",
"4 shows an enlarged cross-sectional view, similar to that of FIG.",
"3, of an orbital implant according to another embodiment of the invention; and FIG.",
"5 shows a portion of the cross-sectional view of FIG.",
"4, enlarged even further.",
"Referring to FIGS.",
"1 to 3, reference numeral 10 generally indicates an orbital implant according to one embodiment of the invention.",
"The implant 10 is substantially spherical, and has a diameter of about 20 mm.",
"It includes a body 12 of synthetic hydroxyapatite having spherical macropores 14 as well as spherical micropores 16.The macropores 14 are all of substantially the same size, and have diameters of 400-1000 μm, typically about 800 μm.",
"The macropores 14 occupy about 60 vol % of the body 12.Some of the macropores 14 are in communication with the outer surface 15 of the body 12, as can be seen in FIG.",
"3.It will be appreciated that at least some adjacent macropores may be interconnected (not shown) by openings or passageways (not shown).",
"The micropores 16 are also all of substantially the same size, and have diameters less than 50 μm, eg about 10-45 μm.",
"The micropores 16 occupy about 40 vol % of the residual hydroxyapatite, ie the hydroxyapatite material between the macropores 14.The body 12 is thus solid save for the macropores and micropores therein.",
"The implant 10 also includes a thin anterior cap 18 of hydroxyapatite material having no macropores.",
"The cap 18 thus contains either no pores at all or only micropores (not shown) having maximum dimensions less than 50 μm, eg having maximum dimensions of about 1 μm.",
"When present, the micropores will occupy about 40% by volume of the cap material.",
"The cap 18 is thus characterized thereby that it contains no pores larger than 50 μm.",
"The cap 18 is in the form of a concave dish, and the rim 20 of the cap 18 has a diameter of about three-quarters that of the implant 10.Thus, when the implant 10 has a diameter of about 20 mm, the rim 20 of the cap 18 has a diameter of about 15 mm.",
"The thickness of the cap 18 is about one-fortieth the diameter of the implant 10.Thus, for an implant 10 having a 20 mm diameter, the thickness of the cap 18 will be about 0.5 mm.",
"The cap 18 thus covers only a portion of the body 12.Referring to FIGS.",
"4 and 5, reference numeral 100 generally refers to an orbital implant according to another embodiment of the invention.",
"Parts of the implant 100 which are the same or similar to those of the orbital implant 10, are indicated with the same reference numerals.",
"The implant 100 is also substantially spherical (not shown), and has a body 12 and an anterior cap (not shown) as hereinbefore described in respect of the implant 10.The body 12 of the implant 100 also has spherical macropores 14; however, apart from some of the macropores 14 of the implant 100 being in communication with the outer surface of the body 12 of the implant 100 (as hereinbefore described in respect of the implant 10) adjacent macropores 14 are interconnected by openings 102.The diameters of the openings 102 are typically about 100 μm or greater.",
"The implant 100 is normally manufactured by a sintering process such as that hereinafter described, and the interconnection of adjacent macropores then typically arises as a result of adjacent macropores coalescing together during the sintering process.",
"As a result of the common openings 102 between adjacent macropores 14 and the macropores 14 which are in communication with the outer surface of the implant body, open paths to the body outer surface are defined by the macropores in the body 12.Thus, the body 12 contains substantially no closed or isolated macropores.",
"The body 12 of the implant 100 also contains spherical micropores 16 (see FIG.",
"5), as hereinbefore described in respect of the implant 10.Moreover, it also contains irregular micropores 104 in the form of interstitial spaces between hydroxyapatite particles 106, resulting from incomplete sintering of hydroxyapatite particles 106 during formation of the body 12 by means of a sintering process such as that hereinafter described.",
"Although the hydroxyapatite particles are shown, in FIG.",
"5, as distinct separate particles, this is for ease of illustration only.",
"In fact, adjacent particles will thus be partially sintered together so that such adjacent particles can no longer be viewed as being distinct particles (as shown in FIG.",
"5) but rather merge so that they are in the form of an agglomerated mass containing the spherical macropores 14, the spherical micropores 16 and the irregular micropores 104.The sizes of the micropores 104 are substantially the same, and are dictated by the sizes of the hydroxyapatite particles 106 used for sintering.",
"Thus, when the particle sizes are about 1 μm, the maximum dimensions of the micropores 104 may be about 1 μm, or smaller.",
"Adjacent micropores 16 and 104 are thus interconnected.",
"Typically, adjacent micropores 16 are interconnected by micropores 104.Additionally, the micropores 104 and/or the micropores 16 are also interconnected to the macropores 14.Thus, the micropores 16, 104 together with the macropores 14, also define open paths to the outer surface of the implant body 12.There are thus substantially no closed or isolated micropores 16, 104 in the implant body.",
"The irregular micropores 104 typically occupy about 40% by volume of the residual hydroxyapatite, ie the macropore free hydroxyapatite, while the spherical micropores 16 typically occupy about 10% by volume of the residual hydroxyapatite.",
"To manufacture the implant 100, a mixture A is prepared by compounding hydroxyapatite powder having a mean particle size of about 1 μm, with a polymeric binder of a type suitable for injection moulding or extrusion; grinding the mixture to less than 300 μm particle size; and mixing stearic acid balls with a size distribution between 500-1000 μm therewith.",
"A mixture B is prepared by compounding hydroxyapatite powder having a mean particle size of about 1 μm with the same polymeric binder as used for mixture A; and grinding the mixture to less than 300 μm particle size.",
"The mixture A is loaded into a die suitable for pressing of a sphere.",
"This die includes a piston which will create a depression on the surface of the sphere during pressing, with the depression having the size and shape of the desired cap 18.The mixture A is lightly pressed to form a sphere containing the said depression.",
"The depression is then filled with a correct amount of the mixture B.",
"Thereafter the structure including the sphere with powder is consolidated by pressing to form a spherical compact comprising mixture A with an intimately bound cap of mixture B.",
"The structure is sintered at a temperature below 1100° C. It will be appreciated that when an implant in accordance with the invention is made by means of a sintering process such as that hereinbefore described, interstitial micropores 104 which result from incomplete sintering of adjacent hydroxyapatite particles, will thus normally be present.",
"Thus, such interstitial micropores will also be present in the body 12 of the implant 10 when it is manufactured by means of such a sintering process.",
"The implants 10, 100 can be implanted into the orbit or eye socket of a human who has had an ocular enucleation or evisceration, or who needs an implant replacement.",
"The implants can be placed according to known procedures for integrated implants.",
"For example, in the case of an evisceration, the implant is implanted to replace the eye contents.",
"Or, in the case of enucleation, the implant is placed without covering or with a covering or wrapping of tissue or artificial material into the eye muscle cone (not shown), and the eye muscles attached directly to the implant 10, 100 or to the implant wrapping.",
"Instead, the eye muscles can be wrapped around the implant 10, 100 and secured together without direct attachment of the eye muscles to the implant 10, 100.The anterior surface of the implant is covered with tissue including the conjunctive.",
"The cap 18 faces the conjunctiva.",
"A healing period is then allowed.",
"During this healing period, fibrovascular tissue ingrowth into the entire body 12 is promoted by the bioactive hydroxyapatite surfaces in conjunction with the open paths provided by the macropores 14, the micropores 16 and the micropores 104.Following this period of healing, the implant is integrated and, due to the muscle attachment, capable of movement.",
"Thereafter the prosthesis, ie an artificial eye, is located in position adjacent the cap 18, to obtain an artificial eye with natural appearance and good motility.",
"It is believed that the orbital implant of this invention addresses two common causes of complications associated with the use of orbital implants of the integrated type.",
"These are incomplete fibrovascular tissue ingrowth into the implant interior and erosion of anterior tissue by rough surface protrusions of a porous body.",
"The orbital implant of this invention addresses the first of these causes of complications by promoting complete ingrowth of fibrovascular tissue into the implant interior.",
"This is achieved by the implant of the invention having three modified material properties, as compared to properties commonly encountered in known porous orbital implants: Firstly, the macropore size is substantially increased, by a factor of 2 to 5, over that commonly encountered in known porous orbital implants.",
"Macropore size is generally restricted in porous orbital implants, to achieve improved mechanical properties and an even external roundness.",
"This is particularly important when the implant is made from materials derived from natural sources such as coral or processed bovine bone, where the external shape has to be achieved by machining.",
"With such materials, the external roundness can be extremely uneven due to the fracture of brittle protrusions and pore edges during machining.",
"It also produces undesirable sharp fracture surfaces.",
"In the orbital implant of this invention, an even roundness is readily achieved due to the entirely synthetic manufacture thereof, which eliminates any need for machining of a brittle surface and thereby avoids protrusions with sharp fracture surfaces.",
"Secondly, this larger macropore size is associated with a corresponding increase in the size of the interconnecting openings between adjacent macropores, to the extent that even the interconnecting openings are larger than the macropores commonly encountered in known porous orbital implants.",
"Thirdly, the orbital implant of the invention can have an engineered distribution of open micropores along the macropore surfaces and in the bulk of the ceramic material.",
"These micropores are present in a very high volume fraction, typically 40 vol % of the macropore-free hydroxyapatite material.",
"This engineered micropore distribution distinguishes the orbital implant of this invention over known bioceramic orbital implant materials, where microporosity is either absent in the material source or regarded as detrimental to mechanical strength and therefore eliminated to a large extent.",
"The small micropore size present at high volume fraction serves to significantly increase surface roughness at the cellular level.",
"It further achieves an increase in surface area, up to a factor of 70, over that of an equivalent material without the micropore distribution.",
"This is desirable in that it increases the bioactivity of the ceramic material.",
"It further achieves a strong associated capillary force exerted by the ceramic bulk, which is absent from materials without the high level of microporosity since the force is proportional to the volume fraction of micropores and inversely proportional to the micropore size.",
"The high degree of surface roughness, the large surface area and the strong capillary force result in immediate and strong adhesion of tissue to the material, avoiding motion and, importantly, micromotion of tissue against the implant.",
"It further results in rapid ingress and retention of fluid with improved cell attachment.",
"When combined with the inherent bioactive property of the hydroxyapatite composition it is further associated with direct tissue apposition, that is direct tissue ongrowth without intervening fibrous tissue as in the case of polymer materials.",
"Finally, it is believed that the combined material properties may be associated with binding and expression of autologous growth factors at the site, which promote early tissue healing.",
"Thus, to summarize, the large macropore size combined with large interconnecting opening size result in open access for fluid and tissue ingrowth to the central regions of the implant.",
"Along the inner macropore surfaces and bulk of the material, the material has been engineered to exhibit high surface roughness, high surface area, strong capillary force and inherent bioactivity.",
"This ensures immediate strong tissue attachment, elimination of micromotion, rapid ingress and retention of fluid with improved cell attachment, direct tissue apposition without intervening fibrous tissue.",
"The orbital implant of this invention further addresses the second cause of complications associated with porous orbital implants of the integrated type.",
"This is the tendency of porous materials to present a rough surface with sharp protrusions to anterior tissue, leading to erosion of the tissue and complications such as exposure of the implant.",
"By incorporating a cap of comparative smoothness, the implant does not present sharp edges to anterior tissue.",
"This serves to avoid erosion of the anterior tissue.",
"The cap is an integral part of the implant structure and is comprised of the same material as the implant body, incorporating the same micropore distribution as described.",
"Hence it exhibits a similar degree of tissue attachment, capillary force and high bioactivity as the porous ceramic body, even in the absence of macropores, since the inherent high bioactivity of the microporous hydroxyapatite allows direct tissue attachment even in the absence of significant tissue ingrowth.",
"From a tissue engineering and materials point of view, it is significant and advantageous that a seamless transition is achieved from porous body to cap, particularly in such a sensitive location where the overlying anterior tissue is relatively thin.",
"This fully incorporated cap serves to further distinguish the material from known orbital implants.",
"Thus, it is different from a cap of different material over the anterior region, which will introduce an artificial transition from a tissue engineering and materials point of view, since two different materials are unlikely to evoke identical response and achieve a seamless transition.",
"It is also different from a polymer cap, in that a polymer cap will exhibit low or no bioactivity and will require some different means to achieve attachment of the anterior tissue.",
"It is also different from a temporary resorbable coating over the implant, such as a polymer- or inorganic cement-based cap, since a resorbable coating will merely delay ingrowth and ongrowth to the ceramic while the underlying roughness of the porous body will eventually present again.",
"Finally, it is extremely difficult or impossible from a ceramic processing point of view to attach and incorporate such a cap on pre-densified material, such as a coral-derived or bone-derived material.",
"Thus, the implant body with incorporated cap does not present sharp and rough edges to the anterior tissue, thereby avoiding erosion of the anterior tissue.",
"Further, full incorporation of the cap is advantageous in that it presents a seamless transition from porous body to cap from a tissue engineering and materials point of view.",
"Further, the cap material exhibits high surface area, suitable roughness at the cellular level only, strong capillary force and inherent high bioactivity.",
"These properties jointly promote tissue attachment, elimination of micromotion, rapid ingress and retention of fluid with improved cell attachment, direct tissue apposition without intervening fibrous tissue."
]
] | Patent_10250525 |
[
[
"Bumper airbag and system",
"An airbag (10) for mounting in the bumper (18) of a motor vehicle (12) is provided.",
"The airbag can have an up-side-down “L” shape or a cylindrical shape.",
"Further, multiple bags can be combined within one system.",
"The airbag is configured to cover substantially the width of the vehicle upon deployment and also provide protection to the occupant of a struck vehicle (36) in the event the occupant is partially expelled from the struck vehicle in the direction of the bag.",
"The airbag is combined with an inflation (23), collision sensor (34) and an electronic control unit (38) to form the airbag system."
],
[
"1.A bumper airbag system comprising: a housing disposed in the bumper of a first vehicle and having a frangible door; a sensor for determining a pending collision between the first vehicle and another object and generating a signal thereof; at least one inflator for dispensing inflation fluid in response to the signal; and an airbag stored in the housing in a folded condition, the airbag member operatively connected to the inflator, whereby upon the firing of the inflator, the airbag inflates opening the frangible door and expanding out from and over the bumper to cover a substantial portion of the width of the vehicle.",
"2.The bumper airbag system of claim 1 further comprising an electronic control unit for processing the signal from the sensor and generating the signal to the inflator.",
"3.The bumper airbag system of claim 1 wherein upon inflation the bumper airbag has an up-side-down “L” shape defined a base portion extending away from the bumper and an arm portion extending downward from the base portion.",
"4.The bumper airbag system of claim 3 wherein the arm portion has a plurality of tubular members.",
"5.The bumper airbag system of claim 1 wherein the bumper airbag is fabricated from a woven polyester laminated material to provide a non-porous enclosure.",
"6.The bumper airbag system of claim 5 wherein the bumper airbag further comprises a nonabrasive, puncture resistant coating on its outer surface.",
"7.The bumper airbag system of claim 1 further comprising at least one pressure relief device.",
"8.The bumper airbag system of claim 7 wherein the pressure relief device is at least one hole in the airbag.",
"9.The bumper airbag system of claim 7 wherein the pressure relief device is a valve mounted in the housing.",
"10.The bumper airbag system of claim 1 further comprising a burst disk disposed between the inflator and the airbag.",
"11.The bumper airbag system of claim 1 wherein upon inflation the airbag takes on a cylindrical shape.",
"12.The bumper airbag system of claim 11 wherein the airbag further comprises a throat member having at least one hole for the inflating gas from the inflator to pass into the interior of the airbag.",
"13.The bumper airbag system of claim 12 wherein the throat member is attached at one end to the housing.",
"14.The bumper airbag system of claim 1 wherein the airbag upon inflation is comprised of a plurality of cylindrically shaped bags.",
"15.The bumper airbag system of claim 14 wherein each of the plurality of cylindrically shaped bags are secured together in such a manner that two of the bags cover the grille of the vehicle and another bag is disposed beyond the other two bags and away from the grille.",
"16.The bumper airbag system of claim 15 wherein the bags are secured by tethers.",
"17.The bumper airbag system of claim 14 wherein each of the bags abuts the others.",
"18.The bumper airbag system of claim 19 further comprising fluid communication ports between the bags.",
"19.A bumper airbag comprising: a first rectangular panel of fabric having four edges, the panel being rolled and two edges being attached to form a cylinder; a second rectangular panel of fabric substantially smaller than the first panel and sewn to the outer surface of the first panel near one of the edges; at least one hole extending through both panels; and a throat member attached to the first panel and encompassing the second panel.",
"20.The bumper airbag of claim 19 wherein the throat member has a flange at one end for attaching to the curved surface of the first panel and opening at its other end for receiving inflating gas.",
"21.The bumper airbag of claim 20 further comprising at least one support tether secured to the inside of the throat member.",
"22.The bumper airbag of claim 19 wherein having a plurality of holes extending through the two panels and a reinforcing member disposed between adjacent holes.",
"23.The bumper airbag of claim 19 further comprising a circular end cap attached at the open ends of the first panel.",
"24.The bumper airbag of claim 23 wherein each end cap has a vent hole."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>For years, the automotive industry has tried various methods and products to reduce the damage to passengers and vehicles in collisions.",
"Of prime importance are the various systems of vehicle airbags that are deployed upon the sensing of an actual collision.",
"These airbags are located in and about the passenger compartment of the motor vehicle and are inflated to surround and protect the occupants from serious injury.",
"Other methods of reducing, to some extent, the forces created in a collision from injuring the occupants are various attempts to provide “crush zones” at the front and the rear of the vehicle to absorb some of the collision forces.",
"Still other methods also deal with design of the vehicle frame, engine mounts and other structural members to absorb the forces by means of controlled structural collapsing.",
"Airbags have been fabricated to the front end of the vehicle that just prior to the instance of a crash, inflate and form a fluid-filled structure between the striking object or vehicle and the struck object or vehicle.",
"PCT application number WO98/50254 “Collision Protection System for Vehicles” teaches airbags mounted to the front of a vehicle.",
"U.S. Pat.",
"No.",
"3,656,791 “Vehicle Impact-Cushioning Device” teaches an airbag mounted to deploy from the front end of a vehicle.",
"U.S. Pat.",
"No.",
"3,708,194 “Vehicle Safety Apparatus” teaches a front-end mounted airbag that includes a fire extinguishing material.",
"Several prior art patents deal with bumper improvements.",
"U.S. Pat.",
"No.",
"4,518,183 “Extendible Safety Impact Bags for Vehicles” teaches mechanisms for extending bumpers outwardly of the vehicle upon the sensing of a potential crash.",
"Air is supplied to airbags to form a somewhat rigid member supporting the bumpers for the duration of the crash and then the airbags are deflated and the bumpers return to their normal position.",
"U.S. Pat.",
"No.",
"4,930,823 “Vehicle Bumper” teaches front and rear bumpers having airbags that are inflated upon contact of the bumper with an object.",
"U.S. Pat.",
"No.",
"5,106,137 “Vehicle Bumper with Combination Foam and Airbag Energy Absorber” teaches a bumper having an internal cavity surrounded by compressible energy absorbing plastic.",
"Inside the cavity is an airbag that is inflated upon the onset of a crash to provide more protection to the front or rear end of the vehicle.",
"U.S. Pat.",
"No.",
"5,651,569 “Inflatable Bumper System” teaches a bumper having an enclosed airbag that is permanently inflated to provide a permanent cushion bumper.",
"U.S. Pat.",
"No.",
"5,725,265 “Airbag System For Vehicle Bumpers” teaches an airbag concealed inside a bumper that is inflated and extends outwardly of the bumper to reduce the effects of the crash.",
"The bumper has an expellable panel on the outer surface of the bumper that is removed by the inflation of the airbag.",
"U.S. Pat.",
"No.",
"6,056,336 “Airbag with Internal Shock Absorber” teaches a bumper airbag having an internal shock absorber.",
"The airbag is deployed in a circular shape.",
"U.S. Pat.",
"No.",
"6,126,214 “Air Bumper” teaches an air inflatable bumper that responds to a crash to provide an air-supported member to protect the car from damages due to collision.",
"Several prior art patents show a system for the detection of a crash and the deployment of airbags.",
"U.S. Pat.",
"No.",
"3,822,076 “Fluid Shock Absorbing Buffer, teaches a front or rear mounted airbags that are inflated when a telescopic rod extending from the vehicle touches a barrier.",
"U.S. Pat.",
"No.",
"4,176,858 “Energy Absorbing Bumper System” teaches a combination of a pneumatic bumper system supporting an airbag system that deploys in response to increased pressure in the pneumatic system as a result of an impact with an object.",
"U.S. Pat.",
"No.",
"5,431,463 “Air Cell Bumper Device” teaches a plurality of air cells containing grouped around a much larger air cell that stores inflation fluid.",
"Upon impact, the material of cells is such that the larger cell ruptures and the fluid therein flows to the smaller cells buffering the impact.",
"The invention is particularly useful on the sides of a vehicle.",
"U.S. Pat.",
"No.",
"5,646,613 “System for Minimizing Automobile Collision Damage” teaches the storage and deployment of various airbags around the vehicle as a result of proximity sensing.",
"The different sides of the vehicle are uniquely controlled.",
"U.S. Pat.",
"No.",
"5,732,785 “Proactive Exterior Airbag System and Its Deployment Method for a Motor Vehicle” teaches a system having a detection unit, a control unit, and a deployment unit together will deploy airbags mounted on the vehicle.",
"This system deploys the airbags before the crash and describes the method used to determine distance and speed between the striking and struck vehicles or objects.",
"European Patent Application EP 1,024,063 “Vehicle Bumper and Hood Airbag System” teaches a bumper and hood bag that is inflated prior to the collision of a pedestrian and the vehicle.",
"The airbag is inflated to absorb the collision forces between the areas from the waist down of a pedestrian and the vehicle.",
"JP 6,144,154 “Shock Relaxing Device” teaches an airbag deployed in front of a bumper to reduce the shock of a pedestrian or bike collision with a car.",
"The increased popularity of sports utility vehicles, passenger trucks and other retail motor vehicles that stand higher than a standard motor vehicle, such as a sedan or sports car, has created new problems in the area of vehicle collisions.",
"Specifically, when one of these higher standing vehicles broadsides a standard vehicle, because of the difference in height between the two vehicles, the bumper of the high vehicle will contact the side window portion of the struck vehicle instead of the door portion.",
"If the collision happens at high speeds, the head of the occupant sitting adjacent the window portion may move outward past the window and into contact with the bumper of the higher vehicle.",
"Accordingly, there is a need for an airbag that can reduce the severity of such collisions."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An advantage of the present invention is that it can reduce the severity of a collision between high standing vehicle and a low standing vehicle.",
"Another advantage of the present invention is that it offers protection to the occupant of the struck vehicle in the event the occupant is partially expelled in the direction of the invention.",
"These and other advantages will be found in the present invention that is directed to an airbag assembly mounted behind a vehicle's bumper coupled with sensors, electronic control units, and inflators to inflate by means of a fluid pressure to expand and provide an interface between a striking and struck motor vehicles.",
"The airbag can have an up-side-down “L” shape or a cylindrical shape.",
"Further, multiple bags can be combined within one system.",
"The airbag is configured to cover substantially the width of the vehicle upon deployment and also provide protection to the occupant of a struck vehicle in the event the occupant is partially expelled from the struck vehicle in the direction of the bag."
],
[
"CROSS REFERENCE TO RELATED APPLICATION This application claims priority to co-pending Provisional Patent Application Ser.",
"No.",
"60/261,056, filed Jan. 11, 2001, which is incorporated herein by reference.",
"TECHNICAL FIELD This invention relates generally to motor vehicle safety devices and in particular to an inflatable airbag for use in the bumper area of a motor vehicle.",
"BACKGROUND OF THE INVENTION For years, the automotive industry has tried various methods and products to reduce the damage to passengers and vehicles in collisions.",
"Of prime importance are the various systems of vehicle airbags that are deployed upon the sensing of an actual collision.",
"These airbags are located in and about the passenger compartment of the motor vehicle and are inflated to surround and protect the occupants from serious injury.",
"Other methods of reducing, to some extent, the forces created in a collision from injuring the occupants are various attempts to provide “crush zones” at the front and the rear of the vehicle to absorb some of the collision forces.",
"Still other methods also deal with design of the vehicle frame, engine mounts and other structural members to absorb the forces by means of controlled structural collapsing.",
"Airbags have been fabricated to the front end of the vehicle that just prior to the instance of a crash, inflate and form a fluid-filled structure between the striking object or vehicle and the struck object or vehicle.",
"PCT application number WO98/50254 “Collision Protection System for Vehicles” teaches airbags mounted to the front of a vehicle.",
"U.S. Pat.",
"No.",
"3,656,791 “Vehicle Impact-Cushioning Device” teaches an airbag mounted to deploy from the front end of a vehicle.",
"U.S. Pat.",
"No.",
"3,708,194 “Vehicle Safety Apparatus” teaches a front-end mounted airbag that includes a fire extinguishing material.",
"Several prior art patents deal with bumper improvements.",
"U.S. Pat.",
"No.",
"4,518,183 “Extendible Safety Impact Bags for Vehicles” teaches mechanisms for extending bumpers outwardly of the vehicle upon the sensing of a potential crash.",
"Air is supplied to airbags to form a somewhat rigid member supporting the bumpers for the duration of the crash and then the airbags are deflated and the bumpers return to their normal position.",
"U.S. Pat.",
"No.",
"4,930,823 “Vehicle Bumper” teaches front and rear bumpers having airbags that are inflated upon contact of the bumper with an object.",
"U.S. Pat.",
"No.",
"5,106,137 “Vehicle Bumper with Combination Foam and Airbag Energy Absorber” teaches a bumper having an internal cavity surrounded by compressible energy absorbing plastic.",
"Inside the cavity is an airbag that is inflated upon the onset of a crash to provide more protection to the front or rear end of the vehicle.",
"U.S. Pat.",
"No.",
"5,651,569 “Inflatable Bumper System” teaches a bumper having an enclosed airbag that is permanently inflated to provide a permanent cushion bumper.",
"U.S. Pat.",
"No.",
"5,725,265 “Airbag System For Vehicle Bumpers” teaches an airbag concealed inside a bumper that is inflated and extends outwardly of the bumper to reduce the effects of the crash.",
"The bumper has an expellable panel on the outer surface of the bumper that is removed by the inflation of the airbag.",
"U.S. Pat.",
"No.",
"6,056,336 “Airbag with Internal Shock Absorber” teaches a bumper airbag having an internal shock absorber.",
"The airbag is deployed in a circular shape.",
"U.S. Pat.",
"No.",
"6,126,214 “Air Bumper” teaches an air inflatable bumper that responds to a crash to provide an air-supported member to protect the car from damages due to collision.",
"Several prior art patents show a system for the detection of a crash and the deployment of airbags.",
"U.S. Pat.",
"No.",
"3,822,076 “Fluid Shock Absorbing Buffer, teaches a front or rear mounted airbags that are inflated when a telescopic rod extending from the vehicle touches a barrier.",
"U.S. Pat.",
"No.",
"4,176,858 “Energy Absorbing Bumper System” teaches a combination of a pneumatic bumper system supporting an airbag system that deploys in response to increased pressure in the pneumatic system as a result of an impact with an object.",
"U.S. Pat.",
"No.",
"5,431,463 “Air Cell Bumper Device” teaches a plurality of air cells containing grouped around a much larger air cell that stores inflation fluid.",
"Upon impact, the material of cells is such that the larger cell ruptures and the fluid therein flows to the smaller cells buffering the impact.",
"The invention is particularly useful on the sides of a vehicle.",
"U.S. Pat.",
"No.",
"5,646,613 “System for Minimizing Automobile Collision Damage” teaches the storage and deployment of various airbags around the vehicle as a result of proximity sensing.",
"The different sides of the vehicle are uniquely controlled.",
"U.S. Pat.",
"No.",
"5,732,785 “Proactive Exterior Airbag System and Its Deployment Method for a Motor Vehicle” teaches a system having a detection unit, a control unit, and a deployment unit together will deploy airbags mounted on the vehicle.",
"This system deploys the airbags before the crash and describes the method used to determine distance and speed between the striking and struck vehicles or objects.",
"European Patent Application EP 1,024,063 “Vehicle Bumper and Hood Airbag System” teaches a bumper and hood bag that is inflated prior to the collision of a pedestrian and the vehicle.",
"The airbag is inflated to absorb the collision forces between the areas from the waist down of a pedestrian and the vehicle.",
"JP 6,144,154 “Shock Relaxing Device” teaches an airbag deployed in front of a bumper to reduce the shock of a pedestrian or bike collision with a car.",
"The increased popularity of sports utility vehicles, passenger trucks and other retail motor vehicles that stand higher than a standard motor vehicle, such as a sedan or sports car, has created new problems in the area of vehicle collisions.",
"Specifically, when one of these higher standing vehicles broadsides a standard vehicle, because of the difference in height between the two vehicles, the bumper of the high vehicle will contact the side window portion of the struck vehicle instead of the door portion.",
"If the collision happens at high speeds, the head of the occupant sitting adjacent the window portion may move outward past the window and into contact with the bumper of the higher vehicle.",
"Accordingly, there is a need for an airbag that can reduce the severity of such collisions.",
"SUMMARY OF THE INVENTION An advantage of the present invention is that it can reduce the severity of a collision between high standing vehicle and a low standing vehicle.",
"Another advantage of the present invention is that it offers protection to the occupant of the struck vehicle in the event the occupant is partially expelled in the direction of the invention.",
"These and other advantages will be found in the present invention that is directed to an airbag assembly mounted behind a vehicle's bumper coupled with sensors, electronic control units, and inflators to inflate by means of a fluid pressure to expand and provide an interface between a striking and struck motor vehicles.",
"The airbag can have an up-side-down “L” shape or a cylindrical shape.",
"Further, multiple bags can be combined within one system.",
"The airbag is configured to cover substantially the width of the vehicle upon deployment and also provide protection to the occupant of a struck vehicle in the event the occupant is partially expelled from the struck vehicle in the direction of the bag.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a perspective view of a high standing vehicle having a one embodiment of a bumper airbag as contemplated by the present invention.",
"FIG.",
"2 is the partial side view of the vehicle and bumper airbag of FIG.",
"1.FIG.",
"3 is an illustration of a collision between the vehicle of FIG.",
"1 and another vehicle.",
"FIG.",
"4 is an illustration of the collision of FIG.",
"3 with the high standing vehicle having a second embodiment of a bumper airbag as contemplated by the present invention.",
"FIG.",
"5 is a perspective view of a third embodiment of a bumper airbag as contemplated by the present invention.",
"FIG.",
"6 is a perspective view of the throat of the bumper airbag of FIG.",
"5.FIG.",
"7 is an end view of the bumper airbag of FIG.",
"5.FIG.",
"8 is a view taken along line 8-8 of FIG.",
"7.FIG.",
"9 is a plan view of the fabric layout of the bumper airbag of FIG.",
"5.FIG.",
"10 is a view taken along line 10-10 of FIG.",
"9.FIG.",
"11 is a perspective view of the bumper airbag of FIG.",
"5 mounted on the bumper of a vehicle.",
"FIG.",
"12 is a cross-sectional side taken along line 12-12 of FIG.",
"11.FIG.",
"13 is a block diagram of a control system for a bumper airbag contemplated by the present invention.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIGS.",
"1-3, a first embodiment of a bumper airbag as contemplated by the present invention is generally denoted by reference numeral 10.An important feature of the airbag 10 is its ability to extend across a substantial portion of the width of the vehicle.",
"By substantial it is meant at least 65% of the width of the vehicle or extending at least between the vehicle's headlamps 14 and 16.It will be appreciated that the airbag can have a number of different shapes as will be described herein.",
"In the first embodiment of the present invention, the airbag 10 has a up-side-down “L” shape and extends forward from the bumper 18 along a base portion 20.An arm portion 22 extends perpendicularly downward from the base portion 20 and includes a plurality of tubular portions 24.In the preferred embodiment, the diameter of each of the tubular portions 24 is about nine inches (23 cm).",
"The airbag member 10 is stored in a folded condition in a housing 26 mounted behind the bumper 18.At least one or more inflators 28 are connected to the folded airbag 10.The airbag member 10 is fixedly mounted to the housing 26 and therefore to the vehicle 12.The housing 26 has a frangible door or cover 30, which in the embodiment illustrated in FIG.",
"1 is located in a direction facing up.",
"As the airbag 10 inflates, it pushes the frangible door 30 open and then proceeds to expand over the bumper 18.The frangible door 30 may be hinged to open against the grille member 32 or toward the bumper 18 or in the alternative the frangible door 30 may be removed.",
"At least one sensor 34 is mounted on the front of the vehicle 12 for sensing an imminent collision with a second vehicle.",
"As shown in FIG.",
"13, the sensor 34 is electrically connected to an electronic control unit, (ECU), 38 that is mounted in a housing 40 on the firewall of the vehicle 12 or inside the vehicle cab.",
"Located in the ECU 38 is a memory unit 42 that contains one or more algorithms 44 at least one of which controls the inflation of the airbag member 10.A power supply 46 is electrically connected to the ECU 38 to electrically power all of the electrical components of the system.",
"There are many types of sensors 34 that are applicable such as a proximity sensor or a crash-responsive sensor.",
"Referring to FIG.",
"2, one end of the base portion 20 is approximately tangentially aligned with the grille member 32.The grille member 32 is typically a curved member and the airbag member 10 is typically a straight tubular member hence the tangential relationship.",
"In some applications this end of the base portion 20 will abut the grille 32 while in other applications it will not touch the grille 32 upon inflation.",
"Of prime importance in the bumper mounted airbags of the present invention is the concern for the occupant of the struck motor vehicle 36, see FIG.",
"3.Tests have shown that the occupant can be partially thrown out of the window of the struck vehicle depending on the severity of the crash.",
"To reduce injury to the thrown occupant, the corners where the end portion 20 meets the arm portion 22 are rounded.",
"Also, the outer surface of the airbag 10 is made smooth and nonabrasive.",
"Hence, when the occupant, more particularly the head of the occupant, is forced out of the window, the head strikes the smooth, nonabrasive, rounded airbag surface thereby reducing the severity of injury.",
"The airbag member 10 is filled with a gas from a gas inflator 28 which is preferably a cold gas inflator so as to avoid the hot has temperatures associated with typical hot gas inflators used in conventional steering wheel or dashboard mounted airbags.",
"The airbag 10 is preferably made from a woven polyester and/or nylon laminated or non-laminated material that is coated in such a manner as to prevent any leakage.",
"The airbag 10 is non-porous.",
"Referring to FIGS.",
"5-12, a second embodiment of a bumper airbag as contemplated by the present invention is generally denoted by reference numeral 58.As with the airbag 10, an important feature of the airbag 58 is its ability, when inflated, to extend across a substantial portion of the width of the vehicle as shown in FIG.",
"11.The bumper airbag 58 when fully inflated is a circular cylindrical bag.",
"Along the side 64 of the airbag 58 is a rectangular connecting housing or throat member 66, shown in FIG.",
"6, that couples the airbag 58 to a housing 96 disposed within the bumper 62 of vehicle 60.The throat member 66 is connected to the airbag 58 by a flange 65 surrounding the throat member.",
"The flange 65 may be sewn or bonded to the airbag.",
"The mating side of the flange 65 is shown in FIG.",
"6.Inside the throat 66 are preferably three inlets or holes 68, 69, 70, (see FIG.",
"8), in the wall of the airbag bag 58.As shown in FIG.",
"7 the connecting housing or throat 66 has parallel walls 74,76 with the wall 76 longer than the wall 74 so as to allow the throat 66 to be mounted to the curved surface of the airbag 58.As shown in FIG.",
"9, the throat member 66 is sewn, (dashed lines) to a sheet or panel of fabric 78 from which the airbag 58 is formed.",
"The throat member 66 is attached to panel 78 near one of its edges 80.To assemble the cylindrical bumper bag 58, the panel 78 is sewn to a panel of fabric 79 that is slightly less than the size of the rectangular connecting throat 66.The seams are tight so as to block any inflation fluid from escaping through these seams when the bumper airbag 58 is inflated.",
"In the preferred embodiment, before the two fabrics are mated, the inlet holes 68,69,70 are punched through the panels 78,79 and then aligned.",
"In an alternate between adjacent inlet holes 68,69,70 is a reinforcing member 82 preferably made of seat belt webbing material.",
"FIG.",
"10 illustrates the reinforcing member 82 on the outer surface of panel 79.Alternatively, the reinforcing member may be mounted between the two panels.",
"After mounting the reinforced members 82, the rectangular connecting member or throat member 66 is sown to the panel 78 so as to encompass the inlet holes.",
"Next the two edges 80, 84 of panel 78 are sown together forming a flat tubular member with open opposed ends.",
"In order to give the bumper bag a cylindrical shape, circular end caps 86 are sown to the open opposed ends.",
"In all cases the thread count and the thread used such as 138 Nylon, is very strong and the stitches are relatively tight.",
"Some of the stitching is accomplished by double needle sewing machines.",
"Optionally in order to keep the throat member 66 of in its rectangular shape, one or more tethers 88 are secured to the inside of the throat member 66 extending between the two long parallel walls 74,76.If tethers 88 are used, they are sown to the throat member 66 before it is sown to the panel 78.Along the axis of each circular end cap 86 and the sides of the airbags 10 and 58 is an open venting hole 90 of such a size to allow the bumper airbags 10, 58 to deflate some time after the crash.",
"Referring to FIGS.",
"11 and 12, an inflated cylindrical bumper airbag 58 is deployed from the bumper 62 of a truck located below the vehicle's grille 98 and supported by the vehicle's frame rails 100.In the center of the bumper 62, is an opening 92 that is enclosed by a frangible door 94.The folded airbag 58 is mounted in an airbag housing 96 disposed in the bumper behind the door 94.Also, mounted in the housing 96 is an inflator 28 separated from the airbag 58 by a burst disk 29.Upon inflation, the airbag 58 forces the door 94 open.",
"The door 94 is hinged to allow it to rotate out of the path of the bumper airbag.",
"The throat member 66 is secured to the housing 96.As the inflated airbag 58 contacts the other vehicle, the pressure in the bag increases.",
"To prevent an over pressure situation where the bag could burst, a relief valve 102 mounted at the rear of the bumper bag housing 96.The valve 102 operates to bleed off pressure above a certain level from the airbag, thus maintaining the bag at its desired pressure.",
"As the crash proceeds, the pressure in the air bag is released through the vent holes 90 causing the bag to deflate.",
"This deflation of the airbag continues the energy attenuation of the crash.",
"Referring to FIG.",
"4, multiple cylindrical airbags 48,49,50 can be combined to operate together.",
"When there are at least two airbags 48, 49, they are secured together by a tether 52 and have a plurality of communication ports 54 therebetween.",
"The communication ports 54 are typically holes surrounded by a reinforcing washer.",
"The washer also functions to secure one airbag to another.",
"The outside surfaces of the airbags 48-50 are nonabrasive and puncture-proof.",
"Puncture-proof is important so that the rubbing of the airbags 48-50 on the struck motor vehicle 36 does not cause tearing.",
"Nonabrasive surface protects the occupants of the struck motor vehicle 36 from being injured in the event the occupant is partially ejected from the vehicle.",
"Thus, a bumper airbag for use in conjunction with a bumper of a vehicle is provided that reduces the transmission of energy in the event of a collision.",
"The bumper airbag is particularly useful with high standing vehicles.",
"The bumper airbag also provides protection to the occupant of the vehicle should the occupant be partially expelled from the struck vehicle in the direction of the bag.",
"Various modifications and alterations to the above-described preferred embodiments will be apparent to those skilled in the art.",
"Accordingly, this description of the invention should be considered exemplary and not as limiting the scope and spirit of the invention as set forth in the following claims."
]
] | Patent_10250602 |
[
[
"5-azido-laevulinic acid, method for the production thereof and its use",
"The present invention relates to 5-azido levulinic acid, a process for its preparation, its use.",
"Using 5-azido levulinic acid as starting material for the synthesis of 5-amino levulinic acid hydrochloride it is possible to obtain the latter in good yield an in pharmaceutical acceptable quality.",
"5-Azido levuliniv acid is synthesized in that methyl 5-bromo levulinate and/or methyl 5-chloro levulinate is converted with aqueous hydrochloric acid and as a result of an incomplete bromine/chlorine exchange at the C-5-postion a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid is obtained, and the obtained 5-chloro levulinic acid, a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid and the pure 5-bromo levulinic acid is transferred into 5-azido levulinic acid by conversion with a nucleophilic azide."
],
[
"1.5-azido levulinic acid 2.Process for the preparation of 5-azidolevuliniv acid, wherein (a) methyl 5-bromo levulinate and/or methyl 5-chloro levulinate is converted with aqueous hydrochloric acid and as a result of an incomplete bromine/chlorine exchange at the C-5-postion a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid is obtained, and (b) the obtained 5-chloro levulinic acid, a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid and the pure 5-bromo levulinic acid is transferred into 5-azido levulinic acid by conversion with a nucleophilic azide.",
"3.Process according to claim 2, wherein in stage (b) the conversion with an azide is carried out in a polar solvent, especially acetone, a C1-C4-alkanol or water as reaction medium.",
"4.Process according to claim 2, wherein in stage (b) an alkali metal azide, in particular sodium azide is used as nucleophilic azide.",
"5.Use of 5-azido levulinic acid for the preparation of 5-amino levulinic acid hydrochloride, whereas 5-azido levulinic acid is converted into 5-amino levulinic acid hydrochloride by catalytic hydrogenation.",
"6.Use according to claim 5, wherein (a) the catalytic hydrogenation ist carried out in aqueous hydrochloric acid, (b) the solvent and excess hydrochloric acid is seperated and (c) 5-amino levulinic acid hydrochloride is obtained by cristallisation from 2-propanol or t-butyl methyl ether/methanol.",
"7.Use of 5-azido levulinic acid as explosive and/or priming fuse.",
"8.Use of 5-azido levulinic acid as explosive and/or priming fuse in airbags in the motor vehicle technique."
],
[
"The present invention relates to 5-azido levulinic acid, a process for its preparation, its use for the preparation of 5-amino levulinic hydrochloride, and its use as explosive.",
"The importance of 5-amino levulinic acid hydrochloride as starting material for the synthesis of various compounds as well as its manifold employment, especially in the field of medicine, will be explained below.",
"5-Amino levulinic acid, storable as its halogen acid addition salt, in particular 5-amino levulinic acid hydrochloride, is a bioorganic compound, which appears as preliminary stage on the tetrapyrrole biosynthetic pathway of porphobilinogene, chlorophylle, haemine, vitamine B12, and cytochrome (K. D. Gibson et al., Biochem.",
"J.",
"1955, 61, 618; S. I. Beale, Plant Physiol.",
"1971, 48, 316; C. A. Rebeiz, Plant Physiol.",
"1970, 46, 543; A. I. Scott, Angew.",
"Chem.",
"1993, 105, 1281-1302; Angew.",
"Chem.",
"Int.",
"Ed.",
"Engl.",
"1993, 32, 1223).",
"As known, these compound or its alkyl esters will be used as starting compound in the synthesis of a multitude of compounds.",
"5-Amino levulinic acid hydrochloride is used as substrate for the assay of 5-amino levulinic acid dehydratase (D. Shemin et al., Methods Enzymol.",
"1970, 17(A), 205; D. L. Coleman, ibid.",
"211; A. M. del C. Beatle et al., ibid., 216; P. Bodlaender et al., Anal.",
"Biochem.",
"1974, 58, 500; D. Gurne, D. Shemin, Methods Enzymol.",
"1976, 44, 844), in acriculture as selective herbicide (5-amino levulinic acid hydrochloride: C. A. Rebeiz et al., Enzyme Microb.",
"Technol.",
"1984, 6, 390; Chem.",
"& Eng.",
"News 1984, 62, 8; S. O. Duke, C. A. Rebeiz, Porphyric Pesticides: Chemistry, Toxikology, and Pharmaceutical Applications.",
"ACS Symposium Series 559 1994; alkyl esters of 5-amino levulinic acid hydrochloride: H. Takeya, Jpn.",
"Kokai Tokkyo Koho JP 0409360, 1992; Chem.",
"Abstract.",
"1992, 116, 19755), as insecticide (C. A. Rebeiz et al., Pestic.",
"Biochem.",
"Physiol.",
"1988, 30, 11; A. I. Scott, Angew.",
"Chem.",
"1993, 105, 1281-1302; Angew.",
"Chem.",
"Int.",
"Ed.",
"Engl.",
"1993, 32, 1223; S. O. Duke, C. A. Rebeiz, Porphyric Pesticides: Chemistry, Toxikology, and Pharmaceutical Applications.",
"ACS Symposium Series 559 1994), and in various fields of medicine.",
"In the field of medicine, in particular the field of oncology, 5-amino levulinic acid hydrochloride is used for the treatment of actinic ceratosis with the help of photodynamic therapy (PDT) (Drugs Fut.",
"2000, 25(1), 74-76 and cited literature therein).",
"But also in photodynamic diagnostics (PDD), this includes some applications of the PDT, i.e.",
"the fluorescence detection of some kinds of cancer like lung cancer, bladder cancer, and prostate cancer (WO 93/20810, WO 96/39188, WO 98/09155; A. G. Hofstetter, Springer Verlag, Berlin, Heidelberg, New York, London, Paris, Tokio, Hong Kong, Barcelona, Budapest, 1995.",
"), as well as in marking brain tumors (W. Stummer et al., Neurosurgery 1998, 42(3), 518-526.)",
"someone fall back on 5-amino levulinic acid hydrochloride.",
"A novel application concerns the photosensitation of arteries in combination with the baloon anglioplastics, which shall make an operative post treatment with hardening of the arteries unnecessary (New Scientist 1999, 162, Nr.",
"2185).",
"Recently, there was reported about the application of 5-amino levulinic acid hydrochloride as hair restorer (Shiseido Co., Ltd.; Cosmo Sogo Kenkyusho K. K., Jpn.",
"Kokai Tokkyo Koho JP 11116, 446 [99116, 446], 1999; Chem.",
"Abstract.",
"1999, 130, 356898z).",
"The big interest in 5-amino levulinic acid hydrochloride is also reflected by the great number of synthetic pathways described in the literature.",
"The discussion of the various processes must follow in view of the transferness to the technical production scale, so that this compound, which is useful in so many fields, can be prepared on a large scale, cost-effective and without pollution of the environment.",
"Some exemplary production processes of 5-amino levulinic acid hydrochloride, starting from different substance classes are discussed in the following.",
"U.S. Pat.",
"No.",
"5,380,935 discloses a process, in which furfuryl amine, the amino function of which was protected, is oxidised by oxygen on the photochemical pathway in the precense of a sensetizer.",
"Catalytic reduction of the intermediate and subsequent acid catalysed deprotection of the resulting product yielded 5-amino levulinic acid hydrochloride.",
"Some other processes make use of furane derivatives as starting material for the synthesis of 5-amino levulinic acid hydrochloride, also (EP-A 0607952; U.S. Pat.",
"No.",
"5,284,973; U.S. Pat.",
"No.",
"5,344,974; L. Cottier, G. Descotes, L. Eymard, K. Rapp, Synthesis 1995, 303-306; K. Suzuki et al., Nippon Kagaku Kaishi 1999, 3, 199-202).",
"All these processes have a great deal in common: The amino function has to be protected first, and in the last step of the synthetic sequence the protecting group has to be removed again.",
"Partly, for the user and the environment, harmful solvents and chemicals are used, which have to be disposed cost-intensive after the reaction.",
"The use of expensive photosensetizers like fullerene (C60) or rose bengal is to be said against a technical application of these processes, too.",
"Alternatively, some succinyl derivatives—by introduction of an carbon-nitrogen moiety—are used for the synthesis of 5-amino levulinic acid hydrochloride.",
"In U.S. Pat.",
"No.",
"3,846,490, e.g.",
"mono methyl succinate mono chloride was reacted together with hippuric acid in γ-picoline as solvent.",
"The obtained oxazolidine derivative is hydrolised with formation of 5-amino levulinic acid hydrochloride.",
"Further processes, which start from succinic acid, are described by A. Pfaltz, Tetrahedron Lett.",
"1984, 25, 2977-2980 and A. Nudelman, A. Nudelman, Synthesis 1999, 5, 568-570.Unfortunately, the single workup steps of these reaction sequences are expensive and result in side products, which have to be seperated.",
"For the preparation of 5-amino levulinic acid hydrochloride according to the processes described here, toxic chemicals (i.e.",
"zinc, copper cyanide) and solvents (i.e.",
"γ-picoline) are used.",
"The toxic side products, built up in parts of the synthesis, have to be disposed.",
"As well, pyridine derivatives are used as starting material for the synthesis of 5-amino levulinic acid hydrochloride.",
"Starting from 2,5-dihydroxy pyridine, C. Herdeis et al.",
"(Arch.",
"Pharm.",
"1984, 317, 304-306) obtained a product by oxidation.",
"Subsequent catalytic hydrogenation yielded 2,5-piperidone, which was converted into 5-amino levulinic acid hydrochloride by acidic hydrolysis.",
"H. Takeya, K. Suzuki, K. Sasaki, Nippon Kagaku Kaishi 1999, 355-358 use 1,5-dihydroxy 2-pyridone as starting material for the preparation of 5-amino levulinic acid hydrochloride.",
"Concerning these reaction pathways, both oxidation and reduction steps are included.",
"The poor yields reached in both processes be in an inconvenient proportion to the expense of the processes.",
"Besides, the provision of large amounts of the needed pyridine derivatives turn into difficult.",
"The processes for the production of 5-amino levulinic acid hydrochloride, listed in the following, are starting with cheap and in large quantities available levulinic acid (production on a ton-scale: J. J. Bozell, L. Moens, D. C. Elliott, Y. Wang, G. G. Neuenschwander, S. W. Fitzpatrick, R. J. Bilski, J. L. Jarnefeld, Res.",
"Cons.",
"Recyc.",
"2000, 28, 227-239).",
"In U.S. Pat.",
"No.",
"5,987,058, methyl 5-bromo levulinate, which is available by bromination of levulinic acid, is transferred to methyl 5-N,N-diformylamino levulinate.",
"Subsequent acid catalysed hydrolysis yields 5-amino levulinic acid hydrochloride.",
"Alternative processes, starting with methyl 5-bromo levulinate have been described.",
"In these processes the bromine at C-5 is exchanged by a nucleophilic nitrogen (potassium phthalimide: E. Benedikt, H.-P. Kost, Z. Naturforsch.",
"1986, 41b, 1593-1594; conversion with sodium azide: H.-J.",
"Ha, S.-K. Lee, Y.-J.",
"Ha, J. W. Park, Synth.",
"Comm.",
"1994, 24(18), 2557-2562).",
"In case of the substitution by an azide, the methyl 5-azido levulinate is obtained as an heavy oil.",
"In view of the further conversion to 5-amino levulinic acid hydrochloride by catalytic hydrogenation and simultaneous ester hydrolysis, it has to be guaranteed, that the azido ester is supplied in pure form.",
"Distillation of the azido ester without any decomposition is not possible and other purification steps, i.e.",
"chromatographic processes, are very expensive, and therefore not recommendable for the technical scale.",
"Further disadvantage of the last described synthetic pathways lie on the difficult handling of methyl 5-bromo levulinate.",
"This compound is a liquid with strong lachrymatory properties.",
"The compound is a strong skin irritant and further, in the presence of traces of acid, it has a tendency to the acid catalysed isomerisation into the compounds methyl 3-bromo-, methyl 3,5-dibromo- and methyl 5-bromo levulinate.",
"This circumstance complicates the storage of the compound.",
"The substitution of bromine in methyl 5-bromo levulinate by an alkali metal imide or alkali azide yields sodium bromide as side product, which moderate to good solubility in organic solvents, especially lower alkanols, is well-known.",
"The production of sodium bromide free 5-amino levulinic acid hydrochloride therefore require partially expensive purification steps.",
"In particular in view of the use of 5-amino levulinic acid hydrochloride for medical purposes it must be secured, that the drug is very pure and free as less as possible from impurities deriving from inorganic salts.",
"A reaction pathway has to be chosen, which yields a pure product by a simple transformation starting with a pure starting material.",
"Side reactions, whereby side products could be formed, and which have to be separated from the major product, should not occur.",
"In comparison to processes, that don't start with levulinic acid, the functionalisation with bromine at the C-5-position of levulinic acid offers the advantage, that 5-amino levulinic acid hydrochloride is available without any problems only in two steps starting with pure methyl 5-bromo levulinate.",
"If pure reagents are used, no organic side products arise, which affect the further reaction pathway and have to be separated expensive.",
"As side products, only potassium bromide and in case of the hydrolysis of methyl 5-phthalimido levulinate and methyl 5-N,N-Diformyl levulinate, phthalic acid and formic acid, respectively, and methanol are build up, the disposition of which is unproblematic.",
"A reaction pathway, starting with pure levulinic acid derivatives like 5-bromo levulinic acid and particulary 5-chloro levulinic acid, transformation of these carboxylic acids in the next step by substitution of the halogen atom with an inorganic azide into 5-azido levulinic acid, and at least reduction of the azido functionality in 5-azido levulinic acid to the amino funtionality, is unknown so far.",
"The carboxylic acids 5-bromo- and 5-chloro levulinic acid are only difficult available on the classic pathway by means of bromination or chlorination of levulinic acid because of the lack of regioselectivity of the halogenation reaction.",
"The desired levulinic acid derivatives are available in only poor yields.",
"But both compounds have the advantageous properties, that they are crystalline, non-lachrymatory and storable.",
"The transformation of 5-chloro levulinic acid with potassium azide is especially advantegeous, because potassium chloride is the only harmless side product formed.",
"It is therefore an object of the present invention to provide a compound and a process for the preparation of this compound, while the employment of this compound for the preparation of 5-amino levulinic acid hydrochloride overcomes the disadvantages mentioned above.",
"This object is achieved by the provision of 5-azido levulinic acid.",
"5-Azido levulinic acid is a so far unknown compound and a new starting compound for the preparation of 5-amino levulinic acid hydrochloride.",
"Further object of the present invention is the use of 5-azido levulinic acid as starting compound for the cost-effective and high-yield preparation of 5-amino levulinic acid hydrochloride of pharmaceutical purity on a technical scale.",
"The provision of 5-azido levulinic acid allows the creation of an improved production process of 5-amino levulinic acid hydrochloride.",
"The process makes the cost-effective and hield-yield production of 5-amino levulinic acid hydrochloride of pharmaceutical purity possible.",
"According to the invention this object is achieved by a process, which makes the preparation of 5-amino levulinic acid hydrochloride in pharmaceutical purity and in a four-step-process with an overall yield of 31-35% possible.",
"According to the inventive process, cost-effective starting compounds are used, and products, which have to be disposed are only hydrobromic acid, methanol and potassium chloride.",
"Surprisingly, a simple way for the preparation of 5-chloro levulinic acid from methyl 5-bromo levulinate was found.",
"Methyl 5-bromo levulinate, which is obtained by bromination of levulinic acid in methanol in a known manner, reacts quantitatively with aqueous hydrochloric acid under ester hydrolysis and simultaneous bromine/chorine-exchange at position C-5 to give 5-chloro levulinic acid.",
"This transformation is unknown so far.",
"This compound represents an unlimited storable and excellently cristallising solid (m.p.",
"75° C.), which posesses no lachrymatory properties.",
"This compound is better suitable for the transformation with a nucleophilic nitrogen like sodium azide, because of the only side product formed is sodium chloride.",
"Sodium chloride itself is practically unsolulable in organic solvents and nontoxic.",
"In the following step, the so obtained 5-chloro levulinic acid will be dissolved in a suitable solvent and stirred with one equivalent of a suitable azide, in particular sodium azide.",
"In particular acetone, but if required also other organic solvents, such as dipolar aprotic solvents, substituted und unsubstituted amides, cylic and acyclic ethers and the like, may in principle be used as solvent for the reaction of the 5-chloro levulinic acid with a suitable azide.",
"However, the work with other solvents having a comparable dissolution behaviour, in particular solvents of high polarity, should not be ruled out.",
"As a new key compound, the so far unknown 5-azido levulinic acid arises in quantitative yield by means of nucleophilic substitution.",
"This new key compound represents a colorless, crystalline (m.p.",
"70-71° C.) and an unlimited and without any decomposition storable compound, if prepared according to the following process.",
"The substance contains no inorganic material, it is not sensitive to impact, and it can be safely handled at room temperature.",
"Because of an uncomplete bromine/chlorine-exchange, 5-chloro levulinic acid may obtain as many as 8% of 5-bromo levulinic acid.",
"But, the presence of small amounts of 5-bromo levulinic acid does not disturb the further reaction pathway, because this compound also reacts with a suitable inorganic azide, in particular sodium azide, in acetone as solvent with clean formation of 5-azido levulinic acid.",
"The conversion of 5-bromo levulinic acid, which was prepared on a different pathway, with sodium azide in acetone, after crystallisation from an organic solvent resulted in the formation of 5-azido levulinic acid in nearly quantitative yield.",
"The compound was free of inorganic salts.",
"In the following step, the key compound 5-azido levulinic acid will be reduced to 5-amino levulinic acid hydrochloride without any difficulties.",
"The reduction is preferably carried out as a catalytic hydrogenation in the presence of a metal catalyst (preferably palladium or platinum on a suitable carrier, such as active carbon) in aqueous hydrochloric acid without any arise of undesireable organic by-products.",
"The catalyst can be regenarated.",
"5-Amino levulinic acid hydrochloride is obtained absolutly pure with an overall yield of 31-35% (starting from levulinic acid).",
"According to the present invention, the problem is solved by making 5-azido levulinic acid available, and providing a process for its preparation.",
"The preparation process of 5-azido levulinic acid implies the conversion of methyl 5-bromo levulinate and/or methyl 5-chlor levulinate with aqueous hydrochloric acid.",
"As a result of an incomplete bromine/chlorine-exchange at the C-5 postion, a mixture of 5-chloro- and 5-bromo levulinic acid is obtained.",
"The obtained mixture of 5-chloro- and 5-bromo levulinic acid, 5-chloro levulinic acid, and pure 5-bromo levulinic acid will be transferred into 5-azido levulinic acid by conversion of the compounds with an nucleophilic azide.",
"It is preferred, according to the present invention, to carry out the conversion with an azide in a polar solvent, especially acetone, an C1-C4-alkanol or water as the reaction medium.",
"Furthermore, according to the present invention, the problem is solved by using 5-azido levulinic acid for the preparation of 5-amino levulinic acid hydrochloride, where 5-azido levulinic acid is transferred to 5-amino levulinic acid hydrochloride by catalytic hydrogenation.",
"In particular it is preferred to carry out the catalytic hydrogenation in aqueous hydrochloric acid, to remove the hydrogenation catalyst, to remove the solvent and excess hydrochloric acid by distillation, and to obtain 5-amino levulinic acid hydrochloride by crystallisation from 2-propanol or tert-butyl methylether/methanol.",
"Surprisingly it was found also, that 5-azido levulinic acid posseses explosive properties.",
"Because of its chemical constitution, only nontoxic gases, namely nitrogen and carbon oxide, will be released as reaction substances.",
"The released amount of gas is very large and will be increased by the exothermic reaction during explosion.",
"5-azido levulinic acid is sensitive to impact and detonates already at an impact energy of 40 J.",
"For this reason, 5-azido levulinic acid is especially suitable as priming fuse and as explosive for the operation of airbags in the motor vehicle industry.",
"The following examples explain the invention.",
"EXAMPLE 1 Preparation of 5-Chloro levulinic acid: A solution of 1 g (4.78 mmole) of methyl 5-bromo levulinate in 20 ml of 3 M hydrochloric acid was stirred at 70° C. until the reaction was finished (24 h, 1H-NMR control spectra showed only product signals).",
"The solvent, excess hydrochloric acid, hydrobromic acid, and methyl alcohol were removed by distillation in in vacuo.",
"To the residue 20 ml of methylene chloride was added and the resulting solution was dried over sodium sulfate.",
"Evaporation of the solvent yielded 702 mg of a pale yellow solid.",
"The solid was dissolved in 2 ml of tert-butyl methylether.",
"Petroleum ether (40-60° C.) was added dropwise until crystallisation occured.",
"The mixture was stirred for 0.5 h at ambient temperature and the crystals were filtered off.",
"Yield: 684 mg, 95% Melting point: 75° C.; an x-ray analysis study of the crystals confirms the structure of the compound.",
"Elemental analysis: Calcd.",
"C 39.89, H 4.69; Found C 39.35, H 4.67.IR (KBr): ν (cm−1)=3500-2100, 1700 (C═O), 1440, 1410, 1370, 1330, 1290, 1210, 1140, 1090, 1030, 950, 865, 765, 730, 610.1H-NMR (CDCl3, 200 MHz): δ (ppm)=2.70 (t, 3J=6.7 Hz, 2H), 2.96 (t, 3J=6.7 Hz, 2H), 4.14 (s, 2H), 8.8 (br.",
"s, 1H).",
"13C-NMR (CDCl3, 50.3 MHz): δ (ppm)=27.7 (HOOC—CH2), 34.0 (CH2—CH2—C═O), 48.0 (CH2—Cl), 178.4 (COOH), 201.2 (C═O).",
"EXAMPLE 2 Preparation of a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid: A mixture of 1 g (4.78 mmole) of methyl 5-bromo levulinate in 20 ml of 3 M hydrochloric acid was stirred for 12 h at 70° C. The solvent, excess hydrochloric acid, hydrobromic acid, and methyl alcohol was removed by distillation in vacuo.",
"To the residue 20 ml of methylene chloride was added and the resulting solution was dried over sodium sulfate.",
"Evaporation of the solvent yielded 740 mg of a pale yellow solid.",
"The solid was dissolved in 2 ml of tert-butyl methylether.",
"Petroleum ether (40-60° C.) was added dropwise until crystallisation occured.",
"The mixture was stirred for 0.5 h at ambient temperature and the crystals filtered off.",
"Yield: 700 mg, 95% 1H-NMR: After integration of the 5-CH2-proton signals of the mixture of 5-chloro levulinic acid and 5-bromo levulinic acid a ratio of 92:8 was found.",
"EXAMPLE 3 Preparation of 5-azido levulinic acid from 5-chloro levulinic acid: 5-Chloro levulinic acid (prepared as described in Example 1) and sodium azide were reacted with one another in a molar ratio of 1:1 in acetone.",
"The reaction mixture was stirred at 50° C. until the end of the reaction (1H-NMR control spectra showed only product signals).",
"To the reaction mixture 20 ml of methylene chloride was added, the solid was filtered off, the filtrate was washed with 3 M aqueous sodium chloride and dried over sodium sulfate.",
"Evaporation of the solvent yielded a pale yellow oil in quantitative yield.",
"Crystalisation of the residue from methylene chloride/petroleum ether (40-60° C.) yielded pale yellow needles consisting of 5-azido levulinic acid.",
"Yield: 700 mg, 95% Melting point: 70-71° C. Elemental analysis: Calcd.",
"C, 38.22; H, 4.49; N, 26.74; Found C, 38.39; H, 4.59; N, 26.07.IR (KBr): ν (cm−1)=3350-2350, 2090 (N3), 1725 (C═O), 1415, 1400, 1370, 1340, 1285, 1260, 1230, 1170, 1080, 1040, 1000, 930, 885, 830, 800, 685, 630.1H-NMR (CDCl3, 200 MHz): δ (ppm)=2.70 (s, 4H), 4.02 (s, 2H), 8.3 (br.",
"s, 1H).",
"13C-NMR (CDCl3, 50.3 MHz): δ (ppm)=27.4 (HOOC—CH2), 34.1 (CH2—CH2—C═O), 57.5 (CH2—N3), 177.3 (COOH), 202.7 (C═O).",
"EXAMPLE 4 Preparation of 5-azido levulinic acid from a mixture of 5-chloro levulinic acid and 5-bromo levulinic acid: A mixture of 5-chloro and 5-bromo levulinic acid (0.5 g, prepared as described in Example 2) and sodium azide were reacted with one another in a molar ration of 1:1 in acetone.",
"The reaction mixture was stirred at 50° C. until the end of the reaction (1H-NMR control spectra showed only product signals).",
"An amount of 20 ml of methylene chloride was added, the solid was filtered off, the filtrate was washed with 3 M aqueous sodium chloride and dried over sodium sulfate.",
"Evaporation of the solvent yielded a yellow oil in quantitative yield.",
"Crystalisation of the residue from methylene chloride/petroleum ether (40-60° C.) yielded pale yellow needles consisting of 5-azido levulinic acid.",
"Yield: 510 mg, 95%; the physical and spectroscopic data are in agreement with those obtained from the material prepared according to Example 3.EXAMPLE 5 Preparation of 5-azido levulinic acid from 5-bromo levulinic acid: 5-bromo levulinic acid (prepared as described in Example 7) was dissolved in acetone.",
"To the resulting solution was added sodium azide to a molar ratio of 1:1 (5-bromo levulinic acid:sodium azide) and the reaction mixture was stirred at 50° C. until the end of the reaction (1H-NMR control).",
"To the reaction mixture 20 ml of methylene chloride was added, the solid was filtered off, the filtrate was washed with 3 M aqueous sodium chloride and dried over sodium sulfate.",
"Evaporation of the solvent yielded a pale yellow oil in quantitative yield.",
"Crystalisation of the residue from methylene chloride/petroleum ether (40-60° C.) yielded pale yellow needles consisting of 5-azido levulinic acid.",
"Yield: 390 mg, 97% Melting point: 70-71° C.; the physical and spectroscopic data are in agreement with those obtained from the prepared according to Example 3.EXAMPLE 6 Preparation of 5-amino levulinic acid hydrochloride by hydrogenation of 5-azido levulinic acid: 5-azido levulinic acid otained according to Example 3 was dissolved in 3 M hydrochloric acid, a hydrogenation catalyst (palladium on carbon) was added and the reaction mixture was hydrogenated for 5 h while passing in hydrogen at a pressure of 6 bar.",
"By monitoring the hydrogenation it was found that the hydrogenation was completed quantitatively after 5 h (1H-NMR control spectra showed only product signals).",
"The catalyst was filtered off from the clear and colorless solution, and the reaction medium, hydrochloric acid, was removed by distillation in vacuo.",
"To the resulting viscous, colorless residue 4 ml of 2-propanol was added while stirring.",
"Alternatively, the residue was dissolved in 5 ml of methanol and an amount of 5 ml of tert-butyl methylether was added while stirring.",
"In both cases colorless crystals were formed after a short time, which were filtered off.",
"The obtained crystals were washed with acetone and dried in vacuo.",
"Yield: 709 mg, 95% Melting point: 150-151° C.; melting point data of various producers: 144-151° C.; Merck: 150-156° C.; Fluka: ˜150° C. (decomposition); Aldrich: 156° C. (decomposition); Acros Organics 156-158° C. (decomposition).",
"The NMR data (A. Nudelman and A. Nudelman, Synthesis 1999, 4, 568-570) and chromatographic data (C. Herdeis, A. Dimmerling, Arch.",
"Pharm.",
"1984, 317, 304-306) are in agreement with those given in the literature.",
"EXAMPLE 7 Preparation of 5-bromo levulinic acid: Levulinic acid (5.0 g, 43.1 mmole) was dissolved in 50 ml of carbon tertrachloride in a three-necked flask with a mechanical stirrer, a dropping funnel, a reflux condenser, and an internal thermometer.",
"To the reaction mixture bromine (6.88 g, 43.1 mmole) was added dropwise during 0.5 h at ambient temperature.",
"After decoloration of the orange reaction mixture 50 ml of water were added.",
"The organic layer was separated, washed with brine, and dried over magnesium sulfate.",
"Evaporation of the solvent yielded a yellow oil.",
"From 50 ml of ether/petroleum ether (40-60° C.) colorless crystals are formed consisting of pure 5-bromo levulinic acid.",
"Yield: 900 mg, 9.5% Melting point: 79-80° C. IR (KBr): ν (cm−1)=3500-2100, 1700 (C═O), 1430, 1400, 1350, 1300, 1280, 1240, 1100, 1050, 970, 920, 860, 760, 720, 610.1H-NMR (CDCl3, 200 MHz): δ (ppm)=2.70 (t, 3J=6.7 Hz, 2H), 2.96 (t, 3J=6.7 Hz, 2H), 3.95 (s, 2H), 8.5 (br.",
"s, 1H).",
"13C-NMR (CDCl3, 50.3 MHz): δ (ppm)=28.0 (HOOC—CH2), 34.1 (CH2—CH2—C═O), 33.9 (CH2—Cl), 178.0 (COOH), 200.4 (C═O)."
]
] | Patent_10250708 |
[
[
"Medical metal implants that can be decomposed by corrosion",
"An in vivo, decomposable medical implant is provided and comprises a metal material that contains, as a main alloying constituent, tungsten or a metal from the group rhenium, osmium, and molybdenum.",
"The method for decomposing the implant, via corrosion in a bio system, includes the step of changing the pH level of the bio system, at least at the site of the implant, from a corrosion-inhibiting level to a corrosion-promoting level."
],
[
"1-11.",
"(cancelled) 12.An in vivo, decomposable medical implant from the group including: stents (coronary stents, peripHeral stents, tracheal stents, bile duct stents, esopHagus stents), surgical clips, osteosynthesis material, biological matrix (foam), metal wiring, metal threads, active substance depots, comprising a metal material, wherein said material contains, as a main alloying constituent, tungsten or a metal selected from the group consisting of rhenium, osmium and molybdenum.",
"13.A medical implant according to claim 12, wherein said material contains, as a secondary constituent, at least one element selected from the group consisting of lanthanides, actinides, iron, osmium, tantalum, platinum, gold, rhenium, gadolinium, yttrium and scandium.",
"14.A medical implant according to claim 13, wherein said lanthanide is cerium.",
"15.A medical implant according to claim 12, wherein said main alloying constituent represents more than 75% of said material, with any remainder, to form 100%, being formed by at least one secondary constituent.",
"16.A medical implant according to claim 15, wherein said main alloying constituent represents 95 to 99.5% of said material.",
"17.A medical implant according to claim 12, wherein said material has a crystalline structure having a particle size of 0.5 to 30 μm.",
"18.A medical implant according to claim 17, wherein said particle size is 0.5 to 5 μm.",
"19.A medical implant according to claim 12, wherein said implant, with the exception of said material, contains metal or non-metal inclusions that comprise an essentially pure alkali or alkaline earth metal, a drug, mRNA or a vector.",
"20.A medical implant according to claim 12, wherein said implant has an essentially tubular base.",
"21.A method for decomposition of the implant of claim 12 via corrosion in a bio system, including the step of: changing the pH level of the bio system, at least at a site of the implant, from a corrosion-inhibiting level to a corrosion-promoting level.",
"22.A method according to claim 21, wherein within the vicinity of a cardiovascular system, the pH level of said bio system is changed to a level of at least 7.4.23.A method according to claim 22, wherein the pH level of said bio system is changed to a level of at least 7.5.24.A method according to claim 22, wherein the pH level of said bio system is changed to a level of at least 7.6.25.A method according to claim 21, wherein within the vicinity of a urine or bio system, the pH level of said bio system is changed from a lower pH level to a higher pH level.",
"26.A method according to claim 21, wherein the pH level of said bio system is changed by supplying or stopping alkalizing or acidifying substances.",
"27.A method according to claim 26, wherein said alkalizing or acidifying substances are at least one of the group consisting of ascorbic acid, sodium bicarbonate, citrates, and diuretics.",
"28.A method according to claim 21, wherein the pH level of said bio system is changed by supplying or stopping drugs that alkalize said bio system.",
"29.A method according to claim 28, wherein said drugs are loop diuretics."
],
[
"The present invention relates to a medical implant that can be decomposed in vivo comprising a metal material and belonging to the group of implants in accordance with the preamble of claim 1.Such an implant is known from DE 19731021.In the case of these implants practically at the same time as the implantation a corrosive action is started, which after a certain time leads to the fact that the implant firstly becomes mechanically unstable and then is completely decomposed.",
"With these implants the material is to be selected in such a way that the corrosion proceeds slowly in order to ensure mechanical stability is maintained to the necessary extent.",
"This also results in correspondingly slow decomposition of the implant material, after this implant has fulfilled its function.",
"In practice complete decomposition will need many times the period, for which the mechanical function should continue to remain in place.",
"Furthermore a process is known for producing so-called coils as vessel sealing systems from a tungsten alloy, which can corrode.",
"Precisely in the case of vessel sealing systems is decomposition of the implant not desirable, in particular if the implantation has not resulted in the vessel closure aimed for.",
"The object therefore of the present invention is to generally improve an implant from the group specified in the preamble of claim 1 so that the mechanical stability of the implant remains in place for as long a period as necessary and after the mechanical function has been fulfilled; corrosion enables the decomposition of the implant to be accelerated.",
"Another object of the present invention is to produce an active substance depot, which allows the active substance to be released in a deliberately controllable way.",
"This object is achieved by an invention with the features of claim 1.Another object of the present invention is to provide a process for the decomposition of a metal implant, which enables the rate of the corrosive decomposition of the implant to be controlled in a purposeful manner.",
"This object is achieved by a process with the features of claim 7.Because it is proposed in the case of the implant embodying the invention that the material contains tungsten as the main alloy or a metal from the group including rhenium, osmium, molybdenum, the implant in the biological environment, for the use of which it is intended, will exhibit corrosion behavior dependent on the pH level, whereby the transition from a non-corrosive condition to a corrosive condition occurs at a pH level, which can be tolerated by the respective biological system.",
"In particular the transition to the corrosive condition is influenced by a process controlling the pH level in the biological system.",
"In the case of the active substance depot the release of the active substance is assisted by the change in the pH level from the corrosion-inhibiting condition to the corrosion-promoting condition.",
"With the process according to the invention decomposition of the metal implant is induced as a result of the pH level of the bio system being changed at least at the place of the implant from the corrosion-inhibiting level to a corrosion-promoting level.",
"Secondary constituents in this case can be a multiplicity of elements, which may also have no influence on the corrosion behavior.",
"With the implant it is advantageous however if the material as the secondary constituent contains one or more elements from the group of lanthanides, in particular cerium, actinides, iron, osmium, tantalum, rhenium, gadolinium, yttrium or scandium.",
"With these alloying elements good corrosion behavior can be achieved for the intended purpose desired.",
"In this case typical compositions are formed so that the main alloying constituent represents more than 75%, in particular 95% to 99.5%, of the material and the remainder to reach 100% is formed from the at least one secondary constituent.",
"Particularly fast decomposition within a certain pH range is possible if the material exhibits a crystalline structure with a particle size of 0.5 μm to 30 μm, in particular 0.5 μm to 5 μm.",
"Then extensive corrosion takes place.",
"However with particle sizes of 10 μm or more inter-crystalline corrosion can also take place, which leads to formation of particles, whereby the body can exude these particles.",
"In addition it is advantageous if the implant contains metal or non-metal inclusions having the nature of sintered metal, which comprise essentially pure alkali or alkaline earth metal, except the alloy material.",
"These inclusions can promote deliberate corrosion in regard to both the start and rate of corrosion.",
"In addition alkali or alkaline earth ions released as a result of corrosion may become physiologically effective in an advantageous way.",
"There results an embodiment advantageous in regard to mechanical stability with good corrosion if the implant has an essentially tubular base.",
"With the process according to the invention the object of changing the pH level of the bio system at least in the place of the implant from the corrosion-inhibiting level to a corrosion-promoting level is achieved.",
"As a result after the implant has fulfilled its mechanical function, fast corrosion can be influenced in a concerted way.",
"In this case advantageously the pH level of the bio system within the vicinity of the cardiovascular system can be increased to a pH level of 7.4 or higher, preferably to a pH level of more than 7.5 and in particular more than 7.6.Likewise it can be advantageous if the pH level of the bio system within the vicinity of the urine or bile system is changed, whereby in the urine system for example the pH level can be raised to over 9 or reduced to levels below 7.The change in the pH level for the promotion of corrosion is advantageously achieved if alkalizing or acidifying substances are supplied to or taken away from the bio system, in particular ascorbic acid, sodium bicarbonate, citrate and/or diuretics (for example frusemide, thiazide, carboanhydrase inhibitors).",
"An advantageous embodiment of the present process proposes that the pH level of the bio system is changed by supplying or stopping drugs alkalizing the bio system, in particular loop diuretics.",
"An embodiment of the present invention is described below.",
"A cardiovascular stent is manufactured with a tubular base from tungsten or a tungsten alloy in the presently known way.",
"The stent is introduced into a restricted blood vessel and is expanded in the region of the vessel restriction.",
"The stent remains there until the vessel has regained sufficient natural stability.",
"Up to this point the pH level in the blood of the patient is maintained at a level of <7.4 by regular administration of ascorbic acid.",
"As soon as it is decided that the support function of the stent is no longer needed, administration of ascorbic acid is stopped and the blood of the patient is alkalized to a pH level of above 7.4 by administering diuretics.",
"In the changed environment the stent will corrode fairly quickly.",
"Relatively fast decomposition of the material results, whereby the material disposed in the blood vessel leads to fast removal of the tungsten particles or tungsten ions arising and thus prevents local build up of any toxic concentration.",
"The material used in this embodiment is an alloy consisting of 99.2% tungsten and 0.8% cerium with a particle size of approximately 1 μm.",
"In this case extensive corrosion, the decomposition rate of which at a pH level of 7.2 amounts to 20 μm per annum, results in the human bloodstream.",
"By increasing the pH level to 7.4 the decomposition rate rises to 50 μm per annum.",
"In the case of a second embodiment an active substance depot is produced from a tungsten alloy, whereby active substances materials having the nature of sintered metal with therapeutically effective characteristics (metal ions, drugs, mRNA, vectors) are introduced into the alloy material.",
"The implant is disposed in a position of the bio system, which can be treated outside the bloodstream.",
"As in the previous embodiment the bio system is firstly kept at a relatively low pH level by administering ascorbic acid or similar active substances.",
"As soon as the active substances are needed, an alkalizing substance is administered so that the pH level rises.",
"The initial corrosion releases the therapeutically effective material and since it is disposed outside the bloodstream, leads to high local concentration of active substance, which is therapeutically effective without impairing the rest of the bio system.",
"In this way tumors, vessel restrictions can be fought by intima proliferation, other vessel reactions such as fibrosis, but also infections or similar can be fought by concerted selectable local and systemic active substance dosages.",
"The same applies to implants in the urinary tracts or bile ducts, whereby for controlling the active substance release in the case of these applications a broader pH spectrum is available.",
"Here it is proposed according to a further embodiment that a urinary tract stent is made from an alloy consisting of 98.5% molybdenum and 1.5% tantalum.",
"This stent is stable at a pH level of more than 2, while by changing the pH level to below 2 the corrosive decomposition is accelerated.",
"Apart from tantalum platinum and gold are also possible here as secondary constituents.",
"As in the first embodiment surgical clips, metal sutures or the like can also be maintained in place until they have fulfilled their function.",
"Afterwards the corrosion and thus decomposition of the material can be induced by deliberately changing the pH level."
]
] | Patent_10250784 |
[
[
"Piezoelectric actuator",
"An apparatus for actuating a brake comprising force transmission means for transmitting force to a friction pad of a brake, at least one piezo-electric device operable when energised to change shape and/or size so as to apply a force to the force transmission means in a direction for actuating the brake, and retaining means operable to resist movement of the force transmission means in an opposite direction"
],
[
"1.An apparatus for actuating a brake comprising: a force transmission means that transmits force to a friction pad of a brake; at least one piezo-electric device operable when energized to change at least one of shape and size so as to apply a force to the force transmission means in a first direction for actuating the brake; and a retaining means operable to resist movement of the force transmission means in a second direction opposite the first direction, wherein the retaining means is a ratchet device.",
"2.An apparatus as defined in claim 1, wherein, wherein the ratchet device comprises a mechanical device.",
"3.An apparatus as defined in claim 2, wherein the mechanical device comprises a ratchet member and a biasing means that biases the ratchet member into engagement with the force transmission means for resisting movement thereof.",
"4.An apparatus as defined in claim 3, wherein the ratchet member has a sharp edge for engaging the force transmission means.",
"5.An apparatus as defined in claim 3, wherein expansion of the piezo-electric device causes the ratchet member to move out of engagement with the force transmission means, and wherein contraction of the piezo-electric device permits the biasing means to move the ratchet member into engagement with the force transmission means.",
"6.An apparatus as defined in claim 1, wherein the apparatus comprises a support, wherein a portion of the force transmission means passes through an opening in the support, the piezo-electric device being interposed between said portion and the support to act against the support when applying force to the force transmission means.",
"7.An apparatus as defined in claim 1, wherein the apparatus further comprises at least one oppositely-acting piezo-electric device operable to apply force to move the force transmission means in the second direction.",
"8.An apparatus as defined in claim 7, wherein the ratchet device comprises a ratchet member and a biasing means that applies a biasing force to bias the ratchet member into engagement with the force transmission means so as to resist movement thereof, wherein the at least one oppositely-acting piezo-electric device is also operable to overcome the biasing force.",
"9.An apparatus for actuating a brake comprising force transmission means for transmitting force to a friction pad of a brake, a structural element, a shaft of the force transmission means passing through an opening in the structural element, and a plurality of piezo-electric devices being interposed between the shaft and the structural element, such that when energized, the piezo-electric devices change shape and/or size so as to act against the structural element and cause a force to be applied to the force transmission means in a direction for actuating the brake, and in a tangential and/or radial direction with respect to the shaft, the piezo-electric devices further being operable to resist movement of the force transmission means in an opposite direction.",
"10.An apparatus for actuating a brake as defined in claim 9, wherein the piezo-electric devices are operable to apply a force to move the force transmission means in the opposite direction.",
"11.An apparatus for actuating a brake as defined in claim 9, wherein sub-sets of the piezo-electric devices are arranged to change size and/or shape when energized so as to act against a radially inner surface of the structural element, the sub-sets of piezo-electric devices capable of being repeatedly energized in sequence so as to apply a large angle of rotation or sustained application of torque to the shaft.",
"12.An apparatus for actuating a brake as defined in claim 9, wherein some of the plurality of piezo-electric devices are arranged to provide axial force to the shaft, and others arranged to provide torque to the shaft, selective expansion of the piezo-electric devices enabling a combined axial and rotational force to be applied to the shaft.",
"13.An apparatus for actuating a brake as defined in claim 9, wherein a first group of the plurality of piezo-electric devices are arranged to provide a lateral force in a first radial direction, and a second group of the plurality of piezo-electric devices are arranged to provide a lateral force in a second radial direction perpendicular to the first radial direction.",
"14.An apparatus for actuating a brake, comprising: a plurality of piezo-electric devices, each device being associated with a respective one of a plurality of device groups, wherein the devices in each group are energizable together; and an oscillating power supply connected to the plurality of piezo-electric devices, wherein the power supply has a phase difference between power supplied to associated groups, to change size and/or shape in groups; sequentially; to apply force directly to a brake pad so as to actuate a brake.",
"15.An apparatus for actuating a brake as defined in claim 14, wherein each group of piezo-electric devices is arranged to apply a force to an associated area of the brake pad so as to vary the force across the brake pad."
],
[
"The present invention relates to an apparatus for actuating a brake.",
"A known vehicle brake arrangement comprises a disc fixed for rotation with a wheel and a brake clamping mechanism comprising a tappet which is mechanically actuated to bring brake pads into contact with the disc and to apply a force between the pads and the disc to provide frictional braking.",
"Piezo-electric devices which change size and/or shape when energised by application of an electric current are known for applying a mechanical actuating force over a short distance.",
"According to the present invention there is provided an apparatus for actuating a brake comprising force transmission means for transmitting force to a friction pad of a brake, at least one piezo-electric device operable when energised to change shape and/or size so as to apply a force to the force transmission means in a direction for actuating the brake, and retaining means operable to resist movement of the force transmission means in an opposite direction.",
"Preferably, the retaining means is a ratchet device.",
"The ratchet device may comprise a mechanical device.",
"The mechanical device may comprise a ratchet member and a biasing means biasing the ratchet member into engagement with the force transmission means for resisting movement thereof.",
"The ratchet member may have a sharp edge for engaging the force transmission means.",
"Preferably, expansion of the piezo-electric device causes the ratchet member to move out of engagement with the force transmission means, and contraction of the piezo-electric device permits the biasing means to move the ratchet member into engagement with the force transmission means.",
"Conveniently, the apparatus comprises a support and a portion of the force transmission means passes through an opening in the support, the piezo-electric device being interposed between said portion and the support so as to act against the support when applying force to the force transmission means.",
"Conveniently, the piezo-electric device extends circumferentially around said portion.",
"The piezo-electric device may comprise a plurality of piezo-electric members arranged around said portion.",
"Preferably, the apparatus comprises at least one oppositely-acting piezo-electric device operable to overcome the biasing force.",
"This permits movement of the force transmission means in the opposite direction for releasing the brake.",
"Preferably, the oppositely-acting piezo-electric device is also operable to apply force to move the force transmission means in the opposite direction.",
"Alternatively, the ratchet device comprises at least one further piezo-electric device operable to change shape and/or size so as to continue the application of force to the force transmission means in the direction for actuating the brake.",
"Conveniently, the apparatus comprises a structural element and a shaft of the force transmission means passes through an opening in the structural element, the or each piezo-electric device being interposed between the shaft and the structural element such that, when energised, the or each piezo-electric device changes shape and/or size so as to act against the structural element and cause force to be applied to the force transmission means in at least one direction.",
"The direction or directions may be axial and/or tangential and/or radial with respect to the shaft.",
"Although the distance that the piezo-electric device moves is small, repeated expansion and contraction of the piezo electric device in conjunction with the ratchet device facilitates sustained application of force and/or much greater movement of the force transmission means.",
"Alternatively, the apparatus comprises a plurality of piezo-electric devices, each device being associated with a respective one of a plurality of device groups, the devices in each group being energisable together, by connection to an oscillating power supply with a phase difference between the supply to associated groups, to change size and/or shape in groups, sequentially, thereby to apply force for actuating a brake.",
"Braking force may be applied continuously by making use of dynamic response characteristics of piezo-electric devices.",
"Phased expansion of groups permits force applied to be sustained at close to the maximum available from each group because as force applied by a first group starts to diminish, force applied by a second group approaches the maximum.",
"The piezo-electric device may be operable to be energised by application of an electric voltage controlled by a controller, for example a microprocessor.",
"The controller may be operable to respond to electrical signals fed back to it from the brake so as to adjust the level of the voltage applied to the piezo-electric device.",
"Preferably, the controller is also operable to adjust the released position of the force transmission means so as to compensate for wear of the brake pads.",
"Piezo-electric devices have a rapid response to application of an energising voltage.",
"This facilitates the provision of high frequency application and removal of voltage to provide rapid actuation of the brake.",
"Frictional braking results in the generation of heat, and piezo-electric elements may be selected which retain their operating characteristics over a wide temperature range.",
"The apparatus described above allows electrical energy to be transformed directly into brake clamping force.",
"A relatively small number of components is required, resulting in a compact, light-weight brake actuator assembly, and reducing manufacturing costs.",
"Energy usage is low as there are few moving components when compared, for example, with electric motor actuators.",
"The use of electrical voltages facilitates rapid and accurate control.",
"Embodiments of the invention will now be described by way of example, with reference to the accompanying drawings in which: FIG.",
"1 is a schematic representation of a portion of a brake having an actuator according to a first embodiment; FIG.",
"2 shows a detail of the actuator of FIG.",
"1; FIGS.",
"3a and 3b respectively show alternative conditions of a portion of an actuator according to a second embodiment; FIG.",
"4a is a sectional elevation of the actuator of FIGS.",
"3a and 3b; FIG.",
"4b is a cross-sectional view along A-A in FIG.",
"4a; FIG.",
"5 shows a brake having an actuator according to a third embodiment; and FIG.",
"6 is a graph showing force applied by the piezo-electric elements of the actuator of FIG.",
"5 against time.",
"Referring to FIG.",
"1, a brake 10 comprises a disc 12 and brake pads 16 operable to clamp the disc 12 to apply a braking force thereto.",
"Brake pads 16 are actuated by force transmission means in the form of a tappet 18 and an actuator 24.Tappet 18 has a reduced diameter portion 20 passing through an opening 21 in a support 22.The support 22 is fixed to a frame 14.Typically, the frame 14 may be slidably mounted to a body member of a vehicle (not shown) with the disc 12 fixed for rotation with a wheel of the vehicle.",
"Referring to FIG.",
"2, the actuator 24 is housed within a circumferentially extending recess 23 in a surface of the support 22 which defines the opening 21.The actuator 24 comprises a first piezo-electric device 26, retaining means in the form of a ratchet member 28, resilient biasing means in the form of a spring 32, and a second piezo-electric device 34.The first piezo-electric device 26 is disposed between the support 22 and the reduced diameter portion 20, abutting a first surface 25 of the recess 23, which surface faces towards the direction of actuation of the brake 10 (to the left as shown in FIG.",
"2).",
"The ratchet member 28 has a sharp edge 30 for engaging the reduced diameter portion 20, permitting movement of the tappet 18 in the direction of actuation and resisting movement of tappet 18 in the opposite direction by biting into the reduced diameter portion 20.The spring 32 biases the ratchet member 28 into engagement with the reduced diameter portion 20.The second piezo-electric device 34 is disposed between the support 22 and the reduced diameter portion 20, in abutment with the ratchet member 28.The second piezo-electric device 34 abuts a second surface 27 of the recess 23, which surface faces away from the direction of actuation of the brake 10.The first and second piezo-electric devices 26, 34 extend around the reduced diameter portion 20 and may comprise a plurality of piezo-electric elements.",
"The piezo-electric devices 26, 34 respond rapidly to application and removal of an energising voltage (or current) controlled by a controller, for example a microprocessor (not shown).",
"Ratchet member 28 is a self-locking device.",
"Many suitable mechanical devices will be apparent to the skilled person.",
"In use, a voltage is applied to the first piezo-electric device 26 causing it to expand, acting against the support 22.The action of the first piezo-electric device 26 against the first surface 25 applies force to the reduced diameter portion 20 so as to move the tappet 18 in the direction of actuation.",
"Removal of the voltage causes the first piezo-electric device 26 to contract, removing force between the support 22 and the tappet 18.Further application of the voltage to the first piezo-electric device 26 results in further application of force to the tappet 18 to further actuate the brake 10.Thus, although each time the first piezo-electric device 26 is expanded only a small movement of tappet 18 is possible, much greater movement is possible by repeatedly expanding and contracting the first piezo-electric device 26.A high frequency of application and removal of the voltage results in rapid actuation of the brake 10.It will be appreciated that a plurality of first piezo-electric devices 26, disposed between the housing and the shaft may be used to provide the actuating force.",
"To release the brake 10, a voltage is applied to the second piezo-electric device 34 causing it to expand and urge the ratchet member 28 to overcome the biasing action of the spring 32 so as to move the ratchet member 28 out of engagement with the reduced diameter portion 20.The action of the first piezo-electric device 26 against the second surface 27 applies force to the reduced diameter portion 20 so as to move the tappet 18 in the opposite direction (to the right in FIG.",
"2).",
"Further movement to release the brake 10 is provided by repeatedly expanding and contracting the second piezo-electric device 34.The controller adjusts the level of the voltages applied to the first and second piezo-electric devices 26, 34, in response to sensor signals from the brake, to ensure that the level of force applied to the tappet 18 is maintained during braking and to enable the position of the tappet 18 on release of the brake to be adjusted to compensate for wear of the brake pads 16.Referring now to FIG.",
"3a, a further brake actuating means comprises a shaft 40 passing through an opening 41 in a structural element 42.Typically, the structural element 42 forms part of a brake and is similar to the support 22 of FIG.",
"1.Shaft 40 forms part of a tappet.",
"A plurality of sets of piezo-electric devices, 43, 44, 45, in this example three, are disposed between a radially outer surface of the shaft 40 and a radially inner surface of the structural element 42 defining the opening 41.The sets of piezo-electric devices 43, 44, 45 respond rapidly to application and removal of an energising voltage.",
"A controller (not shown), for example a microprocessor, is provided for controlling the application and removal of the energising voltage.",
"In use, a first set of piezo-electric devices 43 are energised so as to change shape in a predetermined manner so as to act against the structural element 42 and apply force to the shaft 40 tending to move the shaft 40 in an axial direction as shown in FIG.",
"3a.",
"Subsequently, as shown in FIG.",
"3b, a second set of piezo-electric devices 44 are energised so as to change shape in a predetermined manner so as to apply force to the shaft 40 in the same direction.",
"The energising voltage is removed from the first set of piezo-electric devices 43 so that they return to their original shape, but the shaft 40 is prevented from moving in the opposite direction due to force applied by the second set of piezo-electric devices 44.A third set of piezo-electric devices 45 is then energised to apply force to the shaft 40, and the energising voltage removed from the second set of piezo-electric devices 44.The first set of piezo-electric devices 43 are then energised again, and the sequence repeated for as long as application of force is required.",
"Although each time a set of piezo-electric devices 43, 44, 45 is energised it is only possible to move the shaft 40 a small distance, repeated energisation and de-energisation of the sets of piezo-electric elements 43, 44, 45, in sequence, results in a sustained application of force and/or much larger movement.",
"The rapid response characteristics of the piezo-electric devices 43, 44, 45, enable a high frequency of application and removal of the voltage to be used to provide rapid actuation of the brake.",
"In a modified embodiment shown in FIG.",
"4a, the shaft 40 and opening 41 are substantially coaxially aligned.",
"A plurality of piezo-electric devices, shown generally as 47 are disposed between the radially inner surface, defining the opening 41 in structural element 42, and the radially outer surface of shaft 40.First and second sub-sets of piezo-electric devices 48, 49 are arranged to change size and/or shape when energised so as to act against the radially inner surface of structural element 42 applying a torque to the shaft 40.A large angle of rotation or sustained application of torque is provided by repeated energisation, in sequence, of the sub-sets of piezo-electric devices 48, 49.Some of the plurality of piezo-electric devices 47 may be arranged to provide axial force to the shaft 40 and others arranged to provide torque so that a combined axial and rotational force can be provided by selectively expanding the piezo-electric devices, 47, 48, 49.Additionally, some of the piezo-electric devices 47 may be arranged to provide lateral force in a first radial direction, and others to provide lateral force in a second radial direction perpendicular to the first radial direction.",
"Application of the lateral forces in either or both radial directions facilitates adjustment of the position of the shaft 40 relative to the structural element 42.Rapid response characteristics of the piezo-electric devices 47, 48, 49, enable a high frequency of application and removal of the voltage to be used to provide rapid actuation and adjustment of the brake.",
"Actuation and adjustment is controlled by the controller through the timing and levels of voltages applied to the piezo-electric devices 47, 48, 49, in response to signals from sensors (not shown) on the brake.",
"Referring now to FIG.",
"5, a further brake arrangement 50 comprises a brake disc 51 and brake pads 52 operable to clamp the disc 51 to apply a braking force thereto.",
"The brake pads 52 are actuated by expansion of first and second groups of piezo-electric devices 56, 58 disposed between laterally outer surfaces of the brake pads 52 and laterally inner surfaces of a housing 54.The piezo-electric devices 56, 58 respond rapidly to an applied voltage, which is controlled by a controller, for example a microprocessor (not shown).",
"Each group of piezo-electric devices 56, 58 is connected to an oscillating voltage supply.",
"A phase difference is provided between the voltage supplied to the first group 56 and the voltage supplied to the second group 58.In use, the first group of piezo-electric devices 56 is expanded to apply force to the brake pads 52 as the applied voltage rises.",
"Expansion of the second group of piezo-electric devices 58 to apply force occurs later, due to the phase difference between the applied voltages, and while the first group of piezo-electric devices 56 contracts as the applied voltage falls.",
"Force to the brake pads 52 is sustained by the repeated alternate expansion and contraction of the groups of piezo-electric devices 56, 58, in sequence, due to the phase-shifted oscillating voltages.",
"Rapid response characteristics of the piezo-electric devices 56, 58, enable a high frequency of oscillation of the voltage to be used to provide rapid actuation of the brake.",
"It will be appreciated that more than two groups of piezo-electric devices may be used, the groups being expanded and contracted in sequence by phase-shifted oscillating voltages.",
"FIG.",
"6 is a graph showing the amplitude of the force applied 66 as a function of time 67 in a configuration comprising four groups of piezo-electric devices connected to phase-shifted oscillating voltages.",
"The amplitudes of the forces applied by each group of piezo-electric devices is shown by the four lines 61, 62, 63, 64.The combined effect is to produce an actuating force shown by line 65 which is sustained with only a small variation 68 over a period of time.",
"Thus the braking force may be sustained by making use of the dynamic characteristics of the piezo-electric devices.",
"The sequential expansion of the groups of piezo-electric devices, due to the phase-shifted voltage supplies, ensures that during actuation of the brake, the actuating force is close to the maximum available.",
"The level of the voltage applied to the piezo-electric devices in the groups 56, 58, is controlled by the controller in response to feedback signals from the brake to the controller.",
"Each piezo-electric device in the groups 56, 58 applies force to an associated area of brake pad 52.By adjusting the level of the voltage applied to a piezo-electric device in a group 56, 58, the force applied to the associated area of the brake pad 52 may be adjusted, thereby adjusting the pressure distribution across the brake pad 52 to compensate for the effects of pad wear."
]
] | Patent_10250881 |
[
[
"Autonomous bird predation reduction device",
"A self-guided apparatus for repelling birds through various means of deterrence.",
"The device comprises a chassis, a floatation assembly, a propulsion system, and a guidance and control system.",
"The device may operate in either of at least two modes: passive and active.",
"In the passive mode, the device traverses a predefined area, scaring birds away.",
"In the active mode, the device surveys a designated area and, upon detection of birds, propels itself towards the birds and drives them away."
],
[
"1.A device for traversing a water body and for reducing the number of birds in the vicinity of the water body, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said device above the surface of the water body; (b) an electrically-powered propulsion system adapted to transport said device across the surface of the water body, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said device to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically-powered navigation system adapted to cause said propulsion system to periodically transport the device within the characteristic distance of at least 75% of the surface of the water body at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of the device on the water body causes a reduction in the number of birds in the vicinity of the water body as compared to the number of birds that would be present in the absence of the transport of the device.",
"2.A device as recited in claim 1, wherein said device is adapted to operate in an active and a passive mode; wherein in the active mode, said device autonomously traverses a surveyed area of the water body either continuously or upon detection of at least one bird; and wherein in the passive mode, said device traverses the water body without regard to the location of any birds.",
"3.A device as recited in claim 1, wherein when said collision prevention system detects a potential collision said device is adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either in a passive or active mode.",
"4.A device as recited in claim 1, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel.",
"5.A device as recited in claim 4, wherein said motive source operate said paddle wheels independently to propel and steer said device.",
"6.A device as recited in claim 1, wherein said collision prevention system comprises proximity feelers pivotally mounted on said device and attached to a triggering mechanism.",
"7.A device as recited in claim 6, wherein said triggering mechanism comprises a magnetic switch.",
"8.A device as recited in claim 1, wherein said navigation system is capable of identifying birds and guiding said device in the direction of said birds.",
"9.A device as recited in claim 1, wherein said power source comprises solar panels.",
"10.A device as recited in claim 9, wherein said power source additionally comprises one or more batteries adapted to store energy from said solar panels.",
"11.A device as recited in claim 1, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport the device within the characteristic distance of 90% of the surface of the water body at least once every thirty minutes.",
"12.A collection comprising a plurality of devices for traversing a water body and for reducing the number of birds in the vicinity of the water body, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said devices above the surface of the water body; (b) an electrically-powered propulsion system adapted to transport said devices across the surface of the water body, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said devices to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically powered navigation system adapted to cause the propulsion system to transport said devices across the water body, wherein at least one of said devices in said collection is periodically transported within the characteristic distance of at least 75% of the surface of the water body at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of said devices on the water body causes a reduction in the number of birds in the vicinity of the water body as compared to the number of birds that would be present in the absence of the transport of said devices.",
"13.A collection as recited in claim 12, wherein said devices are adapted to operate in an active and a passive mode; wherein in the active mode, said devices autonomously traverse a surveyed area of the water body either continuously or upon detection of at least one bird; and wherein in the passive mode, said devices traverse the water body without regard to the location of any birds.",
"14.A collection as recited in claim 12, wherein when said collision prevention system detects a potential collision said devices are adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either in a passive or active mode.",
"15.A collection as recited in claim 12, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel.",
"16.A collection as recited in claim 15, wherein said motive source operate said paddle wheels independently to propel and steer said devices.",
"17.A collection as recited in claim 12, wherein said collision prevention system comprises proximity feelers pivotally mounted on said devices and attached to a triggering mechanism.",
"18.A collection as recited in claim 17, wherein said triggering mechanism comprises a magnetic switch.",
"19.A collection as recited in claim 12, wherein said navigation system is capable of identifying birds and guiding said devices in the direction of said birds.",
"20.A collection as recited in claim 12, wherein said power source comprises solar panels.",
"21.A collection as recited in claim 20, wherein said power source additionally comprises one or more batteries adapted to store energy from said solar panels.",
"22.A collection as recited in claim 12, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport said devices within the characteristic distance of 90% of the surface of the water body at least once every thirty minutes.",
"23.A device for traversing a surveyed area and for reducing the number of birds in the vicinity of the surveyed area, said device comprising: (a) one or more floats having sufficient buoyancy to maintain a portion of said device above the surface of a water body when operating the device in water; (b) an electrically-powered propulsion system adapted to transport said device across the surface of the surveyed area, without the need for continuous monitoring or input from a human; (c) an electrically-powered collision prevention system to detect potential collisions before collisions occur, and to cause said propulsion system to alter the direction of transport of said device to avoid at least some detected potential collisions before collisions occur; all without the need for continuous monitoring or input from a human; (d) an electrically-powered navigation system adapted to cause the propulsion system to periodically transport the device within the characteristic distance of at least 75% of the surface of the surveyed area at least once every two hours; and (e) a power source to supply electrical power, directly or indirectly, to said propulsion system, to said collision prevention system, and to said guidance system; whereby: (f) the transport of the device on the surface of the surveyed area causes a reduction in the number of birds in the vicinity of the surveyed area as compared to the number of birds that would be present in the absence of the transport of the device.",
"24.A device as recited in claim 23, wherein said device is adapted to operate in an active and a passive mode; wherein in the active mode, said device autonomously traverses a surveyed area either continuously or upon detection of at least one bird; and wherein in the passive mode, said device traverses the surveyed area without regard to the location of any birds.",
"25.A device as recited in claim 23, wherein when said collision prevention system detects a potential collision said device is adapted to stop, back up, rotate between 0° and 360°, and then proceed forward, while operating either a passive or active mode.",
"26.A device as recited in claim 23, wherein said propulsion system comprises a drive source and a motive source; wherein said drive source comprises at least two paddle wheels; and wherein said motive source comprises at least one electric motor to activate each paddle wheel.",
"27.A device as recited in claim 26, wherein said motive source operate said paddle wheels independently to propel and steer said device.",
"28.A device as recited in claim 23, wherein said collision prevention system comprises proximity feelers pivotally mounted on said device and attached to a triggering mechanism.",
"29.A device as recited in claim 28, wherein said triggering mechanism comprises a magnetic switch.",
"30.A device as recited in claim 23, wherein said navigation system is capable of identifying birds and guiding said device in the direction of said birds.",
"31.A device as recited in claim 23, wherein said power source comprises solar panels.",
"32.A device as recited in claim 23, wherein said power source is a docking station adapted to recharge said device.",
"33.A device as recited in claim 31, wherein said power source additionally comprises one or more batteries adapted to store energy.",
"34.A device as recited in claim 32, wherein said power source additionally comprises one or more batteries adapted to store energy.",
"35.A device as recited in claim 23, wherein said electrically-powered navigation system is adapted to cause the propulsion system to periodically transport the device within the characteristic distance of 90% of the surface of the surveyed area at least once every thirty minutes."
],
[
"<SOH> BACKGROUND ART <EOH>Bird depredation of fish, crawfish, and shrimp in aquaculture ponds poses major problems.",
"For example, pelicans can consume 1 to 3 lb (0.45 to 1.36 kg) of fish per day, and may arrive with hundreds per flock.",
"Cormorants, anhingas, herons, and egrets may also do significant damage to aquaculture ponds.",
"It is estimated that one egret can eat ⅓ lb (0.15 kg) of fish per day, while a great heron can eat ⅔ to ¾ lb (0.30 to 0.34 kg) per day.",
"See G. A. Littauer etal., “Control of Bird Predation at Aquaculture Facilities: Strategies and Cost Estimates,” Southern Regional Aquaculture Center, Publ.",
"No.",
"402 (1997).",
"This problem can be especially troublesome in ponds that have just been stocked with young fish.",
"M. D. Hoy et al., Eastern Wildlife Damage Control Conference, vol.",
"4, pp.",
"109-112 (1989), estimated that wading birds could cause profound losses during fall migration, up to $10,000 per week on bait fish farms.",
"The Louisiana State University Ben Hur Aquaculture Facility in Baton Rouge, La.",
"recently experienced this problem with the white pelican during December 2000, when many fish were eaten and several ponds were completely de-stocked of fish.",
"A. R. Stickley et al., Eastern Wildlife Damage Control Conference, vol.",
"4, pp.",
"105-108 (1989), estimated that in 1988 catfish losses due to double-crested cormorants amounted to $3.3 million.",
"Currently, several different methods are employed to attempt to scare birds from aquaculture ponds.",
"One of the most common methods is the use of sonic cannons.",
"Sonic cannons emit loud bursts of noise.",
"However, birds eventually become accustomed to the noise emitted by sonic cannons.",
"Also, the loud “boom” produced by the sonic cannon can be disturbing to surrounding communities, and typically causes birds to migrate to other parts of the farm where the noise is more tolerable.",
"See M. Bomford et al., “Sonic Deterrents in Animal Damage Control: A Review of Device Test and Effectiveness,” Wildlife Society Bulletin, vol.",
"18, pp.",
"411-422 (1990).",
"Poisons, scarecrows, and nets have also been used.",
"However, these methods have several faults.",
"For example, poisons are usually fatal to birds and may cause casualties in non-target species.",
"Scarecrows are effective for short-term periods only, because birds typically adapt and become accustomed to them.",
"Nets typically have a high initial cost and are not practical for large ponds (>5 acres/˜2 hectares).",
"An unfilled need exists for a cost-effective device and method for effectively reducing bird predation of aquatic organisms over a relatively long period of time.",
"The device should be environmentally friendly, harmless to birds, and capable of withstanding expected environmental elements (e.g., water, wind, sun, and rain) and animal attacks.",
"The device should also be able to endure biological challenges (e.g., wind, weeds, and slime), and should have some level of intelligence to adapt to the evolving conduct of birds."
],
[
"<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG.",
"1 illustrates a front plan view of one embodiment of the predation reduction device.",
"FIG.",
"2 illustrates a perspective view of one embodiment of the predation reduction device with the solar panels removed.",
"FIG.",
"3 is an illustrative trajectory diagram depicting typical paths traversed by one embodiment of the predation reduction device.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"The benefit of the 14 Dec. 2001 filing date of U.S. provisional patent application Ser.",
"No.",
"60/340,511 is claimed under 35 U.S.C.",
"§ 119(e) in the United States, and is claimed under applicable treaties and conventions in all countries.",
"TECHNICAL FIELD This invention pertains to a self-guided, autonomous vehicle designed to provide an inexpensive method of reducing predation of aquatic organisms by birds.",
"BACKGROUND ART Bird depredation of fish, crawfish, and shrimp in aquaculture ponds poses major problems.",
"For example, pelicans can consume 1 to 3 lb (0.45 to 1.36 kg) of fish per day, and may arrive with hundreds per flock.",
"Cormorants, anhingas, herons, and egrets may also do significant damage to aquaculture ponds.",
"It is estimated that one egret can eat ⅓ lb (0.15 kg) of fish per day, while a great heron can eat ⅔ to ¾ lb (0.30 to 0.34 kg) per day.",
"See G. A. Littauer etal., “Control of Bird Predation at Aquaculture Facilities: Strategies and Cost Estimates,” Southern Regional Aquaculture Center, Publ.",
"No.",
"402 (1997).",
"This problem can be especially troublesome in ponds that have just been stocked with young fish.",
"M. D. Hoy et al., Eastern Wildlife Damage Control Conference, vol.",
"4, pp.",
"109-112 (1989), estimated that wading birds could cause profound losses during fall migration, up to $10,000 per week on bait fish farms.",
"The Louisiana State University Ben Hur Aquaculture Facility in Baton Rouge, La.",
"recently experienced this problem with the white pelican during December 2000, when many fish were eaten and several ponds were completely de-stocked of fish.",
"A. R. Stickley et al., Eastern Wildlife Damage Control Conference, vol.",
"4, pp.",
"105-108 (1989), estimated that in 1988 catfish losses due to double-crested cormorants amounted to $3.3 million.",
"Currently, several different methods are employed to attempt to scare birds from aquaculture ponds.",
"One of the most common methods is the use of sonic cannons.",
"Sonic cannons emit loud bursts of noise.",
"However, birds eventually become accustomed to the noise emitted by sonic cannons.",
"Also, the loud “boom” produced by the sonic cannon can be disturbing to surrounding communities, and typically causes birds to migrate to other parts of the farm where the noise is more tolerable.",
"See M. Bomford et al., “Sonic Deterrents in Animal Damage Control: A Review of Device Test and Effectiveness,” Wildlife Society Bulletin, vol.",
"18, pp.",
"411-422 (1990).",
"Poisons, scarecrows, and nets have also been used.",
"However, these methods have several faults.",
"For example, poisons are usually fatal to birds and may cause casualties in non-target species.",
"Scarecrows are effective for short-term periods only, because birds typically adapt and become accustomed to them.",
"Nets typically have a high initial cost and are not practical for large ponds (>5 acres/˜2 hectares).",
"An unfilled need exists for a cost-effective device and method for effectively reducing bird predation of aquatic organisms over a relatively long period of time.",
"The device should be environmentally friendly, harmless to birds, and capable of withstanding expected environmental elements (e.g., water, wind, sun, and rain) and animal attacks.",
"The device should also be able to endure biological challenges (e.g., wind, weeds, and slime), and should have some level of intelligence to adapt to the evolving conduct of birds.",
"DISCLOSURE OF INVENTION We have discovered a reliable and inexpensive device and method for repelling birds through various means of deterrence.",
"The device may be adapted to reduce predation by various bird species, and to operate in different environments.",
"The predation reduction device comprises a chassis, a floatation assembly, a propulsion system, and a guidance and control system.",
"The device may operate in either of at least two modes: passive and active.",
"In the passive mode, the device traverses a predefined area, scaring birds away.",
"In the active mode, the device traverses a surveyed area and, upon detection of birds, propels itself towards the birds and drives them away.",
"Unlike prior devices and methods of reducing bird predation (e.g., poisons, nets, remote control devices, etc.",
"), the novel device is self-guided and employs harassing and intimidating tactics to repel predatory birds.",
"Optionally, a deterrent system (e.g., non-destructive water cannon, scarecrow-type or other disguise, offensive-colored dressing, etc.)",
"is employed to assist in harassing and intimidating the birds.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1 illustrates a front plan view of one embodiment of the predation reduction device.",
"FIG.",
"2 illustrates a perspective view of one embodiment of the predation reduction device with the solar panels removed.",
"FIG.",
"3 is an illustrative trajectory diagram depicting typical paths traversed by one embodiment of the predation reduction device.",
"MODES FOR CARRYING OUT THE INVENTION In the early stages of developing the present invention (unpublished work), a radio-controlled hobby boat (single-propeller drive source and battery-powered) was adapted to operate as a bird predation reduction device.",
"However, several problems were encountered.",
"First, the boat was unable to operate in an autonomous, self-guided manner.",
"While the boat was able to reach places that humans could not, continuous human control was necessary.",
"Second, battery life tended to be less than half an hour, which was insufficient because bird predation typically occurs over an extended time period (an entire day).",
"Third, the propeller easily became entangled in weeds, which caused the boat to either slow or stop, and which increased the propeller's susceptibility to mechanical failure.",
"Fourth, the boat chassis was not designed to carry additional equipment.",
"Some of these problems were addressed by adding a large Styrofoam® (Dow Chemical Company, Midland, Mich.) platform to the boat chassis; however, the boat then became difficult to control.",
"In one embodiment, this invention provides a self-guided, autonomous device for driving birds away from a selected area.",
"The basic design comprises a chassis, a floatation assembly, a propulsion system, and a guidance and control system.",
"The predation reduction device is preferably solar powered and controlled by micro-controllers.",
"The predation reduction device preferably operates in either or both of at least two modes: active and passive.",
"In the active mode, the micro-controllers communicate and function in conformity with a navigation system (e.g., machine vision system, global positioning system, sonar, etc.)",
"and a collision prevention system to autonomously navigate the device around ponds and other waterways, either continuously or upon detection of birds.",
"When operating in the passive mode, the predation reduction device employs the collision prevention system only, allowing the device to move in a pre-determined pattern, or to perform a “random walk” type movement around a specified area.",
"Upon making contact with an obstacle (e.g., shoreline, etc.",
"), the device stops, backs up, rotates between 0° to 360°, and then proceeds forward again.",
"Alternatively, the device can be programmed to traverse a pre-determined pattern.",
"This device has several advantages.",
"First, costs are reduced compared to other predation reduction methods.",
"The predation reduction device is virtually self-sufficient, and thus reduces the need for human intervention.",
"Second, the predation reduction device is less harmful to birds than many existing techniques because it employs harassing and intimidating tactics to drive birds away, instead of poisons or devices that are potentially lethal to birds.",
"Third, the predation reduction device is energy-efficient and environmentally friendly.",
"The device preferably relies on solar power.",
"Fourth, the device can operate extremely quietly.",
"Thus, the device may be employed at various locations, including aquaculture ponds located near residential areas.",
"The device could save as much as hundreds of thousands of dollars per year for farmers by reducing bird predation and environmental impacts.",
"FIG.",
"1 illustrates a front plan view of one embodiment of the predation reduction device 2.This embodiment comprised a chassis 4, a floatation assembly 6, a propulsion system, and a guidance and control system.",
"Optionally, a deterrent system (e.g., non-destructive water cannon, scarecrow-type or other disguise, offensive-colored dressing, lights, sounds, etc.)",
"can be added to the predation reduction device 2 to assist in harassing bird (not shown).",
"The floatation assembly 6 comprised two fixed floats 12 and two movable floats 14.Fixed floats 12 were permanently mounted to chassis 4 on opposite sides of the predation reduction device 2 to provide buoyancy.",
"Each fixed float 12 was enclosed in a casing to prevent damage from floating debris (e.g., sticks, weeds, etc.).",
"Movable floats 14 were adjustably attached to the sides of fixed floats 12 to balance the predation reduction device 2 while in water.",
"(The movable floats 14 were slid forward or backward along the sides of the fixed floats 12 to balance the predation reduction device 2 as equipment was added or removed.)",
"Both fixed and moveable floats, 12 and 14 respectively, were made from a lightweight, buoyant material adapted to resist environmental degradation (e.g., degradation caused by sun, water, and bacteria) while providing sufficient buoyancy to keep predation reduction device 2 afloat, such as Styrofoam® (Dow Chemical Company, Midland, Mich.).",
"The propulsion system comprised a drive source and a motive source.",
"In this embodiment, the drive source comprised two paddle wheels 16.Paddle wheels 16 were chosen because of their ability to traverse shallow water filled with debris, while allowing drive source placement above the water line.",
"(Paddle wheels 16 avoided entanglement with debris because the mechanical drive was located above the water line and the rotational speed was slower than propeller-type drive systems.)",
"Paddle wheels 16 provided operational speeds up to 7 mph (˜11 km/h), which was sufficient to discourage birds, and which incidentally caused additional aeration in the water.",
"Paddle wheels 16 were individually mounted to chassis 4 and centrally positioned between fixed floats 12 at a height sufficient to allow paddle wheels 16 to propel the predation reduction device 2.",
"(Paddle wheels 16 were mounted between fixed floats 12 to avoid unnecessary contact with debris.",
"Fixed floats 12 were adapted to allow approximately 1.5 in (˜3.80 cm) of the surface area of each paddle wheel 16 into the water.)",
"Each paddle wheel 16 independently rotated forward and backward, providing a means for the predation reduction device 2 to be maneuvered to almost any position.",
"For example, to propel the predation reduction device 2 forward or backward, paddle wheels 16 were rotated evenly in the same direction.",
"To change the direction in which the predation reduction device 2 was traveling, paddle wheels 16 were rotated in opposite directions.",
"Each paddle wheel 16 was driven by a motive source capable of providing torque and shaft speed sufficient to accelerate and propel the predation reduction device 2 effectively, such as a Pittman® electric motor, model GM712-31 (Harleysville, Pa.).",
"Electric motors 18 were operated by an electric power source.",
"Electric motors 18 were mounted a safe distance from the water surface and adapted to be attached to paddle wheels 16.Electric motors 18 were chosen for ease of maintenance and the ability to be recharged in an environmentally friendly manner (e.g., solar power).",
"In this embodiment, the electric power source comprised solar power panels 20 mounted on top of the predation reduction device 2.Solar power panels 20 provided a means for energizing electrical systems, and also provided overhead cover for the guidance and control system.",
"Solar power panels 20 provided sufficient power to energize the propulsion system and guidance and control system during daytime operations, and sufficient power to charge batteries for limited nighttime operations.",
"FIG.",
"2 illustrates a perspective view of one embodiment of the predation reduction device.",
"In this embodiment, the guidance and control system comprised an electronic system 22, a collision prevention system, and a navigation system.",
"In this embodiment, the electronic system 22 further comprised a micro-controller system (not shown) having motor controllers capable of supporting program languages and of communicating with the navigation system, such as Lego® Robotic Control X systems (RCX) (Lego Systems, Enfield, Conn.), or Basic II Stamp semiconductor chips (Parallax, Inc., Rocklin, Calif.).",
"The micro-controller system operated by processing guidance information received from either a collision prevention system (mechanical proximity feelers, optical, image, sonar, radar, doppler radar shoreline indicators, etc.)",
"or a navigation system (machine vision, global positioning, local area navigation, etc.",
"), or both.",
"The micro-controller system independently controlled each electric motor 18 by using the guidance information to determine the direction and action needed by the paddle wheels to move the boat.",
"The micro-controller system sent signals to the motor-controllers to actuate electric motors 18.",
"(Motor controllers are not always necessary for operation of the vehicle.",
"In some instances, relays or similar devices can be employed instead.)",
"Programming in software supplied by the micro-controller manufacturer provided a means for the motor controllers to communicate with the navigation system, and to apply variable power to electric motors 18.A collision prevention system was used to prevent the predation reduction device 2 from colliding with obstacles.",
"In one embodiment, the collision prevention system comprised four, ⅜ inch (˜0.9 cm) diameter fiberglass poles (proximity feelers) 28, each mounted on a corner of the predation reduction device 2 and connected to triggering mechanisms 23 operable in moist environments, such as sealed mechanical magnetic switches, Radio Shack Cat.",
"No.",
"49-497 (Tandy Corporation, Fort Worth, Tex.).",
"The collision prevention system communicated with the electronic system 22, and provided a means to operate the predation reduction device 2 in a passive mode.",
"The collision prevention system also prevented the occurrence of collisions when operating the predation reduction device 2 in an active mode.",
"Optionally, a string 25 or wire can be attached between proximity feelers 28, thereby forming a detection zone capable of detecting objects, such as the shoreline, drain pipes, and rocks.",
"Also, a position-indicating system, such as global positioning system, Model “310” or “330” (Magellan Corporation, San Dimas, Calif.), may be employed to track or control the movement of the predation reduction device 2.",
"(not shown) In one embodiment, to facilitate the identification and pursuit of birds, the navigation system employed a machine vision system 30, such as Lego® Mindstorms Vision Command (Lego Systems, Enfield, Conn.).",
"Data from machine vision system 30 was transmitted to a data processing system (not shown) removably mounted underneath solar power panels 20 (not shown) to facilitate an active functional mode, such as a Compaq® Presario® laptop computer, model 275-0005A (Compaq, Houston, Tex.).",
"(The solar power panels 20 also provided overhead cover for the data processing system.)",
"The data processing system processed data gathered by machine vision system 30.Optionally, a deterrent system (e.g., non-destructive water cannon, a rotating laser system, etc.)",
"can be employed to harass and intimidate birds once detected.",
"The rotating laser system scares away birds by creating a moving beam (e.g., line, dot, or strip) on the shore, or on an object sticking out of the water, startling birds as it passes.",
"The laser power range is adapted to avoid adversely affecting the sight of birds.",
"The rotating beam creates a light burst that startles birds and other animals observing the predation reduction device 2.FIG.",
"3 is a trajectory diagram depicting typical paths traversed by one embodiment of the predation reduction device 2.In this embodiment, the predation reduction device 2, operating in passive mode, employed proximity sensors 28 and triggering mechanisms 23 to perform “random walk-type” movements around a pond.",
"Upon detecting the shoreline, the predation reduction device 2 backed away from the shoreline, rotated between 0° to 360°, and headed in a different direction.",
"EXAMPLE 1 Construction of the Prototype The chassis 4 was made from aluminum sheet metal and bar stock.",
"The fixed and moveable floats, 12 and 14, respectively, were fabricated from Styrofoam®, and protected from mechanical damage by 16-gauge aluminum.",
"The chassis 4 contained two fixed floats 12 (volume=660 in3 (˜10.8 L)) and two moveable floats 14 (volume=315 in3 (˜5.2 L)) attached to the side of each fixed float 12 to provide additional buoyancy and balance.",
"The chassis 4 provided approximately 50 lb (˜16 kg) total buoyancy.",
"The total weight of the predation reduction device 2 was between 30-40 lb (˜13-18 kg), depending upon the embodiment being operated.",
"Paddle wheels 16 were constructed from riveted sheet aluminum.",
"Paddle wheels 16 were mounted on each side of the predation reduction device 2 and attached to two 12V Pittman®, Model GM712-31, electric motors 18.Electric motors 18 were connected to paddle wheels 16 by a gear drive with a 1:16 gear ratio, which allowed electric motors 18 to be positioned above fixed floats 12 at a safe distance from the water surface.",
"After determining that electric motors 18 required approximately 0.3 A each, during both forward and backward movement, and about 0.6 A each during directional changes, three solar power panels 20, Model SKU#41144, (LY, Inc., China) (single amorphous silicon cell solar power panels, 12 V dc, 350 mA, 5 Watt), were mounted above paddle wheels 16; these panels also sheltered the data processing system directly underneath.",
"Solar power panels 20 provided 1.3 amperes at 12 V dc in bright sunlight, which provided enough energy to operate the predation reduction device 2 and to charge the batteries.",
"(An additional solar panel was later added to increase the total current to approximately 1.8 amps at 12 V dc, in direct sunlight.)",
"Two 12 V batteries (Elks®, Model 1245, 12 V dc, 5.0 Ah) were used to store power for limited nighttime operations.",
"In one embodiment, the electronic system 22 comprised a single chip micro-controller.",
"Two different micro-controllers were used during initial testing.",
"The first micro-controller was a Lego® Mindstorms RCX.",
"The Mindstorms RCX micro-controller contained three built-in motor controllers (0.5 A each at 9 V dc) and three digital (or switch) inputs.",
"A 9 V regulator was used to power the micro-controller from the 12 V dc batteries.",
"The second micro-controller was a Basic II Stamp semiconductor chip.",
"This chip had 2K bytes of electrical memory, a 20 Hz clock rate (4000 instructions per second), and 16 input/output lines.",
"An H-bridge relay system and motor controllers were operated with the Mindstorms RCX micro-controller to control the paddle wheel motors (since the Mindstorms RCX micro-controller was 9 V dc and main batteries were 12 V dc).",
"The relays used in the H-bridge were Radio Shack Model 275-0005A.",
"Solutions Cubed, Motor Mind B motor controllers were employed with the Basic II Stamp semiconductor chip to operate the paddle wheels 16.This arrangement allowed speed control of electric motors 18 using serial transmissions from the Basis II Stamp micro-controller to the Solutions Cubed, Motor Mind B motor controller, which used pulse width modulation.",
"(Basic II Stamp semiconductor chips have the advantage of applying variable rate power to electric motors 18, using serial transmissions from the Basic II Stamp semiconductor chip and Pulse Width Modulation.)",
"These chips were rated at 2 A continuous current output, with a built-in heat sink.",
"All electronics were protected from the environment using Tupperware® containers or Ziplock®bags.",
"The Mindstorms RCX micro-controller and the Basic Stamp II controller were used to control separately the direction and movement of electric motors 18 for steering and propulsion of the predation reduction device 2.By contrast, when the Vision Command was used, the Mindstorms RCX micro-controller relayed steering commands from the Vision Command software and the Compaq® Presario® laptop computer to the Basic Stamp Controller (which controlled the paddle wheel motors).",
"The Basic Stamp II controller used computer language, basic algorithms and special subroutines to control motor direction and speed when either the proximity feelers 28 were actuated, or when steering commands were sent from the Mindstorms RCX micro-controller and the Vision Command.",
"These routines included special “slow spin-down to a stop” and “slow spin-up from a stop” functions to reduce mechanical wear (i.e., a kind of electronic clutch).",
"In cases where the Mindstorms RCX was used, the same commands were developed using object oriented programming language for the Lego® systems.",
"The collision prevention system was employed to prevent collisions with objects.",
"In facilitate this system, ⅜ inch (˜0.9 cm) diameter fiberglass poles (proximity feelers 28) from a mobile TV antenna, Radio Shack Cat.",
"No.",
"15-1609 (Tandy Corporation, Fort Worth, Tex.",
"), were mounted on each corner, using either a specially bent 4 inch to 6 inch (˜10 cm to 15 cm) standard light tee hinge (National, Sterling, Ill.), which triggered the Radio Shack Cat.",
"No.",
"49-497 (Tandy Corporation, Fort Worth, Tex.)",
"magnetic switches upon collision with the shore, or another object having sufficient inertia.",
"(Magnetic switches 23 were employed because they were sealed and completely waterproof.)",
"A string 25 was run between proximity feelers 28 to detect objects such as drain pipes and rocks.",
"A handheld, waterproof global positioning system (Magellan Corporation Model “310” or “330”) was also added to provide positional information.",
"This system transferred data with the micro-controller through a serial cable using standard NEMA (National Marine Electronics Association) 0183 code.",
"(This system was not used with the Lego® Mindstorms RCX, because serial transmission inputs were not available for it.)",
"EXAMPLE 2 Predation Reduction Tests To confirm that the prototype predation reduction device 2 was effective in repelling predatory birds, tests were conducted using various navigation methods.",
"In one embodiment, a navigation method used proximity feelers 28 to detect the shoreline and then turn the predation reduction device 2.Using this method, the device 2 performed a “random walk” type movement around the pond as the shoreline was encountered.",
"The predation reduction device 2 backed up for 6 seconds to move away from the shoreline, and then turned for 6 seconds to perform a 0° to 360° turn.",
"(Micro-controllers, software and either relays or transistor drivers were adapted and employed to accomplish this “random walk-type” movement.)",
"These functions (i.e., traversing the pond, backing up, and turning) also scared the birds away from the pond and the shoreline.",
"Initial testing was conducted at the Louisiana State University Ben Hur Aquaculture Center in Baton Rouge, La.",
"Another navigation method comprised a global positioning system and programming algorithms to follow coordinates programmed into the global positioning system.",
"Another navigation method employed machine vision system 30 to identify birds.",
"Data from machine vision system 30 was transmitted to the data processing system.",
"The data processing system used a pre-determined grid pattern that divided the image of the pond into sections, and then trained each section to sense light, motion, or color to detect birds.",
"(The image of the pond was photographed by a camera mounted to chassis 4, on the front side of the predation reduction device 2).",
"Motion was detected by comparing subsequent frames from the camera (30 images/sec) to detect differences, such as new occurrences of birds in the frames.",
"Color was detected by “training” the data processing system to recognize the color of a bird (from either a model of the bird or a captured image of a real bird), and then training the data processing system to scan subsequent frames for detection of the color in a certain percentage of the image area (a percentage that could be adjusted).",
"Brightness can also be used to find birds, especially if an infrared camera is employed to acquire images.",
"This approach is especially suitable for work at night.",
"These detection methods can be used separately or they may be combined to detect birds.",
"Image detection was programmed using the Lego® Mindstorms Vision Command system, which contains an object oriented programming language for detection schemes such as these.",
"Navigation routines were also programmed into the Basic II Stamp semiconductor chip to control turning towards the detected birds.",
"Turning data was transmitted to the Basic Stamp II micro-controller using a 5 volt regulator chip, connected between the motor driver output ports of the Lego® Mindstorms RCX.and the micro-controller input pins.",
"Software routines were programmed into the Basic II Stamp chip to control turning towards birds detected in different parts of the image.",
"In addition, a windshield washer (Model P50, Poberk, Brownsville, Tex.)",
"was attached to the Lego® Mindstorms Vision Command system using a 9V dc, single pole, double throw relay, Radio Shack Cat.",
"No.",
"275-005A (Tandy Corporation, Fort Worth, Tex.)",
"to squirt water at a bird (in front of the device 2) when the machine vision system 30 indicated the bird was in the center of the image, approximately 10 to 20 feet away.",
"(This distance can be increased with higher pressure/flow rate water streams and improved image processing equipment).",
"Currently, the water cannon in the prototype delivers a stream of water at 0.25 liters/sec.",
"In some instances, the system also squirted birds on the shore.",
"These operations allowed the predation reduction device 2 to detect, turn, and chase and harass birds.",
"The global positioning system and the Basic II Stamp semiconductor chip have also been programmed to create an imaginary boundary diagram of the pond.",
"However, at the time this application was filed, the “imaginary boundary” system had not yet been tested.",
"It is envisioned that this system would create a box or other shaped area in which the predation reduction device 2 would operate using global positioning system coordinates.",
"When the predation reduction device 2 nears the boundary of the preset area (indicated by the global positioning system coordinates), it would change directions, based on either the random direction algorithm of the predation reduction device 2 or a magnetic compass.",
"This system could be employed when multiple boats are used to patrol separate areas of a large pond.",
"Also, adual stageDoppler radar sensor (Bulldog Security Inc., Wintersville, Ohio) was tested to detect birds close to the predation reduction device 2.This system could be employed to detect objects located near (within 4-5 ft (˜1.2-1.5 m)) of the predation reduction device 2.This system was also able to detect the shoreline and other objects of sufficient mass.",
"Passive infrared sensors are currently being tested to detect birds as well.",
"The “characteristic distance” is defined to be the typical or average distance within which the predation reduction device must approach a bird in order to cause the bird to leave the vicinity of the device.",
"The “characteristic distance” may vary by type of bird, time of day, time of year, weather, hunger or other physical state of the bird, the characteristics of the particular device (e.g., presence of water cannon, color, alligator or other predator-like disguise, etc.",
"), and the bird's degree of familiarity with the device or similar devices.",
"The characteristic distance in prototype tests has typically been on the order of 10 to 30 ft (˜3 to 9 m).",
"EXAMPLE 3 Alternative Embodiment In an alternative embodiment, a docking station (not shown) can be employed to automatically recharge the batteries on the predation reduction device 2.Optionally, batteries with a low charge may be replaced with fully charged batteries.",
"In this embodiment, the docking station comprises a hub platform and output terminal posts.",
"The hub platform has V-shaped guides which extend outwardly to engage the predation reduction device 2.",
"(The docking station can be positioned on the water or moored to the shore-line.)",
"Once a low battery level is detected, the navigation system processes guidance information received from either the collision prevention system (e.g., mechanical proximity feelers, sensory bumper guards, etc.)",
"or the navigation system (machine vision system, differential global positioning system, etc.)",
"to locate and engage the docking station.",
"The machine vision system may be programmed to detect a homing signal (e.g., blinking light) mounted on the docking station.",
"When using the differential global positioning system, the navigation system is provided real time positioning information to locate the docking station.",
"An input terminal post capable of receiving externally provided power and distributing power to the batteries is mounted in front of the predation reduction device 2.Once the predation reduction device 2 locates the docking station, the paddle wheels 16 propel it towards the docking station.",
"Upon engagement, the V-shaped guides lead the predation reduction device 2 to a position where the input terminal post contacts one of the output terminal posts, allowing 12V DC, 110V AC, or 220V AC to be used to recharge the batteries.",
"(Electrical power maybe provided to the docking station by several means, including batteries and a solar power array, an electrical cable connected to an electrical outlet located on the shore, or a gasoline powered electrical generator.)",
"If the predation reduction device 2 initially fails to engage the V-shaped docking port, the collision prevention system (e.g., mechanical proximity feelers, sensory bumper guards, etc.)",
"causes the boat to back up and try again.",
"When charging is completed, a signal is transmitted to the micro-controller system reversing the propulsion of the predation reduction device 2 until it completely disengages from the docking station.",
"The predation reduction device 2 then performs its previous functions until another recharging is needed.",
"RESULTS Reduction in Bird Predation Aquatic predatory birds were not present in large numbers during initial testing, but limited testing was performed on occasional birds (e.g., herons, egrets, geese, and cormorants) that landed in protected ponds.",
"During these tests, birds, were either driven away from the pond or did not fly onto the pond in which the device was located.",
"During one test, an egret watched the predation reduction device 2, and then flew away when the predation reduction device 2 made a turning maneuver near it.",
"In another test, the predation reduction device 2 was placed on a pond and allowed to operate all day.",
"A flock of geese that had usually frequented the pond did not visit the pond while the predation reduction device 2 was present.",
"The birds seemed “hazed” by the predation reduction device 2.Additional tests were conducted on egrets and cormorants, during times of peak bird predation.",
"These tests involved putting up to 2 boats on the surface of a 1 acre pond (˜0.4 hectares) stocked with catfish fingerlings and recording the bird populations with a time-lapse recorder.",
"These tests were run for three days with a boat on the pond, and then three days without a boat.",
"Replications were done for several months between November and December of 2001.Review of the recordings indicated that the boat reduced bird populations by approximately 75%, and in some instances up to 100%.",
"The machine vision system 30 did encounter some initial problems.",
"The system worked well in the laboratory, but had a brightness problem with the intense sunlight found outside; however, the brightness problem may be solved by using a tinted lens, a different camera, or adjusting the camera software.",
"Also, the machine vision system seemed to have problems calibrating to white colors, including sun glare, which appears to be a problem inherent to the Lego® Vision Command software.",
"These problems can be solved by adding software routines to provide a more positive identification of the birds using fuzzy logic, neural networks, maximum likelihood classifier routines and multiple pieces of information from the image or other sensors, such as color, shape, and heat signature combined.",
"Deterrence System Testing of the non-destructive water cannon was effective in scaring birds, but in preliminary tests the predation reduction device 2 was never in close enough proximity to actually spray a bird.",
"Lasers are being investigated for longer range “scaring” of birds.",
"Electrical Power Source The solar power panels 20 worked effectively throughout the day, providing enough energy to operate the predation reduction device 2.Measurements indicated that the predation reduction device 2 consumed approximately 0.6 to 0.8 A, while solar power panels 20 produced over 1.5 A on sunny days.",
"In cloudy conditions, solar power panels 20 supplied enough power to maintain traversing operations for a reduced time (4-6 hours).",
"Nighttime testing has not yet been performed, but it is believed that the prototype predation reduction device 2 will run at least several hours on stored battery power.",
"A future version of the predation reduction device 2 may include batteries with greater storing capacity.",
"Guidance and Control System The collision prevention system proved to be essentially 100% effective.",
"The system allowed the predation reduction device 2 to back up and rotate between 0° to 360°.",
"Proximity feelers 28 and mechanical magnetic sensors 23 provided a means for the predation reduction device 2 to randomly traverse essentially the whole pond.",
"During initial testing, the predation reduction device 2 traversed the entire surface area (i.e., within about 20 ft (˜6.1 m) of 90% of the pond surface) of a 1.5 acre (˜0.6 hectares) pond, with 2 to 5 mph (˜3 to 8 km/h) winds, in about 30 minutes.",
"In addition, during a long duration test, the boat effectively “lived” on the pond without getting caught or snagging on an object.",
"The device can be protected from aerators by placing a protective device around the aerators, such as an anchored plastic tubing ring that sits approximately 1 ft (˜0.3 m) below the surface.",
"Paddle wheels 16 effectively propelled the predation reduction device 2 at a high speed (5 mph) (˜8 km/h), while maintaining low power draw from electrical motors 18.Also, paddle wheels 16 were not jammed or clogged with weeds.",
"(The predation reduction device 2 has not to date been tested in water containing high trash volumes.)",
"Paddle wheel 16 speeds were slow, and water friction was kept to a minimum.",
"Some problems were initially encountered with the gear drive.",
"The Lego® Mindstorms RCX micro-controller and H-relays (which had a sudden “on/off” effect) caused excessive wear in the gear drive and excessive shock loading on shafts and set screws during directional changes.",
"This problem was eliminated by using the Basic II Stamp micro-controller, which was programmed to ramp down the motor speed before a directional change, an effect that could also be implemented in the Lego® RCX programming, i.e., a type of electronic clutch.",
"In some instances, winds in excess of 10 mph (˜16 km/h) caused the predation reduction device 2 to drift, even while power was applied.",
"Several design parameters were changed to reduce wind effects.",
"First, the boat was streamlined.",
"Solar power panels 20 were placed horizontally to reduce wind effects.",
"Floats 12 and 14 were trimmed in size to lower the floatation level of the predation reduction device 2, minimizing the surface area of floats 12 and 14 above the surface of the water.",
"A rudder was added to aid in maintaining direction during windy periods.",
"(not shown) The Lego® RCX micro-controller contained a limited number of input/output lines when used with the machine vision system 30, but it still effectively controlled the propulsion system, including the proximity feelers 28 and the navigation system.",
"The Basic II Stamp semiconductor chip appeared more reliable than the Lego® Mindstorms RCX micro-controller.",
"The global positioning system was accurate to within about 5 to 6 ft (˜1.5 to 1.8 m); however, in some situations, positioning measurements deviated as much as 30 ft (˜9 km/h)(with selective availability turned off).",
"This error is not critical in a large pond (>5 acres/˜2 hectares), but could cause some problems in a small pond (<1 acre/˜0.4 hectares).",
"For this reason, the proximity feeler system and the “random walk” type methods were solely used for navigation.",
"In the future, the global positioning system may be used to guide the predation reduction device 2 between different ponds using, for example, an amphibious-design vehicle capable of traversing land and water through the use of multiple paddle wheels 16.Future testing will explore the interaction between birds and their environment.",
"Birds quickly adapt to “standard” signals, such as the regular movement of equipment or even of other animals.",
"Sudden movements, such as changes in direction or rotation of the predation reduction device 2, seem to startle birds.",
"Active deterrence systems, such as lasers or non-destructive water cannons, seem to reinforce bird intimidation.",
"The addition of highly visible patterns (e.g., large eyes, alligator silhouettes, etc.)",
"may also aid in improving the effectiveness of the deterrence system.",
"It is believed that this device or similar devices could be used for other in-field environmental and biological engineering applications.",
"Possible uses include remote measurement of water quality parameters, crop scouting, site-specific environmental monitoring for various crops, pest predation reduction applications, and possibly crop harvest.",
"Similar systems might be used in wild ecosystems to monitor environmental quality.",
"The addition of a radio modem system to allow data to be sent back to a field station is another future development.",
"Future embodiments of the predation reduction device 2 will include the following: (1) an improved frame design (e.g., a frame built of either plastic, wood or other floatation materials), and mechanical components that generate more power which would allow the predation reduction device 2 to be propelled faster; (2) an improved machine vision system 30 will employ classifier schemes that will help recognize birds using faster algorithms and a combination of information input sources (e.g., color, object size and texture, heat, etc.)",
"to better identify birds; (3) either a swivel drive (U-joint, etc.)",
"or a direct drive coupling to connect electric motors 18 to paddle wheels 16; (4) a differentially-corrected global positioning system may be used to obtain more accurate readings (although not necessary in the current embodiment); (5) multiple paddle wheels 18 capable of traversing levees may be added to the predation reduction device 2 to allow it to move between ponds; and (5) a docking station capable of recharging batteries on the predation reduction device 2, to allow all-night operations and faster speeds if solar panels 20 were removed or reduced.",
"The complete disclosures of all references cited in this specification are hereby incorporated by reference.",
"Also incorporated by reference is the complete disclosure of the inventors own work: S. Hall et al., “Development of an Autonomous Bird Predation Reduction Device,” An ASAE Meeting Presentation, Paper No.",
"01-3131 (Presented Jul.",
"30-Aug. 1, 2001).",
"In the event of an otherwise irreconcilable conflict, however, the present specification shall control."
]
] | Patent_10250973 |
[
[
"Pole climber",
"The pole climber is designed to allow an individual to move vertically up and down a tree or pole with little physical exertion, depending instead on the power and design of the device.",
"An electrically powered motor and pair of metal drums set on opposite ends of a bar-like component, the drums fitted with metal teeth, or spikes would turn, biting into the surface being climbed.",
"The individual stands or sits on the platform while operating the motor, that turns the drums, moving the device and person vertically up & down as desired."
],
[
"1.The device consists of 2 elements a is the climbing assembly b is the power assembly you place a to tree using gravity you hook motor to b power assembly causing device to move up and down tree"
],
[
"<SOH> BACKGROUND OF INVENTION <EOH>Operate, which contributes to their lack of safety.",
"Most of the prior art grips tree on two sides, basically front & rear: which renders the stand unstable if the user places his weight on one side of the platform.",
"All of the prior art share the problems of excess weight awkwardness and noisiness.",
"Each of these problems renders the design inefficient for its intended use."
],
[
"<SOH> SUMMARY OF INVENTION <EOH>The climber is set at the base of tree or pole to be climbed, the two drums seperated, then placed on opposite side of tree.",
"Then adjusted to tree size, bolted together, the the motor is turned on in the up position.",
"The drum turns giping the tree or pole and starts up.",
"The individual then climbs on the platform and up they go.",
"A seat is provided with few parts and folds out of the way when not in use.",
"Further objects and advantages of this invention will become apparent from a consideration of the drawings and ensuing description."
],
[
"BACKGROUND OF INVENTION Operate, which contributes to their lack of safety.",
"Most of the prior art grips tree on two sides, basically front & rear: which renders the stand unstable if the user places his weight on one side of the platform.",
"All of the prior art share the problems of excess weight awkwardness and noisiness.",
"Each of these problems renders the design inefficient for its intended use.",
"SUMMARY OF INVENTION The climber is set at the base of tree or pole to be climbed, the two drums seperated, then placed on opposite side of tree.",
"Then adjusted to tree size, bolted together, the the motor is turned on in the up position.",
"The drum turns giping the tree or pole and starts up.",
"The individual then climbs on the platform and up they go.",
"A seat is provided with few parts and folds out of the way when not in use.",
"Further objects and advantages of this invention will become apparent from a consideration of the drawings and ensuing description.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 Is a perspective view of the perferred embodiment attached to a tree in an opening position.",
"FIG.",
"2 Is a side elevation view attached to a tree of the preferred embodiment.",
"FIG.",
"3 Is a plan view of the frame member and climbing system with preferred embodiment.",
"10 Climbing Assembly 11 Tranverse Element 12 Side Element 13 Horizontal Adjustment 14 Hinged member to adjust platform 15 1″ Shaft 16 1″ Bearing 17 Adjustment Holes for Tree Size 18 1″ Drive Shaft 19 1″ Bearing 20 Spiked Climbing Drum 23 Upper Frame Member 24 1″ Bearing 26 Worm Drive Gear Box 27 ⅜ Shaft where Drill attaches 29 Cordless Drill 30 1″ Bearing 32 Foot Platform Adjustment Holes 33 Foot Platform 35 Lower Frame Member 36 ¼ Bolt 38 Mounting Bracket 39 1½ Tubing 41 Tranverse Element 42 Side Element 44 Side Element 45 Horizontal Adjustment Brace 46 Spiked Trailing Drum 47 Inner Tube 48 Inner Tube Tree Size Adjustment Holes 51 53 55 57 58 61 63 65 67 68 71 72 A 1″ drive shaft 18 which is attached to side element 42 in a bearing 19 other end of shaft 18 is attached to side element 44 in a bearing 24, with the 1″ shaft 18 sticking past side element 44 to engage with worm drive gear box 27 which is mounted to the side of side element 44 with mount bracket 26.A cordless drill 29 is attached to ⅜ shaft 27 which drives worm drive gear box 26 turing 1″ shaft 18 which rotates spiked climbing drum 20 while engaging back side of tree moving climber up or down.",
"Upper frame member 23 also includes a inner tube 47 that slides in side element 42 where side element 42 is cut in half, fixed on one end by 1″ bearing bolt 19 other end of inner tube 47 is drilled with side element 42 with matching holes 17 with inner tube 48 that slides in side element 42 where side element 44 is cut in half, fixed on one end by 1″ bearing bolt 49 other end of inner tube 48 is drilled with side element 44 with matching holes 49.Making the assembling adjustable for different size trees.",
"Frame 23 also includes a foot platform 33 which accommodates the feet of the user.",
"Platform 33 is attached to transverse elements 11, 41, and side elements 42 & 44.Platform 33 is made of a material of sufficient strength to support the user, such as expanded metal.",
"A climbing stand comprised of a single climbing assembly 10 and a foot platform 33, in a climbing configuration is illustrated in FIG.",
"2.In the preferred embodiment, the frames 11, 12, 23, and 35 are made of a rigid material of sufficient strength to support the user; such as steel or aluminum tubing.",
"As illustrated in FIGS.",
"1-3, frame 23 comprises a side element 42 and a side element 44 of predeterming dimensions, each having a section with a plurality of engagement holes 17 and substantially horizontal section 33 forming its distal end.",
"Frame 23 includes a transverse element 11 & 41 and upper frame member of predetermined dimensions, with one end attached to side element 42 and a second end attached to side element 44, these attachments being on the distal side of the forming the inclined sections of side elements 42 and 44, brace 13 together with transverse element 41 and side element 42; as well as brace 45 together with transverse element 41 and side element 44, form triangular relationships in both the horizontal and vertical planes; thereby restraining both lateral and vertical displacement of the inclined sections.",
"Spiked trailing drum 46 and transverse element 41 form an abutment, for tree engagement, on the user's side of the tree.",
"Frame 23 also includes a tree-gripping spiked climbing drum 20.Assembly 20 includes a 1″ drive."
]
] | Patent_10251413 |
[
[
"Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction",
"The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range.",
"The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides.",
"Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units.",
"From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units.",
"The modified collection of multimer units is arranged in a list in order of melting temperature.",
"The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature."
],
[
"1.A method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, wherein the plural capture oligonucleotide probes have melting temperatures within a narrow range, said method comprising: providing a first set of a plurality of tetramers of four nucleotides linked together, wherein (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides; linking groups of 2 to 4 of the tetramers from the first set together to form a collection of multimer units; removing from the collection of multimer units all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than the 4 times the number of tetramers forming a multimer unit, to form a modified collection of multimer units; arranging the modified collection of multimer units in a list in order of melting temperature; randomizing, in 2° C. increments of melting temperature, the order of the modified collection of multimer units; dividing alternating multimer units in the list into first and second subcollections, each arranged in order of melting temperature; inverting the order of the second subcollection; linking in order the first collection of multimer units to the inverted second collection of multimer units in order to form a collection of double multimer units; and removing from the collection of double multimer units those units (1) having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units.",
"2.A method according to claim 1 further comprising: placing the modified collection of double multimer units at positions on a support so that complementary oligonucleotides to be immobilized on the support can be captured at the positions.",
"3.A method according to claim 2, wherein the collection of double multimer units is shown in FIG.",
"26.4.A method according to claim 2, wherein the collection of double multimer units is shown in FIG.",
"27.5.A method according to claim 2, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.",
"6.A method according to claim 1, wherein the multimer units have 12 mers, the double multimer units have 24 mers, and the melting point of the double multimer units is 75-84° C. 7.A method according to claim 1 further comprising: reclaiming double multimer units having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers; unlinking the reclaimed double multimers units to each form a pair of multimer units; selecting multimer units with a melting temperature in ° C. of more than 11 times the number of tetramers and less than 17 times the number of tetramers; and reintegrating the selected multimer units into said method.",
"8.A method according to claim 7, wherein the the method of claim 7 is repeated.",
"9.A method according to claim 1 further comprising: making additional different sets of a plurality of tetramers by producing, base by base, circular permutations of the tetramers within the first set and complements thereof.",
"10.A method according to claim 1, wherein the set of tetramers is shown in Table 1 and complements thereof.",
"11.A method according to claim 10, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"12.A method according to claim 10, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"13.A method according to claim 10, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"14.A method according to claim 1, wherein the collection of double multimer units is shown in FIG.",
"26.15.A method according to claim 1, wherein the modified collection of double multimer units is shown in FIG.",
"27.16.A method according to claim 1, wherein the collection of double multimer units have a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.",
"17.An oligonucleotide array comprising: a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the support can be captured at the different positions, wherein the complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption.",
"18.An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG.",
"26.19.An oligonucleotide array according to claim 17, wherein the collection of double multimer units is shown in FIG.",
"27.20.An oligonucleotide array according to claim 17, wherein the collection of double multimer units has a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.",
"21.An oligonucleotide array according to claim 17, wherein the double multimer units have 24 mers and the melting temperature of the double multimer units is 75-84° C. 22.An oligonucleotide array according to claim 17, wherein the set of tetramers is shown in Table 6 and complements thereof.",
"23.An oligonucleotide array according to claim 17, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 6 and complements thereof.",
"24.A oligonucleotide array according to claim 17, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 6 and complements thereof.",
"25.An oligonucleotide array according to claim 17, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 6 and complements thereof.",
"26.A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising: providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences; providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample; providing a ligase, blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture; subjecting the mixture to one or more ligase detection reaction cycles comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment; providing a support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units; contacting the mixture, after said subjecting, with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide; and detecting the reporter labels of ligated product sequences captured on the support at particular sites, thereby indicating the presence of one or more target nucleotide sequences in the sample.",
"27.A method according to claim 26, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, have a mismatch at a base at the ligation junction which interferes with such ligation.",
"28.A method according to claim 27, wherein the mismatch is at the 3′ base at the ligation junction.",
"29.A method according to claim 26, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base adjacent to a base at the ligation junction which interferes with such ligation.",
"30.A method according to claim 29, wherein the mismatch is at the base adjacent to the 3′ base at the ligation junction.",
"31.A method according to claim 26, wherein the sample potentially contains unknown amounts of one or more of a plurality of target sequences with a plurality of sequence differences, said method further comprising: quantifying, after said detecting, the amount of the target nucleotide sequences in the sample by comparing the amount of captured ligated product sequences generated from the sample with a calibration curve of captured ligated product sequences generated from samples with known amounts of the target nucleotide sequence.",
"32.A method according to claim 26, wherein the sample potentially contains unknown amounts of one or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising: providing a known amount of one or more marker target nucleotide sequence; providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion complementary to capture oligonucleotides on the support, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, there is a mismatch which interferes with such ligation, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture; detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated product generated from the known amount of marker target nucleotide sequences with the amount of captured other ligated product.",
"33.A method according to claim 31, wherein the one or more marker target nucleotide sequences differ from the target nucleotide sequences in the sample at one or more single nucleotide positions.",
"34.A method according to claim 33, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the first oligonucleotide probes have a common target-specific portion, and the second oligonucleotide probes have a differing target-specific portion which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.",
"35.A method according to claim 33, wherein the oligonucleotide probe sets and the marker-specific oligonucleotide probe sets form a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions, which hybridize to a given allele or a marker nucleotide sequence in a base-specific manner.",
"36.A method according to claim 26, wherein the sample potentially contains unknown amounts of two or more of a plurality of target nucleotide sequences with a plurality of sequence differences, said method further comprising: quantifying, after said detecting, the relative amount of each of the plurality of target nucleotide sequences in the sample by comparing the relative amount of captured ligated product sequences generated by each of the plurality of target sequences within the sample, thereby providing a quantitative measure of the relative level of two or more target nucleotide sequences in the sample.",
"37.A method according to claim 26, wherein the target-specific portions of the oligonucleotide probe sets have substantially the same melting temperature so that they hybridize to target nucleotide sequences under similar hybridization conditions.",
"38.A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the support at different sites, are detected, thereby indicating a presence, in the sample of one or more allele at one or more nucleotide position in one or more target nucleotide sequences.",
"39.A method according to claim 38, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation.",
"40.A method according to claim 38, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished with oligonucleotide probe groups having oligonucleotide probes with target-specific portions which overlap.",
"41.A method according to claim 40, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with such ligation.",
"42.A method according to claim 26, wherein multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in a single target nucleotide sequence or multiple allele differences consisting of insertions, deletions, microsatellite repeats, translocations, or other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group, captured on the support at different sites, are detected, thereby indicating a presence, in the sample, of one or more allele differences selected from the group consisting of insertions, deletions, microsatellite repeats, translocations, and other DNA rearrangements in one or more target nucleotide sequences.",
"43.A method according to claim 42, wherein the oligonucleotide probe sets are designed for distinguishing multiple allele differences selected from the group consisting of insertions, deletions, and microsatellite repeats, at one or more nucleotide positions which require overlapping oligonucleotide probe sets, wherein, in the oligonucleotide probe sets of each group, the second oligonucleotide probes have a common target-specific portion, and the first oligonucleotide probes have differing target-specific portions which contain repetitive sequences of different lengths to hybridize to a given allele in a base-specific manner.",
"44.A method according to claim 26, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are distinguished, the oligonucleotide probe sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein one or more sets within a group share common second oligonucleotide probes and the first oligonucleotide probes have differing target-specific portions which hybridize to a given allele excluding the normal allele in a base-specific manner, wherein, in said detecting, the labels of ligated product sequences of each group captured on the support at different sites, are detected, thereby indicating a presence, in the sample, of one or more low abundance alleles at one or more nucleotide positions in one or more target nucleotide sequences.",
"45.A method according to claim 44, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.",
"46.A method according to claim 44, wherein a low abundance of multiple allele differences at multiple adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, in the presence of an excess of normal sequence, or a low abundance of multiple allele differences at multiple nucleotide positions which require overlapping oligonucleotide probe sets in multiple target nucleotide sequences, in the presence of an excess of normal sequence, are quantified in a sample, said method further comprising: providing a known amount of one or more marker target nucleotide sequences; providing a plurality of marker-specific oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe having a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label, wherein the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence, but, when hybridized to any other nucleotide sequence present in the sample or added marker sequences, have a mismatch which interferes with such ligation; providing a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets or marker-specific oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, including marker nucleotide sequences, wherein one or more sets within a group share a common second oligonucleotide probe and the first oligonucleotide probes have different target-specific probe portions which hybridize to a given allele or a marker nucleotide sequence excluding the normal allele, in a base-specific manner, wherein said blending comprises blending the sample, the marker target nucleotide sequences, the plurality of oligonucleotide probe sets, the plurality of marker-specific oligonucleotide probe sets, and the ligase to form a mixture; detecting the reporter labels of the ligated marker-specific oligonucleotide sets captured on the support at particular sites, thereby indicating the presence of one or more marker target nucleotide sequences in the sample; and quantifying the amount of target nucleotide sequences in the sample by comparing the amount of captured ligated products generated from the known amount of marker target nucleotide sequences with the amount of other captured ligated product generated from the low abundance unknown sample.",
"47.A method according to claim 46, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence under selected conditions due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.",
"48.A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of each group, the first oligonucleotide probes have a common target-specific portion and the second oligonucleotide probes have differing target-specific portions which hybridize to a given allele in a base-specific manner, wherein, in said detecting, different reporter labels of ligated product sequences of each group captured on the support at particular sites are detected, thereby indicating a presence, in the sample, of one or more alleles at one or more nucleotide positions in one or more target nucleotide sequences.",
"49.A method according to claim 48, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.",
"50.A method according to claim 48, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a single target nucleotide sequence, or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.",
"51.A method according to claim 50, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, the second oligonucleotide probe has a mismatch at a base at the ligation junction which interferes with such ligation.",
"52.A method according to claim 26, wherein multiple allele differences at one or more nucleotide position in a single target nucleotide sequence or multiple allele differences at one or more positions in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing multiple allele differences at a single nucleotide position, wherein, in the oligonucleotide probes of different groups, the second oligonucleotide probes have a common target-specific portion or the first oligonucleotide probes have a common target-specific portion, wherein, in said detecting, the one of a plurality of labeled ligated product sequences of each group captured on the support at particular sites are detected, thereby indicating a presence of one or more allele at one or more nucleotide positions in one or more target nucleotide sequences in the sample.",
"53.A method according to claim 52, wherein the oligonucleotide probes in a given set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.",
"54.A method according to claim 52, wherein multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in a target nucleotide sequence or multiple allele differences at two or more adjacent nucleotide positions, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences, are distinguished, the oligonucleotide probe groups containing probes with target-specific portions which overlap.",
"55.A method according to claim 54, wherein oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first or second oligonucleotide probes have a mismatch at a base at the ligation junction which interferes with such ligation.",
"56.A method according to claim 53, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, all possible single-base mutations for multiple codons in a single target nucleotide sequence, and all possible single-base mutations for multiple codons in multiple target nucleotide sequences are distinguished, the oligonucleotide sets forming a plurality of oligonucleotide probe groups, each group comprised of one or more oligonucleotide probe sets designed for distinguishing all possible single-base mutations for a single codon, wherein, in the oligonucleotide probes of each group, the second oligonucleotide probes differ only in their 5′ bases at their ligation junction and contain different reporter labels, the first oligonucleotide probes differ only in their 3′ bases at their ligation junction and contain different addressable array-specific portions, or the first oligonucleotide probes differ only in their 3′ bases adjacent to the base at the ligation junction and contain different addressable array-specific portions.",
"57.A method according to claim 53, wherein the oligonucleotide probes in a set are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction, but, when the oligonucleotides in the set are hybridized to any other nucleotide sequence present in the sample, the first oligonucleotide probes have a mismatch at the 3′ base at the ligation junction or the 3′ base adjacent the base at the ligation junction or the second oligonucleotide probes have a mismatch at the 5′ base at the ligation junction which interferes with such ligation.",
"58.A method according to claim 57, wherein all possible single-base mutations for a single codon in a single target nucleotide sequence, or all possible single-base mutations for two or more adjacent codons, or at nucleotide positions which require overlapping oligonucleotide probe sets, in multiple target nucleotide sequences are distinguished, the oligonucleotide probe groups containing oligonucleotide probes with target-specific portions which overlap.",
"59.A method according to claim 26, wherein the denaturation treatment is at a temperature of about 80°-105° C. 60.A method according to claim 26, wherein each cycle, comprising a denaturation treatment and a hybridization treatment, is from about 30 seconds to about five minutes long.",
"61.A method according to claim 26, wherein said subjecting is repeated for 2 to 50 cycles.",
"62.A method according to claim 26, wherein total time for said subjecting is 1 to 250 minutes.",
"63.A method according to claim 26, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.",
"64.A method according to claim 26, wherein the detectable reporter label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.",
"65.A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 20-85° C. 66.A method according to claim 26, wherein the target-specific portions of the oligonucleotide probes are 20 to 28 nucleotides long.",
"67.A method according to claim 26, wherein the mixture further includes a carrier DNA.",
"68.A method according to claim 26 further comprising: amplifying the target nucleotide sequences in the sample prior to said blending.",
"69.A method according to claim 68, wherein said amplifying is carried out by subjecting the sample to a polymerase-based amplifying procedure.",
"70.A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with DNA polymerase.",
"71.A method according to claim 68, when rein said polymerase-based amplifying procedure is carried out with reverse transcriptase.",
"72.A method according to claim 68, wherein said polymerase-based amplifying procedure is carried out with RNA polymerase.",
"73.A method according to claim 68, wherein said amplifying is carried out by subjecting the target nucleotide sequences in the sample to a ligase chain reaction process.",
"74.A method according to claim 26, wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleic acids, modified peptide nucleic acids, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.",
"75.A method according to claim 26, wherein said method is used to detect infectious diseases caused by bacterial, viral, parasitic, and fungal infectious agents.",
"76.A method according to claim 75, wherein the infectious disease is caused by a bacteria selected from the group consisting of Escherichia coli Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes.",
"77.A method according to claim 75, wherein the infectious disease is caused by a fungal infectious agent selected from the group consisting of Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis.",
"78.A method according to claim 75, wherein the infectious disease is caused by a viral infectious agent selected from the group consisting of human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.",
"79.A method according to claim 75, wherein the infectious disease is caused by a parasitic infectious agent selected from the group consisting of Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.",
"80.A method according to claim 26, wherein said method is used to detect genetic diseases.",
"81.A method according to claim 80, wherein the genetic disease is selected from the group consisting of 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Syndrome, thalassemia, Klinefelter's Syndrome, Huntington's Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors in metabolism, and diabetes.",
"82.A method according to claim 26, wherein said method is used to detect cancer involving oncogenes, tumor suppressor genes, or genes involved in DNA amplification, replication, recombination, or repair.",
"83.A method according to claim 82, wherein the cancer is associated with a gene selected from the group consisting of BRCA1 gene, p53 gene, Familial polyposis coli, Her2/Neu amplification, Bcr/Ab1, K-ras gene, human papillomavirus Types 16 and 18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, ENT tumors, and loss of heterozygosity.",
"84.A method according to claim 26, wherein said method is used for environmental monitoring, forensics, and food and feed industry monitoring.",
"85.A method according to claim 26, wherein said detecting comprises: scanning the support at the particular sites and identifying if ligation of the oligonucleotide probe sets occurred and correlating identified ligation to a presence or absence of the target nucleotide sequences.",
"86.A method according to claim 85, wherein said scanning is carried out by scanning electron microscopy, electron microscopy, confocal microscopy, charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging.",
"87.A method according to claim 85, wherein said correlating is carried out with a computer.",
"88.A method according to claim 26, wherein said contacting the mixture with the support is at a temperature of 45-90° C. and for a time period of up to 60 minutes.",
"89.A method according to claim 26, wherein some of the plurality of capture oligonucleotides have identical nucleotide sequences and different labels are used for some different target nucleotide sequence.",
"90.A method according to claim 26, wherein the plurality of capture oligonucleotides each have different nucleotide sequences.",
"91.A method according to claim 89, wherein each capture oligonucleotide has adjacent capture oligonucleotides separated from adjacent capture oligonucleotides by barrier oligonucleotides to which ligated oligonucleotide probe sets will not hybridize during said contacting.",
"92.A method according to claim 26, wherein the oligonucleotide probe sets hybridize to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portion of oligonucleotide probe sets.",
"93.A method according to claim 26 further comprising: treating the mixture chemically or enzymatically, after said subjecting the mixture to a series of ligase detection reaction cycles, to destroy unligated oligonucleotide probes.",
"94.A method according to claim 93, wherein said treating is carried out with an exonuclease.",
"95.A method according to claim 26 further comprising: removing oligonucleotides bound to the capture oligonucleotides to permit reuse of the support with immobilized capture oligonucleotides.",
"96.A method according to claim 26, wherein the support includes different capture oligonucleotides immobilized at different sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are captured and detected at different sites on the support.",
"97.A method according to claim 26, wherein the support includes identical capture oligonucleotides immobilized on the support with the capture oligonucleotides being complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the different oligonucleotide probe sets are detected and distinguished by the different labels.",
"98.A method according to claim 26, wherein the collection of double multimer units is shown in FIG.",
"26.99.A method according to claim 26, wherein the collection of double multimer units is shown in FIG.",
"27.100.A method according to claim 26, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.",
"101.A method according to claim 26, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84° C. 102.A method according to claim 26, wherein the set of tetramers is shown in Table 1 and complements thereof.",
"103.A method according to claim 26, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"104.A method according to claim 26, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"105.A method according to claim 26, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"106.A kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising: a ligase; a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybrided adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample; and a support with different capture oligonucleotides immobilized at different positions, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides, wherein oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.",
"107.A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequences is at a base at a ligation junction at which the oligonucleotide probe of each set ligate together when hybridized to their respective target nucleotide sequences.",
"108.A kit according to claim 106, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′ ends at the ligation junction.",
"109.A kit according to claim 106, wherein the mismatch of oligonucleotide probe sets to nucleotide sequences other than their respective target nucleotide sequence is at a base adjacent to a ligation junction at which the oligonucleotide probes of each set ligate together when hybridized to their respective target nucleotide sequences.",
"110.A kit according to claim 109, wherein the mismatch is on the oligonucleotide probe of the oligonucleotide probe sets which have 3′ ends at the ligation junction.",
"111.A kit according to claim 106, wherein the ligase is selected from the group consisting of Thermus aquaticus ligase, Thermus thermophilus ligase, E. coli ligase, T4 ligase, and Pyrococcus ligase.",
"112.A kit according to claim 106 further comprising: amplification primers suitable for preliminary amplification of the target nucleotide sequences and a polymerase.",
"113.A kit according to claim 106, wherein the support includes different capture oligonucleotides immobilized at different particular sites with different capture oligonucleotides being complementary to different addressable array-specific portions, whereby different oligonucleotide probe sets are hybridized and detected at different sites on the support.",
"114.A kit according to claim 106, wherein the support includes identical capture oligonucleotides immobilized on the support with the capture oligonucleotides complementary to all the addressable array-specific portions and the labels attached to the oligonucleotide probe sets being different, whereby the oligonucleotide probe sets are detected and distinguished by the different labels.",
"115.A kit according to claim 106, wherein the oligonucleotide probe sets and the capture oligonucleotides are configured so that the oligonucleotide probe sets hybridize, respectively, to the target nucleotide sequences at temperatures which are less than that at which the capture oligonucleotides hybridize to the addressable array-specific portions of the oligonucleotide probes sets.",
"116.A kit according to claim 106, wherein the collection of double multimer units is shown in FIG.",
"26.117.A kit according to claim 106, wherein the collection of double multimer units is shown in FIG.",
"27.118.A kit according to claim 106, wherein the collection of double multimer units removed has a melting temperature in ° C. of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers.",
"119.A kit according to claim 106, wherein the double multimer units have 24 mers and the melting point of the double multimer units is 75-84° C. 120.A kit according to claim 106, wherein the set of tetramers is shown in Table 1 or complements thereof.",
"121.A kit according to claim 106, wherein the set of tetramers are one base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"122.A kit according to claim 106, wherein the set of tetramers are two base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"123.A kit according to claim 106, wherein the set of tetramers are three base circular permutations of the tetramers shown in Table 1 and complements thereof.",
"124.A method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support comprising comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Detection of Sequence Differences Large-scale multiplex analysis of highly polymorphic loci is needed for practical identification of individuals, e.g., for paternity testing and in forensic science (Reynolds et al., Anal.",
"Chem., 63: 2-15 (1991)), for organ-transplant donor-recipient matching (Buyse et al., Tissue Antigens, 41: 1-14 (1993) and Gyllensten et al., PCR Meth.",
"Appl, 1: 91-98 (1991)), for genetic disease diagnosis, prognosis, and pre-natal counseling (Chamberlain et al., Nucleic Acids Res., 16: 11141-11156 (1988) and L. C. Tsui, Human Mutat., 1: 197-203 (1992)), and the study of oncogenic mutations (Hollstein et al., Science, 253: 49-53 (1991)).",
"In addition, the cost-effectiveness of infectious disease diagnosis by nucleic acid analysis varies directly with the multiplex scale in panel testing.",
"Many of these applications depend on the discrimination of single-base differences at a multiplicity of sometimes closely space loci.",
"A variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotide sequences in a sample containing a large number of sequence regions.",
"In a simple method, which relies on fragment capture and labeling, a fragment containing a selected sequence is captured by hybridization to an immobilized probe.",
"The captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter moiety.",
"Another widely used method is Southern blotting.",
"In this method, a mixture of DNA fragments in a sample are fractionated by gel electrophoresis, then fixed on a nitrocellulose filter.",
"By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequence can be identified.",
"The method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contain a given probe sequence, and for analyzing restriction-fragment length polymorphisms (“RFLPs”).",
"Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequence(s) by polymerase chain reaction.",
"U.S. Pat.",
"No.",
"4,683,202 to Mullis, et al.",
"and R. K. Saiki, et al., Science 230: 1350 (1985).",
"In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication.",
"The amplified sequence may be readily identified by a variety of techniques.",
"This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample.",
"More recently, methods of identifying known target sequences by probe ligation methods have been reported.",
"U.S. Pat.",
"No.",
"4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4: 560 (1989), U. Landegren, et al., Science 241: 1077 (1988), and E. Winn-Deen, et al., Clin.",
"Chem.",
"37: 1522 (1991).",
"In one approach, known as oligonucleotide ligation assay (“OLA”), two probes or probe elements which span a target region of interest are hybridized with the target region.",
"Where the probe elements match (basepair with) adjacent target bases at the confronting ends of the probe elements, the two elements can be joined by ligation, e.g., by treatment with ligase.",
"The ligated probe element is then assayed, evidencing the presence of the target sequence.",
"In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements.",
"With continued cycles of denaturation, hybridization, and ligation in the presence of the two complementary pairs of probe elements, the target sequence is amplified exponentially, i.e., exponentially allowing very small amounts of target sequence to be detected and/or amplified.",
"This approach is referred to as ligase chain reaction (“LCR”).",
"F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc.",
"Nat'l Acad.",
"Sci.",
"USA, 88: 189-93 (1991) and F. Barany, “The Ligase Chain Reaction (LCR) in a PCR World,” PCR Methods and Applications, 1: 5-16 (1991).",
"Another scheme for multiplex detection of nucleic acid sequence differences is disclosed in U.S. Pat.",
"No.",
"5,470,705 to Grossman et. al.",
"where sequence-specific probes, having a detectable label and a distinctive ratio of charge/translational frictional drag, can be hybridized to a target and ligated together.",
"This technique was used in Grossman, et.",
"al., “High-density Multiplex Detection of Nucleic Acid Sequences: Oligonucleotide Ligation Assay and Sequence-coded Separation,” Nucl.",
"Acids Res.",
"22(21): 4527-34 (1994) for the large scale multiplex analysis of the cystic fibrosis transmembrane regulator gene.",
"Jou, et.",
"al., “Deletion Detection in Dystrophin Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology,” Human Mutation 5: 86-93 (1995) relates to the use of a so called “gap ligase chain reaction” process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.",
"There is a growing need, e.g., in the field of genetic screening, for methods useful in detecting the presence or absence of each of a large number of sequences in a target polynucleotide.",
"For example, as many as 400 different mutations have been associated with cystic fibrosis.",
"In screening for genetic predisposition to this disease, it is optimal to test all of the possible different gene sequence mutations in the subject's genomic DNA, in order to make a positive identification of “cystic fibrosis”.",
"It would be ideal to test for the presence or absence of all of the possible mutation sites in a single assay.",
"However, the prior-art methods described above are not readily adaptable for use in detecting multiple selected sequences in a convenient, automated single-assay format.",
"Solid-phase hybridization assays require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to keep the stringency needed for single-nucleotide mismatch discrimination.",
"Multiplexing of this approach has proven difficult as optimal hybridization conditions vary greatly among probe sequences.",
"Allele-specific PCR products generally have the same size, and a given amplification tube is scored by the presence or absence of the product band in the gel lane associated with each reaction tube.",
"Gibbs et al., Nucleic Acids Res., 17: 2437-2448 (1989).",
"This approach requires splitting the test sample among multiple reaction tubes with different primer combinations, multiplying assay cost.",
"PCR has also discriminated alleles by attaching different fluorescent dyes to competing allelic primers in a single reaction tube (F. F. Chehab, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 86: 9178-9182 (1989)), but this route to multiplex analysis is limited in scale by the relatively few dyes which can be spectrally resolved in an economical manner with existing instrumentation and dye chemistry.",
"The incorporation of bases modified with bulky side chains can be used to differentiate allelic PCR products by their electrophoretic mobility, but this method is limited by the successful incorporation of these modified bases by polymerase, and by the ability of electrophoresis to resolve relatively large PCR products which differ in size by only one of these groups.",
"Livak et al., Nucleic Acids Res., 20: 4831-4837 (1989).",
"Each PCR product is used to look for only a single mutation, making multiplexing difficult.",
"Ligation of allele-specific probes generally has used solid-phase capture (U. Landegren et al., Science, 241: 1077-1080 (1988); Nickerson et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 87: 8923-8927 (1990)) or size-dependent separation (D. Y. Wu, et al., Genomics, 4: 560-569 (1989) and F. Barany, Proc.",
"Natl.",
"Acad.",
"Sci., 88: 189-193 (1991)) to resolve the allelic signals, the latter method being limited in multiplex scale by the narrow size range of ligation probes.",
"The gap ligase chain reaction process requires an additional step—polymerase extension.",
"The use of probes with distinctive ratios of charge/translational frictional drag technique to a more complex multiplex will either require longer electrophoresis times or the use of an alternate form of detection.",
"The need thus remains for a rapid single assay format to detect the presence or absence of multiple selected sequences in a polynucleotide sample.",
"Use of Oligonucleotide Arrays for Nucleic Acid Analysis Ordered arrays of oligonucleotides immobilized on a solid support have been proposed for sequencing, sorting, isolating, and manipulating DNA.",
"It has been recognized that hybridization of a cloned single-stranded DNA molecule to all possible oligonucleotide probes of a given length can theoretically identify the corresponding complementary DNA segments present in the molecule.",
"In such an array, each oligonucleotide probe is immobilized on a solid support at a different predetermined position.",
"All the oligonucleotide segments in a DNA molecule can be surveyed with such an array.",
"One example of a procedure for sequencing DNA molecules using arrays of oligonucleotides is disclosed in U.S. Pat.",
"No.",
"5,202,231 to Drmanac, et. al.",
"This involves application of target DNA to a solid support to which a plurality of oligonucleotides are attached.",
"Sequences are read by hybridization of segments of the target DNA to the oligonucleotides and assembly of overlapping segments of hybridized oligonucleotides.",
"The array utilizes all possible oligonucleotides of a certain length between 11 and 20 nucleotides, but there is little information about how this array is constructed.",
"See also A.",
"B. Chetverin, et.",
"al., “Sequencing of Pools of Nucleic Acids on Oligonucleotide Arrays,” BioSystems 30: 215-31 (1993); WO 92/16655 to Khrapko et.",
"al.",
"; Kuznetsova, et.",
"al., “DNA Sequencing by Hybridization with Oligonucleotides Immobilized in Gel.",
"Chemical Ligation as a Method of Expanding the Prospects for the Method,” Mol.",
"Biol.",
"28(20): 290-99(1994); M. A. Livits, et.",
"al., “Dissociation of Duplexes Formed by Hybridization of DNA with Gel-Immobilized Oligonucleotides,” J. Biomolec.",
"Struct .",
"& Dynam.",
"11(4): 783-812 (1994).",
"WO 89/10977 to Southern discloses the use of a support carrying an array of oligonucleotides capable of undergoing a hybridization reaction for use in analyzing a nucleic acid sample for known point mutations, genomic fingerprinting, linkage analysis, and sequence determination.",
"The matrix is formed by laying nucleotide bases in a selected pattern on the support.",
"This reference indicates that a hydroxyl linker group can be applied to the support with the oligonucleotides being assembled by a pen plotter or by masking.",
"WO 94/11530 to Cantor also relates to the use of an oligonucleotide array to carry out a process of sequencing by hybridization.",
"The oligonucleotides are duplexes having overhanging ends to which target nucleic acids bind and are then ligated to the non-overhanging portion of the duplex.",
"The array is constructed by using streptavidin-coated filter paper which captures biotinylated oligonucleotides assembled before attachment.",
"WO 93/17126 to Chetverin uses sectioned, binary oligonucleotide arrays to sort and survey nucleic acids.",
"These arrays have a constant nucleotide sequence attached to an adjacent variable nucleotide sequence, both bound to a solid support by a covalent linking moiety.",
"The constant nucleotide sequence has a priming region to permit amplification by PCR of hybridized stands.",
"Sorting is then carried out by hybridization to the variable region.",
"Sequencing, isolating, sorting, and manipulating fragmented nucleic acids on these binary arrays are also disclosed.",
"In one embodiment with enhanced sensitivity, the immobilized oligonucleotide has a shorter complementary region hybridized to it, leaving part of the oligonucleotide uncovered.",
"The array is then subjected to hybridization conditions so that a complementary nucleic acid anneals to the immobilized oligonucleotide.",
"DNA ligase is then used to join the shorter complementary region and the complementary nucleic acid on the array.",
"There is little disclosure of how to prepare the arrays of oligonucleotides.",
"WO 92/10588 to Fodor et.",
"al., discloses a process for sequencing, fingerprinting, and mapping nucleic acids by hybridization to an array of oligonucleotides.",
"The array of oligonucleotides is prepared by a very large scale immobilized polymer synthesis which permits the synthesis of large, different oligonucleotides.",
"In this procedure, the substrate surface is functionalized and provided with a linker group by which oligonucleotides are assembled on the substrate.",
"The regions where oligonucleotides are attached have protective groups (on the substrate or individual nucleotide subunits) which are selectively activated.",
"Generally, this involves imaging the array with light using a mask of varying configuration so that areas exposed are deprotected.",
"Areas which have been deprotected undergo a chemical reaction with a protected nucleotide to extend the oligonucleotide sequence where imaged.",
"A binary masking strategy can be used to build two or more arrays at a given time.",
"Detection involves positional localization of the region where hybridization has taken place.",
"See also U.S. Pat.",
"Nos.",
"5,324,633 and 5,424,186 to Fodor et.",
"al., U.S. Pat.",
"Nos.",
"5,143,854 and 5,405,783 to Pirrung, et.",
"al., WO 90/15070 to Pirrung, et.",
"al., A. C. Pease, et.",
"al., “Light-generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis”, Proc.",
"Natl.",
"Acad.",
"Sci USA 91: 5022-26 (1994).",
"K. L. Beattie, et.",
"al., “Advances in Genosensor Research,” Clin.",
"Chem.",
"41(5): 700-09 (1995) discloses attachment of previously assembled oligonucleotide probes to a solid support.",
"There are many drawbacks to the procedures for sequencing by hybridization to such arrays.",
"Firstly, a very large number of oligonucleotides must be synthesized.",
"Secondly, there is poor discrimination between correctly hybridized, properly matched duplexes and those which are mismatched.",
"Finally, certain oligonucleotides will be difficult to hybridize to under standard conditions, with such oligonucleotides being capable of identification only through extensive hybridization studies.",
"The present invention is directed toward overcoming these deficiencies in the art."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range.",
"The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides.",
"Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units.",
"From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units.",
"The modified collection of multimer units is arranged in a list in order of melting temperature.",
"The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature.",
"Alternating multimer units in the list are then divided into first and second subcollections, each arranged in order of melting temperature.",
"After the order of the second subcollection is inverted, the first collection is linked in order to the inverted second collection to form a collection of double multimer units.",
"From the collection of double multimer units those units (1) having a melting temperature in ° C. less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption are removed, to form a modified collection of double multimer units.",
"Another aspect of the present invention relates to an oligonucleotide array which includes a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the solid support can be captured at the different positions.",
"The complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption.",
"Yet another aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.",
"A plurality of oligonucleotide probe sets are also provided with each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label.",
"The oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample.",
"A ligase is also provided with the sample, the plurality of oligonucleotide probe sets, and the ligase being blended to form a mixture.",
"The mixture is subjected to one or more ligase detection reaction cycles comprising a denaturation treatment, where any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, where the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label.",
"The oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment.",
"A support is provided with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units.",
"After subjecting the mixture to one or more ligase detection reaction cycles, the mixture is contacted with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide.",
"The reporter labels of ligated product sequences captured on the support at particular sites are detected, indicating the presence of one or more target nucleotide sequences in the sample.",
"Another aspect of the present invention is directed to a kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"In addition, to a ligase, the kit includes a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample.",
"Also found in the kit is a support with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times then number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.",
"Another aspect of the present invention relates to a method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support.",
"This method includes comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides."
],
[
"This invention was developed with government funding under National Institutes of Health Grant Nos.",
"GM-41337-06, GM43552-05, GM-42722-07, and GM-51628-02.The U.S. Government may have certain rights.",
"FIELD OF THE INVENTION The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range.",
"Other aspects of the present invention relate to a support with the plurality of oligonucleotide probes immobilized on the support, a method of using the support to detect single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences, and a kit for such detection, which includes the support on which the oligonucleotides have been immobilized.",
"BACKGROUND OF THE INVENTION Detection of Sequence Differences Large-scale multiplex analysis of highly polymorphic loci is needed for practical identification of individuals, e.g., for paternity testing and in forensic science (Reynolds et al., Anal.",
"Chem., 63: 2-15 (1991)), for organ-transplant donor-recipient matching (Buyse et al., Tissue Antigens, 41: 1-14 (1993) and Gyllensten et al., PCR Meth.",
"Appl, 1: 91-98 (1991)), for genetic disease diagnosis, prognosis, and pre-natal counseling (Chamberlain et al., Nucleic Acids Res., 16: 11141-11156 (1988) and L. C. Tsui, Human Mutat., 1: 197-203 (1992)), and the study of oncogenic mutations (Hollstein et al., Science, 253: 49-53 (1991)).",
"In addition, the cost-effectiveness of infectious disease diagnosis by nucleic acid analysis varies directly with the multiplex scale in panel testing.",
"Many of these applications depend on the discrimination of single-base differences at a multiplicity of sometimes closely space loci.",
"A variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotide sequences in a sample containing a large number of sequence regions.",
"In a simple method, which relies on fragment capture and labeling, a fragment containing a selected sequence is captured by hybridization to an immobilized probe.",
"The captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter moiety.",
"Another widely used method is Southern blotting.",
"In this method, a mixture of DNA fragments in a sample are fractionated by gel electrophoresis, then fixed on a nitrocellulose filter.",
"By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequence can be identified.",
"The method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contain a given probe sequence, and for analyzing restriction-fragment length polymorphisms (“RFLPs”).",
"Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequence(s) by polymerase chain reaction.",
"U.S. Pat.",
"No.",
"4,683,202 to Mullis, et al.",
"and R. K. Saiki, et al., Science 230: 1350 (1985).",
"In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication.",
"The amplified sequence may be readily identified by a variety of techniques.",
"This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample.",
"More recently, methods of identifying known target sequences by probe ligation methods have been reported.",
"U.S. Pat.",
"No.",
"4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4: 560 (1989), U. Landegren, et al., Science 241: 1077 (1988), and E. Winn-Deen, et al., Clin.",
"Chem.",
"37: 1522 (1991).",
"In one approach, known as oligonucleotide ligation assay (“OLA”), two probes or probe elements which span a target region of interest are hybridized with the target region.",
"Where the probe elements match (basepair with) adjacent target bases at the confronting ends of the probe elements, the two elements can be joined by ligation, e.g., by treatment with ligase.",
"The ligated probe element is then assayed, evidencing the presence of the target sequence.",
"In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements.",
"With continued cycles of denaturation, hybridization, and ligation in the presence of the two complementary pairs of probe elements, the target sequence is amplified exponentially, i.e., exponentially allowing very small amounts of target sequence to be detected and/or amplified.",
"This approach is referred to as ligase chain reaction (“LCR”).",
"F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc.",
"Nat'l Acad.",
"Sci.",
"USA, 88: 189-93 (1991) and F. Barany, “The Ligase Chain Reaction (LCR) in a PCR World,” PCR Methods and Applications, 1: 5-16 (1991).",
"Another scheme for multiplex detection of nucleic acid sequence differences is disclosed in U.S. Pat.",
"No.",
"5,470,705 to Grossman et. al.",
"where sequence-specific probes, having a detectable label and a distinctive ratio of charge/translational frictional drag, can be hybridized to a target and ligated together.",
"This technique was used in Grossman, et.",
"al., “High-density Multiplex Detection of Nucleic Acid Sequences: Oligonucleotide Ligation Assay and Sequence-coded Separation,” Nucl.",
"Acids Res.",
"22(21): 4527-34 (1994) for the large scale multiplex analysis of the cystic fibrosis transmembrane regulator gene.",
"Jou, et.",
"al., “Deletion Detection in Dystrophin Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology,” Human Mutation 5: 86-93 (1995) relates to the use of a so called “gap ligase chain reaction” process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.",
"There is a growing need, e.g., in the field of genetic screening, for methods useful in detecting the presence or absence of each of a large number of sequences in a target polynucleotide.",
"For example, as many as 400 different mutations have been associated with cystic fibrosis.",
"In screening for genetic predisposition to this disease, it is optimal to test all of the possible different gene sequence mutations in the subject's genomic DNA, in order to make a positive identification of “cystic fibrosis”.",
"It would be ideal to test for the presence or absence of all of the possible mutation sites in a single assay.",
"However, the prior-art methods described above are not readily adaptable for use in detecting multiple selected sequences in a convenient, automated single-assay format.",
"Solid-phase hybridization assays require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to keep the stringency needed for single-nucleotide mismatch discrimination.",
"Multiplexing of this approach has proven difficult as optimal hybridization conditions vary greatly among probe sequences.",
"Allele-specific PCR products generally have the same size, and a given amplification tube is scored by the presence or absence of the product band in the gel lane associated with each reaction tube.",
"Gibbs et al., Nucleic Acids Res., 17: 2437-2448 (1989).",
"This approach requires splitting the test sample among multiple reaction tubes with different primer combinations, multiplying assay cost.",
"PCR has also discriminated alleles by attaching different fluorescent dyes to competing allelic primers in a single reaction tube (F. F. Chehab, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 86: 9178-9182 (1989)), but this route to multiplex analysis is limited in scale by the relatively few dyes which can be spectrally resolved in an economical manner with existing instrumentation and dye chemistry.",
"The incorporation of bases modified with bulky side chains can be used to differentiate allelic PCR products by their electrophoretic mobility, but this method is limited by the successful incorporation of these modified bases by polymerase, and by the ability of electrophoresis to resolve relatively large PCR products which differ in size by only one of these groups.",
"Livak et al., Nucleic Acids Res., 20: 4831-4837 (1989).",
"Each PCR product is used to look for only a single mutation, making multiplexing difficult.",
"Ligation of allele-specific probes generally has used solid-phase capture (U. Landegren et al., Science, 241: 1077-1080 (1988); Nickerson et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 87: 8923-8927 (1990)) or size-dependent separation (D. Y. Wu, et al., Genomics, 4: 560-569 (1989) and F. Barany, Proc.",
"Natl.",
"Acad.",
"Sci., 88: 189-193 (1991)) to resolve the allelic signals, the latter method being limited in multiplex scale by the narrow size range of ligation probes.",
"The gap ligase chain reaction process requires an additional step—polymerase extension.",
"The use of probes with distinctive ratios of charge/translational frictional drag technique to a more complex multiplex will either require longer electrophoresis times or the use of an alternate form of detection.",
"The need thus remains for a rapid single assay format to detect the presence or absence of multiple selected sequences in a polynucleotide sample.",
"Use of Oligonucleotide Arrays for Nucleic Acid Analysis Ordered arrays of oligonucleotides immobilized on a solid support have been proposed for sequencing, sorting, isolating, and manipulating DNA.",
"It has been recognized that hybridization of a cloned single-stranded DNA molecule to all possible oligonucleotide probes of a given length can theoretically identify the corresponding complementary DNA segments present in the molecule.",
"In such an array, each oligonucleotide probe is immobilized on a solid support at a different predetermined position.",
"All the oligonucleotide segments in a DNA molecule can be surveyed with such an array.",
"One example of a procedure for sequencing DNA molecules using arrays of oligonucleotides is disclosed in U.S. Pat.",
"No.",
"5,202,231 to Drmanac, et. al.",
"This involves application of target DNA to a solid support to which a plurality of oligonucleotides are attached.",
"Sequences are read by hybridization of segments of the target DNA to the oligonucleotides and assembly of overlapping segments of hybridized oligonucleotides.",
"The array utilizes all possible oligonucleotides of a certain length between 11 and 20 nucleotides, but there is little information about how this array is constructed.",
"See also A.",
"B. Chetverin, et.",
"al., “Sequencing of Pools of Nucleic Acids on Oligonucleotide Arrays,” BioSystems 30: 215-31 (1993); WO 92/16655 to Khrapko et.",
"al.",
"; Kuznetsova, et.",
"al., “DNA Sequencing by Hybridization with Oligonucleotides Immobilized in Gel.",
"Chemical Ligation as a Method of Expanding the Prospects for the Method,” Mol.",
"Biol.",
"28(20): 290-99(1994); M. A. Livits, et.",
"al., “Dissociation of Duplexes Formed by Hybridization of DNA with Gel-Immobilized Oligonucleotides,” J. Biomolec.",
"Struct.",
"& Dynam.",
"11(4): 783-812 (1994).",
"WO 89/10977 to Southern discloses the use of a support carrying an array of oligonucleotides capable of undergoing a hybridization reaction for use in analyzing a nucleic acid sample for known point mutations, genomic fingerprinting, linkage analysis, and sequence determination.",
"The matrix is formed by laying nucleotide bases in a selected pattern on the support.",
"This reference indicates that a hydroxyl linker group can be applied to the support with the oligonucleotides being assembled by a pen plotter or by masking.",
"WO 94/11530 to Cantor also relates to the use of an oligonucleotide array to carry out a process of sequencing by hybridization.",
"The oligonucleotides are duplexes having overhanging ends to which target nucleic acids bind and are then ligated to the non-overhanging portion of the duplex.",
"The array is constructed by using streptavidin-coated filter paper which captures biotinylated oligonucleotides assembled before attachment.",
"WO 93/17126 to Chetverin uses sectioned, binary oligonucleotide arrays to sort and survey nucleic acids.",
"These arrays have a constant nucleotide sequence attached to an adjacent variable nucleotide sequence, both bound to a solid support by a covalent linking moiety.",
"The constant nucleotide sequence has a priming region to permit amplification by PCR of hybridized stands.",
"Sorting is then carried out by hybridization to the variable region.",
"Sequencing, isolating, sorting, and manipulating fragmented nucleic acids on these binary arrays are also disclosed.",
"In one embodiment with enhanced sensitivity, the immobilized oligonucleotide has a shorter complementary region hybridized to it, leaving part of the oligonucleotide uncovered.",
"The array is then subjected to hybridization conditions so that a complementary nucleic acid anneals to the immobilized oligonucleotide.",
"DNA ligase is then used to join the shorter complementary region and the complementary nucleic acid on the array.",
"There is little disclosure of how to prepare the arrays of oligonucleotides.",
"WO 92/10588 to Fodor et.",
"al., discloses a process for sequencing, fingerprinting, and mapping nucleic acids by hybridization to an array of oligonucleotides.",
"The array of oligonucleotides is prepared by a very large scale immobilized polymer synthesis which permits the synthesis of large, different oligonucleotides.",
"In this procedure, the substrate surface is functionalized and provided with a linker group by which oligonucleotides are assembled on the substrate.",
"The regions where oligonucleotides are attached have protective groups (on the substrate or individual nucleotide subunits) which are selectively activated.",
"Generally, this involves imaging the array with light using a mask of varying configuration so that areas exposed are deprotected.",
"Areas which have been deprotected undergo a chemical reaction with a protected nucleotide to extend the oligonucleotide sequence where imaged.",
"A binary masking strategy can be used to build two or more arrays at a given time.",
"Detection involves positional localization of the region where hybridization has taken place.",
"See also U.S. Pat.",
"Nos.",
"5,324,633 and 5,424,186 to Fodor et.",
"al., U.S. Pat.",
"Nos.",
"5,143,854 and 5,405,783 to Pirrung, et.",
"al., WO 90/15070 to Pirrung, et.",
"al., A. C. Pease, et.",
"al., “Light-generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis”, Proc.",
"Natl.",
"Acad.",
"Sci USA 91: 5022-26 (1994).",
"K. L. Beattie, et.",
"al., “Advances in Genosensor Research,” Clin.",
"Chem.",
"41(5): 700-09 (1995) discloses attachment of previously assembled oligonucleotide probes to a solid support.",
"There are many drawbacks to the procedures for sequencing by hybridization to such arrays.",
"Firstly, a very large number of oligonucleotides must be synthesized.",
"Secondly, there is poor discrimination between correctly hybridized, properly matched duplexes and those which are mismatched.",
"Finally, certain oligonucleotides will be difficult to hybridize to under standard conditions, with such oligonucleotides being capable of identification only through extensive hybridization studies.",
"The present invention is directed toward overcoming these deficiencies in the art.",
"SUMMARY OF THE INVENTION The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range.",
"The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides.",
"Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units.",
"From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units.",
"The modified collection of multimer units is arranged in a list in order of melting temperature.",
"The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature.",
"Alternating multimer units in the list are then divided into first and second subcollections, each arranged in order of melting temperature.",
"After the order of the second subcollection is inverted, the first collection is linked in order to the inverted second collection to form a collection of double multimer units.",
"From the collection of double multimer units those units (1) having a melting temperature in ° C. less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption are removed, to form a modified collection of double multimer units.",
"Another aspect of the present invention relates to an oligonucleotide array which includes a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the solid support can be captured at the different positions.",
"The complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption.",
"Yet another aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.",
"A plurality of oligonucleotide probe sets are also provided with each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label.",
"The oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample.",
"A ligase is also provided with the sample, the plurality of oligonucleotide probe sets, and the ligase being blended to form a mixture.",
"The mixture is subjected to one or more ligase detection reaction cycles comprising a denaturation treatment, where any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, where the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label.",
"The oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment.",
"A support is provided with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units.",
"After subjecting the mixture to one or more ligase detection reaction cycles, the mixture is contacted with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide.",
"The reporter labels of ligated product sequences captured on the support at particular sites are detected, indicating the presence of one or more target nucleotide sequences in the sample.",
"Another aspect of the present invention is directed to a kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"In addition, to a ligase, the kit includes a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample.",
"Also found in the kit is a support with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times then number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.",
"Another aspect of the present invention relates to a method to avoid synthesizing ligase detection reaction oligonucleotides that will inappropriately cross-hybridize to capture oligonucleotides on a solid support.",
"This method includes comparing the ligase detection reaction oligonucleotides with the capture oligonucleotides and identifying any capture oligonucleotides likely to cross-hybridize to the ligase detection reaction oligonucleotides.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a flow diagram depicting polymerase chain reaction (“PCR”)/ligase detection reaction (“LDR”) processes, according to the prior art and the present invention, for detection of germline mutations, such as point mutations.",
"FIG.",
"2 is a flow diagram depicting PCR/LDR processes, according to the prior art and the present invention, for detection of cancer-associated mutations.",
"FIG.",
"3 is a schematic diagram depicting a PCR/LDR process, according to the present invention, using addresses on the allele-specific probes for detecting homo- or heterozygosity at two polymorphisms (i.e.",
"allele differences) on the same gene.",
"FIG.",
"4 is a schematic diagram depicting a PCR/LDR process according to the present invention, using addresses on common probes for detecting homo- or heterozygosity at two polymorphisms (i.e.",
"allele differences) on the same gene.",
"FIG.",
"5 is a schematic diagram depicting a PCR/LDR process, according to the present invention, using addresses on the allele-specific probes which distinguishes all possible bases at a given site.",
"FIG.",
"6 is a schematic diagram depicting a PCR/LDR process, according to the present invention, using addresses on the common probes which distinguishes all possible bases at a given site.",
"FIG.",
"7 is a schematic diagram depicting a PCR/LDR process, according to the present invention, using addresses on the allele-specific probes for detecting the presence of any possible base at two nearby sites.",
"FIG.",
"8 is a schematic diagram depicting a PCR/LDR process, according to the present invention, using addresses on the common probes for detecting the presence of any possible base at two nearby sites.",
"FIG.",
"9 is a schematic diagram of a PCR/LDR process, according to the present invention, using addresses on the allele-specific probes for distinguishing insertions and deletions.",
"FIG.",
"10 is a schematic diagram of a PCR/LDR process, according to the present invention, using addresses on the common probes for distinguishing insertions and deletions.",
"FIG.",
"11 is a schematic diagram of a PCR/LDR process, in accordance with the present invention, using addresses on the allele-specific probes to detect a low abundance mutation (within a codon) in the presence of an excess of normal sequence.",
"FIG.",
"12 is a schematic diagram of a PCR/LDR process, in accordance with the present invention, using addresses on the common probes to detect a low abundance mutation (within a codon) in the presence of an excess of normal sequence.",
"FIG.",
"13 is a schematic diagram of a PCR/LDR process, in accordance with the present invention, where the address is placed on the common probe and the allele differences are distinguished by different fluorescent signals F1, F2, F3, and F4.FIG.",
"14 is a schematic diagram of a PCR/LDR process, in accordance with the present invention, where both adjacent and nearby alleles are detected.",
"FIG.",
"15 is a schematic diagram of a PCR/LDR process, in accordance with the present invention, where all possible single-base mutations for a single codon are detected.",
"FIGS.",
"16A-P show the p53 chip hybridization and washing conditions.",
"FIGS.",
"17A-C show two alternative formats for oligonucleotide probe capture.",
"In FIG.",
"17B, the addressable array-specific portions are on the allele-specific probe.",
"Alleles are distinguished by capture of fluorescent signals on addresses Z1 and Z2, respectively.",
"In FIG.",
"17C, the addressable array-specific portions are on the common probe and alleles are distinguished by capture of fluorescent signals F1 and F2, which correspond to the two alleles, respectively.",
"FIG.",
"18 shows the chemical reactions for covalent modifications, grafting, oligomer attachments to solid supports.",
"FIG.",
"19 shows a design in accordance with the present invention using 36 tetramers differing by at least 2 bases, which can be used to create a series of unique 24-mers.",
"FIG.",
"20 shows an outline of the PCR/PCR/LDR method for detection of mutations in BRCA1 and BRCA2.FIGS.",
"21A-B show the multiplex LDR detection of 3 specific mutations in BRCA1 and BRCA2 in a gel-based assay.",
"FIG.",
"22 shows an outline of multiplex LDR detection of 3 specific mutations in BRCA1 and BRCA2 using an universal DNA microarray.",
"FIG.",
"23A-H show the LDR detection of 3 specific mutations in BRCA1 and BRCA2 on an addressable universal microarray.",
"FIG.",
"24A-H show p53 chip hybridization indicating the presence of mutations in DNA from colon tumors.",
"FIG.",
"25 shows a list of 4633 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"26 shows a list of 465 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"27 shows a list of 96 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"28 shows a list of 65 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"29 shows a list of 4633 capture oligonucleotides (in the form of 20 mer PNAs) produced in accordance with the present invention.",
"FIG.",
"30 shows a melting temperature (i.e.",
"Tm) distribution for a list of 96 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"31 shows a melting temperature (i.e.",
"Tm) distribution for a list of 465 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"32 shows a melting temperature (i.e.",
"Tm) distribution for a list of 4633 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"33 shows a sorted melting temperature (i.e.",
"Tm) distribution for a list of 4633 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"34 shows the tetramer usage in the lists of 65, 96, 465, and 4633 capture oligonucleotides produced in accordance with the present invention.",
"FIG.",
"35 sets forth a computer program for comparing a target sequence with an array capture probe to insure that the latter will be designed not to hybridize to the former.",
"FIGS.",
"36A-H show the LDR detection of 7 specific mutations in K-ras on an addressable universal microarray.",
"DETAILED DESCRIPTION OF THE INVENTION AND DRAWINGS The present invention is directed to a method of designing a plurality of capture oligonucleotide probes for use on a support to which complementary oligonucleotide probes will hybridize with little mismatch, where the plural capture oligonucleotide probes have melting temperatures within a narrow range.",
"The first step of the method involves providing a first set of a plurality of tetramers of four nucleotides linked together, where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, (3) no tetramers within a set are palindromic or dinucleotide repeats, and (4) no tetramer within a set has one or less or three or more G or C nucleotides.",
"Groups of 2 to 4 of the tetramers from the first set are linked together to form a collection of multimer units.",
"From the collection of multimer units, all multimer units formed from the same tetramer and all multimer units having a melting temperature in ° C. of less than 4 times the number of tetramers forming a multimer unit are removed to form a modified collection of multimer units.",
"The modified collection of multimer units is arranged in a list in order of melting temperature.",
"The order of the modified collection of multimer units is randomized in 2° C. increments of melting temperature.",
"Alternating multimer units in the list are then divided into first and second subcollections, each arranged in order of melting temperature.",
"After the order of the second subcollection is inverted, the first collection is linked in order to the inverted second collection to form a collection of double multimer units.",
"From the collection of double multimer units, those units (1) having a melting temperature in ° C. less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption are removed, to form a modified collection of double multimer units.",
"Another aspect of the present invention relates to an oligonucleotide array which includes a support and a collection of double multimer unit oligonucleotides at different positions on the support so that complementary oligonucleotides to be immobilized on the solid support can be captured at the different positions.",
"The complementary oligonucleotides will hybridize, within a narrow temperature range of greater than 24° C. with little mismatch, to members of the collection of double multimer unit oligonucleotides, the double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats, and the collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) multimer units with the same 4 tetramers linked together with or without interruption.",
"Yet another aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.",
"A plurality of oligonucleotide probe sets are also provided with each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label.",
"The oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample.",
"A ligase is also provided with the sample, the plurality of oligonucleotide probe sets, and the ligase being blended to form a mixture.",
"The mixture is subjected to one or more ligase detection reaction cycles comprising a denaturation treatment, where any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, where the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions connected together, and (c) the detectable reporter label.",
"The oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment.",
"A support is provided with capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of more than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, to form a modified collection of double multimer units.",
"After subjecting the mixture to one or more ligase detection reaction cycles, the mixture is contacted with the support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide.",
"The reporter labels of ligated product sequences captured on the support at particular sites are detected, indicating the presence of one or more target nucleotide sequences in the sample.",
"Another aspect of the present invention is directed to a kit for identifying one or more of a plurality of sequences differing by single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.",
"In addition, to a ligase, the kit includes a plurality oligonucleotide probe sets, each characterized by (a) a first oligonucleotide probe, having a target sequence-specific portion and an addressable array-specific portion, and (b) a second oligonucleotide probe, having a target sequence-specific portion and detectable reporter label, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a respective target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence, present in the sample.",
"Also found in the kit is a support with different capture oligonucleotides immobilized at different positions, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions and are formed from a collection of double multimer unit oligonucleotides.",
"The oligonucleotide with addressable array-specific portions will hybridize, within a narrow temperature range of greater than 4 times the number of tetramers in the multimer unit with little mismatch, to members of the capture oligonuncleotides.",
"The double multimer unit oligonucleotides are formed from sets of tetramers where (1) each tetramer within the set differs from all other tetramers in the set by at least two nucleotide bases, (2) no two tetramers within a set are complementary to one another, and (3) no tetramers within a set are palindromic or dinucleotide repeats.",
"The collection of double multimer unit oligonucleotides has had the following oligonucleotides removed from it: (1) oligonucleotides having a melting temperature in ° C. of less than 11 times the number of tetramers and more than 15 times then number of tetramers, (2) double multimer units with the same 3 tetramers linked together, and (3) double multimer units with the same 4 tetramers linked together with or without interruption, where the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions.",
"Often, a number of different single-base mutations, insertions, or deletions may occur at the same nucleotide position of the sequence of interest.",
"The method provides for having an oligonucleotide set, where the second oligonucleotide probe is common and contains the detectable label, and the first oligonucleotide probe has different addressable array-specific portions and target-specific portions.",
"The first oligonucleotide probe is suitable for ligation to a second adjacent oligonucleotide probe at a first ligation junction, when hybridized without mismatch, to the sequence in question.",
"Different first adjacent oligonucleotide probes would contain different discriminating base(s) at the junction where only a hybridization without mismatch at the junction would allow for ligation.",
"Each first adjacent oligonucleotide would contain a different addressable array-specific portion, and, thus, specific base changes would be distinguished by capture at different addresses.",
"In this scheme, a plurality of different capture oligonucleotides are attached at different locations on the solid support for multiplex detection of additional nucleic acid sequences differing from other nucleic acids by at least a single base.",
"Alternatively, the first oligonucleotide probe contains common addressable array-specific portions, and the second oligonucleotide probes have different detectable labels and target-specific portions.",
"Such arrangements permit multiplex detection of additional nucleic acid sequences differing from other nucleic acids by at least a single base.",
"The nucleic acids sequences can be on the same or different alleles when carrying out such multiplex detection.",
"The present invention also relates to a kit for carrying out the method of the present invention which includes the ligase, the plurality of different oligonucleotide probe sets, and the solid support with immobilized capture oligonucleotides.",
"Primers for preliminary amplification of the target nucleotide sequences may also be included in the kit.",
"If amplification is by polymerase chain reaction, polymerase may also be included in the kit.",
"FIGS.",
"1 and 2 show flow diagrams of the process of the present invention compared to a prior art ligase detection reaction utilizing capillary or gel electrophoresis/fluorescent quantification.",
"FIG.",
"1 relates to detection of a germline mutation detection, while FIG.",
"2 shows the detection of cancer.",
"FIG.",
"1 depicts the detection of a germline point mutation, such as the p53 mutations responsible for Li-Fraumeni syndrome.",
"In step 1, after DNA sample preparation, exons 5-8 are PCR amplified using Taq (i.e.",
"Thermus aquaticus) polymerase under hot start conditions.",
"At the end of the reaction, Taq polymerase is degraded by treatment with Proteinase K. Products are diluted 20-fold in step 2 into fresh LDR buffer containing allele-specific and common LDR probes.",
"A tube generally contains about 500 fmoles of each primer.",
"In step 3, the ligase detection reaction is initiated by addition of Taq ligase under hot start conditions.",
"The LDR probes ligate to their adjacent probes only in the presence of target sequence which gives perfect complementarity at the junction site.",
"The products may be detected in two different formats.",
"In the first format 4a., used in the prior art, fluorescently-labeled LDR probes contain different length poly A or hexaethylene oxide tails.",
"Thus, each LDR product, resulting from ligation to normal DNA with a slightly different mobility, yields a ladder of peaks.",
"A germline mutation would generate a new peak on the electrophorogram.",
"The size of the new peak will approximate the amount of the mutation present in the original sample; 0% for homozygous normal, 50% for heterozygous carrier, or 100% for homozygous mutant.",
"In the second format 4b., in accordance with the present invention, each allele-specific probe contains e.g., 24 additional nucleotide bases on their 5′ ends.",
"These sequences are unique addressable sequences which will specifically hybridize to their complementary address sequences on an addressable array.",
"In the LDR reaction, each allele-specific probe can ligate to its adjacent fluorescently labeled common probe in the presence of the corresponding target sequence.",
"Wild type and mutant alleles are captured on adjacent addresses on the array.",
"Unreacted probes are washed away.",
"The black dots indicate 100% signal for the wild type allele.",
"The white dots indicate 0% signal for the mutant alleles.",
"The shaded dots indicate the one position of germline mutation, 50% signal for each allele.",
"FIG.",
"2 depicts detection of somatic cell mutations in the p53 tumor suppressor gene but is general for all low sensitivity mutation detection.",
"In step 1, DNA samples are prepared and exons 5-9 are PCR amplified as three fragments using fluorescent PCR primers.",
"This allows for fluorescent quantification of PCR products in step 2 using capillary or gel electrophoresis.",
"In step 3, the products are spiked with a {fraction (1/100)} dilution of marker DNA (for each of the three fragments).",
"This DNA is homologous to wild type DNA, except it contains a mutation which is not observed in cancer cells, but which may be readily detected with the appropriate LDR probes.",
"The mixed DNA products in step 4 are diluted 20-fold into buffer containing all the LDR probes which are specific only to mutant or marker alleles.",
"In step 5, the ligase detection reaction is initiated by addition of Taq ligase under hot start conditions.",
"The LDR probes ligate to their adjacent probes only in the presence of target sequences which give perfect complementarity at the junction site.",
"The products may be detected in the same two formats described in FIG.",
"1.In the format of step 6a, which is used in the prior art, products are separated by capillary or gel electrophoresis, and fluorescent signals are quantified.",
"Ratios of mutant peaks to marker peaks give approximate amount of cancer mutations present in the original sample divided by 100.In the format of step 6b, in accordance with the present invention, products are detected by specific hybridization to complementary sequences on an addressable array.",
"Ratios of fluorescent signals in mutant dots to marker dots give the approximate amount of cancer mutations present in the original sample divided by 100.The ligase detection reaction process, in accordance with the present invention, is best understood by referring to FIGS.",
"3-15.It is described generally in WO 90/17239 to Barany et al., F. Barany et al., “Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene,” Gene, 109: 1-11 (1991), and F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 88: 189-193 (1991), the disclosures of which are hereby incorporated by reference.",
"In accordance with the present invention, the ligase detection reaction can use 2 sets of complementary oligonucleotides.",
"This is known as the ligase chain reaction which is described in the 3 immediately preceding references, which are hereby incorporated by reference.",
"Alternatively, the ligase detection reaction can involve a single cycle which is known as the oligonucleotide ligation assay.",
"See Landegren, et al., “A Ligase-Mediated Gene Detection Technique,” Science 241: 1077-80 (1988); Landegren, et al., “DNA Diagnostics—Molecular Techniques and Automation,” Science 242: 229-37 (1988); and U.S. Pat.",
"No.",
"4,988,617 to Landegren, et al.",
"During the ligase detection reaction phase of the process, the denaturation treatment is carried out at a temperature of 80-105° C., while hybridization takes place at 50-85° C. Each cycle comprises a denaturation treatment and a thermal hybridization treatment which in total is from about one to five minutes long.",
"Typically, the ligation detection reaction involves repeatedly denaturing and hybridizing for 2 to 50 cycles.",
"The total time for the ligase detection reaction phase of the process is 1 to 250 minutes.",
"The oligonucleotide probe sets can be in the form of ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogues, modified peptide nucleotide analogues, modified phosphate-sugar-backbone oligonucleotides, nucleotide analogs, and mixtures thereof.",
"In one variation, the oligonucleotides of the oligonucleotide probe sets each have a hybridization or melting temperature (i.e.",
"Tm) of 66-70° C. These oligonucleotides are 20-28 nucleotides long.",
"It may be desirable to destroy chemically or enzymatically unconverted LDR oligonucleotide probes that contain addressable nucleotide array-specific portions prior to capture of the ligation products on a DNA array.",
"Such unconverted probes will otherwise compete with ligation products for binding at the addresses on the array of the solid support which contain complementary sequences.",
"Destruction can be accomplished by utilizing an exonuclease, such as exonuclease III (L-H Guo and R. Wu, Methods in Enzymology 100: 60-96 (1985), which is hereby incorporated by reference) in combination with LDR probes that are blocked at the ends and not involved with ligation of probes to one another.",
"The blocking moiety could be a reporter group or a phosphorothioate group.",
"T. T. Nikiforow, et al., “The Use of Phosphorothioate Primers and Exonuclease Hydrolysis for the Preparation of Single-stranded PCR Products and their Detection by Solid-phase Hybridization,” PCR Methods and Applications, 3: p. 285-291 (1994), which is hereby incorporated by reference.",
"After the LDR process, unligated probes are selectively destroyed by incubation of the reaction mixture with the exonuclease.",
"The ligated probes are protected due to the elimination of free 3′ ends which are required for initiation of the exonuclease reaction.",
"This approach results in an increase in the signal-to-noise ratio, especially where the LDR reaction forms only a small amount of product.",
"Since unligated oligonucleotides compete for capture by the capture oligonucleotide, such competition with the ligated oligonucleotides lowers the signal.",
"An additional advantage of this approach is that unhybridized label-containing sequences are degraded and, therefore, are less able to cause a target-independent background signal, because they can be removed more easily from the DNA array by washing.",
"The oligonucleotide probe sets, as noted above, have a reporter label suitable for detection.",
"Useful labels include chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.",
"The capture oligonucleotides can be in the form of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogues, modified peptide nucleotide analogues, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.",
"Where the process of the present invention involves use of a plurality of oligonucleotide sets, the second oligonucleotide probes can be the same, while the addressable array-specific portions of the first oligonucleotide probes differ.",
"Alternatively, the addressable array-specific portions of the first oligonucleotide probes may be the same, while the reporter labels of the second oligonucleotide probes are different.",
"Prior to the ligation detection reaction phase of the present invention, the sample is preferably amplified by an initial target nucleic acid amplification procedure.",
"This increases the quantity of the target nucleotide sequence in the sample.",
"For example, the initial target nucleic acid amplification may be accomplished using the polymerase chain reaction process, self-sustained sequence replication, or Q-β replicase-mediated RNA amplification.",
"The polymerase chain reaction process is the preferred amplification procedure and is fully described in H. Erlich, et.",
"al., “Recent Advances in the Polymerase Chain Reaction,” Science 252: 1643-50 (1991); M. Innis, et.",
"al., PCR Protocols: A Guide to Methods and Applications, Academic Press: New York (1990); and R. Saiki, et.",
"al., “Primer-directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase,” Science 239: 487-91 (1988), which are hereby incorporated by reference.",
"J. Guatelli, et.",
"al., “Isothermal, in vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled After Retroviral Replication,” Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 87: 1874-78 (1990), which is hereby incorporated by reference, describes the self-sustained sequence replication process.",
"The Q-β replicase-mediated RNA amplification is disclosed in F. Kramer, et.",
"al., “Replicatable RNA Reporters,” Nature 339: 401-02 (1989), which is hereby incorporated by reference.",
"The use of the polymerase chain reaction process and then the ligase detection process, in accordance with the present invention, is shown in FIG.",
"3.Here, homo- or heterozygosity at two polymorphisms (i.e.",
"allele differences) are on the same gene.",
"Such allele differences can alternatively be on different genes.",
"As shown in FIG.",
"3, the target nucleic acid, when present in the form of a double stranded DNA molecule is denatured to separate the strands.",
"This is achieved by heating to a temperature of 80-105° C. Polymerase chain reaction primers are then added and allowed to hybridize to the strands, typically at a temperature of 20-85° C. A thermostable polymerase (e.g., Thermus aquaticus polymerase) is also added, and the temperature is then adjusted to 50-85° C. to extend the primer along the length of the nucleic acid to which the primer is hybridized.",
"After the extension phase of the polymerase chain reaction, the resulting double stranded molecule is heated to a temperature of 80-105° C. to denature the molecule and to separate the strands.",
"These hybridization, extension, and denaturation steps may be repeated a number of times to amplify the target to an appropriate level.",
"Once the polymerase chain reaction phase of the process is completed, the ligation detection reaction phase begins, as shown in FIG.",
"3.After denaturation of the target nucleic acid, if present as a double stranded DNA molecule, at a temperature of 80-105° C., preferably 94° C., ligation detection reaction oligonucleotide probes for one strand of the target nucleotide sequence are added along with a ligase (for example, as shown in FIG.",
"3, a thermostable ligase like Thermus aquaticus ligase).",
"The oligonucleotide probes are then allowed to hybridize to the target nucleic acid molecule and ligate together, typically, at a temperature of 45-85° C., preferably, 65° C. When there is perfect complementarity at the ligation junction, the oligonucleotides can be ligated together.",
"Where the variable nucleotide is T or A, the presence of T in the target nucleotide sequence will cause the oligonucleotide probe with the addressable array-specific portion Z1 to ligate to the oligonucleotide probe with the reporter label F, and the presence of A in the target nucleotide sequence will cause the oligonucleotide probe with the addressable array-specific portion Z2 to ligate to the oligonucleotide probe with reporter label F. Similarly, where the variable nucleotide is A or G, the presence of T in the target nucleotide sequence will cause the oligonucleotide probe with addressable array-specific portion Z4 to ligate to the oligonucleotide probe with the reporter label F, and the presence of C in the target nucleotide sequence will cause the oligonucleotide probe with the addressable array-specific portion Z3 to ligate to the oligonucleotide probe with reporter label F. Following ligation, the material is again subjected to denaturation to separate the hybridized strands.",
"The hybridization/ligation and denaturation steps can be carried through one or more cycles (e.g., 1 to 50 cycles) to amplify the target signal.",
"Fluorescent ligation products (as well as unligated oligonucleotide probes having an addressable array-specific portion) are captured by hybridization to capture probes complementary to portions Z1, Z2, Z3, and Z4 at particular addresses on the addressable arrays.",
"The presence of ligated oligonucleotides is then detected by virtue of the label F originally on one of the oligonucleotides.",
"In FIG.",
"3, ligated product sequences hybridize to the array at addresses with capture oligonucleotides complementary to addressable array-specific portions Z1 and Z3, while unligated oligonucleotide probes with addressable array-specific portions Z2 and Z4 hybridize to their complementary capture oligonucleotides.",
"However, since only the ligated product sequences have label F, only their presence is detected.",
"FIG.",
"4 is similar to FIG.",
"3 except that in FIG.",
"4, the common oligonucleotide probe has an address-specific portion, while the allele-specific probes have different labels.",
"FIG.",
"5 is a flow diagram of a PCR/LDR process, in accordance with the present invention, which distinguishes any possible base at a given site.",
"Appearance of fluorescent signal at the addresses complementary to addressable array-specific portions Z1, Z2, Z3, and Z4 indicates the presence of A, G, C, and T alleles in the target nucleotide sequence, respectively.",
"Here, the presence of the A and C alleles in the target nucleotide sequences is indicated due to the fluorescence at the addresses on the solid support with capture oligonucleotide probes complementary to portions Z1 and Z3, respectively.",
"Note that in FIG.",
"5 the addressable array-specific portions are on the discriminating oligonucleotide probes, and the discriminating base is on the 3′ end of these probes.",
"FIG.",
"6 is similar to FIG.",
"5, except that in FIG.",
"6, the common oligonucleotide probe has the address-specific portion, while the allele-specific probes have different labels.",
"FIG.",
"7 is a flow diagram of a PCR/LDR process, in accordance with the present invention, for detecting the presence of any possible base at two nearby sites.",
"Here, the LDR probes are able to overlap, yet are still capable of ligating provided there is perfect complementarity at the junction.",
"This distinguishes LDR from other approaches, such as allele-specific PCR where overlapping primers would interfere with one another.",
"In FIG.",
"7, the first nucleotide position is heterozygous at the A and C alleles, while the second nucleotide position is heterozygous to the G, C, and T alleles.",
"As in FIG.",
"5, the addressable array-specific portions are on the discriminating oligonucleotide probes, and the discriminating base is on the 3′ end of these probes.",
"The reporter group (e.g., the fluorescent label) is on the 3′ end of the common oligonucleotide probes.",
"This is possible for example with the 21 hydroxylase gene, where each individual has 2 normal and 2 pseudogenes, and, at the intron 2 splice site (nucleotide 656), there are 3 possible single bases (G, A, and C).",
"Also, this can be used to detect low abundance mutations in HIV infections which might indicate emergence of drug resistant (e.g., to AZT) strains.",
"Returning to FIG.",
"7, appearance of fluorescent signal at the addresses complementary to addressable array-specific portions Z1, Z2, Z3, Z4, Z5, Z6, Z7, and Z8 indicates the presence of the A, G, C, and T, respectively, in the site heterozygous at the A and C alleles, and A, G, C, and T, respectively, in the site heterozygous at the G, C, and T alleles.",
"FIG.",
"8 is similar to FIG.",
"7, except that in FIG.",
"8, the common oligonucleotide probes have the address-specific portions, while the allele specific probes have different labels.",
"FIG.",
"9 is a flow diagram of a PCR/LDR process, in accordance with the present invention, where insertions (top left set of probes) and deletions (bottom right set of probes) are distinguished.",
"On the left, the normal sequence contains 5 A's in a polyA tract.",
"The mutant sequence has an additional 2As inserted into the tract.",
"Therefore, the LDR products with addressable array-specific portions Z1 (representing the normal sequence) and Z3 (representing a 2 base pair insertion) would be fluorescently labeled by ligation to the common probe.",
"While the LDR process (e.g., using a thermostable ligase enzyme) has no difficulty distinguishing single base insertions or deletions in mononucleotide repeats, allele-specific PCR is unable to distinguish such differences, because the 3′ base remains the same for both alleles.",
"On the right, the normal sequence is a (CA)5 repeat (i.e.",
"CACACACACA).",
"The mutant contains two less CA bases than the normal sequence (i.e.",
"CACACA).",
"These would be detected as fluorescent LDR products at the addressable array-specific portions Z8 (representing the normal sequence) and Z6 (representing the 2 CA deletion) addresses.",
"The resistance of various infectious agents to drugs can also be determined using the present invention.",
"In FIG.",
"9, the presence of ligated product sequences, as indicated by fluorescent label F, at the address having capture oligonucleotides complementary to Z1 and Z3 demonstrates the presence of both the normal and mutant poly A sequences.",
"Similarly, the presence of ligated product sequences, as indicated by fluorescent label F, at the address having capture oligonucleotides complementary to Z6 and Z8 demonstrates the presence of both the normal CA repeat and a sequence with one repeat unit deleted.",
"FIG.",
"10 is similar to FIG.",
"9, except that in FIG.",
"10, the common oligonucleotide probes have the address-specific portions, while the allele-specific probes have different labels.",
"FIG.",
"11 is a flow diagram of a PCR/LDR process, in accordance with the present invention, using addressable array-specific portions to detect a low abundance mutation in the presence of an excess of normal sequence.",
"FIG.",
"1I shows codon 12 of the K-ras gene, sequence GGT, which codes for glycine (“Gly”).",
"A small percentage of the cells contain the G to A mutation in GAT, which codes for aspartic acid (“Asp”).",
"The LDR probes for wild-type (i.e.",
"normal sequences) are missing from the reaction.",
"If the normal LDR probes (with the discriminating base=G) were included, they would ligate to the common probes and overwhelm any signal coming from the mutant target Instead, as shown in FIG.",
"11, the existence of a ligated product sequence with fluorescent label F at the address with a capture oligonucleotide complementary to addressable array-specific portion Z4 indicates the presence of the aspartic acid encoding mutant.",
"FIG.",
"12 is similar to FIG.",
"11, except that in FIG.",
"12, the common oligonucleotide probes have address-specific portions, while the allele-specific probes have different labels.",
"FIG.",
"13 is a flow diagram of a PCR/LDR process, in accordance with the present invention, where the addressable array-specific portion is placed on, the common oligonucleotide probe, while the discriminating oligonucleotide probe has the reporter label.",
"Allele differences are distinguished by different fluorescent signals, F1, F2, F3, and F4.This mode allows for a more dense use of the arrays, because each position is predicted to light up with some group.",
"It has the disadvantage of requiring fluorescent groups which have minimal overlap in their emission spectra and will require multiple scans.",
"It is not ideally suitable for detection of low abundance alleles (e.g., cancer associated mutations).",
"FIG.",
"14 is a flow diagram of a PCR/LDR process, in accordance with the present invention, where both adjacent and nearby alleles are detected.",
"The adjacent mutations are right next to each other, and one set of oligonucleotide probes discriminates the bases on the 3′ end of the junction (by use of different addressable array-specific portions Z1, Z2, Z3, and Z4), while the other set of oligonucleotide probes discriminates the bases on the 5′ end of the junction (by use of different fluorescent reporter labels F1, F2, F3, and F4).",
"In FIG.",
"14, codons in a disease gene (e.g.",
"CFTR for cystic fibrosis) encoding Gly and arginine (“Arg”), respectively, are candidates for germline mutations.",
"The detection results in FIG.",
"14 show the Gly (GGA; indicated by the ligated product sequence having portion Z2 and label F2) has been mutated to glutamic acid (“Glu”) (GAA; indicated by the ligated product sequence having portion Z2 and label F1), and the Arg (CGG; indicated by the ligated product sequence having portion Z7 and label F2) has been mutated to tryptophan (“Trp”) (TGG; indicated by the ligated product sequence with portion Z8 and label F2).",
"Therefore, the patient is a compound heterozygous individual (i.e.",
"with allele mutations in both genes) and will have the disease.",
"FIG.",
"15 is a flow diagram of a PCR/LDR process, in accordance with the present invention, where all possible single-base mutations for a single codon are detected.",
"Most amino acid codons have a degeneracy in the third base, thus the first two positions can determine all the possible mutations at the protein level.",
"These amino acids include arginine, leucine, serine, threonine, proline, alanine, glycine, and valine.",
"However, some amino acids are determined by all three bases in the codon and, thus, require the oligonucleotide probes to distinguish mutations in 3 adjacent positions.",
"By designing four oligonucleotide probes containing the four possible bases in the penultimate position to the 3′ end, as well as designing an additional four capture oligonucleotides containing the four possible bases at the 3′ end, as shown in FIG.",
"15, this problem has been solved.",
"The common oligonucleotide probes with the reporter labels only have two fluorescent groups which correspond to the codon degeneracies and distinguish between different ligated product sequences which are captured at the same array address.",
"For example, as shown in FIG.",
"15, the presence of a glutamine (“Gln”) encoding codon (i.e., CAA and CAG) is indicated by the presence of a ligated product sequence containing portion Z1 and label F2.Likewise, the existence of a Gln to histidine (“His”) encoding mutation (coded by the codon CAC) is indicated by the presence of ligated product sequences with portion Z1 and label F2 and with portion Z7 and label F2 There is an internal redundancy built into this assay due to the fact that primers Z1 and Z7 have the identical sequence.",
"A particularly important aspect of the present invention is its capability to quantify the amount of target nucleotide sequence in a sample.",
"This can be achieved in a number of ways by establishing standards which can be internal (i.e.",
"where the standard establishing material is amplified and detected with the sample) or external (i.e.",
"where the standard establishing material is not amplified, and is detected with the sample).",
"In accordance with one quantification method, the signal generated by the reporter label, resulting from capture of ligated product sequences produced from the sample being analyzed, are detected.",
"The strength of this signal is compared to a calibration curve produced from signals generated by capture of ligated product sequences in samples with known amounts of target nucleotide sequence.",
"As a result, the amount of target nucleotide sequence in the sample being analyzed can be determined.",
"This technique involves use of an external standard.",
"Another quantification method, in accordance with the present invention, relates to an internal standard.",
"Here, a known amount of one or more marker target nucleotide sequences are added to the sample.",
"In addition, a plurality of marker-specific oligonucleotide probe sets are added along with the ligase, the previously-discussed oligonucleotide probe sets, and the sample to a mixture.",
"The marker-specific oligonucleotide probe sets have (1) a first oligonucleotide probe with a target-specific portion complementary to the marker target nucleotide sequence and an addressable array-specific portion complementary to capture oligonucleotides on the support and (2) a second oligonucleotide probe with a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label.",
"The oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence.",
"However, there is a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample or added marker sequences.",
"The presence of ligated product sequences captured on the support is identified by detection of reporter labels.",
"The amount of target nucleotide sequences in the sample is then determined by comparing the amount of captured ligated product generated from known amounts of marker target nucleotide sequences with the amount of other ligated product sequences captured.",
"Another quantification method in accordance with the present invention involves analysis of a sample containing two or more of a plurality of target nucleotide sequences with a plurality of sequence differences.",
"Here, ligated product sequences corresponding to the target nucleotide sequences are detected and distinguished by any of the previously-discussed techniques.",
"The relative amounts of the target nucleotide sequences in the sample are then quantified by comparing the relative amounts of captured ligated product sequences generated.",
"This provides a quantitative measure of the relative level of the target nucleotide sequences in the sample.",
"The ligase detection reaction process phase of the present invention can be preceded by the ligase chain reaction process to achieve oligonucleotide product amplification.",
"This process is fully described in F. Barany, et.",
"al., “Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene,” Gene 109: 1-11 (1991) and F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 88: 189-93 (1991), which are hereby incorporated by reference.",
"Instead of using the ligase chain reaction to achieve amplification, a transcription-based amplifying procedure can be used.",
"The preferred thermostable ligase is that derived from Thermus aquaticus.",
"This enzyme can be isolated from that organism.",
"M. Takahashi, et al., “Thermophillic DNA Ligase,” J. Biol.",
"Chem.",
"259: 10041-47 (1984), which is hereby incorporated by reference.",
"Alternatively, it can be prepared recombinantly.",
"Procedures for such isolation as well as the recombinant production of Thermus aquaticus ligase (and Thermus themophilus ligase) are disclosed in WO 90/17239 to Barany, et.",
"al., and F. Barany, et al., “Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA-Ligase Encoding Gene,” Gene 109: 1-11 (1991), which are hereby incorporated by reference.",
"These references contain complete sequence information for this ligase as well as the encoding DNA.",
"Other suitable ligases include E. coli ligase, T4 ligase, Thermus sp.",
"AK16 ligase, Aquifex aeolicus ligase, Thermotoga maritima ligase, and Pyrococcus ligase.",
"The ligation amplification mixture may include a carrier DNA, such as salmon sperm DNA.",
"The hybridization step, which is preferably a thermal hybridization treatment, discriminates between nucleotide sequences based on a distinguishing nucleotide at the ligation junctions.",
"The difference between the target nucleotide sequences can be, for example, a single nucleic acid base difference, a nucleic acid deletion, a nucleic acid insertion, or rearrangement.",
"Such sequence differences involving more than one base can also be detected.",
"Preferably, the oligonucleotide probe sets have substantially the same length so that they hybridize to target nucleotide sequences at substantially similar hybridization conditions.",
"As a result, the process of the present invention is able to detect infectious diseases, genetic diseases, and cancer.",
"It is also useful in environmental monitoring, forensics, and food science.",
"A wide variety of infectious diseases can be detected by the process of the present invention.",
"Typically, these are caused by bacterial, viral, parasite, and fungal infectious agents.",
"The resistance of various infectious agents to drugs can also be determined using the present invention.",
"Bacterial infectious agents which can be detected by the present invention include Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes.",
"Fungal infectious agents which can be detected by the present invention include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis.",
"Viral infectious agents which can be detected by the present invention include human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.",
"Parasitic agents which can be detected by the present invention include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.",
"The present invention is also useful for detection of drug resistance by infectious agents.",
"For example, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multi-drug resistant Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus can all be identified with the present invention.",
"Genetic diseases can also be detected by the process of the present invention.",
"This can be carried out by prenatal screening for chromosomal and genetic aberrations or post natal screening for genetic diseases.",
"Examples of detectable genetic diseases include: 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Syndrome, thalassemia, Klinefelter's Syndrome, Huntington's Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors in metabolism, and diabetes.",
"Cancers which can be detected by the process of the present invention generally involve oncogenes, tumor suppressor genes, or genes involved in DNA amplification, replication, recombination, or repair.",
"Examples of these include: BRCA1 gene, p53 gene, Familial polyposis coli, Her2/Neu amplification, Bcr/Ab1, K-ras gene, human papillomavirus Types 16 and 18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, ENT tumors, and loss of heterozygosity.",
"In the area of environmental monitoring, the present invention can be used for detection, identification, and monitoring of pathogenic and indigenous microorganisms in natural and engineered ecosystems and microcosms such as in municipal waste water purification systems and water reservoirs or in polluted areas undergoing bioremediation.",
"It is also possible to detect plasmids containing genes that can metabolize xenobiotics, to monitor specific target microorganisms in population dynamic studies, or either to detect, identify, or monitor genetically modified microorganisms in the environment and in industrial plants.",
"The present invention can also be used in a variety or forensic areas, including for human identification for military personnel and criminal investigation, paternity testing and family relation analysis, HLA compatibility typing, and screening blood, sperm, or transplantation organs for contamination.",
"In the food and feed industry, the present invention has a wide variety of applications.",
"For example, it can be used for identification and characterization of production organisms such as yeast for production of beer, wine, cheese, yogurt, bread, etc.",
"Another area of use is with regard to quality control and certification of products and processes (e.g., livestock, pasteurization, and meat processing) for contaminants.",
"Other uses include the characterization of plants, bulbs, and seeds for breeding purposes, identification of the presence of plant-specific pathogens, and detection and identification of veterinary infections.",
"Desirably, the oligonucleotide probes are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction.",
"However, when the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with ligation.",
"Most preferably, the mismatch is at the base adjacent the 3′ base at the ligation junction.",
"Alternatively, the mismatch can be at the bases adjacent to bases at the ligation junction.",
"The process of the present invention is able to detect the first and second nucleotide sequences in the sample in an amount of 100 attomoles to 250 femtomoles.",
"By coupling the LDR step with a primary polymerase-directed amplification step, the entire process of the present invention is able to detect target nucleotide sequences in a sample containing as few as a single molecule.",
"Furthermore, PCR amplified products, which often are in the picomole amounts, may easily be diluted within the above range.",
"The ligase detection reaction achieves a rate of formation of mismatched ligated product sequences which is less than 0.005 of the rate of formation of matched ligated product sequences.",
"Once the ligation phase of the process is completed, the capture phase is initiated.",
"During the capture phase of the process, the mixture is contacted with the solid support at a temperature of 25-90° C., preferably 60-80° C., and for a time period of 10-180 minutes, preferably up to 60 minutes.",
"Hybridizations may be accelerated or improved by mixing the ligation mixture during hybridization, or by adding volume exclusion, chaotropic agents, tetramethylammonium chloride, or N,N,N, Trimethylglycine (Betaine monohydrate).",
"When an array consists of dozens to hundreds of addresses, it is important that the correct ligation products have an opportunity to hybridize to the appropriate address.",
"This may be achieved by the thermal motion of oligonucleotides at the high temperatures used, by mechanical movement of the fluid in contact with the array surface, or by moving the oligonucleotides across the array by electric fields.",
"After hybridization, the array is washed with buffer to remove unhybridized probe and optimize detection of captured probe.",
"Alternatively, the array is washed sequentially.",
"The specificity of hybridization may be promoted by the addition of non-specific competitor DNA (e.g.",
"herring sperm DNA) and/or the addition of formamide to the hybridization solution.",
"The stringency of washing may also be augmented by elevating the washing temperature and/or adding formamide to the wash buffer.",
"FIG.",
"16 shows the results of various combinations of the above alterations to standard hybridization and washing conditions.",
"Preferably, the solid support has a porous surface of a hydrophilic polymer composed of combinations of acrylamide with functional monomers containing carboxylate, aldehyde, or amino groups.",
"This surface is formed by coating the support with a polyacrylamide based gel.",
"Suitable formulations include mixtures of acrylamide/acrylic acid and N,N-dimethylacrylamide/glycerol monomethacrylate.",
"A crosslinker, N,N′-methylenebisacryl-amide, is present at a level less than 50:1, preferably less than 500:1.One embodiment of masking negative charges during the contacting of the solid support with the ligation mixture is achieved by using a divalent cation.",
"The divalent cation can be Mg2+, Ca2+, MN2+, or Co2+.",
"Typically, masking with the divalent cation is carried out by pre-hydridizing the solid support with hybridization buffer containing the cation at a minimum concentration of 10 mM for a period of 15 minutes at room temperature.",
"Another embodiment of masking negative charges during the contacting of the solid support with the ligation mixtures is achieved by carrying out the contacting at a pH at or below 6.0.This is effected by adding a buffer to the ligation mixtures before or during contact of it with the solid support.",
"Suitable buffers include 2-(N-morpholino)ethanesulfonic acid (MES), sodium phosphate, and potassium phosphate.",
"Another embodiment of masking negative charges during the contacting of the solid support with the ligation mixture is achieved by capping free carboxylic acid groups with a neutralizing agent while preserving the hydrophillicity of the polymer.",
"Suitable neutralizing agents include ethanolamine diethanolamine, propanolamine, dipropanolamine, isopropanolamine, and diisopropanolamine.",
"Typically, masking with neutralizing agents is carried out by activating the carboxylic acid groups within the solid support with 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide followed by treatment with a solution of the neutralizing agent in a polar aprotic solvent such as chloroform, dichloromethane, or tetrahydrfuran.",
"By masking the negative charges in accordance with the present invention, an enhanced ability to detect the presence of ligated product in the presence of unligated oligonucleotide probes is achieved.",
"In particular, the present invention is effective to detect the presence of ligated product in a ratio to unligated oligonucleotide probes of less than 1:300, preferably less than 1:900, more preferably less than 1:3000, and most preferably less than 1:9000.In addition, by masking the negative charges in accordance with the present invention, an enhanced ability to detect the presence of a target nucleotide sequence from a non-target nucleotide sequence where the target nucleotide sequence differs from a non-target nucleotide sequence by a single base difference is achieved.",
"In particular, the present invention is effective to detect target nucleotide sequence in a ratio of the target nucleotide sequence to non-target nucleotide sequence of less than 1:20, preferably less than 1:50, more preferably less than 1:100, most preferably less than 1:200.It is important to select capture oligonucleotides and addressable nucleotide sequences which will hybridize in a stable fashion.",
"This requires that the oligonucleotide sets and the capture oligonucleotides be configured so that the oligonucleotide sets hybridize to the target nucleotide sequences at a temperature less than that which the capture oligonucleotides hybridize to the addressable array-specific portions.",
"Unless the oligonucleotides are designed in this fashion, false positive signals may result due to capture of adjacent unreacted oligonucleotides from the same oligonucleotide set which are hybridized to the target.",
"The detection phase of the process involves scanning and identifying if ligation of particular oligonucleotide sets occurred and correlating ligation to a presence or absence of the target nucleotide sequence in the test sample.",
"Scanning can be carried out by scanning electron microscopy, confocal microscopy, charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging.",
"Correlating is carried out with a computer.",
"Another aspect of the present invention relates to a method of forming an array of oligonucleotides on a support.",
"This method involves providing a support having an array of positions each suitable for attachment of an oligonucleotide.",
"A linker or support (preferably non-hydrolyzable), suitable for coupling an oligonucleotide to the support at each of the array positions, is attached to the solid support.",
"An array of oligonucleotides on a solid support is formed by a series of cycles of activating selected array positions for attachment of multimer nucleotides and attaching multimer nucleotides at the activated array positions.",
"Yet another aspect of the present invention relates to an array of oligonucleotides on a support per se.",
"The support has an array of positions each suitable for an attachment of an oligonucleotide.",
"A linker or support (preferably non-hydrolyzable), suitable for coupling an oligonucleotide to the support, is attached to the support at each of the array positions.",
"An array of oligonucleotides are placed on a support with at least some of the array positions being occupied by oligonucleotides having greater than sixteen nucleotides.",
"In the method of forming arrays, multimer oligonucleotides from different multimer oligonucleotide sets are attached at different array positions on a support.",
"As a result, the support has an array of positions with different groups of multimer oligonucleotides attached at different positions.",
"The 1,000 different addresses can be unique capture oligonucleotide sequences (e.g., 24-mer) linked covalently to the target-specific sequence (e.g., approximately 20- to 25-mer) of a LDR oligonucleotide probe.",
"A capture oligonucleotide probe sequence does not have any homology to either the target sequence or to other sequences on genomes which may be present in the sample.",
"This oligonucleotide probe is then captured by its addressable array-specific portion, a sequence complementary to the capture oligonucleotide on the addressable support array.",
"The concept is shown in two possible formats, for example, for detection of the p53 R248 mutation (FIGS.",
"17A-C).",
"FIGS.",
"17A-C show two alternative formats for oligonucleotide probe design to identify the presence of a germ line mutation in codon 248 of the p53 tumor suppressor gene.",
"The wild type sequence codes for arginine (R248), while the cancer mutation codes for tryptophan (R248W).",
"The bottom part of the diagram is a schematic diagram of the capture oligonucleotide.",
"The thick horizontal line depicts the membrane or surface containing the addressable array.",
"The thin curved lines indicate a flexible linker arm.",
"The thicker lines indicate a capture oligonucleotide sequence, attached to the solid surface in the C to N direction.",
"For illustrative purposes, the capture oligonucleotides are drawn vertically, making the linker arm in section B appear “stretched”.",
"Since the arm is flexible, the capture oligonucleotide will be able to hybridize 5′ to C and 3′ to N in each case, as dictated by base pair complementarity.",
"A similar orientation of oligonucleotide hybridization would be allowed if the oligonucleotides were attached to the membrane at the N-terminus.",
"In this case, DNA/PNA hybridization would be in standard antiparallel 5′ to 3′ and 3′ to 5′.",
"Other modified sugar-phosphate backbones would be used in a similar fashion.",
"FIG.",
"17B shows two LDR probes that are designed to discriminate wild type and mutant p53 by containing the discriminating base C or T at the 3′ end.",
"In the presence of the correct target DNA and Tth ligase, the discriminating probe is covalently attached to a common downstream oligonucleotide.",
"The downstream oligonucleotide is fluorescently labeled.",
"The discriminating oligonucleotides are distinguished by the presence of unique addressable array-specific portions, Z1 and Z2, at each of their 5′ ends.",
"A black dot indicates that target dependent ligation has taken place.",
"After ligation, oligonucleotide probes may be captured by their complementary addressable array-specific portions at unique addresses on the array.",
"Both ligated and unreacted oligonucleotide probes are captured by the oligonucleotide array.",
"Unreacted fluorescently labeled common probes and target DNA are then washed away at a high temperature (approximately 65° C. to 80° C.) and low salt.",
"Mutant signal is distinguished by detection of fluorescent signal at the capture oligonucleotide complementary to addressable array-specific portion Z1, while wild type signal appears at the capture oligonucleotide complementary to addressable array-specific portion Z2.Heterozygosity is indicated by equal signals at the capture oligonucleotides complementary to addressable array-specific portions Z1 and Z2.The signals may be quantified using a fluorescent imager.",
"This format uses a unique address for each allele and may be preferred for achieving very accurate detection of low levels of signal (30 to 100 attomoles of LDR product).",
"FIG.",
"17C shows the discriminating signals may be quantified using a fluorescent imager.",
"This format uses a unique address where oligonucleotide probes are distinguished by having different fluorescent groups, F1 and F2, on their 5′ end.",
"Either oligonucleotide probe may be ligated to a common downstream oligonucleotide probe containing an addressable array-specific portion Z1 on its 3′ end.",
"In this format, both wild type and mutant LDR products are captured at the same address on the array, and are distinguished by their different fluorescence.",
"This format allows for a more efficient use of the array and may be preferred when trying to detect hundreds of potential germline mutations.",
"The support can be made from a wide variety of materials.",
"The substrate may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, discs, membranes, etc.",
"The substrate may have any convenient shape, such as a disc, square, circle, etc.",
"The substrate is preferably flat but may take on a variety of alternative surface configurations.",
"For example, the substrate may contain raised or depressed regions on which the synthesis takes place.",
"The substrate and its surface preferably form a rigid support on which to carry out the reactions described herein.",
"The substrate and its surface is also chosen to provide appropriate light-absorbing characteristics.",
"For instance, the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, polyethylene, polypropylene, polyvinyl chloride, poly(methyl acrylate), poly(methyl methacrylate), or combinations thereof.",
"Other substrate materials will be readily apparent to those of ordinary skill in the art upon review of this disclosure.",
"In a preferred embodiment, the substrate is flat glass or single-crystal silicon.",
"According to some embodiments, the surface of the substrate is etched using well known techniques to provide for desired surface features.",
"For example, by way of the formation of trenches, v-grooves, mesa structures, raised platforms, or the like, the synthesis regions may be more closely placed within the focus point of impinging light, be provided with reflective “mirror” structures for maximization of light collection from fluorescent sources, or the like.",
"Surfaces on the substrate will usually, though not always, be composed of the same material as the substrate.",
"Thus, the surface may be composed of any of a wide variety of materials, for example, polymers, plastics, ceramics, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or composites thereof.",
"The surface is functionalized with binding members which are attached firmly to the surface of the substrate.",
"Preferably, the surface functionalities will be reactive groups such as silanol, olefin, amino, hydroxyl, aldehyde, keto, halo, acyl halide, or carboxyl groups.",
"In some cases, such functionalities preexist on the substrate.",
"For example, silica based materials have silanol groups, polysaccharides have hydroxyl groups, and synthetic polymers can contain a broad range of functional groups, depending on which monomers they are produced from.",
"Alternatively, if the substrate does not contain the desired functional groups, such groups can be coupled onto the substrate in one or more steps.",
"A variety of commercially-available materials, which include suitably modified glass, plastic, or carbohydrate surfaces or a variety of membranes, can be used.",
"Depending on the material, surface functional groups (e.g., silanol, hydroxyl, carboxyl, amino) may be present from the outset (perhaps as part of the coating polymer), or will require a separate procedure (e.g., plasma amination, chromic acid oxidation, treatment with a functionalized side chain alkyltrichlorosilane) for introduction of the functional group.",
"Hydroxyl groups become incorporated into stable carbamate (urethane) linkages by several methods.",
"Amino functions can be acylated directly, whereas carboxyl groups are activated, e.g., with N,N′-carbonyldiimidazole or water-soluble carbodiimides, and reacted with an amino-functionalized compound.",
"As shown in FIG.",
"18, the supports can be membranes or surfaces with a starting functional group X. Functional group transformations can be carried out in a variety of ways (as needed) to provide group X* which represents one partner in the covalent linkage with group Y*.",
"FIG.",
"18 shows specifically the grafting of PEG (i.e.",
"polyethylene glycol), but the same repertoire of reactions can be used (however needed) to attach carbohydrates (with hydroxyl), linkers (with carboxyl), and/or oligonucleotides that have been extended by suitable functional groups (amino or carboxyl).",
"In some cases, group X* or Y* is pre-activated (isolatable species from a separate reaction); alternatively, activation occurs in situ.",
"Referring to PEG as drawn in FIG.",
"18, Y and Y* can be the same (homobifunctional) or different (heterobifunctional); in the latter case, Y can be protected for further control of the chemistry.",
"Unreacted amino groups will be blocked by acetylation or succinylation, to ensure a neutral or negatively charged environment that “repels” excess unhybridized DNA.",
"Loading levels can be determined by standard analytical methods.",
"Fields, et al., “Principles and Practice of Solid-Phase Peptide Synthesis,” Synthetic Peptides: A User's Guide, G. Grant, Editor, W.H.",
"Freeman and Co.: New York.",
"p. 77-183 (1992), which is hereby incorporated by reference.",
"One approach to applying functional groups on a silica-based support surface is to silanize with a molecule either having the desired functional group (e.g., olefin, amino, hydroxyl, aldehyde, keto, halo, acyl halide, or carboxyl) or a molecule A able to be coupled to another molecule B containing the desired functional group.",
"In the former case, functionalizing of glass- or silica-based supports with, for example, an amino group is carried out by reacting with an amine compound such as 3-aminopropyl triethoxysilane, 3-aminopropylmethyldiethoxysilane, 3-aminopropyl dimethylethoxysilane, 3-aminopropyl trimethoxysilane, N-(2-aminoethyl)-3-aminopropylmethyl dimethoxysilane, N-(2-aminoethyl-3-aminopropyl) trimethoxysilane, aminophenyl trimethoxysilane, 4-aminobutyldimethyl methoxysilane, 4-aminobutyl triethoxysilane, aminoethylaminomethyphenethyl trimethoxysilane, or mixtures thereof.",
"In the latter case, molecule A preferably contains olefinic groups, such as vinyl, acrylate, methacrylate, or allyl, while molecule B contains olefinic groups and the desired functional groups.",
"In this case, molecules A and B are polymerized together.",
"In some cases, it is desirable to modify the silanized surface to modify its properties (e.g., to impart biocompatibility and to increase mechanical stability).",
"This can be achieved by addition of olefinic molecule C along with molecule B to produce a polymer network containing molecules A, B, and C. Molecule A is defined by the following formula: R1 is H or CH3 R2 is (C═O)—O—R6, aliphatic group with or without functional substituent(s), an aromatic group with or without functional substituent(s), or mixed aliphatic/aromatic groups with or without functional substituent(s); R3 is an O-alkyl, alkyl, or halogen group; R4 is an O-alkyl, alkyl, or halogen group; R5 is an O-alkyl, alkyl, or halogen group; and R6 is an aliphatic group with or without functional substituent(s), an aromatic group with or without functional substituent(s), or mixed aliphatic/aromatic groups with or without functional substituent(s).",
"Examples of Molecule A include 3-(trimethoxysilyl)propyl methacrylate, N-[3-(trimethoxysilyl)propyl]-N′-(4-vinylbenzyl)ethylenediamine, triethoxyvinylsilane, triethylvinylsilane, vinyltrichlorosilane, vinyltrimethoxysilane, and vinylytrimethylsilane.",
"Molecule B can be any monomer containing one or more of the functional groups described above.",
"Molecule B is defined by the following formula: (i) R1 is H or CH3, R2 is (C═O), and R3 is OH or Cl.",
"or (ii) R1 is H or CH3 and R2 is (C═O)—O—R4, an aliphatic group with or without functional substituent(s), an aromatic group with or without functional substituent(s), and mixed aliphatic/aromatic groups with or without functional substituent(s); and R3 is a functional group, such as OH, COOH, NH2, halogen, SH, COCl, or active ester; and R4 is an aliphatic group with or without functional substituent(s), an aromatic group with or without functional substituent(s), or mixed aliphatic/aromatic groups with or without functional substituent(s).",
"Examples of molecule B include acrylic acid, acrylamide, methacrylic acid, vinylacetic acid, 4-vinylbenzoic acid, itaconic acid, allyl amine, allylethylamine, 4-aminostyrene, 2-aminoethyl methacrylate, acryloyl chloride, methacryloyl chloride, chlorostyrene, dichlorostyrene, 4-hydroxystyrene, hydroxymethyl styrene, vinylbenzyl alcohol, allyl alcohol, 2-hydroxyethyl methacrylate, or poly(ethylene glycol) methacrylate.",
"Molecule C can be any molecule capable of polymerizing to molecule A, molecule B, or both and may optionally contain one or more of the functional groups described above.",
"Molecule C can be any monomer or cross-linker, such as acrylic acid, methacrylic acid, vinylacetic acid, 4-vinylbenzoic acid, itaconic acid, allyl amine, allylethylamine, 4-aminostyrene, 2-aminoethyl methacrylate, acryloyl chloride, methacryloyl chloride, chlorostyrene, dichlorostyrene, 4-hydroxystyrene, hydroxymethyl styrene, vinylbenzyl alcohol, allyl alcohol, 2-hydroxyethyl methacrylate, poly(ethylene glycol) methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, styrene, 1-vinylimidazole, 2-vinylpyridine, 4-vinylpyridine, divinylbenzene, ethylene glycol dimethacryarylate, N,N′-methylenediacrylamide, N,N′-phenylenediacrylamide, 3,5-bis(acryloylamido)benzoic acid, pentaerythritol triacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetraacrylate, trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate, trimethyolpropane ethoxylate (7/3 EO/OH) triacrylate, triethylolpropane propoxylate (1 PO/OH) triacrylate, or trimethyolpropane propoxylate (2 PO/PH triacrylate).",
"Generally, the functional groups serve as staring points for oligonucleotides that will ultimately be coupled to the support.",
"These functional groups can be reactive with an organic group that is to be attached to the support or it can be modified to be reactive with that group, as through the use of linkers or handles.",
"The functional groups can also impart various desired properties to the support.",
"After functionalization (if necessary) of the support, tailor-made polymer networks containing activated functional groups that may serve as carrier sites for complementary oligonucleotide capture probes can be grafted to the support.",
"The advantage of this approach is that the loading capacity of capture probes can thus be increased significantly, while physical properties of the intermediate solid-to-liquid phase can be controlled better.",
"Parameters that are subject to optimization include the type and concentration of functional group-containing monomers, as well as the type and relative concentration of the crosslinkers that are used.",
"The surface of the functionalized substrate is preferably provided with a layer of linker molecules, although it will be understood that the linker molecules are not required elements of the invention.",
"The linker molecules are preferably of sufficient length to permit polymers in a completed substrate to interact freely with molecules exposed to the substrate.",
"The linker molecules should be 6-50 atoms long to provide sufficient exposure.",
"The linker molecules may be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.",
"According to alternative embodiments, the linker molecules are selected based upon their hydrophilic/hydrophobic properties to improve presentation of synthesized polymers to certain receptors.",
"For example, in the case of a hydrophilic receptor, hydrophilic linker molecules will be preferred to permit the receptor to approach more closely the synthesized polymer.",
"According to another alternative embodiment, linker molecules are also provided with a photocleavable group at any intermediate position.",
"The photocleavable group is preferably cleavable at a wavelength different from the protective group.",
"This enables removal of the various polymers following completion of the syntheses by way of exposure to the different wavelengths of light.",
"The linker molecules can be attached to the substrate via carbon-carbon bonds using, for example, (poly)tri-fluorochloroethylene surfaces or, preferably, by siloxane bonds (using, for example, glass or silicon oxide surfaces).",
"Siloxane bonds with the surface of the substrate may be formed in one embodiment via reactions of linker molecules bearing trichlorosilyl groups.",
"The linker molecules may optionally be attached in an ordered array, i.e., as parts of the head groups in a polymerized monolayer.",
"In alternative embodiments, the linker molecules are adsorbed to the surface of the substrate.",
"As an example of assembling arrays with multimers, such assembly can be achieved with tetramers.",
"Of the 256 (44) possible ways in which four bases can be arranged as tetramers, 36 that have unique sequences can be selected.",
"Each of the chosen tetramers differs from all the others by at least two bases, and no two dimers are complementary to each other.",
"Furthermore, tetramers that would result in self-pairing or hairpin formation of the addresses have been el inated.",
"The final tetramers are listed in Table 1 and have been numbered arbitrarily from 1 to 36.This unique set of tetramers are used as design modules for the sometimes desired 24-mer capture oligonucleotide address sequences.",
"The structures can be assembled by stepwise (one base at a time) or convergent (tetramer building blocks) synthetic strategies.",
"Many other sets of tetramers may be designed which follow the above rules.",
"The segment approach is not uniquely limited to tetramers, and other units, i.e.",
"dimers, trimers, pentamers, or hexamers could also be used.",
"TABLE 1 List of tetramer PNA sequences and complemen- tary DNA sequences, which differ from each other by at least 2 bases.",
"Number Sequence (N-C) Complement (5′-3′) G + C 1.TCTG CAGA 2 2.TGTC GACA 2 3.TCCC GGGA 3 4.TGCG CGCA 3 5.TCGT ACGA 2 6.TTGA TCAA 1 7.TGAT ATCA 1 8.TTAG CTAA 1 9.CTTG CAAG 2 10.CGTT AACG 2 11.CTCA TGAG 2 12.CACG CGTG 3 13.CTGT ACAG 2 14.CAGC GCTG 3 15.CCAT ATGG 2 16.CGAA TTCG 2 17.GCTT AAGC 2 18.GGTA TACC 2 19.GTCT AGAC 2 20.GACC GGTC 3 21.GAGT ACTC 2 22.GTGC GCAC 3 23.GCAA TTGC 2 24.GGAC GTCC 3 25.AGTG CACT 2 26.AATC GATT 1 27.ACCT AGGT 2 28.ATCG CGAT 2 29.ACGG CCGT 3 30.AGGA TCCT 2 31.ATAC GTAT 1 32.AAAG CTTT 1 33.CCTA TAGG 2 34.GATG CATC 2 35.AGCC GGCT 3 36.TACA TGTA 1 Note that the numbering scheme for tetramers permits abbreviation of each address as a string of six numbers (e.g., second column of Table 2 infra).",
"The concept of a 24-mer address designed from a unique set of 36 tetramers (Table 1) allows a huge number of possible structures, 366=2,176,782,336.FIG.",
"19 shows one of the many possible designs of 36 tetramers which differ from each other by at least 2 bases.",
"The checkerboard pattern shows all 256 possible tetramers.",
"A given square represents the first two bases on the left followed by the two bases on the top of the checkerboard.",
"Each tetramer must differ from each other by at least two bases, and should be non-complementary.",
"The tetramers are shown in the white boxes, while their complements are listed as (number)'.",
"Thus, the complementary sequences GACC (20) and GGTC (20′) are mutually exclusive in this scheme.",
"In addition, tetramers must be non-palindromic, e.g., TCGA (darker diagonal line boxes), and non-repetitive, e.g., CACA (darker diagonal line boxes from upper left to lower right).",
"All other sequences which differ from the 36 tetramers by only 1 base are shaded in light gray.",
"Four potential tetramers (white box) were not chosen as they are either all A•T or G•C bases.",
"However, as shown below, the Tm values of A•T bases can be raised to almost the level of G•C bases.",
"Thus, all A•T or G•C base tetramers (including the ones in white boxes) could potentially be used in a tetramer design.",
"In addition, thymine can be replaced by 5-propynyl uridine when used within capture oligonucleotide address sequences as well as in the oligonucleotide probe addressable array-specific portions.",
"This would increase the Tm of an A•T base pair by 1.7° C. Thus, Tm values of individual tetramers should be approximately 15.1° C. to 15.7° C. Tm values for the full length 24-mers should be 95° C. or higher.",
"To illustrate the concept, a subset of six of the 36 tetramer sequences were used to construct arrays: 1=TGCG; 2=ATCG; 3=CAGC; 4=GGTA; 5=GACC; and 6=ACCT.",
"This unique set of tetramers can be used as design modules for the required 24-mer addressable array-specific portion and 24-mer complementary capture oligonucleotide address sequences.",
"This embodiment involves synthesis of five addressable array-specific portion (sequences listed in Table 2).",
"Note that the numbering scheme for tetramers allows abbreviation of each portion (referred to as “Zip #”) as a string of six numbers (referred to as “zip code”).",
"TABLE 2 List of all 5 DNA/PNA oligonucleotide address sequences.",
"Sequence Zip # Zip code (5′ → 3′ or NH2 → COOH) G + C Zip11 1-4-3-6-6-1 TGCG-GGTA-CAGC-ACCT- 15 ACCT-TGCG Zip12 2-4-4-6-1-1 ATCG-GGTA-GGTA-ACCT- 14 TGCG-TGCG Zip13 3-4-5-6-2-1 CAGC-GGTA-GACC-ACCT- 15 ATCG-TGCG Zip14 4-4-6-6-3-1 GGTA-GGTA-ACCT-ACCT- 14 CAGC-TGCG Zip15 5-4-1-6-4-1 GACC-GGTA-TGCG-ACCT- 15 GGTA-TGCG Each of these oligomers contains a hexaethylene oxide linker arm on their 5′ termini [P. Grossman, et al., Nucl.",
"Acids Res., 22: 4527-4534 (1994), which is hereby incorporated by reference], and ultimate amino-functions suitable for attachment onto the surfaces of glass slides, or alternative materials.",
"Conjugation methods will depend on the free surface functional groups [Y. Zhang, et al., Nucleic Acids Res., 19: 3929-3933 (1991) and Z. Guo, et al., Nucleic Acids Res., 34: 5456-5465 (1994), which are hereby incorporated by reference].",
"Synthetic oligonucleotides (normal and complementary directions, either for capture hybridization or hybridization/ligation) are prepared as either DNA or PNA, with either natural bases or nucleotide analogues.",
"Such analogues pair with perfect complementarity to the natural bases but increase Tm values (e.g., 5-propynyl-uracil).",
"In accordance with the present invention, false hybridization signals from DNA synthesis errors are avoided.",
"Addresses can be designed so there are very large differences in hybridization Tm values to incorrect address.",
"In contrast, the direct hybridization approaches depend on subtle differences.",
"The present invention also eliminates problems of false data interpretation with gel electrophoresis or capillary electrophoresis resulting from either DNA synthesis errors, band broadening, or false band migration.",
"The use of a capture oligonucleotide to detect the presence of ligation products, eliminates the need to detect single-base differences in oligonucleotides using differential hybridization.",
"Other existing methods in the prior art relying on allele-specific PCR, differential hybridization, or sequencing-by-hybridization methods must have hybridization conditions optimized individually for each new sequence being analyzed.",
"When attempting to detect multiple mutations simultaneously, it becomes difficult or impossible to optimize hybridization conditions.",
"In contrast, the present invention is a general method for high specificity detection of correct signal, independent of the target sequence, and under uniform hybridization conditions.",
"The present invention yields a flexible method for discriminating between different oligonucleotide sequences with significantly greater fidelity than by any methods currently available within the prior art.",
"The array of the present invention will be universal, making it useful for detection of cancer mutations, inherited (germline) mutations, and infectious diseases.",
"Further benefit is obtained from being able to reuse the array, lowering the cost per sample.",
"The present invention also affords great flexibility in the synthesis of oligonucleotides and their attachment to supports.",
"Oligonucleotides can be synthesized off of the support and then attached to unique surfaces on the support.",
"Segments of multimers of oligonucleotides, which do not require intermediate backbone protection (e.g., PNA), can be synthesized and linked onto to the solid support.",
"Added benefit is achieved by being able to integrate these synthetic approaches with design of the capture oligonucleotide addresses.",
"Such production of solid supports is amenable to automated manufacture, obviating the need for human intervention and resulting contamination concerns.",
"An important advantage of the array of the present invention is the ability to reuse it with the previously attached capture oligonucleotides.",
"In order to prepare the solid support for such reuse, the captured oligonucleotides must be removed without removing the linking components connecting the captured oligonucleotides to the solid support.",
"A variety of procedures can be used to achieve this objective.",
"For example, the solid support can be treated in distilled water at 95-100° C., subjected to 0.01 N NaOH at room temperature, contacted with 50% dimethylformamide at 90-95° C., or treated with 50% formamide at 90-95° C. Generally, this procedure can be used to remove captured oligonucleotides in about 5 minutes.",
"These conditions are suitable for disrupting DNA-DNA hybridizations; DNA-PNA hybridizations require other disrupting conditions.",
"The present invention is illustrated, but not limited, by the following examples.",
"EXAMPLES Example 1 Materials and Methods Oligonucleotide Synthesis and Purification.",
"Oligonucleotides were obtained as custom synthesis products from IDT, Inc. (Coralville, Iowa), or synthesized in-house on an ABI 394 DNA Synthesizer (PE Biosystems Inc.; Foster City, Calif.) using standard phosphoramidite chemistry.",
"Spacer phosphoramidite 18, 3′-amino-modifer C3 CPG, and 3′-fluorescein CPG were purchased from Glen Research (Sterling, Va.).",
"All other reagents were purchased from PE Biosystems.",
"Both labeled and unlabeled oligonucleotides were purified by electrophoresis on 12% denaturing polyacrylamide gels.",
"Bands were visualized by UV shadowing, excised from the gel, and eluted overnight in 0.5 M NaCl, 5 mM EDTA, pH 8.0 at 37° C. Oligonucleotide solutions were desalted on C18 Sep-Paks (Waters Corporation; Milford, Mass.)",
"according to the manufacturer's instructions, following which the oligonucleotides were concentrated to dryness (Speed-Vac) and stored at −20° C. Cleaning of Microscope slides.",
"Glass microscope slides (VWR, precleaned, 3 in.×.1 in.×1.2 mm) were incubated in boiling conc.",
"NH4OH −30% H2O2—H2O (1:1:5, v/v/v) for 10 min and rinsed in distilled water.",
"A second incubation was performed in boiling conc.",
"HCl −30% H2O2—H2O for 10 min.",
"See U. Jönsson, et al., “Absorption Behavior of Fibronetin on Well Characterized Silica Surfaces,” J. Colloid Interface Sci.",
"90: 148-163 (1982), which is hereby incorporated by reference.",
"The slides were rinsed thoroughly in distilled water, methanol, and acetone, and were air-dried at room temp.",
"Polymer Coated Slides.",
"Immediately following cleaning, slides (Fisher Scientific, precleaned, 3 in.×1 in.×1.2 mm) were immersed in 2% methacryloxypropyltrimethoxysilane, 0.2% triethylamine in CHCl3 for 30 min at 25° C., and then washed with CHCl3 (2×15 min).",
"A monomer solution [20 μL: 8% acrylamide, 2% acrylic acid, 0.02% N,N′-methylene-bisacrylamide (500:1 ratio of monomers:crosslinker), 0.8% ammonium persulfate radical polymerization initiator] was deposited on one end of the slides and spread out with the aid of a cover slip (24×50 mm) that had been previously silanized [5% (CH3)2SiCl2 in CHCl3].",
"Polymerization was achieved by heating the slides on a 70° C. hot plate for 4.5 min.",
"Upon removal of the slides from the hot plate, the cover slips were immediately peeled off with aid of a single-edge razor blade.",
"The coated slides were rinsed with deionized water, allowed to dry in an open atmosphere, and stored under ambient conditions.",
"Preparation of Zip-Code Arrays.",
"Polymer-coated slides were pre-activated by immersing them for 30 min at 25° C. in a solution of 0.1 M 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride plus 20 mM N-hydroxysuccinimide in 0.1 M K2HPO4/KH2PO4, pH 6.0.The activated slides were rinsed with water, and then dried in a 65° C. oven; they were stable upon storage for 6 months or longer at 25° C. in a desiccator over Drierite.",
"Arrays were spotted on a Cartesian Technologies Pixsys 5500 robot at 25° C. and 70% relative humidity using 500 μM zip-code oligonucleotide solutions in 0.2 M K2HPO4/KH2PO4, pH 8.3.Each address was spotted in quadruplicate.",
"Additionally, Cy3, Cy5, and fluorescein fiducials were printed along the top and down the right hand side of each array.",
"Following spotting, uncoupled oligonucleotides were removed from the polymer surfaces by soaking the slides in 300 mM bicine, pH 8.0, 300 mM NaCl, 0.1% SDS, for 30 min at 65° C., rinsing with water, and drying.",
"The arrays were stored at 25° C. in slide boxes until needed.",
"PCR Amplification of K-ras DNA Samples.",
"PCR amplifications were carried out under paraffin oil in 50 μL reaction mixtures containing 10 mM Tris.HCl, pH 8.3, 4 mM MgCl2, 50 mM KCl, 800 μM dNTPs, 1 μM forward and reverse primers (50 pmol of each primer; K-rasEx1forward and K-rasEx1reverse (Table 3)), 1 U AmpliTaq Gold, and 100 ng of genomic DNA extracted from paraffin-embedded tumors or from cell lines.",
"Reactions were preincubated for 10 min at 95° C. Amplification was achieved by thermally cycling for 40 rounds of 94° C. for 30 sec; 60° C. for 1 min; and 72° C. for 1 min, followed by a final elongation at 72° C. for 5 min.",
"Following PCR, 1 μL of Proteinase K (18 mg/mL) was added, and reactions were heated to 70° C. for 10 min and then quenched at 95° C. for 15 min.",
"Two μl L of each PCR product was analyzed on a 3% agarose gel to verify the presence of amplification product of the expected size.",
"LDR of K-ras DNA samples.",
"LDR was carried out under paraffin oil in 20 μL volumes containing 20 mM Tris.HCl, pH 8.5-5 mM MgCl2-100 mM KCl, 10 mM DTT, 1 mM NAD+, 10 pmol total LDR probes [500 fmol each of fluorescently-labeled discriminating probes (K-rasc32Wt, labeled with Cy3, Cy5, and fluorescein; K-rasc12.2D labeled with Cy3; K-rasc12.2A labeled with Cy5; K-rasc12.2V labeled with fluorescein; K-rasc12.1S labeled with Cy3; K-rasc12.1R, labeled with Cy5; K-rasc12.1C, labeled with fluorescein; and K-rasc13.4D labeled with Cy3)+5 pmol total common probes; (1500 fmol each of K-rascd32Com9cZip1, K-rascd12Com2cZip2, and K-rascd12ComlcZip3, and 500 fmol of K-rascd13Com4cZip4) (Table 3)], and 2 μL PCR products from the cell line or tumor samples.",
"The reaction mixtures were pre-heated for 2 min at 94° C., and then 25 fmol of wild-type Tth DNA ligase was added.",
"The LDR reaction mixtures were cycled for 20 rounds of 94° C. for 30 sec and 65° C. for 4 min.",
"Hybridization of K-ras LDR Products to DNA Arrays.",
"The LDR reaction mixtures were diluted with 20 μL of 2× hybridization buffer to produce a final buffer concentration of 300 mM MES, pH 6.0, 10 mM MgCl2, 0.1% SDS, denatured at 94° C. for 3 min, and chilled on ice.",
"Arrays were pre-incubated for 15 min at 25° C. in 1× hybridization buffer.",
"Coverwells (Grace, Inc; Sunriver, Oreg.)",
"were attached to the arrays and filled with 30 μL of the diluted LDR reaction mixtures.",
"The arrays were placed in humidified culture tubes and incubated for 1 h at 65° C. and 20 rpm in a rotating hybridization oven.",
"Following hybridization, the arrays were washed in 300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS for 10 min at 25° C. Fluorescent signals were measured using a Scanarray 5000 (GSI Lumonics).",
"LDR detection of 7 specific mutations in K-ras on an addressable universal microarray is shown in FIG.",
"36.The three signals along the top and those down the right hand side of each array are fiducials used for alignment.",
"The next 4 addresses across in the second row correspond to addresses #1, #2, #3, and #4, complements of cZip1, cZip2, cZip3, and cZip4, respectively.",
"The eight cell line (i.e.",
"COLO205, LS180, SW1116, SW480, and DLD1) and tumor samples (G12S, G12R, and G12C) correctly identified the mutations present.",
"Wild-type cell line COLO205 gave Cy3, Cy5, and fluorescein signal at address #1.The wild-type signal at address #1 was used as a control for all experiments.",
"The LS180 cell line containing the Asp12 mutation gave a Cy3 signal at address #2.The SW1116 cell line containing the Ala12 mutation gave a Cy5 signal at address #2.The SW480 cell line containing the Val12 mutation gave a fluorescein signal at address #2.The G12S tumor sample containing the Ser12 mutation gave a Cy3 signal at address #3.The G12R tumor sample containing the Arg12 mutation gave a Cy5 signal at address #3.The G12C tumor sample containing the Cys12 mutation gave a fluorescein signal at address #3.The DLD1 cell line containing the Asp13 mutation gave a Cy3 signal at address #4.The incorrect signals seen at Zip4 in the LS180 and SW1116 samples were due to contamination of the samples.",
"TABLE 3 Primers designed for mutation detection in K-ras by PCR/LDR/Array Hybridiztion.",
"Primer Sequence (5′→3′) K-rasEx1forward AAC CTT ATG TGT GAC ATG TTC TAA TAT AGT CAC K-rasEx1reverse AAA ATG GTC AGA GAA ACC TTT ATC TGT ATC K-rascd32Com9cZip1 PTATGATCCAACAATAGAGGTAAATCTTGTCGCAGATTT TGCGCTGGATTTCAA K-rasc32Wt Cy3-ATTCAGAATCATTTTGTGGACGAA Cy5-ATTCAGAATCATTTTGTGGACGAA Fam-ATTCAGAATCATTTTGTGGACGAA K-rascd12Com2cZip2 PTGGCGTAGGCAAGAGTGCCTTTCGCCGTCGTGTAGGCT TTTCAA K-rasc12.2D Cy3-AAACTTGTGGTAGTTGGAGCTGA K-rasc12.2A Cy5-AAACTTGTGGTAGTTGGAGCTGC K-rasc12.2V Fam-AAACTTGTGGTAGTTGGAGCTGT K-rascd12Com1cZip3 PGTGGCGTAGGCAAGAGTGCCCCGTAAGCCCGTATGGC AGATCAA K-rasc12.1S Cy3-ATATAAACTTGTGGTAGTTGGAGCTA K-rasc12.1R Cy5-ATATAAACTTGTGGTAGTTGGAGCTC K-rasc12.1C Fam-ATATAAACTTGTGGTAGTTGGAGCTT K-rascd13Com4cZip4 PCGTAGGCAAGAGTGCCTTGACATGGCCGTGCTGGGGA CA AGTCAA K-rasc13.4D Cy3-TGTGGTAGTTGGAGCTGGTGA Amplification was achieved by thermal cycling for 40 rounds of 94° C. for 15 sec and 60° C. for 2 min, followed by a final elongation step at 65° C. for 5 mm.",
"Following PCR, 1 μL of Proteinase K (18 mg/mL) was added, and reactions were heated to 70° C. for 10 min and then quenched at 95° C. for 15 min.",
"One μL of each PCR product was analyzed on a 3% agarose gel to verify the presence of amplification product of the expected size.",
"LDR of K-ras DNA samples.",
"LDR reactions were carried out under paraffin oil in 20 μL volumes containing 20 mM Tris.HCl, pH 8.5, 5 mM MgCl2, 100 mM KCl, 10 mM DTT, 1 mM NAD+, 8 pmol total LDR probes (500 fmol each of discriminating probes+4 pmol fluorescently-labeled common probes), and 1 pmol PCR products from cell line or tumor samples.",
"Two probe mixes were prepared, each containing the seven mutation-specific probes, the three common probes, and either the wild-type discriminating probe for codon 12 or that for codon 13 (Table 3).",
"The reaction mixtures were pre-heated for 2 min at 94° C., and then 25 fmol of wild-type Tth DNA ligase was added.",
"The LDR reactions were cycled for 20 rounds of 94° C. for 15 sec and 65° C. for 4 min.",
"An aliquot of 2 μL of each reaction was mixed with 2 L of gel loading buffer [8% blue dextran, 50 mM EDTA, pH 8.0—formamide (1:5)], denatured at 94° C. for 2 min, and chilled on ice.",
"One μL of each mixture was loaded on a 10% denaturing polyacrylamide gel and electrophoresed on an ABI 377 DNA sequencer at 1500 volts.",
"Hybridization of K-ras LDR Products to DNA Arrays.",
"The LDR reactions (17 μL) were diluted with 40 μL of 1.4× hybridization buffer to produce a final buffer concentration of 300 mM MES, pH 6.0, 10 mM MgCl2, 0.1% SDS.",
"Arrays were pre-incubated for 15 min at 25° C. in 1× hybridization buffer.",
"Coverwells (Grace, Inc; Sunriver, Oreg.)",
"were filled with the diluted LDR reactions and attached to the arrays.",
"The arrays were placed in humidified culture tubes and incubated for 1 h at 65° C. and 20 rpm in a rotating hybridization oven.",
"Following hybridization, the arrays were washed in 300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS for 10 min at 25° C. Fluorescent signals were measured using a microscope/CCD, as described in the following paragraph.",
"Image Analysis.",
"Arrays were imaged using a Molecular Dynamics FluorImager 595 (Sunnyvale, Calif.) or an Olympus AX70 epifluorescence microscope (Melville, N.Y.) equipped with a Princeton Instruments TE/CCD-512 TKBM1 camera (Trenton, N.J.).",
"For analysis of fluorescein-labeled probes on the FluorImager, the 488 nm excitation was used with a 530/30 emission filter.",
"The spatial resolution of scans was 100 μm per pixel.",
"The resulting images were analyzed using ImageQuaNT software provided with the instrument.",
"The epifluorescence microscope was equipped with a 100 W mercury lamp, a FITC filter cube (excitation 480/40, dichroic beam splitter 505, emission 535/50), a Texas Red filter cube (excitation 560/55, dichroic beam splitter 595, emission 645/75), and a 100 mm macro objective.",
"The macro objective allows illumination of an object field up to 15 mm in diameter and projects a 7×7 mm area of the array onto the 12.3×12.3 mm matrix of the CCD.",
"Images were collected in 16-bit mode using the Winview32 software provided with the camera Analysis was performed using Scion Image (Scion Corporation, Frederick, Md.).",
"Example 2 Amplification of BRCA1 and BRCA2 Exons for PCR/PCR/LDR Detection of Wild-Type and Mutant Alleles A multiplex assay was used to detect small insertions and deletions using a modified PCR to evenly amplify each amplicon (PCR/PCR) (Belgrader et al., “A Multiplex PCR-Ligase Detection Reaction Assay for Human Identity Testing,” Genome Science and Technology 1: 77-87 (1996), which is hereby incorporated by reference) followed by ligase detection reaction (“LDR”) (Khanna, M. et al., “Multiplex PCR/LDR for Detection of K-ras Mutations in Primary Colon Tumors,” Oncogene 18: 27-38 (1999), which is hereby incorporated by reference).",
"FIG.",
"20 shows how multiplex amplification of the relevant exons is carried out to ensure equal amplification of all products: a limited number of PCR cycles was performed using gene-specific primers, with further rounds of amplification primed from the universal sequences located at the extreme 5′-ends of the PCR primers.",
"This approach minimizes amplification bias due to primer-specific effects.",
"LDR was next used to detect both wild-type and mutant versions of the queried sequence.",
"The ligation oligonucleotides probes hybridize to both wild-type and mutant products, but ligate only when both probes are perfectly matched with no gaps or overlaps.",
"Products can be either eletrophoretically separated or hybridized to a microarray for identification.",
"PCR was carried out as a single-tube, multiplex reaction in order to simultaneously amplify BRCA1 exons 2 and 20 and BRCA2 exon 11.Genomic DNA was extracted from blood samples of Ashkenazi Jewish individuals and amplified in a 25 μl reaction mixture containing 100 ng of DNA, 400 μM of each dNTP, 1×PCR Buffer II (10 mM Tris-HCl pH 8.3 at 25° C., 50 mM KCl) supplemented with 4 mM MgCl2, 1 U AmpliTaq Gold and 2 pmol of each gene-specific primer bearing either universal primer A or B on the 5′ ends.",
"Table 4 shows the primers and probes for detection of BRCA1 and BRCA2 mutations using PCR/LDR/array hybridization as follows: TABLE 4 Primers and Probes designed for mutation de- tection in BRCA1 and BRCA2 by PCR/LDR/Array Hybridization.",
"Primer Sequence (5′->3′) PCR primers: Universal pri- 5′ ggagcacgctatcccgttagac 3′ mer A (Uni A) Universal pri- 5′ cgctgccaactaccgcacatg 3′ mer B (Uni B) BRCA1 exon 2 5′ Uni A- forward TCATTGGAACAGAAAGAAATGGATTTATC 3′ BRCA1 exon 2 5′ Uni B- reverse TCTTCCCTAGTATGTAAGGTCAATTCTGTTC 3′ BRCA1 exon 20 5′ Uni A- forward ACTTCCATTGAAGGAAGCTTCTCTTTC 3′ BRCA1 exon 20 5′ Uni B- reverse ATCTCTGCAAAGGGGAGTGGAATAC 3′ BRCA2 exon 11 5′ Uni A- forward CAAAATATGTCTGGATTGGAGAAAGTTTC 3′ BRCA2 exon 11 5′ Uni B- reverse TTGGAAAAGACTTGCTTGGTACTATCTTC 3′ LDR Gel-Based Assay: Discriminating Oligonucleotide Probes: BRCA1 ex 2 wild- 5′ Fam- type position aaCATTAATGCTATGCAGAAAATCTTAGAG 3′ 185 BRCA1 ex 2 posi- 5′ Tet- tion 185 del AG GTCATTAATGCTATGCAGAAAATCTTAG 3′ mutation BRCA1 ex 20 5′ Fam- wild-type posi- CCAAAGCGAGCAAGAGAATCC 3′ tion 5382 BRCA1 ex 20 po- 5′ Tet- sition 5382 ins aaCAAAGCGAGCAAGAGAATCCC 3′ C mutation BRCA2 ex 11 TTTAGCACAGCAAGT 3′ wild-type posi- tion 6174 BRCA2 ex 11 po- 5′ Tet- sition 6174 del T mutation TACTTGTGGGATTTTTAGCACAGCAAG 3′ LDR Common Oligonucleotide Probes: BRCA1 ex 2 5′ P- position 185 TGTCCCATCTGGTAAGTCAGCACAAAC-B 3′ BRCA1 ex 20 5′ P- position 5382 CAGGACAGAAAGGTAAAGCTCCCTC-B 3′ BRCA2 ex 11 5′ P- position 6174 GGAAAATCTGTCCAGGTATCAGAT-B 3′ LDR Microarray Assay: Discriminating Oligonucleotide Probes: BRCA1 ex 2 5′ Cy3- control TGCATAGGAGATAATCATAGGAATCC 3′ BRCA1 ex 2 posi- 5′ Cy3- ion 185 del AG GTCATTAATGCTATGCAGAAAATCTTAG 3′ mutation BRCA1 ex 20 5′ Cy3- control CCTCTGACTTCAAAATCATGCTG 3′ BRCA1 ex 20 po- 5′ Cy3- sition 5382 ins CAAAGCGAGCAAGAGAATCCC 3′ C mutation BRCA2 ex 11 5′ Cy3- control CTTCCCTATACTACATTTACATATATCTGAAG 3′ BRCA2 ex 11 po- 5′ Cys- sition 6174 del TCTTGTGGGATTTTTAGCACAGCAAG 3′ T mutation Common Oligonu- cleotide Probes for Controls: BRCA1 exon 2 ′P- control + cZip 1 CAAATTAATACACTCTTGTGCTGACTTACCA- cgcagattttgcgctggatttcaa-B 3′ BRCA1 exon 20 5′ P- control + cZip 2 AAAGAAACCAAACACAACCCATCAG- ttcgccgtcgtgtaggcttttcaa-B 3′ BRCA2 exon 11 5′ P- control + cZip 3 TTTCCAAACTAACATCACAAGGTGATATTT- ccgtaagcccgtatggcagatcaa-B 3′ Common Oligonu- cleotide Probes for Mutations: BRCA1 exon 2 5′ P- position 185 + TGTCCCATCTGGTAAGTCAGCACAAAC- cZip 9 catcgtccctttcgatgggatcaa-B 3′ BRCA1 exon 20 5′ P- position 5382 + CAGGACAGAAAGGTAAAGCTCCCTC- cZip 10 caaggcacgtcccagacgcatcaa-B 3′ BRCA2 exon 11 5′ P- position 6174 + GGAAAATCTGTCCAGGTATCAGAT- cZip 11 gcacgggagctgacgacgtgtcaa-B 3′ The reaction was overlaid with mineral oil and preincubated for 10 min at 95° C. Amplification was performed for 15 cycles as follows: 94° C. for 15 sec, 65° C. for 1 min.",
"A second 25 μl aliquot of the reaction mixture was added through the mineral oil containing 25 pmol each of universal primers A and B. Cycling was repeated using 55° C. annealing temperature.",
"The reaction was next digested with a 2 μl solution of 1 mg/ml Proteinase K/50 mM EDTA at 55° C. for 10 min.",
"Proteinase K was eliminated by a final incubation at 90° C. for 15 min.",
"For LDR, oligonucleotide synthesis and purification were carried out as previously described (Khanna et al., “Multiplex PCR/LDR for Detection of K-ras Mutations in Primary Colon Tumors,” Oncogene 18: 27-38 (1999), which is hereby incorporated by reference).",
"Tth DNA ligase was overproduced and purified as described elsewhere (Luo et al., “Identification of Essential Residues in Thermus thermophilus DNA Ligase,” Nucleic Acids Research 24: 3079-3085 (1996) and Barany et al., “Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase Gene,” Gene 109: 1-11 (1991), which are hereby incorporated by reference).",
"LDR was performed in a 20 μl reaction containing 500 fmol of each probe, 2 μl of amplified DNA and 20 mM Tris-HCl, pH 7.6; 10 mM MgCl2; 100 mM KCl; 10 mM DTT; 1 mM NAD+.",
"The reaction was heated to 94° C. for 1.5 min prior to adding 25 fmol of Tth DNA ligase and then subjected to 20 cycles of 15 sec at 94° C. and 4 min at 65° C. (See Table 4).",
"Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) (Rahman et al., “The Genetics of Breast Cancer Susceptibility,” Annu.",
"Rev.",
"Genet.",
"32: 95-121 (1998), which is hereby incorporated by reference) as a model system, the assay readily detected these mutations in multiplexed reactions in over 80 DNA samples.",
"FIG.",
"21A shows a representative LDR gel detecting the three BRCA mutations.",
"By fluorescent end-labeling the discriminating upstream oligonucleotides with either FAM (for wild-type) or TET (for mutant), and by adding different length “tails” to LDR oligonucleotide probes, ligation products were easily distinguished based on label and migration on a polyacrylamide gel.",
"Wild-type products are identified at the right side of the gel.",
"Mutant products are identified at the top of the gel.",
"Electrophoretic separation was performed at 1400 volts using 8 M urea-10% polyacrylamide gels and an ABI 373 DNA sequencer.",
"Fluorescent ligation products were analyzed and quantified using the ABI Gene Scan 672 software.",
"The analysis was next extended to detect the mutations in pooled samples of DNA.",
"DNAs with known mutations were diluted 1:2, 1:5, 1:10, and 1:20 with wild-type DNA prior to PCR amplification and then subjected to LDR These simulation experiments showed PCR/PCR/LDR could successfully detect the presence of all three mutations when known mutant DNA was diluted 1:20 in wild-type DNA prior to amplification.",
"FIG.",
"21B shows BRCA1 and BRCA2 mutation detection on pooled samples of DNA.",
"DNA samples with known mutations were diluted into wild-type DNA prior to amplification.",
"Ligation products from multiplex LDR are shown for each dilution.",
"BRCA1 del AG, BRCA1 ins C, and BRCA2 del T mean that only one mutation is present; multiplex LDR directed against only mutant sequences use 500 fmol of each LDR oligonucleotide probes.",
"3 mutations means that all three mutations are present; multiplex LDR directed against mutant sequences only use 500 fmol of each LDR oligonucleotide.",
"3 mutations+wild-type controls at 1:20 mean that all three mutations are present; multiplex LDR are directed against both mutant and wild-type sequences using 500 fmol and 25 fmol of each LDR oligonucleotide probes, respectively.",
"The pooling experiment was repeated using 249 blinded Ashkenazi Jewish DNA samples.",
"Tubes containing the blinded DNAs were assembled into a 9×9 gridded format and aliquots from each tube were combined across the rows and then down the columns to produce one tube of combined DNA for each row and each column.",
"This was done to uniquely classify each sample using points of intersection on the gridded format.",
"The pooled DNA was then subjected to amplification and LDR as described above.",
"248 of the 249 samples were correctly typed.",
"The single sample that was incorrectly identified as wild-type proved to be too dilute and fell below the limits of detection when mixed with 9 other samples of higher concentration.",
"The number of individual reactions carried out was reduced from 249 to 96 by this strategy (55 pooled samples and 41 individual samples used for confirmation).",
"In addition to gel-based detection, mutation identification was also accomplished by screening reaction products with a universal DNA microarray (Gerry et al., “Universal DNA Microarray Method for Multiplex Detection of Low Abundance Point Mutations,” J Mol Bio.",
"292: 251-262 (1999), which is hereby incorporated by reference).",
"Microarrays were processed and spotted as previously described (Gerry et al., “Universal DNA Microarray Method for Multiplex Detection of Low Abundance Point Mutations,” J Mol Bio.",
"292: 251-262 (1999), which is hereby incorporated by reference) using a Pixsys5500 robot enclosed in a humidity chamber (Cartesian Technologies, Irvine, Calif.).",
"Briefly, LDR reactions were hybridized in 32 μl containing 300 mM MES, pH 6.0, 10 mM MgCl2, 0.1% SDS at 65° C. for 1 h in a rotating chamber.",
"After washing in 300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS for 10 min at 25° C. The array was imaged on an Olympus Provis AX70 microscope using a 100 W mercury burner, a Texas Red filter cube, and a Princeton Instruments TEK512/CCD camera.",
"The 16-bit greyscale images were captured using MetaMorph Imaging System (Universal Imaging Corporation, West Chester, Pa.) and rescaled to more narrowly bracket the LDR signal before conversion to 8-bit greyscale.",
"The 8-bit images were colored using Adobe Photoshop to render the Cy3 signal red.",
"Preliminary microarray experiments using probes designed in the gel-based format (i.e., differentially labeled discriminating probes and identical common probes) demonstrated that wild-type and mutant versions of the three BRCA sequences were readily detected on the array (see WO 97/31256 to Barany et al., which is hereby incorporated by reference).",
"In this version, both types of sequences were directed to the same addresses (e.g., BRCA1 185delAG and BRCA1 185 wild-type were both directed to zip-code 1).",
"Although this format proved successful, PCR/PCR/LDR has the potential of detecting hundreds of mutations in a single-tube reaction and this design does not make optimal use of the array for such large-scale mutation detection experiments.",
"In order to establish the experimental paradigm for future studies, the addressable format was expanded by choosing a sequence in each of the amplicons to use as a control an LDR ligation product.",
"Thus, rather than require detection of wild-type sequences for each mutant LDR product, this format uses a single product to serve as a positive control for multiple different sequence variants within an amplicon (see FIG.",
"22).",
"One advantage of this format is that it minimizes oligonucleotide synthesis; additionally, the use of each of the 64 positions is maximized.",
"Since the number of LDR ligation products that can be detected at a single address is limited by the number of currently available spectrally separated fluorescent labels, confining the control to a specified region of the array permits one more sequence variant to be detected at each remaining address.",
"In the experiments described below, control and mutant LDR ligation products for a queried position were directed to six separate addresses on a 64 position array.",
"All three frameshift mutations were detectable by hybridization to the universal array (FIG.",
"23).",
"FIG.",
"23A shows the assignment of each control and mutant sequence to specific addresses on the array surface.",
"Control signals are directed to the upper three addresses; mutant signals are assigned to the lower three.",
"FIG.",
"23B shows signal produced by a wild-type DNA.",
"FIGS.",
"23C, E, and G show representative hybridizations for individual DNA samples.",
"FIGS.",
"23D, F, and H show representative hybridizations for each mutation using pooled samples of DNA from Ashkenazi individuals.",
"The mutations are identified on the extreme right.",
"Only combinations of the six possible addresses were visible following hybridization and no additional signals were detected at any of the unused addresses.",
"Thus, zip-code hybridization is very specific.",
"Control and mutant signals were clearly present for each of the mutations derived from samples of DNA from single individuals (FIGS.",
"23C, E, and G).",
"Pooled DNA used in analyzing the 249 DNA samples described above produced signals for mutations identical to those found in the gel-based assay (FIGS.",
"23D, F, and H).",
"In each case, the array reproduced the result of the gel.",
"These results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.",
"Example 3 p53 Chip Hybridization and Washing Conditions Three parameters (presence or absence of non-specific DNA competitor, temperature, and wash buffer composition) were varied in different combinations in order to determine which method would produce minimum background noise without significant loss of signal.",
"Hybridization was performed with 100 μg/ml of sheared salmon sperm DNA (FIGS.",
"16B, D, J, and L), 250 μg/ml of sheared salmon sperm DNA (FIGS.",
"16F, H, N, and P), or no non-specific competitor DNA (FIGS.",
"16A, C, E, G, I, K, M, and O).",
"Washing was performed for 10 min using four different conditions: room temperature in standard wash buffer (300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS) (FIGS.",
"16A, B, E, and F); room temperature in standard wash buffer supplemented with 10% formamide (FIGS.",
"16I, J, M, and N); 50° C. in standard wash buffer (FIGS.",
"16C, D, G, and H); or 50° C. in standard wash buffer supplemented with 10% formamide (FIGS.",
"16K, L, O, and P).",
"The numbers on the upper right of each panel indicate the density of pixels for p53 exon 5 control (zip-code 1) for each condition.",
"The percent of loss indicated on the right side of the figure compares the left and right panel directly adjacent to calculated percentage.",
"Fiducials (Cy3, Cy5, and fluorescein) are spotted horizontally on the upper left and vertically on the lower right regions of the chips to give orientation.",
"Zip-code 1 is located directly below the fiducial in the upper left area; subsequent zip-codes are spotted in numerical order in a left to right manner.",
"PCR was carried out as a single-tube, multiplex reaction in order to simultaneously amplify p53 exons 5 and 7.Commercially available genomic DNA from lymphocytes was amplified in a 25 μl reaction mixture containing 100 ng of DNA, 400 μM of each dNTP, 1×PCR Buffer II (10 mM Tris-HCl pH 8.3 at 25° C., 50 mM KCl) supplemented with 4 mM MgCl2, 1 U AmpliTaq Gold and 2 pmol of each gene-specific primer bearing either universal primer A or B on the 5′ ends (see Table 5).",
"The reaction was overlaid with mineral oil and preincubated for 10 min at 95° C. Amplification was performed for 15 cycles as follows: 94° C. for 15 sec, 65° C. for 1 min.",
"A second 25 μl aliquot of the reaction mixture was added through the mineral oil containing 25 pmol each of universal primers A and B. Cycling was repeated using 55° C. annealing temperature.",
"The reaction was next digested with a 2 μl solution of 1 mg/ml Proteinase K/50 mM EDTA at 55° C. for 10 min.",
"Proteinase K was eliminated by a final incubation at 90° C. for 15 min.",
"For LDR, oligonucleotide synthesis and purification were carried out as previously described (Khanna et al., “Multiplex PCR/LDR for Detection of K-ras Mutations in Primary Colon Tumors,” Oncogene 18: 27-38 (1999), which is hereby incorporated by reference).",
"Tth DNA ligase was overproduced and purified as described elsewhere (Luo et al., “Identification of Essential Residues in Thermus thermophilus DNA Ligase,” Nucleic Acids Research 24: 3079-3085 (1996) and Barany et al., “Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase Gene,” Gene 109: 1-11 (1991), which are hereby incorporated by reference).",
"LDR was performed in a 20 μl reaction containing 500 fmol of each probe, 2 μl of amplified DNA and 20 mM Tris-HCl, pH 7.6; 10 mM MgCl2; 100 mM KCl; 10 mM DTT; 1 mM NAD+.",
"The reactants were heated to 94° C. for 1.5 min prior to adding 25 fmol of Tth DNA ligase and then subjected to 20 cycles of 15 sec at 94° C. and 4 min at 65° C. See Table 5 which shows the oligonucleotide primers and probes designed to detect mutations in p53 by PCR/LDR/array hybridization as follows: TABLE 5 Primers and Probes Designed for Mutation Detection in p53 by PCR/LDR/Array Hybridization.",
"Primer/Probe Sequence (5′->3′) Uni A primer GGAGCACGCTATCCCGTTAGAC Uni B2 primer CGCTGCCAACTACCGCACATC p53X5FzipA GGAGCACGCTATCCCGTTAGACCTGTTCACTTGTGCCCTGACTTTC p53X5RzipB CGCTGCCAACTACCGCACATCCCAGCTGCTCACCATCGCTATC K132LA2G Fam-aaaGCCAGTTGGCAAAACATCC K132LA2G3 Cy3-GCCAGTTGGCAAAACATCC K132LA2GCOM pTGTTGAGGGCAGGGGAGTACTGTAaaa-B K132LA2Gzip9 pTGTTGAGGGCAGGGGAGTACTGTA-catcgtccctttcgatgggatcaa-B C135UGA Fam-CTGCCCTCAACAAGATGTTTTA C135UGA3 Cy3-CTGCCCTCAACAAGATGTTTTA C135UGCom pCCAACTGGCCAAGACCTGCCaaaa-B C135UGT Fam-CTGCCCTCAACAAGATGTTTTT C135UGT5 Cy5-CTGCCCTCAACAAGATGTTTTT C135UGzip10 pCCAACTGGCCAAGACCTGCC-caaggcacgtcccagacgcatcaa-B C141UGA Tet-GCCAACTGGCCAAGACCTA C141UGA3 Cy3-GCCAACTGGCCAAGACCTA C141UGCom pCCCTGTGCAGCTGTGGGTTGAaaaaa-B C141UGzip11 pCCCTGTGCAGCTGTGGGTTGA-gcacgggagctgacgacgtgtcaa-B V143UGA Fam-TGGCCAAGACCTGCCCTA V143UGA3 Cy3-TGGCCAAGACCTGCCCTA V143UGACOM pTGCAGCTGTGGGTTGATTCCAaaa-B V143UGAzip12 pTGCAGCTGTGGGTTGATTCCA-agacgcaccgcaacaggctgtcaa-B V143UTC Fam-CCAAGACCTGCCCTGC V143UTC3 Cy3-CCAAGACCTGCCCTGC V143UTCOM pGCAGCTGTGGGTTGATTCCACAaaaa-B V143UTzip13 pGCAGCTGTGGGTTGATTCCACA-catcgctgcaagtaccgcactcaa-B W146UG3A3 Cy3-TGCCCTGTGCAGCTGTGA W146UG3zip pGTTGATTCCACACCCCCGCC-cgatggcttccttacccagattcg-B P152LC2T2 Tet-CGGGTGCCGGGCA P152LC2T23 Cy3-CGGGTGCCGGGCA P152LC2T2COM pGGGGTGTGGAATCAACCCACAaaaaaa-B P152Lzip14 pGGGGTGTGGAATCAACCCACA-ggctgggacgtgcagaccgttcaa-B G154LG1A Fam-atataaCACACCCCCGCCCA G154LG1A3 Cy3-CACACCCCCGCCCA G154LG1ACom pGCACCCGCGTCCGCGatataa-B G154LG1Azip15 pGCACCCGCGTCCGCG-gctggctggcacgcaccagaatca-B V157LGC Tet-GCCATGGCGCGGAG V157LGC5 Cy5-GCCATGGCGCGGAG V157LGCOM pGCGGGTGCCGGGCGaaa-B V157LGT Tet-GCCATGGCGCGGAA V157LGT3 Cy3-GCCATGGCGCGGAA V157LGzip16 pGCGGGTGCCGGGCG-ggctccgtcagaaagcgacaatca-B R158LC1A5 Cy5-GATGGCCATGGCGCT R158LC1zip pGACGCGGGTGCCGGG-acgagggatacccgcaaacgatca-B R158UGA Tet-CGGCACCCGCGTCCA R158UGA3 Cy3-CGGCACCCGCGTCCA R158UGACOM pCGCCATGGCCATCTACAAGC-B R158UGAzip17 pCGCCATGGCCATCTACAAGC-acgagggatacccgcaaacgatca-B A161LC2T5 Cy5-GTGCTGTGACTGCTTGTAGATGA A161LC2Tzip pCCATGGCGCGGACGC-gggaggctgctgtcctttcgatca-B A161UGA Tet-aaaaaaaaGCGTCCGCGCCATGA A161UGA3 Cy3-GCGTCCGCGCCATGA A161UGCOM pCCATCTACAAGCAGTCACAGCACAaaaaaaaa-B A161UGzip18 pCCATCTACAAGCAGTCACAGCACA-gggaggctgctgtcctttcgatca-B V173UGA Fam-CACAGCACATGACGGAGGTTA V173UGA3 Cy3-CACAGCACATGACGGAGGTTA V173UGCOM pTGAGGCGCTGCCCCCAaaaaa-B V173UGT Fam-CACAGCACATGACGGAGGTTT V173UGT5 Cy5-CACAGCACATGACGGAGGTTT V173UGzip19 pTGAGGCGCTGCCCCCA-acagcgtgttcgttgcttgcatca-B R175LC1Com pCCTCACAACCTCCGTCATGTGCT-B R175LC1T Fam-CATGGTGGGGGCAGCA R175LC1T3 Cy3-CATGGTGGGGGCAGCA R175LC1zip20 pCCTCACAACCTCCGTCATGTGCT-atggcgatggtccactcgcaatca-B R175LG2T Fam-CTCATGGTGGGGGCAGT R175LG2T5 Cy5-CTCATGGTGGGGGCAGT R175LG2TCOM pGCCTCACAACCTCCGTCATGTG-B R175LG2Tzip21 pGCCTCACAACCTCCGTCATGTG-gtccgtccatggcaagcgtgatca-B R175UG2A Tet-TGACGGAGGTTGTGAGGCA R175UG2A3 Cy3-TGACGGAGGTTGTGAGGCA R175UG2ACom pCTGCCCCCACCATGAGCGaaaaaa-B R175UG2Azip21 pCTGCCCCCACCATGAGCG-gtccgtccatggcaagcgtgatca-B C176UGA Fam-CGGAGGTTGTGAGGCGCTA C176UGA5 Cy5-CGGAGGTTGTGAGGCGCTA C176UGCom pCCCCCACCATGAGCGCTGaaaaaaa-B C176UGT Fam-CGGAGGTTGTGAGGCGCTT C176UGT3 Cy3-CGGAGGTTGTGAGGCGCTT C176UGzip22 pCCCCCACCATGAGCGCTG-ggctgcacccgttgaggcacatca-B H179LACOM pGGTGGGGGCAGCGCC-B H179LAG Fam-GCTATCTGAGCAGCGCTCAC H179LAG3 Cy3-GCTATCTGAGCAGCGCTCAC H179LAT Fam-GCTATCTGAGCAGCGCTCAA H179LAT5 Cy5-GCTATCTGAGCAGCGCTCAA H179LAzip23 pGGTGGGGGCAGCGCC-tcaacatcggctaacggtccatca-B H179LCT Fam-GCTATCTGAGCAGCGCTCATA H179LCT3 Cy3-GCTATCTGAGCAGCGCTCATA H179LCTCOM pGTGGGGGCAGCGCCTCAC-B H179LCTzip24 pGTGGGGGCAGCGCCTCAC-cgcacgcagtcctcctccgtatca-B X5LCom pCGGGGGTGTGGAATCAACCC-B X5LWT Fam-CGCGGGTGCCGGG X5LWT3 Cy3-CGCGGGTGCCGGG X5LWT5 Cy5-CGCGGGTGCCGGG X5Lzip1 pCGGGGGTGTGGAATCAACCC-cgcagattttgcgctggatucaa-B p53X6FzipA GGAGCACGCTATCCCGTTAGACCCTCTGATTCCTCACTGATTGCTCTTA p53X6RzipB CGCTGCCAACTACCGCACATCGGCCACTGACAACCACCCTTAAC P190LCT Tet-aaaaTCGGATAAGATGCTGAGGAGA P190LCT3 Cy3-TCGGATAAGATGCTGAGGAGA P190LCTCOM pGGCCAGACCCTAAGAGCAATCAGaaaa-B P190LCTzip25 pGGCCAGACCCTAAGAGCAATCAG-ggctcgcaggctggctcatcctaa-B P190LTA5 Cy5-CACTCGGATAAGATGCTGAGGT P190LTAzip pGGGGCCAGACCCTAAGAGCAA-ggctcgcaggctggctcatcctaa-B H193LAG3 Cy3-AATTTCCTTCCACTCGGATAAGAC H193LAGzip pGCTGAGGAGGGGCCAGACC-cgcattcgatggacaggacattcg-B H193LTA5 Cy5-AATTTCCTTCCACTCGGATAAGT H193LTAzip pTGCTGAGGAGGGGCCAGAC-cgcattcgatggacaggacattcg-B R196LCCom pGATAAGATGCTGAGGAGGGGCCA-B R196LCT Fam-CGCAAATTTCCTTCCACTCA R196LCT3 Cy3-CGCAAATTTCCTTCCACTCA R196LCzip26 pGATAAGATGCTGAGGAGGGGCCA-cgcatgaggggaaacgacgagatt-B Y205LAC Fam-AAAAGTGTTTCTGTCATCCAAAG Y205LAC3 Cy3-AAAAGTGTTTCTGTCATCCAAAG Y205LACOM pACTCCACACGCAAATTTCCTTCCAaaaaaa-B Y205LAG Fam-AAAAGTGTTTCTGTCATCCAAAC Y205LAG5 Cy5-AAAAGTGTTTCTGTCATCCAAAC Y205LAzip27 pACTCCACACGCAAATTTCCTTCCA-gcaccgtgaacgacagttgcgatt-B T211LAG Tet-CCACCACACTATGTCGAAAAGC T211LAG3 Cy3-CCACCACACTATGTCGAAAAGC T211LAGCOM pGTTTCTGTCATCCAAATACTCCACACGaaa-B T211LAGzip28 pGTTTCTGTCATCCAAATACTCCACACG-cgcaggtcgctgcgtgtcctgatt-B T211LCT Tet-ACCACCACACTATGTCGAAAAA T211LCT3 Cy3-ACCACCACACTATGTCGAAAAA T211LCTCOM pTGTTTCTGTCATCCAAATACTCCACACaaa-B T211LCTzip29 pTGTTTCTGTCATCCAAATACTCCACAC-cgcaaagcagacacagggtcgatt-B R213LCCom pAAAAGTGTTTCTGTCATCCAAATACTCCa-B R213LCT Tet-GGGCACCACCACACTATGTCA R213LCT3 Cy3-GGGCACCACCACACTATGTCA R213LCzip30 pAAAAGTGTTTCTGTCATCCAAATACTCC-catcgcacttcgctttggctgatt-B Y220LACom pAGGGCACCACCACACTATGTCGA-B Y220LAG Tet-CAGACCTCAGGCGGCTCAC Y220LAG3 Cy3-CAGACCTCAGGCGGCTCAC Y220LAzip31 pAGGGCACCACCACACTATGTCGA-ttgcgggaactcacgaggtcgtat-B X6UCOM pCCTATGAGCCGCCTGAGGTCTaaaa-B X6UWT Fam-aaaTTCGACATAGTGTGGTGGTGC X6UWT3 Cy3-TTCGACATAGTGTGGTGGTGC X6UWT5 Cy5-TTCGACATAGTGTGGTGGTGC X6Uzip2 pCCTATGAGCCGCCTGAGGTCT-ttcgccgtcgtgtaggcttttcaa-B p53X7FzipA GGAGCACGCTATCCCGTTAGACGCCTCATCTTGGGCCTGTGTTATC p53X7RzipB CGCTGCCAACTACCGCACATCGTGGATGGGTAGTAGTATGGAAGAAATC Y234UTA3 Cy3-CTCTGACTGTACCACCATCCACA Y234UTAzip pACAACTACATGTGTAACAGTTCCTGCAT-ggctacgacgcatgtaaacgttcg-B M237UGA Fam-aaaaATAAGTACCACCATCCACTACAACTACATA M237UGA3 Cy3-ATAAGTACCACCATCCACTACAACTACATA M237UGCOM pTGTAACAGTTCCTGCATGGGCGaaaa-B M237UGT Fam-aaaaATAAGTACCACCATCCACTACAACTACATT M237UGT5 Cy5-ATAAGTACCACCATCCACTACAACTACATT M237UGzip32 pTGTAACAGTTCCTGCATGGGCG-gcacggctcgataggtcaagcttt-B C238UGA Tet-CCACCATCCACTACAACTACATGTA C238UGA3 Cy3-CCACCATCCACTACAACTACATGTA C238UGCom pTAACAGTTCCTGCATGGGCGGaaaaa-B C238UGzip33 pTAACAGTTCCTGCATGGGCGG-cgatttcgactcaagcggctcttt-B S241LC2A6 Fam-ATGCCGCCCATGCAGT S241LC2Azip pAACTGTTACACATGTAGTTGTAGTGGATGGT-cgcaatggtaggtgagcaagcaga-B S241LCCom pAACTGTTACACATGTAGTTGTAGTGGATGGTaaa-B S241LCG Fam-TGCCGCCCATGCAGC S241LCG5 Cy5-TGCCGCCCATGCAGC S241LCT Fam-TGCCGCCCATGCAGA S241LCT3 Cy3-TGCCGCCCATGCAGA S241LCzip34 pAACTGTTACACATGTAGTTGTAGTGGATGGT-cgcaatggtaggtgagcaagcaga-B G244UG1T Fam-aaaaaCATGTGTAACAGTTCCTGCATGT G244UG1T3 Cy3-CATGTGTAACAGTTCCTGCATGT G244UG1TCOM pGCGGCATGAACCGGAGGCaaaaaa-B G244UG1Tzip35 pGCGGCATGAACCGGAGGC-gtccccgttacctaggcgatcaga-B G244UG2A Fam-aaaaaaTGTGTAACAGTTCCTGCATGGA G244UG2A3 Cy3-TGTGTAACAGTTCCTGCATGGA G244UG2COM pCGGCATGAACCGGAGGCCaaaaaa-B G244UG2T Fam-aaaaaaTGTGTAACAGTTCCTGCATGGT G244UG2T5 Cy5-TGTGTAACAGTTCCTGCATGGT G244UG2zip36 pCGGCATGAACCGGAGGCC-atgggtccacagtaccgctgcaga-B G245UG1A Tet-AACAGTTCCTGCATGGGCA G245UG1A3 Cy3-AACAGTTCCTGCATGGGCA G245UG1ACom pGCATGAACCGGAGGCCCAaaaa-B G245UG1Azip37 pGCATGAACCGGAGGCCCA-ccgtgggagattaggtggctcaga-B G245UG2A Fam-CAGTTCCTGCATGGGCGA G245UG2A3 Cy3-CAGTTCCTGCATGGGCGA G245UG2ACom pCATGAACCGGAGGCCCATCaaa-B G245UG2Azip38 pCATGAACCGGAGGCCCATC-gggaatggaggtgggaacgagaca-B G245UG2T Tet-aaaaaaaaAGTTCCTGCATGGGCGA G245UG2T5 Cy5-CAGTTCCTGCATGGGCGT G245UG2TCOM pCATGAACCGGAGGCCCATCaaaaaaaaa-B R248LCCom pGTTCATGCCGCCCATGCAaa-B R248LCT Tet-GGTGAGGATGGGCCTCCA R248LCT3 Cy3-GGTGAGGATGGGCCTCCA R248LCzip39 pGTTCATGCCGCCCATGCA-cgtggctgactcgctgcgatgaca-B R248UGA Tet-TGGGCGGCATGAACCA R248UGA3 Cy3-TGGGCGGCATGAACCA R248UGCom pGAGGCCCATCCTCACCATCATaa-B R248UGzip40 pGAGGCCCATCCTCACCATCAT-ttgcgcaccatcaggttagggaca-B R249LACom pCCGGTTCATGCCGCCCAa-B R249LAG Fam-TGATGGTGAGGATGGGCCC R249LAG5 Cy5-TGATGGTGAGGATGGGCCC R249LAT Fam-TGATGGTGAGGATGGGCCA R249LAT3 Cy3-TGATGGTGAGGATGGGCCA R249LAzip41 pCCGGTTCATGCCGCCCA-gcaccgatatggagaccgcagaca-B R249LG3C Tet-GATGATGGTGAGGATGGGG R249LG3C3 Cy3-GATGATGGTGAGGATGGGG R249LG3Com pCTCCGGTTCATGCCGCC-B R249LG3zip42 pCTCCGGTTCATGCCGCC-catcgacaaggtaacgcgtggaca-B P250LC2T3 Cy3-AGTGTGATGATGGTGAGGATGA P250LC2Tzip pGCCTCCGGTTCATGCCG-gtcccaagttgcggctcactttcg-B I254LAG Fam-CTGGAGTCTTCCAGTGTGATGAC I254LAG3 Cy3-CTGGAGTCTTCCAGTGTGATGAC I254LAGCOM pGGTGAGGATGGGCCTCCG-B I254LAGzip43 pGGTGAGGATGGGCCTCCG-tgagcgcaaggtcagagcacgaca-B X7LCom pCATGCAGGAACTGTTACACATGTAGTTGTAa-B X7LWT Tet-TCCGGTTCATGCCGCC X7LWT3 Cy3-TCCGGTTCATGCCGCC X7LWT5 Cy5-TCCGGTTCATGCCGCC X7Lzip3 pCATGCAGGAACTGTTACACATGTAGTTGTA-ccgtaagcccgtatggcagatcaa-B p53X8FzipA GGAGCACGCTATCCCGTTAGACGGACAGGTAGGACCTGATTTCCTTAC p53X8RzipB CGCTGCCAACTACCGCACATCCGCTTCTTGTCCTGCTTGCTTAC F270UTA Tet-aaaaaATCTACTGGGACGGAACAGCA F270UTA3 Cy3-ATCTACTGGGACGGAACAGCA F270UTCOM pTTGAGGTGCGTGTTTGTGCCTaaaaaa-B F270UTzip44 pTTTGAGGTGCGTGTTTGTGCCT-aagccgcagcacgattccgtgaca-B V272UGA Fam-aaaaaaGGACGGAACAGCTTTGAGA V272UGA3 Cy3-GGACGGAACAGCTTTGAGA V272UGCOM pTGCGTGTTTGTGCCTGTCCTGGaaaaaaa-B V272UGT Fam-aaaaaaGGACGGAACAGCTTTGAGT V272UGT5 Cy5-GGACGGAACAGCTTTGAGT V272UGzip45 pTGCGTGTTTGTGCCTGTCCTGG-tgagaagcgtccaagccagaacga-B R273LCCom pCACCTCAAAGCTGTTCCGTCCCaa-B R273LCT Tet-CCAGGACAGGCACAAACACA R273LCT3 Cy3-CCAGGACAGGCACAAACACA R273LCzip46 pCACCTCAAAGCTGTTCCGTCCC-catccaaggtccgacacgcaacga-B R273UCA5 Cy5-ACGGAACAGCTTTGAGGTGA R273UCAzip pGTGTTTGTGCCTGTCCTGGGAGA-catccaaggtccgacacgcaacga-B R273UGA Tet-CGGAACAGCTTTGAGGTGCA R273UGA3 Cy3-CGGAACAGCTTTGAGGTGCA R273UGCom pTGTTTGTGCCTGTCCTGGGAGaaaaaa-B R273UGzip47 pTGTTTGTGCCTGTCCTGGGAG-ttcgacgattcgcatcaacgcaag-B C275UGA Tet-aaaaaaaAGCTTTGAGGTGCGTGTTTA C275UGA3 Cy3-CAGCTTTGAGGTGCGTGTTTA C275UGCOM pTGCCTGTCCTGGGAGAGACCaaaaaaa-B C275UGT Tet-aaaaaaaaCAGCTTTGAGGTGCGTGTTTT C275UGT5 Cy5-CAGCTTTGAGGTGCGTGTTTT G275UGzip48 pTGCCTGTCCTGGGAGAGACC-aacggggaaggttgagcgtgacag-B R280UGA Tet-TTTGTGCCTGTCCTGGGAA R280UGA3 Cy3-TTTGTGCCTGTCCTGGGAA R280UGCOM pAGACCGGCGCACAGAGGAAGaaaaaa-B R280UGT Tet-TTTGTGCCTGTCCTGGGAT R280UGT5 Cy5-TTTGTGCCTGTCCTGGGAT R280UGzip49 pAGACCGGCGCACAGAGGAAG-cactgcacacgaaacggcacacag-B D281UCA3 Cy3-GTGCCTGTCCTGGGAGAGAA D281UCAGzip pCGGCGCACAGAGGAAGAGAA-aagcaagccaaggtatggctttgc-B D281UCG5 Cy5-GTGCCTGTCCTGGGAGAGAG D281UGA3 Cy3-TTGTGCCTGTCCTGGGAGAA D281UGACzip pACCGGCGCACAGAGGAAGAG-cgtgcgcacactcactgtccttcg-B D281UGC5 Cy5-TTGTGCCTGTCCTGGGAGAC R282LCCom pGTCTCTCCCAGGACAGGCACAAAaaa-B R282LCT Fam-TCTCTTCCTCTGTGCGCCA R282LCT3 Cy3-TCTCTTCCTCTGTGCGCCA R282LCzip50 pGTCTCTCCCAGGACAGGCACAAA-taccgacatcctgggattgcatgg-B R282UG2A5 Cy5-CCTGTCCTGGGAGAGACCA R282UG2Azip pGCGCACAGAGGAAGAGAATCTCC-taccgacatcctgggattgcatgg-B E286UGA3 Cy3-AGACCGGCGCACAGAGA E286UGAzip pAAGAGAATCTCCGCAAGAAAGGG-ttcggctgttcgtaggcaagaggt-B R306LCT Cy3-TTGTCCTGCTTGCTTACCTCA R306LCT Fam-aaaaTTGTCCTGCTTGCTTACCTCA R306LCTCOM pCTTAGTGCTCCCTGGGGGCAGaaaaa-B R306LCTzip51 pCTTAGTGCTCCCTGGGGGCAG-actccgcattgccagagctgatgg-B X8UCOM pCTCACCACGAGCTGCCCCC-B X8UWT Fam-TCCGCAAGAAAGGGGAGC X8UWT3 Cy3-TCCGCAAGAAAGGGGAGC X8UWT5 Cy5-TCCGCAAGAAAGGGGAGC X8Uzip4 pCTCACCACGAGCTGCCCCC-atggccgtgctggggacaagtcaa-B The PCR primers are specifically designed to amplify regions within and surrounding the p53 gene.",
"After 15 rounds of amplification at high Tm's (i.e.",
"65° C.) using the longer gene-specific primers (at 1-2 pmoles per reaction), the two universal primers (bold upper case) are added at 50 pmoles in 50 μl and products cycled for an additional 20 rounds of amplification.",
"The allele-specific LDR probes contained fluorescent labels on the 5′-ends (Fam, Tet, Cy3 or Cy5) and the discriminating bases on their 3′-ends.",
"Non-genomic sequence was added to the 5′-ends of some probes (designated by bold lower case) to control the final ligation product size for gel-based assays.",
"The common LDR probes contained 5′-phosphates (p) and C-3 blocking (B) groups on their 3′-ends.",
"Common LDR probes used in array-based detection have zipcode sequences #(designated by lower case) on their 3′-ends.",
"LDR reactions were hybridized in 32 μl containing 300 mM MES, pH 6.0, 10 mM MgCl2, 0.1% SDS with or without 100 μg/ml sheared salmon sperm DNA at 65° C. for 1 h in a rotating chamber.",
"After washing in 300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS with or without 10% formamide for 10 min at 25° C. or for 10 min at 50° C. The array was imaged on an Olympus Provis AX70 microscope using a 100 W mercury burner, a Texas Red filter cube, and a Princeton Instruments TEK512/CCD camera.",
"The 16-bit greyscale images were captured using MetaMorph Imaging System (Universal Imaging Corporation) and rescaled to more narrowly bracket the LDR signal before conversion to 8-bit greyscale.",
"The 8-bit images were inverted using Adobe Photoshop to render the Cy3 signal black.",
"Example 4 p53 Chip Hybridization Showing the Presence of Mutations in DNA from Colon Tumors A p53 chip can detect the presence of 75 different mutations in exons 5, 6, 7 and 8 and uses 144 LDR oligonucleotides (see Table 5).",
"FIG.",
"24 is an example of microarray-based p53 mutation detection using DNA derived from colon tumors.",
"The mutation status of each sample and the zip-codes expected to capture signal are indicated to the right of each panel.",
"The figure shows Cy3 background in the lowest panel on the right that is due to contaminating fluorescence in the spotted zip-codes (FIG.",
"24H).",
"PCR was carried out as a single-tube, multiplex reaction in order to simultaneously amplify p53 exons 5, 6, 7, and 8.Genomic DNA extracted from colon tumors was amplified in a 25 μl reaction mixture containing 100 ng of DNA, 400 μM of each dNTP, 1×PCR Buffer II (10 mM Tris-HCl pH 8.3 at 25° C., 50 mM KCl) supplemented with 4 mM MgCl2, 1 U AmpliTaq Gold and 2 pmol of each gene-specific primer bearing either universal primer A or B on the 5′ ends (see Table 5).",
"The reaction was overlaid with mineral oil and preincubated for 10 min at 95° C. Amplification was performed for 15 cycles as follows: 94° C. for 15 sec, 65° C. for 1 min.",
"A second 25 μl aliquot of the reaction mixture was added through the mineral oil containing 25 pmol each of universal primers A and B. Cycling was repeated using 55° C. annealing temperature.",
"The reaction was next digested with a 2 μl solution of 1 mg/ml Proteinase K/50 mM EDTA at 55° C. for 10 min.",
"Proteinase K was eliminated by a final incubation at 90° C. for 15 min.",
"For LDR, oligonucleotide synthesis and purification were carried out as previously described (Khanna et al., “Multiplex PCR/LDR for Detection of K-ras Mutations in Primary Colon Tumors,” Oncogene 18: 27-38 (1999), which is hereby incorporated by reference).",
"Tth DNA ligase was overproduced and purified as described elsewhere (Luo et al., “Identification of Essential Residues in Thermus thermophilus DNA Ligase,” Nucleic Acids Research 24: 3079-3085 (1996) and Barany et al., “Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase Gene,” Gene 109: 1-11 (1991), which are hereby incorporated by reference).",
"LDR was performed in a 20 μl reaction containing 500 fmol of each probe, 2 μl of amplified DNA and 20 mM Tris-HCl, pH 7.6; 10 mM MgCl2; 100 mM KCl; 10 mM DTT; 1 mM NAD+.",
"Two reactions were performed for each sample containing LDR probes that were designed to hybridize to the upper strand or lower strand of p53 sequence.",
"The reaction was heated to 94° C. for 1.5 min prior to adding 25 fmol of Tth DNA ligase and then subjected to 20 cycles of 15 sec at 94° C. and 4 min at 65° C. (See Table 5) LDR reactions were hybridized in 32 μl containing 100 μg/ml sheared salmon sperm DNA 300 mM MES, pH 6.0, 10 mM MgCl2, 0.1% SDS at 65° C. for 1 h in a rotating chamber.",
"After washing in 300 mM bicine, pH 8.0, 10 mM MgCl2, 0.1% SDS for 10 min at 50° C. The array was imaged on an Olympus Provis AX70 microscope using a 100 W mercury burner, a Texas Red filter cube, and a Princeton Instruments TEK512/CCD camera.",
"The 16-bit greyscale images were captured using MetaMorph Imaging System (Universal Imaging Corporation) and rescaled to more narrowly bracket the LDR signal before conversion to 8-bit greyscale.",
"Using Adobe Photoshop, the 8-bit images were first inverted to render the Cy3 signal black and then the images for each sample derived from hybridization using LDR targeted to the upper strand and lower strand of the p53 sequence were overlaid and merged.",
"The results of this procedure are shown in FIG.",
"24.Example 5 Optimized Zipcode Sequence Construction Using Tetramers The universal DNA array is designed on the concept of using divergent sequences to uniquely tag LDR products such that each one is captured at a unique site.",
"The heart of the concept is the design of 36 tetramers, each of which differs from any other by at least 2 bases (See Table 6).",
"TABLE 6 Original tetramer Tetramer Tetramer New tetramer designation sequence complement designation (See Table 1) 5′-3′ 5′-3′ G + C bases 1 6 TTGA TCAA 1 2 7 TGAT ATCA 1 3 8 TTAG CTAA 1 4 26 AATC GATT 1 5 31 ATAC GTAT 1 6 32 AAAG CTTT 1 7 36 TACA TGTA 1 8 1 TCTG CAGA 2 9 2 TGTC GACA 2 10 5 TCGT ACGA 2 11 9 CTTG CAAG 2 12 10 CGTT AACG 2 13 11 CTCA TGAG 2 14 13 CTGT ACAG 2 15 15 CCAT ATGG 2 16 16 CGAA TTCG 2 17 17 GCTT AAGC 2 18 18 GGTA TACC 2 19 19 GTCT AGAC 2 20 21 GAGT ACTC 2 21 23 GCAA TTGC 2 22 25 AGTG CACT 2 23 27 ACCT AGGT 2 24 28 ATCG CGAT 2 25 30 AGGA TCCT 2 26 33 CCTA TAGG 2 27 34 GATG CATC 2 28 3 TCCC GGGA 3 29 4 TGCG CGCA 3 30 12 CACG CGTG 3 31 14 CAGC GCTG 3 32 20 GACC GGTC 3 33 22 GTGC GCAC 3 34 24 GGAC GTCC 3 35 29 ACGG CCGT 3 36 35 AGCC GGCT 3 By combining these 36 tetramers in sets of six, addresses that are 24 bases long can be constructed.",
"A 1296 array can be designed based on the concept of alternating tiling of given sets of tetramers.",
"These capture oligonucleotides differed from their neighbors at three alternating positions, but were the same at the other three positions, i.e.",
"(First=A, third=C, and fifth=E positions).",
"Thus, each capture oligonucleotide differed from any other one by at least 6 out of 24 positions.",
"Moreover, these differences were distributed across the length of the capture oligonucleotides.",
"When aligning a correct capture oligonucleotide with an incorrect address, the Tm differences were predicted to be greater than 24° C. Nevertheless, one of the possibilities with this type of design is for three contiguous tetramers in a given set of positions (i.e.",
"ABC) to match another capture oligonucleotide, but at a different set of positions (i.e.",
"BCD).",
"Since optimal surfaces are three-dimensional porous surfaces, a given LDR product has numerous opportunity to be captured at the correct address.",
"Even if an LDR product transiently dissociates from a given oligonucleotide within the correct address, it will rapidly find and hybridize to another oligonucleotide within the same address.",
"In preliminary studies, it was found that changes which would be expected to alter Tm, (i.e.",
"use of propynyl derivatives) did not markedly affect yield of correctly hybridized products.",
"Thus, hybridization may be kinetically controlled.",
"In order to minimize the possibility of even low levels of cross-hybridization between two closely related capture oligonucleotides, the sequences can be designed to maximize differences among the tetramer order with a 24 mer capture oligonucleotide.",
"The process for designing such sequences is outlined below: 1.Create three columns containing all 46,656 (=36×36×36) permutations of three sets of the 36 tetramers.",
"2.Compute the Tm of the 46,656 12-mers using the Oligo 6.0 program from Molecular Biology Insights, Inc. (Cascade, Colo.) and sort the list according to predicted Tm values.",
"3.Remove 12-mers that contain one GC base (Tetramers #1-7) in each tetramer or contain three GC bases in each tetramer (Tetramers #28-36).",
"This process removes the extremes in Tm range.",
"Remove 12-mers with Tm values less than 24° C. Remove the remaining 12-mers that have three repeated tetramers (i.e.",
"9-9-9) 4.Group the set of 12-mers by Tm with a new group for each 2 degrees increase in Tm.",
"Values were set by dividing Tm by two and truncating to whole numbers.",
"5.Randomize list and split into odd and even 12-mers.",
"Invert second list and append to the end of first list to form 6 tetramers=12,880 address candidates.",
"Concatenate sequences and determine Tm values of 24-mers.",
"6.Select only hexa-tetramers with Tm values between 75 and 84.Reclaim unused trimers and make new hexa-tetramers with increased Tm and add back to list.",
"7.The lists were pruned using the 13 selection conditions as described in Table 7: A “1” indicates a match at that position, a “0” indicates no match.",
"Anytime two candidate addresses matched at one of the conditions, it was removed from the candidate list and returned to the unused trimer list.",
"TABLE 7 Condition Tetramer 1 Tetramer 2 Tetramer 3 Tetramer 4 Tetramer 5 Tetramer 6 L Four in a row 1 1 1 1 0 0 M Four in a row 0 1 1 1 1 0 R Four in a row 0 0 1 1 1 1 L Three in a row 1 1 1 0 0 0 M Three in a row 0 1 1 1 0 0 M Three in a row 0 0 1 1 1 0 R Three in a row 0 0 0 1 1 1 Interrupted 4-1 1 1 0 1 1 0 Interrupted 4-2 1 1 0 0 1 1 Interrupted 4-3 0 1 1 0 1 1 Interrupted 4-4 1 0 1 0 1 1 Interrupted 4-5 1 1 0 1 0 1 Interrupted 4-6 1 0 1 1 0 1 8.The above 13 selection conditions reduced the list to 9,650 address candidates.",
"9.The above 13 selection conditions remove matches of four in a row and three in a row which are in the same alignment as one another; however, they do not remove sequences which are similar but shifted over by one or two tetramer units.",
"In order to eliminate those kinds of artifacts, the sequences were copied below the original 6 tetramers, offset by a tetramer or two as in Table 8 below: TABLE 8 Condition Position A Position B Position C Position D Position E Position F Four in a row + 1 Position A Position B Position C Position D Four in a row + 2 Position A Position B Position C Position D Four in a row − 1 Position C Position D Position E Position F Four split − 1 Position B Position C Position E Position F 10.These three selections culled the list to 8,894 candidate capture oligonucleotides, 4,798 more than the target of 4,096 for a 64×64 address array.",
"These capture oligonucleotide sequences have the following properties: (1) there are no cases of 4 tetramers in a row which are identical, either when capture oligonucleotide sequences are aligned with each other, or when they are offset with respect to each other or split with respect to each other; (2) there are no cases of 3 tetramers in a row which are identical, when capture oligonucleotide seqences are aligned with each other from one end; and (3) there are no cases where four out of six tetramers are in the same position.",
"11.For further selection, sequences of three in a row which were offset were eliminated as in Table 9 below.",
"TABLE 9 Condition Position A Position B Position C Position D Position E Position F Three in a row + 1 Position A Position B Position C Three in a row + 2 Position A Position B Position C Three in a row + 3 Position A Position B Position C Three in a row + 1 Position B Position C Position D Three in a row + 2 Position B Position C Position D Three in a row + 1 Position C Position D Position E 12.These six selections culled the list to 3,038 candidate capture oligonucleotides, more than a more selective target of 2,500 for a 50×50 address array.",
"These capture oligonucleotide sequences have the following properties: (1) there are no cases of 4 tetramers in a row which are identical, either when capture oligonucleotide seqences are aligned with each other, or when they are offset with respect to each other; (2) there are no cases of 3 tetramers in a row which are identical, when capture oligonucleotides seqences are aligned with each other from one end, or when they are offset with respect to each other; and (3) there are no cases where four out of six tetramers are in the same position.",
"13.The order of tetramers were reversed and new Tm values were calculated, added back in (6,076), and then pruned as described in steps 7-11 above.",
"The candidate list increased only marginally to 3,270.Therefore, an approach which enriched the unused trimers was needed.",
"14.A list of used trimers in all positions was used to determine available (unused) trimers and construct new sets of hexa-tetramers.",
"To increase the percent of hexa-tetramers with Tm values in the 75-84° C. range, trimers with predicted Tm values of 34-50° C. were inverted with respect to each other and used (i.e.",
"trimer ABC with Tm of 34 was fused to trimer DEF with Tm of 50, trimer ABC with Tm of 38 was fused to trimer DEF with Tm of 46, etc.).",
"15.Sets of hexa-tetramers were constructed and the trimers generated at the junction (i.e.",
"positions BCD and CDE) were retested against the used trimer list, and those hexa-tetramers which conflicted were recycled.",
"Those hexa-tetramers which did not conflict were added to the 3,270 candidate list and resorted and pruned as described in steps 7-11.The candidate list was expanded to 4,035.16.The process was reiterated two more times to generate the final list of 4,633 capture oligonucleotide (FIG.",
"25, which refers to the tetramers in Table 6), 537 sequences more than the target of 4,096 for a 64×64 address array.",
"These capture oligonucleotide sequences have the following properties: (1) there are no cases of 4 tetramers in a row which are identical, either when capture oligonucleotide seqences are aligned with each other, or when they are offset with respect to each other; (2) there are no cases of 3 tetramers in a row which are identical, when capture oligonucleotide seqences are aligned with each other from one end, or when they are offset with respect to each other; and (3) there are no cases where four out of six tetramers are in the same position.",
"17.Using the 4,633 capture oligonucleotide list, smaller lists for an 8×8=64 address array, 8×12=96 address array, 16×24=384 address array, and 20×20=400 address array were created.",
"As selection criteria, capture oligonucleotides which shared pairs of tetramers in common were selectively removed from the list.",
"A culling of all dimer pairs which were the same in given positions (i.e.",
"AB=AB) reduced the list to 465 capture oligonucleotide sequences (FIG.",
"26, which refers to the tetramers in Table 6).",
"A second culling of dimer pairs similar among neighboring positions (i.e.",
"AB=BC, BC=CD, etc.)",
"and removal of all dimer pairs used more than twice reduced the set to 96 capture oligonucleotide sequences (FIG.",
"27, which refers to the tetramers in Table 6).",
"Finally, ensuring that no dimer was used more than once generated a list of 65 capture oligonucleotide sequences (FIG.",
"28, which refers to the tetramers in Table 6).",
"18.The capture oligonucleotides can also be in the form of PNA (i.e.",
"Peptide Nucleotide Analogues), as shown in FIG.",
"29 (which refers to the tetramers in Table 6), which contains a list of 4633 such capture oligonucleotides.",
"These PNA capture oligonucleotides are in the form of 20 mer units.",
"PNA provides the advantage of increasing the Tm of the oligonucleotide, on average 1.0° C. to 1.5° C. per base, so the Tm values of the oligomers listed in FIG.",
"29 would be on average 20° C. (or more) higher when synthesized as the PNA form.",
"Thus, the addresses would only need to be 20mers or less in the PNA form.",
"These sequences are amenable to a more rapid synthesis by considering two alternative approaches.",
"In the first approach, the 36 tetramers listed in Table 6 are initially synthesized, and then 5 tetramers linked in the correct order to form the sequences listed in FIG.",
"29.Alternatively, the PNA oligomers would be synthesized using a lithographic synthesis approach.",
"A standard lithographic synthesis would use the 4 bases over and over again, i.e.",
"A-C-G-T for the first position, A-C-G-T for the second position, etc., and would require 4×20=80 masks.",
"The current sequences listed in FIG.",
"29 are amenable to synthesis in 62 masks, or less by altering the order of masks.",
"The 62 masks would allow attachment of the PNA monomers in the following order: T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G For a given sequence, the mask at the next base which allows that sequence is opened over that address.",
"By way of example, if the sequence were T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A, it could be finished using just 20 masks.",
"Different sequences will require more masks.",
"Thus, the 20-mers are finished with different numbers of masks.",
"Examples are provided below for synthesis of 5 different addresses Zip ID#s 1, 2, 3, 4, and 26 which require less than 62 masks.",
"In these examples, use of a mask is designated by an underlining of that base to achieve the correct sequence.",
"Zip ID#1.AATCCAGCGCAAAATCTGCG = 45 masks T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G Zip ID#2.AAAGCCTACACGACGGCGAA = 56 masks T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G Zip ID#3.TCTGCCATACGGGCTTACGG = 50 masks T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G Zip ID#4.CTTGTCCCCAGCACGGCCAT = 49 masks T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G Zip ID#26.TCGTCGTTTCCCCTCATGCG = 54 masks T-G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T- G-C-A-T-G-C-A-G-T-C-A-T-G-C-A-T-G-C-A-G-T-C-A-T-G- C-A-T-G-C-A-G-T-C-A-T-G This demonstrates that PNA addresses of 20 mers may be synthesized using a lithographic approach with no more than 62 masks, far less than the 80 masks required by the standard approach to synthesize a 20mer, and even less then the 64 masks required to make a standard PNA 16 mer.",
"All addresses were selected to have Tm values between 75 and 84° C. The distribution of Tm values is more or less independent of capture oligonucleotide number.",
"While addresses with higher G+C content in general gave higher Tm values, the simple Tm=4(G+C)+2(A+T) rule was off by up to 10° C. in many cases.",
"The values for the 96, 465, and 4633 capture oligonucleotides is shown in FIGS.",
"30, 31, and 32, respectively.",
"Sorted Tm values of the 4633 list of capture oligonucleotide probes are shown in FIG.",
"33.The gradient of Tm values was relatively even, with the majority of capture oligonucleotides (80%) having Tm values from and including 75° C. to 80° C. FIG.",
"34 shows tetramer usage in the lists of 65, 96, 465, and 4633 capture oligonucleotides produced in accordance with the present invention.",
"If tetramer distribution was completely random, each tetramer should be represented 2.7% of the time.",
"However, selection was biased towards higher Tm capture oligonucleotide probes.",
"Thus, tetramers which tend to increase the Tm values, i.e.",
"#29=TGCG are over-represented, while tetramers which tend to decrease the Tm values, i.e.",
"#7=TACA are under-represented.",
"The present approach to mutation detection has three orthogonal components: (i) primary PCR amplification; (ii) solution-phase LDR detection; and (iii) solid-phase hybridization capture.",
"Therefore, background signal from each step can be minimized, and, consequently, the overall sensitivity and accuracy of the present method is significantly enhanced over those provided by other strategies.",
"For example, “sequencing by hybridization” methods require: (i) multiple rounds of PCR or PCR/T7 transcription; (ii) processing of PCR amplified products to fragment them or render them single-stranded; and (iii) lengthy hybridization periods (10 h or more) which limit their throughput (Guo, et al., Nucleic Acids Res.",
"22: 5456-5465 (1994); Hacia, et.",
"al., Nat.",
"Genet., 14441-447 (1996); Chee, et al., Science, 274: 610-614 (1996); Cronin, et al., Human Mutation, 7: 244-255 (1996); Wang, et al., Science, 280: 1077-1082 (1998); Schena, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 93: 10614-10619 (1996); and Shalon, et al., Genome Res., 6: 639-645 (1996), which are hereby incorporated by reference).",
"Additionally, since the immobilized probes on these arrays have a wide range of Tm's, it is necessary to perform the hybridizations at temperatures from 0° C. to 44° C. The result is increased background noise and false signals due to mismatch hybridization and non-specific binding, for example on small insertions and deletions in repeat sequences (Hacia, et.",
"al., Nat.",
"Genet., 14441-447 (1996); Cronin, et al., Human Mutation, 7: 244-255 (1996); Wang, et al., Science 280: 1077-1082 (1998); and Southern, E. M., Trends in Genet., 12: 110-115 (1996), which are hereby incorporated by reference).",
"In contrast, the approach of the present invention allows multiplexed PCR in a single reaction (Belgrader, et al., Genome Sci.",
"Technol., 1: 77-87 (1996), which is hereby incorporated by reference), does not require an additional step to convert product into single-stranded form, and can readily distinguish all point mutations including slippage in repeat sequences (Day, et al., Genomics, 29: 152-162 (1995), which is hereby incorporated by reference).",
"Alternative DNA arrays suffer from differential hybridization efficiencies due to either sequence variation or to the amount of target present in the sample.",
"By using the present approach of designing divergent address sequences with similar thermodynamic properties, hybridizations can be carried out at 65° C., resulting in a more stringent and rapid hybridization.",
"The decoupling of the hybridization step from the mutation detection stage offers the prospect of quantification of LDR products, as has already been achieved using gel-based LDR detection.",
"Arrays spotted on polymer surfaces provide substantial improvements in signal capture, as compared with arrays spotted or synthesized in situ directly on glass surfaces (Drobyshev, et al., Gene, 188: 45-52 (1997); Yershov, et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 93: 4913-4918 (1996); and Parinov, et al., Nucleic Acids Res., 24: 2998-3004 (1996), which are hereby incorporated by reference).",
"However, the polymers described by others are limited to using 8- to 10-mer addresses while the polymeric surface of the present invention readily allows 24-mer capture oligonucleotides to penetrate and couple covalently.",
"Moreover, LDR products of length 60 to 75 nucleotide bases are also found to penetrate and subsequently hybridize to the correct address.",
"As additional advantages, the polymer gives little or no background fluorescence and does not exhibit non-specific binding of fluorescently-labeled oligonucleotides.",
"Finally, capture oligonucleotides spotted and coupled covalently at a discrete address do not “bleed over” to neighboring spots, hence obviating the need to physically segregate sites, e.g., by cutting gel pads.",
"The present invention relates to a strategy for high-throughput mutation detection which differs substantially from other array-based detection systems presented previously in the literature.",
"In concert with a polymerase chain reaction/ligase detection reaction (PCR/LDR) assay carried out in solution, the array of the present invention allows for accurate detection of single base mutations, whether inherited and present as 50% of the sequence for that gene, or sporadic and present at 1% or less of the wild-type sequence.",
"This sensitivity is achieved, because thermostable DNA ligase provides the specificity of mutation discrimination, while the divergent addressable array-specific portions of the LDR probes guide each LDR product to a designated address on the DNA array.",
"Since the address sequences remain constant and their complements can be appended to any set of LDR probes, the addressable arrays of the present invention are universal.",
"Thus, a single array design can be programmed to detect a wide range of genetic mutations.",
"Robust methods for the rapid detection of mutations at numerous potential sites in multiple genes hold great promise to improve the diagnosis and treatment of cancer patients.",
"Noninvasive tests for mutational analysis of shed cells in saliva, sputum, urine, and stool could significantly simplify and improve the surveillance of high risk populations, reduce the cost and discomfort of endoscopic testing, and lead to more effective diagnosis of cancer in its early, curable stage.",
"Although the feasibility of detecting shed mutations has been demonstrated clearly in patients with known and genetically characterized tumors (Sidransky, et al., Science, 256: 102-105 (1992), Nollau, et al., Int.",
"J.",
"Cancer, 66: 332-336 (1996); Calas, et al., Cancer Res.",
"54: 3568-73 (1994); Hasegawa, et al., Oncogene 10: 1413-16 (1995); and Wu et al., Early Detection of Cancer Molecular Markers (Lippman, et al.",
"ed.)",
"(1994), which are hereby incorporated by reference), effective presymptomatic screening will require that a myriad of potential low frequency mutations be identified with minimal false-positive and false-negative signals.",
"Furthermore, the integration of technologies for determining the genetic changes within a tumor with clinical information about the likelihood of response to therapy could radically alter how patients with more advanced tumors are selected for treatment.",
"Identification and validation of reliable genetic markers will require that many candidate genes be tested in large scale clinical trials.",
"While costly microfabricated chips can be manufactured with over 100,000 addresses, none of them have demonstrated a capability to detect low abundance mutations (Hacia, et.",
"al., Nat.",
"Genet., 14441-447 (1996); Chee, et al., Science, 274: 610-614 (1996); Kozal, et al., Nat.",
"Med., 2: 753-759 (1996); and Wang, et al., Science, 280: 1077-1082 (1998), which are hereby incorporated by reference), as required to accurately score mutation profiles in such clinical trials.",
"The universal addressable array approach of the present invention has the potential to allow rapid and reliable identification of low abundance mutations in multiple codons in numerous genes, as well as quantification of multiple gene deletions and amplifications associated with tumor progression.",
"In addition, for mRNA expression profiling, the LDR-universal array can differentiate highly homologous genes, such as K-, N-, and H-ras.",
"Moreover, as new therapies targeted to specific genes or specific mutant proteins are developed, the importance of rapid and accurate high-throughput genetic testing will undoubtedly increase.",
"Example 6 Computer Software For Designing Addressable Array to Avoid Binding to Target Sequence In designing an addressable array, it is important to insure that the target sequence does not hybridize to capture probes on the array.",
"As described below, a computer program has been designed for this purpose.",
"The program locates stretches of sequence that match any of the array sequences at N-x of N adjacent nucleotide positions.",
"The parameters x and N are set by the user.",
"The program sends output to the screen and to a file.",
"The screen output summarizes the number of sequences comparisons where the longest match was i of M bases, where M is greater than or equal to N, and where i is greater than or equal to M−x.",
"The output file shows the actual match for each sequence pair, as well as giving the summary information provided on the screen.",
"An example of the file output is shown below.",
"Input file 1: kraspoly.dos Input file 2: zip64.dos Minimum number of sites that must match: 7 Maximum number of mismatches allowed: 2 7 out of 8 attcagaatc (ATTTTGtG) gacgaa K-rasc32Wt ZIP1 cgcag (ATTTTGcG) ctggatttcaa 7 out of 9 attcagaatcattt (TGtGGACgA) a K-rasc32Wt ZIP4 atggccgtgc (TGgGGACaA) gtcaa < .",
".",
".",
"deleted output .",
".",
".",
"> 7 out of 8 tgtggtagt (TGgAGCTG) gtga K-rasc13.4D ZIP61 ggctcgtg (TGtAGCTG) ccgttcct 7 out of 8 tgtggtagttg (GAGcTGGT) ga K-rasc13.4D Z1P62 ggtcaagcgct (GAGgTGGT) ccatc SUMMARY OF ANALYSIS Comparisons with 1 mismatch 8 out of 9 bases matching: 10 7 out of 8 bases matching: 9 Comparisons with 2 mismatches 9 out of 11 bases matching: 2 8 out of 10 bases matching: 27 7 out of 9 bases matching: 36 A total of 84 out of 520 sequence comparisons met the match criteria.",
"The area within the parentheses represents the longest identified match, with upper-case alleles representing the actual matched sites and lower-case alleles representing the allowed mismatches.",
"The program has been written in ANSI C for the purpose of portability across platforms.",
"The precise software used is set forth in FIG.",
"35.The program accepts input files in straight text format.",
"Sequences may include standard ambiguity codes (e.g., the code Y corresponds to either C or T).",
"Each of F1 sequences in Input File #1 is compared to each of F2 sequence in File #2, for a total of F1×F2 comparisons.",
"For each pair, the two sequences are compared N consecutive sites at a time in all possible alignments.",
"If a match of at least N−x out of N adjacent sites is detected, the number of sites compared is incremented by one (i.e., after i increments, the match criteria become N−x+i out of N+i sites).",
"This process is repeated until no matches meeting the match criteria are found.",
"The longest match for a sequence pair is defined as the match involving the longest value of N+i, as opposed to the longest value of N−x+i.",
"Therefore, users are explicitly should repeat all analyses with different levels of stringency (i.e., x=0, x=1, x=2, .",
".",
".",
").",
"This is important if the user is concerned with the possibility that a perfect, or near-perfect, match might be masked by a less perfect match over a longer stretch.",
"For example, 7 out of 7 matched sites would not be reported if (i) a pair of sequences matched at 8 out of 10 sites and (ii) if the match criterion allowed 2 mismatches.",
"Although the invention has been described in detail for the purpose of illustration, it is understood that such details are solely for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims."
]
] | Patent_10257158 |
[
[
"Progressive distributor comprising displaceable pistons",
"The invention relates to a progressive distributor comprising three or more control pistons (SA to SD), each of which can be displaced in a respective housing bore (A to D) of a distributor housing (G).",
"According to the invention, said pistons are depressed alternately into their two final positions in the corresponding housing bore (A to D) by a pressurised lubricant, which is supplied via a housing inlet (E), whereby a specific quantity of said lubricant (VSA to VSD) is delivered by means of an outlet (A1 to A8) on a respective front face.",
"The outlet leads from one of two annular grooves (R) of the housing bore (A to D) of the respective preceding control piston (SA to SD).",
"The control pistons (SA to SD) are controlled sequentially by the lubricant as a result of the two respective annular grooves (R) in such a way that the following control piston (SA to SD) can only be displaced by the lubricant, if the piston displacement of the preceding control piston (SA to SD) has been fully or nearly completed.",
"The invention is characterised in that the housing bores (A to D) are connected to the inlet (E) by means of a central bore (M), a respective channel (K1 to K8) connecting an annular groove (R) of the housing bore (A to D) of the preceding control piston (SA to SD) to the front face of the respective housing bore (A to D) of the following control piston (SA to SD), whereby for all but one (SD) of the control pistons, the annular groove (R) and the front face lie on the same side, but for said one control piston (SD) the annular groove (R) and the front face lie on opposite sides.",
"A respective connection channel (V1 to V8) interconnects the respective front faces of all or at least several housing bores (A to D) with a front face of the respective corresponding bore (BA to BD) of a metering piston (DA to DD) that can be displaced back and forth by the lubricant between two stops (AS1 to AS8), one of said stops being axially displaceable and/or interchangeable.",
"The displacement resistance of each metering piston (DA to DD) that is allocated to a respective control piston (SA to SD) is less than the displacement resistance of the respective following control piston (SA to SD)."
],
[
"1.Progressive distributor comprising three or more control pistons (SA to SD), each of which can be displaced in a housing bore (A to D) of a distributor housing (G) and whereby these pistons are pushed alternately into their two final positions in the corresponding housing bore (A to D) by a lubricant which is supplied under pressure via a housing inlet (E) whereby a specific quantity of lubricant (VSA to VSD) is delivered by means of an outlet (A1 to A8) on a respective front face, said outlet leads from one or two annular grooves (R) of the housing bore (A to D) of the respective preceding control pistons (SA to SD), said control pistons (SA to SD) are controlled sequentially by the lubricant as a result of the two respective annular grooves (R) in such a way that the next control piston (SA to SD) cannot be displaced by the lubricant until the piston displacement of the preceding control piston (SA to SD) has been fully or almost fully completed, said housing bores (A to D) are connected to the inlet (E) via a central bore (M), whereby in each case a channel (K1 to K8) connects an annular groove (R) of the respective housing bore (A to D) of the preceding control piston (SA to SD) to the front face of the respective housing bore (A to D) of the following control piston (SA to SD), whereby for all but one (SD) of the control pistons, the annular groove (R) and the front face lie on the same side, but for said control piston (SD) the annular groove (R) and the front face lie on opposite sides, a respective connection channel (Vt to V8) interconnects the respective front faces of all or at least several housing bores (a to D) with a front face of the respective corresponding bore (BA to BD) of a metering piston (DA to DD) that can be moved to and fro by the lubricant between two stops (AS 1 to ASS), at least one of the said stops being axially displaceable and/or interchangeable and whereby the displacement resistance of each metering piston (DA to DD) assigned to a control piston (SA to SD) is smaller than the displacement resistance of the respective following control piston (SA to SD).",
"2.Progressive distributor as claimed in claim 1, wherein the displacement resistance of the respective metering piston (DA to DD) is smaller than the displacement resistance of its assigned control piston (SA to SD).",
"3.Progressive distributor as claimed in claim 1, wherein the displacement resistance of the respective metering piston (DA to DD) is adjusted and adjustable by means of an O-ring arrangement and/or fitting adjustment 4.Progressive distributor as per claim 1, wherein the respective metering piston (DA to DD) has a larger effective area than its assigned control piston (SA to SD).",
"5.Progressive distributor as claimed in claim 1, wherein the respective metering piston (DA to DD) is axis parallel to its assigned control piston (SA to SB) and lies on the side of the distributor housing (G) assigned to the inlet and, specifically, with the exception of one control piston (SA) which lies closest to the inlet and whose metering piston (DA) lies furthest away from it.",
"6.Progressive distributor as claimed in claim 1, wherein the stops (AS1 to AS8) are arranged on one front face of the distributor housing (G) immediately adjacent to the exit ports of the corresponding outlet (A1 to AS).",
"7.Progressive distributor as claimed in claim 1, wherein in opposite front faces of the distributor housing (G) in each case every second outlet (A1 to A8), every second assigned stop (ASI to ASS) and every assigned housing bore (A to D) for the control pistons ($A to SD) are arranged in one of three rows in such a manner that the corresponding outlets (A1 to AS), stops (AS 1 to ASS) and housing bores (A to D) for the control pistons (SA to SD) are positioned immediately adjacent to each other.",
"8.Progressive distributor as claimed in claim 1, wherein the exit ports of the outlets (A1 to A8) are in each case equipped with a non-return valve (RV1 to RV8)."
],
[
"The invention refers to a progressive distributor with three or more control pistons each of which can be displaced in a housing bore of a distributor housing, which progressive distributor each time with delivery of a specific quantity of lubricant through a frontal outlet, which outlet leads from one of two annular grooves of the housing bore of the respective preceding control piston, from which lubricant fed in under pressure through a housing inlet is alternately pressed into its two final positions in the respective housing bore, whereby the control pistons through the two annual grooves are sequentially controlled by the lubricant in such a manner that the next control piston cannot be displaced by the lubricant until the piston displacement of the preceding control piston has been completed or nearly completed, whereby the housing bores are connected with the inlet through a central bore, whereby in each case a channel connects an annular groove of the respective housing bore of the preceding control piston with the front face of the particular housing bore of the next control piston and specifically, except for one control piston, with annular groove and front face on the same side, but in the case of the one control piston with annular groove and front face on opposite sides.",
"Such a progressive distributor is known from DE 34 05 690 C2.To design such a progressive distributor with additional functions and thereby make it suitable for novel applications, especially also in those cases where machines must be lubricated with very small quantities or where some lubricating points must be attended to frequently, but others e.g.",
"only daily, weekly or monthly, it is proposed that in each case a connecting channel connects the respective front faces of at least one housing bore although not of all housing bores with a front face of an additional piston whereby an additional piston is mounted so that it can be moved to and fro between two stops by the lubricant, whereby at least one piston must act against an outlet pressure that is higher than the outlet pressure against which the additional piston acts and whereby for the production of the higher outlet pressure the corresponding outlets feature an overflow valve or a non-return valve with counterpressure.",
"In an alternative solution the arrangement with the overflow valves or non-return valves with counterpressure, the additional piston can have at least one circumferential groove which only releases an outlet of a piston in the two final positions of the additional piston.",
"With such progressive distributors when the respective piston to whose housing bore the additional cylinder is connected, is moved from one side to the other through load on one front face, the outlet assigned to this piston not only delivers the quantity of lubricant displaced by the opposite front face of this piston, but also through displacement of the additional piston from its final position at one end to the opposite end an additional quantity of lubricant corresponding to the free volume of the additional cylinder.",
"With the one solution the displacement cylinder of the individual pistons pauses long enough until the entire contents of the additional cylinder are emptied, because at least one of these other pistons had to displace itself against an outlet pressure higher than the outlet pressure that the additional piston must overcome allowing for friction losses.",
"With the other solution the additional piston blocks the required outlet for the continuation of the displacement cycle of the pistons long enough until the additional piston has been displaced from its one final position to the other and its circumferential groove releases the required outlet for the continuation of the displacement cycle.",
"The first solution variant has the advantage that the piston can be varied without interrupting the stroke.",
"The second solution variant has the advantage that it works reliably under all pressure conditions, because it does not depend on increased counterpressure at at least two opposite outlets of a piston.",
"In both cases the progressive distributor works with the one additional cylinder as a hydraulic timing element, whereby the delay for the continuation of the displacement cycle is determinable or determined by the volume of the additional cylinder.",
"The purpose of this invention is to propose a progressive distributor from all of whose outlets or at least several outlets different lubricant volumes can be delivered combined with minimum constructional effort and certain operation.",
"This purpose is met with a progressive distributor of the type described in the introduction by the fact that in each case a connection channel interconnects the respective front faces of all or at least several housing bores with a front face of the respective assigned bore of a metering piston that can be displaced to and fro by the lubricant between two stops of which at least one is axially adjustable and/or exchangeable and that the displacement resistance of each metering piston assigned to a control piston is smaller than the displacement resistance of the respective following piston.",
"Advantageously the displacement resistance of the respective metering piston is also smaller than the displacement resistance of its assigned control piston.",
"A determinant for the displacement resistance of the metering piston is its front face under lubricant pressure and therefore also the cross-section of the bores in which the metering pistons are displaceable.",
"In particular, however, the displacement resistance of the respective metering piston can be set by an O-ring arrangement and/or fitting adjustment in its bores.",
"A constructionally favourable configuration of the progressive distributor invented is obtained if the respective metering piston lies parallel to its assigned control piston and on the side of the distributor housing assigned to the inlet and, specifically, with the exception of one control piston which lies closest to the inlet and whose metering piston lies furthest away from it.",
"For simple operability it is advantageous if the stops on one front face are immediately adjacent to the exit ports of the corresponding outlet.",
"It is thereby particularly provided that in opposite front faces of the distributor housing in each instance every second outlet, every second assigned stop and every assigned housing bore for the control pistons are arranged in one of three rows in such a manner that the corresponding distributor housing, outlets and housing bores for the control pistons are positioned immediately adjacent to each other.",
"To avoid undesired refluxes and/or fouling of the progressive distributor the exit ports of the outlets can be equipped with a non-return valve.",
"Further aims, features, advantages and application possibilities of the invention will be evident from the following description of embodiments with reference to the drawings.",
"All features described and/or pictorially illustrated constitute in themselves or in any combination the subject of the invention, even independent of their summary in individual claims and their cross-reference.",
"FIG.",
"1 Sectional diagram of an embodiment of the progressive distributor, and FIG.",
"2 Diagrammatic view of a front face of the distributor housing The embodiment shown in FIG.",
"1 refers to a progressive distributor with a distributor housing G in which in four housing bores A to D four control pistons SA to SD can be displaced to and fro through lubricant supplied through the inlet E of a central bore M e.g.",
"by a pump (not shown).",
"The housing bores A to D and the control pistons SA to SD are associated with eight (four on each side) outlets A1 to A8.Each control piston SA to SD has two annular grooves R axially interspaced from each other.",
"The respective front faces of the housing bores A to D are in flow-through connection with the annular groove R of the corresponding control pistons SA to SD of the preceding housing bore A to D by means of channels K1 to K8.For the top three housing bores A to C the area of the annular groove R and the front face of the following housing bore D are situated on the same side of the central longitudinal plane (extending perpendicular to the drawing plane from top to bottom).",
"However, the housing bore D of the bottom control piston SD is in flow-through connection in the area of the annular groove R through the channels K1 and K2 with the front face of the housing bore A of the top control piston A lying opposite in relation to the said central longitudinal plane.",
"The outlets A1 to A8 in each case run by way of the housing bore A to D and the annular groove R of the respective preceding control piston SA to SD.",
"The housing bores A to D are all in communication with the inlet E by way of the central bore M. This achieves that the control pistons SA to SD are alternately pushed into their two end positions in their housing bore A to D through the effect of the lubricant delivered on the one front face and through delivery of a specific lubricant volume VSA to VSD via one of the outlets A1 to A8 on the other side.",
"The respective next control piston SA to SD cannot be displaced by the lubricant until the piston displacement of the preceding control piston SA to SD has been fully or almost fully completed.",
"Each time a connecting channel V1 connects the respective front face of the housing bores A to D with a front face of the respective assigned bore BA to BD with two end stops AS1 to AS8 of which at least one is axially adjustable and/or exchangeable.",
"In the bores BA to BD one of the metering pistons BA to BD can in each instance be displaced to and fro by the lubricant between the stops AS1 to AS8.Thereby the displacement resistance of the particular metering piston DA to DD assigned to the corresponding control piston SA to SD smaller than the displacement resistance of the respective following control piston SA to SD.",
"Furthermore the displacement resistance of the respective metering piston DA to DD is smaller than the displacement resistance of its corresponding control piston SA to SD.",
"The displacement resistance of the respective metering piston DA to DD is in the case illustrated set through fitting adjustment, whereby the respective metering piston DA to DD has a larger frontal effective area than the corresponding control pistons SA to SD.",
"The displacement resistance can also be set through an O-ring arrangement on the particular metering piston DA to DD.",
"In the embodiment shown the respective metering piston DA to DD is axis parallel to its corresponding control piston SA to SD and situated on the side of the distributor housing assigned to the inlet E, with the exception of one control piston SA which lies closest to the inlet E and whose metering piston DA is furthest away from the inlet E. As can be seen in FIG.",
"2 each second outlet A1 to A8, each second corresponding stop AS1 to AS 8 and each opening of the corresponding housing bores A to D for the control pistons SA to SD are in each case arranged in one of three rows in opposite front faces of the distributor housing G in such a manner that the mutually corresponding outlets A1 to A8, stops AS1 to AS8 and housing bores A to D for the control pistons SA to SD are immediately adjacent to each other for easy operation.",
"FIG.",
"1 shows that the exit ports of the outlets A1 to A8 may be equipped with a non-return valve RV1 to RV8.If e.g.",
"in the position of control pistons SA to SD and metering pistons DA to DD shown in FIG.",
"1, lubricant is fed into the central bore M via the inlet E, it moves to the right front face of the control piston SB via the annular groove R of the control piston SA and via the connecting channel V8 to the right front face of the metering piston DB which is consequently pushed to the left in its respective bores B and BB.",
"The displaced lubricant volume VSB and VDB is delivered to the outlet A7 by means of the connecting channel V7 via the channel K7 and the left annular groove R of the control piston SA.",
"As can be seen, the lubricant volume VDB that can be displaced by the metering piston DB is determined by the variable axial position of the stop A7 and is therefore variable.",
"If both the control piston SB and the metering piston DB have been displaced as far as possible to the left, the same movement of the control piston SC and the metering piston DC is continued by means of the annular groove of the next control piston SB and the channel K6 etc.",
"Because of the transposition of the channels K1 and K2 to the respective annular groove R of the control piston SD on the opposite side, the movements of all pistons reverse as soon as all pistons SA to SD and metering pistons DA to DD in the drawing have been displaced to the left etc.",
"It is obvious that an adjustable stop can also be provided on the right side of the bores BA to BD for the metering pistons DA to DD shown in the drawing in order to provide on this side also a variable displaced lubricant volume.",
"REFERENCE LIST A to D Housing bores for control pistons SA to SD A1 to A8 Outlets AS1 to AS8 Stops BA to BD Bores of the metering pistons DA to DD DA to DD Metering pistons E Inlet G Distributor housing K1 to K8 Channels M Central bore R Annular grooves RV1 to RV8 Non-return valve SA to SD Control pistons V1 to V8 Connection channels VDA to VDD Lubricant volumes (metering piston) VSA to VSD Lubricant volume (control piston)"
]
] | Patent_10257417 |
[
[
"Lactic acid bacteria overproducing exopolysaccharides",
"The invention concerns lactic acid bacteria overproducing exopolysaccharides following mutation in the gene coding for α-phosphoglucomutase.",
"Said mutants are useful, in particular for preparing fermented products or for producing exopolysaccharides."
],
[
"1.A lactic acid bacteria mutant overproducing exopolysaccharides, in which the pgm gene of alpha-phosphoglucomutase is totally or partially inactivated.",
"2.The mutant as claimed in claim 1, in which the galU gene is overexpressed.",
"3.The mutant as claimed in either one of claims 1 and 2, characterized in that said lactic acid bacterium is Streptococcus thermophilus.",
"4.The mutant as claimed in claim 3, characterized in that it is obtained from a Streptococcus thermophilus strain capable of using galactose.",
"5.The use of a lactic acid bacterium mutant as claimed in any one of claims 1 to 4, for producing a fermented product.",
"6.The use of a lactic acid bacterium mutant as claimed in any one of claims 1 to 4, for producing an exopolysaccharide.",
"7.A nucleic acid encoding an α-phosphoglucomutase the amino acid sequence of which exhibits at least 70% identity or at least 85% similarity with the α-phosphoglucomutase represented by the sequence SEQ ID NO: 2."
],
[
"The invention relates to the regulation of exocellular heteropolysaccharide production by lactic acid bacteria.",
"In general, polysaccharides are used a great deal as additives in food, but also in cosmetics and pharmaceutical products, for example as thickeners and/or gelling agents, texture stabilizers, fat substitutes, etc.",
"Among the polysaccharides used in this way, mention will in particular be made of those produced by microorganisms, in particular bacteria, such as dextrans, xanthans, gellans, pullulans, etc.",
"Many species of lactic acid bacteria, in particular of lactococci, such as Lactococcus lactis, of leuconostocs, such as Leuconostoc mesenteroide, of streptococci, such as Streptococcus thermophilus, and of lactobacilli, such as Lactobacillus casei, Lactobacillus sake, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp.",
"bulgaricus and Lactobacillus helveticus, etc., produce polysaccharides.",
"These polysaccharides can be grouped into 2 categories: homopolysaccharides, such as dextrans, which result from the polymerization of a single sugar, and heteropolysaccharides, which have a complex structure, combining basic units consisting of two or more different sugars (commonly D-galactose, D-glucose and L-rhamnose).",
"The heteropolysaccharides of lactic acid bacteria are conventionally designated under the general term EPS (for exopolysaccharides), which will also be used hereinafter.",
"They play a major role in the development of the texture, of the mouthfeel and of the rheology of fermented dairy products.",
"In addition, it has been observed that some of them have biological activities by which they might exert diverse effects beneficial to the health [for review, cf.",
"DE VUYST and DEGEEST, FEMS Microbiology Reviews, 23, 153-177, (1999)].",
"However, the amount of heteropolysaccharides produced by lactic acid bacteria is generally low (of the order of 10 to 200 mg per liter of fermented product).",
"To improve the texture, manufacturers of fermented products add other texturing agents, such as stabilizers (modified starches, carrageenan, guar, pectin, gelatin, etc.).",
"However, these additions are not always authorized (for example in natural yogurt), and generally affect the taste and aroma of the product.",
"Optimized production of exocellular heteropolysaccharides (EPS) in the product is therefore preferable.",
"Given the importance of EPS in the agrofoods industries, mentioned above, many studies have related to methods for increasing the production thereof by optimizing the biotechnological processes by acting on the temperature, the pH and the composition of the medium.",
"These approaches are, however, sometimes difficult to apply in the context of certain agrofoods processes, such as the production of fermented dairy products for which the medium and the fermentation conditions are specific to each type of product.",
"These production constraints therefore limit the use of conventional methods for optimizing the processes in order to improve EPS production.",
"An alternative to these methods would be to use strains suitable for these processes, i.e.",
"strains capable of producing EPS in greater quantity, and/or in which the EPS production may be controlled under the conditions for producing fermented products.",
"One of the main limitations of EPS production by lactic acid bacteria may come from competition between the biosynthesis of these EPS and other metabolic pathways for the use of available sugars.",
"In fact, although homopolysaccharides are mainly produced by specific extracellular enzymes from substrates present in the medium, the synthesis of heteropolysaccharides takes place at least in part inside the cell.",
"In particular, the formation of heteropolysaccharide precursors (nucleotide sugars), consisting of sugars activated by reaction with triphosphate nucleotides, might involve intracytoplasmic enzymes which also contribute to other metabolic pathways, and in particular to glycolysis.",
"Now, due to the fermentative metabolism of lactic acid bacteria, the glycolysis reactions are more active than those relating to EPS synthesis.",
"The inventors set themselves the aim of obtaining lactic acid bacteria mutants in which it is possible to control and in particular increase the capacity to use sugars available in the medium, and in particular galactose, to produce EPS.",
"With this aim, they have investigated the genes involved in the synthesis of nucleotide sugars, the precursors of EPS, and/or the genes involved at the crossroads between the pathways of glycolysis and of EPS biosynthesis.",
"Among the latter, they were more particularly interested in the genes for phosphoglucomutase (PGM), which is involved in the transformation of metabolic derivatives of galactose into glycolysis intermediates, and for glucose-1-phosphate uridyltransferase, which catalyzes the formation of nucleotide sugars, the precursors of EPS.",
"DE VUYST and DEGEEST (abovementioned publication) put forward the hypothesis that phosphoglucomutase might play an important linking role between glycolysis and EPS biosynthesis, and that diverting part of the carbon flow to this enzyme might make it possible to increase EPS production.",
"However, they also underline that it remains to be seen whether this can effectively be done.",
"It has recently been reported [HARDY et al., J Bacteriol., 1854-1863, (2000)] that inactivation in Streptococcus pneumoniae of a gene [GENBANK AF165218] encoding a phosphoglucomutase leads to a dramatic decrease in the production of EPS forming the bacterial capsule, and is also highly damaging to cell viability, although this bacterium possesses another pgm gene.",
"The existence of an α-PGM and a β-PGM has been reported in L. lactis; however, only the gene corresponding to β-PGM has been isolated [QIAN et al., Microbiology, 143, 855-865, (1997)].",
"No gene capable of encoding an α-PGM of a lactic acid bacterium has been genetically characterized to date.",
"The inventors have now succeeded in cloning and characterizing a pgm gene encoding an α-PGM of Streptococcus thermophilus.",
"This gene is represented in the attached sequence listing under the number SEQ ID NO: 1, and the corresponding polypeptide is represented under the number SEQ ID NO: 2.A subject of the present invention is a nucleic acid sequence encoding an α-PGM the amino acid sequence of which exhibits at least 70% identity or at least 85% similarity, preferably 80% identity or at least 90% similarity, advantageously at least 90% identity or at least 95% similarity, and most preferably at least 95% identity or at least 99% similarity, with the α-PGM represented by the sequence SEQ ID NO: 2, and also any fragment of more than 20 bp of said sequence.",
"The percentage identity of a sequence with a reference sequence is defined herein as the percentage of residues of this sequence which are identical to those of the reference sequence when the 2 sequences are aligned so that the positions of the residues correspond to a maximum.",
"A polypeptide the amino acid sequence of which exhibits at least X% identity with a reference sequence can thus comprise up to 100-X modifications per 100 amino acids of the reference sequence.",
"These modifications include the deletion, substitution or insertion of amino acid residues, which may or may not be consecutive.",
"The percentage similarity of a sequence with a reference sequence is defined herein as the percentage of residues of this sequence which are identical with those of the reference sequence, or which differ therefrom only by one conservative substitution, when the 2 sequences are aligned so that the positions of the residues correspond to a maximum.",
"The term “conservative substitution” is intended to mean the substitution of an amino acid residue with another residue having similar physicochemical characteristics (size, charge or polarity) which do not change the functional properties of the protein.",
"A polypeptide the amino acid sequence of which exhibits at least X% similarity with a reference sequence can thus comprise up to 100-X nonconservative modifications per 100 amino acids of the reference sequence.",
"These modifications include the deletion, nonconservative substitution, or insertion of amino acid residues, which may or may not be consecutive.",
"The polypeptides thus exhibiting the greatest percentage identities or similarities with the sequence SEQ ID NO: 2, identified by searching on the “GENBANK nr” database using the BLASTp program [ALTSCHUL et al., Nucleic Acids Res., 25, 3389-3402, (1997)], with the default parameters, are as follows: the ybbT protein of Bacillus subtilis: 57% identity and 69% similarity; the femD protein of Staphylococcus aureus: 53% identity and 69% similarity; the hypothetical phosphoglucomutase of Streptomyces coelicolor: 42% identity and 55% similarity; the mrsA homologue of Pseudomonas syringae: 41% identity and 54% similarity; the mrsA homologue of Mycobacterium leprae: 41% identity and 54% similarity.",
"No significant homology with the pgm gene of L. lactis described by QIAN et al.",
"(publication cited above) was observed.",
"The percentage identity with the pgm gene of S. pneumoniae described by HARDY et al.",
"(publication cited above) is less than 31%.",
"The inventors have performed site-directed mutagenesis of the pgm gene of S. thermophilus, and have noted that, surprisingly, total or partial inactivation of this gene leads to an increase in EPS production.",
"A subject of the present invention is also a lactic acid bacteria mutant overproducing EPS, in which the pgm gene of alpha-phosphoglucomutase is totally or partially inactivated.",
"Said lactic acid bacterium will preferably be a mesophilic or thermophilic bacterium, chosen from streptococci and lactobacilli.",
"By way of example, it may be Lactococcus lactis, Streptococcus thermophilus, Leuconostoc mesenteroide, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus sake, etc.",
"Inactivation of the pgm gene may be obtained by making one or more mutations in the sequence encoding α-PGM, and/or in sequences controlling its expression.",
"Use may in particular be made of site-directed mutagenesis techniques, which are in themselves known to those skilled in the art and which make it possible to introduce a defined mutation into a gene, at the desired position.",
"It is thus possible, for example, to inactivate the pgm gene by introducing into the coding sequence, or the regulatory sequences, an exogenous sequence, for example a transposon.",
"Advantageously, it is also possible to replace the wild-type sequence of the pgm, by homologous recombination, with the mutated sequence.",
"In this case, one or more modifications by insertion, deletion or substitution of one or more nucleotides, which may or may not be consecutive, are made in a sequence identical to that of the region of the gene intended to be mutated.",
"According to a preferred embodiment of the present invention, the mutated sequence is inserted into a vector which allows integration by recombination (simple crossing-over) between the bacterial DNA fragment cloned into the vector and the homologous region of the bacterial genome.",
"Excision of the vector sequences can then occur following a second recombination event (double crossing-over), which results in substitution of the wild-type chromosomal form by the modified form.",
"Vectors which allow the integration of an exogenous sequence into the chromosome of a lactic acid bacterium are known in themselves, and are available for most species of lactic acid bacteria; they may, for example, be nonreplicating vectors, unstable replicative vectors or vectors which replicate conditionally, vectors carrying insertion sequences, etc.",
"In certain cases, it is also possible to transform the bacterium directly with the DNA carrying the mutated sequence intended to be inserted.",
"Mutants of the pgm gene in accordance with the invention can also be obtained by random mutagenesis (for example by chemical mutagenesis or radiation); they may also be natural mutants selected from cultures of lactic acid bacteria by screening on the basis of their phenotypic properties.",
"In fact, the mutants in which the α-PGM is partially or totally inactive have normal growth on lactose, and very slow growth on glucose or galactose alone.",
"Such mutants, whether they are natural mutants or are derived from mutagenesis, can therefore be selected directly on the basis of this property.",
"The presence of mutations in the pgm gene, and the nature of these mutations, can be easily verified with conventional molecular biology techniques, using nucleic acid primers or probes or strains derived from the sequence SEQ ID NO: 1.If desired, it is also possible to increase or decrease EPS production at will, as a function, for example, of the culturing conditions, by placing the pgm gene under the transcriptional control of an inducible promoter.",
"The inventors have also cloned and characterized the galU gene which encodes S. thermophilus glucose-1-phosphate uridyltransferase.",
"This gene is represented in the attached sequence listing under the number SEQ ID NO: 3.The GalU protein encoded by this gene is represented under the number SEQ ID NO: 4.A subject of the present invention is also a nucleic acid sequence encoding a glucose-1-phosphate uridyltransferase the amino acid sequence of which exhibits at least 90% identity or at least 95% similarity, and advantageously at least 95% identity or at least 99% similarity, with the glucose-1-phosphate uridyltransferase represented by the sequence SEQ ID NO: 4.The polypeptides thus exhibiting the greatest percentage identities or similarities with the sequence SEQ ID NO: 4, identified by searching on the “GENBANK nr” database using the BLASTp program [ALTSCHUL et al., Nucleic Acids Res., 25, 3389-3402, (1997)], are as follows: the glucose-1-phosphate uridyltransferase of Streptococcus mutans: 87% identity and 93% similarity; the GalU protein of Streptococcus pneumoniae: 87% identity and 93% similarity; the UDP-glucose pyrophosphorylase of Streptococcus pyogenes: 84% identity and 90% similarity; the Cap3C protein of Streptococcus pneumoniae: 76% identity and 88% similarity; the UDP-glucose pyrophosphorylase of Bacillus subtilis: 55% identity and 74% similarity.",
"Overexpression of the galU gene makes it possible to increase EPS synthesis by increasing the amount of precursors thereof.",
"A subject of the present invention is therefore also a lactic acid bacterium mutant overproducing EPS, in which the galU gene is overexpressed.",
"Such a mutant may in particular be obtained by introducing one or more copies of this gene into a lactic acid bacterium, and/or replacing its promoter with a strong promoter.",
"Preferably, said mutant is obtained from a lactic acid bacterium also having a partially or totally inactive α-PGM.",
"Mutant strains in accordance with the invention may in particular be obtained from S. thermophilus, and in particular from S. thermophilus strains selected for their ability to grow on galactose.",
"In fact, in this bacterium, another factor which is limiting for EPS synthesis may come from the fact that a large number of strains do not effectively use galactose, whether for growth or EPS synthesis.",
"The inventors have noted that gal+ strains of S. thermophilus, selected for their ability to grow on galactose, can synthesize a larger amount of EPS.",
"Using these clones capable of using galactose for their growth, it is possible to carry out a second selection of the strains producing colonies which appear to be larger in volume, which reflects greater synthesis of EPS.",
"The use of these gal+ strains of S. thermophilus, as starting material for the production of mutants in accordance with the invention, therefore further improves the ability thereof to produce EPS.",
"Lactic acid bacterial strains in accordance with the invention can advantageously be used for producing fermented products, in particular food products, in which it is desired to control, and in particular increase, the EPS content, and also for producing EPS.",
"The present invention will be clearly understood by virtue of the further description which follows, which refers to examples of obtaining and using lactic acid bacterial strains in accordance with the invention.",
"EXAMPLE 1 Selecting S. Thermophilus Strains Using Galactose more Effectively for EPS Synthesis A) Strains capable of effectively using galactose were selected using cultures of industrial leavens of S. thermophilus from the RHODIA and DANONE collections: RD488 (JIM7446) (RHODIA collection) and ext 1.5 (JIM7459) (DANONE collection).",
"For this, a 12-hour culture in M17 medium [TERZAGHI and SANDINE, Appl.",
"Microbiol., 29, 807-813, (1975)] containing 1% of lactose is deposited onto M17 agar plates containing 1% of galactose, and is incubated at 42° C. for 2 days.",
"The colonies thus obtained are streaked onto an M17 agar plate containing 1% of galactose, and are incubated at 42° C. overnight.",
"The colonies thus growing on M17 galactose are recovered and grown in liquid M17 containing 1% galactose at 42° C. for 6 hours before being placed in a collection.",
"Other media, such as the chemically defined medium described by SISSLER et al.",
"[Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 96, No.",
"16, 8985-8990, (1999)], in which the sugar source can be controlled, can also be used for this selection.",
"In order to verify that the strains obtained are indeed capable of more effectively using galactose, the growth of these strains and of the parents thereof are compared.",
"The growths are carried out as follows: overnight precultures in 10 ml of BELLIKER medium (DIFCO Laboratories) to which 10 g of beef extract/1 (DFICO) are added, and containing 1% of galactose, are diluted 100-fold and grown in 2 ml of M17 medium containing 2% of sugar (sucrose, lactose, glucose or galactose) for 4 hours at 42° C. They are then inoculated at 2% into 200 μl of M17 medium containing 1% of sugar.",
"The growth of the strains is then followed using the BIOSCREEN system (LABSYSTEMS).",
"FIG.",
"1 represents the growth, on various sugars, of the industrial strain JIM7459 (1A) and of a gal+mutant selected from this strain (1B).",
"B) It is possible to carry out a further selection from clones already capable of using galactose for growth.",
"Clones producing colonies which appear to have a greater volume are then sought on the plates.",
"These strains use galactose even more effectively.",
"FIG.",
"2 represents the growth, on various sugars, of a gal+ mutant (2A) selected as described in A above, from a culture of an industrial strain (JIM7446, DANONE collection), and of a gal++ mutant (2B) selected from a culture of this gal+mutant on the basis of colony size.",
"After growth of these various strains in chemically defined medium, or in M17 medium, the bacterial pellet of the gal+ mutants is more filamentous than that of the wild-type strains, which may reflect a difference in EPS production between the mutants obtained and their parental strain.",
"The EPS production of these strains was determined from a culture in 80 ml of chemically defined medium [SISSLER et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 96, No.",
"16, 8985-8990, (1999)].",
"After growth, the cultures are centrifuged at 16 000 g for 10 min at 4° C. The cell pellet is removed and the supernatant is precipitated (2 volumes of 100% ethanol per volume of supernatant) for 24 hours at 4° C. After precipitation, a centrifugation is performed at 16 000 g for 15 minutes at 4° C. The supernatant is removed and the pellet is resuspended in 60 ml of water.",
"Dialysis is performed against water for 4 days, exchanging the water 3 to 4 times per day.",
"The EPS are assayed using the phenol/sulfuric acid method [DUBOIS et al., Analytical Chemistry, 28, 350-356, (1956)].",
"The results obtained in the case of the 7446 Gal+ strain, and of a mutant of this strain capable of using galactose more effectively, are shown in table I below.",
"TABLE I STRAINS EPS (mg/l) 7446 Gal+ 4 7446 Gal++ 8 After growth in the presence of galactose, the strains using galactose more effectively produce increased amounts of EPS.",
"EXAMPLE 2 Cloning the PGM Gene of Streptococcus Thermophilus and Constructing Mutants in which this Gene is Inactive Cloning the Gene: The pgm gene of Streptococcus thermophilus can be obtained by reverse PCR (OCHMAN et al., Biotechnology (NY), 8, No.",
"8, 759-760, (1990)] from the chromosomal DNA of S. thermophilus.",
"The chromosomal DNA of S. thermophilus is digested with restriction enzymes (BamHi, EcoRI, HindIII, NcoI, PsI, XhI) and the cleavage products are then circularized then amplified by PCR using the primers complementary to the opposite strand: OST15, OST16, OSY23 to OST26 (table II).",
"TABLE II Primer Sequence OST15 ACATAACCACCAAAACGACCTAA OST16 GGAAGCTGAGATGGCTGGTA OST23 TCTTCAAGAACTGCACGGTCA OST24 CAAAATTCTTCGTCTTTACCG OST25 GCTAGCCTTATCGCCAGTCAA OST26 TTGAGTAAATCAGTTCCAGTT The bands obtained are extracted from the gel and sequenced.",
"The sequence of the cloned fragment, comprising the pgm gene and its flanking regions, is represented in the attached sequence listing under the number SEQ ID NO: 1.The length of the open reading frame of the pgm gene of Streptococcus thermophilus is 1350 bp.",
"Constructing Mutants of the pgm Gene In order to decrease the activity of the pgm gene, the inventors adopted a strategy of inactivation by insertion of a vector into the gene by homologous recombination.",
"The general strategy of inactivation is represented in FIG.",
"3.Initially, thermosensitive replication plasmids containing fragments internal to the pgm gene were constructed using the thermosensitive replication vector pG+host [BISWAS et al., J.",
"Bacteriol., 175, 11, 3628-3635, (1993); PCT application WO/181164].",
"Two PCR fragments produced with the oligonucleotides OST29+OST31 and OST28+OST31, and representing, respectively, 650 and 835 bp of the pgm gene, were cloned into pG+host, generating the plasmids pST28 and pST29.TABLE III Primer Sequence OST28 TAAGGGCCCTAAATATTTTGGAA CAGA OST29 CCGGGGCCCACTTCTCTCAGTAG GTAT OST31 AATATCGATATTTTCATCAACGGC AAT The 650 bp fragment carried by pST28 is central to the gene, it is therefore expected that the insertion thereof should produce total inactivation of the gene.",
"The 835 bp fragment carried by pST29 contains the portion located in the 5′ position of the gene with the translation initiation codon, but without its ribosome binding site.",
"It is expected that the insertion of pST29 into the chromosome will cause a very large decrease in translation of pgm, allowing only a very low base expression.",
"The plasmids pG+host, pST28 and pST29 were introduced by transformation into the S. thermophilus strains JIM7446 and JIM7459 at 30° C., and then integrated by simple crossing-over, as shown diagrammatically in FIG.",
"3.The integration by simple crossing-over was carried out according to the protocol described by BISWAS et al.",
"[publication cited above, (1993)] with the following modification: The Streptococcus thermophilus strains containing the plasmid pG+host or its derivatives are grown overnight at 30° C. in the presence of erythromycin, and then diluted 50-fold in the same medium.",
"They are then transferred to 42° C. for 6 hours.",
"The samples are then diluted and plated out, firstly, at 42° C. on M17 plates containing erythromycin in order to detect the integration events and, secondly, at 30° C. on M17 plates without antibiotic in order to detect the total number of viable cells.",
"The frequency of integration per cell is calculated from the ratio of these counts.",
"The frequencies of integration of these plasmids are, respectively, 3.5×10−3, 3×10−3 and 10−2 for pG+host, pST28 and pST29.Clones derived from each of these integrations were isolated to give the strains STJ3, STJ1 and STJ2, respectively.",
"The STJ1 (pST28), STJ2 (pST29) and STJ3 (pG+host) strains were grown on plates of chemically defined medium containing glucose and galactose or on plates of the same media containing either glucose alone or galactose alone.",
"The results obtained are given in table IV.",
"TABLE 4 Growth on CDM Growth on CDM Growth on CDM Plasmid strains Glu Gal Ery Glu Ery Gal Ery STJ1 pST28 Normal No growth No growth STJ2 pST29 Normal No growth No growth STJ3 pG + host Normal Normal Normal The STJ1 and STJ2 clones do not therefore grow on glucose or galactose alone, but grow normally in lactose or on a glucose and galactose mixture.",
"This shows that glucose and galactose metabolism has indeed been uncoupled in this strain and that the gene whose activity has been affected is indeed pgm.",
"These results show that the inactivated gene clearly encodes an enzyme which connects the EPS pathway and glycolysis.",
"It therefore probably encodes α-PGM, the sequence of which had not yet been characterized experimentally in lactic acid bacteria.",
"Assaying the EPS Produced by the Streptococcus thermophilus Strain STJ2 Carrying a Mutation in the pgm Gene The EPS are assayed as described in example 1 above.",
"The EPS production in the strain affected in terms of expression of the pgm gene is 20 mg/l.",
"This production, compared to the 8 mg/l obtained for the parental strain 7446 Gal++, therefore corresponds to a two and a half-fold increase.",
"EXAMPLE 3 EPS Production by pgm Gene Mutants Derived From Industrial Strains The plasmid pST29 described in example 2 above was used to transform the industrial strains (DANONE collection) Ext 1.1 (JIM7455) and Ext 1.10 (JIM7464).",
"These strains produce EPS with different compositions (1-glu:2-gal: 1-galNac for Ext 1.1 and 3-gal: 1-rha for Ext 1.10), and which are also different from that produced by the JIM7446 strain (3-glu:4-gal).",
"After electroporation, the cells are plated out at 30° C. for 12 h and the plates are then placed at 42° C. in order to force integration of the plasmid pST29 into the pgm gene by homologous recombination between the pgm sequences present on the plasmid and those present on the chromosome.",
"Colonies of mutated bacteria capable of growing at 42° C. in the presence of erythromycin were obtained.",
"These bacteria are also capable of growing on CDM (chemically defined medium), in the presence of glucose and galactose, but not in the presence of glucose alone or galactose alone.",
"The transformed or untransformed strains were cultured in 10 ml of CDM containing 2% lactose, 1.5% casitone and 10 mM urea, in the presence of erythromycin (5 μg/ml) for the strains transformed with the plasmid pST29.The EPS produced by the various strains were assayed after 67 h 30 min of culturing at 42° C. The results obtained are given in table V below.",
"TABLE V Strain OD600 pH mg EPS/l mg EPS/l/OD 7455 (Ext 1.1) 1.566 4.6 12.5 8 7455::pST29 0.98 4.6 14.68 16 7464 (Ext 1.10) 1.29 4.24 23.6 18.29 7464::pST29 0.99 4.2 44 44 It is noted that the strains in which the pgm gene is inactivated in accordance with the invention produce, at equal biomass, 2 to 2.5 times more EPS than the strains of origin.",
"EXAMPLE 4 Constructing a Mutant of the pgm Gene by Double Crossing-Over A vector named pSTJ6, containing a pgm gene inactivated by internal mutation, was constructed by reverse PCR amplification using the plasmid pST29 as matrix and the following primers, derived from the sequence of the pgm gene with point mutations, inducing in particular the creation of an NsiI site: GACATGCATCGCTTGTCGATTAGCCAGAAGGTC GCGATGCATGTCATTAAGCTGATGGGCGTCGAAG The mutations introduced into the primers are underlined: the NsiI site created is indicated in italics.",
"The PCR product is cleaved with NsiI and religated on itself to give the vector pSTJ6.This vector is introduced by transformation into the S. thermophilus strain JIM7446.The colonies are plated out at 30° C. for 12 h, and then grown at 42° C. for 24 h in order to force the integration of the vector into the chromosome via a first crossing-over by recombination between the pgm sequences present on the plasmid and those present on the chromosome.",
"The colonies thus obtained, erythromycin resistant and capable of growing at 42° C., are then grown at 30° C. in order to promote excision of the pG+host plasmid sequences via a second crossing-over by recombination between the pgm sequences present on either side of the pG+host sequences.",
"pG+host is then removed from the cell by a step of growth at 42° C. in the absence of selection with erythromycin.",
"The strain thus obtained no longer contains any sequences of the vector pG+host and, in particular, it does not contain a gene for resistance to antibiotics.",
"It differs from the S. thermophilus strain of origin, JIM7446, only by the presence of the mutations in a portion of the pgm gene.",
"The initial sequence and the mutated sequence of this portion of the pgm gene are shown below; the mutations are underlined.",
"Initial sequence: GCTGAAGGACTTGGAACGCTTGTCGATTATCCA Mutated sequence: GCTTAATGACATGCATCGCTTGTCGATTAGCCA The mutated strain has the phenotype characteristic of the pgm mutants, described in examples 2 and 3 above.",
"It is capable of growing on CDM in the presence of glucose and galactose but not in the presence of glucose alone or galactose alone.",
"The increase in EPS production by this strain, relative to the parental strain, is comparable to that observed in the case of the strains transformed with pST29, described in examples 2 and 3.EXAMPLE 5 Constructing Mutants Overproducing galU Cloning the galU gene encoding glucose-1-phosphate uridyltransferase The entire galU gene can be obtained by reverse PCR [OCHMAN et al.",
"(1990), publication cited above] from the chromosomal DNA of S. thermophilus, using primers complementary to the opposite strand (OST13, OST14, OST21 and OST22) (table VI).",
"TABLE VI Primer Sequence OST13 CCAAGATATCTTCAATACCAGAC OST14 GAAAACACAACGCGTCTTTGC OST21 GCACCAGCACCGACTGCGATA OST22 ACTGGGTAGTTAAACGGTAAT The bands obtained are extracted from the gel and sequenced.",
"The sequence of the cloned fragment, comprising the galU gene, is represented in the attached sequence listing under the number SEQ ID NO: 3.The size of the galU gene is 914 bp.",
"Overexpression of GalU The galU gene (with its terminator and its ribosome binding site) was amplified with the oligonucleotides OST44 and OST45 (table VI) and cloned downstream of the p45 promoter [SIBAKOV et al., Appl.",
"Environ.",
"Microbiol., 57, 341-348, (1991)] of L. lactis in the ApaI site.",
"TABLE VI Primer Sequence OST44 AATGGGCCCAAAAATAAAAAATCTAAG GAG OST45 AAGCTGCAGACTTATCTTTAAATTAAAT GA A SmaI fragment of the resulting plasmid, carrying P45 followed by galU, was purified and inserted into the SmaI site of the vector pGKV259 which replicates in S. thermophilus.",
"The plasmid pGKV259 [VAN DER VOSSEN et al., Appl.",
"Environ.",
"Microbiol., 53, 2452-2457, (1987)] expressing the galU gene under the control of the p45 promoter is called pSTJ4 (FIG.",
"4).",
"This plasmid is introduced by transformation into the S. thermophilus strain JIM7446 or JIM7459 or into the strain containing a mutation in the pgm gene (STJ2)."
]
] | Patent_10257737 |
[
[
"Method for measuring cylinder specific parameters in a combustion engine",
"The invention concerns a method to measure parameters in the combustion chamber of a piston engine that comprises an ignition system.",
"An alternating voltage is applied across the secondary winding of the ignition coil and the value of the current that arises in a measurement circuit that co-operates with the secondary winding is measured.",
"The value of the current depends on the resistance (R1) of the measurement circuit, on the inductance (L1) and resistance of the secondary winding, and on the impedance of the combustion chamber, i.e.",
"on its capacitance (C1) and its resistance.",
"For example, top dead centre, the pressure in the cylinder, analysis of the ionic current and change of the burning time can be determined by means of the method."
],
[
"1.A method to measure and determine cylinder-specific parameters such as pressure, piston position and impedance in a combustion chamber of a piston engine having an ignition system with an ignition coil and spark plugs, characterised in that measurement occurs with the aid of the ignition system through the application of an alternating voltage, that can be changed by a control program, across a secondary winding of the ignition coil, after which the value of the current that arises is detected as a function of time in a measurement circuit that co-operates with the secondary winding, whereby the value of current detected depends on a measurement resistance of the measurement circuit, on an inductance and resistance of the secondary winding and on a characteristic impedance of the combustion chamber, which comprises a capacitance and a resistance, and whereby in addition, the pressure in a cylinder and/or a position of a piston can be determined by means of the value of the current.",
"2.The method according to claim 1, characterised in that the measurement is carried out during revolutions of the piston engine in which combustion does not take place.",
"3.The method according to claim 2, characterised in that the measurement is carried out while the piston engine is mechanically motored.",
"4.The method according to claim 2 or 3, characterised in that the maximum value of the current that arises in the measurement circuit that co-operates with the secondary winding is measured, whereby the highest value of the capacitance of the combustion chamber and this maximum value of the secondary current, that is, the top dead centre, is determined.",
"5.The method according to claim 2 or 3, characterised in that the frequency of the alternating voltage is varied, whereby the value of the resistance of the combustion chamber is determined, which value of the resistance is equivalent to the insulation resistance in the high-tension section of the ignition system.",
"6.The method according to claim 1, characterised in that the measurement is carried out during revolutions of the engine during which combustion takes place.",
"7.The method according to claim 6, characterised in that the measurement is carried out during that part of the engine revolution during which combustion does not take place, whereby the frequency of the alternating voltage is varied and the value of the resistance of the combustion chamber is determined, which value of the resistance is equivalent to the insulation resistance of the high-tension section of the ignition system.",
"8.The method according to claim 7, characterised in that the burning time of the ignition spark can be changed by means of applying a changed voltage level across the measurement circuit.",
"9.The method according to claim 7, characterised in that an ionic current from the combustion process is analysed by means of the measurement circuit.",
"10.The method according to claim 7, characterised in that both analysis of an ionic current and increased burning time of the spark are generated by the measurement circuit.",
"11.The method according to any one of the preceding claims, characterised in that the alternating voltage that can be changed is applied through a primary winding of an existing ignition coil.",
"12.The method according to claim 11, characterised in that the ignition coil comprises more than one primary winding.",
"13.The method according to any one of claims 1-10, characterised in that the alternating voltage that can be changed is applied through a primary winding of a separate transformer, the secondary winding of which is placed in series with the secondary winding of the ignition coil.",
"14.The method according to any one of claims 1-10, characterised in that the alternating voltage that can be changed is applied via a primary winding of a separate transformer, the secondary winding of which is connected in series with the secondary windings of several ignition coils, which coils are connected in parallel.",
"15.The method according to any one of claims 1-14, characterised in that the ignition voltage of the ignition system is generated in an inductive ignition system.",
"16.The method according to any one of claims 1-14, characterised in that an ignition voltage of the ignition system is generated in an inductive ignition system."
],
[
"<SOH> TECHNICAL FIELD <EOH>The present invention concerns a method for measuring and determining cylinder-specific parameters in a combustion chamber in a piston engine that comprises an ignition system."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The method of measuring and determining cylinder-specific parameters in a combustion chamber of a piston engine that comprises an ignition system is characterised in that the measurement occurs with the aid of the ignition system in that an alternating voltage is applied across the secondary winding of the ignition coil, after which the value of the current that arises in a co-operating measurement circuit on the secondary side is detected as a function of time.",
"The absolute value of the current depends on the measurement resistance of the measurement circuit, on the inductance and the resistance of the secondary winding and on the characteristic impedance of the combustion chamber, which comprises a capacitance and a resistance.",
"The capacitance of the combustion chamber changes when the position of the piston changes and it has its highest value when the piston is situated at the top dead centre.",
"The secondary current has its highest value when the capacitance reaches its maximum.",
"The measurement according to the invention can be carried out during revolutions of the engine when no combustion takes place.",
"The measurement can even be carried out by “motoring” the engine before it is started.",
"The measurement can also be carried out during revolutions of the engine when combustion does take place.",
"In this case, by applying a suitable voltage level across the measurement circuit, the ionic current from the combustion process can also be analysed and, in the same way, the burning time of the spark can be increased, or multisparks can be supplied through the application of a voltage across the measurement circuit that is suitable for this purpose.",
"Other characteristics of the invention are specified in the following claims."
],
[
"TECHNICAL FIELD The present invention concerns a method for measuring and determining cylinder-specific parameters in a combustion chamber in a piston engine that comprises an ignition system.",
"PRIOR ART For several functions of internal combustion engines it is important to be able to measure and determine various parameters in the combustion chamber in a reliable manner.",
"Vital information such as IMEP and HEAT RELEASE can be determined based on the cylinder pressure and an accurate determination of top dead centre.",
"IMEP is an acronym for “Indicated Mean Effective Pressure” and is a measure of the work that the cylinder performs during one working cycle without taking friction losses into account.",
"HEAT RELEASE is the heat that is released by the combustion.",
"Furthermore, it is important to be able to localise top dead centre (“the upper turning point”) of the engine in a reliable manner in order to make possible spark formation of the spark plug when the piston is in the position that is most advantageous for making the ignition of the fuel-air mixture as efficient as possible, both with respect to function and from the point of view of emissions.",
"The detection of top dead centre must be possible rapidly and correctly for the ignition to be controlled in an optimal manner.",
"The detection must also be possible during operation of the engine.",
"The methods that are currently used to calibrate the position of top dead centre are time-consuming.",
"For example, a dial indicator can be arranged in the cylinder, and the piston displaced such that a minimum distance between the piston and the cylinder head is achieved.",
"It is subsequently cumbersome to fine-adjust the position that has been adjusted in this manner.",
"Cylinder pressure is currently determined either with a piezoelectric sensor or with the aid of ionic currents from the combustion process.",
"The present invention solves the problem with the determination of top dead centre and can be used both before the engine has been started and under revolution of the engine with or without combustion.",
"One advantage is that reading off of the position of top dead centre according to the invention is more exact than when using the previously known methods.",
"The invention also means that the cylinder pressure can be measured even during revolutions in which no combustion takes place.",
"The present invention also has the advantage that measurement of ion currents, modification of burning time and a multispark function can be carried out in the ignition system in an efficient manner.",
"“Multispark function” is used to denote the delivery by the ignition system of a new spark as soon as the system has been recharged to the correct level.",
"Furthermore, the method according to the invention provides information about the appearance of the pressure curve not only during combustion but also during revolutions without combustion.",
"This means that an extensive analysis of the combustion process, such as, for example, facts about the current IMEP and HEAT RELEASE, can be obtained with the aid of the invention.",
"Since the invention uses impedance identification, it is also possible to detect leakage currents, soot formation and other defects of the spark plug of the internal combustion engine.",
"The invention also has the advantage that the measurement voltage level can be directly changed from, for example, a control program without requiring any modification of the hardware of the ignition system.",
"This means increased opportunities with respect to detecting ionic currents.",
"SUMMARY OF THE INVENTION The method of measuring and determining cylinder-specific parameters in a combustion chamber of a piston engine that comprises an ignition system is characterised in that the measurement occurs with the aid of the ignition system in that an alternating voltage is applied across the secondary winding of the ignition coil, after which the value of the current that arises in a co-operating measurement circuit on the secondary side is detected as a function of time.",
"The absolute value of the current depends on the measurement resistance of the measurement circuit, on the inductance and the resistance of the secondary winding and on the characteristic impedance of the combustion chamber, which comprises a capacitance and a resistance.",
"The capacitance of the combustion chamber changes when the position of the piston changes and it has its highest value when the piston is situated at the top dead centre.",
"The secondary current has its highest value when the capacitance reaches its maximum.",
"The measurement according to the invention can be carried out during revolutions of the engine when no combustion takes place.",
"The measurement can even be carried out by “motoring” the engine before it is started.",
"The measurement can also be carried out during revolutions of the engine when combustion does take place.",
"In this case, by applying a suitable voltage level across the measurement circuit, the ionic current from the combustion process can also be analysed and, in the same way, the burning time of the spark can be increased, or multisparks can be supplied through the application of a voltage across the measurement circuit that is suitable for this purpose.",
"Other characteristics of the invention are specified in the following claims.",
"SHORT DESCRIPTION OF THE DRAWINGS In the following description of embodiments of the invention reference will be made to the accompanying drawings, in which: FIG.",
"1 shows the construction in principle of the secondary circuit according to the invention; FIG.",
"2 shows examples of connection of circuits to detect top dead centre according to the invention; FIG.",
"3 shows a measurement circuit with a transformer; FIG.",
"4 shows a curve for determination of top dead centre; FIG.",
"5 shows a measurement circuit with a separate transformer, the secondary winding of which is connected in series with the secondary windings of several ignition coils that are connected in parallel.",
"DESCRIPTION OF EMBODIMENTS Thus, the secondary current of the ignition coil is used, according to the invention, in order to be able to detect when the piston is located at top dead centre.",
"FIG.",
"1 shows the construction in principle of a secondary circuit for the detection.",
"The measurement circuit comprises, in addition to a measurement resistance R1, the inductance L1 of the secondary winding of the ignition coil, the resistance R2 of the secondary circuit and the cylinder impedance that consists of the parallel connection of R3, R4 and C1.A combustion procedure means that the impedance of the combustion chamber is influenced.",
"In order to be able simply to determine the values of R3, R4 and C1, the measurement for a 4-stroke engine should take place during two revolutions of the engine, that is, during one working cycle.",
"During the cycle in which combustion does not take place, which is usually denoted as the “waste”-cycle, the impedance is influenced only by the position of the piston and not by the pressure or by the ionic current (R4).",
"During the combustion phase, the impedance is influenced also by the compressive pressure.",
"When compression and leakage effects remain but no combustion takes place, the compression and the leakage resistance (R3) can be measured.",
"By changing the frequency of the measurement signal, the value of the capacitance C1 can be separated from the value of the leakage resistor R3, on the condition that no ionic current is present (combustion is not taking place).",
"The separation of the various resistances for a 2-stroke engine can take place by producing a deliberate misfiring, for example, by switching off the ignition or fuel supply, or by turning the engine over manually or with the starter motor before combustion is started.",
"A further alternative for doing this is to measure during that part of the working cycle during which ionic current is not present.",
"Based on the circuit if FIG.",
"1, the voltage across the measurement resistor can be expressed with the aid of a Laplace transform according to (1): where.",
"V meas = - I s ( R 2 + sL 1 + R 3 R 4 C 1 R 4 sC 1 + R 3 sC 1 + R 3 R 4 ) where ( 1 ) I s = V meas R 1 ( 2 ) and where Vmeas is the voltage across the measurement resistor, Is is the current in the secondary circuit, R1 is the measurement resistance, sL1 is the impedance of the secondary winding, R2 is the resistance of the secondary circuit and 1/sC1, R3 and R4 comprise the characteristic impedance of the combustion chamber.",
"The capacitance of the combustion chamber C1 depends on, among other factors, the position of the piston, the composition of the gas, the temperature and the cylinder pressure.",
"When the piston in the cylinder moves, the cylinder capacitance C1 changes and thus affects the impedance of the secondary circuit.",
"The change can be measured by means of the voltage Vmeas.",
"Top dead centre can be detected in this way, since the capacitance of the cylinder reaches its highest value at top dead centre.",
"The absolute value of the secondary current Is thus has its highest value at top dead centre, if no combustion takes place.",
"It can be seen from equations (1) and (2) that it is desirable to use an alternating voltage in the secondary circuit in order to be able to measure the change in impedance during motion of the piston.",
"One method for generating alternating current for the detection of top dead centre of a piston cylinder is shown in FIG.",
"2.In addition to the secondary circuit, which agrees with that shown in FIG.",
"1, a battery of voltage V1 is shown on the primary side of an ignition coil T1 connected to one end of the primary winding of the ignition coil, the second end of which is connected to a switch SW that is controlled by a control signal t. Thus, according to the invention, the primary winding of the ignition coil can be used to apply an alternating voltage across the spark plug.",
"The frequency of the alternating voltage can be varied, whereby the resistance of the combustion chamber is determined, which value of resistance is equivalent to the insulation resistance in the high-tension section of the ignition system.",
"According to one embodiment of the invention, top dead centre can be detected during revolutions of the engine when combustion is taking place.",
"The same measurement circuit can then be used to analyse the ionic current from the combustion process.",
"In this case, the primary winding is used to generate not only a spark, but also alternating voltage for the detection of top dead centre.",
"The use of the same primary winding both for high tension and for measurement of the ionic current, which is thus accomplished by a special connection, means that the available space within the engine compartment is used more efficiently since no extra coils need to be added.",
"Furthermore, the invention can be applied to existing ignition coils.",
"In another embodiment, an ignition coil is used that has double primary windings whereby one winding is used for spark voltage and one winding is used for the detection of top dead centre and for analysis of the ionic current.",
"An alternative method for the analysis of the ionic current is to use an ionic current transformer T2 with a separate primary winding, the secondary winding of which is placed in series with the secondary winding of the ignition coil T1, see FIG.",
"3.In this way, a triangularly shaped voltage, for example, can be applied to the primary winding of the ionic current transformer.",
"This leads to a constant voltage across the output of the ionic current transformer and thus also across the secondary winding of the ignition coil.",
"A variable DC voltage is obtained with which the signal from the ionic current is analysed, see FIG.",
"3.The circuit according to FIG.",
"3 can also be used together with curves that have other shapes.",
"According to one embodiment, the voltage can be applied via the primary winding of a separate transformer, the secondary winding of which is connected in series with the secondary windings of several ignition coils, which coils are connected in parallel, see FIG.",
"5.One advantage of the circuit according to FIG.",
"3 is that no inductive connection between the ignition coil T1 and the ionic current transformer T2 is necessary.",
"This is particularly advantageous in designs in which the space available for the ignition coil is small, since the ionic current transformer T2 can be placed where more space is available.",
"The measurement of ionic current according to the invention is more dynamic than measurement with known methods, since it makes possible measurement during change of the level of ionic current if the composition of the fuel is changed.",
"With conventional technology for the measurement of ionic currents, this can lead to the ionic current becoming too small or too large for analysis to be possible.",
"With conventional ionic current technology using a fixed voltage level, changes of the system must be accomplished in equivalent cases.",
"With the method according to the invention, the signal amplitude can be changed by, for example, adjusting the frequency of the control signal.",
"A further advantage of the embodiment according to the invention is that the leakage effects that arise at high temperatures are avoided.",
"These arise in the conventional measurement circuit for ionic current with Zener diodes.",
"The measurement voltage decays at these high temperatures, and the measurement result becomes poorer, something that can be remedied through the method according to the invention.",
"The circuit according to the invention can also be used to prolong the burning time of the spark or both to prolong the burning time of the spark and to analyse the ionic current.",
"This combination is advantageous since a problem that is currently present with ignition coils with long burning times is that they risk disturbing the measurement of ionic current.",
"The burning time can be extended with the aid of the invention also for those cycles in which measurement of the ionic current is not necessary.",
"The possibility also exists, only by altering the frequency of the switching, for a very rapid spark generation with high energy and with a short time interval between each spark, something that is known as multisparking.",
"According to one embodiment of the invention, the working pressure in the cylinder can be detected by means of the measuring circuit, whereby the maximum pressure lies at the top dead centre of the piston, if combustion does not take place.",
"FIG.",
"4 shows an example of measurement results for the detection of top dead centre, in which the signal of the alternating voltage measured across R1 has been processed in order to measure the amplitude of the pressure curve and the position of the pressure maximum.",
"The method according to the invention can be applied in a capacitive ignition system or in an inductive ignition system."
]
] | Patent_10257820 |
[
[
"Method and apparatus to measure flow rate",
"A method and apparatus for measuring the flow rate of a single phase fluid, particularly a gas, through a conduit.",
"A pulse of tracer fluid is discharged or injected into a first fluid flowing through a conduit, the concentration of the tracer fluid is measured as a function of time at a pont downstream from where it is injected, and the flow rate of the first fluid is determined based on the concentration measurements.",
"The concentration of tracer fluid is preferably sampled substantially continuously from when the tracer gas first passes the measuring point until substantially all of the tracer fluid has passed the measuring point and the sampled concentrations integrated.",
"The concentration of tracer fluid is preferably determined by measuring the thermal conductivity of the mixture of the first fluid and tracer fluid which is dependent upon the concentration of the tracer fluid."
],
[
"1.A method of measuring the flow rate of a first single phase fluid flowing through a conduit, the method comprising discharging a known or determinable molar quantity of tracer fluid into a first fluid flowing through a conduit; determining the concentration of the tracer fluid at a suitable point downstream from where it is discharged; and determining the flow rate of the first fluid through the conduit dependent upon the molar quantity of tracer fluid discharged and the measured concentration of the tracer fluid at the suitable point downstream.",
"2.A method according to claim 1, wherein the concentration of the tracer fluid is determined by measuring the change in thermal conductivity of the mixture of first fluid and tracer fluid compared with the thermal conductivity of the first fluid alone, which is a function of the concentration of the tracer fluid.",
"3.A method according to claim 2, wherein the concentration of the tracer fluid is determined from the measured thermal conductivity using a processing means using suitable algorithms.",
"4.A method according to claim 2, wherein the concentration of the tracer fluid is determined from the measured thermal conductivity using a look-up table.",
"5.A method according to any of the preceding claims wherein a pulse of tracer fluid is injected into a first fluid flowing through a conduit.",
"6.A method according to claim 1, wherein the concentration of the tracer fluid is measured a plurality of times as it passes the measuring point.",
"7.A method according to claim 2, wherein the concentration of the tracer fluid is sampled substantially continuously as it passes the measuring point.",
"8.A method according to claim 6 or claim 7, wherein the concentration of the tracer fluid is measured or sampled from when the tracer gas first passes the measuring point until substantially all of the tracer fluid has passed the measuring point.",
"9.A method according to any of claims 6 to 8, wherein the measured or sampled concentrations of tracer fluid are summed.",
"10.A method according to any of claims 6 to 8, wherein the measured or sampled concentrations of tracer fluid are integrated with respect to time.",
"11.A method according to any of the preceding claims, wherein the measured or sampled concentrations are adjusted to take account of background tracer fluid concentrations.",
"12.A method according to any of the preceding claims, wherein the flow rate of the first fluid through the conduit is determined dependent upon the molar quantity of tracer fluid injected into the first fluid.",
"13.A method according to any of the preceding claims, wherein the flow rate of the first fluid through the conduit is determined dependent upon the concentration of tracer fluid injected into the first fluid.",
"14.A method according to any of the preceding claims, wherein the tracer fluid includes helium.",
"15.A method according to any of the preceding claims, wherein the first fluid is a gas.",
"16.A method according to claim 15, wherein the first fluid is natural gas.",
"17.A method of measuring the flow rate of a first fluid flowing through a conduit according to claim 1, wherein the flow rate is determined independent of information about the conduit geometry.",
"18.An apparatus for measuring the flow rate of a first single phase fluid flowing through a conduit, the apparatus comprising a device for discharging a pulse of tracer fluid into a first fluid flowing in a conduit; determining means for determining the concentration of the tracer fluid at a point downstream from where the discharging device is arranged to discharge the tracer fluid; and control means for determining the flow rate of a first fluid through a conduit dependent upon the known or determinable molar quantity of tracer fluid discharged and the concentration of the tracer fluid determined by the determining means.",
"19.An apparatus according to claim 18, wherein the determining means includes a measuring means for measuring the change in thermal conductivity of the mixture of first fluid and tracer fluid compared with the thermal conductivity of the first fluid alone which is dependent upon the concentration of the tracer gas and conversion means for converting the measured change in thermal conductivity into a value corresponding to the tracer fluid concentration.",
"20.An apparatus according to claim 19, wherein the conversion means converts the measured change in thermal conductivity into a corresponding tracer fluid concentration using suitable algorithms.",
"21.An apparatus according to claim 19, wherein the conversion means converts the measured change in thermal conductivity into a corresponding tracer fluid concentration using a look-up table.",
"22.An apparatus according to claim 18, wherein the tracer fluid is injected into the first fluid.",
"23.An apparatus according to claim 18, wherein the control means is arranged to receive a plurality of concentration measurements taken as the tracer fluid passes the measuring means.",
"24.An apparatus according to claim 23, wherein the control means is arranged to receive substantially continuous concentration samples as the tracer fluid passes the measuring means.",
"25.An apparatus according to claim 23 or claim 24, wherein the control means is arranged to receive concentration measurements or samples from at least when the tracer fluid first passes the measuring point until substantially all of the tracer fluid has passed the measuring point.",
"26.An apparatus according to any of claims 23 to 25, wherein the control means sums the measured or sampled concentrations of tracer fluid.",
"27.An apparatus according to any of claims 23 to 25, wherein the control means integrates the measured or sampled concentrations of tracer fluid with respect to time.",
"28.An apparatus according to any of claims 23 to 27, wherein the control means is arranged to take account of background tracer fluid concentrations in its determination of the flow rate of a first fluid.",
"29.An apparatus according to any of claims 18 to 28, wherein the control means is arranged to take account of the molar quantity of tracer fluid injected into the first fluid by the discharging device in its determination of the flow rate of a first fluid.",
"30.An apparatus according to any of claims 18 to 29, wherein the control means is arranged to take account of the concentration of the tracer fluid discharged into the first fluid by the discharging device in its determination of the flow rate of a first fluid.",
"31.An apparatus according to any of claims 18 to 30, wherein the measuring means includes a thermal conductivity detector for detecting the thermal conductivity of the passing fluid which is a function of its concentration of tracer fluid.",
"32.An apparatus according to claim 18, wherein the flow rate of a fluid through a conduit is determined independent of information about the geometry of the conduit through which the first fluid flows."
],
[
"The present invention relates to a method and apparatus for measuring the rate of flow of a fluid, particularly a gas, through a conduit.",
"The flow rate of a fluid through a pipe can be measured using ‘time of flight’ methods in which a marker is introduced into the fluid flowing in the pipe and the time taken for the marker to travel a known distance along the pipe is measured.",
"Such a method is disclosed in U.S. Pat.",
"No.",
"5,646,354 in which microwave radiation is injected into a flowing stream of material to heat the material at that point.",
"A temperature sensor is positioned a known distance from the point at which the flowing material is heated and the time taken by the heated material to reach the temperature sensor is measured.",
"However, a number of problems arise with ‘time of flight’ methods of measuring flow rate.",
"For example, the volume between the point at which the marker is introduced and the point at which the marker is detected must be known precisely.",
"This can be difficult to determine if the conduit through which the fluid flows is difficult to access, such as if it is underground.",
"Furthermore, the conduit through which the marker travels should be straight as any bends would lead to uncertainty as to the actual distance travelled by the marker which could follow one of a number of paths around the bend.",
"Thus such a ‘time of flight’ meter would be difficult to apply in many circumstances such as a local transmission network supplying gas to consumers as the pipes of such a network are generally concealed underground and have many bends.",
"It is an object of the present invention to be able to measure the flow rate of a fluid through a conduit whilst overcoming one or more of the problems previously mentioned.",
"According to a first aspect of the present invention, a method of measuring the flow rate of a first substantially single phase fluid flowing through a conduit comprises: discharging a known or determinable molar quantity of a tracer fluid into a first fluid flowing through a conduit; measuring the concentration of the tracer fluid as a function of time at a suitable point downstream from where it is discharged, and determining the flow rate of the first fluid through the conduit dependent upon the molar quantity of a tracer fluid discharged and the measured concentration of the tracer fluid at the suitable point downstream.",
"Using this technique, the flow rate of a fluid may be determined independent of or without the need for details of the conduit geometry or a long straight length of conduit.",
"The concentration of the tracer fluid may be measured more than once or substantially continually monitored or sampled during the passage of the tracer fluid past the measuring point and the measured concentrations integrated or summed.",
"The concentration of the tracer fluid is preferably measured from when the tracer fluid first passes the sampling point until all of the injected tracer fluid has passed.",
"The flow rate of the first fluid is preferably determined dependent on the molar quantity of the injected tracer fluid as well as its measured concentration at a point downstream.",
"The first fluid and the tracer fluid are preferably single phase fluids, more preferably gases at ambient temperatures.",
"The concentration of the tracer gas is preferably determined by measuring the thermal conductivity of the first fluid and tracer fluid mixture.",
"According to a second aspect of the present invention, an apparatus for measuring the flow rate of a first substantially single phase fluid through a conduit comprises: a device for discharging a known or determinable molar quantity of tracer fluid into a first fluid flowing in a conduit; means for measuring the concentration of the tracer fluid at a point downstream from where the discharging device is arranged to discharge the tracer fluid, and control means for determining the flow rate of a first fluid through a conduit dependent upon the concentration of the tracer fluid measured by the measuring means.",
"The means for measuring the concentration of the tracer fluid preferably comprises means for measuring the thermal conductivity of the mixture of first fluid and tracer fluid and means for converting the thermal conductivity measurement into a corresponding value indicative of the concentration of tracer fluid in the mixture.",
"The invention is described further by way of example with reference to the accompanying drawings, in which FIG.",
"1 diagrammatically shows an arrangement for measuring the flow rate of a fluid through a conduit; FIG.",
"2 shows the arrangement of FIG.",
"1 in more detail; FIG.",
"3 shows a preferred form of fluid concentration detector and FIG.",
"4 shows a measured tracer concentration profile.",
"As shown in FIG.",
"1, a conduit for containing a fluid flowing therethrough with a flow rate Q is provided with a device 2 to discharge or inject a known or determinable molar quantity of tracer fluid into the conduit 1 and a detector 3, downstream of the injector 2, to measure the concentration of the tracer fluid as it passes.",
"A control means 4 connected to the detector 3 is arranged to determine the flow rate Q of the fluid flowing through the conduit 1 based upon the molar quantity of tracer fluid injected and the concentration of the tracer fluid measured by the detector 3.FIG.",
"2 shows the flow rate measuring apparatus of FIG.",
"1 in more detail.",
"In this example the conduit 1 is a pipe arranged to convey fuel gas.",
"However, the invention is applicable to the measurement of the flow rate of any fluid such as air or other gases.",
"The injector 2 comprises a charge vessel 21 arranged to be charged with tracer fluid of a known concentration, in this case 100% helium, from a suitable source 22 such as a helium cylinder via a valve 23.Valve 23 is controlled by control means 4, which may be a portable computer or a processing means for example, via control line 41 to supply the charge vessel 21 with helium when required.",
"The charge vessel 21 is provided with a pressure sensor 24 and a temperature sensor 25 to measure the pressure and temperature respectively of the tracer gas within the charge vessel 21.The control means 4 measures the pressure and temperature from sensors 24, 25 via lines 42 and 43 respectively.",
"Using these sensors and knowing the volume of the charge vessel 21, the control means 4 is able to determine the molar quantity of helium in the charge vessel 21.The control means 4 can fill the charge vessel 21 with a desired quantity of helium by monitoring the pressure and temperature sensors 24, 25 and controlling valve 23.When it is desired to make a measurement of the flow rate Q of the fuel gas flowing through conduit 1, the control means 4 opens valve 26 via control line 44 for a quantity of tracer gas to pass into the conduit 1 to mix with the gas the flow rate of which is to be measured.",
"The tracer gas is injected into conduit 1 through an injection unit to ensure good mixing with the fuel gas to obtain precise measurements with the detector 3.The molar quantity of tracer injected can then be determined by relating the initial and final pressures and temperatures, and the previously determined volume of the charge vessel 21.The detector 3 may be any device capable of measuring the concentration of the passing tracer fluid.",
"In this case the detector 3 measures the change in thermal conductivity of the passing mixture of fuel gas and tracer gas compared with the thermal conductivity of the fuel gas alone.",
"Since thermal conductivity sensors such as those produced by Hartman & Braun of Germany are compact, reliable and inexpensive, their use in the present invention to determine the concentration of tracer gas produces a correspondingly compact, reliable and inexpensive flow rate measurement device.",
"The thermal conductivity measured by sensor 3 is passed to control means 4 via line 45.Control means 4 determines a value corresponding to the concentration of tracer gas from the measured thermal conductivity.",
"In this example, the control means 4 converts the measured thermal conductivity into a value corresponding to the concentration of tracer gas that would produce that change in the thermal conductivity measurement, using a predetermined concentration stored in correspondence to each of various possible measured changes in thermal conductivity.",
"A table of measured changes in thermal conductivity with corresponding values of tracer gas concentration is prepared in advance by making measurements of the thermal conductivities of mixtures of a first fluid in this case natural gas, with various quantities of tracer fluid, in this case Helium.",
"For even greater accuracy measurements of temperature and pressure may also be made at the point where the thermal conductivity is measured and look-up tables produced for each combination of temperature and pressure.",
"Use of such a so-called look-up table considerably reduces the processing power required which reduces the cost and size of the device and increases its speed.",
"Look-up tables of any suitable size may be used depending upon the precision required for the device.",
"In another example the control means 4 is arranged to determine a value corresponding to the concentration of tracer gas from the thermal conductivity measurements of the detector 3 using suitable functions or algorithms.",
"For even greater accuracy measurements may be made of the temperature and pressure at which the thermal conductivity is measured and these measurements taken into consideration by the suitable functions or algorithms which determine a value corresponding to the concentration of tracer gas at that temperature and pressure.",
"In this example, the conduit 1 is arranged to convey natural gas and the tracer gas is 100% helium.",
"A measurement is also made of the thermal conductivity of the first fluid with no added tracer to provide an indication of the thermal conductivity of the natural gas which can vary with composition.",
"The background thermal conductivity is then used as a baseline from which a change in thermal conductivity can be determined.",
"The arrangement of the detector 3 used in this example is shown more clearly in FIG.",
"3.Gas from conduit 1 is sampled by a tube 31 arranged diametrically across the conduit 1 with a number of holes 32 arranged along the length of the tube 31 to receive gas flowing through the conduit 1.The sampled gas is conveyed up the tube 31 to a sampling chamber 33.The sampling chamber 33 presents the sampled gas to a thermal conductivity sensor 34 as is well known in the art which conveys an electrical signal indicative of the measured thermal conductivity to control means 4 via line 45.The sampled gas is returned to conduit 1 via return tube 35 which in this example surrounds tube 31.The continual flow of sampled fluid through the detector arrangement 3 enables a continual sampling of the concentration of the passing tracer gas to be made.",
"Alternatively the thermal conductivity sensor 34 could be positioned in the conduit 1 directly in the gas flow such that the tubing arrangement is not required.",
"After the injector 2 has injected the tracer gas into the conduit 1, the control means 4 monitors the measurements of detector 3, which are indicative of the concentration of the passing tracer gas, for sufficient time to ensure that substantially all of the tracer gas has passed the sampling point.",
"This results in a tracer concentration profile of the form shown in FIG.",
"4 with tracer concentration C plotted against time t. The tracer gas passes the detector between times t1 and t2 and a background level of tracer gas is indicated by Co.",
"The control means 4 determines the actual volumeric flow rate Q of fluid through the conduit 1 dependent upon the measured concentration of tracer fluid C using the following function: Q = VC 1 ∫ t 1 t 2 ( C - Co ) ⅆ t Where V is the volume of the injected tracer fluid corrected to line conditions at the measuring point C1 is the concentration of the injected tracer fluid and Co is the measured background level of tracer fluid (note: C is often taken as the increased concentration of tracer and Co is taken as zero) In practice, to integrate the measured concentration of tracer gas, measurements of concentration are sampled at regular intervals, in this case every millisecond, and the sampled measurements summed.",
"Any suitable number and frequency of samples of the measured concentration may be taken depending upon the precision required.",
"Flow rate tests using the above apparatus with a measured concentration sampling period of every millisecond have produced flow rate results accurate to within 1%.",
"The volumetric flow rate Q determined by control means 4 may be displayed on a display means associated with the control means 4 or communicated to a suitable remote device.",
"The determined volumetric flow rates Q may be stored, preferably electronically, for subsequent analysis.",
"The flow meter described above is particularly suitable for use with subterranean local gas transmission pipes which deliver gas to consumers as the pipe geometry does not need to be known and a straight length of pipe is not required, and also to the in-situ testing of meters where components such as regulators make determining internal volumes difficult and where pipe lengths are short.",
"Many modifications may be made to the example described above without departing from the scope of the invention.",
"For example, the invention may be used to determine the flow rate of any fluid such as natural gas or air.",
"Furthermore any tracer fluid may be used provided its concentration may be measured and any technique for measuring the concentration of the tracer fluid may be used."
]
] | Patent_10257953 |
[
[
"Closing system for plastic shaped parts",
"The present invention relates to a seal system for plastic moldings and to the use of the seal system in the engine fluid sector."
],
[
"1.A seal system for plastic moldings, comprising.",
"a plastic molding, wherein said plastic molding has an opening; a plastic screw; and an internal thread which is molded into the plastic molding and receives the plastic screw, said thread being made of plastic.",
"2.The seal system according to claim 1, wherein the thread is integrated in the plastic material of the plastic molding.",
"3.The seal system according to claim 1, wherein the plastic screw, plastic molding and thread are made of the same material.",
"4.The seal system according to claim 3, wherein the plastic screw, plastic molding and thread are made of polyamide 6.6 GF 35%.",
"5.The seal system according to claim 1, wherein a seal element is arranged between the screw made of plastic and an opening in the plastic molding.",
"6.The seal system according to claim 1, wherein the plastic screw has a tightening contour.",
"7.The seal system according to claim 6, wherein the tightening contour is selected from the group consisting of a hexagon socket, hexagon projection and groove contour.",
"8.The seal system according to claim 1, wherein a rib partly or wholly surrounding the plastic molding opening is provided on the plastic molding.",
"9.The seal system according to claim 1, wherein a catch is applied to the plastic screw and a catch is applied to the plastic molding, thus forming a positive engagement between the plastic screw and the plastic molding.",
"10.The seal system according to claim 9, wherein the catch is applied to a rib that partly or wholly surrounds an opening in the plastic molding.",
"11.The seal system according to claim 9, wherein the catch is applied to the head of the plastic screw.",
"12.The seal system according to claim 8, wherein the tightening torque of the plastic screw is up to 8 Nm due to the catches.",
"13.The seal system according to claim 9, wherein the releasing torque of the plastic screw is up to 8 Nm due to the catches.",
"14.The seal system according to claim 9, wherein the releasing torque of the plastic screw is up to 5 Nm due to the catches.",
"15.Use of the seal system according to claim 1 in the engine fluid sector.",
"16.A seal system for plastic moldings, comprising plastic molding means, wherein said plastic molding means has an opening; plastic screw means; and internal thread means which is molded into the plastic molding means and receives the plastic screw means, said thread means being made of plastic.",
"17.The seal system according to claim 16, wherein the plastic screw means has a tightening contour.",
"18.The seal system according to claim 16, wherein a rib partly or wholly surrounding the opening of the plastic molding means, is provided on the plastic molding means.",
"19.The seal system according to claim 16, wherein a catch is applied to the plastic screw means and a catch is applied to the plastic molding means, thus forming a positive engagement between the plastic screw and the plastic molding."
],
[
"The present invention relates to a seal system for plastic moldings and to the use of the seal system in the engine and transmission fluid sector.",
"On systems in the engine or transmission compartment that come into direct contact with oil, seal systems for filling or draining the oil are required now and then.",
"For the design of such seal systems a construction is usually chosen based on a combination of metal and plastic.",
"This involves the use of profile seals or O-ring seals that ensure sealing between themselves or with the component being sealed.",
"Even on housing components made of plastic, the filling and draining screws and their threads of the prior art are made of metal.",
"Oil pans with seal systems of the prior art, for example, are produced by coating threaded bushings made of metal, usually of steel or brass, in the thermoplastic injection mold or subsequently pressing them with heat or with the aid of ultrasound into a location pre-molded in the plastic housing.",
"Another process involves pressing threaded bushings with a collar into the plastic container.",
"This means that the embedded metal bushing has to satisfy exacting requirements in terms of retaining strength and positional accuracy.",
"When threaded bushes are coated and the tolerances on the bushing or in the injection mold or the injection molding parameters are changed, there is a risk of the molten plastic penetrating at the ends of the bushing or even into the thread turns.",
"On plastic moldings whose bushings are pressed in with heat or with the aid of ultra-sound there is the problem that molten plastic can also escape at the places mentioned above.",
"Another problem concerns the recycling of such plastic moldings with embedded metal parts, since separating these different materials is a very complicated process.",
"The technical object was therefore to provide seal systems for sealing housing components made of plastic that avoid the above-mentioned problems and permit a reduction in cost, weight and process risks.",
"The technical object is achieved by a seal system for plastic moldings comprising a plastic screw 2 and an internal thread 4 which is molded into the plastic molding and receives the plastic screw 2, said thread being made of plastic.",
"The present invention permits the direct integration of a seal system in the housing component being sealed and ensures the functions required for such components throughout their service life.",
"The elimination of additional metal parts such as threaded bushings to transmit the tightening forces in the area of the thread advantageously yields a marked reduction in the number of individual components, production time, production costs and weight.",
"As a result of direct integration in the contours of the housing component, the design of the seal system in plastic also permits the choice and use of the optimum position, shape and orientation.",
"The inventive seal system also satisfies the demands of the automotive industry for a recycling-friendly design that eliminates the cost-intensive and complex separation of plastic and metal parts.",
"In a preferred embodiment, the thread 4 is integrated in the plastic material of the plastic molding 1.In another preferred embodiment, the plastic screw 2, plastic molding 1 and thread 4 are made of the same material.",
"In a particularly preferred fashion, the plastic screw 2, plastic molding 1 and thread 4 are made of polyamide 6.6 GF 35%.",
"In another especially preferred embodiment, a seal element 3 is arranged between the screw made of plastic 2 and the opening 9 in the plastic molding.",
"In another embodiment the plastic screw 2 is given a tightening contour 5.The tightening contour 5 is preferably a hexagon socket, hexagon projection or groove contour.",
"In another especially preferred embodiment, a rib 6 partly or wholly surrounding the opening 9 is provided on the plastic molding.",
"A catch 7 is preferably applied to the plastic screw 2 and a catch 8 to the plastic molding, thus achieving a positive engagement between the plastic screw 2 and the plastic molding.",
"The catch 8 is especially preferably applied to the rib 6.In an especially preferred embodiment, the catch 7 is applied to the head of the plastic screw 2.The positive engagement is achieved through the interaction of the catches 7, 8 molded onto the screw head and the housing respectively.",
"This prevents the plastic screw from becoming released or loosening or falling out by itself due to vibrations to or a temperature change during its service life.",
"By adapting the two catches 7, 8 to each other, the screw can only be turned with the application of extra force.",
"In an especially preferred embodiment, the tightening torque of the plastic screw 2 is up to 8 Nm due to the catches 7, 8.The releasing torque of the plastic screw 2 is preferably is up to 8 Nm due to the catches 7, 8.In an especially preferred embodiment, the releasing torque of the plastic screw 2 is up to 5 Nm due to the catches 7, 8.Due to the settling and creep properties of plastic during its service life and because of temperature, a loss of tightening torque of up to about 3 Nm usually occurs.",
"The remaining tightening torque of up to 5 Nm is sufficient to ensure the plastic screw's tight fit with the retention of its function.",
"The overmeasure of the catch ensures that the release or loosening of the screw and the associated loss of liquid or leakage are prevented.",
"The inventive seal system is used in the engine fluid sector.",
"Oil pans of the transmission and of the engine and the tank for cooling fluid or brake fluid can thus be equipped with the seal system.",
"The purpose of the plastic screw is to permit the filling and draining of liquids, preferably of oil or cooling water.",
"A seal element held between the plastic screw and the housing, preferably an 0-ring seal, performs the sealing task.",
"The sealing function is designed and ensured independently of the releasing torque in the thread and in the catch.",
"The filling and draining screw keeps the seal element in position independently of the releasing or tightening torques existing in the thread or in the catch.",
"The inventive device is explained in detail with reference to the following figures: FIG.",
"1 shows a cross section of the inventive seal system.",
"FIG.",
"2 shows a top view of the plastic screw.",
"FIG.",
"3 shows a detail of the edge area of the plastic screw.",
"FIG.",
"1 shows a cross section of the inventive seal system.",
"The plastic screw 2 is fully screwed into the thread 4 of the plastic molding 1 and thus fully seals the opening 9 of the plastic molding 1.In this embodiment a rib 6 surrounds the opening 9 on the plastic molding 1.The plastic screw 2 has a screw head whose dimensions are adapted to the rib 6.Catches 7 and 8 are applied to the rib 6 and to the screw head.",
"A seal element 3 is arranged between the screw and the plastic is molding and seals the opening.",
"FIG.",
"2 shows a top view of the plastic screw.",
"In this figure the plastic screw 2 is given a tightening contour 5, which in this case is designed as a hexagon socket.",
"FIG.",
"3 shows a detail of the edge area of the plastic screw.",
"The rib 6 with the catch 8 can be seen as well as the edge of the plastic screw and its catch 7.Key to the drawings: 1 Plastic molding 2 Plastic screw 3 Seal element 4 Thread 5 Tightening contour 6 Rib 7 Catch on the plastic screw 8 Catch on the rib 9 Opening"
]
] | Patent_10258177 |
[
[
"Substituted Benzoic Acid Amides and Use thereof for the Inhibition of Angiogenesis",
"Substituted benzoic acid amides of formula (I) and their use as pharmaceutical agents for treating diseases that are triggered by persistent angiogenesis as well as their intermediate products for the production of benzoic acid amides are described."
],
[
"1.Compounds of general formula I in which A stands for the group ═NR7, W stands for oxygen, sulfur, two hydrogen atoms or the group ═NR8, Z stands for a bond, the group ═NR10 or ═N—, for branched or unbranched C1-12-alkyl or for the group m, n and o stand for 0-3, Ra, Rb, Rc, Rd, Re, Rf, independently of one another, stand for hydrogen, fluorine, C1-4-alkyl or the group ═N10, and/or Ra and/or Rb can form a bond with Rc and/or Rd or Rc can form a bond with Re and/or Rf, or up to two of radicals Ra-Rf can close a bridge with up to 3 C atoms each to form R1 or to form R7, R1 stands for branched or unbranched C1-6-alkyl, C2-12-alkenyl or C3-12-alkinyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for C3-10-cycloalkyl or C3-10-cycloalkenyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for aryl or hetaryl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy, or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means monocyclic aryl, bicyclic aryl or heteroaryl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy, or hydroxy, R4, R5 and R6 stand for hydrogen, halogen, or C1-6-alkoxy, C1-6-alkyl or C1-6-carboxyalkyl that is unsubstituted or that is optionally substituted in one or more places with halogen, or R4 and R5 together form the group R7 stands for hydrogen or C1-6-alkyl or forms a bridge with up to 3 ring members with Ra-Rf from Z or to form R1, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, and R1 must not be thiazole, as well as isomers and salts thereof.",
"2.Compounds of general formula I, according to claim 1, in which A stands for the group ═NR7, W stands for oxygen, sulfur or two hydrogen atoms, Z stands for a bond, the group ═NR10 or for branched or unbranched C1-12-alkyl, R1 stands for branched or unbranched C1-6-allyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for C3-10-cycloalkyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for phenyl, pyridyl, naphthyl, quinolyl, isoquinolyl, indanyl, tetralinyl, indolyl, thienyl, indazolyl or benzothiazolyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means phenyl, pyrimidinyl or pyridyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkoxy or hydroxy, R4, R5, R6 and R7 stand for hydrogen, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, as well as isomers and salts thereof.",
"3.Compounds of general formula I, according to claims 1 and 2, in which A stands for the group ═NR7, W stands for oxygen, or for one or two hydrogen atoms, Z stands for a bond, the group ═NR10 or for branched or unbranched C1-12-alkyl, R1 stands for branched or unbranched C1-6-alkyl; or for C3-10-cycloalkyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for phenyl, pyridyl, naphthyl, quinolyl, indenyl, tetralinyl, indolyl, thienyl, indazolyl, or benzothiazolyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkenyl, R11 stands for phenyl, pyrimidinyl or pyridyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkoxy or hydroxy, R4, R5, R6 and R7 stand for hydrogen, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, as well as isomers and salts thereof.",
"4.Use of the compounds of general formula I, according to claims 1 to 3, for the production of a pharmaceutical agent for treating tumors, psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, and injuries to nerve tissue.",
"5.Pharmaceutical agents that contain at least one compound according to claims 1 to 3.6.Pharmaceutical agents according to claim 5, for treating tumors, psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, and injuries to nerve tissue.",
"7.Compounds, according to claims 1 to 3, with suitable formulations and vehicles.",
"8.Use of the compounds of formula I according to claims 1 to 3 as inhibitors of the tyrosine kinase KDR and FLT.",
"9.Use of the compounds of general formula I, according to claims 1 to 3, in the form of a pharmaceutical preparation for enteral, parenteral and oral administration.",
"10.Intermediate compounds of general formula II in which R2 and R3 mean hydrogen or the group X11, X means C1-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means phenyl or pyridyl that is optionally substituted by C1-6-alkoxy, whereby R2 and R3 stand for hydrogen, although not simultaneously, as well as isomers and salts thereof, as intermediate products for the production of the compounds of general formula I.",
"11.Compounds of general formula II, according to claim 10, for the production of a pharmaceutical agent for treating tumors, psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, and injuries to nerve tissue."
],
[
"The invention relates to substituted benzoic acid amides and their use as pharmaceutical agents for treating diseases that are triggered by persistent angiogenesis as well as their intermediate products for the production of benzoic acid amides.",
"Persistent angiogenesis can be the cause of various diseases, such as psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases and arteriosclerosis or can result in an aggravation of these diseases.",
"Direct or indirect inhibition of the VEGF receptor (VEGF=vascular endothelial growth factor) can be used for treating such diseases and other VEGF-induced pathological angiogenesis and vascular permeable conditions, such as tumor vascularization.",
"For example, it is known that the growth of tumors can be inhibited by soluble receptors and antibodies against VEGF.",
"Persistent angiogenesis is induced by the factor VEGF via its receptor.",
"So that VEGF can exert this action, it is necessary that VEGF bind to the receptor, and a tyrosine phosphorylation is induced.",
"Only derivatives of the compounds claimed here that have been removed were described as calpain inhibitors (WO 9823581, WO 9825883), phospholipase A2 inhibitors (WO 9700583), prostaglandin D2 antagonists (WO 9700853), neurokinin A antagonists (WO 95 16682), tranquilizers (U.S. Pat.",
"No.",
"3,892,752) or anorexigenics (FR 1600541).",
"An action of these known compounds in connection with VEGF was not previously described.",
"It has now been found that compounds of general formula I in which A stands for the group ═NR7, W stands for oxygen, sulfur, two hydrogen atoms or the group ═NR8, Z stands for a bond, the group ═NR10 or ═N—, for branched or unbranched C1-12-alkyl or for the group m, n and o stand for 0-3, Ra, Rb, Rc, Rd, Re, Rf, independently of one another, stand for hydrogen, fluorine, C1-4-alkyl or the group ═NR10, and/or Ra and/or Rb can form a bond with Rc, and/or Rd or Rc can form a bond with Re and/or Rf, or up to two of radicals Ra-Rf can close a bridge with up to 3 C atoms each to form R1 or to form R7, R1 stands for branched or unbranched C1-6-alkyl, C2-12-alkenyl or C3-12-alkinyl that is optionally substituted in one or more places with halogen or C1-6-alkyl or for C3-10-cycloalkyl or C3-10-cycloalkenyl that is optionally substituted in one or more places with halogen or C1-6-alkyl, or for aryl or heteroaryl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy, or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means monocyclic aryl, bicyclic aryl or heteroaryl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy, or hydroxy, R4, R5 and R6 stand for hydrogen, halogen, or C1-6-alkoxy, C1-6-alkyl or C1-6-carboxyalkyl that is unsubstituted or that is optionally substituted in one or more places with halogen, or R4 and R5 together form the group R7 stands for hydrogen or C1-6-alkyl or forms a bridge with up to 3 ring members with Ra-Rf from Z or to form R1, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, and R1 must not be thiazole, as well as isomers and salts thereof, stop tyrosine phosphorylation or the persistent angiogensis and thus prevent the growth and propagation of tumors.",
"If R7 forms a bridge to R1, heterocyclic compounds result to which R1 is fused.",
"For example, there can be mentioned: If Ra, Rb, Rc, Rd, Re, Rf, independently of one another, represent hydrogen or C1-4 alkyl, Z thus forms an alkyl chain.",
"If Ra, and/or Rb form a bond with Rc and/or Rd or Rc and/or Rd form a bond with Re and/or Rf, Z stands for an alkenyl or alkinyl chain.",
"If Ra-Rf forms a bridge with itself, Z represents a cycloalkyl or cycloalkenyl group.",
"If up to two of radicals Ra-Rf form a bridge with up to 3 C atoms to form R1, Z together with R1 is a benzocondensed or hetaryl-condensed (Ar) cycloalkyl.",
"For example, there can be mentioned: If one of radicals Ra-Rf closes a bridge to form R7, a nitrogen heterocyclic compound is formed that can be separated from R1 by a group.",
"For example, there can be mentioned: Alkyl is defined in each case as a straight-chain or branched alkyl radical, such as, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, isopentyl, or hexyl, whereby C1-4-alkyl radicals are preferred.",
"Cycloalkyl is defined in each case as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl.",
"Cycloalkenyl is defined in each case as cyclobutenyl, cylopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl or cyclodecenyl, whereby the linkage can be carried out both to the double bond and to the single bonds.",
"Halogen is defined in each case as fluorine, chlorine, bromine or iodine.",
"The alkenyl and alkinyl substituents are in each case straight-chain or branched and contain 2-6, preferably 2-4 C atoms.",
"For example, the following radicals can be mentioned: vinyl, propen-1-yl, propen-2-yl, but-1-en-1-yl, but-1-en-2-yl, but-2-en-1-yl, but-2-en-2-yl, 2-methyl-prop-2-en-1-yl, 2-methyl-prop-1 but-1-en-3-yl, ethinyl, prop-1-in-1-yl, but-1-in-1-yl, but-2-in-1-yl, but-3-en-1-yl, and allyl.",
"The aryl radical in each case has 6-12 carbon atoms, such as, for example, naphthyl, biphenyl and especially phenyl.",
"The heteroaryl radical in each case can be benzocondensed.",
"For example, as 5-ring heteroaromatic compounds, there can be mentioned; thiophene, furan, oxazole, thiazole, imidazole, and benzo derivatives thereof, and as 6-ring heteroaromatic compounds, there can be mentioned: pyridine, pyrimidine, triazine, quinoline, isoquinoline and benzo derivatives.",
"The aryl radical and the heteroaryl radical in each case can be substituted in the same way or differently in 1, 2 or 3 places with halogen, C1-4-alkoxy, nitro, trifluoromethyl, trifluoromethoxy, cyano, SOqR5 or C1-4-alkyl, whereby q stands for 0-2.If an acid group is included, the physiologically compatible salts of organic and inorganic bases are suitable as salts, such as, for example, the readily soluble alkali salts and alkaline-earth salts as well as N-methyl-glucamine, dimethyl-glucamine, ethyl-glucamine, lysine, 1,6-hexadiamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxy-methyl-amino-methane, aminopropanediol, Sovak base, and 1-amino-2,3,4-butanetriol.",
"If a basic group is included, the physiologically compatible salts of organic and inorganic acids are suitable, such as hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, fumaric acid, i.a.",
"Those compounds of general formula I in which A stands for the group ═NR7, W stands for oxygen, sulfur or two hydrogen atoms, Z stands for a bond, the group ═NR10 or for branched or unbranched C1-12-alkyl, R1 stands for branched or unbranched C1-6-alkyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for C3-10-cycloalkyl that is optionally substituted in one or more places with halogen or C1-6-alkyl, or for phenyl, pyridyl, naphthyl, quinolyl, isoquinolyl, indanyl, tetralinyl, indolyl, thienyl, indazolyl or benzothiazolyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means phenyl, pyrimidinyl or pyridyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkoxy or hydroxy, R4, R5, R6 and R7 stand for hydrogen, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, as well as isomers and salts thereof, have proven especially effective.",
"Those compounds of general formula I in which A stands for the group ═NR7, W stands for oxygen, or for one or two hydrogen atoms, Z stands for a bond, the group ═NR10 or for branched or unbranched C1-12-alkyl, R1 stands for branched or unbranched C1-6-alkyl, or for C3-10-cycloalkyl that is optionally substituted in one or more places with halogen or C1-6-alkyl; or for phenyl, pyridyl, naphthyl, quinolyl, isoquinolyl, indenyl, tetralinyl, indolyl, indazolyl, benzothiazolyl or thienyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkyl, C1-6-alkoxy or C1-6-alkyl or C1-6-alkoxy that is substituted in one or more places with halogen, R2 and R3 stand for hydrogen, an OH group or the group XR11, X stands for C2-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 stands for phenyl, pyrimidinyl or pyridyl that is unsubstituted or that is optionally substituted in one or more places with halogen, C1-6-alkoxy or hydroxy, R4, R5, R6 and R7 stand for hydrogen, R8 and R10 stand for hydrogen or C1-6-alkyl, whereby R2 and R3 stand for hydrogen, although not simultaneously, and if R2 stands for an OH group, R3 does not stand for hydrogen, and if R3 stands for an OH group, R2 does not stand for hydrogen, as well as isomers and salts thereof, have proven quite especially effective.",
"The compounds according to the invention prevent a phosphorylation, i.e., certain tyrosine kinases can be selectively inhibited, whereby the persistent angiogenesis can be stopped.",
"Thus, for example, the growth and the propagation of tumors is prevented.",
"The compounds of general formula I according to the invention also contain the possible tautomeric forms and comprise the E- or Z-isomers or, if a chiral center is present, also the racemates and enantiomers.",
"The compounds of formula I as well as their physiologically compatible salts can be used as pharmaceutical agents based on their inhibitory activity relative to the phosphorylation of the VEGF receptor.",
"Based on their profile of action, the compounds according to the invention are suitable for treating diseases that are caused by persistent angiogenesis.",
"Since the compounds of formula I are identified as inhibitors of the tyrosine kinase KDR and FLT, they are suitable in particular for treating those diseases that are caused by persistent angiogenesis that is triggered via the VEGF receptor or by an increase in vascular permeability.",
"The subject of this invention is also the use of the compounds according to the invention as inhibitors of the tyrosine kinase KDR and FLT.",
"Subjects of this invention are thus also pharmaceutical agents for treating tumors or use thereof.",
"The compounds according to the invention can be used either alone or in a formulation as pharmaceutical agents for treating psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis and injuries to nerve tissue.",
"In treating injuries to nerve tissue, quick scar formation on the injury sites can be prevented with the compounds according to the invention, i.e., scar formation is prevented from occurring before the axons reconnect.",
"A reconstruction of the nerve compounds was thus facilitated.",
"The formation of ascites in patients can also be suppressed with the compounds according to the invention.",
"VEGF-induced edemas can also be suppressed.",
"Such pharmaceutical agents, their formulations and uses, are also subjects of this invention.",
"The invention thus also relates to the use of compounds of general formula I for the production of a pharmaceutical agent for treating tumors, psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, and injuries to nerve tissue.",
"To use the compounds of formula I as pharmaceutical agents, the latter are brought into the form of a pharmaceutical preparation, which in addition to the active ingredient for enteral or parenteral administration contains suitable pharmaceutical, organic or inorganic inert carrier materials, such as, for example, water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, etc.",
"The pharmaceutical preparations can be present in solid form, for example as tablets, coated tablets, suppositories, capsules or in liquid form, for example as solutions, suspensions or emulsions.",
"They optionally contain, moreover, adjuvants such as preservatives, stabilizers, wetting agents or emulsifiers, salts for changing osmotic pressure or buffers.",
"For parenteral administration, especially injection solutions or suspensions, especially aqueous solutions of the active compounds in polyhydroxyethoxylated castor oil, are suitable.",
"As carrier systems, surface-active adjuvants such as salts of bile acids or animal or plant phospholipids, but also mixtures thereof as well as liposomes or components thereof can also be used.",
"For oral administration, especially tablets, coated tablets or capsules with talc and/or hydrocarbon vehicles or binders, such as for example, lactose, corn starch or potato starch, are suitable.",
"The administration can also be carried out in liquid form, such as, for example, as juice, to which optionally a sweetener is added.",
"The dosage of the active ingredients can vary depending on the method of administration, age and weight of the patient, type and severity of the disease to be treated and similar factors.",
"The daily dose is 0.5-1000 mg, preferably 50-200 mg, whereby the dose can be given as a single dose to be administered once or divided into 2 or more daily doses.",
"The above-described formulations and forms for dispensing are also subjects of this invention.",
"The production of the compounds according to the invention is carried out according to methods that are known in the art.",
"For example, compounds of formula I are obtained, in that a) in a compound of general formula II in which R4 to R6 have the above-mentioned meaning and A is halogen or OR11, whereby R11 means hydrogen, C1-4-alkyl or C1-4-acyl, and R2′ and R3′ mean hydrogen, aldehyde, halogen or OH, O-triflate, O-tosylate or O-mesylate, first R2′ or R3′ is converted into an alkenyl or alkinyl, optionally saturated in the corresponding alkane, and then COA is converted into an amide, or b) a compound of general formula III in which R4 to R6 have the above-mentioned meaning and T means a protective group, is acylated and then optionally the keto group is reduced to alcohol or alkane, the protective group is cleaved off, the amine is converted into a nitrile, and the nitrile is saponified and converted into an amide.",
"The sequence of steps can be interchanged in all cases.",
"The amide formation is carried out according to methods that are known in the literature.",
"For amide formation, it is possible to start from a corresponding ester.",
"The ester is reacted according to J. Org.",
"Chem.",
"1995, 8414 with aluminum trimethyl and the corresponding amine in solvents such as toluene at temperatures of 0° C. up to the boiling point of the solvent.",
"If the molecule contains two ester groups, both are converted into the same amide.",
"When nitriles are used instead of ester, amidines are obtained under analogous conditions.",
"For amide formation, however, all processes that are known from peptide chemistry are also available.",
"For example, the corresponding acid can be reacted with the amine in aprotic polar solvents, such as, for example, dimethylformamide, via an activated acid derivative that can be obtained with, for example, hydroxybenzotriazole and a carbodiimide, such as, for example, diisopropylcarbodiimide, or else with preformed reagents, such as, for example, HATU (Chem.",
"Comm.",
"1994, 201) or BTU, at temperatures of between 0° C. and the boiling point of the solvent, preferably at 80° C. For the amide formation, the process can also be used with the mixed acid anhydride, imidazolide or azide.",
"Salicylamides are obtained if the corresponding phenol is reacted in the presence of a Friedel-Crafts catalyst, such as boron trichloride, with isocyanates or isothiocyanates in solvents, such as, for example, toluene, at temperatures of 0° C. up to the boiling point of the solvent.",
"If various amide groups are to be introduced into the molecule, for example, the second ester group must be introduced into the molecule after the production of the first amide group and then amidated, or there is a molecule in which one group is present as an ester, the other is present as an acid, and the two groups are amidated in succession according to various methods.",
"Thioamides can be obtained from the anthranilamides by reaction with diphosphadithianes according to Bull Soc.",
"Chirp.",
"Belg.",
"87, 229, 1978 or by reaction with phosphorus pentasulfide in solvents such as pyridine or even quite without solvent at temperatures of 0° C. to 200° C. The products can also be subjected to an electrophilic aromatic substitution.",
"The substitution then takes place on compounds of formula III in the ortho- or para-position into the or one of the amino group(s, into compounds of formula II in the meta-position) to form the carbonyl group.",
"Thus, acylation can be done by Friedel-Crafts acylation with acid chlorides in the presence of Friedel-Crafts catalysts, such as, for example, aluminum trichloride in solvents such as nitromethane, carbon disulfide, methylene chloride or nitrobenzene at temperatures of between 0° C. and the boiling point of the solvent, preferably at room temperature.",
"According to processes that are known in the literature, one or more nitro groups can be introduced without solvent, for example by nitrating acid, nitric acid of various concentrations, or by metal nitrates, such as, for example, copper(II) nitrate or iron(III) nitrate in polar solvents such as ethanol or glacial acetic acid or else in acetic anhydride.",
"The introduction of halogens is carried out according to processes that are known in the literature, e.g., by reaction with bromine, N-bromo- or N-iodosuccinimide or utropin hydrotribromide in polar solvents such as tetrahydrofuran, acetonitrile, methylene chloride, glacial acetic acid or dimethylformamide.",
"The reduction of the nitro group is performed in polar solvents at room temperature or elevated temperature.",
"As catalysts for the reduction, metals such as Raney nickel or noble-metal catalysts such as palladium or platinum or else palladium hydroxide optionally on vehicles are suitable.",
"Instead of hydrogen, for example, ammonium formate, cyclohexene or hydrazine can also be used in a known way.",
"Reducing agents such as tin(II) chloride or titanium(III) chloride can also be used, such as complex metal hydrides, optionally in the presence of heavy metal salts.",
"Iron can also be used as a reducing agent.",
"The reaction is then performed in the presence of an acid, such as, e.g., acetic acid or ammonium chloride, optionally with the addition of a solvent, such as, for example, water, methanol, iron/ammonia, etc.",
"With an extended reaction time, an acylation of the amino group can occur in this variant.",
"If an alkylation of an amino group is desired, alkylation can be done according to commonly used methods—for example with alkyl halides—or according to the Mitsonubo variant by reaction with an alcohol in the presence of, for example, triphenylphosphine and azodicarboxylic acid ester.",
"The amine can also be subjected to reductive alkylation with aldehydes or ketones, whereby the reaction is performed in the presence of a reducing agent, such as, for example, sodium cyanoborohydride in a suitable inert solvent, such as, for example, ethanol, at temperatures of 0° C. up to the boiling point of the solvent.",
"If a start is made from a primary amino group, the reaction can be performed optionally in succession with two different carbonyl compounds, whereby mixed derivatives are obtained [Literature, e.g., Verardo et al.",
"Synthesis (1993), 121; Synthesis (1991), 447; Kawaguchi, Synthesis (1985), 701; Micovic et al.",
"Synthesis (1991), 1043].",
"It can be advantageous first to form the Schiff base by reaction of the aldehyde with the amine in solvents such as ethanol or methanol, optionally with the addition of adjuvants such as glacial acetic acid, and then to add only reducing agents, such as, e.g., sodium cyanoborohydride.",
"The hydrogenation of alkene or alkine groups in the molecule is carried out in the usual way by, for example, catalytically activated hydrogen.",
"As catalysts, heavy metals such as palladium or platinum, optionally on a vehicle, or Raney nickel can be used.",
"The procedure is performed at temperatures of 0° C. up to the boiling point of the solvent and at pressures of up to 20 bar, but preferably at room temperature and normal pressure.",
"By the use of catalysts, such as, for example, a Lindlar catalyst, triple bonds can be partially hydrogenated to double bonds, whereby preferably the Z-form is produced.",
"This hydrogenation is preferably performed in pyridine as a solvent with palladium on calcium carbonate as a catalyst.",
"In the same way, the Z-double bond can be produced from the triple bond by reduction with diimine, produced, for example, according to R. M. Moriatry et al.",
"Synth.",
"Comm.",
"17, 703, 1987.The acylation of an amino group is carried out in the usual way, with, for example, acid halide or acid anhydride optionally in the presence of a base such as dimethylaminopyridine in solvents such as methylene chloride, tetrahydrofuran or pyridine, according to the Schotten-Baumann variant in aqueous solution at weakly alkaline pH or by reaction with an anhydride in glacial acetic acid.",
"A reduction of a keto group is carried out according to processes that are known in the art.",
"Thus, by complex metal hydrides, such as, for example, sodium borohydride in solvents such as methanol or isopropanol, the keto group, in addition to the amide group or ester group, can be reduced selectively to alcohol.",
"A reduction of a keto group to the methylene group can be carried out according to Clemmensen with zinc in hydrochloric acid or else, for example, with silanes in trifluoroacetic acid.",
"The introduction of the halogens chlorine, bromine, iodine or the azido group via an amino group can be carried out, for example, also according to Sandmeyer by the diazonium salts that are intermediately formed with nitrites being reacted with copper(I) chloride or copper(I) bromide in the presence of the corresponding acid, such as hydrochloric acid or hydrobromic acid or with potassium iodide.",
"If an organic nitrite is used, the halogens can be introduced into a solvent, such as, for example, dimethylformamide, e.g., by adding methylene iodide or tetrabromomethane.",
"The removal of the amino group can be achieved either by reaction with an organic nitrite in tetrahydrofuran or by diazotization and reductive boiling-down of the diazonium salt with, for example, phosphorous acid, optionally with the addition of copper(I) oxide.",
"The introduction of fluorine can be accomplished, for example, by Balz-Schiemann reaction of the diazonium tetrafluoroborate or according to J. Fluor.",
"Chem.",
"76, 1996, 59-62 by diazotization in the presence of HFxpyridine and subsequent boiling-down optionally in the presence of a fluoride ion source, such as, e.g., tetrabutylammonium fluoride.",
"The introduction of the azido group is accomplished after diazotization by reaction with sodium azide at room temperature.",
"Ether cleavages are performed according to processes that are common in literature.",
"In this case, a selective cleavage can also be achieved in several groups that are present in the molecule.",
"In this case, the ether is treated, for example, with boron tribromide in solvents such as dichloromethane at temperatures of between −100° C. up to the boiling point of the solvent, preferably at −78° C. It is also possible, however, to cleave the ether by sodium thiomethylate in solvents such as dimethylformamide.",
"The temperature can be between room temperature and the boiling point of the solvent, preferably at 150° C. The introduction of the alkenyl group is carried out with the corresponding vinyl compounds under the conditions of the Heck reaction.",
"For the introduction of the ethinyl groups, the Songashira reaction is used.",
"As a leaving group R2′, halogens such as fluorine, chlorine, bromine, iodine or O-mesylate, O-tosylate, O-triflate or O-nonaflate are suitable.",
"The nucleophilic substitution for introducing ethinyl or ethenyl radicals is performed under the catalysis of transition metal complexes such as Pd(O), e.g., palladium tetrakis triphenylphosphine or Pd(2+), such as palladium-bis-tri-o-tolylphosphine-dichloride, nickel (II) or nickel (O) according to methods that are known in the literature, optionally in the presence of a base and optionally under co-catalysis of a salt, such as, for example, copper(I) iodide or lithium chloride.",
"As nucleophiles, for example, vinyl or ethinyl compounds, tin-organic compounds or zinc-organic compounds are suitable.",
"The reaction can be performed in polar solvents, such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, acetonitrile, in hydrocarbons such as toluene or in ethers such as tetrahydrofuran, dimethoxyethane or diethyl ether.",
"As bases, inorganic bases such as alkali or alkaline-earth hydroxides or bicarbonates, carbonates, phosphates or organic bases such as cyclic, alicyclic and aromatic amines, such as pyridine, triethylamine, DBU and Hünig base, are suitable, whereby in many cases, bases such as diethylamine or piperidine can also simultaneously be solvents.",
"The application of pressure can be beneficial to the reaction.",
"If a trimethylsilylethinyl group is introduced, the trimethylsilyl group can by reaction with fluorides, such as, for example, potassium fluoride or tetrabutylammonium fluoride in solvents such as tetrahydrofuran, methylene chloride, or acetonitrile at temperatures of 0° C. up to the boiling point of the solvent.",
"An alkenyl group can also be introduced, however, by olefination reactions, such as, e.g., the Peterson olefination, the Wittig reaction or the Wittig-Homer reaction.",
"To this end, the aldehyde is reacted with the anion that was already produced, e.g., a correspondingly substituted phosphonium salt or phosphonic acid ester in solvents such as toluene, tetrahydrofuran, diethyl ether or dimethoxyethane.",
"As bases, e.g., alkali hydrides, alkali amides, alkali alcoholates, such as, for example, potassium tert-butylate, alkali and alkaline-earth carbonates or hydroxides optionally are suitable in the presence of phase-transfer catalysts, such as, e.g., crown ethers or else organic bases such as triethylamine diisopropylethylainine or diazabicycloundecane, optionally in the presence of salts such as lithium bromide.",
"The isomer mixtures can be separated into enantiomers or E/Z isomers according to commonly used methods, such as, for example, crystallization, chromatography or salt formation.",
"The production of the salts is carried out in the usual way by a solution of the compound of formula I being mixed with the equivalent amount or an excess of a base or acid, which optionally is in solution, and the precipitate being separated or the solution being worked up in the usual way.",
"If the production of the intermediate compounds is not described, the latter are known or can be produced analogously to known compounds or processes that are described here.",
"The intermediate compounds that are described are especially suitable for the production of benzoic acid amides according to the invention.",
"Especially suitable are those intermediate compounds of general formula II in which R2 and R3 mean hydrogen or the group XR11, X means C1-6-alkyl, C2-6-alkenyl or C2-6-alkinyl, R11 means phenyl or pyridyl that is optionally substituted by C1-6-alkoxy, whereby R2 and R3 stand for hydrogen, although not simultaneously, as well as isomers and salts thereof.",
"These intermediate compounds are also subjects of this invention.",
"The intermediate products are themselves partially active and thus can also be used for the production of a pharmaceutical agent for treating tumors, psoriasis; arthritis, such as rheumatoid arthritis, hemangioma, angiofibroma; eye diseases, such as diabetic retinopathy, neovascular glaucoma; renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathic syndrome, transplant rejections and glomerulopathy; fibrotic diseases, such as cirrhosis of the liver, mesangial cell proliferative diseases, arteriosclerosis, and injuries to nerve tissue.",
"The following examples explain the production of the compounds according to the invention without the scope of the claimed compounds being limited to these examples.",
"EXAMPLE 1.0 Production of (N-4-chlorophenyl)-2-(4-pyridylethyl)Benzoic Acid Amide 105 mg of 2-(4-pyridylethyl)benzoic acid methyl ester is mixed in 7.5 ml of toluene with 56 mg of 4-chloroaniline, cooled to 4° C. and mixed under argon and in a moisture-free environment with 0.22 ml of trimethylaluminum (2 m solution in hexane).",
"Then, the mixture was heated for 2 hours to a bath temperature of 120° C. After cooling, it is mixed with 30 ml of a dilute sodium bicarbonate solution and extracted twice with 25 ml each of ethyl acetate.",
"The organic phase is washed with water, dried, filtered and concentrated by evaporation.",
"The residue is chromatographed on silica gel with ethyl acetate:cyclohexane=1:1 as an eluant.",
"133 mg (89% of theory) of (N-4-chlorophenyl)-2-(4-pyridylethyl)-benzoic acid amide is obtained as an oil.",
"Produced in a way similar to Example 1.0 are: Melting Point Example —Z—R1 R7 R2 R3 ° C. 1.1 H H 1.2 H H 1.3 H H 98-99 1.4 H H Oil 1.5 H H Oil 1.6 H H Oil 1.7 H H 1.8 H H Oil 1.9 H H 1.10 H H 1.11 H H Oil 1.12 H H 1.13 H H 1.14 H H 1.15 H H 1.16 H H 1.17 H H Oil 1.18 H H 1.19 H H 1.20 H H 121-122 1.21 H H 1.22 H H 1.23 H H 1.24 H H 130-131 1.25 H H 1.26 H H 1.27 H H 1.28 H H 1.29 H H 1.30 H H 1.31 H H 105-107 1.32 —(CH2)11—Me H H 1.33 —(CH2)6—Me H H 1.34 H H 1.35 —(CH2)3—CF3 H H 1.36 —(CH2)2-t-Bu H H 1.37 —(CH2)2-i-Prop H H 1.38 H H 1.39 H H 105.5 1.40 H H 136.2 1.41 H H Oil 1.42 H H 181.2 1.43 H H Oil 1.44 H H 1.45 H H 156.4 1.46 H H 1.47 H H 1.48 H H 1.49 H H 191.6 1.50 H H 106.5 1.51 H H 128.5 1.52 H H 191.5 1.53 H H 212.7 1.54 H H 179 1.55 H H >300 1.56 H H 163.5 1.57 H H 137.9 1.58 H H 115-118 1.59 H H 151-153 1.60 H H 1.61 H H 1.62 H H 1.63 H H 1.64 n-heptyl H H Oil 1.65 H H 120.2 1.66 H H 108.6 1.67 H H 113.7 1.68 H H 1.69 H H 1.70 H H 1.71 H H 1.72 H H 1.73 H H 1.74 H H 204.7 1.75 H H >300 1.76 H H 1.77 H H 126.6 1.78 H H 133.2 1.79 H H 139.5 1.80 H H 126.3 1.81 H H 1.82 H H 163.4 1.83 H H 185.4 1.84 H H Oil 1.85 H H 1.86 H H 136.8 1.87 H H 131.1 1.88 H H 140.6 1.89 H H Oil 1.90 H H 194.6 1.91 H H 188.9 1.92 H H 1.93 H H 146.4 1.94 H H EXAMPLE 2.0 Production of E-N-4-chlorophenyl-3-(2-pyridylethenyl)Benzoic Acid Amide 179 mg of N-4-chlorophenyl-3-(2-pyridylethinyl)benzoic acid amide is mixed in 7 ml of pyridine with 20 mg of palladium on calcium carbonate (5%), and it is hydrogenated for 1.5 hours under normal hydrogen pressure at room temperature.",
"After the catalyst is suctioned off on diatomaceous earth, the filtrate is concentrated by evaporation.",
"The residue is chromatographed on silica gel with ethyl acetate:hexane=1:1 as an eluant.",
"123 mg (68% of theory) of (Z)—N-4-chlorophenyl)-3-(2-pyridylethenyl)-benzoic acid amide is obtained as an oil.",
"Produced in a way similar to Example 2.0 are also the following compounds: Melting Point Example —Z—R1 R7 R2 R3 ° C. 2.1 H H 2.2 H H 2.3 H H 120.1 2.4 H H 157.9 2.5 H H 95.2 2.6 H H 116.2 2.7 H H 123 EXAMPLE 3.0 Production of (E)-N-4-chlorophenyl-3-(2-pyridylethenyl)Benzoic Acid Amide 120 mg of (Z)—N-4-chlorophenyl-3-(2-pyridylethenyl)benzoic acid amide is mixed in toluene with iodine and refluxed for 7 hours.",
"After concentration by evaporation, the residue is chromatographed on silica gel with ethyl acetate:hexane=1:1 as an eluant.",
"60 mg (50% of theory) of (E)-N-4-chlorophenyl-3-(2-pyridylethenyl)benzoic acid amide with a melting point of 212.7° C. is obtained.",
"Similarly produced are: Melting Point Example —Z—R1 R7 R2 R3 ° C. 3.1 H H 173.2 3.2 H H 146.9 3.3 H H 178 EXAMPLE 4.0 Production of N-(4-chlorophenyl)-2-(3-[4-hydroxyphenyl)propyl)]Benzoic Acid Amide 90 mg of N-(4-chlorophenyl)-2-(3-[4-methoxyphenyl)propyl)]benzoic acid amide is mixed in 8 ml of methylene chloride at −78° C. drop by drop with 1.2 ml of boron tribromide, and after the addition is completed, it is stirred overnight at room temperature.",
"Then, it is mixed with water, the methylene chloride is distilled off in a vacuum, and the water is shaken out with ethyl acetate.",
"The ethyl acetate phase is concentrated by evaporation, and the residue is chromatographed on silica gel with hexane:ethyl acetate=8:2 as an eluant.",
"24 mg (28% of theory) of N-(4-chlorophenyl)-2-(3-[4-hydroxyphenyl)propyl)]-benzoic acid amide is obtained.",
"Similarly produced are: Melting Point Example —Z—R1 R7 R2 R3 ° C. 4.1 H H 4.2 H H 4.3 H H 4.4 H H 4.5 H H 4.6 H H 4.7 H OH 164.1 4.8 H OH 115.3 4.9 H OH 137.4 4.10 H OH Oil 4.11 H OH 203.7 4.12 H OH EXAMPLE 5.0 Production of N-(4-chlorophenyl)-3-(4-methoxystyryl)Salicylic Acid Amide 904 mg of 4-methoxy-2′-hydroxystyrene is introduced into 40 ml of toluene and mixed at 4° C. with 4 ml of a solution of boron trichloride (1 mol in hexane).",
"It is then stirred at room temperature for 1 hour, mixed with 614 mg of 4-chlorophenyl isocyanate and heated for 1.5 hours to 120° C. Then, it is mixed with 5 ml of methanol and concentrated by evaporation.",
"The residue is chromatographed twice on silica gel, first with ethyl acetate:hexane=1:1, and a second time with toluene:ethyl acetate=100:3.5 as an eluant.",
"150 mg (10% of theory) of N-(4-chlorophenyl)-3-(4-methoxystyryl)salicylic acid amide is obtained as an oil.",
"Similarly produced from the corresponding starting materials are: Melting Point Example R3 ° C. 5.1 3-MeO—Ph 116.3 5.2 3-MeO—Ph—CH2 5.3 4-MeO—Ph—CH2 163.6 Production of the Intermediate Compounds EXAMPLE Z-5.0 Production of 4-methoxy-2′-hydroxystyrene 2.44 g of salicyl aldehyde is mixed in 200 ml of toluene first with 12.5 g of 4-methoxy-benzyltriphenylphosphonium chloride.",
"2.24 g of potassium-tert-butylate is then added while being cooled with ice.",
"It is then stirred first for 1 hour at this temperature and then for 3.5 hours at room temperature.",
"After mixing with 100 ml of water and acidification with 1N hydrochloric acid, it is extracted three times with 50 ml of ethyl acetate.",
"The collected organic phase is washed with saturated sodium chloride solution, dried, filtered and concentrated by evaporation.",
"The residue is chromatographed on silica gel with ethyl acetate:hexane=2:8 as an eluant.",
"3.1 g (68% of theory) of 4-methoxy-2′-hydroxystyrene is obtained.",
"Similarly produced are also the following compounds: Melting Point Example R3 ° C. Z-5.1 3-MeO—Ph Oil Z-5.2 3-MeO—Ph—CH2 Oil Z-5.3 4-MeO—Ph—CH2 Oil EXAMPLE 6.0 Production of N-(4-chlorophenyl)-3-(4-methoxyphenethyl)Salicylic Acid Amide 813 mg of 4-methoxy-2′-hydroxy 1,2-diphenylethane is reacted analogously to Example Z-5.0.The residue that is obtained after the working-up that is described there is chromatographed on silica gel with ethyl acetate:hexane=1:1 as an eluant, and the corresponding fractions are concentrated by evaporation and stirred with ethyl acetate/hexane in crystalline form.",
"375 mg (27.6% of theory) of N-(4-chlorophenyl)-3-(4-methoxyphenylethyl)salicylic acid amide with a melting point of 141° C. is obtained.",
"Similarly produced are also the following compounds: Melting Point Example R3 ° C. 6.1 3-MeO—Ph 130.2 6.2 3-MeO—Ph—CH2 Oil 6.3 4-MeO—Ph—CH2 158 Production of the Intermediate Compounds EXAMPLE Z-6.0 Production of 4-methoxy-2′-hydroxy-1,2-diphenylethane 905 mg of 4-methoxy-2′-hydroxystyrene is mixed in 50 ml of ethanol with 1.3 g of palladium on carbon (10) and hydrogenated for 70 minutes at room temperature under normal hydrogen pressure.",
"After the catalyst is suctioned off and after concentration by evaporation, 830 mg of 4-methoxy-2′-hydroxy 1,2-diphenylethane is obtained.",
"Similarly produced are also the following compounds: Melting Point Example R3 ° C. Z-6.1 3-MeO—Ph Oil Z-6.2 3-MeO—Ph—CH2 Oil Z-6.3 4-MeO—Ph—CH2 EXAMPLE 7.0 Production of Intermediate Products The examples below explain the production of the intermediate products according to the invention that are especially suitable for the production of the compounds of general formula I according to the invention, without the invention being limited to these examples.",
"Method A Production of 2-(4-pyridiylethenyl)Benzoic Acid Methyl Ester A mixture of 2.10 g of 2-iodobenzoic acid methyl ester and 0.97 g of 4-vinylpyridine in 24 ml of dimethylformamide is mixed with 1.04 g of triethylamine and 40 mg of palladium(II) acetate as well as 24 mg of tri-o-tolylphosphine under argon, and it is heated for 5 hours in a glass pressure vessel to 100° C. After concentration by evaporation in a vacuum, the residue is chromatographed on silica gel with hexane:ethyl acetate=1:1 as an eluant.",
"1.8 g (94% of theory) of 2-(4-pyridiylethenyl)-benzoic acid methyl ester is obtained.",
"Method B Production of 2-(4-pyridylethinyl)Benzoic Acid Methyl Ester 2.10 g of 2-iodobenzoic acid methyl ester is mixed in 25 ml of dimethylformamide under argon with 2.94 g of triethylamine, 179 mg of bis(triphenylphosphine)palladium(II) chloride, 111 mg of copper(I) iodide and 900 mg of 4-ethinylpyridine, and it is heated in a glass pressure vessel for 3.5 hours to a bath temperature of 80° C. After concentration by evaporation in a vacuum, the residue is chromatographed on silica gel with hexane:acetone=1:1 as an eluant.",
"1.08 g (45% of theory) of 2-(4-pyridiylethinyl)-benzoic acid methyl ester is obtained.",
"Method C Production of 2-(4-pyridylethyl)Benzoic Acid Methyl Ester 237 mg of 2-(4-pyridylethinyl)benzoic acid methyl ester, is mixed in 30 ml of ethanol with 200 mg of palladium on carbon (10%) and hydrogenated at normal pressure and at room temperature for 20 minutes.",
"Then, catalyst is suctioned off on diatomaceous earth, and the filtrate is concentrated by evaporation.",
"220 mg of 2-(4-pyridylethyl)benzoic acid methyl ester is obtained.",
"Instead of the ethinyl compound, the corresponding ethenyl compound can also be used.",
"Method D According to the method that is described in Example 2.0, the corresponding esters can also be converted into the Z compounds.",
"Method E According to the method that is described in Example 3.0, in the case of the esters, the E compounds can also be produced from the corresponding Z compound.",
"Method F According to the method that is described in Method B, 2-trimethylsilylethinylbenzoic acid methyl ester can also be produced from 2-iodobenzoic acid ethyl ester with ethinyltrimethylsilane in 83% yield.",
"Method G 464 mg of 2-trimethylsilylethinylbenzoic acid methyl ester is mixed in 15 ml of absolute methylene chloride with 2.75 ml of tetrabutylammonium fluoride (1 M in tetrahydrofuran) and stirred for 2.5 hours at room temperature.",
"After washing with dilute ammonia, the organic phase is dried, filtered and concentrated by evaporation and used without further purification in the next stage.",
"Method H 440 mg of 2-ethinylbenzoic acid methyl ester is reacted with 1.94 g of 3-iodoanisole according to Method B, and 680 mg (55.3% of theory) of 2-carbethoxymethyl-3′-methoxydiphenylacetylene is produced after column chromatography on silica gel with ethyl acetate:hexane=2.8 as an eluant.",
"Similarly produced are also the following compounds: Example R2 R3 Method 7.0 H C 7.1 H C 7.2 H C 7.3 H A 7.4 H A 7.5 H E 7.6 H B 7.7 H B 7.8 H B 7.9 H C 7.10 H C 7.11 H C 7.12 H A 7.13 H A 7.14 H E 7.15 H B 7.16 H B 7.17 H B 7.18 H D 7.19 H B 7.20 H C 7.21 H D 7.22 H A 7.23 H A 7.24 H D 7.25 H D 7.26 H C 7.27 H C 7.28 H E 7.29 H A 7.30 H F-H 7.31 H B 7.32 H C 7.33 H B 7.34 H F-H 7.35 H C 7.36 H C 7.37 H F-H 7.38 H C The sample applications below explain the biological action and the use of the compounds according to the invention without the latter being limited to the examples.",
"Solutions Required for the Tests Stock solutions Stock solution A: 3 mmol of ATP in water, pH 7.0 (−70° C.) Stock solution B: g-33P-ATP 1 mCi/100 μl Stock solution C: poly-(Glu4Tyr) 10 mg/ml in water Solution for dilutions Substrate solvent: 10 mmol of DTT, 10 mmol of manganese chloride, 100 mmol of magnesium chloride Enzyme solution: 120 mmol of tris/HCl, pH 7.5, 10 μM of sodium vanadium oxide Sample Application 1 Inhibition of the KDR- and FLT-1 Kinase Activity in the Presence of the Compounds According to the Invention In a microtiter plate (without protein binding) that tapers to a point, 10 μl of substrate mix (10 μl of volume of ATP stock solution A+25 μCi of g-33P-ATP (about 2.5 μl of stock solution B)+30 μl of poly-(Glu4Tyr) stock solution C+1.21 ml of substrate solvent), 10 μl of inhibitor solution (substances corresponding to the dilutions, 3% DMSO in substrate solvent as a control) and 10 μl of enzyme solution (11.25 μg of enzyme stock solution (KDR or FLT-1 kinase) are added at 4° C. in 1.25 ml of enzyme solution (dilute).",
"It is thoroughly mixed and incubated for 10 minutes at room temperature.",
"Then, 10 μl of stop solution (250 mmol of EDTA, pH 7.0) is added, mixed, and 10 μl of the solution is transferred to a P 81 phosphocellulose filter.",
"Then, it is washed several times in 0.1 M phosphoric acid.",
"The filter paper is dried, coated with Meltilex and measured in a microbeta counter.",
"The IC50 values are determined from the inhibitor concentration, which is necessary to inhibit the phosphate incorporation to 50% of the uninhibited incorporation after removal of the blank reading (EDTA-stopped reaction).",
"The results of the kinase inhibition IC50 in μM are presented in the table below: Example No.",
"VEGFR I (FLT) VEGFR II (KDR) 1.60 2 0.5 1.31 0.2 0.4 1.89 2 0.3 1.54 0.05 0.5 1.57 0.2 0.2 1.64 0.2 0.3 1.67 KH 5 1.1 0.2 0.2 KH = No inhibition"
]
] | Patent_10258345 |
[
[
"Conductive gasket and material therefor",
"A conductive gasket and a paste for the electrical industry based on at least one elastomer (10) and on an admixture of conductive particles in the form of fibers (20), in which the fibers (20) are flexible and have been embedded in the elastomer (10) in random orientation and with formation of a large number of contact points between fibers."
],
[
"1.A conductive paste, comprising: at least one elastomeric material and a plurality of conductive particles in the form of elongated fibers, wherein the fibers are flexible and are substantially uniformly distributed in the elastomeric material in random orientation and with formation of a large number of contact points between contacting fibers.",
"2.The paste as claimed in claim 1, wherein the proportion of fibers, based on the total volume of the paste, is less than 50% by volume.",
"3.The paste as claimed in claim 1 wherein the proportion of fibers is less than 25% by volume of the total volume of the paste.",
"4.The paste as claimed in claim 1, wherein the fibers have a length:diameter ratio of more than 2.5.The paste as claimed in claim 1, wherein the fibers have a length:diameter ratio of more than 10.6.The paste as claimed in claim 1, wherein the fibers have a length:diameter ratio of more than 50.7.The paste as claimed in claim 5, wherein the fibers have a diameter less than about 0.1 mm.",
"8.The paste as claimed in claim 1, wherein the paste is composed of two or more components which cure immediately after they are combined.",
"9.The paste as claimed in claim 1, wherein the paste comprises monomers which polymerize at room temperature.",
"10.The paste as claimed in any of claim 1, wherein the paste comprises monomers which polymerizes on exposure to heat.",
"11.The paste as claimed in claim 1, wherein the paste has a sufficiently low viscosity to be applied in the form of a strand about 1-5 mm wide, directly in the region of a joint, to form an electrically shielding gasket for a housing.",
"12.The paste as claimed in claim 1, wherein the paste has a sufficiently high viscosity to be moldable by injection molding or extrusion to provide a sealing element.",
"13.The paste of claim 1, wherein the fibers comprise metal covered polymer fibers.",
"14.The paste of claim 1, wherein the elastomer comprises a silicone based elastomer.",
"15.A conductive EMI shielding gasket having a top and a bottom surface, comprising an elastomeric binder having elongated and flexible electrically conductive fibers substantially evenly distributed therein and substantially each fiber is in contact with another fiber to provide a conductive network of fibers from the top surface to the bottom surface of the gasket.",
"16.The gasket of claim 15, wherein the binder comprises a silicone based elastomer.",
"17.The gasket of claim 15, constructed for use in a cellular phone.",
"18.The gasket of claim 15, wherein the fibers are metal covered polymer fibers.",
"19.The gasket of claim 15, wherein the fibers have a length to diameter ratio of over about 10.20.A method of forming a gasket, comprising depositing a gasket shaped strand of a curable paste of elastomeric material having flexible conductive fibers evenly distributed therein and permitting the paste to cure."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The invention relates to gaskets having electrically conductive properties for use in shielding the emission of electromagnetic waves from electronic devices and to a conductive paste for use in the electrical industry that can be used to form such gaskets.",
"Conventional electrically conductive shielding gaskets can be formed with glass spheres and pure metallic powders and can be used for shielding at input/output (I/O) panels in electronic devices such as computers, to prevent leakage of electromagnetic waves and radio frequency interference (RFI).",
"Gaskets can be formed with a foam inner body and conductive fabric disposed around the foam inner body.",
"Others are formed of conductive polymers and conductive elastomers or with foam bodies coated with conductive elastomer or polymer materials.",
"For many fine detailed operations, it is advantages to provide the gasket material in paste form.",
"Materials of this type are widely used and are required by a wide variety of applications.",
"They have gained particular importance, for example, in connection with the sealing of electromagnetically shielded housings in electronic devices which emit electromagnetic radiation or can be disturbed by electromagnetic radiation penetrating from outside.",
"To give EMI (electromagnetic interference) shielding or, respectively, RFI (radio frequency interference) shielding, and to improve electromagnetic compatibility (EMC), the housings are produced from a material which is electrically conducting or has been coated with an electrical conductor.",
"It is known that gaskets made from an electrically conductive flexible material can be used so that the region of the joints at which the parts of the housing are joined together is also given shielding.",
"An example of a material of this type is known from U.S. Pat.",
"No.",
"4,011,360, the contents of which are incorporated by reference.",
"This known material is based on an elastomer, specifically a silicone rubber material, which has an admixture of electrically conducting particles therein.",
"This material polymerizes (cures) when exposed to atmospheric moisture at room temperature.",
"DE 43 19 965 C2, the contents of which are incorporated herein by reference discloses the use of a material of this type for producing the housings described above.",
"The material is extruded as a strand of paste directly in the region of the joint onto one of the parts of the housing, and is cured there to form the gasket.",
"This process is also known by the skilled worker as the formed-in-place-gasket process.",
"Existing gaskets, such as formed-in-place gaskets lack sufficient flexibility for certain applications.",
"Others, can lack sufficient EMI shielding properties.",
"Still others are difficult to use.",
"Accordingly, it is desirable to provide an improved conductive shielding gasket and a paste for forming a gasket in place, which overcomes drawbacks of the prior art."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The invention is based on the concept of using conductive particles in the form of fibers which are flexible.",
"These are admixed in random orientation with the elastomer in a way which produces a large number of contact points.",
"The embedding of the fibers in the elastomer therefore takes the form of an irregular three-dimensional matrix.",
"The contact points ensure ideal and statistically random distribution conductivity within the material.",
"Depending on the nature of the fibers used, one preferred embodiment allows the selected proportion of fibers to be less than 50%, based on the total volume of the material.",
"It is preferably possible to reduce the proportion of fibers to less than 25% by volume.",
"Compared with materials previously available, this gives a significant improvement in mechanical properties, in particular in the flexibility of the polymerized final product, which is moreover very inexpensive.",
"It is preferable for the fibers used to have a diameter/length ratio of more than 2.Ideal results can be achieved if the diameter/length ratio is more than 10 and even more than 50.The shape of fibers of this type makes them highly flexible, and they therefore give an ideal result with regard to the mechanical properties of the final product.",
"An embodiment further optimized in this respect provides for the use of fibers with a diameter of less than 0.1 mm.",
"These extremely thin fibers can be embedded into the elastomer in an ideal manner, such that the embedded fibers have virtually no adverse effect on the flexibility of the elastomer.",
"It is also possible to design the material in a manner known as a two- or multicomponent material such as reactive materials in which a cure agent is mixed with a polymerizable or otherwise curable resin.",
"The two components are not mixed until immediately prior to processing.",
"This method can give a very inexpensive material which is easy to process.",
"Depending on the application, the material selected may be one which polymerizes (cures) at room temperature.",
"On the other hand it is also possible to provide a material which polymerizes on exposure to heat, so that the polymerization procedure can be controlled by controlling temperature.",
"This is of particular importance with a view to automated mass production.",
"The formulation of the material is preferably such that it has low viscosity.",
"It is therefore particularly suitable for forming an electrically shielding gasket for a housing, the material for the gasket being applied in the form of a strand directly in the region of a joint of a housing.",
"Typical applications for a material of this type are the formation of a gasket for a mobile telephone housing or the like.",
"However, the viscosity should not be too low, or it will run and not stay in place until cured.",
"Typical strands of paste and gaskets formed therefrom can be about 1-5 mm wide.",
"In other embodiments of the invention, the strand can be wider or thinner than this range.",
"It is also possible to design a material with high viscosity, for example in order to produce sealing elements in the form of sealing strips, sealing pads, sealing tubes or O-rings, by injection molding or extrusion.",
"One specific example of an application provides for the use of the conductive paste for producing a flexible gasket for an electromagnetically shielded housing.",
"A paste of the material is applied by means of a path-controlled nozzle directly to a part of the housing in the region where the housing has a joint to be sealed.",
"The nozzle is moved over the part of the housing by means of computer-controlled metering equipment while the plastics material is being discharged.",
"The velocity of relative movement of the nozzle and the part of the housing is determined by the viscosity of the paste, the amount and velocity of the paste emerging from the nozzle, the cross-sectional area of the nozzle passage, the desired cross-sectional profile of the gasket to be produced and the makeup of the material.",
"The strand made from the paste and dispensed in this way polymerizes under ambient conditions at room temperature.",
"This procedure takes a relatively long time, but can be accelerated by controlled exposure to heat or through the use of catalysts or accelerators.",
"The present invention provides a material of the type which can be used as an EMI gasket.",
"The material provided should have improved mechanical properties and moreover be inexpensive, in order for example to open up even those application sectors in which economic reasons have hitherto prevented the use of large elastomer gaskets.",
"Accordingly, it is an object of the invention to produce a gasket and materials for producing the gasket which are highly conductive, highly flexible and easy to use."
],
[
"BACKGROUND OF THE INVENTION The invention relates to gaskets having electrically conductive properties for use in shielding the emission of electromagnetic waves from electronic devices and to a conductive paste for use in the electrical industry that can be used to form such gaskets.",
"Conventional electrically conductive shielding gaskets can be formed with glass spheres and pure metallic powders and can be used for shielding at input/output (I/O) panels in electronic devices such as computers, to prevent leakage of electromagnetic waves and radio frequency interference (RFI).",
"Gaskets can be formed with a foam inner body and conductive fabric disposed around the foam inner body.",
"Others are formed of conductive polymers and conductive elastomers or with foam bodies coated with conductive elastomer or polymer materials.",
"For many fine detailed operations, it is advantages to provide the gasket material in paste form.",
"Materials of this type are widely used and are required by a wide variety of applications.",
"They have gained particular importance, for example, in connection with the sealing of electromagnetically shielded housings in electronic devices which emit electromagnetic radiation or can be disturbed by electromagnetic radiation penetrating from outside.",
"To give EMI (electromagnetic interference) shielding or, respectively, RFI (radio frequency interference) shielding, and to improve electromagnetic compatibility (EMC), the housings are produced from a material which is electrically conducting or has been coated with an electrical conductor.",
"It is known that gaskets made from an electrically conductive flexible material can be used so that the region of the joints at which the parts of the housing are joined together is also given shielding.",
"An example of a material of this type is known from U.S. Pat.",
"No.",
"4,011,360, the contents of which are incorporated by reference.",
"This known material is based on an elastomer, specifically a silicone rubber material, which has an admixture of electrically conducting particles therein.",
"This material polymerizes (cures) when exposed to atmospheric moisture at room temperature.",
"DE 43 19 965 C2, the contents of which are incorporated herein by reference discloses the use of a material of this type for producing the housings described above.",
"The material is extruded as a strand of paste directly in the region of the joint onto one of the parts of the housing, and is cured there to form the gasket.",
"This process is also known by the skilled worker as the formed-in-place-gasket process.",
"Existing gaskets, such as formed-in-place gaskets lack sufficient flexibility for certain applications.",
"Others, can lack sufficient EMI shielding properties.",
"Still others are difficult to use.",
"Accordingly, it is desirable to provide an improved conductive shielding gasket and a paste for forming a gasket in place, which overcomes drawbacks of the prior art.",
"SUMMARY OF THE INVENTION The invention is based on the concept of using conductive particles in the form of fibers which are flexible.",
"These are admixed in random orientation with the elastomer in a way which produces a large number of contact points.",
"The embedding of the fibers in the elastomer therefore takes the form of an irregular three-dimensional matrix.",
"The contact points ensure ideal and statistically random distribution conductivity within the material.",
"Depending on the nature of the fibers used, one preferred embodiment allows the selected proportion of fibers to be less than 50%, based on the total volume of the material.",
"It is preferably possible to reduce the proportion of fibers to less than 25% by volume.",
"Compared with materials previously available, this gives a significant improvement in mechanical properties, in particular in the flexibility of the polymerized final product, which is moreover very inexpensive.",
"It is preferable for the fibers used to have a diameter/length ratio of more than 2.Ideal results can be achieved if the diameter/length ratio is more than 10 and even more than 50.The shape of fibers of this type makes them highly flexible, and they therefore give an ideal result with regard to the mechanical properties of the final product.",
"An embodiment further optimized in this respect provides for the use of fibers with a diameter of less than 0.1 mm.",
"These extremely thin fibers can be embedded into the elastomer in an ideal manner, such that the embedded fibers have virtually no adverse effect on the flexibility of the elastomer.",
"It is also possible to design the material in a manner known as a two- or multicomponent material such as reactive materials in which a cure agent is mixed with a polymerizable or otherwise curable resin.",
"The two components are not mixed until immediately prior to processing.",
"This method can give a very inexpensive material which is easy to process.",
"Depending on the application, the material selected may be one which polymerizes (cures) at room temperature.",
"On the other hand it is also possible to provide a material which polymerizes on exposure to heat, so that the polymerization procedure can be controlled by controlling temperature.",
"This is of particular importance with a view to automated mass production.",
"The formulation of the material is preferably such that it has low viscosity.",
"It is therefore particularly suitable for forming an electrically shielding gasket for a housing, the material for the gasket being applied in the form of a strand directly in the region of a joint of a housing.",
"Typical applications for a material of this type are the formation of a gasket for a mobile telephone housing or the like.",
"However, the viscosity should not be too low, or it will run and not stay in place until cured.",
"Typical strands of paste and gaskets formed therefrom can be about 1-5 mm wide.",
"In other embodiments of the invention, the strand can be wider or thinner than this range.",
"It is also possible to design a material with high viscosity, for example in order to produce sealing elements in the form of sealing strips, sealing pads, sealing tubes or O-rings, by injection molding or extrusion.",
"One specific example of an application provides for the use of the conductive paste for producing a flexible gasket for an electromagnetically shielded housing.",
"A paste of the material is applied by means of a path-controlled nozzle directly to a part of the housing in the region where the housing has a joint to be sealed.",
"The nozzle is moved over the part of the housing by means of computer-controlled metering equipment while the plastics material is being discharged.",
"The velocity of relative movement of the nozzle and the part of the housing is determined by the viscosity of the paste, the amount and velocity of the paste emerging from the nozzle, the cross-sectional area of the nozzle passage, the desired cross-sectional profile of the gasket to be produced and the makeup of the material.",
"The strand made from the paste and dispensed in this way polymerizes under ambient conditions at room temperature.",
"This procedure takes a relatively long time, but can be accelerated by controlled exposure to heat or through the use of catalysts or accelerators.",
"The present invention provides a material of the type which can be used as an EMI gasket.",
"The material provided should have improved mechanical properties and moreover be inexpensive, in order for example to open up even those application sectors in which economic reasons have hitherto prevented the use of large elastomer gaskets.",
"Accordingly, it is an object of the invention to produce a gasket and materials for producing the gasket which are highly conductive, highly flexible and easy to use.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention is further described below using the example shown diagrammatically in the figures.",
"FIG.",
"1 is a cross sectional view of a strand of paste which can be used to form gasket material in accordance with preferred embodiments of the invention; FIG.",
"2 shows the gasket strand of FIG.",
"1 after deformation; FIG.",
"3 shows a cross sectional view of a strand of conventional gasket material; and FIG.",
"4 shows the gasket strand of FIG.",
"3 after deformation.",
"DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An elastomeric material containing conductive particles or fibers and method of formation is provided for use as low impedance gasket materials including electromagnetic interference (EMI) gaskets formed from the materials.",
"The particles can be formed from Teflon®, nylon, acrylics, polyesters and other polymer fibers which can be plated, coated or otherwise covered or embedded with conductive material or otherwise rendered conductive, so as to yield highly flexible electrically conductive additives which can be used in the formation of electrically conductive gasket material.",
"In a preferred embodiment of the invention, the particles are in the nature of fibers of the above materials, coated or plated with electrically conductive materials.",
"One way of rendering the particles electrically conductive is through an electroplating process to provide the particles with a metal surface covering and thereby provide a highly electrically conductive surface on the flexible particles.",
"Suitable metals include silver, copper, nickel or alloys of silver, copper and/or nickel, as well as other combinations and materials which provide low impedance surfaces for the particles.",
"By way of nonlimiting example, preferred particles are in the range of about 10 to 100 microns in average diameter with a length of 20 microns to 1000 microns.",
"In certain embodiments of the invention, the lengths and diameters of the particles could be higher or lower than this range.",
"In other embodiments of the invention the length of the fibers can advantageously range from about 30 microns to even several centimeters in length.",
"Preferred particles should also have a low specific gravity to ensure that they do not settle out of a paste before curing, preferably ranging from about 1.15 to 1.5.The conductive particles can be combined with compatible elastomeric compounds and formed into appropriate shielding material.",
"Suitable elastomers include compatible silicones, foamed and unfoamed fluoropolymers, EPDM (ethylene-propylene-diene monomer) neoprene, SANTOPRENE™ and other elastomeric materials which are suitably combined with conductive particles/fibers as described herein for use as shielding gaskets as described herein.",
"The conductive particle portion of the elastomeric material is advantageously from about 5% to 50% by weight of the elastomer/compound combination.",
"One advantageous feature of the conductive shield (gasket) is to provide suitable distribution of the conductive particle fibers, so that sufficient conductive material is present at the surface of the gasket, to provide low impedance and high shielding.",
"It is also important for the particles to be in contact with each other to transmit current from one surface to the other.",
"Thus a large percentage of the particles must be in contact with other particles to present a continuous network of conductive particles from surface to surface.",
"The paste (uncured material) can be used in compression molding, injection molding and extrusion processes to form preformed gaskets, sleeves, strips, o-rings, tubes and other images.",
"Electrically conductive particles have exhibited various shapes.",
"Particles frequently encountered in conventional gaskets have the shape of flakes, spheres, irregularly shaped bodies or fibers.",
"In the simplest case, the particles are manufactured directly from conductive material.",
"It is moreover possible to prepare particles from nonconductive materials and then to cover or coat these with conductive material.",
"It has been determined that these particles reduce the elasticity of the cured final product.",
"The elasticity here is given solely by the elastomer into which the particles have been embedded.",
"Forming the particles as flexible fiber strands can therefore yield highly flexible conductive gaskets.",
"To achieve good conductivity it has generally been necessary to provide more than 50% by volume of conductive particles to ensure the required contact of the particles with one another.",
"The resultant proportion of elastomer has been comparatively small.",
"It has been determined that this also leads to a rigid final product with disadvantageous mechanical properties and high cost.",
"FIG.",
"3, for comparison, shows a section of a strand 1′, which is composed of an elastomer 10′ and of an admixture of conductive particles 20′.",
"The conductive particles 20′ have been packed tightly within the elastomer 10′ in order to ensure the required good electrical conductivity via a large number of contact points.",
"The proportion of the particles 20′ by volume, based on the total volume, is about 80%.",
"In FIG.",
"4, for comparison, the strand 1′ has been exposed to a force F, as arises in practice for example at a joint of a housing.",
"The deformation shown diagrammatically is possible only if each of the particles 20′ can be displaced to the side.",
"Otherwise deformation is possible only to a very small extent, specifically until the particles 20′ have their maximum close-packed density.",
"It has been determined that this can be improved by using less particles, but making the particles in the form of longer, flexible, conductive strands.",
"FIGS.",
"1 and 2 show a strand 1 according to preferred embodiments of the invention.",
"A large number of long, thin, flexible fibers 20 have been embedded in the elastomer material 10.They have random orientation, and the fibers 20 contact one another at a large number of contact points to form a conductive network.",
"Even without deformation, this ensures electrical conductivity, as indicated in FIG.",
"1.It can be seen directly from FIG.",
"2 that relatively problem-free deformation can occur on exposure to a force F. In this case the flexibility of the strand 1 is almost exclusively a function of the flexibility of the elastomer, since the proportion by volume of the fibers 20 is less than 50%.",
"The fibers 20 provide only slight hindrance to the deformation of the strand 1.There is substantial freedom in the choice of fibers 20.It is preferable to use extremely thin fibers which are relatively long and have a cross section of less than 0.1 mm.",
"The length is at least twice the diameter, but the full effect of the advantage achievable is not seen until very much longer fibers are used, such as these in which the length is 10, 50 and even more than 50 times the diameter.",
"The diameter data above should not be understood as a restriction implying that the only fibers which can be used are those with approximately circular cross-section.",
"Rather, the cross-sectional shapes which may be used include others, and may per se be as desired.",
"Diameter should be understood to refer to the average cross sectional side to side distance regardless whether the particle has a round cross section.",
"The fibers 20 may be composed of the materials commonly used for particles of this type, including, for example, naturally occurring materials.",
"If the starting material for the fibers is nonconducting, the fibers are covered or coated with conductive material.",
"The strand 1 shown in FIGS.",
"1 and 2 has thus been permeated by a type of three-dimensional matrix of fibers 20, and the distribution of the fibers 20 and their contact points with one another, determined statistically over the cross section, is at least approximately constant.",
"It is apparent from the above that it is possible to form a conductive paste and an EMI shielding gasket from such paste, which has ideal mechanical and electrical properties and is also inexpensive.",
"It is relatively easy here to achieve the desired processing properties, in particular via suitable choice of the elastomer, which may also be in the form of a two- or multicomponent material."
]
] | Patent_10258426 |
[
[
"Photovoltaic cell",
"A photovoltaic cell is described having a photoactive layer (4) made of two components, namely a conjugated polymer component as an electron donor and a fullerene component as an electron acceptor.",
"In order to provide advantageous conditions, it is suggested that the two components and their mixed phases have an average largest grain size smaller than 500 nm in at least some sections of photoactive layer (4)."
],
[
"1.A photovoltaic cell having a photoactive layer made of two components, namely a conjugated polymer component as an electron donor and a fullerene component as an electron acceptor, characterized in that the conjugated polymer component comprises a PPV polymer derivative, and the two components and their mixed phases have an average largest grain size smaller than 500 nm in at least some sections of the photoactive layer.",
"2.A method for producing a photovoltaic cell according to claim 1, comprising applying a mixture made of the two components and a solvent on a carrier layer, which is provided with an electrode layer, to form a film, and disposing a counterelectrode on this film, characterized in that a dispersion agent, is added as a solvent to the mixture made of the two components.",
"3.The photovoltaic cell of claim 1, wherein the fullerene component comprises a functionalized fullerene.",
"4.The photovoltaic cell of claim 3, wherein the functionalized fullerene is PCBM.",
"5.The photovoltaic cell of claim 1, further comprising a carrier layer.",
"6.The photovoltaic cell of claim 5, wherein the carrier layer comprises PEDOT.",
"7.The photovoltaic cell of claim 1, further comprising an electrode and a counterelectrode.",
"8.The photovoltaic cell of claim 7, wherein the electrode comprises indium tin oxide.",
"9.The photovoltaic cell of claim 7, wherein the counterelectrode comprises aluminum.",
"10.A photovoltaic cell, comprising a photoactive layer containing a conjugated polymer component as an electron donor and a functionalized fullerene as an electron acceptor, wherein the conjugated polymer component, the functionalized fullerene, and their mixed phases have an average largest grain size smaller than 500 nm in at least some sections of the photoactive layer.",
"11.The photovoltaic cell of claim 10, wherein the conjugated polymer component comprises a PPV derivative.",
"12.The photovoltaic cell of claim 10, wherein the functionalized fullerene is PCBM.",
"13.The photovoltaic cell of claim 10, further comprising a carrier layer.",
"14.The photovoltaic cell of claim 14, wherein the carrier layer comprises PEDOT.",
"15.The photovoltaic cell of claim 10, further comprising an electrode and a counterelectrode.",
"16.The photovoltaic cell of claim 15, wherein the electrode comprises indium tin oxide.",
"17.The photovoltaic cell of claim 15, wherein the counterelectrode comprises aluminum.",
"18.The method of claim 2, wherein the dispersion agent is chlorobenzene.",
"19.The method of claim 2, wherein the mixture is applied on the carrier layer by spin coating or dripping."
],
[
"The present invention relates to a photovoltaic cell having a photoactive layer made of two components, namely a conjugated polymer component as an electron donor and a fullerene component as an electron acceptor.",
"Plastics having extensive π-electron systems, in which single and double bonds follow one another alternately in sequence, are referred to as conjugated plastics.",
"These conjugated plastics have energy bands which are comparable with semiconductors in regard to electron energy, so that they may also be transferred from the non-conductive state into the metallically conductive state through doping.",
"Examples of such conjugated plastics are polyphenylenes, polyvinylphenylenes (PPV), polythiophenes, or polyanilines.",
"The efficiency of energy conversion of photovoltaic polymer cells made of a conjugated polymer is, however, typically between 10−3 and 10−2%.",
"To improve this efficiency, heterogeneous layers made of two conjugated polymer components have already been suggested (U.S. Pat.",
"No.",
"5,670,791 A), one polymer component being used as an electron donor and the other polymer component as an electron acceptor.",
"By using fullerenes, particularly buckminsterfullerenes C60, as electron acceptors (U.S. Pat.",
"No.",
"5,454,880 A), the charge carrier recombination otherwise typical in the photoactive layer may be largely avoided, which leads to an efficiency of 0.6% to 1% under AM (air mass) 1.5 conditions.",
"In spite of this, the achievable efficiency generally remains too low for a cost-effective, technical use of such photoactive layers for constructing photovoltaic cells.",
"The present invention is therefore based on the object of designing a photovoltaic cell of the type initially described in such a way that a further increase of the efficiency of energy conversion is possible.",
"The present invention achieves the object described in that both components and their mixed phases have an average largest grain size smaller than 500 nm in at least some sections of the photoactive layer.",
"The present invention is based on the knowledge that effective charge separation may only be ensured in the contact region between the electron donor and the electron acceptor, so that after photoexcitation of the conjugated polymer components, the excitation energy is only relayed to the fullerene components in the form of electrons in the contact regions with the fullerene components.",
"If the average largest grain size of the components and mixed phases in the photoactive layer is kept smaller than 500 nm, then, due to the enlargement of the surface connected therewith, the proportion of contact between the two components may be increased accordingly, which leads to a significant improvement of the charge separation.",
"The efficiency, which is a function of this charge separation, rose to a characteristic 2.5% under simulated AM 1.5 conditions.",
"To manufacture photovoltaic cells having a photoactive layer whose average grain size is smaller than 500 nm, a mixture made of the two components and a solvent may be applied as a film to a carrier layer provided with an electron layer, before this film, which forms the photoactive layer, is covered with a counter electrode, as is typical.",
"However, it must be ensured that an appropriate dispersion agent is used as a solvent for both components, in order to ensure the desired fine grain of the photoactive layer.",
"Chlorobenzene may particularly advantageously be used as a dispersion agent in this case.",
"The effect of the fine-grained structure of the photoactive layer of a photovoltaic cell according to the present invention will be described more detail with reference to the drawing.",
"FIG.",
"1 shows the basic construction of a photovoltaic cell according to the present invention in section, FIG.",
"2 shows the surface structure of a typical photoactive layer, FIG.",
"3 shows the surface structure of a photoactive layer according to the present invention, FIG.",
"4 shows the current-voltage characteristic of a typical photovoltaic cell and a photovoltaic cell according to the present invention, and FIG.",
"5 shows the charge yield per incident luminous intensity in relation to the wavelength of the photoexcitation, for a typical photovoltaic cell and for a photovoltaic cell according to the present invention.",
"As shown in FIG.",
"1, the photovoltaic cell comprises a transparent glass carrier 1, onto which an electrode layer 2 made of indium/tin oxide (ITO) is applied.",
"This electrode layer 2 generally has a comparatively rough surface structure, so that it is covered with a smoothing layer 3 made of a polymer, typically PEDOT, which is made electrically conductive through doping.",
"Photoactive layer 4, which is made of two components, each having a layer thickness of, for example, 100 nm to a few μm depending on the application method, is applied onto this smoothing layer 3 before counterelectrode 5 is applied.",
"If ITO is used as a hole-collecting electrode, aluminum, which is vapor deposited onto photoactive layer 4, is used as an electron-collecting electrode.",
"The photoactive layer is made of a conjugated polymer, preferably a PPV derivate, as an electron donor and a fullerene, particularly functionalized fullerene PCBM, as an electron acceptor.",
"The concept of polymer is to be understood to mean both high polymers and oligomers.",
"These two components are mixed with a solvent and applied as a solution onto smoothing layer 3 by, for example, spin coating or dripping.",
"Toluene is used as a typical solvent, however, it cannot ensure the desired fine structure of photoactive layer 4, as is shown in FIG.",
"2, in which the typical surface structure of such a photoactive layer using toluene as a solvent is illustrated.",
"The grain structure of fullerene components 6 and/or a mixed phase may particularly be seen in atomic force microscopy (tapping-mode AFM images), as is schematically reproduced in FIGS.",
"2 and 3, while the polymer components and/or a further mixed phase essentially fill up the intervals between the distinct grains.",
"As is shown by the length unit illustrated, a maximum grain size significantly greater than 500 nm results.",
"However, if a dispersion agent, preferably chlorobenzene, is used as a solvent according to the present invention, then a significantly finer structure is obtained for active layer 4, with an otherwise corresponding composition, which accordingly results in a smoother surface structure, as shown in FIG.",
"3.The average grain size of less than 500 nm of photoactive layer 4 achievable with the aid of the dispersion agent produces a significant increase of the number of contact points between the electron donor and the electron acceptor and therefore a significantly improved charge separation and reduced charge recombination, which may be read directly from the voltage-current characteristic.",
"In FIG.",
"4, current density I of the photovoltaic cells to be compared is graphed over voltage U, at an excitation energy of 80 mW/cm2 under simulated AM 1.5 conditions.",
"If one compares characteristic 7 of the photovoltaic cell having the coarse-grained structure of photoactive layer 4 to characteristic 8, which was recorded for a photovoltaic cell having a fine-grained structure of photoactive layer 4, one immediately recognizes the improved ratios in a photovoltaic cell according to the present invention as shown in characteristic 8.The short-circuit current measured at voltage 0 V was 2.79 mA/cm2 for the known cell, and was 5.24 mA/cm2 for the cell according to the present invention.",
"Since the no-load voltage also increased from 710 mV to 770 mV, an increase in efficiency from approximately 1% to 2.6% could be achieved, it being taken into consideration that the bulk factor increased from 0.40 to 0.52 due to the finer structure of the photoactive layer according to the present invention.",
"The effects according to the present invention may be seen particularly clearly in FIG.",
"5, in which the charge yield per incident luminous intensity IPCE[%]=1240.Ik[μA/cm2]/A[nm].Il[W/m2] is graphed over wavelength λ for the photovoltaic cells to be compared.",
"The short-circuit current is to be entered in the formula above with Ik, and the luminous intensity with Il.",
"It is shown that, according to characteristic 9 for the cell according to the present invention in comparison to characteristic 10 of the typical cell, approximately double the charge yield per incident luminous intensity results if the fine structure of heterogeneous photoactive layer 4 has an average grain smaller than 500 nm."
]
] | Patent_10258713 |
[
[
"Method and apparatus for manufacturing composite sheet-like materials",
"A method of producing a composite laminate comprises the steps of feeding ground reclaimed material, for example carpet backing, to a dispersing station (16), dispersing the reclaimed material on a moving web (10), bringing an intermediate web (20) into engagement with the layer of reclaimed material, melting the reclaimed material to fuse the reclaimed material to the intermediate web, passing the laminate through compression rollers (28, 30) to control the thickness of the laminate, and cooling the laminate.",
"The moving web (10) may comprise carpeting material in which case the melted reclaimed material is fused to the carpeting material (10) and intermediate web (20)."
],
[
"1.A method of producing a composite laminate, comprising the steps of feeding ground reclaimed material to a dispersing station, dispersing the reclaimed material on a moving web, bringing an intermediate web into engagement with the layer of reclaimed material, melting the reclaimed material to fuse the reclaimed material to the intermediate web, passing the laminate through compression rollers to control the thickness of the laminate, and cooling the laminate.",
"2.A method of producing a composite laminate according to claim 1, wherein said moving web comprises a carpeting type material and wherein the melted reclaimed material is fused to said carpeting type material and intermediate web.",
"3.A method of producing a composite laminate according to claim 2, wherein said reclaimed material is ground backing material from carpeting.",
"4.A method of producing a composite laminate according to claim 1, wherein said melting step comprises passing the reclaimed materials and intermediate web through an oven having heating units on opposite sides of the laminate, the heating units being independently controllable.",
"5.A method of producing a composite laminate according to claim 2, wherein said melting step comprises passing the reclaimed materials and intermediate web through an oven having heating units on opposite sides of the laminate, the heating units being independently controllable."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Briefly, in accordance with the invention, a method of producing a composite laminate comprises the steps of feeding ground reclaimed material to a dispersing station, dispersing the reclaimed material on a moving web, bringing an intermediate web into engagement with the layer of reclaimed material, melting the reclaimed material to fuse the reclaimed material to the intermediate web, passing the laminate through compression rollers to control the thickness of the laminate, and cooling the laminate."
],
[
"This invention relates to a method and apparatus for manufacturing composite layered sheets or tiles from reclaimed waste carpet materials and the like.",
"Historically, extrusion processes have been used to produce multilayered composite sheets from reground materials, fiber waste, and/or virgin materials.",
"An extrusion process requires that a high quality consistent material be fed into the extruder to obtain a quality sheet product.",
"Extrusion processes are sensitive to variations in material, bulk density, flow times and melt indices.",
"These variations affect the material flow which can either starve or flood the screw resulting in variations in the extruded sheet in width, thickness, line speed, tensile strength, and surface tension, among other things.",
"The present invention provides a method for manufacturing composite layered sheets or tiles from reclaimed materials which is less sensitive to the quality and consistency of the input material than an extrusion process.",
"SUMMARY OF THE INVENTION Briefly, in accordance with the invention, a method of producing a composite laminate comprises the steps of feeding ground reclaimed material to a dispersing station, dispersing the reclaimed material on a moving web, bringing an intermediate web into engagement with the layer of reclaimed material, melting the reclaimed material to fuse the reclaimed material to the intermediate web, passing the laminate through compression rollers to control the thickness of the laminate, and cooling the laminate.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a partially schematic illustration of an apparatus and process for producing composite layered sheets and/or tiles from reclaimed waste carpet materials in accordance with a preferred embodiment of the invention; and FIG.",
"2 is a sectional view of a composite layer sheet produced in accordance with the invention.",
"DETAILED DESCRIPTION U.S. Pat.",
"No.",
"6,029,916 of White discloses a method and apparatus for reclaiming backing and fiber materials from waste carpet or the like.",
"U.S. Pat.",
"No.",
"6,029,916 is hereby incorporated by reference into this specification.",
"The apparatus and process disclosed in the '916 patent produce granulated reclaimed backing material together with fiber waste products.",
"The present invention uses the reclaimed backing material to produce various different types of sheets, including single layer sheets and composite layered sheets or tiles which can be used in the carpet, auto and/or construction industries as well as others.",
"The process is described with reference to FIG.",
"1 which is a partially schematic illustration of an apparatus for producing a multi-layered composite material.",
"For purposes of this description, the invention is described in connection with the production of a four-layer composite carpet tile.",
"Referring to FIG.",
"1, a roll of carpet fiber material is shown at 10.Any other material can be used.",
"This material may form the visible or upper surface of the carpet and is provided without a backing.",
"The carpet fiber material is fed as a web to an endless conveyor belt 12 which conveys the carpet fiber web through the various operating stations of the apparatus.",
"The carpet feed station may be a simple carpet unwind station (e.g.",
"form Menzel Corporation) or it may consist of a sew-in station, accumulator and/or a J-box/scray to allow time to splice rolls of fabric, carpet, etc.",
"together to allow uninterrupted flow.",
"The first station comprises a mixer or blender 14 which combines the reclaimed materials (e.g.",
"from hopper 294 of the '916 patent) with virgin material (if necessary) and other additives (such as coloring agents) according to a specified “recipe”.",
"The reclaimed backing material and other input materials may be fed to the mixer by a conventional material handling system such as a Flexicon auger system, conveyor belt, or vacuum (e.g.",
"the vacuum system from Process Control Corp).",
"The blended material within the mixer 14 is dispersed by gravity onto a displacement roller at a scattering station 16.A suitable mixer and scattering unit is sold by Schott & Meissner Co.",
"The scattering station 16 places the granulated material from mixer 14 onto the fiber web 10 as it moves with the conveyor belt 12.The scattering rate is a function of the final product specifications and depends on the desired thickness of the product and the line speed.",
"The materials can be preblended off line and/or a volumetric or gravimetric blender can be added to feed the scattering unit if needed.",
"In one practical embodiment, a tufted carpet fiber may be conveyed past station 16 at a belt speed of about 1.5 meters per minute.",
"For a reclaimed carpet tile, scattering may be at a rate of about 0.5% per minute.",
"The percent of volume is based on the quantity measuring means of a Schott and Meissner (1 meter) scattering unit.",
"If a single layer sheet, for example 1.1 mm thick, is to be produced, the material from station 16 can be scattered directly onto the belt 12 at a rate of 0.6% per minute.",
"The web is then passed through an infrared heating station 18 which preheats the reclaimed material prior to the melting and fusing stage described below.",
"In the example described above, an infrared heater from Glenro, Inc. set to a temperature of 100-110° C. was used.",
"In the illustrated embodiment, a laminate feed station is provided.",
"In this station, an intermediate sheet such as a fiberglass sheet 20 can be introduced to the laminate.",
"Any other intermediate sheet such as a prelaminated sheet or a woven reinforced sheet may also be used.",
"Typically, the intermediate sheet functions as a reinforcement to enhance the strength of the final product.",
"Its incorporation at this stage of the process avoids the need to use separate machines for each step in the production of a composite sheet.",
"The laminate feed station includes suitable control rollers which bring the intermediate (fiberglass) sheet 20 into engagement with the preheated layer of reclaimed backing material on the fiber web 10.From the laminate feed station, the three layer laminate is introduced to an oven which includes a calibration roller 22 which is preset for each product specification.",
"The roller gap depends on the heat, time of contact and pressure and ensures an even distribution of the reclaimed material over the desired width of the web.",
"For carpet tile, the precalibration roller may be compressed to a gap of 4.0 mm up to 8 mm specific to the thickness or sculpture in the top layer 10.For the single layer sheet, the setting may be 1.3 mm.",
"The oven may comprise a Thermofix contact heat oven from Schott & Meissner or a Glenro laminating oven.",
"An endless belt (not shown) keeps the material level and even as it moves through the oven.",
"After the precalibration, the material enters the heating section of the oven where heat is applied through heat plates 24 positioned as shown above and below the conveyor belt 12.The ability to apply heat from the top independently of the heat applied by the bottom plates through the conveyor belt allows multiple formulas for multiple product configurations.",
"The number of heaters used depends on the final product specification.",
"For carpet tile, the top heating units may be at 220° C. and the bottom heating units at 0° C. Three units are used for a one meter wide laminate.",
"For the single layer sheet, both top and bottom heaters may be set at 220° C. During this heating stage, which melts and fuses the reclaimed material to the fiber web 10 and the intermediate (fiberglass) sheet 20, the gasses and smoke produced are exhausted through a vent 26 and ducted to an acceptable point of discharge.",
"After the laminate has been fused it is passed through two pairs of calibrated compression or nip rollers 28 and 30 which are used to complete the desired thickness setting while the material is still soft.",
"The setting on the calibration rollers 28, 30 corresponds to that of the precalibration roller 22.If desired, the upper roller(s) can be engraved in order to emboss the product to meet the specification of a particular customer.",
"After final compression by the nip rollers 28,30, the laminate flows through a group of parallel cooling plates 32.The cooling plates are cooled by an exterior chilling system for exact temperature control and can be controlled independently to permit flexibility for a wide range of product specifications.",
"A cooling range between 10 and 15° C. may be used.",
"In the illustrated embodiment, the three layer composite is then fed through a second laminating process which is the same as the process described above.",
"Thus, another layer of reclaimed material can be introduced to the three layer laminate by means of a second mixer 34 and scattering station 36.In this case, the reclaimed material is deposited on top of the intermediate (fiberglass) web 20.It is then preheated by infrared heaters 38 and, if desired, aligned with a further intermediate sheet in a second laminate feed station.",
"In the illustrated embodiment which consists of four layers only, a second intermediate laminate is not employed.",
"The four layer laminate is then passed through a second oven which also includes a calibration roller 42, heating plates 44, calibration rollers 48,50 and cooling plates 52.The composite laminate which exits from the second oven thus consists of four layers, namely the carpet fiber web 10, reclaimed material, intermediate (fiberglass) web 20, and reclaimed material.",
"(see FIG.",
"2) By way of further example, when a second laminating process is employed, the reclaimed material may be deposited at a rate of 0.5% per minute on top of the intermediate (fiberglass) web 20.The infrared heaters 38 may be set at approximately 110° C. In the second oven, calibration roller 42 may be set at 0.5 mm.",
"The heating plates 44 may be heated to a temperature of 200° C. with the bottom heating units at 0° C. The finished product can then be passed to an accumulator 54 and then sized and packaged in station 56 in a conventional way.",
"In the preferred embodiment, the operator has the capability of determining the formula for the reclaimed material (i.e.",
"the percentage of reclaimed material, virgin material and other additives), the lines speed, heat, pressure and thickness in order to create a final composite product such as carpeting, carpet or plastic floor tiles, liner materials, mats of all description, awnings, billboards and numerous other products.",
"Typically, the reclaimed material comprises polyvinyl chloride, rubber, polycarbite waste or nylon fiber."
]
] | Patent_10258838 |
[
[
"Method and device for the mechanical-thermal separation of different materials",
"The invention relates to a method and system for the mechanical-thermal separation of different materials such as textile flat structures, threads, and plastic films by heating and simultaneously compacting the material.",
"According to the invention, the material is fed through a gap which is formed between a tool (2) and an opposing surface (3) that touches the tool while forming a point.",
"An adjustable electrical current is conducted over the contact point (5) in such a manner that a temperature profile (14), which is adapted to the density of the material, in the area of the contact point (5) is set such that the temperature of the tool (2) and opposing surface (3), independent of their shape and cross-section up to the contact point, is, in the advance direction of the material (1), increased to the same maximum value, and the contact point is heated in a defined manner."
],
[
"1.Method for the mechanical/thermal severing of different materials, such as textile fabrics, yarns and plastic films by heating and simultaneously compacting the material, the material being passed in the transporting direction through a gap, which is closed at one end and formed between a tool and a counter surface, contacting the tool at one point, a controllable electric current being passed through this contact point so that a temperature profile, adapted to the thickness of the material, develops in the area of the contact point, the temperatures of the tool and of the counter surface, independently of their shape and cross-section, increasing in the direction of the contact point, that is, in the transporting direction of the material, to the same maximum value.",
"2.The method of claim 1, characterized in that the gap is wedge-shaped and that the tool and/or the counter surface can be constructed to be stationary and/or rotating and/or oscillating.",
"3.The method of claim 1, characterized in that a directly contacting or contactless temperature sensor is used at the contact point for shortening the adjustment times.",
"4.The method of claim 1, characterized in that the tool and/or the counter surface are preheated to a basic temperature, which is related to maximum value of the temperature, which is to be attained at the contact point.",
"5.The method of claim 1, characterized in that the current flowing through the contact point is used as an error signal.",
"6.Arrangement for mechanically/thermally severing certain materials such textile fabrics, yarns or plastic sheets, by means of a tool (2) and a counter surface (3), the tool (2) being disposed with respect to the counter surface (3), so that a wedge-shaped gap (4) is formed, which is closed at one end by a contact point (5) and through which the material (1) is passed, the tool (2) and the counter surface (3) being connected with a controllable circuit (6), so that a controllable current flows through the contact point (5).",
"7.The arrangement of claim 6, characterized in that, for obtaining am error signal and/or for controlling the temperature of the contact site (5), a current detector (8) is disposed in the lead from or to the contact site (5) and a temperature sensor (7) is disposed at the contact site (5).",
"8.The arrangement of claim 6, characterized in that the geometry and material properties as well as the surface treatment of the tool (2) and of the counter surface (3) are matched to one another, in order to obtain a desired temperature profile in the area of the contact point (5)."
],
[
"The invention relates to a method and a device for mechanically-thermally severing different materials such as yarns, textile fabrics and plastic sheets by simultaneously heating and compacting as defined in claims 1 and 6.According to the known state of the art, methods and devices are known, for which indirectly heated, stationary or rotating knives as well as also directly heated, current-carrying knives or wire hoops press on a counter surface and, in so doing, melt and sever materials passing through.",
"In the DE 40 11 293 A1, a device for thermally severing textile fabrics is described, for which a heated wire hoop acts with a flattened crest on the textile fabric, severs it by a combined thermal and mechanical action and fuses the severed edge.",
"The DE 30 27 440 A1 discloses a severing device for textile labels, which consists of a knife, which can be heated, and a pressure pad, which can be pressed against the label tape in the direction of the severing edge of the knife.",
"The DE 22 14 554 discloses a device for cutting tapes, for which a cutting tool is provided, which has several cutting knives, which are disposed next to one another and press against a heated knife contact roller.",
"The sheet of fabric is brought into contact with the knife contact roller only in the regions, in which the cutting too, which is not heated, presses against the knife contact roller.",
"From the DE 25 02 724, it is known that the temperature of the blade for severing label tape is kept above the melting temperature of the tape and the temperature of the plate opposite to the blade, is maintained at, a significantly lower level.",
"From the DE 195 36 963 A1 and 196 04 735 A1, a device is known for producing textile tape from a wide sheet, for which a family of melting/cutting edges is provided, which consists of two elements, which are acted upon against one another by a force and one of which is heated.",
"The wide sheet is pulled between the cutting elements, which are in contact with one another.",
"In order to obtain a soft, melted edge at the tapes, a clamping pad is disposed, which is pressed against the face of the wide sheet in the region of the melt cut.",
"From the EP 0549748 B1, a method is known for cutting thermally, for which the active part of the hot cutting wire is disposed in a direction at right angles to the direction of movement of the sheet of textile and held inclined at an angle between 0° and 90° against the guiding plane.",
"By these means, an inclined cut is achieved, which assigns a portion of the molten mass to a scrap strip and for which the melted edge is at the back of the textile sheet.",
"It is a disadvantage of the known methods and devices that, for this method, in each case the whole severing organ is heated and that the temperature of the severing organ decreases in the direction of the contacting or severing site.",
"An optimum edge quality is obtained if the material is compacted mechanically and the melting temperature of the material is attained or exceeded briefly at one point, in order to fix the edge in this state.",
"However, because of the higher temperature of conventional severing organs the melting presses commences already with the contact between the severing organ and the material, so that a mechanical compaction cannot take place.",
"Moreover, the energy required increases out of proportion as the requirements for acceptation increase, for example, at higher speeds, in order to ensure that sufficient heat flows to the severing site.",
"Even for very thin materials, for which the energy required is actually a minimum, the whole of the severing organ must always be brought to the necessary melting or severing temperature.",
"There are applications, for which only a severing is required as an alternative for cutting, melting of the edges not being necessary.",
"For this purpose the severing organ should be as thin as possible.",
"For conventional methods, however, this is limited by the heat flow required while, at the same time, mechanical stability is maintained.",
"It is an object of the invention to develop a method and a device of the type described, with which the disadvantages of the state of the art are avoided and the material-specific severing task is ensured with a num amount of energy and a combination of temperature increase and mechanical compaction, which can be varied within wide limits.",
"This objective is accomplished with a method and a device, defined by the characterizing features of claims 1 and 2.According to the method for the mechanical/thermal severing of different materials, such as textile fabrics, yarns and plastic sheets, by heating and simultaneously compacting the material, the latter is passed through a gap, which is formed between a tool and a counter surface touching the tool punctually, a controllable electric current being passed over the point of contact so that a temperature profile, adapted the thickness of the material, is produced in the region of the point of contact in such a manner, that the temperature of the tool and the counter surface, independently of their shape and cross section, increases in the direction of the point of contract, that is in the feeding direction of the material, to the same maximum value and the point of contact is heated in a defined manner.",
"A manually adjustable or automatically controlled current flows though the contact point, heating it.",
"Due to the geometry of the tool and the counter surface, as well due to their material properties (thermal conductivity, electric conductivity, material combinations, coating surface treatment, etc.",
"), the desired temperature as well as the material properties (compaction, friction, squeezing, etc.)",
"can be affected within wide limits.",
"It is advantageous to work with very low voltages of not more than 1 volt and moderate to high currents, since this expands the selection of materials in relation to those with a moderate to good conductivity.",
"For the arrangement for mechanically/thermally severing different materials, such as textile fabrics, yarns and plastic sheets, by means of a tool and a counter surface, the tool is disposed with respect to the counter surface so that a wedge-shaped gap with a punctual contacting site is formed, through which the material is passed, the tool and the counter surface being connected with a controllable circuit, so that a controllable current flows thought the contact site.",
"The tool and the counter surface may be stationary or one of the two may rotate or oscillate or both may rotate or oscillate.",
"In the rotating version, profiled tools (zigzag) or perforated tools are, for example, also conceivable, provided that it is insured that the contact is always maintained.",
"Since the highest temperature always exists at the point of contact, a temperature sensor can be mounted there directly with contacting or contactless (IR) detection, as a result of which very short adjustment times can be realized.",
"The current flowing through the point of contact can be used, at the same time, as an error signal.",
"Each interruption in the current, even if it is of very short duration, necessarily results in materials not being severed.",
"If necessary or desirable, a soft severing edge in the material is attained particularly owing to the fact that only a heated point is used for the cutting, the heat is developed in the severing site and the temperature of the material before the severing is still below the melting point.",
"Advantageous developments of the invention are given in the dependent claims.",
"The invention is described in greater detail in the following by an example of the device severing polyester material.",
"In the associated drawing, FIG.",
"1 shows a diagrammatic representation of an inventive device, FIG.",
"2 shows the diagrammatic representation of the temperature profile at the severing site with a device of the state of the art and FIG.",
"3 shows a diagrammatic representation of the temperature profile at the severing site (in region of the point of contact) when the inventive method is used As shown in FIG.",
"1, the device for mechanically/thermally severing different materials pursuant to the invention consists essentially of a tool 2 and a counter surface 3.The tool 2 and the counter surface 3 are positioned with respect to one another so that a wedge-shaped gap 4 with a punctual contacting site 5, though which the material 1 is passed (FIGS.",
"2 and 3), is formed between them.",
"The tool 2 and the counter surface 3 are connected with a controllable circuit 6, over which the contacting site 5 can be heated in a controlled manner.",
"For controlling the temperature of the contacting site 5, a temperature sensor 7, which can measure the temperature by direct contact or in a contactless manner, is connected at the contacting site 5.For obtaining an error signal, a current detector 8 is disposed in the lead to or from the contact point 5.The tool 2 and the counter surface 3 can have different geometric shapes.",
"In order to explain the inventive method, the temperature profile at the severing site or contacting site 5 when the known method and devices are used (FIG.",
"2) is compared with that of the inventive method and device (FIG.",
"3).",
"For a direct comparison, the same masses and geometry of the severing organs (tool 2) were selected.",
"With these, a somewhat compressible polyester material was to be severed, for example.",
"The melting point 10 of the material 1 is at 260° C. For the conventional method of FIG.",
"2, the melting process commences when the first fibers of material 1 come into contact with the current-carrying severing hoop 2, because the temperature of the latter, due to the design of the device, must already be appreciably above the melting temperature 10 of the material 1.For this reason also, mechanical compaction is not possible and the displacement of the material 1 by the hoop 2 results in the formation of a slight bead.",
"Since the heat is supplied over the hoop 2 and the contacting site 5 provides heat to the counter surfaced 3 and the material 1, the hoop 2 has the lowest temperature at the place 5 where it contacts the counter surface 3.However, this temperature must still always be higher than the melting temperature 10 of the material 1, in order to sever the latter reliably.",
"As the speed of the material increases (material is transported in the direction of the arrow), the temperature difference becomes larger, because only little thermal energy is absorbed at the sections of the hoop not in contact with the material.",
"In contrast to this, the melting temperature 10 is exceeded less and for a shorter time for the inventive method according to the representation in FIG.",
"3.In the plasticization zone 12, mechanical compaction 11 can take place, which compensates for the displacement of material in the melting zone 13 and avoids the formation of a bead.",
"As the speed of the material increases (material transported in the direction of the arrow), additional thermal energy is supplied over the contact point 5 to the melting zone 13 and absorbed there by the material 1.By controlling the temperature at the contact point 5, it is possible to ensure that the amount of energy supplied is only sufficient for maintaining the temperature level.",
"According to an advantageous embodiment of the method, the tool 2 and/or the counter surface 3 are preheated to an average temperature level in relation to the maximum temperature, which is to be attained in the region of the contact point 5.The basic temperature may amount, for example, to 80% of the melting temperature of the material 1, so that only the thermal energy difference for raising the temperature from the basic to the maximum and necessary for the cutting/melting, must still be introduced into the contact point 5.The combination of conventionally heating (basic temperature) the tool 2 and/or the counter surface 3 and of heating the contact point 5 by the method, expands the range of applications of the method with regard to material thicknesses, which are to be processed, and the feeding speeds of the material 1.When the inventive method is used for materials, which essentially cannot be compressed, it should be noted that, in order to avoid excessive bead formation, the thickness of the tool 2 must be changed or minimized correspondingly.",
"If more melting of the edges is desired, this can be achieved by the appropriate geometric shaping of the tool 2 or of the counter surface 3.The inventive method ensures further advantageous applications.",
"The counter surface 3 can be constructed so that, by active heating over the contact point 5, it brings a considerable amount of energy from the opposite side into the material 1.If melting of the edges is undesirable (melting usually is always associated with some hardening), a plasticized compaction of the edge can be achieved at the contact point 5 at a temperature level below the melting point of the material by increased mechanical squeezing.",
"With that, materials, containing portions, which are not thermoplastic, can also be severed.",
"The following can be severed particularly advantageously with this method: very thin, thermoplastic materials, since the thermal energy is a minimum, thin to average thermoplastic materials, which are to be fused easily without thickening the edges, up to average thicknesses of compressible, thermoplastic materials and materials, suitable for squeezing/cutting, for which the cutting forces can be reduced by the action of heat and/or the edges are to be fused."
]
] | Patent_10258986 |
[
[
"Heterocyclic cyclopentyl tetrahydroisoquinoline and tetrahydropyridopyridine modulators of chemokine receptor activity",
"The present invention is directed to compounds of the formula I: Wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, X, n and the broken lines are as defined herein which are useful as modulators of chemokine receptor activity.",
"In particular, these compounds are useful as modulators of the chemokine receptor CCR-2."
],
[
"1.A compound of formula I: wherein: X is selected from the group consisting of: C, N, O, S and SO2; Y is N or C; R1 is selected from the group consisting of: hydrogen, —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, —C0-6alkyl-S—C1-6alkyl, —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), hydroxy, heterocycle, —CN, —NR12R12, —NR12COR13, —NR12SO2R14, —COR11, —CONR12R12, and phenyl, where R11 is independently selected from the group consisting of: hydroxy, hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R12 is selected from the group consisting of: hydrogen, C1-6 alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R13 is selected from the group consisting of: hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R14 is selected from the group consisting of: hydroxy, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl and C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where said substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where said alkyl and said cycloalkyl are unsubstituted or substituted with 1-7 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-13alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-3alkyl, (h) —COR11, (i) —SO2R14, (j) —NHCOCH3, (k) —NHSO2CH3, (l) -heterocycle, (m) ═O and (n) —CN, and where said phenyl and heterocycle are unsubstituted or substituted with 1-3 substituents where said substituents are independently selected from the group consisting of: halo, hydroxy, C1-3alkyl, C1-3alkoxy and trifluoromethyl; R2 is selected from the group consisting of: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where the alkyl is unsubstituted or substituted with 1-6 substituents independently selected from fluoro and hydroxy, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j) —NR12CONR12R12, (k) -heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (p) —SO2—NR12R12, and (p) ═O, where R2 is connected to the ring via a double bond; R3 is oxygen or is absent when Y is N; R3 is selected from when Y is C: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where said alkyl is unsubstituted or substituted with 1-6 substituents independently selected from: fluoro, hydroxy, and —COR11, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j) —NR12CONR12R12, (k) -heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (o)—SO2—NR12R12 and (p) nitro; R4 is selected from the group consisting of: (a) hydrogen, (b) C1-6alkyl, (c) trifluoromethyl, (d) trifluoromethoxy, (e) chloro, (f) fluoro, (g) bromo, and (h) phenyl; R5 is selected from the group consisting of: (a) C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro and optionally substituted with hydroxyl, (b) —O—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (c) —CO—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (d) —S—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (e)-pyridyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (f) fluoro, (g) chloro, (h) bromo, (i) —C4-6cycloalkyl, (j) —O—C4-6cycloalkyl, (k) phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (l) —O-phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (m) —C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (n) —O—C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (o) -heterocycle, (p) —CN, and (q) —COR11; R6 is selected from: (a) hydrogen, (b) C1-6alkyl, (c) trifluoromethyl, (d) fluoro, (e) chloro, and (f) bromo; R7 is selected from: hydrogen, (C0-6alkyl)-phenyl, (C0-6alkyl)-heterocycle, (C0-6alkyl)-C3-7cycloalkyl, (C0-6alkyl)-COR11, (C0-6alkyl)-(alkene)-COR11, (C0-6alkyl)-SO3H, (C0-6alkyl)—W—C0-4alkyl, (C0-6alkyl)-CONR12-phenyl, (C0-6alkyl)-CONR15—V—COR11, and nothing (when X is O, S, or SO2), where V is C1-6alkyl or phenyl, where W is selected from the group consisting of: a single bond, —O—, —S—, —SO—, —SO2—, —CO—, —CO2—, —CONR12— and —NR12—, where the R15 can be hydrogen, C1-4alkyl, or where R15 is joined via a 1-5 carbon tether to one of the carbons of V to form a ring, where the C0-6alkyl is unsubstituted or substituted with 1-5 substituents, where said substituents are independently selected from: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, where said phenyl, heterocycle, cycloalkyl, and C0-4alkyl is unsubstituted or substituted with 1-5 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where the phenyl and heterocycle may be fused to another heterocycle, which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl, and where alkene is unsubstituted or substituted with 1-3 substituents which are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) C1-3alkyl, (d) phenyl, and (e) heterocycle; R8 is selected from the group consisting of: (a) hydrogen, (b) nothing when X is either O, S, SO2 or N or when a double bond joins the carbons to which R7 and R10 are attached, (c) hydroxy, (d) C1-6alkyl, (e) C1-6alkyl-hydroxy, (f) —O—C1-3alkyl, (g) —COR11, (h) —CONR12R12, and (i) —CN; or where R7 and R8 are joined together to form a ring which is selected from the group consisting of: (a) 1H-indene, (b) 2,3-dihydro-1H-indene, (c) 2,3-dihydro-benzofuran, (d) 1,3-dihydro-isobenzofuran, (e) 2,3-dihydro-benzothiofuran, (f) 1,3-dihydro-isobenzothiofuran, (g) 6H-cyclopenta[d]isoxazol-3-ol (h) cyclopentane, and (i) cyclohexane, where said ring formed may be unsubstituted or substituted with 1-5 substituents independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where R7 and R9 or R8 and R10 may be joined together to form a ring which is phenyl or heterocycle, wherein said ring is unsubstituted or substituted with 1-7 substituents where said substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-13alkyl, (f) —COR11 (g) —CN, (h) —NR12R12, and (i) —CONR12R12; R9 and R10 are independently selected from the group consisting of: (a) hydrogen, (b) hydroxy, (c) C1-6alkyl, (d) C1-6alkyl-COR11, (e) C1-6alkyl-hydroxy, (f) —O—C1-3alkyl, (g)═O, when R9 or R10 is connected to the ring via a double bond (h) halo; n is selected from 0, 1 and 2; the dashed line represents a single or a double bond; and pharmaceutically acceptable salts thereof and individual diastereomers thereof.",
"2.The compound of claim 1 having formula Ia: wherein R16 and R17 are independently selected from the group consisting of: (a) hydrogen, (b) halo, (c) trifluoromethyl, (d) hydroxy, (e) C1-3alkyl, (f) —O—C1-3alkyl, (g) —C0-3—CO2H, (h) —C0-3—CO2C1-3alkyl, (i) —CN, and (j) —C0-3-heterocycle, or where the R16 and R17 are joined together to form a heterocycle which is fused to the phenyl ring, and which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"3.The compound of claim 1 having the formula Ib: and pharmaceutically acceptable salts and individual diastereomers thereof.",
"4.The compound of claim 1 having formula: and pharmaceutically acceptable salts and individual diastereomers thereof.",
"5.The compound of claim 1 having formula Id: wherein said C1-4 carbon chain may be unsubstituted, or substituted with 1-4 substituents which are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, where said C1-4 carbon chain optionally is included within a C3-7cycloalkyl ring, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"6.The compound of claim 1 having formula Ie: wherein the dotted lines represent a single or double bond, o is 1 or 2, and A, B, and D are independently selected from C, N, O, or S, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"7.The compound of claim 1 having formula If: wherein X is N or O, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"8.The compound of claim 1 having formula Ig: wherein R16 and R17 optionally join to form a heterocycle which is fused to the phenyl ring, and wherein said ring is optionally substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"9.The compound of claim 1 having formula Ih: and pharmaceutically acceptable salts and individual diastereomers thereof.",
"10.The compound of claim 1 having formula Ii: wherein Het is a heterocycle, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"11.The compound of claim 1 having formula Ij: wherein said C1-4 carbon chain is optionally substituted with 1-4 substituents independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, and pharmaceutically acceptable salts and individual stereoisomers thereof.",
"12.The compound of claim 1 having formula Ik: and pharmaceutically acceptable salts and individual diastereomers thereof.",
"13.The compound of claim 1 wherein R1 is selected from the group consisting of: —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, and —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), where the alkyl and the cycloalkyl are unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-3alkyl, (h) —COR11, (i) —CN, (j) —NR12R12, and (k) —CONR12R12, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"14.The compound of claim 1 wherein R1 is selected from the group consisting of: (1) —C1-6alkyl, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, and (e) —COR11, (2) —C0-6alkyl-O—C1-6alkyl-, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) trifluoromethyl, and (c) —COR11, (3) and —(C3-5cycloalkyl)-(C0-6alkyl), which is unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from the group consisting of: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, and (e) —COR11.15.The compound of claim 14 wherein R1 is selected from the group consisting of: (a) C1-6alkyl, (b) C1-6alkyl substituted with hydroxy and (c) C1-6alkyl substituted with 1-6 fluoro.",
"16.The compound of claim 15 wherein R1 is selected from the group consisting of: (a) —CH(CH3)2 (b) —CH(OH)CH3, and (c) —CH2CF3.17.A compound selected from: 18.A pharmaceutical composition which comprises an inert carrier and a compound of claim 1.19.A method for modulation of chemokine receptor activity in a mammal which comprises the administration of an effective amount of the compound of claim 1.20.A method for treating, ameliorating, controlling or reducing the risk of an inflammatory and immunoregulatory disorder or disease which comprises the administration to a patient of an effective amount of the compound of claim 1.21.A method for treating, ameliorating, controlling or reducing the risk of rheumatoid arthritis which comprises the administration to a patient of an effective amount of the compound of claim 1.22.A method for treating, ameliorating, controlling or reducing the risk of stroke which comprises the administration to a patient of an effective amount of the compound of claim 1.23.A method for treating, ameliorating, controlling or reducing the risk of cancer which comprises the administration to a patient of an effective amount of the compound of claim 1."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The chemokines are a family of small (70-120 amino acids), proinflammatory cytokines, with potent chemotactic activities.",
"Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract various cells, such as monocytes, macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in Schall, Cytokine, 3, 165-183 (1991) and Murphy, Rev.",
"Immun., 12, 593-633 (1994)).",
"These molecules were originally defined by four conserved cysteines and divided into two subfamilies based on the arrangement of the first cysteine pair.",
"In the CXC-chemokine family, which includes IL-8, GROα, NAP-2 and IP-10, these two cysteines are separated by a single amino acid, while in the CC-chemokine family, which includes RANTES, MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β and eotaxin, these two residues are adjacent.",
"The α-chemokines, such as interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils, whereas β-chemokines, such as RANTES, MIP-1α, MIP-1β, monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and eotaxin are chemotactic for macrophages, monocytes, T-cells, eosinophils and basophils (Deng, et al., Nature, 381, 661-666 (1996)).",
"The chemokines are secreted by a wide variety of cell types and bind to specific G-protein coupled receptors (GPCRs) (reviewed in Horuk, Trends Pharm.",
"Sci., 15, 159-165 (1994)) present on leukocytes and other cells.",
"These chemokine receptors form a sub-family of GPCRs, which, at present, consists of fifteen characterized members and a number of orphans.",
"Unlike receptors for promiscuous chemoattractants such as C5a, fMLP, PAF, and LTB4, chemokine receptors are more selectively expressed on subsets of leukocytes.",
"Thus, generation of specific chemokines provides a mechanism for recruitment of particular leukocyte subsets.",
"On binding their cognate ligands, chemokine receptors transduce an intracellular signal though the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration.",
"There are at least seven human chemokine receptors that bind or respond to β-chemokines with the following characteristic pattern: CCR-1 (or “CKR-1” or “CC-CKR-1”) [MIP-1α, MIP-1β, MCP-3, RANTES] (Ben-Barruch, et al., J. Biol.",
"Chem., 270, 22123-22128 (1995); Beote, et al, Cell, 72, 415-425 (1993)); CCR-2A and CCR-2B (or “CKR-2A”/“CKR-2A” or “CC-CKR-2A”/“CC-CKR-2A”) [MCP-1, MCP-2, MCP-3, MCP-4]; CCR-3 (or “CKR-3” or “CC-CKR-3”) [Eotaxin, Eotaxin 2, RANTES, MCP-2, MCP-3] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-4 (or “CKR-4” or “CC-CKR-4”) [MIP-1α, RANTES, MCP-1] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-5 (or “CKR-5” or “CC-CKR-5”) [MIP-1α, RANTES, MIP-1β] (Sanson, et al., Biochemistry, 35, 3362-3367 (1996)); and the Duffy blood-group antigen [RANTES, MCP-1] (Chaudhun, et al., J. Biol.",
"Chem., 269, 7835-7838 (1994)).",
"The β-chemokines include eotaxin, MIP (“macrophage inflammatory protein”), MCP (“monocyte chemoattractant protein”) and RANTES (“regulation-upon-activation, normal T expressed and secreted”) among other chemokines.",
"Chemokine receptors, such as CCR-1, CCR-2, CCR-2A, CCR-2B, CCR-3, CCR-4, CCR-5, CXCR-3, CXCR-4, have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma, rhinitis and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.",
"Humans who are homozygous for the 32-basepair deletion in the CCR-5 gene appear to have less susceptibility to rheumatoid arthritis (Gomez, et al., Arthritis & Rheumatism, 42, 989-992 (1999)).",
"A review of the role of eosinophils in allergic inflammation is provided by Kita, H., et al., J. Exp.",
"Med.",
"183, 2421-2426 (1996).",
"A general review of the role of chemokines in allergic inflammation is provided by Lustger, A. D., New England J.",
"Med., 338(7), 426-445 (1998).",
"A subset of chemokines are potent chemoattractants for monocytes and macrophages.",
"The best characterized of these is MCP-1 (monocyte chemoattractant protein-1), whose primary receptor is CCR2.MCP-1 is produced in a variety of cell types in response to inflammatory stimuli in various species, including rodents and humans, and stimulates chemotaxis in monocytes and a subset of lymphocytes.",
"In particular, MCP-1 production correlates with monocyte and macrophage infiltration at inflammatory sites.",
"Deletion of either MCP-1 or CCR2 by homologous recombination in mice results in marked attenuation of monocyte recruitment in response to thioglycollate injection and Listeria monocytogenes infection (Lu et al., J. Exp.",
"Med., 187, 601-608 (1998); Kurihara et al.",
"J. Exp.",
"Med., 186, 1757-1762 (1997); Boring et al.",
"J. Clin.",
"Invest., 100, 2552-2561 (1997); Kuziel et al.",
"Proc.",
"Natl.",
"Acad.",
"Sci., 94, 12053-12058 (1997)).",
"Furthermore, these animals show reduced monocyte infiltration into granulomatous lesions induced by the injection of schistosomal or mycobacterial antigens (Boring et al.",
"J. Clin.",
"Invest., 100, 2552-2561 (1997); Warmington et al.",
"Am J.",
"Path., 154, 1407-1416 (1999)).",
"These data suggest that MCP-1-induced CCR2 activation plays a major role in monocyte recruitment to inflammatory sites, and that antagonism of this activity will produce a sufficient suppression of the immune response to produce therapeutic benefits in immunoinflammatory and autoimmune diseases.",
"Accordingly, agents which modulate chemokine receptors such as the CCR-2 receptor would be useful in such disorders and diseases.",
"In addition, the recruitment of monocytes to inflammatory lesions in the vascular wall is a major component of the pathogenesis of atherogenic plaque formation.",
"MCP-1 is produced and secreted by endothelial cells and intimal smooth muscle cells after injury to the vascular wall in hypercholesterolemic conditions.",
"Monocytes recruited to the site of injury infiltrate the vascular wall and differentiate to foam cells in response to the released MCP-1.Several groups have now demonstrated that aortic lesion size, macrophage content and necrosis are attenuated in MCP-1−/− or CCR24−/− mice backcrossed to APO-E −/−, LDL-R −/− or Apo B transgenic mice maintained on high fat diets (Boring et al.",
"Nature, 394, 894-897 (1998); Gosling et al.",
"J. Clin.",
"Invest., 103, 773-778 (1999)).",
"Thus, CCR2 antagonists may inhibit atherosclerotic lesion formation and pathological progression by impairing monocyte recruitment and differentiation in the arterial wall."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is further directed to compounds which are modulators of chemokine receptor activity and are useful in the prevention or treatment of certain inflammatory and immunoregulatory disorders and diseases, allergic diseases, atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and asthma, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.",
"The invention is also directed to pharmaceutical compositions comprising these compounds and the use of these compounds and compositions in the prevention or treatment of such diseases in which chemokine receptors are involved.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"BACKGROUND OF THE INVENTION The chemokines are a family of small (70-120 amino acids), proinflammatory cytokines, with potent chemotactic activities.",
"Chemokines are chemotactic cytokines that are released by a wide variety of cells to attract various cells, such as monocytes, macrophages, T cells, eosinophils, basophils and neutrophils to sites of inflammation (reviewed in Schall, Cytokine, 3, 165-183 (1991) and Murphy, Rev.",
"Immun., 12, 593-633 (1994)).",
"These molecules were originally defined by four conserved cysteines and divided into two subfamilies based on the arrangement of the first cysteine pair.",
"In the CXC-chemokine family, which includes IL-8, GROα, NAP-2 and IP-10, these two cysteines are separated by a single amino acid, while in the CC-chemokine family, which includes RANTES, MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β and eotaxin, these two residues are adjacent.",
"The α-chemokines, such as interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils, whereas β-chemokines, such as RANTES, MIP-1α, MIP-1β, monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and eotaxin are chemotactic for macrophages, monocytes, T-cells, eosinophils and basophils (Deng, et al., Nature, 381, 661-666 (1996)).",
"The chemokines are secreted by a wide variety of cell types and bind to specific G-protein coupled receptors (GPCRs) (reviewed in Horuk, Trends Pharm.",
"Sci., 15, 159-165 (1994)) present on leukocytes and other cells.",
"These chemokine receptors form a sub-family of GPCRs, which, at present, consists of fifteen characterized members and a number of orphans.",
"Unlike receptors for promiscuous chemoattractants such as C5a, fMLP, PAF, and LTB4, chemokine receptors are more selectively expressed on subsets of leukocytes.",
"Thus, generation of specific chemokines provides a mechanism for recruitment of particular leukocyte subsets.",
"On binding their cognate ligands, chemokine receptors transduce an intracellular signal though the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration.",
"There are at least seven human chemokine receptors that bind or respond to β-chemokines with the following characteristic pattern: CCR-1 (or “CKR-1” or “CC-CKR-1”) [MIP-1α, MIP-1β, MCP-3, RANTES] (Ben-Barruch, et al., J. Biol.",
"Chem., 270, 22123-22128 (1995); Beote, et al, Cell, 72, 415-425 (1993)); CCR-2A and CCR-2B (or “CKR-2A”/“CKR-2A” or “CC-CKR-2A”/“CC-CKR-2A”) [MCP-1, MCP-2, MCP-3, MCP-4]; CCR-3 (or “CKR-3” or “CC-CKR-3”) [Eotaxin, Eotaxin 2, RANTES, MCP-2, MCP-3] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-4 (or “CKR-4” or “CC-CKR-4”) [MIP-1α, RANTES, MCP-1] (Rollins, et al., Blood, 90, 908-928 (1997)); CCR-5 (or “CKR-5” or “CC-CKR-5”) [MIP-1α, RANTES, MIP-1β] (Sanson, et al., Biochemistry, 35, 3362-3367 (1996)); and the Duffy blood-group antigen [RANTES, MCP-1] (Chaudhun, et al., J. Biol.",
"Chem., 269, 7835-7838 (1994)).",
"The β-chemokines include eotaxin, MIP (“macrophage inflammatory protein”), MCP (“monocyte chemoattractant protein”) and RANTES (“regulation-upon-activation, normal T expressed and secreted”) among other chemokines.",
"Chemokine receptors, such as CCR-1, CCR-2, CCR-2A, CCR-2B, CCR-3, CCR-4, CCR-5, CXCR-3, CXCR-4, have been implicated as being important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma, rhinitis and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.",
"Humans who are homozygous for the 32-basepair deletion in the CCR-5 gene appear to have less susceptibility to rheumatoid arthritis (Gomez, et al., Arthritis & Rheumatism, 42, 989-992 (1999)).",
"A review of the role of eosinophils in allergic inflammation is provided by Kita, H., et al., J. Exp.",
"Med.",
"183, 2421-2426 (1996).",
"A general review of the role of chemokines in allergic inflammation is provided by Lustger, A. D., New England J.",
"Med., 338(7), 426-445 (1998).",
"A subset of chemokines are potent chemoattractants for monocytes and macrophages.",
"The best characterized of these is MCP-1 (monocyte chemoattractant protein-1), whose primary receptor is CCR2.MCP-1 is produced in a variety of cell types in response to inflammatory stimuli in various species, including rodents and humans, and stimulates chemotaxis in monocytes and a subset of lymphocytes.",
"In particular, MCP-1 production correlates with monocyte and macrophage infiltration at inflammatory sites.",
"Deletion of either MCP-1 or CCR2 by homologous recombination in mice results in marked attenuation of monocyte recruitment in response to thioglycollate injection and Listeria monocytogenes infection (Lu et al., J. Exp.",
"Med., 187, 601-608 (1998); Kurihara et al.",
"J. Exp.",
"Med., 186, 1757-1762 (1997); Boring et al.",
"J. Clin.",
"Invest., 100, 2552-2561 (1997); Kuziel et al.",
"Proc.",
"Natl.",
"Acad.",
"Sci., 94, 12053-12058 (1997)).",
"Furthermore, these animals show reduced monocyte infiltration into granulomatous lesions induced by the injection of schistosomal or mycobacterial antigens (Boring et al.",
"J. Clin.",
"Invest., 100, 2552-2561 (1997); Warmington et al.",
"Am J.",
"Path., 154, 1407-1416 (1999)).",
"These data suggest that MCP-1-induced CCR2 activation plays a major role in monocyte recruitment to inflammatory sites, and that antagonism of this activity will produce a sufficient suppression of the immune response to produce therapeutic benefits in immunoinflammatory and autoimmune diseases.",
"Accordingly, agents which modulate chemokine receptors such as the CCR-2 receptor would be useful in such disorders and diseases.",
"In addition, the recruitment of monocytes to inflammatory lesions in the vascular wall is a major component of the pathogenesis of atherogenic plaque formation.",
"MCP-1 is produced and secreted by endothelial cells and intimal smooth muscle cells after injury to the vascular wall in hypercholesterolemic conditions.",
"Monocytes recruited to the site of injury infiltrate the vascular wall and differentiate to foam cells in response to the released MCP-1.Several groups have now demonstrated that aortic lesion size, macrophage content and necrosis are attenuated in MCP-1−/− or CCR24−/− mice backcrossed to APO-E −/−, LDL-R −/− or Apo B transgenic mice maintained on high fat diets (Boring et al.",
"Nature, 394, 894-897 (1998); Gosling et al.",
"J. Clin.",
"Invest., 103, 773-778 (1999)).",
"Thus, CCR2 antagonists may inhibit atherosclerotic lesion formation and pathological progression by impairing monocyte recruitment and differentiation in the arterial wall.",
"SUMMARY OF THE INVENTION The present invention is further directed to compounds which are modulators of chemokine receptor activity and are useful in the prevention or treatment of certain inflammatory and immunoregulatory disorders and diseases, allergic diseases, atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and asthma, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.",
"The invention is also directed to pharmaceutical compositions comprising these compounds and the use of these compounds and compositions in the prevention or treatment of such diseases in which chemokine receptors are involved.",
"DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to compounds of the formula I: X is selected from: C, N, O, S and SO2; Y is selected from N or C. R1 is selected from: hydrogen, —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, —C0-6alkyl-S—C1-6alkyl, —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), hydroxy, heterocycle, —CN, —NR12R12, —NR12COR13, —NR12SO2R14, —COR11, —CONR12R12, and phenyl, where R11 is independently selected from: hydroxy, hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl, C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-16 alkyl, and trifluoromethyl, and where R12 is selected from: hydrogen, C1-6 alkyl, benzyl, phenyl, C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R13 is selected from: hydrogen, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl, C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where R14 is selected from: hydroxy, C1-6 alkyl, —O—C1-6alkyl, benzyl, phenyl, C3-6 cycloalkyl where the alkyl, phenyl, benzyl, and cycloalkyl groups can be unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: halo, hydroxy, C1-3alkyl, C1-3alkoxy, —CO2H, —CO2—C1-6 alkyl, and trifluoromethyl, and where the alkyl and the cycloalkyl are unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-3alkyl, (h) —COR11, (i) —SO2R14, (j) —NHCOCH3, (k) —NHSO2CH3, (l) -heterocycle, (m) ═O, (n) —CN, and where the phenyl and heterocycle are unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: halo, hydroxy, C1-3alkyl, C1-3alkoxy and trifluoromethyl; R2 is selected from: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where the alkyl is unsubstituted or substituted with 1-6 substituents independently selected from: fluoro, and hydroxy, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j)—NR12CONR12R12, (k) -heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (o)—SO2—NR12R12, and (p) ═O, where R2 is connected to the ring via a double bond; R3 is oxygen or is absent when Y is N; R3 is selected from the following list when Y is C: (a) hydrogen, (b) hydroxy, (c) halo, (d) C1-3alkyl, where the alkyl is unsubstituted or substituted with 1-6 substituents independently selected from: fluoro, hydroxy, and —COR11, (e) —NR12R12, (f) —COR11, (g) —CONR12R12, (h) —NR12COR13, (i) —OCONR12R12, (j)—NR12CONR12R12, (k)-heterocycle, (l) —CN, (m) —NR12—SO2—NR12R12, (n) —NR12—SO2—R14, (o)—SO2—NR12R12 and (p) nitro; R4 is selected from: (a) hydrogen, (b) C1-6alkyl, (c) trifluoromethyl, (d) trifluoromethoxy, (e) chloro, (f) fluoro, (g) bromo, and (h) phenyl; R5 is selected from: (a) C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro and optionally substituted with hydroxyl, (b) —O—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (c) —CO—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (d) —S—C1-6alkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (e)-pyridyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (f) fluoro, (g) chloro, (h) bromo, (i) —C4-6cycloalkyl, (j) —O—C4-6cycloalkyl, (k) phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (l) —O-phenyl, which may be unsubstituted or substituted with one or more substituents selected from the group consisting of: halo, trifluoromethyl, C1-4alkyl, and COR11, (m) —C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (n) —O—C3-6cycloalkyl, where alkyl may be unsubstituted or substituted with 1-6 fluoro, (o)-heterocycle, (p) —CN, and (q) —COR11; R6 is selected from: (a) hydrogen, (b) C1-6alkyl, and (c) trifluoromethyl (d) fluoro (e) chloro, and (f) bromo; R7 is selected from: nothing (when X═O), hydrogen, (C0-6alkyl)-phenyl, (C0-6alkyl)-heterocycle, (C0-6alkyl)-C3-7cycloalkyl, (C0-6alkyl)-COR11, (C0-6alkyl)-(alkene)-COR11, (C0-6alkyl)-SO3H, (C0-6alkyl)-W—C0-4alkyl, (C0-6alkyl)-CONR12-phenyl, (C0-6alkyl)-CONR15—V—COR11, and nothing (when X is O, S, or SO2), where V is selected from C1-6alkyl or phenyl, and where W is selected from: a single bond, —O—, —S—, —SO—, —SO2—, —CO—, —CO2—, —CONR12— and —NR12—, and where the R15 can be hydrogen, C1-4alkyl, or where R15 is joined via a 1-5 carbon tether to one of the carbons of V to form a ring, and where the C0-6alkyl is unsubstituted or substituted with 1-5 substituents, where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, and where the phenyl, heterocycle, cycloalkyl, and C0-4alkyl is unsubstituted or substituted with 1-5 substituents where the substituents are independently selected from: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-13alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where the phenyl and heterocycle may be fused to another heterocycle, which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl, and where alkene is unsubstituted or substituted with 1-3 substituents which are independently selected from: (a) halo, (b) trifluoromethyl, (c) C1-3alkyl, (d) phenyl, and (e) heterocycle; R8 is selected from: (a) hydrogen, (b) nothing when X is either O, S, SO2 or N or when a double bond joins the carbons to which R7 and R10 are attached, (c) hydroxy, (d) C1-6alkyl, (e) C1-6alkyl-hydroxy, (f) —O—C1-3alkyl, (g) —COR11, (h) —CONR12R12, and (i) —CN; or where R7 and R8 may be joined together to form a ring which is selected from: (a) 1H-indene, (b) 2,3-dihydro-1H-indene, (c) 2,3-dihydro-benzofuran, (d) 1,3-dihydro-isobenzofuran, (e) 2,3-dihydro-benzothiofuran, (f) 1,3-dihydro-isobenzothiofuran, (g) 6H-cyclopenta[d]isoxazol-3-ol (h) cyclopentane, and (i) cyclohexane, where the ring formed may be unsubstituted or substituted with 1-5 substituents independently selected from: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —C0-3—COR11, (g) —CN, (h) —NR12R12, (i) —CONR12R12, and (j) —C0-3-heterocycle, or where R7 and R9 or R8 and R10 may be joined together to form a ring which is phenyl or heterocycle, wherein the ring is unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-13alkyl, (f) —COR11, (g) —CN, (h) —NR12R12, and (i) —CONR12R12; R9 and R10 are independently selected from: (a) hydrogen, (b) hydroxy, (c) C1-6alkyl, (d) C1-6alkyl-COR11, (e) C1-6alkyl-hydroxy, (f) —O—C1-13alkyl, (g) ═O, when R9 or R10 is connected to the ring via a double bond (h) halo; n is selected from 0, 1 and 2; the dashed line represents a single or a double bond; and pharmaceutically acceptable salts thereof and individual diastereomers thereof.",
"Another embodiment of the present invention includes compounds of formula Ia: wherein R1, R2, R3, R5, R9, Y, and n are defined herein, and wherein R16 and R17 are independently selected from: (a) hydrogen, (b) halo, (c) trifluoromethyl, (d) hydroxy, (e) C1-3alkyl, (f) —O—C1-3alkyl, (g) —C0-3—CO2H, (h) —C0-3—CO2C1-3alkyl, (i) —CN, and —(j) —C0-3-heterocycle, or where the R16 and R17 are joined together to form a heterocycle which is fused to the phenyl ring, and which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"Another embodiment of the present invention also includes compounds of formula Ib: wherein the dashed line represents a single or a double bond and R1, R2, R3, R5, R9, R16, R17, Y, and n are defined herein; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"A still further embodiment of the present invention includes compounds of formula Ic: wherein R1, R2, R3, R5, R9, R16, R17, Y, and n are defined herein, and where Het is a heterocycle and pharmaceutically acceptable salts and individual diastereomers thereof.",
"Another embodiment of the present invention include compounds of formula Id: wherein R1, R2, R3, R5, R9, R11, Y, W, and n are defined herein and where the C1-4 carbon chain may be unsubstituted, or substituted with 1-4 substituents which are independently selected from: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-13alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, or where the C1-4 carbon chain may be included within a C3-7cycloalkyl ring, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"A further embodiment of the present invention includes compounds of formula Ie: wherein R1, R2, R3, R5, R9, R16, R17, X, Y, and n are defined herein, and where the dotted lines can represent either a single or double bond, and where o can be 1 or 2, and where A, B, and D, can independently be selected from C, N, O, or S, to make a phenyl ring (when X, A, B, D, are all C, and o=2) or to make a heterocycle when at least one of X, A, B, D are N, O, or S and not C, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"A still further embodiment of the present invention includes compounds of formula If: wherein R1, R2, R3, R5, R7, R9, R10, Y, and n are defined herein, and X is either N, or O (in which case R7 is nothing) and pharmaceutically acceptable salts and individual diastereomers thereof.",
"Another embodiment of the present invention includes compounds of formula Ig: wherein R1, R5, R9, R16, R17, and Y are defined herein, or where the R16 and R17 are joined together to form a heterocycle which is fused to the phenyl ring, and which itself may be unsubstituted or substituted with 1-2 substituents independently selected from hydroxy, halo, —COR11, and —C1-3alkyl; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"A further embodiment of the present invention includes compounds of formula Ih: wherein the dashed line represents a single or a double bond and R1, R5, R9, R16, R17, and Y are defined herein; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"An additional embodiment of the present invention includes compounds of formula II: wherein R1, R5, R9, R16, R17, and Y are defined herein, and where Het is a heterocycle; and pharmaceutically acceptable salts and individual diastereomers thereof.",
"A still further embodiment of the present invention includes compounds of formula Ij: wherein R1, R5, R9, R11, y, and W are defined herein and where the C1-4 carbon chain may be unsubstituted, or substituted with 1-4 substituents which are independently selected from: (a) halo, (b) hydroxy, (c) —C0-6alkyl (d) —O—C1-3alkyl, (e) trifluoromethyl, and (f) —C0-2alkyl-phenyl, and pharmaceutically acceptable salts and individual stereoisomers thereof.",
"Another embodiment of the present invention includes compounds of formula Ik: wherein R1, R5, R9, R10, and Y are defined herein, and pharmaceutically acceptable salts and individual diastereomers thereof.",
"In a still further embodiment of the present invention X is C, O or N. In another embodiment of the present invention X is C or O.",
"In another embodiment of the present invention R1 is selected from: —C1-6alkyl, —C0-6alkyl-O—C1-6alkyl, and —(C0-6alkyl)-(C3-7cycloalkyl)-(C0-6alkyl), where the alkyl and the cycloalkyl are unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-13alkyl, (d) trifluoromethyl, (f) C1-3alkyl, (g) —O—C1-13alkyl, (h) —COR11, (i) —CN, (i) —NR12R12, and (k) —CONR12R12.In another aspect of the present invention R1 is selected from: (1) —C1-6alkyl, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-13alkyl, (d) trifluoromethyl, and (e) —COR11, (2) —C0-6alkyl-O—C1-6alkyl-, which is unsubstituted or substituted with 1-6 substituents where the substituents are independently selected from: (a) halo, (b) trifluoromethyl, and (c) —COR11, (3) —(C3-5cycloalkyl)-(C0-6alkyl), which is unsubstituted or substituted with 1-7 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) —O—C1-3alkyl, (d) trifluoromethyl, and (e) —COR11.In still another aspect of the present invention R1 is selected from: (a) C1-6alkyl, (b) C1-6alkyl substituted with hydroxy (c) C1-6alkyl substituted with 1-6 fluoro.",
"In a still further aspect of the present invention R1 is selected from: (a) —CH(CH3)2, (b) —CH(OH)CH3, and (c) —CH2CF3.In another aspect of the present invention R2 is selected from: (a) hydroxy (b) hydrogen (c) ═O, where R2 is connected to the ring via a double bond.",
"In another aspect of the present invention R2 is hydrogen.",
"In a still further aspect of the present invention when Y is N, R3 is nothing or O (to give a N-oxide) In a further aspect of the present invention when Y is N, R3 is nothing.",
"In a still further aspect of the present invention when Y is C, R3 is selected from: (a) hydrogen (b) halo (c) hydroxy (d) C1-3alkyl, where the alkyl is unsubstituted or substituted with 1-6 substituents independently selected from: fluoro, and hydroxy, (e) —COR11, (f) —CONR12R12, (g) -heterocycle, (h) —NR12—SO2—NR12R12, (i) —NR12—SO2—R14, (j)—SO2—NR12R12, (k) -nitro, and (l) —NR12R12; In another aspect of the present invention when Y is C, R3 is hydrogen.",
"In another aspect of the present invention R4 is hydrogen.",
"In another aspect of the present invention R5 is selected from: (a) C1-6alkyl substituted with 1-6 fluoro, (b) —O—C1-6alkyl substituted with 1-6 fluoro, (c) chloro, (d) bromo, and (e) phenyl.",
"In another aspect of the present invention R5 is selected from: (a) trifluoromethyl, (b) trifluoromethoxy, (c) chloro, (d) bromo, and (e) phenyl.",
"In another aspect of the present invention R5 is trifluoromethyl.",
"In another aspect of the present invention R6 is hydrogen In another aspect of the present invention R7 is phenyl, heterocycle, C3-7cycloalkyl, C1-6alkyl, —COR11, and —CONH—V—COR11, where V is selected from C1-6alkyl or phenyl, and where the phenyl, heterocycle, C3-7cycloalkyl, and C1-6alkyl is unsubstituted or substituted with 1-5 substituents where the substituents are independently selected from: (a) halo, (b) trifluoromethyl, (c) hydroxy, (d) C1-3alkyl, (e) —O—C1-3alkyl, (f) —COR11, (g) —CN, (h)-heterocycle, and (i) —CONR12R12.In still another aspect of the present invention (when X is not O) that R7 is phenyl, heterocycle, C1-4alkyl, —COR11, and —CONH—V—COR11, where V is selected from C1-6alkyl or phenyl, and where the phenyl, heterocycle, and C1-4alkyl is unsubstituted or substituted with 1-3 substituents where the substituents are independently selected from: (a) halo, (b) hydroxy, (c) C1-3alkyl, (d) —O—C1-13alkyl, (e) —COR11, and (f)-heterocycle In still another aspect of the present invention (when X is C), R7 is selected from: In another aspect of the present invention when X is C, R8 is selected from: (a) hydrogen, (b) hydroxy, (c) —CN, and (d) —F In another aspect of the present invention R7 and R8 may be joined together to form a ring which is selected from: (a) 1H-indene, (b) 2,3-dihydro-1H-indene, where the ring formed may be unsubstituted or substituted with 1-3 substituents independently selected from: (a) halo, (b) hydroxy, (c) C1-3alkyl, (d) —O—C1-13alkyl, (e) —COR11, and (f)-heterocycle.",
"In another aspect of the present invention R9 and R10 are independently selected from: (a) hydrogen, (b) hydroxy, (c) —CH3, (d) —O—CH3, and (e) ═O (where R9 and/or R10 are joined to the ring via a double bond).",
"In yet another aspect of the present invention n=1 or 2.Representative compounds of the present invention include those presented in the Examples and pharmaceutically acceptable salts and individual diastereomers thereof.",
"The compounds of the instant invention have at least two asymmetric centers at the 1- and 3-positions of the cyclopentyl ring.",
"Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule.",
"Each such asymmetric center will independently produce two optical isomers and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the ambit of this invention.",
"The absolute configurations of one aspect of the compounds of this invention, where the substituents on the cyclopentyl ring (amide and amine units) are cis, as depicted: The absolute configurations of a still further aspect of the compounds of this invention are those of the orientation as depicted: wherein the carbon bearing the amine substituent is designated as being of the (R) absolute configuration and the carbon bearing the amide subunit can be designated as being of either the (S) or (R) absolute configuration depending on the priority for R1.For example if R is isopropyl then the absolute stereochemistry at the carbon bearing the amide subunit would be (S) since the amide and amine units are preferred to have the cis arrangement on the cyclopentyl ring.",
"The independent syntheses of diastereomers and enantiomers or their chromatographic separations may be achieved as known in the art by appropriate modification of the methodology disclosed herein.",
"Their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.",
"As appreciated by those of skill in the art, halo or halogen as used herein are intended to include chloro, fluoro, bromo and iodo.",
"As used herein, “alkyl” is intended to mean linear, branched and cyclic structures having no double or triple bonds.",
"Thus C1-6alkyl is defined to identify the group as having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, such that C1-6alkyl specifically includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, pentyl and hexyl.",
"“Cycloalkyl” is an alkyl, part or all of which which forms a ring of three or more atoms.",
"C0 or C0alkyl is defined to identify the presence of a direct covalent bond.",
"The term “heterocycle” as used herein is intended to include the following groups: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof.",
"The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.",
"As used herein, “pharmaceutically acceptable salts” refer to derivatives wherein the parent compound is modified by making acid or base salts thereof.",
"Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.",
"The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.",
"For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.",
"The pharmaceutically acceptable salts of the present invention can be prepared from the parent compound which contains a basic or acidic moiety by conventional chemical methods.",
"Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.",
"Suitable salts are found, e.g.",
"in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418.Exemplifying the invention is the use of the compounds disclosed in the Examples and herein.",
"Specific compounds within the present invention include a compound which selected from the group consisting of: the title compounds of the Examples; and pharmaceutically acceptable salts thereof and individual diastereomers thereof.",
"The subject compounds are useful in a method of modulating chemokine receptor activity in a patient in need of such modulation comprising the administration of an effective amount of the compound.",
"The present invention is directed to the use of the foregoing compounds as modulators of chemokine receptor activity.",
"In particular, these compounds are useful as modulators of the chemokine receptors, in particular CCR-2.The utility of the compounds in accordance with the present invention as modulators of chemokine receptor activity may be demonstrated by methodology known in the art, such as the assay for chemokine binding as disclosed by Van Riper, et al., J. Exp.",
"Med., 177, 851-856 (1993) which may be readily adapted for measurement of CCR-2 binding.",
"Receptor affinity in a CCR-2 binding assay was determined by measuring inhibition of 125I-MCP-1 to the endogenous CCR-2 receptor on various cell types including monocytes, THP-1 cells, or after heterologous expression of the cloned receptor in eukaryotic cells.",
"The cells were suspended in binding buffer (50 mM HEPES, pH 7.2, 5 mM MgCl2, 1 mM CaCl2, and 0.50% BSA) with and added to test compound or DMSO and 125I-MCP-1 at room temperature for 1 h to allow binding.",
"The cells were then collected on GFB filters, washed with 25 mM HEPES buffer containing 500 mM NaCl and cell bound 125I-MCP-1 was quantified.",
"In a chemotaxis assay chemotaxis was performed using T cell depleted PBMC isolated from venous whole or leukophoresed blood and purified by Ficoll-Hypaque centrifugation followed by rosetting with neuraminidase-treated sheep erythrocytes.",
"Once isolated, the cells were washed with HBSS containing 0.1 mg/ml BSA and suspended at 1×107 cells/ml.",
"Cells were fluorescently labeled in the dark with 2 μM Calcien-AM (Molecular Probes), for 30 min at 37° C. Labeled cells were washed twice and suspended at 5×106 cells/ml in RPMI 1640 with L-glutamine (without phenol red) containing 0.1 mg/ml BSA.",
"MCP-1 (Peprotech) at 10 ng/ml diluted in same medium or medium alone were added to the bottom wells (27 μl).",
"Monocytes (150,000 cells) were added to the topside of the filter (30 μl) following a 15 min preincubation with DMSO or with various concentrations of test compound.",
"An equal concentration of test compound or DMSO was added to the bottom well to prevent dilution by diffusion.",
"Following a 60 min incubation at 37° C., 5% CO2, the filter was removed and the topside was washed with HBSS containing 0.1 mg/ml BSA to remove cells that had not migrated into the filter.",
"Spontaneous migration (chemokinesis) was determined in the absence of chemoattractant In particular, the compounds of the following examples had activity in binding to the CCR-2 receptor in the aforementioned assays, generally with an IC50 of less than about 3 μM, preferably less than about 11M.",
"Such a result is indicative of the intrinsic activity of the compounds in use as modulators of chemokine receptor activity.",
"Mammalian chemokine receptors provide a target for interfering with or promoting eosinophil and/or lymphocyte function in a mammal, such as a human.",
"Compounds which inhibit or promote chemokine receptor function, are particularly useful for modulating eosinophil and/or lymphocyte function for therapeutic purposes.",
"Accordingly, compounds which inhibit or promote chemokine receptor function would be useful in treating, preventing, ameliorating, controlling or reducing the risk of a wide variety of inflammatory and immunoregulatory disorders and diseases, allergic diseases, atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and asthma, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis.",
"For example, an instant compound which inhibits one or more functions of a mammalian chemokine receptor (e.g., a human chemokine receptor) may be administered to inhibit (i.e., reduce or prevent) inflammation.",
"As a result, one or more inflammatory processes, such as leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes, histamine) or inflammatory mediator release, is inhibited.",
"In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention.",
"For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated.",
"However, the method can also be practiced in other species, such as avian species (e.g., chickens).",
"Diseases and conditions associated with inflammation and infection can be treated using the compounds of the present invention.",
"In one embodiment, the disease or condition is one in which the actions of lymphocytes are to be inhibited or promoted, in order to modulate the inflammatory response.",
"Diseases or conditions of humans or other species which can be treated with inhibitors of chemokine receptor function, include, but are not limited to: inflammatory or allergic diseases and conditions, including respiratory allergic diseases such as asthma, particularly bronchial asthma, allergic rhinitis, hypersensitivity lung diseases, COPD, hypersensitivity pneumonitis, eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilic pneumonia), delayed-type hypersentitivity, interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis or dermatomyositis); systemic anaphylaxis or hypersensitivity responses, drug allergies (e.g., to penicillin, cephalosporins), insect sting allergies; autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease; graft rejection (e.g., in transplantation), including allograft rejection or graft-versus-host disease; inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis; spondyloarthropathies; scleroderma; psoriasis (including T-cell mediated psoriasis) and inflammatory dermatoses such an dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); eosinphilic myositis, eosinophilic fasciitis; cancers with leukocyte infiltration of the skin or organs, stroke, Other diseases or conditions in which undesirable inflammatory responses are to be inhibited can be treated, including, but not limited to, reperfusion injury, atherosclerosis, certain hematologic malignancies, cytokine-induced toxicity (e.g., septic shock, endotoxic shock), polymyositis, dermatomyositis.",
"Diseases or conditions of humans or other species which can be treated with modulators of chemokine receptor function, include, but are not limited to: immunosuppression, such as that in individuals with immunodeficiency syndromes such as AIDS or other viral infections, individuals undergoing radiation therapy, chemotherapy, therapy for autoimmune disease or drug therapy (e.g., corticosteroid therapy), which causes immunosuppression; immunosuppression due to congenital deficiency in receptor function or other causes; and infections diseases, such as parasitic diseases, including, but not limited to helminth infections, such as nematodes (round worms), (Trichuriasis, Enterobiasis, Ascariasis, Hookworm, Strongyloidiasis, Trichinosis, filariasis), trematodes (flukes) (Schistosomiasis, Clonorchiasis), cestodes (tape worms) (Echinococcosis, Taeniasis saginata, Cysticercosis), visceral worms, visceral larva migraines (e.g., Toxocara), eosinophilic gastroenteritis (e.g., Anisaki sp., Phocanema sp.",
"), and cutaneous larva migraines (Ancylostona braziliense, Ancylostoma caninum).",
"In addition, treatment of the aforementioned inflammatory, allergic and autoimmune diseases can also be contemplated for promoters of chemokine receptor function if one contemplates the delivery of sufficient compound to cause the loss of receptor expression on cells through the induction of chemokine receptor internalization or delivery of compound in a manner that results in the misdirection of the migration of cells.",
"The compounds of the present invention are accordingly useful in treating, preventing, ameliorating, controlling or reducing the risk of a wide variety of inflammatory and immunoregulatory disorders and diseases, allergic conditions, atopic conditions, as well as autoimmune pathologies.",
"In a specific embodiment, the present invention is directed to the use of the subject compounds for treating, preventing, ameliorating, controlling or reducing the risk of autoimmune diseases, such as rheumatoid arthritis or psoriatic arthritis.",
"In another aspect, the instant invention may be used to evaluate putative specific agonists or antagonists of chemokine receptors, including CCR-2.Accordingly, the present invention is directed to the use of these compounds in the preparation and execution of screening assays for compounds that modulate the activity of chemokine receptors.",
"For example, the compounds of this invention are useful for isolating receptor mutants, which are excellent screening tools for more potent compounds.",
"Furthermore, the compounds of this invention are useful in establishing or determining the binding site of other compounds to chemokine receptors, e.g., by competitive inhibition.",
"The compounds of the instant invention are also useful for the evaluation of putative specific modulators of the chemokine receptors, including CCR-2.As appreciated in the art, thorough evaluation of specific agonists and antagonists of the above chemokine receptors has been hampered by the lack of availability of non-peptidyl (metabolically resistant) compounds with high binding affinity for these receptors.",
"Thus the compounds of this invention are commercial products to be sold for these purposes.",
"The present invention is further directed to a method for the manufacture of a medicament for modulating chemokine receptor activity in humans and animals comprising combining a compound of the present invention with a pharmaceutical carrier or diluent.",
"The present invention is further directed to the use of the present compounds in treating, preventing, ameliorating, controlling or reducing the risk of infection by a retrovirus, in particular, herpes virus or the human immunodeficiency virus (HIV) and the treatment of, and delaying of the onset of consequent pathological conditions such as AIDS.",
"Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection: AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV.",
"For example, the compounds of this invention are useful in treating infection by HIV after suspected past exposure to HIV by, e.g., blood transfusion, organ transplant, exchange of body fluids, bites, accidental needle stick, or exposure to patient blood during surgery.",
"In an aspect of the present invention, a subject compound may be used in a method of inhibiting the binding of a chemokine to a chemokine receptor, such as CCR-2, of a target cell, which comprises contacting the target cell with an amount of the compound which is effective at inhibiting the binding of the chemokine to the chemokine receptor.",
"The subject treated in the methods above is a mammal, for instance a human being, male or female, in whom modulation of chemokine receptor activity is desired.",
"“Modulation” as used herein is intended to encompass antagonism, agonism, partial antagonism, inverse agonism and/or partial agonism.",
"In an aspect of the present invention, modulation refers to antagonism of chemokine receptor activity.",
"The term “therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.",
"The term “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.",
"By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.",
"The terms “administration of” and or “administering a” compound should be understood to mean providing a compound of the invention to the individual in need of treatment.",
"As used herein, the term “treatment” refers both to the treatment and to the prevention or prophylactic therapy of the aforementioned conditions.",
"The term “substituted” in reference to substitution on alkyl, cycloalkyl, phenyl, heterocycle, or some other chemical group is intended to include mono- and poly-substitution by a named substituent to the extent such single and multiple substitution is chemically allowed in any of the named chemical groups.",
"It is understood that the definition of a substituent at a particular location in a molecule is independent of its definition at other locations in the molecule.",
"Thus, for example, when R3=alkyl substituted with 1-5 of R12 (defined elsewhere), each R12 is independently selected from the possible values thereof; i.e., each R12 can be the same as or different from any other R 12.The term “optionally substituted” is intended to include both substituted and unsubstituted.",
"Thus, for example, optionally substituted aryl could represent a pentafluorophenyl or a phenyl ring.",
"Combined therapy to modulate chemokine receptor activity for thereby treating, preventing, ameliorating, controlling or reducing the risk of inflammatory and immunoregulatory disorders and diseases, including asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis, and those pathologies noted above is illustrated by the combination of the compounds of this invention and other compounds which are known for such utilities.",
"For example, in treating, preventing, ameliorating, controlling or reducing the risk of inflammation, the present compounds may be used in conjunction with an antiinflammatory or analgesic agent such as an opiate agonist, a lipoxygenase inhibitor, such as an inhibitor of 5-lipoxygenase, a cyclooxygenase inhibitor, such as a cyclooxygenase-2 inhibitor, an interleukin inhibitor, such as an interleukin-1 inhibitor, an NMDA antagonist, an inhibitor of nitric oxide or an inhibitor of the synthesis of nitric oxide, a non-steroidal antiinflammatory agent, or a cytokine-suppressing antiinflammatory agent, for example with a compound such as acetaminophen, aspirin, codeine, embrel, fentanyl, ibuprofen, indomethacin, ketorolac, morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic, sufentanyl, sunlindac, tenidap, and the like.",
"Similarly, the instant compounds may be administered with a pain reliever; a potentiator such as caffeine, an H2-antagonist, simethicone, aluminum or magnesium hydroxide; a decongestant such as phenylephrine, phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxy-ephedrine; an antiitussive such as codeine, hydrocodone, caramiphen, carbetapentane, or dextramethorphan; a diuretic; and a sedating or non-sedating antihistamine.",
"Likewise, compounds of the present invention may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of the present invention are useful.",
"Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present invention.",
"When a compound of the present invention is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the present invention is typically employed.",
"Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of the present invention.",
"Examples of other active ingredients that may be combined with a compound of the present invention, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) VLA-4 antagonists such as those described in U.S. Pat.",
"No.",
"5,510,332, WO95/15973, WO96/01644, WO96/06108, WO96/20216, WO96/22966, WO96/31206, WO96/40781, WO97/03094, WO97/02289, WO 98/42656, WO98/53814, WO98/53817, WO98/53818, WO98/54207, and WO98/58902; (b) steroids such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistamines (H1-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilamine, astemizole, terfenadine, loratadine, desloratadine, cetirizine, fexofenadine, descarboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such as β2-agonists (terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, bitolterol, and pirbuterol), theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (zafirlukast, montelukast, pranlukast, iralukast, pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005); (f) non-steroidal antiinflammatory agents (NSAIDs) such as propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors; (h) inhibitors of phosphodiesterase type IV (PDE-IV); (i) other antagonists of the chemokine receptors, especially CCR-1, CCR-2, CCR-3, CXCR-3 and CCR-5; (j) cholesterol lowering agents such as HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, rosuvastatin, and other statins), sequestrants (cholestyramine and colestipol), cholesterol absorption inhibitors (ezetimibe), nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), and probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides (metformin), α-glucosidase inhibitors (acarbose) and glitazones (troglitazone and pioglitazone); (1) preparations of interferon beta (interferon beta-1α, interferon beta-1β); (m) other compounds such as 5-aminosalicylic acid and prodrugs thereof, antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic agents.",
"The weight ratio of the compound of the present invention to the second active ingredient may be varied and will depend upon the effective dose of each ingredient.",
"Generally, an effective dose of each will be used.",
"Thus, for example, when a compound of the present invention is combined with an NSAID the weight ratio of the compound of the present invention to the NSAID will generally range from about 1000:1 to about 1:1000, or from about 200:1 to about 1:200.Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.",
"In such combinations the compound of the present invention and other active agents may be administered separately or in conjunction.",
"In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).",
"The compounds of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.",
"In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, monkeys, etc., the compounds of the invention are effective for use in humans.",
"The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy.",
"All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients.",
"In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.",
"In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.",
"As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.",
"The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.",
"Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.",
"Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.",
"These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.",
"The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.",
"For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.",
"They may also be coated by the techniques described in the U.S. Pat.",
"Nos.",
"4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.",
"Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.",
"Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.",
"Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.",
"The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.",
"Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.",
"The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.",
"Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.",
"These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.",
"Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.",
"Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.",
"Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.",
"The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.",
"The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.",
"Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.",
"The emulsions may also contain sweetening and flavoring agents.",
"Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose.",
"Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.",
"The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.",
"This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.",
"The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.",
"Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.",
"In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.",
"For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides.",
"In addition, fatty acids such as oleic acid find use in the preparation of injectables.",
"The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug.",
"These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.",
"Such materials are cocoa butter and polyethylene glycols.",
"For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed.",
"(For purposes of this application, topical application shall include mouthwashes and gargles.)",
"The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions.",
"In treating, preventing, ameliorating, controlling or reducing the risk of conditions which require chemokine receptor modulation an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses.",
"The dosage level will be about 0.1 to about 250 mg/kg per day; or about 0.5 to about 100 mg/kg per day.",
"A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day.",
"Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day.",
"For oral administration, the compositions may be provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, 2.0 to 500, 3.0 to 200, or 1, 5, 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 750, 800, 900 and/or 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.",
"The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.",
"It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.",
"Several methods for preparing the compounds of this invention are illustrated in the following Schemes and Examples.",
"Starting materials are commercially available, made by known procedures, or prepared as illustrated herein.",
"One of the principal routes used for preparation of compounds within the scope of the instant invention which bear a 1,1,3-trisubstituted cyclopentane framework 1-5 is depicted in Scheme 1.According to this route, keto acids 1-1 (preparation described in Schemes 2A, 2B, 2C, and 2D) is coupled to amines 1-2 (preparation described in Schemes 3A-G).",
"This can be accomplished in various ways, including by first converting the acid to its acid chloride with a reagent such as oxalyl chloride, and then combining with amine 1-2 in the presence of a base such as triethylamine.",
"Reductive amination of 1-3 with an amine 1-4 using, for example, NaB(OAc)3H or NaBH3CN as the reducing agent gives chemokine receptor modulators 1-5.The compounds 1-9, which can be synthesized according to the chemistry described in Scheme 1 represent stereoisomeric mixtures (Eliel, E. E., Wilen, S. H., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York).",
"In particular, compounds 1-5 are often obtained as a mixture of cis and trans isomers.",
"When 1-1 is a single stereoisomer (1-1a) only 2 possible isomers of 1-5 can result (cis and trans); these can be separated by a variety of methods, including by preparative TLC, flash chromatography, MPLC, or by HPLC using a column with a chiral stationary phase.",
"When 1-1 is racemic, a total of 4 possible isomers of 1-5 can be obtained.",
"Again, these may be separated by HPLC using a column with a chiral stationary phase, or by a combination of the methods above.",
"The synthesis of racemic 1-1 is detailed in Scheme 2A, while syntheses of the chiral 1-1a are described in Schemes 2B and 2C.",
"Furthermore, compounds 1-5 can themselves be modified to give new chemokine receptor modulators 1-5.1.For example, an ester functional group within a compound 1-5 can be hydrolyzed to the corresponding carboxylic acid, which also can be a chemokine receptor modulator.",
"As depicted in Scheme 1A, the keto-ester 1-6 could be reductively aminated with amine 1-4 to form the amino ester 1-7 under a variety of conditions, including sodium triacetoxyborohydride or sodium cyanoborohydride.",
"Alkylation of the ester 1-7 with an alkylating agent such as an alkyl chloride, bromide or iodide in the presence of an appropriate base such as lithium bis(trimethylsilyl)amide, affords the intermediate esters 1-8.These esters formed in the above mentioned transformations represent in general a mixture of 1,3-cis- and 1,3-trans-diastereoisomers, which could be separated into respective diastereoisomeric pairs using column chromatography.",
"A similar diastereoisomeric separation could be also accomplished later, after the esters 1-8 were hydrolytically cleaved to yield the respective acids 1-9.This hydrolysis was readily accomplished under usual conditions, including lithium, sodium or potassium hydroxide, at ambient to elevated temperatures, depending on the nature of the ester group and substituent R1.These diastereoisomers could be separated by crystallization from a variety of solvents, taking advantage of the finding, that the cis-diastereoisomeric acids are less soluble, when compared to their trans-epimers.",
"The compounds of formula I-5 are then formed from the acids 1-9 and tetrahydroisoquinoline derivatives 1-2 under standard amide-bond forming reaction conditions, including carbodimide reagents, such as DCC, EDC and catalysts such as DMAP, HOAT or HOBT.",
"Intermediate 1-3 can also be resolved by Chiral HPLC to give 1-3a and 1-3b (Scheme 1B).",
"This then would give cis/trans isomers 1-5a and 1-5b.",
"One of the principal routes used for preparation of Intermediate 1-1 and Intermediate 1-6 is outlined in Scheme 2A.",
"According to this route, 3-oxocyclopentanecarboxylic acid (2-1), which can be synthesized following a known procedure (Stetter, H., Kuhlman, H., Liebigs Ann.",
"Chim., 1979, 944) is esterified under standard conditions.",
"When R18 represents a tert-Butyl group, the respective ester 1-6 can be prepared by reacting the appropriate alcohol, in this case tert-butanol, with acid 2-1 in the presence of sulfuric acid.",
"Protection of the oxo-group in 2-1 can be achieved by a number of ways (Greene, T., Wuts, P. G. M., Protective Groups in Organic Chemistry, John Wiley & Sons, Inc., New York, N.Y. 1991).",
"The particularly suitable dimethyl acetal protecting group can be introduced using trimethyl orthoformate as a reagent in a suitable solvent such as dichloromethane and methyl alcohol in the presence of an acidic catalyst.",
"Alternatively, in the case of R18 being a methyl group, the acid 2-1 can be converted to 2-3 directly by using trimethyl orthoformate and an acidic catalyst, such as para-toluenesulfonic acid.",
"An alkylation of esters 2-3 with an alkylating agent such as an alkyl chloride, bromide or iodide in the presence of an appropriate base such as lithium diisopropylamide, produces compounds 2-4.The ester protecting group present in 2-4 can be removed in a number of ways, depending on the nature of the ester.",
"Methyl esters (R18=methyl) can be hydrolyzed in the presence of an acid or base at ambient or elevated temperatures, whereas tert-butyl esters (R18=tert-butyl) can be easily cleaved under acidic conditions.",
"Under these conditions, the dimethyl acetal is simultaneously deprotected to give 1-1.Intermediate 1-1 can be prepared as a single stereoisomer (1-1a) in various ways including those depicted in Schemes 2B and 2C.",
"According to Scheme 2B, racemic 1-1 can be converted to its benzyl ester.",
"There are many ways to effect this esterification, one of which being by a sequence involving conversion to the corresponding acid chloride with, for example oxalyl chloride, followed by treatment with benzyl alcohol in the presence of a base such as triethylamine.",
"Then the racemic benzyl ester 2-5 can be separated by chiral preparative HPLC to give 2-5a as a single stereoisomer.",
"Removal of the benzyl group to give the chiral ketoacid 1-1a can be accomplished in several ways.",
"One convenient way is by hydrogenolysis in the presence of a catalyst such as Pd/C.",
"According to Scheme 2C, chiral ketoacid compound 1-1a can be prepared starting from commercially available optically pure amino acid 2-6.Protection of the carboxylic acid group can be achieved in a variety of ways.",
"When R18 is methyl, esterification can be accomplished by treatment with methanol in the presence of an acid catalyst such as HCl.",
"Treatment with Boc2O results in protection of the amine group of 2-7.Stereoselective alkylation of ester 2-8 with an alkylating agent such as an alkyl chloride, bromide or iodide in the presence of an appropriate base such as lithium bis(trimethylsilyl)amide, produces compounds 2-9.Hydrogenation in the presence of a catalyst such as Pd/C affords 2-10.Hydrolysis of the ester to give 2-11 can be achieved under standard conditions depending on the R18 group.",
"For example, when R18 is methyl (methyl ester), hydrolysis can be accomplished by treatment with a base such as sodium hydroxide, lithium hydroxide, or potassium hydroxide, with or without heating.",
"The Boc protecting group can be removed under standard acidic conditions, such as with HCl in a solvent such as dioxane, or with TFA.",
"Oxidation of 2-12 to give 1-1a (as a single stereoisomer if constituent R1 is achiral, or as a mixture of stereoisomers if constituent R1 has a chiral center) can be achieved in several ways, including by treatment with NBS, followed by treatment with sodium methoxide.",
"The enolate generated from ester 2-3 (R18 being a benzyl or tert-Butyl group) in the presence of a strong base such as lithium diisopropylamide can be reacted with aldehydes (R1aCHO) or ketones (R1aR2aCO) to produce the appropriate hydroxyalkyl substituted compounds 2-4.1 as indicated in Scheme 2D.",
"The resulting hydroxy group can be protected in various ways, including by treatment with acetic anhydride in the presence of a base such as triethylamine to give compounds 24.2.Once again the ester protecting group is removed under conditions suitable for the particular protecting group.",
"In the case of the tert-butyl esters (R18 is t-butyl), deprotection is achieved under acidic conditions.",
"The latter usually induces cleavage of the acetal protecting group as well, and the keto acids 1-1.1 can be prepared this way in an one-pot procedure.",
"Their conversion to the final modulators of chemokine activity 1-9 can be achieved as described previously, with minor modifications to accommodate the protected hydroxy in 1-1.1.Amines 1-2 can be prepared in several ways as shown in schemes 3A-3G.",
"The 5-aza-tetrahydroisoquinoline fragment can be prepared in accordance to the literature methods of MarCoux, J-F. et al.",
"(J. Chem.",
"Lett., 2000, 2 (15), 2339-2341).",
"Alternatively, it can be prepared as outlined in Scheme 3A.",
"Compound 3-1, normally obtained from commercial sources, is brominated (Br2, AcOH) to give 3-2.Metal halogen exchange (NaH, t-butyl lithium) followed by treatment with DMF provides aldehyde 3-3.Conversion of the aldehyde group to a nitrile can be achieved with sodium formate, hydroxylamine hydrochloride and formic acid.",
"The resulting nitrile 3-4 can be treated with phosphorous oxychloride to give 2-chloropyridine 3-5.Displacement of the chloro group can be achieved with the sodium salt of a dialkylmalonate.",
"Reduction of the nitrile group of 3-6 with hydrogen and Raney Ni catalyst is accompanied by cyclization to afford compound 3-7.Decarboxylation can be achieved in a variety of ways depending on the ester.",
"In the case represented in Scheme 3A, the t-butyl ester was decarboxylated with TFA to give 3-8.Reduction (BH3), followed by protection of the resulting amine using Boc2O, gives 3-9, which can be conveniently purified.",
"Removal of the Boc protecting group to give 1-2a can be achieved in various ways, including by treatment with anhydrous HCl in dioxane or some other solvent.",
"Compounds of the type 1-2a could also be prepared according to Scheme 3B.",
"Commercially available 3-10 can be methylated with methyl iodide in the presence of a base such as K2CO3 to give 3-11.Cycloaddition with a protected piperidinone in the presence of NH3 in methanol furnishes 5-azatetrahydroiso-quinoline 3-12 (R10c can be various protecting groups such as benzyl or benzoyl).",
"Hydrogenation of the nitro group of compound 3-12 with hydrogen and a catalyst such as Pd/C gives 3-13.Diazonium salt formation followed by warming with sulfuric acid provides 5-aza-7-hydroxytetrahydroisoquinoline 3-14.Removal of the protecting group R10c is achieved in different ways depending upon the nature of R10c.",
"If R10c is benzyl, hydrogenation in the presence of HCl and a catalyst such as Pd/C can be applied.",
"If R10c is benzoyl, hydrolysis can be achieved by heating in concentrated HCl solution.",
"Installation of a Boc protecting group on to 3-15 can be easily achieved with Boc2O to give 3-16.Various R10d can then be incorporated generating ethers (see Scheme 3C).",
"The Boc protecting group on the resulting compounds 3-17 can finally be removed with HCl or TFA to give 1-2b.",
"Alternatively, Compound 3-14 itself can be converted to ethers (according to Scheme 3C).",
"The resulting ether 3-18 can be converted to compound 3-19 by removal of R10c as described above.",
"The 5-aza-7-hydroxytetrahydroisoquinolines 3-14 and 3-16 in Scheme 3B can be converted to various ethers (see Scheme 3C).",
"Alkyl ethers can be generated from an alkyl halide and a base (such as K2CO3, NaOH, or NaH) giving compounds 3-19 and 3-22.A trifluoromethyl ether can be prepared by initial methyl xanthate formation (NaH, CS2; MeI), followed by sequential treatment with 1,3-dibromo-5,5-dimethylhydantoin (or NBS) and HF/pyridine solution giving 3-20.Aryl ethers can be prepared by a number of methods, including reaction of arylboronic acids in the presence of copper (II) acetate and triethylamine, to give compounds 3-21.Compounds 1-2c where R5 is a halide (IVc) can be prepared according to Scheme 3D.",
"Compound 3-13 can be converted to the halide 3-22 according to classical procedures via the diazonium salt.",
"Alternatively the known cycloaddition reaction to a suitably protected piperidinone can be applied.",
"Removal of the protecting group R10c can be achieved as described previously.",
"Furthermore, after incorporation into advanced intermediates, fragments 1-2c can be further modified so as to prepared 7-aryl-5-azotetrahydroisoquinoline containing analogs.",
"This can be accomplished by coupling of the 5-aza-7-halotetrahydroisoquinoline intermediates to aryl boronic acids (or aryl stannanes), mediated by transition metal catalysts such as Pd(OAc)2.The preparation of tetrahydroisoquinoline amine components is outlined in Schemes 3E-3G.",
"The tetrahydroisoquinolines incorporated into the amide portion of 1-5 often contain one or two substituted groups on various positions.",
"Most of these are not available commercially, however, can be obtained through synthesis, representative examples of which are shown in Schemes 3F and 3G.",
"An example of a synthesis of the simple tetrahydroisoquinoline (1-2d) is depicted in Scheme 3E.",
"According to this, the commercially available 4-trifluoromethyl phenylacetonitrile (3-23) is converted to the corresponding amine (3-24) using hydrogenation in the presence of Ra—Ni, and trifluoroacetic anhydride is then used to cap the amine.",
"The resultant amide (3-25) is treated with formaldehyde in the presence of sulfuric acid to give the cyclic compound (3-26) which is further converted into tetrahydroisoquinoline (1-2d).",
"Many 5-substituted atetrahydroisoquinolines can also be prepared based on 3-26 (Scheme 3F).",
"Iodonization on the 5-position yields compound 3-28.After conversion to the cyano compound (3-29) under palladium (0) catalyzed conditions, the amide is cleaved to the amine 3-30 which can be converted into amino ester 1-2ein high yield by a two step sequence.",
"The iodo compound 3-28 can also be converted to other compounds as shown in the experimental section.",
"Modification of these substituents can also be made after the assembly of the final framework (1-5) to make new chemokine modulators of the form 1-5.1 (see Scheme 1).",
"An example of this would be the hydrolysis of a methyl ester (from 1-2e) to make the corresponding carboxylic acid (see examples).",
"Heterocyclic 7-substituted tetrahydroisoquinolines could be obtained by utilizing commercially available tetrahydroisoquinoline.",
"As described is Scheme 3G, tetrahydroisoquinoline (3-32) is nitrated by treatment with potassium nitrate in the presences of concentrated sulfuric acid.",
"The 7-nitro-tetrahydroisoquinoline 3-33 is treated with trifluoroacetic anhydride to protect the amine and the resulting amide then hydrogenated with 10% palladium on carbon under hydrogen at 50 psi pressure to afford the aniline derivative 3-34.Base hydrolysis yields a 7-amino substituted tetrahydroisoquinoline (3-35) which could be used in the synthesis of further CCR2 antagonist or other tetrahydroisoquinoline derivatives.",
"Protection of 3-35 with benzylchloroformate in the presence of an organic base such as triethylamine or diisopropylethyl amine affords the carbamate 3-37.This intermediate could be utilized to make the tetrazole and substituted tetrazoles such as 1-2 g. Intermediate 3-37 is treated with trifluoroacetic anhydride to form the trifluoroacetyl protected amide which then is converted to the trifluoromethyl substituted tetrazole by sequential reactions with triphenylphosphine heated to reflux for 15 hours followed by sodium azide in DMF at room temperature.",
"Hydrogenation with 10% palladium on carbon under hydrogen atmosphere affords the heterocyclic substituted tetrahydroisoquinoline 1-2 g. Alternatively 3-34 can be directly derivatized as shown to the triazole 3-36 with N,N-dimethylforamide azine in the presences of a catalytic amount of an acid such as toluene sulfonic acid heated to reflux for 24 to 48 hours.",
"Basic hydrolysis of this intermediate gives amine component 1-2f.",
"Additional examples of tetrhydroisoquinolines incorporated into the amide portion of compounds within the scope of the instant invention, as well as their syntheses are further described in the Experimental section.",
"Amines 1-4 were obtained from various sources.",
"Most were commercially available, some were known from the literature and could be prepared according to published procedures, and some were prepared as described herein.",
"Since their structures and the methods for their preparation are diverse, only two schemes will be outlined in this section; individual syntheses of amines 1-4 can be found in the Experimental Section.",
"Scheme 4A shows one method for the synthesis of 4-aryl substituted piperidines.",
"Enol triflate 4-1 (prepared according to Wustrow, D. J., Wise, L. D., Synthesis, (1991), 993-995.)",
"could be coupled to boronic acids 4-2 as described by Wustrow and Wise.",
"Hydrogenation of the olefin in 4-3 could be achieved using hydrogen in the presence of a catalyst such as Pd(OH)2/C.",
"Removal of the Boc protecting group could be achieved using standard acidic conditions, such as HCl in dioxane or TFA/DCM to afford piperidine 14.1.Another example of the synthesis of amine 14 is shown in Scheme 4B.",
"Commercially available alcohol (4-5) is first sulfonylated with methanesulfonyl chloride to give compound 4-6 which can be directly substituted with tetrazole to give heteroaryl piperidine 4-7.Removal of the Boc protecting group under the standard conditions gives the amine hydrochloride 4-8.Another principal route for the synthesis of chemokine receptor modulators is depicted in Scheme 5.According to this route, compound 2-11 (described in Scheme 2C) is condensed with amine 1-2 (described in Scheme 1) using a peptide coupling reagent such as EDC to give 5-1.The Boc protecting group is removed under standard conditions such as with HCl in a solvent such as dioxane followed by treatment of the resulting amine 5-2 with a dialdehyde 5-3 in the presence of a reducing agent such as sodium triacetoxyborohydride leads to a double reductive alkylation sequence with concomitant cyclization to give 1-5.2.In accord with Scheme 1, further modifications, such as hydrolysis of an ester group present within 1-5.2 can be effected to give new chemokine receptor modulators 1-5.3.One way of preparing dialdehydes 5-3 is outlined in Scheme 6.According to this route, a cycloalkene 6-1 is oxidatively cleaved with, for example, ozone followed by dimethylsulfide, to give the dialdehyde.",
"Alternatively, in place of the dialdehydes 5-3 the intermediate ozonides 6-2 can themselves be used directly in the double reductive amination reaction leading to 1-5.2.In some cases the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.",
"The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.",
"Concentration of solutions was generally carried out on a rotary evaporator under reduced pressure.",
"Flash chromatography was carried out on silica gel (230-400 mesh).",
"MPLC refers to medium pressure liquid chromatography and was carried out on a silica gel stationary phase unless otherwise noted.",
"NMR spectra were obtained in CDCl3 solution unless otherwise noted.",
"Coupling constants (J) are in hertz (Hz).",
"Abbreviations: diethyl ether (ether), triethylamine (TEA), N,N-diisopropylethylamine (DIEA) saturated aqueous (sat'd), room temperature (rt), hour(s) (h), minute(s) (min).",
"The following are representative procedures for the preparation of the compounds used in the following Examples or which can be substituted for the compounds used in the following Examples which may not be commercially available.",
"In some cases the order of carrying out the foregoing reaction schemes may be varied to facilitate the reaction or to avoid unwanted reaction products.",
"The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.",
"Concentration of solutions was generally carried out on a rotary evaporator under reduced pressure.",
"Flash chromatography was carried out on silica gel (230-400 mesh).",
"NMR spectra were obtained in CDCl3 solution unless otherwise noted.",
"Coupling constants (J) are in hertz (Hz).",
"Abbreviations: diethyl ether (ether), triethylamine (TEA), N,N-diisopropylethylamine (DIEA) saturated aqueous (sat'd), room temperature (rt), hour(s) (h), minute(s) (min).",
"The following are representative Procedures for the preparation of the compounds used in the following Examples or which can be substituted for the compounds used in the following Examples which may not be commercially available.",
"INTERMEDIATE 1 Step A A solution of 4-trifluoromethylphenylacetonitrile (10 g, 49 mmol) in a mixture of ethanol (100 mL) and ammonium hydroxide (20 mL of a 29.3% aqueous solution) was hydrogenated over Raney nickel (1 g) for 16 h. The catalyst was removed by filtration through celite and the filtrate evaporated to dryness.",
"The neat residue was added in a dropwise manner to trifluoroacetic anhydride (25 mL, 180 mmol) cooled at 0° C. and the resulting mixture stirred at 0° C. for 30 minutes.",
"The reaction mixture was poured onto ice (250 mL) and the resulting mixture stirred for 30 minutes after which the precipitate was removed by filtration and air dried to give the product as a white solid (13.4 g, 90%).",
"Step B To a mixture of the product from step A (13.4 g, 44.0 mmol) and paraformaldehyde (2 g, 50 mmol) was added in one portion a mixture of concentrated sulfuric acid (90 mL) and glacial acetic acid (60 mL) and the resulting mixture stirred at room temperature for 16 hours.",
"The reaction mixture was poured onto a mixture of ice and water (1 L) and extracted with ethyl acetate (3×150 mL); the combined ethyl acetate layers were washed with water (3×500 mL), saturated NaHCO3 (200 mL), and sat NaCl (100 mL), dried over MgSO4, filtered and evaporated in vacuo.",
"The residue was purified by column chromatography on silica elution with 10% Et2O in hexanes to give the product (8.29 g, 60%).",
"Step C To a solution of the trifluoroacetamide formed in Step B (8.29 g, 26.0 mmol) in ethanol (200 mL) was added a solution of potassium carbonate (20 g, 150 mmol) in water (50 mL), and the resulting mixture stirred at reflux for 1 hour.",
"The ethanol was removed by rotary evaporation and water (150 mL) was added to the residue.",
"Extracted with CH2Cl2 (3×100 mL), the combined CH2Cl2 layers were washed with sat NaCl (100 mL), dried over Na2SO4, filtered and evaporated in vacuo to give the product (5.2 g, 91%); 1H NMR 500 MHz (CDCl3) δ=1.81 (1H, br s), 2.84 (2H, d, J=6.0 Hz), 3.15 (2H, t, J=6.0 Hz), 4.05 (2H, s), 7.19 (1H, d, J=8.0 Hz), 7.27 (1H, s), 7.37 (1H, d, J=8.0 Hz).",
"INTERMEDIATE 2 Step A To a solution of 5-trifluoromethyl-2-pyridinol (51.0 g, 307 mmol) and sodium acetate (26.2 g, 319 mmol) in glacial acetic acid (200 mL) was added bromine (16.7 mL, 325 mmol) and the resulting mixture was heated at 80° C. for 2.5 hours.",
"The reaction was allow to cool to room temperature and then was evaporated under reduced pressure.",
"The residue was neutralized with saturated NaHCO3 solution and extracted with ethyl acetate (3×200 mL).",
"The organic layers were combined, dried over MgSO4, filtered, and evaporated in vacuo to yield 74.45 g (98.7%) of the crude product.",
"1H NMR (400 MHz, CDCl3) δ 8.04 (d, J=2.6 Hz, 1H), 7.89 (m, 1H).",
"Step B Under nitrogen, the substituted pyridine, described in Step A (48.8 g, 202 mmol) was added by small portions to a suspension of NaH (8.9 g, 220 mmol) in anhydrous THF (500 mL).",
"After complete addition of the intermediate, the reaction mixture was cooled to −78° C. and treated with tert-butyllithium (260 mL, 444 mmol) added dropwise via syringe.",
"After stirring for 5 minutes, DMF (50 mL, 707 mmol) was added slowly to maintain the temperature below −50° C. The resulting mixture was then stirred for 10 hours allowing to warm to room temperature.",
"The mixture was quenched with 2N HCl and then diluted with ethyl acetate (1000 mL).",
"The organic layer was separated, washed with brine, dried over MgSO4, and evaporated in vacuo.",
"The desired product was precipitated out of ethyl acetate and hexane and filtered to yield a light brown solid (28.55 g, 73.8%).",
"1H NMR (500 MHz, CD3OD) δ 10.13 (s, 1H), 8.21 (s, 2H).",
"Step C A mixture of the compound from Step B (18 g, 95 mmol), sodium formate (7.1 g, 105 mmol), hydroxylamine hydrochloride (7.3 g, 110 mmol), and formic acid (150 mL) was stirred at room temperature for 2 hours and then refluxed overnight.",
"The reaction mixture was cooled and let stand at room temperature for 7 days.",
"The reaction was poured into water and extracted with ethyl acetate (3×).",
"Combined organic layers were washed with water (2×), saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated in vacuo to yield the desired product as a brown powder (17.84 g, 89.8%).",
"1H NMR (400 MHz, CD3OD) δ 8.37 (d, J=2.7 Hz, 1H), 8.19 (q, J=0.7 Hz, 0.3?",
"?/Hz, 1H).",
"Step D To a mixture of phosphorous oxychloride (13.4 mL, 144 mmol) and quinoline (8.7 mL, 73.4 mmol) was added the product from Step C (24.6 g, 131 mmol) and the resulting mixture was refluxed for 3 hours.",
"The reaction was cooled to 100° C. before water (70 mL) was slowly added.",
"The mixture was further cooled to room temperature and neutralized carefully with saturated NaHCO3 solution.",
"The aqueous layer was extracted with ethyl acetate (3×) and the organic layers were combined, dried over MgSO4, filtered, and evaporated in vacuo.",
"The crude product was purified by flash chromatography to afford (23.5 g, 87.0%) of the desired compound.",
"1H NMR (500 MHz, CDCl3) δ 8.88 (d, J=2.0 Hz, 1H), 8.26 (d, J=2.5 Hz, 1H).",
"Step E To a suspension of NaH (7.8 g, 200 mmol) in THF (100 mL) under nitrogen was added dropwise a solution of tert-butyl methyl malonate (20 mL, 120 mmol) in anhydrous THF (100 mL) via syringe.",
"The reaction mixture was stirred for 0.5 h before a solution of the compound prepared in Step D (20.1 g, 97.6 mmol) in THF (200 mL) was added slowly via syringe.",
"The reaction was stirred at room temperature overnight, then quenched with a saturated solution of NH4Cl.",
"The organic layer was separated and the aqueous layer extracted with ethyl acetate (3×).",
"The combined organic layers were washed with water (3×), dried over Na2SO4, filtered, and evaporated in vacuo.",
"Flash chromatography afforded 31.76 g (94.6%) of the pure desired product.",
"1H NMR (500 MHz, CDCl3) δ 9.03 (d, J=1.5 Hz, 1H), 8.25 (d, J=2.0 Hz, 1H), 5.25 (s, 1H), 3.86 (s, 3H), 1.52 (s, 9H).",
"Step F A suspension of Raney Ni (1 g) and the product from Step E (18.2 g, 52.9 mmol) in ethanol (130 mL) was placed on a Parr Apparatus and hydrogenated at 40 psi overnight.",
"The suspension was filtered through celite and the filtrate evaporated in vacuo to afford 16.35 g (97.8%) of crude product.",
"1H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H), 7.89 (s, 1H), 7.82 (s, 1H), 4.83 (d, J=16 Hz, 1H), 4.72 (s, 1H), 4.49 (d, J=16 Hz, 1H), 1.45 (s, 9H).",
"Step G To the mixture of the product from Step F (16 g, 51 mmol) in DCM (60 mL) was added TFA (30 mL) and the resulting mixture stirred at room temperature for 0.5 h. The solution was evaporated under reduced pressure and the residue was dissolved in DCM.",
"The mixture was neutralized by slow addition of a solution of saturated sodium bicarbonate and the organic layer removed.",
"The aqueous was extracted with DCM (4×) and then all organic layers were combined, dried over Na2SO4, filtered, and evaporated in vacuo to afford 10.42 g (95.2%) of the desired product.",
"1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 7.78 (s, 1H), 7.30 (s, 1H), 4.63 (s, 2H), 3.90 (s, 2H).",
"Step H: To a solution of the product from Step G (18.0 g, 83.3 mmol) in THF (50 mL) was added 1.0 M Borane in THF (417 mL, 420 mmol) and the resulting solution stirred at room temperature overnight.",
"The solution was evaporated under reduced pressure and then the residue was treated with 1% HCl/MeOH solution in which the resulting mixture was heated at 50° C. overnight to breakdown the borane complex.",
"Treatment with acidic methanol was repeated twice to insure that the borane complex was eliminated.",
"The crude product from this reaction was then immediately used for the next reaction.",
"A solution of crude product described immediately above (83.3 mmol, assuming 100% conversion) and DIEA (43 mL, 250 mmol) in DCM was treated with di-tert-butyl dicarbonate (36.4 g, 167 mmol) and the resulting mixture stirred at room temperature overnight.",
"The solution was washed with saturated sodium bicarbonate solution, water, and brine.",
"The aqueous layers were combined and back-washed with DCM (2×).",
"The combined organic layers were then dried over Na2SO4, filtered, and evaporated to dryness.",
"The crude product was purified by flash chromatography and MPLC to afford (11.89 g, 47.2% for last two steps) as a yellow solid.",
"1H NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 7.66 (s, 1H), 4.67 (s, 2H), 3.79 (t, J=6.0 Hz, 2H), 3.08 (t, J=5.5 Hz, 2H), 1.51 (s, 9H).",
"Step I The product described in Step H (11.89 g) was treated with a solution of 4 M HCl in dioxane.",
"The solution was stirred at room temperature for 2 hours and then evaporated in vacuo to afford Intermediate 2 (10.85 g, 99%) as a yellow powder.",
"LC-MS for C9H10F3N2[M+H+] calculated 202.07, found 203.0.INTERMEDIATE 3 Step A A solution of methyl-3-oxocyclopentane-carboxylate (20 g, 160 mmol) and trimethyl orthoformate (85 mL, 780 mmol) in methanol was treated with a catalytic amount of p-toluenesulfonic acid (3.00 g, 15.6 mmol) and the resulting solution was stirred for 4 h at room temperature.",
"The solvent was evaporated under reduced pressure and the residue was then dissolved in ether (600 mL).",
"The solution was washed with saturated sodium bicarbonate (2×200 mL), water (150 mL), brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the solvent evaporated as before.",
"Purification by flash column chromatography (eluant: 25% ether/pentane) afforded 21.52 g (73%) of the desired product as a clear oil.",
"1H NMR (500 MHz, CDCl3) δ 3.68 (s, 3H), 3.21 (d, J=9.9 Hz, 6H), 2.89 (p, J=8.5 Hz, 1H), 2.14-2.05 (m, 2H), 2.02-1.80 (m, 4H).",
"Step B A flame dried 500 mL round bottom flask was charged with 150 mL of dry THF, and then, set under nitrogen and cooled to −78° C. using an acetone/dry ice bath.",
"Diisopropylamine (19.2 mL, 137 mmol) was added to the cooled solvent via syringe followed by the slow addition of 2.5M n-butyllithium in hexane (55 mL, 140 mmol).",
"After 5 minutes stirring, the methyl ketal described in Step A, Intermediate 3 (21.52 g, 114.4 mmol) in 50 mL of THF was added dropwise via syringe and the resulting mixture stirred at −78° C. for 2 hours.",
"2-Iodopropane (34.3 mL, 343 mmol) was then added dropwise via syringe and the resulting mixture was stirred overnight allowing to warm slowly to room temperature.",
"The reaction was quenched with a solution of 10% citric acid and the organics separated.",
"The aqueous layer was extracted with ether (3×150 mL) and all the organics combined, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure.",
"The crude product was purified by flask column using an eluant of 20% ether/pentane to afford 16.74 g (64%) of the desired product.",
"1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3H), 3.18 (d, J=20.5 Hz, 6H), 2.57 (d, J=13.9 Hz, 1H), 2.29-2.20 (m, 1H), 1.90 (p, J=6.8 Hz, 1H), 1.88-1.80 (m, 2H), 1.69-1.61 (m, 2H), 0.89 (dd, J=11.9 Hz, 6.8 Hz, 6H).",
"Step C A solution of the ester (described in Step B, Intermediate 3, 16.7 g, 72.7 mmol) in ethanol (30 mL) was treated with 5 M NaOH (55 mL) and the resulting mixture heated to reflux for three days.",
"The mixture was then cooled to room temperature and acidified with concentrated hydrochloric acid.",
"The organic solvent was evaporated under reduced pressure and the aqueous layer was then extracted with DCM (5×100 mL).",
"The organic extracts were combined, dried over anhydrous magnesium sulfate, filtered, and evaporated in vacuo to yield the crude 3-oxocyclopentane carboxylic acid (11.07 g, 90%) as a yellow oil.",
"1H NMR (500 MHz, CDCl3) δ 2.70 (d, J=18.1 Hz, 1H), 2.44-2.39 (m, 1H), 2.30-2.15 (m, 2H), 2.14 (dd, J=18.1, 1.0 Hz, 1H), 2.06 (p, J=6.9 Hz, 1H), 1.98 (m, 1H), 0.98 (dd, J=11.4, 6.9 Hz, 6H).",
"Step D Procedure A: To a solution of acid (described in Step C, Intermediate 3, 2.00 g, 11.8 mmol) in DCM (50 mL) was added oxalyl chloride (1.54 mL, 17.6 mmol) followed by 2 drops of DMF.",
"The solution was stirred at room temperature for 80 minutes and then evaporated under reduced pressure.",
"The residue was dissolved in DCM (2 mL) and added via syringe to a prepared solution of Intermediate 1 (2.36 g, 11.8 mmol) and triethylamine (2.13 mL, 15.3 mmol) in DCM (40 mL).",
"The resulting mixture was stirred at room temperature for 18 hours and then quenched with water (25 mL).",
"The organics were separated, washed with 1 N HCl, saturated sodium bicarbonate, and brine, dried over anhydrous magnesium sulfate, filtered, and evaporated.",
"The crude product was purified by MPLC using an eluant of 60% ethyl acetate/hexane to afford Intermediate 3 (3.18 g, 77%).",
"1H NMR (500 MHz, CDCl3) δ 7.46 (d, J=7.3 Hz, 1H), 7.39 (s, 1H), 7.29 (d, J=7.7 Hz, 1H), 4.81 (m, 2H), 3.93 (m, 1H), 3.82 (m, 1H), 2.94 (m, 3H), 2.54 (m, 1H), 2.43 (d, J=8.5 Hz, 1H), 2.32 (m, 2H), 2.26 (p, J=6.6 Hz, 1H), 2.16 (m, 1H), 0.93 (dd, J=19.7 Hz, 6.8 Hz, 6H).",
"LC-MS for C19H23F3NO2 calculated 353.16, found [M+H+] 354.25.Procedure B: A mixture of the acid prepared in Step C, Intermediate 3 (1.0 g, 5.9 mmol), Intermediate 1 (1.18 g, 5.88 mmol), DMAP (71 mg, 0.59 mmol), and N,N-diisopropyl ethylamine (1.02 mL, 5.88 mmol) in dichloromethane (20 mL) was treated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 2.25 g, 11.7 mmol) and stirred at room temperature overnight.",
"The reaction mixture was diluted with dichloromethane (30 mL), washed with water (2×20 mL), brine (1×30 mL), dried over anhydrous sodium sulfate and the solvent was evaporated.",
"The pure compound was obtained by MPLC purification (eluant 60% ethyl acetate/hexane), 1.08 g (52%).",
"LC-MS for C19H23F3NO2 calculated 353.16, found [M+H+] 354.25.INTERMEDIATE 4 To a solution of acid (described in Step C, Intermediate 3, 540 mg, 3.20 mmol) in DCM (50 mL) was added oxalyl chloride (0.834 mL, 9.60 mmol) followed by 2 drops of DMF.",
"The solution was stirred at room temperature for 80 minutes and then evaporated under reduced pressure.",
"The residue was dissolved in DCM (2 mL) and added via syringe to a prepared solution of Intermediate 2 (880 mg, 3.20 mmol) and triethylamine (0.820 mL, 6.50 mmol) in DCM (20 mL).",
"The resulting mixture was stirred at room temperature for 18 hours and then quenched with water (25 mL).",
"The organics were separated, washed with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered, and evaporated.",
"The crude product was purified by MPLC using a step-wise gradient eluant of 0-70% ethyl acetate/hexane to afford Intermediate 2 (720 mg, 64%).",
"ESI-MS calculated for C18H21F3N2O2: 354.16; found 355 (M+H) INTERMEDIATE 5 Resolution of Intermediate 4 to its individual enantiomers was accomplished by chiral separation using HPLC equipped with a Preparative ChiralPak AD column.",
"The separation was completed by injecting 100 mg/run and using an eluant of 25% isopropanol and 75% heptane with a flow rate of 9 mL/min.",
"INTERMEDIATE 6 Step A Procedure A: A solution of 3-oxo-cyclopentane carboxylic acid (Stetter, H., Kuhlmann, H. Liebigs Ann.",
"Chem., 1979, 7, 944-9) (5.72 g, 44.6 mmol) in dichloromethane (30 mL) was treated with N,N′-diisopropyl-O-tert-butyl-iso-urea (21.2 mL, 89.3 mmol) and the reaction mixture was stirred at ambient temperature overnight.",
"The precipitated N,N′-diisopropyl urea was filtered off, the filtrate concentrated in vacuo and the residue was purified by distillation (bp: 125-129° C.@ 18 mmHg) to yield 4.74 g (58%) of the pure product.",
"1H NMR (500 MHz, CDCl3): δ 3.02 (p, J=7.8 Hz, 1H), 2.05-2.50 (m, 6H), 1.45 (s, 9H).",
"13C NMR (125 MHz, CDCl3): δ 217.00, 173.47, 80.99, 41.88, 41.14, 27.94, 26.57.Procedure B: A 2 L round RBF was charged with anhydrous magnesium sulfate (113 g, 940 mmol) and dichloromethane (940 mL) was added.",
"While stirring, the suspension was treated with concentrated sulfuric acid (12.5 mL, 235 mmol) followed in 15 minutes by 3-oxo-cyclopentane carboxylic acid (30.1 g, 235 mmol).",
"After stirring for 15 minutes, tert-butanol (87 g, 1.2 mol) was added.",
"The reaction vessel was closed with a stopper to aid retention of isobutylene, and stirred at ambient temperature for 72 hours.",
"The solid was filtered off through a plug of celite, volume of the filtrate was reduced to approximately 500 mL, and washed with saturated solution of sodium bicarbonate (2×150 mL).",
"The organic phase was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation at reduced pressure (180 mmHg).",
"The crude product was purified by distillation to yield 39.12 g (90%) of pure product.",
"Step B A solution of tert-Butyl 3-oxocyclopentane carboxylate (11.54 g, 62.64 mmol) in dichloromethane (200 mL) was treated with trimethyl orthoformate (41.4 mL, 251 mmol) in the presence of p-toluenesulfonic acid (400 mg) and stirred at room temperature for 48 hours.",
"The dark reaction mixture was poured onto saturated solution of sodium bicarbonate, and the crude product was extracted with dichloromethane.",
"The combined organic extracts were dried with anhydrous magnesium sulfate, the solvent was removed in vacuo, and the crude product was purified by distillation (bp.",
": 104° C.@ 4 mmHg) to yield 12.32 g (85%) of the desired product.",
"1H NMR (500 MHz, CDCl3): δ 3.21 (s, 3H), 3.20 (s, 3H), 2.80 (m, 1H), 2.10 to 1.80 (bm, 6H), 1.46 (s, 9H).",
"13C NMR (125 MHz, CDCl3): δ 174.9, 111.2, 80.3, 67.8, 49.2, 42.5, 37.4, 33.8, 28.3, 22.0.Step C A flame dried 500 mL round bottom flask was charged with 100 mL of dry THF, and then, set under nitrogen and cooled to −78° C. using an acetone/dry ice bath.",
"Diisopropylamine (7.9 mL, 56 mmol) was added to the cooled solvent via syringe followed by the slow addition of 2.5 M n-butyllithium in hexane (22.6 mL, 56.45 mmol).",
"After 5 minutes stirring, the acetal (described in Step B, Intermediate 6, 10.0 g, 43.4 mmol) in 50 mL of THF was added dropwise via syringe and the resulting mixture stirred at −78° C. for 2 hours.",
"Acetylaldehyde (7.3 mL, 130 mmol) was then added dropwise via syringe and the resulting mixture was stirred for 2 h at −78° C. The reaction was quenched by pouring the mixture into a solution of 10% citric acid (300 mL) and then extracting with dichloromethane (2×150 mL).",
"The organics were combined, dried over anhydrous magnesium sulfate, filtered, and evaporated under reduced pressure.",
"During the reaction or work-up some of the acetal was hydrolyzed to the ketone, therefore, the crude mixture was taken onto the next step without purification.",
"Step D The crude intermediate (described in Step C, Intermediate 6, 56.45 mmol assumed 100% conversion for Step C) was treated with a solution of 10% trifluoroacetic acid in dichloromethane and the resulting mixture stirred overnight at room temperature.",
"The reaction was concentrated in vacuo, then diluted with water, and extracted with dichloromethane.",
"The organics were combined, dried over anhydrous magnesium sulfate, filtered, and evaporated under reduced pressure to afforded 8.04 g (83%) of the crude product that was used without further purification.",
"Step E A mixture of the acid (described in Step D, Intermediate 6, 300 mg, 1.74 mmol), Intermediate 2 (486, 1.74 mmol), HOAt (237 mg, 1.74 mmol), and N,N-diisopropyl ethylamine (0.606 mL, 3.48 mmol) in dichloromethane (15 mL) was treated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 667 mg, 3.48 mmol) and stirred at room temperature for five days.",
"The reaction mixture was diluted with dichloromethane (30 mL), washed with water (20 mL), brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated in vacuo.",
"The product, Intermediate 6, was obtained by preparative plate purification (eluant 100% ethyl acetate), 260 mg (42%).",
"INTERMEDIATE 7 Step A A solution of 2-fluoro-4-trifluoromethylphenylacetonitrile (10 g, 49 mmol) in a mixture of ethanol (100 mL) and ammonium hydroxide (20 mL of a 29.3% aqueous solution) was hydrogenated over Raney nickel (1 g) for 16 h. The catalyst was removed by filtration through celite and the filtrate evaporated to dryness.",
"The neat residue was added in a dropwise manner to trifluoroacetic anhydride (25 mL, 180 mmol) cooled at 0° C. and the resulting mixture was stirred at 0° C. for 30 minutes.",
"The reaction mixture was poured onto ice (250 g) and the resulting mixture was stirred for 30 minutes after which time the precipitate was removed by filtration and air dried to give the product as a white solid (13.4 g, 90%); 1H NMR 500 MHz (CDCl3) δ=3.02 (2H, t, J=7.0 Hz), 3.66 (2H, q, J=6.6 Hz), 6.44 (1H, br s), 7.34 (2H, m), 7.41 (1H, d, J=7.8 Hz).",
"Step B To a mixture of the product from Step A (13 g, 44 mmol) and paraformaldehyde (2.0 g, 48 mmol) was added in one portion a mixture of concentrated sulfuric acid (90 mL) and glacial acetic acid (60 mL) and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured onto a mixture of ice and water (1 L) and extracted with ethyl acetate (3×150 mL); the combined ethyl acetate layers were washed with water (3×500 mL), saturated NaHCO3 (200 mL), and saturated NaCl (100 mL), dried over MgSO4, filtered and evaporated in vacuo.",
"The residue was purified by column chromatography on silica elution with 10% Et2O in hexanes to give the product (8.29 g, 60%); 1H NMR 500 MHz (CDCl3) δ=3.01 (2H, m), 3.91 and 3.97 (2H, t, J=6.2 Hz), 4.83 and 4.88 (2H, s), 7.21-7.28 (3H, m).",
"Step C To a solution of the trifluoroacetamide formed in Step B (8.3 g, 26 mmol) in ethanol (200 mL) was added a solution of potassium carbonate (20 g, 150 mmol) in water (50 mL), and the resulting mixture was stirred at reflux for 1 h. The ethanol was removed under reduced pressure and water (150 mL) was added to the residue and extracted with CH2Cl2 (3×100 mL).",
"The combined CH2Cl2 layers were washed with saturated NaCl (100 mL), dried over Na2SO4, filtered and evaporated in vacuo to give the product (5.2 g, 91%); 1H NMR 500 MHz (CDCl3) δ=1.74 (1H, br s), 2.78 (2H, d, J=6.0 Hz), 3.17 (2H, t, J=6.0 Hz), 4.05 (2H, s), 7.04-7.14 (3H, m).",
"INTERMEDIATE 8 Step A A 3-neck round bottomed flask equipped with an addition funnel and condenser and containing zinc dust (2.45 g, 37.4 mmol) was flame dried.",
"After cooling, and purging the system with nitrogen gas, 6 mL of THF was added, followed by 1,2-dibromoethane (0.298 mL, 3.46 mmol).",
"The mixture was warmed to a vigorous reflux using a heat gun and stirred at reflux for ˜30 seconds (gas evolution was observed), then cooled to room temperature.",
"The warming and cooling was repeated two more times.",
"Then chlorotrimethylsilane (0.402 mL, 3.17 mmol) was added and the mixture was stirred at room temperature for 20 minutes.",
"N-t-butoxycarbonyl-4-iodo-piperidine (known: Billotte, S. Synlett (1998), 379, 8.97 g, 28.8 mmol) in 15 mL of THF was added over a period of about 1 minutes.",
"The reaction mixture was stirred at 50° C. for 1.5 h, then was cooled to room temperature.",
"Meanwhile, a mixture of tri-2-furylphosphine (267 mg, 1.15 mmol) and Tris(dibenzylideneacetone)-dipalladium(0) chloroform adduct (298 mg, 0.288 mmol) was dissolved in 6 mL of THF under a nitrogen atmosphere, stirred for 15 minutes at room temperature, and added to the organozinc solution.",
"Then a solution of 2-bromopyrimidine (5.50 g, 34.6 mmol) in a mixture of 58 mL of THF and 20 mL of N,N-dimethylacetamide was added.",
"The reaction mixture was warmed to 80° C. and stirred for 3.5 h, then was cooled to room temperature and stirred for 36 h. The reaction mixture was filtered through celite and the filter cake was washed with ethyl acetate.",
"The filtrate was diluted further with ethyl acetate, and washed with saturated NaHCO3 solution.",
"The aqueous layer was back extracted with ethyl acetate, the organic layers were combined and washed twice with water and once with brine.",
"The organic phase was dried over anhydrous MgSO4, filtered, and concentrated.",
"Purification by flash chromatography (silica, stepwise gradient: 25% ethyl acetate/hexane, 40% ethyl acetate/hexane, 60% ethyl acetate/hexane, 80% ethyl acetate/hexane, 100% ethyl acetate) to afford 4.92 g of pure 4-(2-pyrimidyl)-piperidine product (65%).",
"1H NMR (500 MHz, CDCl3): δ 8.70 (d, J=5.0 Hz, 2H), 7.16 (app t, J=4.5 Hz, 1H), 4.24 (br s, 2H), 3.05 (m, 1H), 2.89 (br m, 2H), 2.01 (br d, J=13 Hz, 2H), 1.84 (dq, J=4.5, 12.5 Hz, 2H), 1.49 (s, 9H).",
"Step B The N-t-butoxycarbonylpiperidine prepared in Step A (4.64 g, 17.6 mmol) was dissolved in 4 N HCl in dioxane (50 mL) and stirred at room temperature for 2.25 h. The reaction mixture was concentrated to afford 4.16 g of piperidine hydrochloride (100%) which required no further purification.",
"1H NMR (500 MHz, CD3OD): δ 8.95 (d, J=5.5 Hz, 2H), 7.60 (t, J=5.0 Hz, 1H), 3.53 (dt, J=13, 3.5 Hz, 2H), 3.35 (tt, J=4.0, 11.0 Hz, 1H), 3.20 (br t, J=13.8 Hz, 2H), 2.30 (br d, J=14.0 Hz, 2H), 2.11-2.20 (m, 2H); ESI-MS calc.",
"for C9H13N3: 163; Found: 164 (M+H).",
"INTERMEDIATE 9 This intermediate was prepared using the procedure described for Intermediate 8 except that 4-bromo pyrimidine was used in place of 2-bromo pyrimidine.",
"LC-MS for C9H13N3 calculated 163.28, found [M+H]+ 164.INTERMEDIATE 10 4-(1H-1,2,4-triazol-1-yl)piperidine hydrochloride Step A tert-butyl 4-hydroxypiperidine-1-carboxylate To a stirred solution of 4-hydroxypiperidine (60.8 g) in dichloromethane (500 mL) was added a solution of di-ter-butyl dicarbonate (19 g, 0.55 mol) in dichloromethane (500 mL) very slowly.",
"After the addition, which took 1 h, the resulted mixture was stirred at ambient temperature for 5 h. The mixture was then washed with saturated NaHCO3, 3 N HCl, brine, dried and evaporated to give tert-butyl 4-hydroxypiperidine-1-carboxylate as a thick oil (90 g).",
"Step B tert-butyl 4-[(methylsulfonyl)oxy]piperidine-1-carboxylate To a stirred solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (21.1 g, 100 mmol) and triethyl amine (22 mL) in dichloromethane (250 mL) at 0° C. was slowly added methanesulfonyl chloride (9.0 mL, 1.1 equiv.).",
"The resulting mixture was stirred for an additional 1 h and during this time white solid was formed.",
"The mixture was then washed with 3 N HCl, dried over Na2SO4 and evaporated to give: tert-butyl 4-[(methylsulfonyl)oxy]piperidine-1-carboxylate as a white solid (29.2 g).",
"1H NMR (400 MHz, CDCl3): δ 4.92-4.87 (m, 1H), 3.75-3.69 (m, 2H), 3.34-3.28 (m, 2H), 3.05 (s, 3H), 2.01-1.94 (m, 2H), 1.87-1.78 (m, 2H).",
"Step C 4-(1H-1,2,4-triazol-1-yl)piperidine hydrochloride To a stirred solution of: tert-butyl 4-[(methylsulfonyl)oxy]piperidine-1-carboxylate (5.9 g, 21 mmol) and 1,2,4-triazole (1.8 g, 25 mmol equiv.)",
"in DMF at ambient temperature was added sodium hydride (60% in mineral oil, 1.0 g, 25 mmol).",
"The mixture was stirred at 60° C. for 5 days, and the TLC showed no starting mesylate left.",
"The mixture was poured into ice water and extracted with ethyl acetate (3×).",
"The organic layer was dried, evaporated and purified by silica flash column eluting with 0-10% methanol in ethyl acetate to give tert-butyl 4-(1H-1,2,4-triazol-1-yl)piperidine-1-carboxylate as a white solid.",
"The solid was then treated with hydrogen chloride in dioxane (4 N, 10 mL) for 2 h. The mixture was then evaporated to remove most of the dioxane to give a white solid, which was washed with ethyl acetate to give the desired 4-(1H-1,2,4-triazol-1-yl)piperidine hydrochloride salt (5.55 g).",
"1H NMR (300 MHz, CD3OD): δ 10.00 (s, 1H), 8.97 (s, 1H), 5.10-5.00 (m, 1H), 3.63-3.58 (br.",
"d, 2H), 3.33-3.26 (br.",
"d, 2H), 2.50-2.30 (m, 4H).",
"The following compounds 10-16 were prepared in a similar fashion to Intermediate 10 using: tert-butyl 4-[(methylsulfonyl)oxy]piperidine-1-carboxylate and the appropriate heterocycles.",
"INTERMEDIATE 11 4-(1H-pyrazol-1-yl)piperidine hydrochloride Prepared using pyrazole according to the procedure for Intermediate 10.INTERMEDIATE 12 4-(1H-imidazol-1-yl)piperidine hydrochloride Prepared from imidazole according to the procedure for Intermediate 10: 1H NMR (400 MHz, CD3OD): δ 9.18 (s, 1H), 7.86 (s, 1H), 7.65 (s, 1H), 4.9-4.8 (hidden under CD3OD peak, 1H), 3.61-3.61 (br.",
"d., 2H), 3.33-3.26 (m, 2H), 2.49-2.45 (br.",
"d, 2H), 2.39-2.28 (m, 2H).",
"INTERMEDIATE 13 4-(1H-1,2,3-triazol-1-yl)piperidine hydrochloride Prepared from 1,2,3-triazole according to the procedure for Intermediate 10.4-(1H-1,2,3-triazol-1-yl)piperidine hydrochloride: 1H NMR (400 MHz, CD3OD): δ 8.77 (s, 1H), 8.54 (s, 1H), 5.26-5.19 (m, 1H), 3.65-3.59 (m, 2H), 3.37-3.29 (m, 2H), 2.60-2.54 (m, 2H), 2.50-2.39 (m, 2H).",
"INTERMEDIATE 14 4-(2H-1,2,3-triazol-2-yl)piperidine hydrochloride Prepared from 1,2,3-triazole according to the procedure for Intermediate 10.4-(2H-1,2,3-triazol-2-yl)piperidine hydrochloride: 1H NMR (400 MHz, CD3OD): δ 7.72 (s, 2H), 4.94-4.87 (m, 1H), 3.54-3.48 (m, 2H), 3.28-3.22 (m, 2H), 2.46-2.32 (m, 4H).",
"INTERMEDIATE 15 4-(1H-tetraazol-1-yl)piperidine hydrochloride Prepared from tetrazole according to the procedure for Intermediate 10.4-(1H-tetraazol-1-yl)piperidine hydrochloride: 1H NMR (400 MHz, CD3OD): δ 8.77 (s, 1H), 5.30-5.23 (m, 1H), 3.58-3.53 (m, 2H), 3.35-3.29 (m, 2H), 2.58-2.2.52 (m, 2H), 2.48-2.38 (m, 2H).",
"INTERMEDIATE 16 4-(2H-tetraazol-2-yl)piperidine hydrochloride Prepared from tetrazole according to the procedure for Intermediate 10.4-(2H-tetraazol-2-yl)piperidine hydrochloride: 1H NMR (400 MHz, CD3OD): δ 9.32 (s, 1H), 5.08-5.00 (m, 1H), 3.61-3.57 (m, 2H), 3.33-3.28 (m, 2H), 2.52-2.47 (m, 2H), 2.42-2.32 (m, 2H).",
"INTERMEDIATE 17 Prepared from 5-methyltetrazole according to the procedure for Intermediate 10.1H NMR (400 MHz, CD3OD): δ 5.08-5.00 (m, 1H), 3.61-3.57 (m, 2H), 3.33-3.28 (m, 2H), 2.52-2.47 (m, 2H), 2.42-2.32 (m, 2H), 1.68 (s, 3H).",
"INTERMEDIATE 18 Step A Hydroxylamine hydrochloride (8.26 g, 119 mmol) and triethylamine (16.6 mL, 119 mmol) were combined in 50 mL of DMSO.",
"The suspension was filtered to remove triethylamine hydrochloride and the filter cake was washed with THF.",
"The filtrate was partially concentrated to remove the THF.",
"Then commercially available 1-tert-butoxycarbonyl-4-cyanopiperidine (5.0 g, 24 mmol) was added to the DMSO solution and the resulting reaction mixture was stirred at 75° C. for 3 h, and then at room temperature overnight.",
"The reaction mixture was diluted with ethyl acetate and washed with water.",
"The aqueous layer was back-extracted with more ethyl acetate and the combined organic layers were washed four times with water and once with brine.",
"The organic layer was dried over anhydrous MgSO4, filtered, and concentrated to give 3.51 g of product.",
"Step B A solution of the compound from Step A (1.02 g, 4.19 mmol) in 20 mL THF was treated with thiocarbonyldiimidazole (897 mg, 5.03 mmol), whereupon gas evolution and an exotherm were noted.",
"The reaction mixture was stirred at room temperature for 1 h, then was transferred to a suspension of silica gel #60 (20 g) in 180 mL of 5:1 CHCl3/methanol.",
"The reaction mixture was stirred at room temperature for 5 days, then filtered and concentrated.",
"Purification by MPLC (silica, 50% ethyl acetate/hexane) afforded 143 mg of thiodiazolone.",
"Boc intermediate 1H NMR (500 MHz, CD3OD): δ 4.16 (m, 2H), 2.86 (t, J=11.5 Hz, 2H), 2.77 (tt, J=4.0, 11.0 Hz, 1H), 1.98 (dd, J=2.0, 13.0 Hz, 2H), 1.73 (dq, J=4.5, 12.0 Hz, 2H), 1.47 (s, 9H).",
"The Boc intermediate (139 mg, 0.487 mmol) was dissolved in 4 N HCl in dioxane (5 mL) and stirred at room temperature for 1.5 h. The reaction mixture was concentrated to give 94.3 mg of piperidine hydrochloride product.",
"INTERMEDIATE 19 4-(1H-pyrazol-3-yl)piperidine Step A 4-(1H-pyrazol-3-yl)pyridine To a mixture of 4-acetylpyridine (75 mL, 0.68 mol) and ethyl formate (109 mL) in anhydrous benzene (1 L) was added sodium methoxide (73 g) and the resulting mixture was refluxed for 18 h. The mixture was cooled and benzene decanted from a sticky solid, which had formed during the reaction.",
"The crude product was dissolved in water (700 mL) and hydrazine dihydrochloride was added and the resulting mixture was stirred at room temperature for 2 h. The mixture was redissolved by addition of 5 N NaOH.",
"Precipitate formed which was removed by filtration and dried to give 4-(1H-pyrazol-3-yl)pyridine (35 g).",
"Step B 1-benzyl-4-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine To a hot (80° C.) solution of 4-(1H-pyrazol-3-yl)pyridine (9.6 g) in 2-propanol (60 mL) was added benzyl bromide (20 mL, 2.5 equiv.)",
"and the resulting mixture was heated at reflux for 10 minutes.",
"After cooling in an ice bath, the precipitate was filtered and washed with more 2-propanol and air dried.",
"The solid was suspended in ethanol at 0° C. and sodium borohydride (13 g) was added in several portions over 30 minutes, and the mixture was stirred for an additional 30 minutes.",
"The reaction was quenched by the careful addition of water, the ethanol was removed by evaporation, and the residue was partitioned between dichloromethane and water.",
"The organic layer was dried over MgSO4, filtrated and evaporated to give 1-benzyl-4-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine (16 g) Step C 4-(1H-pyrazol-3-yl)piperidine A solution of 1-benzyl-4-(1H-pyrazol-3-yl)-1,2,3,6-tetrahydropyridine (16 g) was hydrogenated over palladium on carbon (10%, 1 g) at 40 psi overnight.",
"The catalyst was removed by filtration through celite and the filtrate was evaporated.",
"NMR shows the product is 1-benzyl-4-(1H-pyrazol-3-yl)-piperidine (16 g).",
"To a solution of 1-benzyl-4-(1H-pyrazol-3-yl)-piperidine (16 g) and formic acid (30 mL) in ethanol (400 mL) was added palladium on carbon (10%, 2 g) and the resulting mixture was stirred at room temperature overnight.",
"The catalyst was removed by filtration and the filtrate evaporated.",
"The product was purified by adding di-tert-butyl dicarbonate (2 equiv.)",
"and triethyl amine (1.5 equiv.)",
"in dichloromethane to give a Boc protected intermediate.",
"Evaporated and purification by column chromatography on silica eluting with 20-40% ethyl acetate in hexane give pure tert-butyl 4-(1H-pyrazol-3-yl)piperidine-1-carboxylate.",
"The Boc intermediate was then treated with methanolic HCl to give 4-(1H-pyrazol-3-yl)piperidine hydrochloride salt (3.5 g).",
"Loss of material was due to the formation of the di-Boc product, which was not collected.",
"1H NMR (400 MHz, CDCl3): δ 8.00 (s, 2H), 3.48 (br.",
"d, J=13 Hz, 2H), 3.28-3.20 (m, 1H), 3.13 (br.",
"t, J=13 Hz, 2H), 2.23 (br.",
"d, J=14 Hz, 2H), 1.97-1.85 (m, 2H).",
"INTERMEDIATE 20 Step A To a mixture of 4-bromophenethylamine hydrobromide (25 g, 89 mmol) and pyridine (36 mL, 445 mmol) in CH2Cl2 (100 mL) cooled at 0° C. was added dropwise trifluoroacetic anhydride (18.8 mL, 133 mmol).",
"After complete addition the mixture was stirred at room temperature for 48 hours then poured onto ice (500 g).",
"The mixture was extracted with CH2Cl2 (4×100 mL), the combined CH2Cl2 layers were washed with 1N HCl (4×100 mL), sat NaCl (100 mL), dried over MgSO4, filtered and evaporated in vacuo to give the product (26.13 g, 100%); 1H NMR 500 MHz (CDCl3) □=2.86 (2H, t, J=7.1 Hz), 3.59 (2H, q, J=6.6 Hz), 6.57 (1H, br s), 7.09 (2H, d, J=8.5 Hz), 7.43 (2H, d, J=8.5 Hz).",
"Step B To a mixture of the product from Step A (26 g, 88 mmol) and paraformaldehyde (5.6 g, 130 mmol), was added in one portion a mixture of concentrated sulfuric acid (130 mL) and glacial acetic acid (195 mL), and the resulting mixture stirred at room temperature for 17 hours.",
"The reaction mixture was poured onto ice/water (1.5 L) and extracted with ethyl acetate (3×300 mL), the combined ethyl acetate layers were washed with water (2×600 mL), sat NaHCO3 (300 mL), and saturated NaCl (150 mL), dried over MgSO4, filtered and evaporated in vacuo.",
"The residue was dissolved in ethanol (450 mL), and a solution of potassium carbonate (60 g, 434 mmol) in water (150 ml) was added.",
"The mixture was heated to reflux for 1 hour then cooled and evaporated in vacuo.",
"Water (500 mL) was added to the residue and extracted with CH2Cl2 (3×300 mL); the combined CH2Cl2 layers were washed with water (500 mL), sat NaCl (150 mL), dried over Na2SO4, filtered and concentrated in vacuo.",
"The residue was purified by column chromatography on silica elution with 5% CH3OH in CH2Cl2 containing 0.5% NH4OH to give the product (10 g, 54%); 1H NMR 500 MHz (CDCl3) δ=1.77 (1H, br s), 2.77 (2H, d, J=6.0 Hz), 3.11 (2H, t, J=6.0 Hz), 3.97 (2H, s), 6.95 (1H, d, J=8.0 Hz), 7.15 (1H, s) 7.23 (1H, dd, J=1.2 and 8.2 Hz).",
"INTERMEDIATE 21 Step A To a solution of 3-cyclopentene-1-carboxylic acid (Org.",
"Synth.",
"75, p195-200, 1998) (31.5 g, 281 mmol) in anhydrous N,N-dimethylformamide (300 mL), under an atmosphere of nitrogen, was added potassium carbonate (97 g, 710 mmol), and iodomethane (35 mL, 560 mmol).",
"The resulting mixture was stirred at room temperature for 16 h, then poured into water (1 L), and extracted with diethyl ether (3×400 mL).",
"The combined diethyl ether layers were washed with water (3×500 mL), saturated NaCl (200 mL), dried over MgSO4, filtered and concentrated in vacuo, to give 34 g (96%).",
"1H NMR (CDCl3, 500 MHz): δ 5.64 (s, 2H), 3.68 (s, 3H), 3.11 (quintet, J=8.5 Hz, 1H), 2.63 (d, J=8.3 Hz, 4H).",
"Step B To a cooled (−78° C.) solution of diisopropylamine (34.4 mL, 250 mmol) in anhydrous tetrahydrofuran (250 mL) under an atmosphere of nitrogen was added slowly n-butyllithium (100 mL of a 2.5M solution in hexanes, 250 mmol), and the resulting mixture was stirred at −78° C. for 10 minutes.",
"To this mixture was added methyl-3-cyclopentenecarboxylate (25.8 g, 200 mmol), after stirring for a further 15 minutes 2-iodopropane (41 mL, 410 mmol) was added, and the mixture stirred at −78° C. for 30 minutes then allowed to rise to +4° C. and was left standing at this temperature for 72 h. The reaction mixture was poured in 5% citric acid (700 mL) and extracted with diethyl ether (3×300 mL).",
"The combined diethyl ether layers were washed with water (2×500 mL), saturated NaCl (1×100 mL), dried over MgSO4, filtered and concentrated in vacuo.",
"The residue was purified by vacuum distillation 50° C.@ 5 mmHg to provide 28.9 g (86%) of product.",
"1H NMR (CDCl3, 500 MHz): δ 5.54 (s, 2H), 3.67 (s, 3H), 2.85 (d, J=15.1 Hz, 2H), 2.30 (dd, J=14.9 Hz, 2H), 2.07 (t, J=6.6 Hz, 1H), 0.82 (d, J=6.6 Hz, 6H).",
"Step C To a cooled (0° C.) solution of borane-methyl sulfide (20 mL, 200 mmol) in anhydrous tetrahydrofuran (100 mL), under an atmosphere of nitrogen, was added, using a double ended needle, a solution of the cyclopentene ester prepared in Step B (28.9 g, 172 mmol).",
"After complete addition the reaction mixture was stirred at room temperature for 20 h. The mixture was cooled in an ice bath and sodium hydroxide (60 mL of a 3 N solution, 181 mmol) was added dropwise, followed by 30% hydrogen peroxide (65 mL) and the resulting mixture was stirred at 40° C. for 1 h. The mixture was poured into water (600 mL) and extracted with diethyl ether (3×200 mL), the combined diethyl ether layers were washed with water (3×500 mL), saturated NaCl (100 mL), dried over MgSO4, filtered and concentrated in vacuo.",
"The residue was purified by column chromatography on silica elution with 20% EtOAc/hexanes to give 18.5 g (58%) of product.",
"Step D To a (−78° C.) solution of oxalyl chloride (55 mL, 110 mmol) in anhydrous dichloromethane (300 mL) under an atmosphere of nitrogen was added in a dropwise manner dimethyl sulfoxide (15.5 mL, 219 mmol), and the resulting mixture stirred at −78° C. for 10 minutes.",
"To this mixture was added, using a double ended needle, a solution of the product from Step C (18.5 g, 100 mmol) in anhydrous dichloromethane (100 mL).",
"The reaction mixture was stirred at −78° C. for a further 15 minutes, then triethylamine (69 mL, 500 mmol) was added and the resulting mixture was allowed to rise to room temperature over 2 h. The reaction mixture was washed with water (500 mL), saturated NaCl (150 mL), dried over MgSO4, filtered and concentrated in vacuo, to give 18 g of product which was used in the next step without further purification.",
"Step E To a solution of the cyclopentanone prepared in Step D (18 g, 98 mmol) in anhydrous 1,2-dichloroethane (500 mL), under an atmosphere of nitrogen, was added 4-(4-fluorophenyl)piperidine hydrochloride (25 g, 120 mmol), diisopropylethylamine (20.4 mL, 116 mmol), sodium triacetoxyborohydride (112 g, 531 mmol), and 4 Å molecular sieves (powder 10 g).",
"The mixture was stirred at room temperature for 48 h, and then diluted with dichloromethane (500 mL), and filtered through celite.",
"The filtrate was washed with saturated NaHCO3 solution (500 mL), water (500 mL), saturated NaCl (200 mL), dried over Na2SO4, filtered and concentrated in vacuo to give 28 g (82%) of product.",
"This material was used in the next step without further purification.",
"Step F 1-isopropyl-3-(4-(4-fluorophenyl)piperidin-1-yl)cyclopentanecarboxylic acid To a solution of the cyclopentane methyl ester prepared in Step E (28 g, 81 mmol) in ethanol (500 mL), was added a solution of potassium hydroxide (30 g, 540 mmol) in water (100 mL), and the resulting mixture was heated at reflux for 18 h. The cooled mixture was concentrated in vacuo to remove the ethanol, and water (200 mL) was added to the residue.",
"The mixture was extracted with diethyl ether (3×200 m/L), and the aqueous layer was neutralized by the addition of concentrated hydrochloric acid.",
"The mixture was extracted with a mixture of 9/1 chloroform/2-propanol (3×150 mL), and the combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo.",
"To the residue was added acetone (70 mL) and the mixture was heated to reflux briefly and then was left standing at +5° C. for 16 h. The acetone was decanted way from the white solid, and the remaining solid was dried to give 11.5 g (43%) of product which was a 10:1 mixture of cis and trans isomers.",
"ESI-MS calc.",
"for C20H28FNO2: 333; Found: 334 (M+H).",
"INTERMEDIATE 22 2,5-Dimethyl-3-pyrroline (3.128 g, 32.19 mmol) was dissolved in triethylamine (8.97 mL, 64.4 mmol) and cooled to 0° C. Carbobenzyloxychloride (10.11 mL, 70.83 mmol) in a minimal amount of dichloromethane was added dropwise.",
"The reaction mixture was slowly warmed to room temperature and stirred for 48 h. The reaction was quenched with saturated sodium bicarbonate solution (150 mL), and the organic layer was then washed with saturated sodium bicarbonate solution (2×100 mL) and brine (1×100 mL), dried over MgSO4, filtered, and concentrated.",
"Intermediate 1 (3.844 g) was obtained in a 52% yield through purification by silica gel flash column chromatography using a gradient solvent system of 5% EtOAc in hexanes to 10% EtOAc in hexanes.",
"ESI-MS calculated for C14H17NO2: 31.29, found 232 (M+H).",
"INTERMEDIATE 23 Procedure A: Step A A mixture of (1S)-(+)-2-azabicyclo[2.2.1]hept-5-en-3-one (10.3 g, 94.4 mmol) in ethyl acetate (200 mL) and 10% Pd/C (0.5 g), was hydrogenated at room temperature.",
"After 24 h the reaction mixture was filtered and evaporated leaving behind 10.4 g (100%) of the product that was taken in 250 mL methanol and HCl (12 M, 6 mL).",
"The resultant mixture was stirred at room temperature, until the reaction was complete (72 h).",
"Evaporation of methanol followed by drying under high vacuum, yielded title compound as an off white solid (16.0 g, 96%).",
"1H NMR (500 MHz, D2O): δ 3.70 (s, 3H), 3.01 (m, 1H), 2.38 (m, 1H), 2.16-1.73 (m, 6H).",
"Step B To a suspension of the intermediate from Step A (10.2 g, 56.8 mmol) in dry dichloromethane (200 mL) was added benzophenone imine (10.2 g, 56.8 mmol) at room temperature and the resultant mixture was stirred for 24 h. The reaction mixture was filtered and the filtrate was evaporated, to leave behind a yellow oil that was triturated with ether (100 mL), filtered and evaporated.",
"This operation was repeated twice to ensure that the product was free of ammonium chloride impurities.",
"The resultant oil was thoroughly dried under vacuum to yield the title compound (18.03 g, >100%) and required no further purification.",
"1H NMR (500 MHz, CDCl3): δ 7.5-7.18 (m, 10H), 3.75 (m, 1H), 3.7 (s, 3H), 2.78 (m, 1H), 2.26-1.71 (m, 6H).",
"Step C To a solution of lithium diisopropylamide (prepared from diisopropylamine (7.7 g, 76 mmol) and n-butyllithium (30.4 mL, 2.5 M in hexanes, 76 mmol) in tetrahydrofuran (120 mL) at −78° C. was added the ester from Step B (18.0 g, 58.6 mmol).",
"The resultant burgundy colored solution was stirred for 20 min after which it was quenched with 2-iodopropane (14.9 g, 88.0 mmol).",
"The reaction mixture was gradually warmed over 3 h to 0° C. and this temperature was maintained for an additional 3 h. Reaction was quenched with water and extracted with ethyl acetate.",
"The organic layer was washed with water, brine, dried (anhydrous magnesium sulfate) and concentrated to yield an oil.",
"To the solution of the crude Schiff base (20.0 g) in tetrahydrofuran (100 mL) was added HCl (5.0 mL, 12 M).",
"The resulting reaction mixture was allowed to stir at room temperature for 3 h. After the removal of all volatiles, the hydrochloride salt was taken up into dichloromethane (250 mL), saturated solution of sodium bicarbonate (250 mL) and di-tert-butyl dicarbonate (26.0 g, 1.4 Eq.)",
"were added.",
"The resultant mixture was vigorously stirred overnight at room temperature.",
"The organic layer was separated and washed with water, brine, dried (anhydrous magnesium sulfate) and concentrated to yield an oil.",
"Purification by flash column chromatography (eluent: hexanes/ethyl acetate 19:1) gave the desired product (4.91 g, 30%).",
"1H NMR (500 MHz, CDCl3): 4.79 (br, 1H), 4.01 (m, 1H), 3.71 (s, 3H), 2.18-1.60 (m, 6H), 1.44 (s, 9H), 0.87 (d, J=6.9 Hz, 3H), 0.86 (d, J=6.9 Hz, 3H).",
"Step D To a solution of the ester from Step C (4.91 g, 17.2 mmol) in methanol (100 mL) was added a solution of LiOH (3.6 g, 85 mmol) in water (20 mL) and tetrahydrofuran (10 mL).",
"The resultant mixture was heated at 80° C. until the reaction was complete (18 h).",
"The methanol was removed in vacuo and the crude product was taken up with water/ethyl acetate (200 mL, 1:4) and cooled to 0° C. The acidity of the mixture was adjusted to pH 6.The ethyl acetate layer was separated, washed with water, brine, dried (anhydrous magnesium sulfate) and concentrated to yield an oil.",
"Purification by flash column chromatography (eluent: hexanes/ethyl acetate 1:1+2% AcOH) gave Intermediate 11 (3.9 g, 84%).",
"1H NMR (500 MHz, CDCl3): 11.36 (br, 1H), 6.49 (br, 1H), 4.83 (m, 1H), 3.71 (s, 3H), 2.30-1.55 (m, 6H), 1.46 (s, 9H), 0.94 (d, J=6.9 Hz, 3H), 0.933 (d, J=6.9 Hz, 3H).",
"Procedure B: Step A Commercially available (1R,4S)-4-amnocyclopent-2-ene-1-carboxylic acid was converted to its methyl ester hydrochloride salt via classical procedures.",
"Step B To a suspension of amine from Step A (6.31 g, 35.5 mmol) in acetone (40 mL) and water (20 mL) was added solid NaHCO3 (6.6 g, 78 mmol) in portions.",
"After 5 min, a solution of di-tert-butyl dicarbonate (8.5 g, 39 mmol) in acetone (60 mL) was added and the reaction mixture was stirred at room temperature.",
"After 3 h, acetone was removed in vacuo and the residue was partitioned between ether (500 mL) and saturated aqueous NaHCO3 solution (120 mL).",
"The ether layer was further washed with aqueous NaHCO3 solution (1×100 mL), brine (1×10 mL), dried over anhydrous Na2SO4, concentrated and purified by flash chromatography (15% ethyl acetate/hexanes) to afford the product (7.25 g, 85%).",
"Step C To a solution of lithium bis(trimethylsilyl)amide (10.4 g, 62.1 mmol) in tetrahydrofuran (100 mL) was added a solution of the intermediate from Step B (6.71 g, 27.8 mmol) in tetrahydrofuran (10 mL) over 10 min at −78° C. The resulting solution was stirred at −78° C. for 30 min before isopropyl iodide (3.3 mL, 33 mmol) was added in one portion.",
"The reaction was allowed to warm up to −25° C. and this temperature was maintained overnight.",
"The reaction was then quenched with an aqueous saturated NH4Cl solution (250 mL).",
"The organic layer was separated and the aqueous layer was further extracted with diethyl ether (3×100 mL).",
"The combined organic layers were then washed with brine (1×100 mL), dried over anhydrous Na2SO4, filtered, concentrated and purified by flash chromatography (5-10% ethyl acetate/hexanes) to give the product (5.66 g, 72%) as a clear oil (cis/trans=4.3/1).",
"1H NMR (500 MHz, CDCl3) cis-isomer: δ 5.79 (s, 2H), 4.75 (m, 1H), 3.72 (s, 3H), 2.28-2.20 (m, 2H), 2.0 (dd, J=15, 4 Hz, 1H), 1.45 (s, 9H), 0.85 (d, J=6.6 Hz, 3H), 0.81 (d, J=7 Hz, 3H).",
"Step D To a solution of the product from Step C (1.6 g, 5.7 mmol) in tetrahydrofuran (50 mL), methanol (50 mL) and water (10 mL) was added LiOH monohydrate (400 mg) and the reaction was heated to reflux overnight until the TLC indicated that the reaction was complete.",
"The organic solvents were removed in vacuo and the aqueous layer was washed with ether (1×) and then acidified slowly with concentrated HCl until the pH reached 4.The resulting suspension was extracted with CH2Cl2 (3×).",
"The combined organic layers were dried over anhydrous MgSO4, filtered and concentrated to give the product as a mixture of two cis/trans isomers (1.5 g) as a foaming yellow solid.",
"This solid was dissolved in ethyl acetate (2 mL) with heating and diluted with hexanes (50 mL) to give a clear solution.",
"This solution was allowed to cool to room temperate slowly over 1 h and then maintained at −25° C. in a freezer overnight.",
"The trans-isomer was crystallized out along with some of the desired cis-isomer (500 mg total).",
"The mother solution was collected and concentrated to give the title compound (1 g, 66%, cis-isomer only).",
"1H NMR (500 MHz, CDCl3) cis-isomer: δ 5.80 (m, 2H), 4.80 (m, 1H), 2.40-2.20 (m, 2H), 2.15-2.0 (m, 1H), 1.5 (m, 9H), 1.0-0.8 (m, 3H).",
"Step E To a solution of the product from Step D (1 g) in ethanol (30 mL) was added 10% Pd/C (100 mg) and the resulting mixture was agitated on a Parr apparatus at 501b pressure of H2 overnight.",
"The mixture was filtered through celite and concentrated in vacuo to afford the title compound (1 g, 99%).",
"1H NMR (500 MHz, CDCl3): 11.36 (br, 1H), 6.49 (br, 1H), 4.83 (m, 1H), 3.71 (s, 3H), 2.30-1.55 (m, 6H), 1.46 (s, 9H), 0.94 (d, J=6.9 Hz, 3H), 0.933 (d, J=6.9 Hz, 3H).",
"INTERMEDIATE 24 Step A Intermediate 2 (4.6 g, 16 mmol) and Intermediate 23 (4.0 g, 14 mmol) were first dried by azeotropic distillation with toluene (3×50 mL) and placed under high vacuum for 30 min.",
"Under nitrogen, 4-dimethylaminopyridine (1.08 g, 8.60 mmol), anhydrous dichloromethane (40 mL), and diisopropylethylamine (7.0 mL, 40 mmol) were added sequentially.",
"After the intermediates were in solution, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (6.80 g, 14.3 mmol) was added, immediately followed by additional diisopropylethylamine (7.0 mL, 40 mmol).",
"The reaction mixture was stirred at room temperature overnight and then quenched with saturated NaHCO3.The aqueous layer was back washed with dichloromethane (3×50 mL) and the organic layers were combined, dried over Na2SO4, filtered, and evaporated in vacuo.",
"The crude product was purified by flash chromatography (stepwise gradient 0-60%, ethyl acetate/hexanes) to afford the product (4.80 g, 74%) as a yellow foam.",
"1H NMR (500 MHz, CDCL3) δ 8.72 (s, 1H), 7.70 (s, 1H), 4.88 (br d, J=17.0 Hz, 1H), 4.78 (d, J=17.6 Hz, 1H), 4.04-3.84 (m, 2H), 3.52 (br s, 1H), 3.12 (br t, J=5.6 Hz, 1H), 2.32-2.06 (m, 3H), 1.98-1.70 (m, 4H), 1.64-1.54 (m, 1H), 1.44 (s, 9H), 0.92-0.82 (m, 6H).",
"LC-MS for C23H32F3N3O3 calculated 455.24, found [M+H]+ 456.2.Step B The product from Step A (1.2 g, 2.6 mmol) was dissolved in 4 M HCl in dioxane (50 mL) and the resulting solution was stirred at room temperature for 1 h. The reaction mixture was evaporated under vacuum to afford the product (904 mg, 97%) as a white powder.",
"LC-MS calculated for C18H24F3N3O is 355.20, found [M+H]+ 356.2.INTERMEDIATE 25 Step A To a flask was added Intermediate 23 (1.1 g, 4.0 mmol), Intermediate 1 (0.944 g, 4.00 mmol), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (1.85 g, 4.00 mmol), DMAP (0.29 g, 2.4 mmol), DIEA (2.77 mL, 16 mmol) and DCM (20 mL).",
"The resulting mixture was stirred for 36 h under nitrogen.",
"The entire mixture was applied onto a silica gel column and eluted with 20% EtOAc/Hexane.",
"The desired Boc-amide was obtained as a gummy solid (1.5 g, 82%).",
"ESI-MS calc.",
"for C24H33F3N2O3: 454; Found: 455 (M+H).",
"Step B The Boc amino amide from Step A was treated with 10 mL of 4 N HCl/Dioxane for 1 h. the reaction mixture was evaporated and the product was dried under vacuum.",
"Intermediate 25 was obtained as a yellow solid (1.2 g).",
"ESI-MS calc.",
"for C19H25F3N2O: 354; Found: 355 (M+H).",
"INTERMEDIATE 26 Step A To a flask containing Intermediate 1 (10 g, 50 mmol) was added 30 mL of 70% nitric acid.",
"The mixture was cooled at 0° C., 30 mL of concentrated sulfuric acid was added over 30 min.",
"The resulting solution was stirred at RT overnight, poured into an ice-water mixture, adjusted to pH>10 with solid LiOH—H2O at 0° C. Under vigorous stirring, a solution of di-tert-butyl carbonate (21.8 g, 100 mmol) in 500 mL of DCM was added.",
"The mixture was stirred for 30 min, the organic layer was separated, the aqueous layer was extracted with DCM (2×200 mL).",
"The combined extracts were washed with water (500 mL), dried over Na2SO4, and evaporated.",
"The crude product was purified by flash chromatography (silica gel, 20% EtOAc/Hexane) to afford the title compound as a white solid (17.0 g, 98%).",
"1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.62 (s, 1H), 4.72 (s, 2H), 3.67 (t, J=6.0 Hz, 2H), 3.13 (t, J=6.0 Hz, 2H), 1.49 (s, 9H).",
"Step B The above intermediate from Step A (17.0 g) was dissolved in 100 mL of 4 M HCl/dioxane, stirred for one hour, evaporated and dried under vacuum.",
"Intermediate 26 was obtained as white solid.",
"1H NMR (400 MHz, CD3OD) δ 8.75 (s, 1H), 8.00 (s, 1H), 2.58 (s, 2H), 3.57 (t, J=6.0 Hz, 2H), 3.42 (t, J=6.0 Hz, 2H).",
"INTERMEDIATE 27 Step A To a flask was added Intermediate 27 (1.10 g, 4.00 mmol), Intermediate 23 (1.15 g, 4.00 mmol), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (1.85 g, 4.00 mmol), DMAP (0.29 g, 2.4 mmol), DIEA (2.7 mL, 16 mmol) and DCM (20 mL).",
"The resulting mixture was stirred for 36 h under nitrogen.",
"The entire mixture was applied onto a silica gel column and eluted with 20% EtOAc/Hexane to yield the title compound as a gummy solid (1.5 g, 75%).",
"ESI-MS calc.",
"for C24H32F3N3O5: 499; Found: 500 (M+H).",
"Step B The coupling product from the previous step (1.5 g) was treated with 10 mL of 4N HCl/Dioxane for 1 h, evaporated and dried under high vacuum to yield the title compound as a yellow solid (1.2 g).",
"1H NMR (400 MHz, CD3OD) δ 8.20 (s, 1H), 7.95 (wide, 1H), 4.98 (s, 2H), 4.00 (dd, 2H), 3.90 (t, 2H), 3.68 (m, 1H), 3.45 (m, 3H), 3.20 (s, 2H), 2.15-2.50 (m, 3H), 1.80-2.10 (m, 2H), 1.80 (m, 2H), 0.90 (m, 6H).",
"ESI-MS calc.",
"for C19H24F3N3O3: 399; Found: 400 (M+H).",
"INTERMEDIATE 28 Step A To a stirring mixture of N-trifluoroacetyl-7-trifluoromethyl-1,2,3,4-tetrahydroisoquinoline, from Intermediate 1, Step B (6.0 g, 20 mmol), NIS (6.9 g, 30 mmol) and TFA (15 mL) was added dropwise concentrated sulfuric acid (1.5 mL).",
"A large amount of solid was formed.",
"The mixture was stirred overnight at RT, poured into an ice-water mixture, extracted with ethyl acetate (3×).",
"The combined organic phases were washed with water and brine, dried over Na2SO4, evaporated.",
"The residue was purified on silica gel (eluted with 10% EtOAc/Hexane).",
"The combined fractions were washed with sat.",
"NaHSO3, dried over Na2SO4, evaporated and dried under vacuum to afford the title compound as a white solid (5.0 g).",
"1H NMR (400 MHz, CDCl3) δ 8.02 (d, J=2.5 Hz, 1H), 7.42 (d, j=3.0 Hz, 1H), 4.85, 4.79 (ss, 2H), 3.95, 3.90 (tt, J=1.5, 1.5 Hz, 2H), 2.97 (m, 2H).",
"ESI-MS calc.",
"For C12H8F6INO: 423; Found: 424 (M+H).",
"Step B A mixture of the iodo compound (Step A, 4.2 g, 10 mmol), zinc cyanide (2.3 g, 20 mmol) and tetrakis-triphenyl phosphene palladium (0) complex (0.4 g) in 50 mL of DMF was purged with nitrogen several times, then heated at 85° C. overnight under nitrogen.",
"LC-MS showed a complete conversion.",
"The insoluble material was removed by filtration.",
"The filtrate was diluted with water and extracted with ethyl acetate (3×).",
"The ethyl acetate layers were combined, filtered through celite, then washed with water, dried over Na2SO4, and evaporated.",
"The residue was purified on silica gel (eluted with 10% EtOAc/Hex) to yield the title compound as a white solid (2.5 g).",
"1H NMR (400 MHz, CDCl3) δ 7.85 (d, J=2.1 Hz, 1H), 7.65 (d, J=2.6 Hz, 1H), 4.91, 4.86 (ss, 2H), 4.00 (m, 2H), 3.25 (m, 2H).",
"ESI-MS calc.",
"For C13H8F6N2O: 323; Found: 323 (M+H).",
"Step C A mixture of the amide Intermediate from Step B (500 mg, 1.55 mmol), potassium carbonate (1.5 g), ethanol (20 mL) and water (0.5 mL) was heated at 80° C. until TLC showed complete cleavage.",
"The solvent was evaporated, diluted with water, extracted with DCM (3×), dried over Na2SO4, evaporated, and dried under vacuum.",
"The title product was obtained as a white solid (0.41 g).",
"ESI-MS calc.",
"For C11H9F3N2: 226; Found: 227 (M+H).",
"Step D The above cyano intermediate from Step C (1.9 g, 8.5 mmol) was refluxed with 50 mL of concentrated aq.",
"HCl for 48 h. LC-MS showed a complete hydrolysis.",
"The mixture was cooled to RT and the resultant precipitate was collected by filtration and washing with concentrated aq.",
"HCl.",
"The desired product as its HCl salt (1.75 g, 73%) was obtained after drying under high vacuum.",
"1H NMR (CD3OD, 400 MHz): δ 8.20 (s, 1H), 7.80 (s, 1H), 4.51 (s, 2H), 3.55 (m, 4H).",
"LC-MS for C11H10F3NO2 calculated 245, found [M+H]+ 246.Step E To a suspension of the above amino acid HCl salt from Step D (1.75 g, 6.25 mmol) in 50 mL of methanol was added slowly a neat solution of acetyl chloride (5 mL).",
"The resultant mixture was refluxed until LC-MS showed a complete esterification (˜3 h), then the solvent was evaporated and dried under high vacuum to yield the title compound as a white solid (1.85 g, 100%).",
"1H NMR (CD3OD, 400 MHz): 8.19 (s, 1H), 7.82 (s, 1H), 4.50 (s, 2H), 3.94 (s, 3H), 3.53 (s, 4H).",
"LC-MS for C12H12F3NO2 calculated 259, found [M+H]+ 260.INTERMEDIATE 29 Procedure A: Step A H2SO4 (conc., 15.3 g, 8.30 mL, 156 mmol) was added dropwise to a vigorously stirred suspension of MgSO4 (75 g, 620 mmol) in DCM (650 mL).",
"The mixture was stirred for 0.5 h, then known cyclopentanone-3-carboxylate (20.0 g, 156 mmol) was added, followed by t-butanol (58 g, 780 mmol).",
"The reaction vessel was tightly sealed and the mixture was stirred overnight at room temperature.",
"The next morning another 30 mL of t-butanol was added.",
"Again the reaction vessel was tightly sealed, and the reaction mixture was stirred over the weekend.",
"The reaction mixture was then filtered through celite.",
"The filtrate was washed with 2 N NaOH.",
"The aqueous layer was back-washed with DCM.",
"The organic layers were combined, washed with water, then brine, dried over anhydrous MgSO4, filtered, and concentrated to afford 19.9 g (69%) of tert-butyl 3-oxocyclopentanecarboxylate.",
"The reaction progress was monitored by TLC using 50% ethyl acetate/hexane and staining with anisaldehyde stain (SM and product stain purple).",
"1H NMR (500 MHz, CDCl3): 3.02 (p, J=7.8 Hz, 1H), 2.05-2.50 (m, 6H), 1.45 (s, 9H).",
"13C NMR (125 MHz, CDCl3): 217.00, 173.47, 80.99, 41.88, 41.14, 27.94, 26.57.Step B To a solution of tert-butyl 3-oxocyclopentanecarboxylate (19.8 g, 107 mmol) in 1:1 DCM/methanol (150 mL) was added trimethylorthoformate (46.8 mL, 428 mmol), followed by TsOH.H2O (˜0.5 g).",
"The reaction mixture was stirred at room temperature for 2 h. Then more TsOH.H2O (˜0.25 g) was added and the reaction mixture was stirred overnight.",
"The reaction mixture was concentrated at room temperature and the resulting residue was dissolved in ether and washed with saturated NaHCO3 solution, then with brine.",
"The ethereal layer was dried over anhydrous MgSO4, filtered, and concentrated.",
"Purification by flash chromatography (silica, 15% ethyl acetate/hexane) gave 22.2 g (90%) of tert-butyl 3,3-dimethoxycyclopentanecarboxylate.",
"1H NMR (500 MHz, CDCl3): 3.21 (s, 3H), 3.20 (s, 3H), 2.80 (m, 1H), 2.10 to 1.80 (bm, 6H), 1.46 (s, 9H).",
"13C NMR (125 MHz, CDCl3): 174.9, 111.2, 80.3, 67.8, 49.2, 42.5, 37.4, 33.8, 28.3, 22.0.Step C To a cooled (−78° C.) solution of LDA (1.5 M in cyclohexane, 41 mL, 61 mmol) in THF (150 mL) was added dropwise over 10 min tert-butyl 3,3-dimethoxycyclopentanecarboxylate (9.37 g, 40.7 mmol) in 25 mL of THF.",
"The resulting mixture was stirred at −78° C. for 30 min, then was treated dropwise with 2-iodopropane (16.3 mL, 163 mmol).",
"After stirring for an additional 10 min, the reaction mixture was permitted to warm to room temperature.",
"After stirring overnight, the reaction mixture was diluted with ether and washed with brine.",
"The ethereal layer was dried over anhydrous MgSO4, filtered, and concentrated.",
"After storing the crude product under vacuum overnight, it was purified by MPLC (silica, 20% ethyl acetate/hexane) to give 8.32 g of tert-butyl 1-isopropyl-3,3-dimethoxycyclopentanecarboxylate (75%).",
"1H NMR (500 MHz, CDCl3) δ 3.21 (s, 3H), 3.18 (s, 3H), 2.56 (app d, J=14 Hz, 1H), 2.26 (m, 1H), 1.78-1.89 (m, 3 Step D tert-Butyl 1-isopropyl-3,3-dimethoxycyclopentanecarboxylate (8.32 g, 30.5 mmol) was dissolved in 4 N anhydrous HCl in dioxane (50 mL) and water (10 mL) was added.",
"The reaction mixture was stirred at room temperature overnight, then was concentrated.",
"The residue was dissolved in DCM, dried over anhydrous MgSO4, filtered, and concentrated to give 5.44 g of 1-isopropyl-3-oxocyclopentanecarboxylic acid (used without purification).",
"1H NMR (500 MHz, CDCl3) δ 2.70 (d, J=18.1 Hz, 1H), 2.44-2.39 (m, 1H), 2.30-2.15 (m, 2H), 2.14 (dd, J=18.1, 1.0 Hz, 1H), 2.06 (p, J=6.9 Hz, 1H), 1.98 (m, 1H), 0.98 (dd, J=11.4, 6.9 Hz, 6H).",
"Step E A cooled (0° C.) solution of 1-isopropyl-3-oxocyclopentanecarboxylic acid (5.44 g, 32.0 mmol) in DCM (75 mL) was treated with oxalyl chloride (8.36 mL, 95.9 mmol), followed by 3 drops of DMF.",
"The reaction mixture was permitted to warm to room temperature and stir for 1.75 h. The reaction mixture was then concentrated and stored under vacuum for 30 min.",
"The resulting acid chloride was dissolved in DCM (75 mL), cooled to 0° C., and treated with benzyl alcohol (8.28 mL, 80.0 mmol), followed by triethyl amine (8.92 mL, 64.0 mmol, dropwise).",
"Then approximately 100 mg of DMAP was added and the reaction mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was diluted with DCM and washed with 1 N HCl solution, saturated NaHCO3 solution, and brine.",
"The organic layer was dried over anhydrous MgSO4, filtered, and concentrated.",
"Purification by MPLC (silica, 50% ethyl acetate/hexane) gave 6.11 g (73%) of benzyl 1-isopropyl-3-oxocyclopentanecarboxylate.",
"1HNMR (CDCl3, 500 MHz): δ 7.36 (m, 5H), 5.17 (d, J=2.5 Hz, 2H), 2.85 (d, J=18.5 Hz, 1H), 2.48 (m, 1H), 2.29 (dd, J=10.0, 3.0 Hz, 1H), 1.98-2.23 (m, 3H), 1.93 (m, 1H), 0.95 (m, 6H).",
"Resolution of the racemic product was accomplished by chiral HPLC using a chiralcel OD column, and eluting with 15% 2-propanol/hexane (100 mg/injection; was accomplished using a programmed Gilson HPLC system).",
"2.11 g Of the desired faster eluting isomer, benzyl (1S)-1-isopropyl-3-oxocyclopentanecarboxylate, were obtained.",
"Step F Benzyl (1S)-1-isopropyl-3-oxocyclopentanecarboxylate (1.27 g, 4.88 mmol) was combined with Pd/C (10% Degussa, 500 mg) in 20 mL of methanol and stirred under a hydrogen atmosphere (balloon) for 2 h. The reaction had only proceeded part way (˜30% conversion) so the reaction mixture was filtered, another portion of Pd/C (500 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 5 h. Since the reaction had now gone to completion, the reaction mixture was filtered through celite and concentrated to afford 704 mg of (1S)-1-isopropyl-3-oxocyclopentanecarboxylic acid that did not require further purification.",
"Note that the large quantities of catalyst were used because the ester obtained after chiral separation must have been poisoned by an impurity.",
"This was unique to this particular sample.",
"Normally much smaller quantities of catalyst are used.",
"1H NMR was identical to that of the racemic acid above (Step D).",
"Procedure B: To a solution of (1S,3R)-3-[(tert-butoxycarbonyl)amino]-1-isopropylcyclopentanecarboxylic acid (7.46 g, 27.5 mmol) in dioxane (10 mL) was added 4 N HCl in dioxane (30 mL).",
"The reaction mixture was stirred at room temperature for 2 hours, then concentrated in vacuo to give the corresponding aminoacid salt as a white solid.",
"This solid was then dissolved in CH2Cl2 (100 mL) and solid NaHCO3 (7.0 g, 82.5 mmol) was added.",
"After cooled to 0° C., a solution of NBS (20.0 g, 110 mmol) in CH2Cl2 (200 mL) was slowly added to the reaction over 4 hours.",
"After the addition, the reaction was concentrated to dryness in vacuo and then dissolved in ethanol (100 mL).",
"To this ethanol solution was added NaOMe (4.45 g, 82.5 mmol) and the reaction was heated to reflux.",
"After 1 hour at reflux, the reaction was cooled to 0° C. and 2N aqueous H2SO4 (50 mL) was added.",
"The mixture was stirred at room temperature for 1 hour before concentrating in vacuo to about 60 mL in volume.",
"The remaining mixture was partitioned between water (150 mL) and ethyl acetate (100 mL).",
"The aqueous layer was further extracted with ethyl acetate twice.",
"The organic layers were combined and dried over anhydrous MgSO4, concentrated and purified by flash chromatography (silca, ethyl acetate/hexanes) to give (1S)-1-isopropyl-3-oxocyclopentanecarboxylic acid (3.00 g, 64%).",
"INTERMEDIATE 30 Procedure A Step A To a stirred solution of tert-butyl 4-[3-(ethoxycarbonyl)phenyl]piperidine-1-carboxylate (48 g, 220 mmol) in chloroform (900 mL) was added ruthenium (IV) oxide hydrate (6.0 g, 45 mmol) followed by a solution of sodium periodate (150 g, 700 mmol) in water (900 mL).",
"The resulting heterogenous reaction mixture was stirred at room temperature for 11 days before being filtered through a short column of celite.",
"The organic layer was removed and the aqueous layer was extracted twice with DCM.",
"The combined organic layers were washed with a 10% solution of sodium thiosulfate in water twice, and once with brine.",
"This solution was dried over MgSO4, filtered, and concentrated under reduced pressure.",
"The product was purified by flash chromatography (silica gel, 20% EA/hexanes) to give 22.5 g (64.8 mmol) of tert-butyl 4-[3-(ethoxycarbonyl)phenyl]-2-oxopiperidine-1-carboxylate (29%).",
"ESI-MS calculated for C19H25NO5: 347.17; found 370.1 (M+Na) Step B Potassium bis(trimethylsilyl)amide (14 g, 71 mmol) was mixed with 300 mL of THF in a 1000 mL flame-dried round bottomed flask and the resulting mixture was cooled to −78° C. tert-butyl 4-[3-(ethoxycarbonyl)phenyl]-2-oxopiperidine-1-carboxylate (22.5 g, 64.8 mmol) dissolved in 150 mL of THF was added slowly to the mixture, via an addition funnel, and the resulting reaction mixture was stirred at −78° C. for 30 min.",
"Methyl iodide (12.1 mL, 195 mmol) was then added dropwise and the reaction mixture was allowed to stir at −78° C. for 4 h before being allowed to warm to room temperature overnight.",
"The reaction was quenched with saturated ammonium chloride and extracted 3 times with ether.",
"The combined ethereal layers were washed with brine and dried over MgSO4, filtered, and concentrated under reduced pressure.",
"The product was purified by flash chromatography (10-20% EA/hexanes) to give 6.1 g of the trans racemate of tert-butyl 4-[3-(ethoxycarbonyl)phenyl]-3-methyl-2-oxopiperidine-1-carboxylate (26%).",
"ESI-MS calculated for C20H27NO5: 361.19; found 384.25 (M+Na).",
"Step C The product from Step B (6.1 g, 17 mmol) was dissolved in 4.0 M HCl in dioxane and stirred at room temperature for 2 h before being concentrated under reduced pressure to give the desired product as an orange solid which was sued directly in the next step without further purification.",
"ESI-MS calculated for C15H19NO3: 261.14; found 262.1 (M+H).",
"Step D The product from the previous step (entire amount ˜17 mmol) was dissolved in THF (100 mL) and treated dropwise with 2.0 M borane-methyl sulfide solution in THF (31 mL, 62 mmol).",
"The resulting solution was stirred at room temperature for 4 h before being stored at 4° C. for 72 h. The solvent was removed under reduced pressure and the resulting residue was dissolved in 0.5 M HCl (aqueous ˜38%) in ethanol.",
"This solution was heated to 50° C. and stirred for 4 h. The solvent was removed and the procedure was repeated again to ensure the break up of the borane complex.",
"The solvent was removed and the product was purified by MPLC (0-15% (10% NH4OH/MeOH)/DCM) to give the desired product which was 80% pure.",
"This crude material was dissolved in DCM (100 mL) and treated with di-tert-butyl dicarbonate (2.95 g, 13.5 mmol), diisopropylethylamine (2.30 mL, 13.5 mmol) and DMAP (10 mg).",
"The resulting reaction mixture was stirred overnight at room temperature before being diluted with DCM and washed with 1 N aqueous, aqueous saturated sodium bicarbonate, and brine.",
"The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure.",
"The intermediate was purified by MPLC (0-40% EA/hexanes).",
"The resulting colorless oil was dissolved in 4.0 M HCl in dioxane and the resulting reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated to dryness to give 2.13 g (7.52 mmol) of the desired HCl salt.",
"ESI-MS calculated for C15H21NO2: 247.16; found 248.15 (M+H) Procedure B Step A A solution of 1-benzyl-3-methylpiperidin-4-one (48 g, 0.24 mol) in THF (200 mL) was added dropwise to a cooled (−78° C.) solution of sodium bis(trimethylsilyl)amide (2M in THF, 141 mL, 0.28 mol) in THF (100 mL).",
"The resulting dark orange mixture was stirred at −78° C. for 2 hr after which a solution of N-phenyl-bis(trifluoromethanesulfonimide) (100 g, 0.28 mol) in THF (300 mL) was added dropwise.",
"The mixture was allowed to warm to r.t. and stirred for 3 hr.",
"Most of the THF was removed in vacuo.",
"The residue was partitioned between Ether and aqueous 1M NaOH.",
"The organic layer was then washed repeatedly with aqueous 1M NaOH, dried over sodium sulfate, and evaporated to dryness.",
"The dark orange residue was then dissolved in CH2Cl2 and filtered through a silica gel plug eluting with CH2Cl2.The desired 1-benzyl-3-methyl-1,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (79 g) was obtained as a yellow oil.",
"Step B A mixture of 1-benzyl-3-methyl-1,2,3,6-tetrahydropyridin-4-yl trifluoromethanesulfonate (3 g, 9.0 mmol), [3-(ethoxycarbonyl)phenyl]boronic acid (2.26 g, 11.6 mmol), PdCl2(PPh3)2 (315 mg, 0.44 mmol), and potassium carbonate (2.5 g, 18.0 mmol) in Toluene:EtOH (10:1, 50 mL) were heated to 100° C. for 18 hr.",
"The cooled reaction mixture was partitioned between EtOAC and H2O and the aqueous layer was extrated with EtOAc (4×).",
"The organics were combined, dried over sodium sulfate, filtered and evaporated to dryness.",
"The residue was purified by column chromatography on silica gel eluting with a gradient of hexane:EtOAc to yield ethyl 3-(1-benzyl-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)benzoate as a clear oil.",
"Step C Ethyl 3-(1-benzyl-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)benzoate (32.5 g, 98 mmol) was dissolved in EtOAc (400 mL) and was hydrogenated for 18 hr with a hydrogen balloon in the presence of a catalytic amount (1.1 g) of platinum oxide.",
"Platinum oxide was filtered through a celite plug and the filtrate was evaporated to dryness.",
"The resulting yellow oil was purified by column chromatography on silica gel eluting with a gradient of hexane:EtOAc.",
"Less polar compound: ethyl 3-[(3R,4R)-1-benzyl-3-methylpiperidin-4-yl]benzoate as a mixture of two diastereomers More polar compound: desired ethyl 3-[(3R,4S)-1-benzyl-3-methylpiperidin-4-yl]benzoate) as a mixture of two diastereomers (9 g, 24%).",
"Step D Ethyl 3-[(3S,4R)-1-benzyl-3-methylpiperidin-4-yl]benzoate (9 g, 26.7 mmol) was dissolved in EtOH: 1M HCl (1:1, 160 mL) and was hydrogenated at 50 psi in the presence of a catalytic amount of palladium on carbon (1 g) for 36 hr.",
"Palladium was filtered through a celite plug and the filtrate was evaporated to dryness to give 7.2 g of ethyl 3-[(3S,4R)-3-methylpiperidin-4-yl]benzoate as an HCl salt.",
"INTERMEDIATE 31 Ethyl 3-[(3R,4R)-3-methylpiperidin-4-yl]benzoate was synthesized as described for INTERMEDIATE 30 (procedure B, step D) using ethyl 3-[(3R,4R)-1-benzyl-3-methylpiperidin-4-yl]benzoate as starting material.",
"INTERMEDIATE 32 Step A To a mixture of tert-butyl 4-{[(trifluoromethyl)sulfonyl]oxy}-3,6-dihydropyridine-1(2H)-carboxylate (prepared according to Wustrow, D. J., Wise, L. D., Synthesis, (1991), 993-995.; 10.5 g, 31.6 mmol), 3-(ethoxycarbonyl)phenylboronic acid (8.59 g, 44.3 mmol), lithium chloride (3.98 g, 94.8 mmol), and 2 M Na2CO3 solution (44 mL) in DME (107 mL) was added Pd(PPh3)4 (1.82 g, 1.58 mmol), and the resulting mixture was stirred at reflux under a nitrogen atmosphere for 3.5 h. The reaction mixture was cooled to rt, stirred overnight, then partially concentrated to remove most of the DME.",
"To the remaining aqueous mixture was added DCM, 2M Na2CO3 solution, and ˜10mL of 28% NH4OH solution.",
"The layers were separated and the organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated.",
"Purification by flash chromatography (silica, 10% ethyl acetate/hexanes eluent) afforded tert-butyl 4-[3-(ethoxycarbonyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate.",
"1HNMR (CDCl3, 500 MHz): δ 8.07 (s, 1H), 7.95 (d, J=7.5 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 6.13 (br s, 1H), 4.41 (q, J=7.0 Hz, 2H), 4.12 (br s, 2H), 3.68 (t, J=5.5 Hz, 2H), 2.58 (br s, 2H), 1.52 (s, 9H), 1.43 (t, J=7.0 Hz, 3H).",
"Step B A mixture of tert-butyl 4-[3-(ethoxycarbonyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate (6.48 g, 19.6 mmol) and Pd(OH)2/C (20% Pd, 1 g) in 50 mL of methanol was stirred under a hydrogen atmosphere (balloon) for 18 h. The reaction mixture was then filtered through a celite plug, and the filtrate was concentrated to give tert-butyl 4-[3-(ethoxycarbonyl)phenyl]piperidine-1-carboxylate which did not require further purification.",
"1HNMR (CDCl3, 500 MHz): δ 7.91 (m, 2H), 7.40 (m, 2H), 4.40 (q, J=7.0 Hz, 2H), 4.28 (br s, 2H), 2.83 (m, 2H), 2.73 (tt, J=12.5, 4.0 Hz, 1H), 1.85 (br d, J=13.0 Hz), 1.67 (dq, J=4.0, 12.5 Hz, 2H), 1.51 (s, 9H), 1.42 (t, J=7.0 Hz).",
"Step C tert-Butyl 4-[3-(ethoxycarbonyl)phenyl]piperidine-1-carboxylate (3.24 g, 9.72 mmol) was dissolved in anhydrous 4N HCl in dioxane (ca.",
"30 mL) and stirred at rt for 1.5 h. The reaction mixture was concentrated to give ethyl 3-piperidin-4-ylbenzoate hydrochloride as a pale yellow solid that required no further purification.",
"ESI-MS calc.",
"for C14H19NO2: 233; Found: 234 (M+H).",
"INTERMEDIATE 33 Step A A mixture of 5-bromo-2-fluorobenzoic acid (25 g, 0.11 mol), methyl iodide (8.5 mL, 0.14 mol), and potassium carbonate (31 g, 0.23 mol) in DMF (200 mL) was heated to 50° C. for 18 hr.",
"The cooled reaction mixture was diluted with EtOAc (200 mL) and the organic phase was washed with a saturated aqueous NaCl solution (4×).",
"The organic phase was then dried over sodium sulfate and evaporated to dryness to give 24 g of methyl 5-bromo-2-fluorobenzoate as a yellow oil.",
"Step B tert-Butyl 4-[4-fluoro-3-(methoxycarbonyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate was synthesized as described for INTERMEDIATE 32 using tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (prepared according to Eastwood, P. R. Tetrahedron Lett., 41, 19, 2000, 3705-3708) and methyl 5-bromo-2-fluorobenzoate as starting material and PdCl2dppf.CH2Cl2 as a catalyst.",
"Step C tert-Butyl 4-[4-fluoro-3-(methoxycarbonyl)phenyl]piperidine-1-carboxylate was synthesized as described for INTERMEDIATE 30 (procedure B, step C) using tert-butyl 4-[4-fluoro-3-(methoxycarbonyl)phenyl]-3,6-dihydropyridine-1(2H)-carboxylate as starting material.",
"Step D tert-Butyl 4-[4-fluoro-3-(methoxycarbonyl)phenyl]piperidine-1-carboxylate (471 mg, 1.40 mmol) was dissolved in anhydrous 4N HCl in dioxane (5mL) and stirred at r.t. for 40 min.",
"The white solid was filtered and washed with Et2O to afford methyl 2-fluoro-5-piperidin-4-ylbenzoate.",
"INTERMEDIATE 34 Methyl 3-fluoro-5-piperidin-4-ylbenzoate.HCl was synthesized as described for INTERMEDIATE 33 using 3-bromo-5-fluorobenzoic acid as starting material.",
"INTERMEDIATE 35 Methyl 2-methyl-3-piperidin-4-ylbenzoate.HCl was synthesized as described for INTERMEDIATE 33 using 3-bromo-2-methylbenzoic acid as starting material.",
"INTERMEDIATE 36 Methyl 2-methoxy-5-piperidin-4-ylbenzoate.HCl was synthesized as described for INTERMEDIATE 33 using 3-bromo-6-methoxylbenzoic acid as starting material.",
"INTERMEDIATE 37 Methyl 4-fluoro-3-piperidin-4-ylbenzoate.HCl was synthesized as described for INTERMEDIATE 33 using 3-bromo-4-fluorobenzoic acid as starting material.",
"INTERMEDIATE 38 Methyl (3-piperidin-4-ylphenyl)acetate HCl was synthesized as described for INTERMEDIATE 33 using (3-bromophenyl)acetic acid as starting material.",
"INTERMEDIATE 39 A mixture of methyl 5-bromo-2-fluorobenzoate (6.5 g, 27.9 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi-1,3,2-dioxaborolane (8.5 g, 33.5 mmol), PdCl2dppf.CH2Cl2 (1.1 g, 1.4 mmol), dppf (0.77 g, 1.4 mol), and KOAc (5.4 g, 55.8 mmol) in DMF (100 mL) was heated to 90° C. for 18 hr.",
"The cooled reaction mixture was diluted with EtOAc (200 mL) and the organic phase was washed with a saturated aqueous NaCl solution (4×).",
"The organic phase was then dried over sodium sulfate and evaporated to dryness.",
"The dark residue was dissolved in CH2Cl2, filtered through a silica gel plug, and washed with CH2Cl2 (500 mL).",
"The filtrate was evaporated to dryness and the residue was purified by column chromatography on silica gel eluting with a gradient of EtOAc and hexane to afford methyl 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (5 g) as a green solid.",
"INTERMEDIATE 40 Methyl 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate was synthesized as described for INTERMEDIATE 39 using 3-bromo-5-fluorobenzoic acid as starting material.",
"INTERMEDIATE 41 The HCl salt of methyl 2-fluoro-5-[(3R,4S)-3-methylpiperidin-4-yl]benzoate was synthesized as described for INTERMEDIATE 30 (procedure B) using methyl 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate as starting material.",
"INTERMEDIATE 42 The HCl salt of methyl 3-fluoro-5-[(3R,4S)-3-methylpiperidin-4-yl]benzoate was synthesized as described for INTERMEDIATE 30 (procedure B) using methyl 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate as starting material.",
"INTERMEDIATE 43 Step A Methyl magnesium bromide (1.4M in toluene/THF, 8 mL, 11 mmol) was added to tert-butyl 4-[3-(methoxycarbonyl)phenyl]piperidine-1-carboxylate (710 mg, 2.22 mmol) in THF (8 mL).",
"The resulting mixture was stirred at r.t. for 18 hr.",
"The reaction mixture was quenched with H2O and the aqueous was extracted with EtOAc (3×).",
"The organics were combined, dried over sodium sulfate, filtered, and evaporated to dryness to yield tert-butyl 4-[3-(1-hydroxy-1-methylethyl)phenyl]piperidine-1-carboxylate as a clear oil.",
"Step B 2-(3-Piperidin-4-ylphenyl)propan-2-ol.HCl and 4-(3-isopropenylphenyl)piperidine.HCl were obtained as a mixture following the procedure for INTERMEDIATE 35 (step D).",
"INTERMEDIATE 44 Step A A cooled (0° C.) solution of Boc-isonipecotic acid (5.97 g, 26.0 mmol) and Meldrum's acid (3.75 g, 26.0 mmol) in DMF (54 mL) was treated dropwise with diethyl cyanophosphonate (4.34 mL, 28.6 mmol) and triethylamine (11.2 mL, 80.7 mmol).",
"After stirring at 0° C. for an additional 30 min the reaction mixture was permitted to warm to rt and stir over 3 days.",
"The reaction mixture was concentrated under reduced pressure and ether and 1N HCl solution were added.",
"The aqueous layer was extracted again with ether and the ethereal layers were combined and washed with 1N HCl solution, twice with water, and lastly with brine.",
"He ethereal layer was then dried over anhydrous MgSO4, filtered, and concentrated to give tert-butyl 4-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)(hydroxy)methyl]piperidine-1-carboxylate which contained approximately 10% of Boc-isonipecotic acid.",
"1H NMR (500 MHz, CDCl3) δ 4.24 (br m, 2H), 3.97 (tt, J=12, 3.5 Hz, 1H), 2.84 (br m, 2H), 1.83 (m, 2H), 1.76 (s, 6H), 1.69-1.75 (m, 2H), 1.48 (s, 9H).",
"Step B A solution of tert-butyl 4-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)(hydroxy)methyl]piperidine-1-carboxylate (8.99 g, 25.3 mmol) and t-butyl N-(t-butoxycarbonyloxy)carbamate (5.90 g, 25.3 mmol) in toluene (200 mL) was stirred at 65° C. for 14 h, then at rt for 36 h. The reaction mixture was concentrated and the residue was purified by flash chromatography (silica, 25% ethyl acetate/hexanes) to afford tert-butyl 4-(3-{(tert-butoxycarbonyl)[(tert-butoxycarbonyl)oxy]amino}-3-oxopropanoyl)piperidine-1-carboxylate.",
"1H NMR analysis was consistent with product but complex due to carbamate rotamers.",
"Step C A solution of tert-butyl 4-(3-{(tert-butoxycarbonyl)[(tert-butoxycarbonyl)oxy]amino}-3-oxopropanoyl)piperidine-1-carboxylate (6.2 g, 12.7 mmol) in 100 mL of methanol was treated with 4N HCl solution (150 mL) and the resulting suspension was stirred overnight at rt.",
"In the morning the reaction mixture had become clear.",
"The reaction mixture was concentrated.",
"Since the crude product could not be easily purified it was used as is in the subsequent step.",
"ESI-MS calc.",
"for C8H12N2O2: 168; Found: 169 (M+).",
"EXAMPLE 1 To a solution of Intermediate 3 (150 mg, 0.425 mmol), 4-carbethoxypiperidine (125 mg, 0.425 mmol), and DCM (25 mL) was added molecular sieves (4 Å) and NaBH(OAc)3 (450 mg, 2.12 mmol).",
"The reaction mixture was stirred at room temperature for 18 h before being filtered through celite, diluted with saturated NaHCO3, and extracted with DCM (3×).",
"The combined organic layers were dried over Na2SO4 and purified by preparative TLC (3/96.7/0.3, MeOH/DCM/NH4OH) to yield Example 1 (220 mg, 97.8%).",
"LC-MS for C27H38F3N2O3 [M+H+] calculated 495.28, found 495.25.A number of compounds were prepared as detailed in Example 1 using various amines.",
"These compounds are summarized in the table below.",
"TABLE 1 (EXAMPLES 2 to 6) Ex- Calcu- am- Molecular lated Found ple R Formula [M+H+] [M+H+] 2 C27H38F3N2O3 495.28 495.15 3 C27H38F3N2O3 485.28 495.15 4 C28H40F3N2O3 509.29 509.35 5 C25H34F3N2O3 467.24 467.1 6 C26H36F3N2O3 481.26 481.2 EXAMPLE 7 A mixture of the product from Example 1 (185 mg, 0.349 mmol), 5 N NaOH (200 μL, 1.04 mmol), and MeOH (5 mL) was heated at 60° C. for 3 hours before adding a solution of 4N HCl in dioxane to neutralize the base.",
"The reaction solution was concentrated and purified by reverse phase HPLC to yield Example 7 (115 mg, 65.7%).",
"LC-MS for C25H34F3N2O3 [M+H+] calculated 467.24, found 467.35.Examples 8-12 were prepared as detailed in Example 7 using Examples 2-6 as starting materials.",
"These compounds are summarized in the table below.",
"TABLE 2 (EXAMPLES 8 to 12) Ex- am- Molecular Calculated Found ple R Formula [M+H+] [M+H+] 8 C26H36F3N2O3 481.26 481.3 9 C25H34F3N2O3 467.24 467.3 10 C26H36F3N2O3 481.26 481.3 11 C24H32F3N2O3 453.23 453.25 12 C25H33F3N2O3 467.24 467.25 EXAMPLE 13 To a solution of Intermediate 4 (50 mg, 0.14 mmol) in methylene chloride (20 mL) was added 1-ethoxycarbonylpiperazine (23 mg, 0.14 mmol).",
"After adding powdered 4 Å molecular sieves (25 mg), sodium triacetoxyborohydride (180 mg, 0.84 mmol) was added and the reaction mixture was stirred overnight.",
"The mixture was diluted with methylene chloride, washed with aqueous saturated sodium bicarbonate, dried under sodium sulfate and concentrated in vacuo.",
"The crude product was purified by preparative TLC (7/92.31.7, methanol/methylene chloride/ammonium hydroxide) to yield Example 13 as a mixture of 4 diastereomers (55 mg, 79%).",
"LC-MS: MW calculated 496.27, found 497.3.A number of compounds were prepared as detailed in Example 13 using various piperazines instead of 1-ethoxycarbonylpiperazine.",
"These compounds, prepared as mixtures of 4 diastereomers each, are summarized in the table below.",
"TABLE 3 (EXAMPLES 14 to 16) Ex- Calcu- am- Molecular lated Found ple R Formula [M+H+] [M+H+] 14 C28H36F3N4O 501.28 501.25 15 C29H37F3N4O 515.29 515.3 16 C29H35F3N4O 528.27 529.25 EXAMPLE 17 To a solution of Intermediate 4 (50 mg, 0.14 mmol) in methylene chloride (20 mL) was added 4-benzoylpiperidine hydrochloride (32 mg, 0.14 mmol) and N,N-diisopropylethylamine (73 μL, 0.42 mmol).",
"After adding 4 Å powdered molecular sieves (25 mg), sodium triacetoxyborohydride (180 mg, 0.84 mmol) was added and the reaction mixture was stirred overnight.",
"The mixture was extracted with methylene chloride, washed with sodium bicarbonate, dried under sodium sulfate and concentrated in vacuo.",
"The crude product was purified by preparative TLC (7/92.3/0.7, methanol/methylene chloride/ammonium hydroxide) to yield Example 17 (30 mg, 43%) as a mixture of 4 diastereomers.",
"ESI-MS: calculated MW: 527.28, found 528.25.EXAMPLE 18 A mixture of the Intermediate 4 (176 mg, 0.5 mmol), the spiropiperidine (as HCl salt, 115 mg, 0.6 mmol), DIEA (100 mg, 0.8 mmol), molecular sieves (4 Å, 200 mg) and sodium triacetoxyborohydride (212 mg, 1.0 mmol) in dichloromethane (10 mL) was stirred overnight.",
"The reaction was quenched with sat.",
"aq.",
"sodium carbonate.",
"The solid was removed by filtration through celite.",
"The crude product was extracted into dichloromethane and purified on preparative TLC (1000 micron, 10%[aq.",
"NH4OH/MeOH 1/9]/DCM).",
"The title compound was obtained as a mixture of cis and trans racemic isomers (155 mg, 63%).",
"LC-MS calc.",
"for C26H35F3N4O2: 492; Found: 493 (M+H).",
"C. ZHOU EXAMPLE 19 To a stirred solution of Intermediate 5 (50 mg, 0.14 mmol) and piperidine (28 μL, 0.28 mmol), in DCM (10 mL), was added 4 Å powdered molecular sieves (50 mg) and sodium triacetoxyborohydride (150 mg, 0.71 mmol).",
"The resulting solution was allowed to stir at room temperature for 3 days before being filtered through celite and washed with saturated aqueous sodium bicarbonate and brine.",
"The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude oil that was purified by preparative TLC (0.5% NH4OH/4.5% MeOH/95% DCM) to give 16 mg of a colorless solid as a mixture of 2 diastereomers.",
"A small portion of this free base was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"ESI-MS calc.",
"for C23H32F3N3O: 423.25; found 424 (M+H).",
"Several other examples were made according to the procedure described in Example 19 except that various substituted piperidines where used as the amine component in place of piperidine.",
"The examples are compiled in Table 4.TABLE 4 (EXAMPLES 20 to 28) Molecular Calculated Found MW Example Structure Formula MW [M + H] 20 C25H35F3N4O2 480.27 481 21 C26H36F3N3O3 495.27 496 22 C26H36F3N3O3 495.27 496 23 C24H34F3N3O 437.27 438 24 C24H34F3N3O 437.27 438 25 C25H36F3N3O 451.28 452 26 C23H32F3N3O2 439.24 440 27 C23H32F3N3O2 439.24 440 28 C24H32F3N3O 435.25 436 EXAMPLE 29 and EXAMPLE 30 The free base of the product prepared in Example 22 (55 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 25% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"27 mg of the faster eluting diastereomer (Example 29) and 20 mg of the slower eluting diastereomer (Example 30) were recovered.",
"EXAMPLE 29 ESI-MS calc.",
"for C26H36F3N3O3: 495.27; found 496 (M+H).",
"EXAMPLE 30 ESI-MS calc.",
"for C26H36F3N3O3: 495.27; found 496 (M+H).",
"EXAMPLE 31 Example 29 (10 mg, 0.018 mmol), was dissolved in a mixture of methanol (1 mL) and THF (1 mL), and treated with a solution of lithium hydroxide monohydrate (5 mg, 0.12 mmol) in water (1 mL).",
"The resulting solution was stirred for 18 h at room temperature before being concentrated under reduced pressure.",
"The product was purified by reverse phase HPLC (C18, 20-100% MeCN/H2O) and converted to its hydrochloride salt by addition of 2 N HCl in ethyl ether to give 6.8 mg of product (70 ESI-MS calc.",
"for C24H32F3N3O3: 467.24; found 468 (M+H).",
"EXAMPLE 32 Example 30 (10 mg, 0.018 mmol), was dissolved in a mixture of methanol (1 mL) and THF (1 mL), and treated with a solution of lithium hydroxide monohydrate (5 mg, 0.12 mmol) in water (1 mL).",
"The resulting solution was stirred for 18 h at room temperature before being concentrated under reduced pressure.",
"The product was purified by reverse phase HPLC (C18, 20-100% MeCN/H2O) and converted to its hydrochloride salt by addition of 2 N HCl in ethyl ether to give 3.8 mg of product (39 ESI-MS calc.",
"for C24H32F3N3O3: 467.24; found 468 (M+H).",
"EXAMPLE 33 To a solution of Example 30 (15 mg, 0.026 mmol), in THF (2 mL) was added lithium triethylborohydride (1.0 M solution in THF, 150 μL, 0.15 mmol).",
"After 18 h at room temperature an additional portion of lithium triethylborohydride (100 μL) was added and the resulting mixture was stirred for 24 h before being concentrated under reduced pressure.",
"The resulting residue was dissolved in DCM and washed with aqueous saturated sodium bicarbonate, 1 N aqueous HCl, and then brine.",
"The organic layer was dried over Na2SO4, filtered, treated with 2 N HCl in ether followed by hexanes and concentrated under reduced pressure to give 1.5 mg of the desired product as a hydrochloride salt (11%).",
"ESI-MS calc.",
"for C24H34F3N3O2: 453.26; found 454 (M+H).",
"EXAMPLE 34 and EXAMPLE 35 The free base of the product prepared in Example 20 (60 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 25% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"26 mg of the faster eluting diastereomer (Example 34) and 17 mg of the slower eluting diastereomer (Example 35) were recovered.",
"EXAMPLE 34 ESI-MS calc.",
"for C25H35F3N4O2: 480.27; found 481 (M+H).",
"EXAMPLE 35 ESI-MS calc.",
"for C25H35F3N4O2: 480.27; found 481 (M+H).",
"EXAMPLE 36 and EXAMPLE 37 The free base of the product prepared in Example 22 (54 mg) was resolved into 2 mixtures of 2 diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 13% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"33 mg of the faster eluting diastereomers (Example 36) and 10 mg of the slower eluting diastereomers (Example 37) were recovered.",
"EXAMPLE 36 ESI-MS calc.",
"for C26H36F3N3O3: 495.27; found 496 (M+H).",
"EXAMPLE 37 ESI-MS calc.",
"for C26H36F3N3O3: 495.27; found 496 (M+H).",
"EXAMPLES 38-41 The free base of the product prepared in Example 24 (40 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 20% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"15 mg of the fastest eluting diastereomer (Example 38), 1.5 mg of diastereomer 2 (Example 39), 7 mg of diastereomer 3 (Example 40), and 6 mg of the slowest eluting diastereomer (Example 41) were recovered.",
"EXAMPLE 38 ESI-MS calc.",
"for C24H34F3N3O: 437.27; found 438 (M+H).",
"EXAMPLE 39 ESI-MS calc.",
"for C24H34F3N3O: 437.27; found 438 (M+H).",
"EXAMPLE 40 ESI-MS calc.",
"for C24H34F3N3O: 437.27; found 438 (M+H).",
"EXAMPLE 41 ESI-MS calc.",
"for C24H34F3N3O: 437.27; found 438 (M+H).",
"EXAMPLES 42-46 The free base of the product prepared in Example 25 (40 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 20% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"All 6 diastereomers were resolved, but peaks 2 and 6 were combined in overlapping runs to give 4 pure diatereomers and one mixture of 2.EXAMPLE 42 Peak 1: ESI-MS calc.",
"for C25H36F3N3O: 451.28; found 452 (M+H).",
"EXAMPLE 43 Peak 2/6: ESI-MS calc.",
"for C25H36F3N3O: 451.28; found 452 (M+H).",
"EXAMPLE 44 Peak 3: ESI-MS calc.",
"for C25H36F3N3O: 451.28; found 452 (M+H).",
"EXAMPLE 45 Peak 4: ESI-MS calc.",
"for C25H36F3N3O: 451.28; found 452 (M+H).",
"EXAMPLE 46 Peak 5: ESI-MS calc.",
"for C25H36F3N3O: 451.28; found 452 (M+H).",
"EXAMPLE 47 and EXAMPLE 48 The free base of the product prepared in Example 27 (20 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 25% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"7 mg of the faster eluting diastereomer (Example 47) and 6 mg of the slower eluting diastereomer (Example 48) were recovered.",
"EXAMPLE 47 ESI-MS calc.",
"for C23H32F3N3O2: 439.24; found 440 (M+H).",
"EXAMPLE 48 ESI-MS calc.",
"for C23H32F3N3O2: 439.24; found 440 (M+H).",
"EXAMPLE 49 To a stirred solution of Intermediate 5 (50 mg, 0.14 mmol) and morpholine (25 μL, 0.28 mmol), in DCM (10 mL), was added 4 Å powdered molecular sieves (50 mg) and sodium triacetoxyborohydride (150 mg, 0.71 mmol).",
"The resulting solution was stirred at room temperature for 3 days before being filtered through celite and washed with saturated aqueous sodium bicarbonate and brine.",
"The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude oil that was purified by preparative TLC (0.5% NH4OH/4.5% MeOH (95% DCM) to give 57 mg of a colorless solid as a mixture of 2 diastereomers.",
"A small portion of this free base was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"ESI-MS calc.",
"for C22H30F3N3O0: 425.23; found 426 (M+H).",
"Several other examples were made according to the procedure described in Example 49 except that various substituted morpholines where used as the amine component in place of morpholine.",
"The examples are compiled in Table 5.TABLE 5 (EXAMPLES 50 to 53) Molecular Calculated Found MW Example Structure Formula MW [M + H] 50 C24H34F3N3O2 453.26 454 51 C29H36F3N3O3 531.27 532 52 C23H30F3N3O2 437.23 438 EXAMPLE 53 and EXAMPLE 54 The free base of the product prepared in Example 50 (60 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 20% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"26 mg of the faster eluting diastereomer (Example 53) and 27 mg of the slower eluting diastereomer (Example 54) were recovered.",
"EXAMPLE 53 ESI-MS calc.",
"for C24H34F3N3O2: 453.26; found 454 (M+H).",
"EXAMPLE 54 ESI-MS calc.",
"for C24H34F3N3O2: 453.26; found 454 (M+H).",
"EXAMPLE 55 and EXAMPLE 56 The free base of the product prepared in Example 52 (48 mg) was resolved into its individual diastereomers using an HPLC equipped with a ChiralCel OD column, eluting with 25% ethanol/hexanes.",
"Each compound was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"18 mg of the faster eluting diastereomer (Example 53) and 23 mg of the slower eluting diastereomer (Example 54) were recovered.",
"EXAMPLE 55 ESI-MS calc.",
"for C23H30F3N3O2: 437.23; found 438 (M+H).",
"EXAMPLE 56 ESI-MS calc.",
"for C23H30F3N3O2: 437.23; found 438 (M+H).",
"EXAMPLE 57 To a stirred solution of Intermediate 5 (50 mg, 0.14 mmol) and pyrrolidine (23 μL, 0.28 mmol), in DCM (10 mL), was added 4 Å powdered molecular sieves (50 mg) and sodium triacetoxyborohydride (150 mg, 0.71 mmol).",
"The resulting solution was stirred at room temperature for 3 days before being filtered through celite and washed with saturated aqueous sodium bicarbonate and brine.",
"The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude oil that was purified by preparative TLC (0.5% NH4OH/4.5% MeOH/95% DCM) to give 15 mg of a higher running diastereomer and 35.5 mg of a lower running isomer both of which where recoved as colorless solid.",
"A small portion of the free base was converted to its hydrochloride salt by the addition of 2 N HCl in ethyl ether.",
"Top: ESI-MS calc.",
"for C22H30F3N3O: 409.23; found 410 (M+H).",
"Bottom: ESI-MS calc.",
"for C22H30F3N3O: 409.23; found 410 (M+H).",
"Several other examples were made according to the procedure described in Example 57 except that various substituted pyrollidines where used as the amine component in place of pyrrolidine.",
"The examples are compiled in Table 6.TABLE 6 (EXAMPLES 58 to 62) Molecular Calculated Found MW Example Structure Formula MW [M + H] 58 C27H36F3N3O2 491.28 492 59 C27H33F3N4O 486.26 487 60 C27H33F3N4O 486.26 487 61 C27H33F3N4O 486.26 487 62 C28H40F3N3O3 523.30 524 EXAMPLE 63 A mixture of the Intermediate 3 (55 mg, 0.15 mmol), (1S,4S)-(−)-2-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1]heptane (as HBr salt, 85 mg, 0.3 mmol), DIEA (65 mg, 0.5 mmol), molecular sieves (4 Å, 250 mg) and sodium triacetoxyborohydride (212 mg, 1.0 mmol) in dichloromethane (5 mL) was stirred overnight.",
"The reaction was quenched with sat.",
"aq.",
"sodium carbonate.",
"The solid was removed by filtration through celite.",
"The crude product was extracted into dichloromethane and purified on preparative TLC (1000 micron, 5%[aq.",
"NH4OH/MeOH 1/9]/DCM).",
"The title compound was obtained as a mixture of cis and trans racemic isomers (75 mg, 95%).",
"LC-MS calc.",
"for C30H35F4N3O: 529; Found: 530 (M+H).",
"EXAMPLE 64 A mixture of the Intermediate 3 (55 mg, 0.15 mmol), (1S,4S)-(−)-2-(2-methyl)-2,5-diazabicyclo[2.2.1]heptane (as maleic acid salt, 100 mg, 0.3 mmol), DIEA (65 mg, 0.5 mmol), molecular sieves (4 Å, 250 mg) and sodium triacetoxyborohydride (212 mg, 1.0 mmol) in dichloromethane (5 mL) was stirred overnight.",
"The reaction was quenched with sat.",
"aq.",
"sodium carbonate.",
"The solid was removed by filtration through celite.",
"The crude product was extracted into dichloromethane and purified on preparative TLC (1000 micron, 5%[aq.",
"NH4OH/MeOH 1/9]/DCM).",
"The title compound was obtained as a mixture of cis and trans racemic isomers (52 mg, 66%).",
"LC-MS calc.",
"for C31H38F3N3O: 525; Found: 526 (M+H).",
"EXAMPLE 65 Step A A neat mixture of 54 g (0.29 mole) ethyl (2-aminothiazol-4-yl)acetate and 50 g (0.276 mole) benzophenone imine was stirred at 190° C. for 5 h and then cooled at RT and diluted with 100 mL of CH2Cl2.The entire mixture was transferred onto a silica gel column and eluted with 20% EtOAc/Hexane.",
"The title compound was obtained as light-yellow solid (70 g, 69% yield).",
"1H NMR (300 MHz, CDCl3): 1.26 (t, 3H), 3.74 (s, 2H), 4.15 (q, 2H), 6.87 (s, 1H), 77.25-7.86 (m, 10H); Mass Spectrum (NH3—Cl): m/z 351 (M+1).",
"Step B To a mixture of 35 g (0.10 Mole) of the Schiff base ester form Step A, cis-1,3-dichloro-2-butene (13 mL, 0.11 Mole) in 500 mL of DME at RT was added in multiple portions solid NaH (60% oil, 10.0 g, 0.25 Mole).",
"The resulting mixture was stirred for 2 days, poured into 2000 mL of ice-water, extracted with 1500 mL of ether.",
"The ether layer was washed with water (3×500 mL), dried over Na2SO4 and evaporated.",
"FC (Silica Gel, 5% EtOAc/Hexane) afforded the title compound as an oil (24 g, 59%).",
"1H NMR (300 MHz, CDCl3): δ 1.20 (t, 3H), 2.87 (d, 2H), 3.19 (d, 2H), 4.14 (q, 2H), 5.29 (s, 2H), 6.71 (s, 1H), 7.26-7.81 (m, 10H).",
"Mass Spectrum (NH3—Cl): m/z 403 (M+1).",
"Step C 24.0 g (0.059 mol) of the cyclopentene Schiff base from Step B was dissolved in 100 mL of 4 N HCl/dioxane.",
"After 1 h, 1.8 mL of water was added.",
"The mixture was stirred for 3 h, evaporated to dryness.",
"The residue was dissolved in 100 mL of CH2Cl2 and added 15 mL of DIEA.",
"The entire mixture was dumped onto a silica gel column, eluted with 20% EtOAc/Hexane to remove benzophenone, then eluted with 40% EtOAc/Hexane to give the title compound as a light yellow solid (12.0 g, 85%).",
"1H NMR (300 MHz, CDCl3): δ 1.19 (t, 3H), 2.79 (d, 2H), 3.15 (d, 2H), 4.13 (q, 2H), 5.66 (s, 2H), 5.82 (wide, 2H), 6.19 (s, 1H).",
"Step D A mixture of 12 g (50 mmol) of the aminothiazole from Step C, 28 g (130 mmol) of di-tert-butyl dicarbonate and 0.6 g of DMAP in 250 mL of DCM was stirred overnight, and evaporated.",
"The title compound (21.0 g, 96%) was obtained as a yellow oil after flash chromatography purification on silica gel (10% EtOAc/Hexane).",
"1H NMR (300 MHz, CDCl3): 1.18 (t, 3H), 1.49 (d, 18H), 2.88 (d, 2H), 3.18 (d, 2H), 4.13 (q, 2H), 5.65 (s, 2H), 6.83 (s, 1H).",
"Mass Spectrum (NH3—CI): mz 439 (M+1).",
"Step E To a solution of 13.1 g (30.0 mmol) of the ester from Step D in 50 mL of anhydrous ether at −78° C. was added dropwise a solution of BH3.DMS in THF (14 mL, 24 mmol).",
"The cooling bath was removed and the mixture was stirred at room temperature for 3 h, diluted with 250 mL of DCM, added 25 g of sodium acetate and 55 g of PCC.",
"The mixture was stirred overnight.",
"The entire mixture was applied to a silica gel column and eluted with in 10% EtOAc/Hexane and then 30% EtOAc/Hexane.",
"Two components were obtained.",
"The fast-eluted isomer (yellow oil, 6.0 g) was identified as the title compound.",
"1H NMR (300 MHz, CDCl3): 1.21 (t, 3H), 1.50 (s, 18H), 2.33 (t, 2H), 2.42-2.70 (m, 2H), 2.78-3.10 (dd, 2H), 4.18 (q, 3H), 6.88 (s, 1H).",
"Mass Spectrum (NH3-CI): m/z 455 (M+1).",
"Step F The slow-eluted component from above was proved to be the title compound (gummy material, 1.80 g).",
"1H NMR (300 MHz, CDCl3): (t, 3H), 1.46 (s, 9H), 2.27 (3, 2H), 2.38-2.62 (m, 2H), 2.64-3.00 (dd, 2H), 4.11 (q, 2H), 6.66 (s, 1H).",
"Mass Spectrum (NH3-CI): m/z 355 (M+1).",
"Step G A mixture of 1.40 g (4.00 mmol) of the keto ester from Step F and 0.82 g (13 mmol) of lithium hydroxide monohydrate in a solution of 20 mL of MeOH and 2 mL of water was stirred at room temperature overnight.",
"The entire mixture was applied to a silica gel column and eluted with 10% MeOH/CH2Cl2.Evaporation in vacuo afforded a light yellow solid.",
"1.30 g of the title product was obtained as a fluffy solid.",
"1H NMR (300 MHz, CDCl3): (t, 9H), 2.10-3.20 (m, 8H), 6.60 (s, 1H).",
"Step H A mixture of the keto acid prepared in Step G (1.0 g, 3.0 mmol), Intermediate 1 (as HCl salt, 0.66 g, 3.0 mmol) and EDC (0.95 g, 5.0 mmol) in dichloromethane (10 mL) was stirred for 2 days.",
"The mixture was diluted with dichloromethane and washed with 1 N aq.",
"HCl, dried over sodium sulfate and evaporated.",
"The residue was purified on preparative TLC (1500 micron, 10%[aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the title product as a yellow solid (0.52 g, 34%).",
"LC-MS calc.",
"for C25H27F3N2O4S: 508; Found: 509 (M+H).",
"Step I The product from step H (510 mg, 1.0 mmol) was mixed with TFA (10 mL) for 30 min.",
"TFA was removed and the residue was purified on preparative TLC (10%[aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the desired product as a white solid (223 mg, 55%).",
"LC-MS calc.",
"for C20H19F3N2O2S: 408; Found: 409 (M+H).",
"Step J A mixture of the product from Step I (210 mg, 0.50 mmol), morpholine (440 mg, 5.0 mmol), molecular sieves (4 Å, 500 mg) and sodium triacetoxyborohydride (420 mg, 2.0 mmol) in dichloromethane (15 mL) was stirred overnight.",
"The reaction was quenched with sat.",
"aq.",
"sodium carbonate.",
"The solid was removed by filtration through celite.",
"The crude product was extracted into dichloromethane and purified on preparative TLC (1000 micron, 10%[aq.",
"NH4OH/MeOH 1/9]/DCM).",
"The title compound was obtained as a mixture of cis and trans racemic isomers (200 mg, 84%).",
"LC-MS calc.",
"for C24H28F3N3O2S: 479; Found: 480 (M+H).",
"EXAMPLE 66 A mixture of the Intermediate 3 (70 mg, 0.20 mmol), cis-2,6-dimethylmorpholine (23 μL, 0.20 mmol), molecular sieve (4 μL, 200 mg), and sodium triacetoxyborohydride (210 mg, 0.99 mmol) in DCM was stirred for 5 days.",
"The reaction mixture was diluted by DCM, filtered, and washed with sat.",
"aq.",
"NaHCO3, water and brine.",
"DCM layers were dried over Na2SO4, filtered and concentrated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 3% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final title compound as a free base.",
"Its HCl salt (62.2 mg) was formed by treatment with 4 N HCl/dioxane.",
"ESI-MS calc.",
"for C25H35F3N2O2: 452.27; Found: 453 (M+H).",
"The diastereoisomers were separated into one mixture of 2 cis diastereoisomers and two single diastereomers using HPLC (AD column, 5% EtOH/hexane).",
"EXAMPLE 67 Example 68 was prepared starting from Intermediate 3 and (1S,4S)-(+)-2-aza-5-oxabiclclo[2.2.1]heptane hydrochloride as detailed in Example 66.The cis and trans isomers were resolved on preparative TLC (4/95.6/0.4, MeOH/DCM/NH4OH).",
"Top spot: ESI-MS calc.",
"for C24H31F3N2O2: 436.23; Found: 437 (M+H).",
"Bottom spot: ESI-MS calc.",
"for C24H31F3N2O2: 436.23; Found: 437 (M+H).",
"EXAMPLE 68 Example 68 was prepared starting from piperidine and Intermediate 3 as detailed in Example 49.ESI-MS calc.",
"for C24H33F3N2O: 422.25; Found: 423 (M+H).",
"EXAMPLE 69 Intermediate 22 (563 mg, 2.43 mmol) was dissolved in dichloromethane and cooled to −78° C. Ozone was bubbled into the solution until a blue color persisted.",
"Nitrogen gas was then bubbled through the solution until disappearance of the blue color.",
"Na2SO4 was added into the stirring solution.",
"The reaction mixture was filtered into a flask containing Intermediate 23.Dichloromethane (20 mL), triethylamine (136 μL, 0.974 mmol), and NaB(OAc)3H (929 mg, 4.38 mmol) were added to the reaction flask.",
"The reaction mixture was stirred at room temperature under N2 gas for two hours and then diluted with dichloromethane and washed with saturated sodium bicarbonate solution (2×100 mL) and brine (1×100 mL).",
"The organic layers were dried over MgSO4, filtered, and concentrated.",
"The crude products were divided into three batches and loaded onto three ion exchange column.",
"Impurities were flushed away with using 40% MeOH/hexanes (100 mL).",
"The desired product was then eluted from the columns with a 30% solution 2N NH3 in MeOH further diluted in dichloromethane.",
"Example 69 (131 mg, 0.223 mmol, 46% yield) was purified by preparatory TLC using 32% EtOAc/hex.",
"The diastereomers were then isolated through preparatory TLC using 45% EtOAc/hex (top isomer: 20 mg, 0.034 mmol, 7% yield; middle isomer: 45 mg, 0.076 mmol, 16% yield; bottom isomer: 54 mg, 0.092 mmol, 19% yield).",
"ESI-MS calculated for C32H41F3N4O3: 586.69, found 587 (M+H).",
"Several other piperazines and homo-piperazines where made according to the same procedure described in Example 69.Table 7 summarizes these compounds.",
"TABLE 7 (EXAMPLES 70 to 72) Molecular Calculated Found MW Example Structure Formula MW [M + H] 70 C26H37F3N4O 478.29 479 71 C25H35F3N4O 464.28 465 72 C27H35F3N6O 516.28 517 EXAMPLE 73 The above compound was prepared from Intermediate 5 and 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-]pyridine according to the procedure described in Example 49.ESI-MS calc.",
"for C24H30F3N5O: 461.24; found 462 (M+H).",
"EXAMPLE 74 The above compound was prepared from Intermediate 5 and 1,4-diazepan-5-one according to procedure described in Example 49.ESI-MS calc.",
"for C23H31F3N4O2: 452.24; found 453 (M+H).",
"EXAMPLE 75 The above compound was prepared from Intermediate 5 and 2,3,4,5-tetrahydro-1,4-benzoxazepine according to the procedure described in Example 57.Top spot: ESI-MS calc.",
"for C27H32F3N3O2: 487.24; found 488 (M+H).",
"Bottom spot: ESI-MS calc.",
"for C27H32F3N3O2: 487.24; found 488 (M+H).",
"EXAMPLE 76 Palladium catalyst on carbon (top: 6 mg, middle: 18 mg, bottom: 20 mg) was added to three flasks each containing one of the three diastereomers of Example 70 (top: 29.6 mg, middle: 89.9 mg, bottom: 102.3 mg).",
"The mixtures were dissolved in MeOH (3-6 mL), and the reaction vessels were flushed repeatedly with hydrogen gas.",
"The reaction was stirred at room temperature under a hydrogen atmosphere for 4.5 hours and then passed through an Acrodisc® syringe filter with a 0.45 μm PTFE membrane.",
"Compound 76 (top isomer: 16.0 mg, 0.0354 mmol, 71% yield; middle isomer: 42.7 mg, 0.0943 mmol, 62% yield; bottom isomer: 34.5 mg, 0.0762 mmol, 44% yield) was isolated through preparatory TLC using a 10% NH4OH/MeOH (1:9) in dichloromethane solvent system.",
"ESI-MS calculated for C24H35F3N4O: 452.56, found 453 (M+H).",
"EXAMPLE 77 Example 76 (middle stereoisomer on TLC, 11 mg, 0.024 mmol) was dissolved in dichloromethane (3 mL) and cooled to 0° C. Ethylchloroformate (5 μL, 0.05 mmol), triethylamine (10 μL, 0.07 mmol), and a catalytic amount of DMAP (1-2 mg) was added to the reaction flask.",
"The reaction was warmed to room temperature and stirred for two hours under a nitrogen atmosphere.",
"Thereafter, the reaction was diluted with DCM and washed with saturated sodium bicarbonate solution (1×25 mL) and brine (1×25 mL).",
"The organic layer was dried over MgSO4, filtered, and concentrated.",
"Example 77 (4.2 mg, 0.0080 mmol, 33% yield) was purified through preparatory TLC using a 2% NH4OH/MeOH (1:9) in dichloromethane solvent system.",
"ESI-MS calculated for C27H39F3N4O3: 524.62, found: 525 (M+H).",
"EXAMPLE 78 Example 76 (middle stereoisomer on TLC, 11 mg, 0.024 mmol) was dissolved in dichloromethane (3 mL) and cooled to 0° C. Acetic anhydride (11 μL, 0.12 mmol), triethylamine (23 μL, 0.17 mmol), and a catalytic amount of DMAP (1-2 mg) was added to the reaction flask.",
"The reaction was warmed to room temperature and stirred for 2.5 hrs.",
"under a nitrogen atmosphere.",
"The reaction was then diluted with DCM and washed with saturated sodium bicarbonate solution (1×25 mL) and brine (1×25 mL).",
"The organic layer was dried over MgSO4, filtered, and concentrated.",
"Example 78 (5.3 mg, 0.011 mmol, 45% yield) was purified through preparatory TLC using a 2% NH4OH/MeOH (1:9) in dichloromethane solvent system.",
"ESI-MS calculated for C26H37F3N4O2: 494.59, found: 495 (M+H).",
"EXAMPLE 79 Dichloromethane (2-3 mL per flask) was added to three separate flasks containing the three diastereomers of Example 77 (top: 16 mg, 0.035 mmol, middle: 11 mg, 0.023 mmol, bottom: 14 mg, 0.031 mmol).",
"Methylisocyanate (top: 21 μL, 0.35 mmol, middle: 14 μL, 0.23 mmol, bottom: 19 μL, 0.31 mmol) was added to each reaction vessel.",
"The reactions were stirred for three hours and then concentrated before isolating Example 79 (top isomer: 12.8 mg, 0.0251 mmol, 72% yield; middle isomer: 10.4 mg, 0.0204 mmol, 89% yield; bottom isomer: 11.1 mg, 0.0218 mmol, 70% yield) by preparatory TLC using a 2% NH4OH/MeOH (1:9) in dichloromethane solvent system.",
"ESI-MS calculated for C26H38F3N5O2: 509.61, found: 510 (M+H).",
"EXAMPLE 80 Step A A solution of cis-hydroxy-D-proline (2.98 g, 22.7 mmol) in methanol (20 mL) was treated with thionyl chloride (1.78 mL, 24.4 mmol) and stirred at room temperature for 1 h. The reaction mixture was then heated to 65° C. overnight.",
"Evaporation of the volatiles gave the crude methyl ester (4.0816 g), which was dissolved in dichloromethane (50 mL) and diisopropyl ethyl amine (9.59 mL, 55.1 mmol) was added.",
"The reaction mixture was cooled to 0° C. and neat benzyl chloroformate (3.71 mL, 26.0 mmol) was added via syringe.",
"After stirring at 0° C. for 30 minutes the cooling bath was removed.",
"The reaction was quenched by pouring into an aqueous solution of citric acid (10%, 50 mL) and the product was extracted into dichloromethane.",
"The combined organic phases were back-washed with brine, dried with anhydrous sodium sulfate, and the solvent was removed in vacuo.",
"The crude product was purified by flash chromatography (silica gel, ethyl acetate:hexanes/4:6) to yield 4.0717 g (69%) of the pure product.",
"1H NMR (500 MHz, CDCl3): δ 7.35 m (5H), 5.21 (d, J=12.6 Hz, 2H), 5.10 (d, J=13 Hz, 1H), 5.06 (d, J=12.35 Hz, 1H), 4.45 (m, 2H), 3.80 (s, 3H), 2.35 (m, 1H), 2.15 (m, 1H).",
"Step B A solution of the alcohol from the previous step (2.63 g, 9.42 mmol) and diisopropylethylamine (3.28 mL, 18.8 mmol) in dichloromethane 40 mL was cooled to −78° C. and tert-butyldimethylsilyl trifluoromethane sulfonate (2.596 mL, 11.30 mmol) was added via syringe.",
"The reaction mixture was stirred at −78° C. for 2 h, and quenched by pouring onto 50 mL of saturated solution of sodium bicarbonate.",
"The organic phase was separated, dried with magnesium sulfate and the volatiles were removed in vacuo.",
"The residue (6.0 g) was further purified by column chromatography (silica gel, ethyl acetate:hexanes/1:4) to afford 2.8355 g (76%) of the pure product.",
"1H NMR (500 MHz, CDCl3): 7.35 m (5H), 5.20 (m=2H), 4.45 (m, 2H), 3.70 (m, 4H), 3.45 (m, 1H), 2.23 (m, 2H) 0.87 (bs, 9H), 0.05 (m, 6H).",
"Step C A solution of the ester from previous step (2.83 g, 7.19 mmol) in THF (10 mL) was treated with lithium borohydride (250 mg, 11.5 mmol) at 0° C. and the reaction mixture was stirred at room temperature for 72 h. It was then diluted with diethyl ether, washed with water and a 1 M solution of phosphoric acid.",
"The combined organic extracts were dried (sodium sulfate) and the solvent was removed in vacuo.",
"The crude product was further purified by column chromatography (silica gel, ethyl acetate:hexanes/3: 7) to yield 1.5636 g (60%) of the pure product.",
"LC MS: for C19H31NO4Si calculated 365.20, found 366.25 [M+H ]+.",
"Step D A solution of the alcohol from the previous step (1.56 g, 4.27 mmol) and diisopropylethylamine (1.49 mL, 8.53 mmol) in dichloromethane (20 mL) was treated with methanesulfonyl chloride (496 μL, 6.41 mmol) at 0° C. The reaction mixture was stirred at cold for 15 minutes, then at room temperature for another 30 minutes.",
"It was diluted with dichloromethane, washed with water and dried with magnesium sulfate.",
"The solvent was removed in vacuo and the crude product was used without any delay in the next step as obtained.",
"LC MS: for C20H33NSO6Si calculated 443.18, found 444.25 [M+H]+.",
"Step E A solution of the TBS-ether from the previous step (1.82 g, 4.10 mmol) in THF (50 mL) was treated with tetrabutylammonium fluoride (4.51 mL, 1 M solution in THF).",
"And the reaction mixture was stirred at room temperature until LC MS analysis indicated complete removal of the TBS group, about 15 minutes.",
"The reaction mixture was then cooled to 0° C. and sodium hydride (180 mg, 60% suspension, 45.1 mmol) was added.",
"Stirring at room temperature was continued for 24 h. The reaction was quenched by pouring onto water, and the crude product was extracted into ethyl acetate.",
"The solvent was removed in vacuo, and the crude product was purified by flash chromatography (dichloromethane: ether/7: 3) to afford 595 mg (59%) of the desired product.",
"1H NMR (500 MHz, CDCl3): 7.38 (m, 5H), 5.30 (m, 2H), 4.52 (m, 2H), 3.80 (m, 2H), 3.40 (m, 2H), 1.70 (m, 2H).",
"Step F A solution of the CBZ-amine from the previous step (590 mg, 2.53 mmol) in ethyl alcohol (30 mL) was hydrogenated in the presence of Pd/C (142 mg, 10%) under balloon pressure.",
"The reaction was monitored by LC by the disappearance of the starting material and appearance of toluene.",
"The catalyst was filtered off and the filtrate was evaporated to dryness to leave 321 mg (94%) of the desired product.",
"Step G A solution of Intermediate 3 (120 mg, 0.340 mmol), amine hydrochloride from previous step (46 mg, 0.34 mmol), diisopropyl ethylamine (60 μL, 0.34 mmol) and 4 Å molecular sieves (crushed, 360 mg) in dichloroethane (10 mL) was treated with sodium triacetoxyborohydride, and stirred at room temperature for 24 h. The reaction was quenched by pouring onto saturated solution of sodium bicarbonate and the crude product was extracted with dichloromethane.",
"The volatiles were removed in vacuo, and preparative TLC (ethyl acetate: ethyl alcohol: ammonium hydroxide/90: 8: 2 gave the pure product (118 mg, 80%).",
"LC MS: for C24H31N2F3O2 calculated 436.23, found 437.20 [M+H]+.",
"EXAMPLE 81 To a solution of Intermediate 21 (150 mg, 0.43 mmol) in dichloromethane (10 mL) was added EDC (414 mg, 2.16 mmol), HOAt (59 mg, 0.43 mmol) and Intermediate 1 (87 mg, 0.43 mmol) and the resulting mixture was stirred at room temperature for 4 days.",
"The reaction was quenched with water and diluted with 20 mL of dichloromethane.",
"The organic layer was separated and the aqueous layer was extracted with DCM (2×20 mL).",
"The organics were combined, dried over sodium sulfate, filtered and evaporated under reduced pressure.",
"The residue was purified by preparative TLC (eluant: 10% methanol: 89.5% dichloromethane: 0.5% NH4OH) to yield 40 mg (17%) of the final pure desired product as a mixture of two cis isomers.",
"LC-MS for C30H36N2OF4 calculated 516.28, found [M+H]+517.EXAMPLE 82 The hydrochloride salt of the pyrimidyl piperidine (Intermediate 8) (67 mg, 0.34 mmol) was combined with Intermediate 4 (100 mg, 0.28 mmol), triethylamine (46 μL, 0.35 mmol), and 4 Å powdered molecular sieves (100 mg) in DCM.",
"After 15 minutes at room temperature, sodium triacetoxyborohydride (240 mg, 1.13 mmol) was added and the resulting mixture was stirred for 3 days before being filtered through celite, diluted with DCM and washed with saturated sodium bicarbonate and brine.",
"The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give a crude oil that was purified by preparative TLC (silica gel, 0.3% NH4OH/2.7% MeOH/97% DCM) to give 110 mg of a colorless oil.",
"Resolution of the individual diastereomers was accomplished by HPLC using a ChiralPak AD column eluting with 30% isopropanol/hexanes to give 2 single diastereomers and a single mixture of the 2 other diastereomers.",
"First peak 10 mg: ESI-MS calc.",
"for C28H35F3N4O: 500.28; found 504 (M+H).",
"Second peak 11 mg: ESI-MS calc.",
"for C28H35F3N4O: 500.28; found 504 (M+H).",
"Third peak 7.0 mg ESI-MS calc.",
"for C28H35F3N4O: 500.28; found 504 (M+H).",
"EXAMPLES 83-91 Several other examples where prepared in a similar fashion to Example 82, utilizing different piperidine intermediates.",
"These Examples (83-91) are shown below.",
"Molecular Calculated Found Example R1 Formula [M] [M + H] 83 C27H36F3N4O 488.27 489 84 C27H36F3N4O 488.27 489 85 C27H36F3N4O 488.27 489 86 C26H35F3N5O 489.27 490 87 C26H35F3N5O 489.27 490 88 C26H35F3N5O 489.27 490 89 C25H34F3N6O 490.26 491 90 C25H34F3N6O 490.26 491 91 C26H36F3N6O 504.26 505 EXAMPLE 92 This product was prepared in an analogous fashion to that of Example 81, except Intermediate 21 was replaced with commercially available 4-cyano-4-phenylpiperidine.",
"The crude product was purified by preparative TLC (eluant: 5% methanol: 94.5% DCM: 0.5% NH4OH) to yield Example 92 as a mixture of four isomers.",
"LC-MS for C31H36F3N3O calculated 523.28, found [M+H]+524.EXAMPLE 93 This product was prepared in an analogous fashion to that of Example 81, except Intermediate 21 was replaced with commercially available 4-phenylpiperidine.",
"The crude product was purified by preparative TLC (eluant: 10% methanol: 89.5% DCM: 0.5% NH4OH) to yield Example 93 as a mixture of four isomers.",
"LC-MS for C30H37F3N2O calculated 498.29, found [M+H]+ 499.EXAMPLE 94 Step A To a solution of the cyclopentanone carboxylic acid (from Step C, Intermediate 3) (2.3 g, 14 mmol) in dichloromethane (70 mL) was added oxalyl chloride (1.8 mL, 21 mmol) followed by 2 drops of DMF.",
"The solution was stirred at room temperature for 80 minutes and then evaporated under reduced pressure.",
"The residue was dissolved in DCM (20 mL) and added via syringe to a prepared solution of Intermediate 3 (3.0 g, 14 mmol) and triethylamine (2.1 mL, 15 mmol) in DCM (50 mL).",
"The resulting mixture was stirred at room temperature for 18 h and then quenched with water (25 mL).",
"The organics were separated, washed with 1 N HCl, saturated sodium bicarbonate solution, and brine, dried over anhydrous magnesium sulfate, filtered, and evaporated.",
"The residue was purified on a Biotage Flash 40 (eluant: 40% ethyl acetate/60% hexane) to afford 2.2 g (43%) of the title compound.",
"Step B A solution of the product described in Step A (200 mg, 0.54 mmol) commercially available 4-fluorophenylpiperidine hydrochloride (120 mg, 0.54 mmol), diisopropylethylamine (94 μL, 0.54 mmol) and crushed molecular sieves (4 Å, 100 mg) in dichloromethane (10 mL) was treated with sodium triacetoxyborohydride (343 mg, 1.60 mmol) and stirred at room temperature overnight.",
"The reaction was quenched with saturated sodium bicarbonate (10 mL) and diluted with an additional 10 mL of DCM.",
"The organic layer was separated and the aqueous washed with dichloromethane (2×5 mL).",
"The organics were combined, dried over anhydrous sodium sulfate, filtered and evaporated.",
"The residue was purified by preparative TLC (eluant: 8% ethanol: 90% ethyl acetate: 2% NH4OH) to yield two isomers, higher eluting (25 mg, 5%) and lower eluting (37.2 mg, 7.6%) of unknown absolute stereochemistry.",
"LC-MS for C30H35N2OF5 calculated 534.28, found [M+H]+ 535.EXAMPLE 95 This product was prepared in an analogous fashion to that of Example 94, except 4-fluorophenylpiperidine hydrochloride was replaced with Intermediate 9.The crude product was purified by preparative TLC (eluent: 90% ethyl acetate: 8% ethanol: 2% NH4OH) to yield 450 mg (66%) of the title product as a mixture of four isomers.",
"LC-MS for C28H34N4OF4 calculated 518.27, found [M+H]+ 519.EXAMPLES 96-104 Several other examples where prepared in a similar fashion to Example 94, utilizing different piperidine intermediates.",
"These Examples (96-104) are shown below.",
"Molecular Calculated Found Example R1 Formula [M] [M + H] 96 C25H33F4N6O 508.26 509 97 C25H33F4N6O 508.26 509 98 C27H35F4N4O 506.27 507 99 C26H34F4N5O 507.27 507 100 C26H34F4N5O 507.27 508 101 C26H34F4N5O 507.27 508 102 C27H35F4N4O 506.27 507 103 C27H35F4N4O 506.27 507 104 C26H35F4N6O 522.28 523 EXAMPLE 105 To a solution of Intermediate 21 (150 mg, 0.43 mmol) in dichloromethane (10 mL) was added EDC (170 mg, 0.86 mmol), HOAt (59 mg, 0.43 mmol) and Intermediate 21 (91 mg, 0.43 mmol) and the resulting mixture was stirred at room temperature for 3 days.",
"The reaction was quenched with water and diluted with 20 mL of dichloromethane.",
"The organic layer was separated and the aqueous layer was extracted with DCM (2×20 mL).",
"The organics were combined, dried over sodium sulfate, filtered and evaporated under reduced pressure.",
"The residue was purified by preparative TLC (eluant: 7% ethanol: 92% dichloromethane: 1.0% NH4OH) to yield 121 mg (50%) of the final desired product as a mixture of two cis isomers.",
"LC-MS for C29H36BrFN2O calculated 526.28, found [M+H]+ 527 and [(M+2)+H]+ 529.EXAMPLE 106 To a solution of Example 105 (100 mg, 0.210 mmol), phenylboronic acid (30 mg, 0.23 mmol), toluene (1.4 mL), and MeOH (0.6 mL) was added a solution of Na2CO3 (80 mg, 0.74 mmol) and Pd(PPh3)2Cl2 (8 mg, 0.01 mmol) in H2O (0.4 μL).",
"The reaction mixture was heated at 80° C. in a high pressure tube for 12 h before filtered through celite and concentrated to dryness.",
"The concentrate was diluted with DCM, washed with 1 N NaOH solution (3×), dried over Na2SO4, and concentrated in vacuo.",
"The crude product was purified by preparative TLC (eluent: 5/94.5/0.5, MeOH/DCM/NH4OH) to yield Example 106 (63.3 mg, 63.3%) a mixture of 2 cis isomers.",
"LC-MS for C35H41FN2O calculated 524.29, found [M+H]+ 525.4.EXAMPLE 107 This product was prepared in an analogous fashion to that of Example 106, except phenylboronic acid was replaced with 4-pyridylboronic acid.",
"The crude product was purified by preparative TLC (eluent: 10/89/1.0, MeOH/DCM/NH4OH) to yield Example 107 as a mixture of two cis isomers.",
"LC-MS for C34H40F3O calculated 525.26, found [M+H]+ 526.3.EXAMPLE 108 This product was prepared in an analogous fashion to that of Example 106, except phenylboronic acid was replaced with 3-pyridylboronic acid.",
"The crude product was purified by preparative TLC (eluent: 10/89/1.0, MeOH/DCM/NH4OH) to yield Example 108 as a mixture of two cis isomers.",
"LC-MS for C34H40FN3O calculated 525.26, found [M+H]+ 526.3.EXAMPLE 109 This product was prepared in an analogous fashion to that of Example 106, except phenylboronic acid was replaced with 2-pyridylboronic acid.",
"The crude product was purified by preparative TLC (eluent: 10/89/1.0, MeOH/DCM/NH4OH) to yield Example 109 as a mixture of two cis isomers.",
"LC-MS for C34H40FN3O calculated 525.26, found [M+H]+ 526.3.EXAMPLE 110 Step A To ice cold concentrated sulfuric acid was added in a dropwise manner 1,2,3,4-tetrahydroisoquinoline (23 mL, 170 mmol), followed by potassium nitrate (18.8 g, 186 mmol) at such a rate that the temperature did not rise above 5° C. After complete addition the mixture was stirred at room temperature for 18 h then poured onto a stirred mixture of ice (700 g) and NH4OH (150 mL).",
"The mixture was extracted with CHCl3 (3×300 mL).",
"The combined CHCl3 layers were washed with saturated NaCl (200 mL), dried over Na2SO4, filtered and concentrated in vacuo.",
"The residue was dissolved in ethanol (130 mL) and cooled in an ice bath as concentrated hydrochloric acid (22 mL) was added.",
"The formed precipitate was removed by filtration and recrystallized from methanol to give the product (12.45 g, 34%); 1H NMR 500 MHz (DMSO-d6) δ=3.13 (2H, t, J=6.2 Hz), 3.35 (2H, t, J=6.2 Hz), 4.35 (2H, s), 7.50 (1H, d, J=8.5 Hz), 8.07 (1H, dd, J=2.3 and 8.5 Hz), 8.19 (1H, d, J=2.3 Hz) 10.02 (2H, br s).",
"Step B To a suspension of the product from Step A (12 g, 58 mmol), and pyridine (23.7 mL, 293 mmol) in anhydrous CH2Cl2 (50 ml) cooled at 0° C. was added in a dropwise manner trifluoroacetic anhydride (12 mL, 87 mmol), and the resulting mixture was stirred at room temperature for 18 h. The reaction mixture was poured onto ice (500 g) and extracted with CH2Cl2 (4×150 mL).",
"The combined CH2Cl2 layers were washed with 1 N HCl (4×100 mL), saturated NaCl (100 mL), dried over Na2SO4, filtered and evaporated in vacuo to give the product (15.92 g, 89%); 1H NMR 500 MHz (CDCl3) δ=3.07 (2H, m), 3.91 and 3.94 (2H, t, J=6.2 Hz), 4.85 and 4.88 (2H, s), 7.36 (1H, dd, J=8.7 and 11.9 Hz), 8.07 (1H, dd, J=2.3 and 8.5 Hz), 8.01-8.08 (2H m).",
"Step C A solution of the product from Step B (16 g, 58 mmol) in ethanol (200 mL) was hydrogenated in a Parr Apparatus at 50 psi over PtO2 until hydrogen uptake ceased.",
"The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo to give to the product (14.2 g, 100%); 1H NMR 500 MHz (CDCl3) δ=2.84 (2H, t, J=5.7 Hz), 3.35 (2H, br s), 3.80 and 3.84 (2H, t, J=6.0 Hz), 4.64 and 4.69 (2H, s), 6.45 (1H, d, J=10.0 Hz), 6.57 (1H, t d, J=2.4 and 8.5 Hz), 6.95 (1H, d, J=8.5 Hz).",
"Step D To a solution of the product from Step C (2.0 g, 8.7 mmol) in toluene (50 mL) was added N,N-dimethylformamide azine (2.3 g, 16 mmol) and a spatula end of toluene sulfonic acid, and the resulting mixture was heated at reflux for 24 h. The mixture was concentrated in vacuo and the residue was partitioned between CH2Cl2 (50 mL) and water (50 mL).",
"The organic layer was separated and washed with saturated NaHCO3 (25 mL) and saturated NaCl (25 mL), dried over MgSO4, filtered and concentrated in vacuo.",
"The residue was triturated with CH2Cl2 (4 mL), filtered and dried to give the product (1.0 g, 39%); 1H NMR 500 MHz (CDCl3) δ=3.05 (2H, t, J=5.7 Hz), 3.90 and 3.95 (2H, t, J=6.0 Hz), 4.83 and 4.89 (2H, s), 7.20 (1H, d), 7.26 (1H, t), 7.38 (1H, t) 8.45 (2H, s).",
"Step E To a solution of the product from Step D (1.0 g, 3.4 mmol) in ethanol (50 mL) was added a solution of potassium carbonate (2.3 g, 17 mmol) in water (10 mL) and the resulting mixture was heated at reflux for 90 minutes.",
"The cooled reaction mixture was concentrated in vacuo and the solid residue was extracted with CH2Cl2 (3×10 mL).",
"The combined CH2Cl2 layers were evaporated in vacuo and the residue was purified by column chromatography on silica eluting with 10% CH3OH in CH2Cl2 containing 0.5% NH4OH to give the product (550 mg, 82%); 1H NMR 500 MHz (CDCl3) δ=2.69 (1H, br s), 2.85 (2H, t, J=5.9 Hz), 3.17 (2H, t, J=6.0 Hz), 4.07 (2H, s), 7.20 (1H, d, J=1.8 Hz), 7.14 (1H, dd, J=1.8 and 8.0 Hz), 7.23 (1H, d, J=8.0 Hz), 8.43 (2H, s).",
"Step F This product was prepared in an analogous fashion to that of Example 81, except Intermediate I was replaced with the product described in Step E. The crude product was purified by preparative TLC (eluant: 10% methanol: 89.5% DCM: 0.5% NH4OH) to yield Example 30 as a mixture of two cis isomers.",
"LC-MS for C31H39FN5O calculated 515.31, found [M+H]+ 516.EXAMPLE 111 Step A To a solution of the trifluoroacetamide produced in Step C of Example 110 (7.5 g, 31 mmol) in ethanol (200 mL) was added a solution of potassium carbonate (17 g, 120 mmol) in water (50 mL), and the resulting mixture was heated at reflux for 90 minutes.",
"The cooled reaction mixture was concentrated in vacuo, and the residue was diluted with water (200 mL).",
"The mixture was extracted with CH2Cl2 (3×100 mL).",
"The combined CH2Cl2 layers were dried over Na2SO4, filtered and concentrated to give the product (3.76 g, 83%); 1H NMR 500 MHz (CDCl3) δ=2.69 (2H, t, J=6.0 Hz), 3.11 (2H, t, J=6.0 Hz), 3.45 (2H, br s), 3.92 (2H, s), 6.36 (1H, d, J=2.3 Hz), 6.52 (1H, dd, J=2.3 and 8.0 Hz), 6.89 (1H, d, J=8.0 Hz).",
"Step B To a solution of the product from Step A (3.76 g, 25.4 mmol) in tetrahydrofuran (100 mL) was added triethylamine (4.24 mL, 30.5 mmol), and benzyl chloroformate (4.0 mL, 28 mmol), and the resulting mixture was stirred at room temperature for 4 h. Ethyl acetate (100 mL) was added to the reaction mixture and the whole was washed with water (250 mL), 5% citric acid solution (150 mL), saturated NaHCO3 (150 mL), and saturated NaCl (100 mL), dried over MgSO4, filtered and concentrated in vacuo to give the product (7.2 g, 100%).",
"Step C To a solution of the product from Step B (7.2 g, 25 mmol) and pyridine (5.1 mL, 64 mmol) in CH2Cl2 (150 mL) cooled in an ice bath was added trifluoroacetic anhydride (5.38 mL, 38.1 mmol), and the resulting mixture was stirred at room temperature for 5 h. The mixture was poured onto ice (150 g) and extracted with CH2Cl2 (4×100 mL).",
"The combined CH2Cl2 layers were washed with 1 N HCl (4×75 mL), saturated NaCl (100 mL), dried over MgSO4, filtered and evaporated.",
"The residue was dissolved in CCl4 (200 mL) and triphenylphosphine (10 g, 38 mmol) was added and the resulting mixture was heated at reflux for 15 h, cooled and concentrated in vacuo.",
"The residue was dissolved in anhydrous N,N-dimethyl formamide (150 mL) and this solution was added to a solution of sodium azide (1.65 g, 25.4 mmol) in anhydrous N,N-dimethyl formamide (75 mL), and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was poured into water (500 mL) and extracted with Et2O (3×100 mL).",
"The combined Et2O layers were washed with water (2×250 mL), saturated NaCl (100 mL), dried over MgSO4, filtered and concentrated in vacuo.",
"The residue was purified by column chromatography on silica eluting with a gradient ranging from 10% EtOAc in hexanes to 20% EtOAc in hexanes to give the product (3.4 g, 33%); 1H NMR 500 MHz (CDCl3) δ=2.99 (2H, br m), 3.80 (2H, t, J=6.0 Hz), 4.75 (2H, s), 5.21 (2H, s), 7.20-7.45 (8H, m).",
"Step D To a nitrogen flushed solution of the product from step C (3.4 g, 8.4 mmol) in methanol (100 mL) was added 10% palladium on carbon (500 mg) and the resulting mixture was stirred under a balloon of hydrogen for 5 h. The catalyst was removed by filtration through celite and the filtrate was evaporated in vacuo to give the product (2.2 g, 97%); 1H NMR 500 MHz (CDCl3) δ=2.33 (1H, br s), 2.91 (2H, t, J=6.0 Hz), 3.19 (2H, t, J=6.0 Hz), 4.08 (2H, s), 7.14 (1H, d, 1.8 Hz), 7.22 (1H, dd, J=1.8 and 8.2 Hz), 7.31 (1H, d, J=8.2 Hz).",
"Step E This product was prepared in an analogous fashion to that of Example 81, except Intermediate I was replaced with the product described in Step D. The crude product was purified by preparative TLC (eluant: 10% methanol: 89.5% DCM: 0.5% NH4OH) to yield Example 31 as a mixture of two cis isomers.",
"LC-MS for C31H36F3N6O calculated 584.29, found [M+H]+ 585.EXAMPLE 112 This product was prepared in an analogous fashion to that of Example 81, except Intermediate 1 was replaced with commercially available 6,7-diethoxy-tetrahydroisoquinoline.",
"The crude product was purified by preparative TLC (eluant: 5% ethanol: 94% DCM: 1.0% NH4OH) to yield Example 32 as a mixture of two cis isomer.",
"LC-MS for C33H45FN2O3 calculated 536.34, found [M+H]+ 537.EXAMPLE 113 Step A This product was prepared in a similar manner to Intermediate 1, except 2-Fluoro-4-trifluoromethyl phenylacetonitrile was replaced with 3,4-di-fluoromethyl phenylacetonitrile.",
"LC-MS for C9H9F2N calculated 169.07, found [M+H]+ 170.1 Step B This product was prepared in an analogous fashion to that of Example 81, except Intermediate 1 was replaced with the product described in Step A.",
"The crude product was purified by preparative TLC (eluant: 6% ethanol: 93% DCM: 1.0% NH4OH) to yield Example 33 as a mixture of two cis isomers.",
"LC-MS for C29H35F3N2O calculated 484.27, found [M+H]+ 485.EXAMPLE 114 This product was prepared in an analogous fashion to that of Example 82, except Intermediate 8 was replaced with commercially available 4-hydroxy-4-phenylpiperidine.",
"The crude product was purified by preparative TLC (eluant: 10% methanol: 89% DCM: 1.0% NH4OH) to yield Example 114 as a mixture of four isomers.",
"LC-MS for C30H37F3N2O2 calculated 514.28, found [M+H]+ 515.EXAMPLE 115 and 116 A solution of Intermediate 5 (100 mg, 0.22 mmol), 4-fluorophenylpiperidine hydrochloride (57 mg, 0.26 mmol), diisopropylethylamine (45 μL, 0.26 mmol) and crushed molecular sieves (4 Å, 50 mg) in dichloroethane (5 mL) was treated with sodium triacetoxyborohydride (233 mg, 1.10 mmol) and stirred at room temperature overnight.",
"The reaction was quenched with saturated sodium bicarbonate (10 mL) and diluted with an additional 10 mL of DCE.",
"The organic layer was separated and the aqueous layer was washed with dichloromethane (2×5 mL).",
"The organics were combined, dried over anhydrous sodium sulfate, filtered and evaporated.",
"The residue was purified by preparative TLC (eluent: 0.5% NOH: 5% MeOH: 94.5% CH2Cl2) to yield 72.3 mg (63%) of the final product as a mixture of two diastereomers.",
"The separation was accomplished by using a HPLC equipped with a preparatory ChiralCel OD column eluting with an eluant of 15% ethanol and 85% hexane with a flow rate of 9 mL/min.",
"This afforded the undesired trans isomer (35 mg, 50%) and the desired cis isomer (25 mg, 36%).",
"Total recovery 86%.",
"LC-MS for both: C29H35N3OF4 calculated 517.28, found [M+H]+ 518.3.EXAMPLE 115 1H NMR (1st isomer, undesired) (500 MHz, CDCl3) δ 8.73 (s, 1H), 7.69 (s, 1H), 7.20 (app dd, J=5.5, 8.6 Hz, 2H) 6.98 (app t, J=8.6 Hz, 2H), [4.88 (d, J=17.5 Hz, 1H), 4.80 (d, J=17.5 Hz, 1H) (ABx q)]4.05-3.96 (m, 1H), 3.88 (app dt, J=5.9, 13.1 Hz, 1H), 3.27 (br d, J=11.4 Hz, 1H), 3.12 (br t, J=5.6 Hz, 3H), 2.83 (dd, J=5.7, 11.9 Hz, 1H), 2.52-2.44 (m, 1H), 2.43-2.34 (m, 1H), 2.16-1.95 (m, 6H), 1.86-1.74 (m, 4H), 1.45 (br t, J=10.0, 3H), 1.03 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H).",
"EXAMPLE 116 1H NMR (2nd isomer, desired) (500 MHz, CDCl3) δ 8.72 (s, 1H), 7.69 (s, 1H), 7.19 (app dd, J=5.5, 8.6 Hz, 2H) 6.98 (app t, J=8.6 Hz, 2H), [4.94 (br s, 1H), 4.69 (br d, J=17.6 Hz, 1H) (ABx q)]4.05-3.80 (m, 1H), 3.20-3.08 (m, 4H), 2.68 (dd, J=6.6, 12.8 Hz, 1H), 2.52-2.43 (m, 2H), 2.28 (dd, J=7.3, 12.8 Hz, 1H), 2.14-2.00 (m, 3H), 1.97-1.87 (m, 2H), 1.83 (br d, J=12.8 Hz, 2H) 1.75 (br d, 12.4 Hz, 2H), 1.56-1.48 (m, 1H), 1.42-1.34 (m, 1H), 1.01 (d, J=6.5 Hz, 3H), 0.80 (d, J=6.6 Hz, 3H).",
"EXAMPLE 117 The hydrochloride salt of the pyrimidyl piperidine (Intermediate 8, 133 mg, 0.564 mmol) was combined with Intermediate 5 (100 mg, 0.282 mmol), DIEA (240 μL, 1.40 mmol), and 4 Å powdered molecular sieves (200 mg) in DCM.",
"After 15 minutes at room temperature, sodium triacetoxyborohydride (300 mg, 1.41 mmol) was added and the resulting mixture was stirred for 3 days before being filtered through celite, diluted with DCM and washed with saturated sodium bicarbonate and brine.",
"The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude oil that was purified by preparative TLC (silica gel, 0.5% NH40OH/4.5% MeOH/95% DCM) to give 126 mg of a colorless oil.",
"Resolution of the cis/trans isomers was accomplished by HPLC using a ChiralPak OD column eluting with 20% ethyl alcohol/hexanes to give 57 mg of the trans isomer and 45 mg of the cis isomer.",
"First peak 57 mg: ESI-MS calc.",
"for C27H34F3N5O: 501.27; found 502 (M+H).",
"Second peak 45 mg: ESI-MS calc.",
"for C27H34F3N5O: 501.27; found 502 (M+H).",
"EXAMPLES 118-129 Several other examples were prepared in a similar fashion to Example 117, utilizing different piperidine intermediates.",
"These Examples (118-129) are shown below.",
"Molecular Calculated Found Example R1 R2 Formula [M] [M + H] 118 H C27H34F3N5O 501.27 502 119 H C24H32F3N7O 491.26 492 120 H C26H34F3N5O 489.27 490 121 H C25H33F3N6O 490.27 491 122 H C25H33F3N6O 490.27 491 123 H C26H34F3N5O 489.27 490 124 H C25H34F3N7O 505.28 506 125 H C26H33F3N4OS 506.23 507 126 H C32H43F3N3O3 574.33 575 127 H C30H39F3N3O3 546.29 547 128 H C25H32F3N5O2S 523.22 524 129 H C26H35F3N6O 504.28 505 EXAMPLE 130 Step A This ketone was obtained from the racemic Intermediate 6 by resolution on the ChiralCel OD preparative column, eluting with 15% ethyl alcohol in hexanes at 9.0 mL/min.",
"The faster eluting enantiomers retention time under analogous analytical conditions (1.0 mL/min) was 11.25 minutes.",
"LC MS for C17H19F3N2O3 calculated 356.13, found 357.05 [M+H]+.",
"Step B The final compound was synthesized starting from the faster eluting ketone described in Step A of this example and Intermediate 10 according to the procedure described in Example 19.The respective cis- and trans-diastereoisomeric mixtures were separated by preparative TLC.",
"LC MS for C24H31F3N6O2 calculated 492.25, found 493.30 [M+H]+.",
"EXAMPLE 131 This compound was synthesized starting from the ketone preparation of which was described in Example 130, Step A and Intermediate 9 according to the procedure described in Example 19.LC MS for C26H32F3N5O2 calculated 503.25, found 504.25 [M+H]30 .",
"EXAMPLE 132 Step A This ketone was synthesized following procedures described in Intermediates 6, except that methoxymethyl chloride was used instead of acetaldehyde in Step C, Intermediate 6.The respective enantiomers were obtained by HPLC separation using a ChiralCel OD preparative column (eluent hexane:ethyl alcohol/85: 15, 9.0 mL/min).",
"LC MS for C26H32F3N5O2 calculated 503.25, found 504.25 [M+H]+.",
"Step B The final compound was prepared starting from the ketone from Step A and 4-(4-fluorophenyl)piperidine according to the procedure described in Example 19.The respective isomers were obtained by preparative TLC (eluent ethyl acetate: ethyl alcohol: ammonium hydroxide/90:9: 1).",
"LC MS for C26H32F3N5O2 calculated 503.25, found 504.70 [M+H]+.",
"EXAMPLE 133 Step A To a mixture of 2-(trifluorormethyl)acrylic acid (20.0 g, 143 mmol) and benzyl alcohol (14.8 mL, 142 mmol) in DCM (150 mL), was added EDC (40.93 g, 214.2 mmol) in portions.",
"The reaction mixture was stirred for 2 h, diluted by DCM, washed with water and brine, dried over Na2SO4, filtered and concentrated.",
"The residue was purified by flash column chromatography (silica gel, 5% EtOAc/hexane) to yield the product (13.7 g, 42%) as a viscous oil.",
"1H-NMR (400 MHz, CDCl3) δ 7.36-7.43 (m, 5H), 6.78 (d, 1H), 6.48 (d, 1H), 5.32 (s, 2H).",
"Step B To a flamed dried flask was added 2-[(trimethylsilyl)methyl]-2-propen-1-yl acetate (12.18 mL, 57.35 mmol) and the intermediate described in Step A, Example 52 (13.2 g, 57.4 mmol), and tetrakis(triphenylphosphine)palladium(0) (13.3 g, 11.5 mmol) in THF (200 mL) under nitrogen.",
"The reaction mixture was refluxed overnight, diluted by DCM (150 mL), filtered, and concentrated.",
"The residue was purified by flash column chromatography (silica gel, 100% hexane to 2.5% EtOAc/hexane to 5% EtOAc/hexane) to afford the product (11.68 g, 71.7%).",
"1H-NMR (400 MHz, CDCl3) δ 7.33-7.42 (m, 5H), 5.23 (s, 2H), 4.93 (m, 2H), 3.03 (m, 1H), 2.78 (m, 1H), 2.36-2.52 (m, 3H), 2.12-2.24 (m, 1H).",
"Step C A solution of the product described in Step B (11.6 g, 40.8 mmol) in DCM (150 mL) was cooled to −78° C. and saturated with nitrogen.",
"Ozone was bubbled into the reaction mixture until the solution became blue, then triphenylphosphine (12.8 g, 49.0 mmol) was added to the mixture.",
"The reaction mixture was stirred overnight, and then evaporated under reduced pressure.",
"The residue was purified by flash column chromatography (silica gel, 30% EtOAc/hexane) to yield the title compound (8.49 g, 72.7%).",
"1H-NMR (400 MHz, CDCl3) δ 7.33-7.42 (m, 5H), 5.26 (s, 2H), 2.92 (m, 1H), 2.65 (m, 1H), 2.35-2.54 (m, 4H).",
"Step D To a solution of the intermediate described in Step C (1.00 g, 3.49 mmol) in ethanol (60 mL), was added Pd—C (10%, 100 mg).",
"The reaction mixture was placed in a Parr-shaker and shaken under 501b pressure of H2 for 1.5 h. The solution was diluted by methanol, filtered through celite and evaporated under vacuum to afford the acid (692 mg, 100%) as a yellow oil.",
"LC-MS calc.",
"for C7H7F3O3: 196.03; Found: 197 (M+H).",
"Step E To a mixture of the product described in Step D (684 mg, 3.49 mmol), EDC (2.01 g, 10.5 mmol), and Intermediate 2 (916 mg, 3.84 mmol) in DCM (30 mL) was added DIEA (670 μL, 3.84 mmol) and the resulting solution was stirred overnight at room temperature.",
"The reaction mixture was diluted with DCM, washed by water and brine, dried over Na2SO4, filtered and evaporated in vacuo.",
"The residue was purified by column chromatography (silica gel, 50% EtOAc/hexane) to afford the title compound.",
"1H-NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 7.71 (s, 1H), 4.86 (d, J=5.5 Hz, 2H), 3.91-4.08 (m, 2H), 3.18 (t, J=5.0 Hz, 2H), 2.98 (s, 2H), 2.64-2.85 (m, 2H), 2.43-2.56 (m, 2H).",
"LC-MS calc.",
"for C16H14F6N2O2: 380.10; Found: 381 (M+H).",
"Step F To a mixture of the compound described in Step E (100 mg, 0.263 mmol), 4-phenylpiperidine HCl salt (52 mg, 0.26 mmol), molecular sieve (4 Å, 180 mg), DIEA (46 μL, 0.26 mmol) in DCM (5 mL), was added sodium triacetoxyborohydride (167 mg, 0.789 mmol) and the resulting mixture was stirred overnight at room temperature.",
"The reaction was diluted with DCM, filtered through celite, and evaporated under reduced pressure.",
"The residue was purified by preparative TLC (1000 micron, eluant: 0.4% aqueous NH4OH: 4% MeOH: 95.6% DCM) to yield a mixture of cis- and trans-isomers as a free base.",
"The cis- and trans-isomers were separated by preparative chiral HPLC (chiral OD column, eluant: 5% EtOH/hexane) to yield the final product of the title compound as the desired cis isomers.",
"Its HCl salt (20.4 mg) was formed by treatment with 4 N HCl/dioxane.",
"1H-NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 7.70 (s, 1H), 7.20-7.35 (m, 5H), 4.85 (m, 2H), 3.99 (m, 2H), 3.14-3.24 (m, 5H), 2.46-2.56 (m, 3H), 2.02-2.22 (m, 5H), 1.70-1.91 (m, 5H).",
"LC-MS calc.",
"for C27H29F6N3O: 525.22; Found: 526 (M+H).",
"EXAMPLE 134 Step A To a stirred, −78° C. solution of cyclohexene (15 mL) in 100 mL of dichloromethane was bubbled in ozone until light blue color appeared.",
"Excessive ozone was removed by a nitrogen flow, then 60 mL of dimethyl sulfide was added.",
"The mixture was left overnight, dried over sodium sulfate.",
"The solvent and DMS were removed under low vacuum.",
"The crude product was used in next step without further purification.",
"Step B A mixture of Intermediate 27 (135 mg, 0.3 mmol), the 1,6-hexane-dialdehyde from Step A (˜100 mg), molecular sieve (4 Å, 50 mg), DIEA (130 mg, 1.0 mmol) in DCM (10 mL crude material) was stirred for 5 min.",
"Then sodium triacetoxyborihydride (424 mg, 2.0 mmol) was added.",
"The resulting mixture was stirred for 2 h, quenched with sat.",
"aq.",
"Na2CO3, filtered, washed with DCM.",
"The filtrates were separated, the aq.",
"solution was extracted with DCM.",
"The combined DCM layers were dried over Na2SO4, evaporated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 10% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final product of the title compound as a free base.",
"Its HCl salt (42 mg) was formed by treatment with 4 M HCl/dioxane.",
"ESI-MS calc.",
"For C25H34F3N3O3: 481; Found: 482 (M+H).",
"EXAMPLE 135 A mixture of the Example 134 (35 mg), Pd/C (5%, 5 mg) and methanol (20 mL) was hydrogenated on a Parr Apparatus for one hour.",
"The catalyst was removed by filtration.",
"The filtrate was evaporated to afford the title product as a white solid (32 mg).",
"ESI-MS calc.",
"For C25H36F3N3O: 451; Found: 452 (M+H).",
"EXAMPLE 136 Step A To a stirred, −78° C. solution of cycloheptene (5 mL) in 50 mL of dichloromethane was bubbled in ozone until light blue color appeared.",
"Excessive ozone was removed by a nitrogen flow, then 20 mL of dimethyl sulfide was added.",
"The mixture was left overnight, dried over sodium sulfate.",
"The solvent and DMS were removed under low vacuum.",
"The crude product was used in next step without further purification.",
"Step B A mixture of the Intermediate 27 (135 mg, 0.3 mmol), 1,7-heptane-dialdehyde from Step A) (˜100 mg), molecular sieve (4 Å, 50 mg), DIEA (130 mg, 1.0 mmol) in DCM (10 mL crude material) was stirred for 5 min.",
"Then sodium triacetoxyborohydride (424 mg, 2.0 mmol) was added.",
"The resulting mixture was stirred for 2 h, quenched with sat.",
"aq.",
"Na2CO3, filtered, washed with DCM.",
"The filtrates were separated, the aq.",
"solution was extracted with DCM.",
"The combined DCM layers were dried over Na2SO4, evaporated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 10% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final product of the title compound as a free base.",
"Its HCl salt (50 mg) was formed by treatment with 4 M HCl/dioxane.",
"ESI-MS calc.",
"For C26H36F3N3O3: 485; Found: 486 (M+H).",
"EXAMPLE 137 Step A 5-Norbornene-2-carboxylic acid (5.4 g, 40 mmol), benzyl alcohol (4.3 g, 40 mmol), EDAC.HCl (9.5 g, 50 mmol), DIEA (5.2 g, 40 mmol) were weighed into a flask.",
"50 mL of dichloromethane was added.",
"The mixture was stirred overnight, washed with 2 M aq.",
"HCl, water and sat.",
"aq.",
"sodium carbonate, dried over sodium sulfate, evaporated.",
"The residue was purified on MPLC (10% EtOAc/Hexane).",
"The title compound was obtained as a mixture of exo and endo isomers (5.2 g).",
"Step B To a stirred, −78° C. solution of benzyl 5-norbornene-2-carboxylate (1.2 g, 5 mmol) in 50 mL of dichloromethane was bubbled in ozone until light blue color appeared.",
"Excessive ozone was removed by a nitrogen flow, then 20 mL of dimethyl sulfide was added.",
"The mixture was left overnight, dried over sodium sulfate.",
"The solvent and DMS were removed under low vacuum.",
"The crude product was used in next step without further purification.",
"Step C A mixture of the Intermediate 27 (86 mg, 0.2 mmol), the di-aldehyde ester from Step B (˜200 mg), molecular sieve (4 Å, 500 mg), DIEA (130 mg, 1.0 mmol) in DCM (10 mL crude material) was stirred for 5 min.",
"Then sodium triacetoxyborohydride (420 mg, 2.0 mmol) was added.",
"The resulting mixture was stirred for 2 h, quenched with sat.",
"aq.",
"Na2CO3, filtered, washed with DCM.",
"The filtrates were separated, the aq.",
"solution was extracted with DCM.",
"The combined DCM layers were dried over Na2SO4, evaporated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 10% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final product of the title compound as a free base.",
"Its HCl salt (62 mg) was formed by treatment with 4N HCl/dioxane.",
"ESI-MS calc.",
"For C34H40F3N3O5: 627; Found: 428 (M+H).",
"Step D A mixture of the amino ester from Step C (60 mg), Pd/C (5%, 100 mg) and methanol (20 mL) was hydrogenated on a Parr Aparatus under 50 lbs of hydrogen for one hour.",
"The catalyst was removed by filtration.",
"The filtrate was evaporated, the residue was purified on preparative TLC (developed by methanol) to afford the title product as a white solid (18 mg).",
"ESI-MS calc.",
"For C27H36F3N3O: 507; Found: 508 (M+H).",
"EXAMPLE 138 Step A A mixture of Intermediate 26 (as HCl salt, 1.2 g, 4.0 mmol), Intermediate 23 (1.1 g, 4.0 mmol), PyBrOP (1.9 g, 4.0 mmol), DMAP (0.29 g, 2.4 mmol) and DIEA (2.8 mL, 16 mmol) in 10 mL of dichloromethane was stirred at room temperature overnight.",
"The entire mixture was applied to a silica gel column and eluted with 20% ethyl acetate/hexane.",
"The title compound was obtained as a white solid (1.7 g, 83%).",
"LC-MS for C26H35F3N2O5 calculated 512, found [M+H−100]30 413.Step B The above amide from Step A (1.7 g, 3.3 mmol) was mixed with 20 mL of 4 M HCl/dioxane.",
"The resulting solution was stirred for one hour, evaporated and dried under high vacuum to yield the title product as a white solid (1.45 g, 100%).",
"LC-MS for C21H27F3N2O3 calculated 412, found [M+H]+ 413.Step C A mixture of the above intermediate from Step B (140 mg, 0.30 mmol), glutaric dialdehyde (50% H2O, 120 mg, 0.60 mmol), molecular sieve (4 Å, 1500 mg), DIEA (52 mg, 0.40 mmol) in DCM (10 mL) was stirred for 5 min.",
"Then sodium triacetoxyborohydride (212 mg, 1.00 mmol) was added.",
"The resulting mixture was stirred for one hour, quenched with sat.",
"aq.",
"Na2CO3, filtered, washed with DCM.",
"The filtrates were separated, the aq.",
"solution was extracted with DCM.",
"The combined DCM layers were dried over Na2SO4 and evaporated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 10% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final product of the title compound as a free base.",
"Its HCl salt (80 mg) was formed by treatment with 4N HCl/dioxane.",
"ESI-MS calc.",
"For C26H35F3N2O3: 480; Found: 481 (M+H).",
"EXAMPLE 139 A mixture of Example 138 (45 mg, 0.090 mmol), lithium hydroxide monohydrate (50 mg), water (0.1 mL) and methanol (1.0 mL) was stirred at room temperature overnight, the entire mixture was loaded on preparative TLC and developed with methanol.",
"The title compound as obtained as white solid (34 mg).",
"LC-MS for C25H33F3N2O3 calculated 466, found [M+H]+467.EXAMPLE 140 A mixture of the Intermediate from Example 138, Step B (140 mg, 0.30 mmol), 1,6-hexane dialdehyde from Example 136, Step A (˜100 mg), molecular sieve (4 Å, 500 mg), DIEA (130 mg, 1.00 mmol) in DCM (20 mL) was stirred for 5 min.",
"Then sodium triacetoxyborohydride (424 mg, 2.00 mmol) was added.",
"The resulting mixture was stirred for one hour, quenched with sat.",
"aq.",
"Na2CO3, filtered, washed with DCM.",
"The filtrates were separated, the aq.",
"solution was extracted with DCM.",
"The combined DCM layers were dried over Na2SO4, evaporated.",
"The residue was purified on preparative TLC (1000 micron) (developed by 10% [aq.",
"NH4OH/MeOH 1/9]/DCM) to yield the final product of the title compound as a free base.",
"Its HCl salt (75 mg) was formed by treatment with 4 M HCl/dioxane.",
"ESI-MS calc.",
"For C27H37F3N2O3: 494; Found: 495 (M+H).",
"EXAMPLE 141 A mixture of the Example 140 (20 mg, 0.04 mmol), lithium hydroxide monohydrate (50 mg), water (0.1 mL) and methanol (1.0 mL) was stirred at room temperature overnight, the entire mixture was loaded on preparative TLC and developed with methanol.",
"The title compound as obtained as white solid (15 mg).",
"LC-MS for C26H35F3N2O3 calculated 480, found [M+H]+ 481.EXAMPLE 142 Step A To a stirred solution of Intermediate 29 (190 mg, 1.1 mmol) in 1 mL of dichloromethane was added a solution of oxalyl chloride (2 M, 0.70 mL, 1.4 mmol) in dichloromethane, then a trace of DMF.",
"The mixture was stirred at room temperature for 30 min.",
"before being evaporated to remove the solvent and excessive reagent under vacuum.",
"The residue was dissolved in 1 mL of dichloromethane and added into a solution of the Intermediate 2 (295 mg, 1.00 mmol) and DIEA (260 mg, 2.0 mmol) in dichloromethane (2 mL).",
"The reaction was stirred for 2 h. The entire mixture was loaded onto preparative TLC plate (1000 micron) and developed with 10% MeOH/DCM.",
"The title compound was obtained as yellow solid (300 mg).",
"LC-MS for C21H24F3NO4 calculated 411, found [M+H]+ 412.Step B A mixture of the above ketone from Step A (300 mg, 0.73 mmol), 4-fluorophenylpiperidine hydrochloride (214 mg, 1.00 mmol), DIEA (129 mg, 1.00 mmol), molecular sieves (4 Å, 500 mg) and sodium triacetoxyborohydride (212 mg, 1.00 mmol) in 10 mL of dichloromethane was stirred at room temperature over the weekend, quenched with sat.",
"aq.",
"sodium carbonate, extracted with dichloromethane and purified on preparative TLC (1000 micron), eluting with 10% MeOH/DCM.",
"The title compound was obtained as a mixture of cis and trans isomers (270 mg).",
"LC-MS for C32H38F4N2O3 calculated 574, found [M+H]+ 575.EXAMPLE 143 A mixture of the Example 142 (22 mg, 0.04 mmol), lithium hydroxide monohydrate (30 mg) in MeOH/H2O (9/1, 0.5 mL) was stirred at 60° C. for 2 h., the entire mixture was loaded on a preparative TLC plate and developed with methanol.",
"The title compound as obtained as white solid (12 mg).",
"LC-MS for C31H34F4N2O3 calculated 560, found [M+H]+ 561.EXAMPLE 144 Step A Intermediate 29 (100 mg, 0.588 mmol) was dissolved in DCM (20 mL) and treated sequentially with oxalyl chloride (153 μL, 1.76 mmol) and DMF (1 drop).",
"The resulting solution was stirred at room temperature for 2 h, before being concentrated to dryness and dried under high vacuum for 30 min.",
"The resulting residue was dissolved in DCM (5 mL) and added dropwise to a stirred solution of Intermediate 1 (177 mg, 0.882 mmol) in DCM (5 mL) and triethylamine (5 mL).",
"The resulting reaction mixture was stirred at room temperature overnight, before being diluted with DCM and washed with bicarb, 1 N aqueous HCl, and brine.",
"The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to give 230 mg of the desired crude product, which was used in the next step without further purification.",
"Step B A mixture of the product from the previous step (115 mg, 0.326 mmol), ethyl 3-piperidin-4-ylbenzoate hydrochloride (131 mg, 0.489 mmol), DIEA (83 μL, 0.49 mmol), molecular sieves (4 Å, 100 mg) and sodium triacetoxyborohydride (346 mg, 1.63 mmol) in 10 mL of dichloromethane was stirred at RT over weekend, quenched with sat.",
"aq.",
"sodium bicarbonate, extracted with dichloromethane and purified on preparative TLC (silica gel, eluting with 40% THF/hexanes).",
"The title compound was obtained as a mixture of cis and trans isomers (200 mg).",
"LC-MS calc.",
"for C33H41F3N2O3: 570.3; found [M+H]+ 571.6.The individual stereosiomers were obtained by resolution on a ChiralCel OD column eluting with 10% ethanol/hexanes: Peak 1: LC-MS calc.",
"for C33H41F3N2O3: 570.3; found [M+H]+ 571.6.Peak 2: LC-MS calc.",
"for C33H41F3N2O3: 570.3; found [M+H]+ 571.6.EXAMPLE 145 A mixture of the Example 144 Peak 2 (80 mg,), lithium hydroxide monohydrate (30 mg) in EtOH/H2O (4/1, 4 mL) was stirred at room temperature overnight.",
"The product was purified by reverse phase HPLC and converted to an HCl salt in the usual fashion.",
"The title compound as obtained as white solid (45 mg).",
"LC-MS calculated for C31H37F3N2O3: 542.28; found [M+H]+ 543.EXAMPLE 146 A mixture of the Example 144 Peak 1 (78 mg,), lithium hydroxide monohydrate (30 mg) in EtOH/H2O (4/1, 4 mL) was stirred at room temperature overnight.",
"The product was purified by reverse phase HPLC and converted to an HCl salt in the usual fashion.",
"The title compound as obtained as white solid (49 mg).",
"LC-MS calculated for C31H37F3N2O3: 542.28; found [M+H]+ 543.TABLE 8 Analogs Prepared in an Analogous Fashion to EXAMPLE 144 ESI-MS observed M + H+ EX.",
"Amine X Formula/calc.",
"MW (M + 1) 147 C C34H43F3N2O3584 585 148 C C34H43F3N2O3584 585 149 C C34H43F3N2O3584 585 150 C C34H43F3N2O3584 585 151 N C33H42F3N3O3585 586 152 N C33H42F3N3O3585 586 153 N C33H42F3N3O3585 586 154 N C33H42F3N3O3585 586 155 N C31H38F3N3O3557 558 156 N C32H40F3N3O3571 572 157 N C32H38F3N3O4585 586 158 N C32H38F3N3O3569 570 159 N C32H42F3N3O2557 558 160 N C32H40F3N3O539 540 161 N C31H37F4N3O3575 576 162 N C31H37F4N3O3575 576 163 N C28H34F3N3O485 486 164 N C31H37F4N3O3575 576 165 N C29H36F3N3O499 500 166 N C32H40F3N3O3571 572 167 C C32H38F4N2O3574 575 168 C C32H38F4N2O3574 575 169 N C26H33F3N4O3506 507 170 N C23H31F4N3O441 442 171 C C24H32F4N2O440 441 172 C C23H30F4N2O426 427 173 C C23H30F4N2O426 427 174 C C24H33F3N2O422 423 175 C C26H33F6N3O2533 534 176 C C23H29F5N2O444 445 177 C C24H30F6N2O476 477 178 C C23H31F3N2O408 409 179 C C25H32F6N2O490 491 180 C C24H31F5N2O458 459 181 C C24H31F5N2O458 459 182 C C25H32F6N2O490 491 In many cases the analogs listed in TABLE 8 could be further modified to generate new target chemokine receptor modulators.",
"For example, the ester groups of the analogs in this table were hydrolyzed to give the corresponding carboxylic acids which were themselves potent modulators.",
"These hydrolyses were usually accomplished under the conditions shown in EXAMPLE 145 or with minor modifications to those conditions.",
"A representative list of the resulting carboxylic acid containing chemokine receptor modulators is presented in TABLE 9.TABLE 9 Carboxylic Acid Containing Analogs From Esters in Table 8 ESI-MS observed M + H+ EX.",
"Amine X Formula/calc.",
"MW (M + 1) 183 C C32H39F3N2O3556 557 184 C C32H39F3N2O3556 557 185 C C32H39F3N2O3556 557 186 C C32H39F3N2O3556 557 187 N C31H38F3N3O3556 558 188 N C31H38F3N3O3557 558 189 N C31H38F3N3O3557 558 190 N C31H38F3N3O3557 558 191 N C30H36F3N3O3543 544 192 N C31H38F3N3O3557 558 193 N C30H35F4N3O3561 562 194 N C30H35F4N3O3561 562 195 N C30H35F4N3O3561 562 196 N C31H38F3N3O3557 558 197 N C31H36F4N2O3560 561 198 N C31H36F4N2O3560 561 EXAMPLE 199 Example 199 was prepared using intermediate 24 and 4-hydroxy-4-(6-methoxypyridin-3-yl)cyclohexanone (Xue, C.; et. al.",
"WO2004/050024) in an analogous fashion to example 82.LC-MS calculated for C30H39F3N4O3: 560.67; found [M+H]+ 562.EXAMPLE 200 Example 200 was prepared using intermediate 25 and 4-hydroxy-4-(6-methoxypyridin-3-yl)cyclohexanone in an analogous fashion to example 82.LC-MS calculated for C31H40F3N3O3: 559.68; found [M+H]+ 561.EXAMPLE 201 Step A This product was prepared in an analogous fashion to that of Example 65, except the intermediate from Step A was replaced with commercially available 3-nitrophenyl acetic acid ethyl ester.",
"The crude product was purified via silica gel chromatography, eluting with a 1% to 10% gradient of MeOH in DCM.",
"LC/MS (ESI) 561.4 (M+H+).",
"1.15 g (2.05 mmol) of the product from step A was stirred in MeOH at room temperature with N,N-dimethyl hydrazine (20.5 mmol, 1.23 g) and 60 mg activated charcoal.",
"To this was added ˜5-10% FeCl3 (˜20-50 mg, spatula tip addition), and reaction was warmed to reflux.",
"After 2 hrs, additional portions of N,N-dimethyl hydrazine (20.5 mmol, 1.23 g) and FeCl3 (−20-50 mg, spatula tip addition) were added.",
"TLC analysis 2 hrs later showed complete reaction.",
"Crude mixture was filtered through celite and concentrated in vacuo with no further purification necessary.",
"LC/MS (ESI) 531.5 (M+H+).",
"EXAMPLE 202 The aniline product from example 201 (265 mg, 0.5 mmol) was stirred with DIEA (260 μL, 1.5 mmol) in DCM (5 mL).",
"To this was added acetic anhydride (72 μL, 0.75 mmol) neat, via syringe.",
"TLC analysis after 20 min showed complete conversion to product.",
"The crude reaction is diluted with 50 mL DCM and washed 2× with H2O (25 mL each).",
"The DCM layer was dried over MgSO4, filtered, and concentrated in vacuo.",
"The residue was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 573.5 (M+H+).",
"EXAMPLE 203 The aniline product from example 201 (265 mg, 0.5 mmol) was stirred at room temperature in 8 mL of 1:1 AcOH:H2O.",
"To this was added 81 mg (1.0 mmol) of potassium cyanate.",
"After 30 minutes, TLC showed complete reaction.",
"The resulting mixture was concentrated to dryness in vacuo, and the crude residue was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 574.4 (M+H+).",
"EXAMPLE 204 The aniline product from example 201 (265 mg, 0.5 mmol) was stirred at room temperature in dioxane.",
"To this was added 120 μL (1.5 mmol) of ethyl isocyanate.",
"After 18 hrs, TLC analysis showed complete reaction.",
"The resulting mixture was concentrated to dryness in vacuo, and the crude residue was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 602.7 (M+H+).",
"EXAMPLE 205 This product was prepared in an analogous fashion to that of example 65, except the intermediate from Step A was replaced with commercially available 3-methoxyphenyl acetic acid ethyl ester.",
"The crude product was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 546.3 (M+H+).",
"EXAMPLE 206 The TFA salt (583 mg, 0.88 mmol) of the methoxyphenyl product from example 205 was stirred in dry DCM (20 mL) at room temperature.",
"To this was added boron tribromide (360 μL, 3.5 mmol) neat, via syringe.",
"After 20 min, analysis of the reaction via LC/MS showed complete conversion to product.",
"The reaction was quenched with MeOH (5 mL), then diluted with 100 mL DCM.",
"The crude mixture was washed 2× with H2O (50 mL each), then dried over MgSO4, filtered, and concentrated in vacuo.",
"The residue was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 532.6 (M+H+).",
"EXAMPLE 207 This product was prepared in an analogous fashion to that of example 65 using methyl piperidine-4-carboxylate.",
"The crude product was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 580.5 (M+H+).",
"Selected analogs above (Examples 201, 205, 206, 207) could be further modified to generate new target chemokine receptor modulators.",
"For example, the ester groups of these analogs were hydrolyzed to give the corresponding carboxylic acids which were themselves potent modulators.",
"These hydrolyses were usually accomplished under the conditions shown in example 145.A representative list of the resulting carboxylic acid containing chemokine receptor modulators is presented in Table 10.TABLE 10 Analogs Prepared in an Analogous Fashion to EXAMPLE 145 ESI-MS observed EX.",
"Aryl Formula/Calc.",
"MW M + H+ (M + 1) 208 C27H31F3N4O3516.6 517.5 209 C28H32F3N3O4531.6 532.4 210 C27H30F3N3O4517.6 518.5 211 C26H30F3N5O4S565.6 566.6 EXAMPLE 212 This product was prepared in an analogous fashion to that of example 65 using 4-hydroxypiperidine.",
"The crude product was purified by reverse phase preparative HPLC, to afford the product as a colorless solid.",
"LC/MS (ESI) 538.4 (M+H+).",
"EXAMPLE 213 Step A Compound was synthesized using commercially available piperidin-4-yl-acetic acid methyl ester in an analogous fashion to example 19.To a solution of methyl [1-((3S)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopentyl)piperidin-4-yl]acetate (470 mgs, 0.92 mmol) in methanol (5 ml) was added 2N Lithium Hydroxide solution (1.8 ml, 3.7 mmol), the reaction was then stirred overnight at room temperature.",
"The reaction was neutralized to pH=5 using 3N HCl.",
"The crude was purified on reverse phase HPLC using 5%-100% ACN/H2O to yield [1-((3S)-3-{[3-(1,1-difluoroethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}-3-isopropylcyclopentyl)piperidin-4-yl]acetic acid (320 mgs, 91%), LC-MS 482 (M++1).",
"EXAMPLE 214 Compound was synthesized using commercially available 4-piperidine butyric acid hydrochloride in an analogous fashion to example 19.LCMS 511 (M++1).",
"EXAMPLE 215 Step A To a solution of N-Boc-4-piperidinylpropionic acid methyl ester (300 mg, 1.1 mmol) in dichloromethane (2 ml) was added a solution of 1N HCl in ethyl ether (5 ml, 5 mmol), and the resulting mixture was stirred for 2 hours.",
"Solvent was removed under reduced pressure to afford crude desired methyl 3-piperidin-4-ylpropanoate (242 mg, 100%) Step B This compound was synthesized using the reductive amination procedure already described in previous examples by reacting the product from step A with (3S)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopentanone to yield desired compound.",
"Step C The desired compound was prepared in the same procedure from the product in step B as in example 204.",
"(310 mgs, 60% overall).",
"LC-MS 497 (M++1).",
"EXAMPLE 216 Compound was synthesized using the in an analogous fashion to example 215 using methyl 3-methylpiperidine-4-carboxylate LC-MS 482 (M++1).",
"EXAMPLES 217-219 Examples 217-219 were prepared using the recemic form of example 32 and commercially available amino ester intermediates by the following procedure: To a solution of example 32 (250 mgs, 0.54 mmol) in dichloromethane (2 ml), was added corresponding amino ester (1.2 mmol), BOP (240 mgs, 1.0 mmol), triethyl amine (90 mgs, 0.89 mmol), the reaction was stirred overnight.",
"The reaction was added sat.",
"NaHCO3 as well as ethyl acetate (50 ml).",
"The organic layer was washed with brine (10 ml), dried, concentrated, the residue was purified on HPLC to afford the desired compound.",
"(Table 11) TABLE 11 Examples 218-220 EXAMPLE R M.F.",
"M.W.",
"Found (M + H) 217 C27H37F3N4O4 538.62 539 218 C31H37F3N4O4 586.66 587 219 C31H37F3N4O4 586.66 587 EXAMPLE 220 Step A At −78° C., the solution of 1-Benzyl-3-methyl-4-piperidone (4.5 g, 22.6 mmol), was added dropwise to LDA (2N solution in THF/n-Heptane, 12.5 ml, 24.8 mmol)), the reaction was stirred for a half hour, then a solution of N-phenylbis(trifluoromethanesulfonimide) (8.9 g, 24.8 mmol) in THF(50 ml) added to it.",
"The reaction was then warmed to room temperature for two hours.",
"THF was removed under reduced pressure.",
"The residue was purified on silica gel using 5% ethyl acetate/hexane as eluant to afford the desired compound as yellow oil (6.5 g, 87%), LC-MS 336 (M++1).",
"Step B At 0° C., to a solution of above compound (2.5 g, 7.5 mmol) in THF (30 ml), was added tetrakis(triphenyl phosphine) palladium (560 mg, 0.38 mmol), 4-ethoxy-4-oxobutylzinc bromide (0.5N in THF, 23 ml, 11.2 mmol).",
"The reaction was stirred for 2 hours.",
"The reaction was quenched using Sat.NaHCO3 (20 ml) and extracted with ethyl acetate (100 ml).",
"The organic layer was washed with Brine (30 ml), concentrated and the residue was purified on Biotage using 10% ethyl acetate/hexane as eluant to afford the desired product (0.75 g, 33%).",
"LC-MC 302 (M++1).",
"Step C To the solution of above product (0.75 mg, 2.5 mmol) in methanol (10 ml) was added palladium hydroxide (300 mg, 1.9 mmol).",
"The reaction was stirred in H2 1 atm overnight.",
"The catalyst was filtered and the filtrate was concentrated to afford the desired product as yellow oil (490 mg, 100%).",
"Step D The final product was obtained by reacting the product from step C in an analogous fashion as in example 82 then hydrolyzing using 2N lithium hydroxide as in example 145 to afford the final product as off-white solid (0.75 g, 52%).",
"LC-MS 552(M++1).",
"EXAMPLE 221 Compound was synthesized in an analogous manner to example 201 using 3-fluorophenyl acetic acid ethyl ester.",
"ESI MS (M+H+) calc 534.6 found 534.3 EXAMPLE 222 Compound was synthesized in an analogous manner to example 208 using example 221.ESI MS (M+H+) calc 520.5 found 520.4 EXAMPLE 223 Step A Methyl (1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (0.44 g, 2 mmol) and LHMDS (1.8 mL of 20% solution in THF, 2.2 mmol) were stirred at −78° C. for 30 minutes.",
"To this solution was added 4 bromo-t-butyl crotonate (0.48 g, 2.2 mmol).",
"The solution was allowed to warm to room temperature over 1 hour, quenched with water and extracted with ethyl acetate.",
"The solvent was dried, filtered and evaporated to yield a brown oil.",
"ESI MS (M+H+) calc 360.5 found 360.2 Step B Methyl (1R,4S)-1-[2-(tert-butoxycarbonyl)cyclopropyl]-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylate (0.63 g, 1.8 mmol) and LiOH (0.09 g, 3.9 mmol) were stirred in methanol/water at 60° C. for 30 minutes.",
"The solution was neutralized and extracted with ethyl acetate and water.",
"The organic layer was dried, filtered, and evaporated to yield a brown solid.",
"Step C To (1R,4S)-1-[2-(tert-butoxycarbonyl)cyclopropyl]-4-(2,5-dimethyl-1H-pyrrol-1-yl)cyclopent-2-ene-1-carboxylic acid (0.46 g, 1.8 mmol) in DMF, was added HATU (0.73 g, 1.9 mmol), and N-methyl morpholine (0.55 g. 5.4 mmol) and was allowed to stir for 30 minutes.",
"3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridine (0.46 g, 1.93 mmol) was added and stirred at room temperature for 12 hours.",
"The solution was extracted with ethyl acetate and water, dried, filtered and evaporated.",
"The crude material was chromatographed on silica with ethyl acetate/hexanes (10-50%) to yield and off white solid.",
"ESI MS (M+H+) calc 530.6 found 530.4 Step D tert-Butyl 2-((1R,4S)-4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopent-2-en-1-yl)cyclopropanecarboxylate (0.9 g, 1.7 mmol), hydroxylamine (50% in water, 0.7 g, 10.5 mmol), hydroxylamine hydrochloride (0.7 g, 10.5 mmol) and methanol (10 mL) were stirred at 70° C. for 6 hours.",
"The solution was cooled to ambient temperature, extracted with ethyl acetate and water.",
"The organic layer was dried, filtered and evaporated to yield an off white solid.",
"ESI MS (M+H+) calc 452.5 found 452.3 Step E tert-Butyl 2-((1R,4S)-4-amino-1-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopent-2-en-1-yl)cyclopropanecarboxylate (0.15 g, 0.33 mmol), THF (2 mL), H2SO4 (0.3 mL), glutardialdehyde (50% in water, 0.64 g, 3.2 mmol), and sodium triacetoxy borohydride (0.7 g, 3.3 mmol) were stirred at room temperature for 1 hour.",
"The solution was basified with sodium hydroxide, extracted with ethyl acetate, the organic layer dried, and evaporated.",
"The crude material was purified on RPHPLC to yield a white solid.",
"ESI MS (M+H+) calc 520.6 found 520.7 Step F 6-({(1R,4S)-1-[2-(tert-butoxycarbonyl)cyclopropyl]-4-piperidinium-1-ylcyclopent-2-en-1-yl}carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-1-ium bis(trifluoroacetate) (0.1 g), Pd/C (0.05 g) in methanol (10 mL) was added hydrogen.",
"The solution was stirred at room temperature for 5 hours.",
"The solution was filtered and evaporated to yield and off white solid.",
"ESI MS (M+H+) calc 522.6 found 522.5 Step G 6-({(1S,3R)-1-[2-(tert-butoxycarbonyl)cyclopropyl]-3-piperidinium-1-ylcyclopentyl}carbonyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-1-ium bis(trifluoroacetate) (0.1 g) in methylene chloride (5 mL) was added TFA (2 mL).",
"The solution was stirred at room temperature for 2 hours, then the solvent was evaporated to yield an off white solid.",
"ESI MS (M+H+) calc 466.5 found 466.4 EXAMPLE 224 Step A Intermediate tert-Butyl 2-((1R,4S)-4-amino-1-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopent-2-en-1-yl)cyclopropanecarboxylate (0.37 mmol), triacetoxy borohydride (0.087 g, 0.41 mmol), and triethylamine (0.05 mL) in DCE (5 mL) were stirred at room temperature for 18 hours, filtered, evaporated and purified by RPHPLC to yield a white solid.",
"ESI MS (M+H+) calc 566.7 found 566.5 Step B 1,5-anhydro-3-[((1S,4R)-4-[2-(tert-butoxycarbonyl)cyclopropyl]-4-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-1-ium-6(5H)-yl]carbonyl}cyclopent-2-en-1-yl)ammonio]-2,3-dideoxy-4-O-methyl-D-glycero-pentitol bis(trifluoroacetate) (0.15 g), Pd/C (0.05 g) were stirred under hydrogen for 2 hours, filtered and evaporated.",
"The residue was dissolved in methylene chloride (4 mL) and TFA (2 mL) was added and the solution stirred for 2 hours.",
"The solvent was evaporated and the product purified by RPHPLC to yield an off white solid.",
"ESI MS (M+H+) calc 512.6 found 512.4 EXAMPLE 225 Step A (3S)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopentanone (0.12 g, 0.34 mmol), methyl 2-piperidin-4-ylcyclopropanecarboxylate (0.1 g, 0.34 mmol), sodium triacetoxy borohidride (0.14 g, 0.67 mmol), triethylamine (0.05 mL), in DCE were stirred at room temperature for 18 hours.",
"The solution was filtered, evaporated and purified on RPHPLC to an off white solid.",
"ESI MS (M+H+) calc 522.6 found 522.5 Step B methyl 2-[1-((3S)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naphthyridin-6(5H)-yl]carbonyl}cyclopentyl)piperidin-4-yl]cyclopropanecarboxylate (0.08 g), LiOH (0.025 g) were stirred in water/methanol for 3 hours.",
"The solution was neutralized, extracted with ethyl acetate, dried, filtered and evaporated to yield an off white solid.",
"ESI MS (M+H+) calc 508.6 found 508.5 EXAMPLE 226 Step A To a solution of 3-{[5-amino-7-(trifluoromethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}-3-isopropylcyclopentanone (500 mg, 1.35 mmol) in dry methylene chloride (10 mL) was added diisopropylethylamine (0.8 mL, 4.0 mmol).",
"The reaction was stirred at room temperature for 5 minutes and then methane sulfonyl chloride was added via syringe (219 mg, 1.9 mmol).",
"After 2 hrs the reaction was diluted with ethyl acetate.",
"The solution was washed with aqueous NaHCO3 and the organic layer was dried (anhydrous Na2SO4), filtered and the solvent evaporated.",
"The residue was chromatographed on silica gel eluting with a 30-70% ethyl acetate/hexane gradient to give the title compound as a pale yellow oil (420 mg) 69% yield.",
"1H-NMR (500 MHz, MeOD): 8.17 (1H, s), 7.95 (1H, s), 4.93 (2H, s), 3.93-3.80 (3H, m3), 3.49 (3H, s), 3.36-3.21 (3H, m), 3.28-3.31 (3H, m), 2.73-2.68 (1H, m), 2.22-2.19 (1H, d, m), 1.50 (1H, s), 0.85-0.82 (6H, d, J=10 Hz).",
"Step B A similar procedure as in example 82 was followed with the product obtained from step A to give the desired compound.",
"1H-NMR (500 MHz, MeOD): 7.90-7.86 (2H, m), 7.50 (1H, s), 7.49 (1H, m), 7.49-7.43 (2H, m), 3.87-3.85 (6H, m), 3.68 (1H, s), 3.49-3.48 (6H, m), 3.27-2.94 (5H, m) 2.6 (2H, m), 2.23-1.50 (10H, m), 1.05-0.70 (6H, m).",
"TABLE 12 Analogs are Prepared in an Analogous Fashion to EXAMPLE 145 (followed by ester hydrolysis as described in EXAMPLE 145) Amines: TABLE 13 Analogs are Prepared in an Analogous Fashion to EXAMPLE 145 (followed by ester hydrolysis as described in EXAMPLE 145) Amines: TABLE 15 Analogs are Prepared in an Analogous Fashion to EXAMPLE 199 Amines: TABLE 16 Analogs are Prepared in an Analogous Fashion to EXAMPLE 200 Amines:"
]
] | Patent_10260008 |
[
[
"Production of high grade and high concentration of free fatty acids from residual residual oils, fats and greases",
"The present invention relates to a process for the production of unsaturated and saturated free fatty acids.",
"The process comprises the steps of: (a) selecting a starting material from the group consisting of trap oils, trap greases, yellow greases and brown greases, (b) pre-treating the oils and/or greases selected in step (a) in order to separate the oils and/or greases, from residual solids and water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, (c) bleaching the mixture of free fatty acids obtained in step (b) in order to obtain a suitable coloration thereof, (d) fractionating the bleached free fatty acids obtained in step (c) in two fractions: saturated and unsaturated fatty acids, (e) purifying the unsaturated fatty acids obtained from step (d), and purifying the saturated fatty acids obtained from step (d)."
],
[
"1.A process for producing unsaturated and saturated free fatty acids, the process being characterized in that it comprises the steps of: a) selecting a starting material from the group consisting of trap oils, trap greases, yellow greases and brown greases, b) pre-treating the oils and/or greases selected in step a) in order to separate the oils and/or greases from water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, c) bleaching the mixture of free fatty acids obtained in step b) in order to obtain a suitable coloration thereof, d) fractionating the bleached free fatty acids obtained in step c) in two fractions: saturated and unsaturated, e) purifying the unsaturated fatty acids obtained from step d), and f) purifying the saturated fatty acids obtained from step d).",
"2.A process according to claim 1, characterized in that it further comprises a fat splitting step of the mixture of saturated and unsaturated free fatty acids obtained in step b), prior to the bleaching step c).",
"3.A process according to claim 2, characterized in that the fat splitting step is carried out by hydrolysis or saponification of the oil and/or greases obtained in step b), 4.A process according to claim 3, characterized in that the fat splitting is carried out by hydrolysis at a temperature varying between 150° C. to 260° C. and at a pressure varying between 76 psi and 500 psi.",
"5.A process according to claim 3, characterized in that the fat splitting is carried out by saponification at a temperature varying between 100° C. and 150° C. and at a pressure varying between 20 psi and 50 psi.",
"6.A process according to any one of claims 1 to 5, characterized in that in step b), the pretreatment comprises hot filtration and decantation of oils or/and greases selected in step a).",
"7.A process according to any one of claims 2 to 6, characterized in that in step c), the bleaching is carried out by adsorption with an adsorbent selected from the group consisting of silica gel, crystalline silica, bentonite, Fuller's earth and a mixture thereof.",
"8.A process according to claim 7, characterized in that the bleaching of the free fatty acids is carried out in a batch or continuous mode by percolation in different columns.",
"9.A process according to claim 8, characterized in that the bleaching is carried out at a temperature ranging from 100° C. to 150° C. for a period ranging from 15 minutes to 1 hour.",
"10.A process according to claim 8 or 9, characterized in that the bleaching is carried in a batch mode under vacuum.",
"11.A process according to claim 8 or 9, characterized in that the bleaching is carried out in continuous mode under nitrogen atmosphere.",
"12.A process according to any one of claims 2 to 6, characterized in that the bleaching step c) is carried by a treatment with a sufficient amount of hydrogen peroxide at a temperature varying from 60° C. to 90° C. for a period varying from 20 minutes to 3 hours.",
"13.A process according to claim 12, characterized in that the concentration of the hydrogen peroxide ranges from 10 to 30% by weight 14.A process according to claim 12 or 13, characterized in that the bleaching step is carried out at a temperature of 80° C. for a period of 1 hour.",
"15.A process according to any one of claims 2 to 6, characterized in that the bleaching step c) is carried out by molecular distillation by a vacuum thin-film distillation step at a temperature varying from 150° C. to 200° C. and at a pressure varying from 0.1 to 5 mm Hg.",
"16.A process according to claim 15, characterized in that the bleaching step is carried out at a temperature varying from 165° C. to 185° C. and at a pressure varying from 0.2 to 0.5 mm Hg.",
"17.A process according to claim 1, characterized in that the bleaching step is carried out by molecular distillation by a vacuum thin-film distillation step."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Most commercial unsaturated acids (i.e.",
"oleic acid) are derived from animal tallow (by-product of the meat industry), tall oil (by-product of paper mills) or natural vegetable oils.",
"Fat splitting processes are well known in the art.",
"The most common methods are: 1) Twichell process; 2) Batch autoclave process; 3) Continuous process; and 4) Enzymatic process.",
"In Twichell process, the fat is hydrolyzed at a temperature of 100° C. to 105° C. and at atmospheric pressure for 12 to 48 hours.",
"Alkyl-aryl acid or cycloaliphatic sulfonic acid with sulfuric acid (0.75-1.25% w/w) are used as catalysts.",
"Yields of 85%-95% are obtained.",
"The main inconvenients of this process are the catalyst handling, long reaction time, tendency to form dark-colored acid and high labor cost.",
"In the batch autoclave operations, the fat is hydrolyzed in the presence or absence of a catalyst.",
"Live steam is injected continuously at the bottom while venting a small amount to maintain the desired agitation and operating pressure.",
"After settling and formation of an aqueous and a fatty acids phase, the fatty acids phase is treated with mineral acid to separate the soap formed.",
"The fatty acids phase is further washed with water to remove traces of the mineral acid.",
"Under catalytic conditions (i.e.",
"zinc, calcium or magnesium oxides) the fatty acids phase is reacted for a period of 5 to 10 hours at 150° C. -175° C. A high yield of about 85%-95% is obtained.",
"Without catalyst the fatty acids phase is reacted for a period of 2 to 4 hours at a high temperature (240° C.) to give similar yields.",
"The principal inconvenient of this process is the catalyst handling, and high labor cost.",
"In continuous operations also known as the Colgate—Emery process, a single-stage countercurrent high pressure splitting is carried out for fat hydrolysis.",
"The fat is introduced by means of a sparge ring from the bottom of the splitting tower while water is introduced by the top.",
"The crude fat passes as a coherent phase from the bottom to the top, while heavier splitting water travels downward as a dispersed phase through the mixture of fat and fatty acids.",
"The high temperatures involved (250° C. -260° C.) associated to high pressures (725 psi) assures degrees of splitting up to 98% in only 2 to 3 hours.",
"The principal inconvenient of this process is the high cost associated with the equipment and the restriction to relative clean starting materials.",
"In enzymatic operations, the lipase from Candida rugosa, Aspergillus niger ; and Rhizopus arrhizus had been studied at temperatures of 26° C. to 46° C. for periods of 48 to 72 hours.",
"Even though 98% of splitting is claimed there is no commercial process available until now.",
"The principal inconvenient of this process is that because enzymes work very well over a specific substrate under specific conditions, when the starting material is composed of more than one product, the reaction is less selective.",
"Long reaction times and great volumes required to satisfy the optimal concentration are also current problems involved in this kind of procedure.",
"Fractionation of free fatty acids is commonly performed by distillation of tall oil.",
"Tall oil is recovered in most paper mills by acidulation of the soap skimming from black liquor.",
"Crude tall oil (CTO) consists of a mixture of fatty acids (40%-45%), resin acid (40%-45%) and various neutral components (i.e.",
"hydrocarbons, wax alcohols, sterols, esters and residues).",
"About 40% to 50% of the fatty acids contained in tall oil are oleic acid, while another 35% to 45% are linoleic acid.",
"Higher quality of tall oil fatty acids, TOFA, (less than 2% of resins acid) can be obtained by distillation through two columns: a rosin column and a fatty acids column.",
"Oleic acid is probably the most important unsaturated fatty acids (UFA) because many applications have been developed for its use in different fields (i.e.",
"cosmetics, chemicals, lubricants, textiles, etc.).",
"Separation of oleic acid form tall oil distillates requires additional refining steps.",
"Best known-process for fractionation of fatty acids by crystallization from solvent is the “Emersol” process, developed by Emery Industries Inc. in 1934.Different American patents used different solvents (methanol: U.S. Pat.",
"No.",
"2,421,157; acetone: U.S. Pat.",
"No.",
"2,450,235 and methyl formate: U.S. Pat.",
"No.",
"3,755,389) to separate saturated fatty acids from unsaturated fatty acids.",
"The process was optimized by addition of crystallizing promoters (neutral fats, tallow, and glycerol tri-stearate).",
"One more refined promoter is described in Australian patent AU-28434/92.It is the reaction product of: 1) a polyhydric alcohol (i.e.",
"glycerol, pentaerythritol, trimethylol pentane, etc.",
"), 2) a dicarboxylic acid (i.e.",
"adipic, oxalic, succinic, azelaic, glutaric and tartaric) and 3) a fatty acids.",
"All these process require explosion proof installations and low temperature refrigeration systems.",
"Other methods for producing oleic acid involve separation over molecular sieves (U.S. Pat.",
"No.",
"4,529,551 and U.S. Pat.",
"No.",
"4,529,551); lithium soap separation (U.S. Pat.",
"No.",
"4,097,507), urea complexation (U.S. Pat.",
"No.",
"2,838,480 and U.S. Pat.",
"No.",
"4,601,856) and complexation with dienophiles (U.S. Pat.",
"No.",
"5,194,640).",
"All these process have the inconvenient of a high cost operation associated to the use of chemicals required.",
"Dry fractionation technology was originally developed for treatment of animal fat (i.e.",
"beef tallow) in the 60's.",
"Since this time, many improvements were performed in response to the ever-increasing demand of the industry for new products with very specific requirements.",
"Two main sources are now the target of this technology: 1) vegetable oils such as palm oil, soybean oil, sunflower oil, rapeseed oil, groundnuts oil, cottonseed oil and palm kernel oil and 2) animal fats such as beef tallow, milk fat, lard and fish oil.",
"These fats and oils are mainly composed of triglycerides, diglycerides and monoglycerides (i.e.",
"a broad range of melting points) constituting a large number of intersoluble glycerides that are very difficult to separate by dry fractionation (i.e.",
"solvent free crystallization).",
"The separation of a liquid fraction (i.e.",
"olein, used in food oil) and a solid fraction (i.e.",
"stearin, used in shortening and margarine) can be achieved through dry fractionation.",
"In the present invention, dry fractionation was used to separate purified free fatty acid obtained by splitting the residual oils and greases recuperated from industrial and commercial operations (i.e.",
"trap greases, yellow greases and brown greases).",
"The free fatty acids obtained from these starting materials are mainly constituted by unsaturated fatty acids, such as mainly oleic acid, linoleic acid, linolenic acid and saturated fatty acids such as palmitic acid and stearic acid.",
"The range of melting points for these limited number of products, in comparison with all the possible combinations presented by glycerides, was shown to be wide enough to perform a highly selective separation."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a process for the production of free fatty acids that uses residual oils, fats and greases as the starting material.",
"Another object of the present invention is to provide an inexpensive and simple way to produce high grade and high concentration of free fatty acids.",
"A further object of the invention is to overcome most of the drawbacks mentioned hereinabove.",
"More precisely, the objects of the present invention are provided by a process for producing unsaturated and saturated free fatty acids, the process comprising the steps of: a) selecting a starting material from the group consisting of trap oils and greases, yellow greases and brown greases, b) pre-treating the oils and/or greases selected in step a) in order to separate the oils and/or greases, from residual solids and water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, c) bleaching the mixture of free fatty acids obtained in step b) in order to obtain a suitable coloration thereof, d) fractionating the bleached mixture of free fatty acids obtained in step c) in two fractions: saturated and unsaturated fatty acids, e) purifying the unsaturated fatty acids obtained from step d), and f) purifying the saturated fatty acids obtained from step d).",
"The process of the present invention has the, advantage of using inexpensive starting material thereby reducing the cost all the while allowing the recycling of the starting material that is normally eliminated through costly treatments thereof.",
"The process of the present invention also has the advantage of giving the option of eliminating a hydrolysis step in the production of the free fatty acids, thereby simplifying the process for the production of fatty acids and reducing the production cost of same."
],
[
"FIELD OF THE INVENTION The present invention relates to the production of free fatty acids from residual oils, fats and greases.",
"More precisely, the present invention relates to a process for the production of unsaturated and saturated free fatty acids from residual oils, fats and greases.",
"BACKGROUND OF THE INVENTION Most commercial unsaturated acids (i.e.",
"oleic acid) are derived from animal tallow (by-product of the meat industry), tall oil (by-product of paper mills) or natural vegetable oils.",
"Fat splitting processes are well known in the art.",
"The most common methods are: 1) Twichell process; 2) Batch autoclave process; 3) Continuous process; and 4) Enzymatic process.",
"In Twichell process, the fat is hydrolyzed at a temperature of 100° C. to 105° C. and at atmospheric pressure for 12 to 48 hours.",
"Alkyl-aryl acid or cycloaliphatic sulfonic acid with sulfuric acid (0.75-1.25% w/w) are used as catalysts.",
"Yields of 85%-95% are obtained.",
"The main inconvenients of this process are the catalyst handling, long reaction time, tendency to form dark-colored acid and high labor cost.",
"In the batch autoclave operations, the fat is hydrolyzed in the presence or absence of a catalyst.",
"Live steam is injected continuously at the bottom while venting a small amount to maintain the desired agitation and operating pressure.",
"After settling and formation of an aqueous and a fatty acids phase, the fatty acids phase is treated with mineral acid to separate the soap formed.",
"The fatty acids phase is further washed with water to remove traces of the mineral acid.",
"Under catalytic conditions (i.e.",
"zinc, calcium or magnesium oxides) the fatty acids phase is reacted for a period of 5 to 10 hours at 150° C. -175° C. A high yield of about 85%-95% is obtained.",
"Without catalyst the fatty acids phase is reacted for a period of 2 to 4 hours at a high temperature (240° C.) to give similar yields.",
"The principal inconvenient of this process is the catalyst handling, and high labor cost.",
"In continuous operations also known as the Colgate—Emery process, a single-stage countercurrent high pressure splitting is carried out for fat hydrolysis.",
"The fat is introduced by means of a sparge ring from the bottom of the splitting tower while water is introduced by the top.",
"The crude fat passes as a coherent phase from the bottom to the top, while heavier splitting water travels downward as a dispersed phase through the mixture of fat and fatty acids.",
"The high temperatures involved (250° C. -260° C.) associated to high pressures (725 psi) assures degrees of splitting up to 98% in only 2 to 3 hours.",
"The principal inconvenient of this process is the high cost associated with the equipment and the restriction to relative clean starting materials.",
"In enzymatic operations, the lipase from Candida rugosa, Aspergillus niger; and Rhizopus arrhizus had been studied at temperatures of 26° C. to 46° C. for periods of 48 to 72 hours.",
"Even though 98% of splitting is claimed there is no commercial process available until now.",
"The principal inconvenient of this process is that because enzymes work very well over a specific substrate under specific conditions, when the starting material is composed of more than one product, the reaction is less selective.",
"Long reaction times and great volumes required to satisfy the optimal concentration are also current problems involved in this kind of procedure.",
"Fractionation of free fatty acids is commonly performed by distillation of tall oil.",
"Tall oil is recovered in most paper mills by acidulation of the soap skimming from black liquor.",
"Crude tall oil (CTO) consists of a mixture of fatty acids (40%-45%), resin acid (40%-45%) and various neutral components (i.e.",
"hydrocarbons, wax alcohols, sterols, esters and residues).",
"About 40% to 50% of the fatty acids contained in tall oil are oleic acid, while another 35% to 45% are linoleic acid.",
"Higher quality of tall oil fatty acids, TOFA, (less than 2% of resins acid) can be obtained by distillation through two columns: a rosin column and a fatty acids column.",
"Oleic acid is probably the most important unsaturated fatty acids (UFA) because many applications have been developed for its use in different fields (i.e.",
"cosmetics, chemicals, lubricants, textiles, etc.).",
"Separation of oleic acid form tall oil distillates requires additional refining steps.",
"Best known-process for fractionation of fatty acids by crystallization from solvent is the “Emersol” process, developed by Emery Industries Inc. in 1934.Different American patents used different solvents (methanol: U.S. Pat.",
"No.",
"2,421,157; acetone: U.S. Pat.",
"No.",
"2,450,235 and methyl formate: U.S. Pat.",
"No.",
"3,755,389) to separate saturated fatty acids from unsaturated fatty acids.",
"The process was optimized by addition of crystallizing promoters (neutral fats, tallow, and glycerol tri-stearate).",
"One more refined promoter is described in Australian patent AU-28434/92.It is the reaction product of: 1) a polyhydric alcohol (i.e.",
"glycerol, pentaerythritol, trimethylol pentane, etc.",
"), 2) a dicarboxylic acid (i.e.",
"adipic, oxalic, succinic, azelaic, glutaric and tartaric) and 3) a fatty acids.",
"All these process require explosion proof installations and low temperature refrigeration systems.",
"Other methods for producing oleic acid involve separation over molecular sieves (U.S. Pat.",
"No.",
"4,529,551 and U.S. Pat.",
"No.",
"4,529,551); lithium soap separation (U.S. Pat.",
"No.",
"4,097,507), urea complexation (U.S. Pat.",
"No.",
"2,838,480 and U.S. Pat.",
"No.",
"4,601,856) and complexation with dienophiles (U.S. Pat.",
"No.",
"5,194,640).",
"All these process have the inconvenient of a high cost operation associated to the use of chemicals required.",
"Dry fractionation technology was originally developed for treatment of animal fat (i.e.",
"beef tallow) in the 60's.",
"Since this time, many improvements were performed in response to the ever-increasing demand of the industry for new products with very specific requirements.",
"Two main sources are now the target of this technology: 1) vegetable oils such as palm oil, soybean oil, sunflower oil, rapeseed oil, groundnuts oil, cottonseed oil and palm kernel oil and 2) animal fats such as beef tallow, milk fat, lard and fish oil.",
"These fats and oils are mainly composed of triglycerides, diglycerides and monoglycerides (i.e.",
"a broad range of melting points) constituting a large number of intersoluble glycerides that are very difficult to separate by dry fractionation (i.e.",
"solvent free crystallization).",
"The separation of a liquid fraction (i.e.",
"olein, used in food oil) and a solid fraction (i.e.",
"stearin, used in shortening and margarine) can be achieved through dry fractionation.",
"In the present invention, dry fractionation was used to separate purified free fatty acid obtained by splitting the residual oils and greases recuperated from industrial and commercial operations (i.e.",
"trap greases, yellow greases and brown greases).",
"The free fatty acids obtained from these starting materials are mainly constituted by unsaturated fatty acids, such as mainly oleic acid, linoleic acid, linolenic acid and saturated fatty acids such as palmitic acid and stearic acid.",
"The range of melting points for these limited number of products, in comparison with all the possible combinations presented by glycerides, was shown to be wide enough to perform a highly selective separation.",
"SUMMARY OF THE INVENTION An object of the present invention is to provide a process for the production of free fatty acids that uses residual oils, fats and greases as the starting material.",
"Another object of the present invention is to provide an inexpensive and simple way to produce high grade and high concentration of free fatty acids.",
"A further object of the invention is to overcome most of the drawbacks mentioned hereinabove.",
"More precisely, the objects of the present invention are provided by a process for producing unsaturated and saturated free fatty acids, the process comprising the steps of: a) selecting a starting material from the group consisting of trap oils and greases, yellow greases and brown greases, b) pre-treating the oils and/or greases selected in step a) in order to separate the oils and/or greases, from residual solids and water and obtain a mixture consisting principally of saturated and unsaturated free fatty acids, c) bleaching the mixture of free fatty acids obtained in step b) in order to obtain a suitable coloration thereof, d) fractionating the bleached mixture of free fatty acids obtained in step c) in two fractions: saturated and unsaturated fatty acids, e) purifying the unsaturated fatty acids obtained from step d), and f) purifying the saturated fatty acids obtained from step d).",
"The process of the present invention has the, advantage of using inexpensive starting material thereby reducing the cost all the while allowing the recycling of the starting material that is normally eliminated through costly treatments thereof.",
"The process of the present invention also has the advantage of giving the option of eliminating a hydrolysis step in the production of the free fatty acids, thereby simplifying the process for the production of fatty acids and reducing the production cost of same.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a graphical representation of the differential scanning calorimetry of free fatty acids of trap oils and greases used in the process of the present invention.",
"FIG.",
"2 is a graphical representation showing the cooling curve of the free fatty acids produced by the process of the present invention.",
"DETAILED DESCRIPTION OF THE INVENTION According to a preferred embodiment of the present invention, the process can be achieved in the following sequential step: 1) selecting a starting material from the group consisting of: trap oils, greases and fats; 2) pre-treating the selected oils, fats and greases in order to separate residual solids and water therefrom so to obtain a mixture consisting principally of saturated and unsaturated free fatty acids; 3) fat splitting of the pretreated mixture by hydrolysis or saponification 4) bleaching the hydrolysed or saponified free fatty acids; 5) fractionating the bleached free fatty acids so to obtain an unsaturated and a saturated fraction of fatty acids; 6) purifying the unsaturated fraction of fatty acids; and 7) purifying the saturated fraction of free fatty acids.",
"In step 1), the starting material is selected from the group consisting of: residual oils, fats and greases.",
"This step is crucial and constitutes the gist of the present invention.",
"The residual oils, fats and greases are post-consumers and/or by-products of industrial and commercial operations.",
"Three principal sources are trap oils and greases, yellow greases and brown greases.",
"Trap oils and greases are collected in the traps installed on the sewage water outlet of restaurants and food industries.",
"These traps allow the collection of the oils and greases carried over with the wastewater of washing operations, before they reach the municipal sewage network.",
"These greases are collected by dedicated trucks and sent to pre-treatment plants.",
"Yellow and brown greases are residual oils and greases from cooking operations.",
"They are mainly collected in restaurants and food industries.",
"Yellow greases have a low concentration of free fatty acids (acid value of about 5 to 15 mg KOH/g) as a result of its short and limited contact with water (i.e.",
"moisture of food).",
"As in the case of trap oils and greases used in frying process (i.e.",
"high temperature in presence of air) principal alteration lead to oxidized monomers, dimers, oligomers, volatile compounds, cyclic monomers and non-polar compounds.",
"Trap oils and greases are mainly constituted of a mixture of oils and greases (3 to 10%), water (90 to 95%) and residual solids (1 to 5%).",
"At room temperature, trap oils and greases form a non-homogeneous and unstable emulsion.",
"They have a strong odor characteristic of acetic and/or butyric fermentation (i.e.",
"fermentation of olives before oil extraction).",
"In step 2), once the starting material is selected, it undergoes a pre-treatment step for eliminating the water and residual solid present in the raw material.",
"Different known methods may be used for eliminating water and residual solids.",
"Such known methods may include hot filtration, in order to separate the suspended solids, and a hot decantation, in order to separate oils and greases from water.",
"Decantation can be done in a batch mode in heated decanting tanks or in a continuous mode in a three phase dynamic separator where oils and greases are recovered in the upper phase (i.e.",
"floating phase), the solids being decanted at the bottom of the separator and the water been extracted in the middle of the separator.",
"Any temperature between 50° C. and 100° C. could be used for decanting, but preferably the temperature should range between 60° C. and 80° C. The recovered oily phase is a mixture of free fatty acids, tri, di, and monoglycerides, trimers and dimer acids, oxidized monomers, unsaponifiables and other colored long chain oxidized products.",
"A typical composition of pre-treated trap oil is: 98% oil, 2% residual solids and traces of water.",
"The oil is mainly constituted by free fatty acids (acid value of about 130 to 160 mg KOH/g) coming from the natural enzymatic hydrolysis, which occurs during the lying time of the oils and greases in traps.",
"A typical composition of pretreated trap oils and greases is presented in Table No1.In step 3), the mixture principally comprising unsaturated and saturated free fatty acid obtained from the pre-treatment step may undergo fat splitting in order to complete the hydrolysis of the non-hydrolysed compounds (i.e.",
"tri-, di- and monoglycerides).",
"Fat splitting can be achieved by hydrolysis at high temperature and pressure.",
"Typical temperature ranges from 150° C. to 260° C., and more preferably from 200° C. to 240° C. Typical pressure ranges from 75 psi to 500 psi and more preferably from 150 psi to 450 psi.",
"The reaction time can vary between 1 to 6 hours and more preferably between 2 to 4 hours.",
"Metal oxide catalysts, such as zinc, calcium, and magnesium could favor the reaction at a concentration by weight of 1% to 5% and more preferably 2% to 4%.",
"A typical composition of free fatty acids (FFA) obtained by hydrolysis is presented in Table No1.Typical color values are shown in Table No2.Fat splitting could also be achieved by saponification under controlled temperature and pressure conditions.",
"Suitable temperature or pressure conditions for saponification are about 100° C. to 150° C. and about 20 to 50 psi respectively.",
"After saponification, the mixture is cooled to about 85° C. to 95° C. Neutralization is carried out with a mineral acid selected from the group consisting of H2SO4, H3PO4, HCl and the like, at a pH of about 5 to 7.Separation of the aqueous phase leads to the oily phase containing free fatty acids (FFA).",
"In step 4), the so-obtained mixture of unsaturated and saturated free fatty acids is bleached in order to give a suitable coloration thereof.",
"Various known bleaching procedures namely adsorption, treatment with hydrogen peroxide (H2O2) or various distillation techniques may be used.",
"Bleaching by adsorption is carried out with one adsorbent or a combination of adsorbents of the group consisting of: silica gel, crystalline silica, bentonite, Fuller's earth, diatomaceous earth and activated carbon.",
"Bleaching could be performed in a batch or continuous mode by percolation in different columns.",
"This step may further be carried out at a temperature varying from 100° C. to 150° C. and more preferably from 115° C. to 130° C. The time reaction may vary from 15 minutes to 1 hour and more preferably from 30 minutes to 45 minutes.",
"When the bleaching step is carried out by adsorption, inert atmosphere is strongly recommended.",
"Under batch mode it could be successfully achieved under vacuum and in continuous mode it can be achieved under nitrogen atmosphere.",
"A typical composition for bleached free fatty acids (FFA) obtained under batch conditions is presented in Table No1.Typical color values are shown in Table No2.Bleaching by treatment with a hydrogen peroxide solution (H2O2 at 35% w/w) may be achieved at a temperature of 80° C. for one hour.",
"Concentration by weight of the peroxide solution could be at 1%, 10% or 30% but more preferably at 10%.",
"Bleaching by distillation techniques such as the ones selected from the group consisting of falling film evaporation, wiped film evaporation, fractional distillation and molecular distillation may also be carried out.",
"One particular method for bleaching (known as “short path”) is based in the separation of the colorful products by molecular distillation.",
"Industrially this application could be done by a vacuum thin-film distillation process, which permits distillation at very reduced pressure (i.e.",
"between 0.1 mm Hg −5 mm Hg) and at a temperature ranging between 150°-200° C. The equipment used for the molecular distillation comprises essentially a vertical which one double jacketed cylinder with an internal condenser and a rotating roller wiper system.",
"Free fatty acids (FFA) are heated until complete homogenization.",
"They are then continuously fed onto the rotating distributor and thrown by centrifugal force on a heated wall.",
"They are further uniformly distributed by wiping elements while flowing downwards.",
"The internal condenser and film of the product to be evaporated (i.e.",
"1 mm thick) are so close that condensation is almost instantaneous.",
"The very short residence time (i.e.",
"about 1 minute) avoid degradation reactions and limit the risk of fouling in the internal wall of the cylinder.",
"The absence of oxygen (i.e.",
"high vacuum) also contributes to prevent most common degradations associated to air oxidation.",
"In another preferred embodiment, the bleaching of the free fatty acids is carried out by molecular distillation.",
"The main interest of this bleaching technique is to furnish a high quality material that could be directly fractionated without the fat splitting operation.",
"Table No1 shows the composition of trap oils and greases pretreated and distilled by molecular distillation.",
"It is evident that the quality is as good as, if not better, than those bleached free fatty acids (FFA) obtained by other techniques such as clay and hydrogen peroxide.",
"Thus the fat splitting step 3) is eliminated when the mixture are bleached by molecular distillation.",
"In this case the process is reduced to the following steps: a) Pre-treatment of the trap oil and greases.",
"b) Molecular distillation of the pretreated trap oils and greases.",
"c) Fractionation of the resulting distillate in two fractions: saturated and unsaturated.",
"d) Purification of unsaturated fatty acids (UFA).",
"e) Purification of saturated fatty acids (SFA).",
"TABLE N1 Typical properties and compositions for different products obtained at different stages of the process Properties Trap Oil & Greases Pretreated* & Free Fatty Bleached Free Fatty Acids Molecular Acids Free Fatty Acids Unsaturated Saturated Pretreated* Distilled (FFA) Clay H2O2 Mol.",
"Dist.",
"(UFA)** (SFA)** Acid Value (mg KOH/g) 157 189 183 183 180 186 169 188 Volatile (%) 1.8 1.2 2.1 0.9 0.1 Composition Capric Acid (%) 10:00 0.2 0.2 0.15 0.15 0.19 Lauric Acid (%) 12:00 0.2 0.4 0.33 0.33 0.2 0.2 0.41 0.2 Myristic Acid (%) 14:00 1.1 1.4 1.28 1.38 1.1 1.2 1.24 1.68 Palmitic Acid (%) 16:00 15.7 19.4 17.85 17.77 15.8 19.0 5.44 42.78 Margaric Acid (%) 17:00 0.1 0.2 0.26 0.26 0.3 — 0.75 Stearic Acid (%) 18:00 7.8 8.8 9.2 7.81 7.8 7.8 1.44 21.02 Arachldic Acid (%) 20:00 0.86 0.76 — — Myristoleic Acid (%) 14:01 0.2 — — 0.1 Palmltoleic Acid (%) 16:01 2.4 3.0 2.42 2.52 2.0 2.5 3.27 0.95 Heptadecenoic Acid (%) 17:01 0.20 0.19 0.25 — Oleic Acid (%) 18:1c 45.4 44.2 43.3 44.97 40.6 45.5 57.78 20.60 Elaldic Acid (%) 18:1t 5.29 5.31 5.47 4.89 Linoleic Acid (%) 18:2c 13.2 14.2 14.17 13.17 10.3 12.3 17.40 — Linolelaidic Acid (%) 18:2t 1.67 1.87 2.11 5.18 Linolenic Acid (%) 18:03 1.8 1.8 1.82 1.62 0.9 1.3 0.96 0.59 Others (%) 0.8 0.7 0.8 1.3 Total FAA (%) 88.7 94.5 98.8 98.11 79.5 91.5 95.96 98.64 Total Unsaturated (UFA) (%) 62.8 63.4 68.87 69.65 53.8 61.7 87.24 32.2 Total Saturated (SFA) (%) 25.1 30.4 29.93 28.46 24.9 28.5 8.72 66.43 *Hot decanted and filtered **Fractionated after clay bleaching TABLE NO 2 Typical color for different products obtained at different stages of the process.",
"Pretreated Fat Free Fatty Bleached Free Free Fatty Acids Oils & Greases Acids Fatty Acids Unsaturated Saturated Molecular Dist.",
"** (FFA) Clay H2O2 (UFA)* (SFA)* Color FAC >45 <7 <11 B <11 <1 (AOCS CC 13a-43) Yellow 1″ Lovibond 25 Out of range 10 50 21 3 (AOCS CC 13b-45) Red 1″ Lovibond 3 Out of range 3.2 6 4.8 1 (AOCS CC 13b-45) *Fractionated after clay bleaching.",
"**Measured in the 5¼″ Lovibond's scale.",
"***After hydrolysis (without any molecular distillation treatment).",
"In step 5), fractionation of bleached free fatty acids or pretreated and molecular distilled trap oils and greases could be achieved by different methods: a) By quenching the bleached free fatty acids oils in a solvent at low temperatures.",
"Solvent could be one selected from the group consisting of hexane, acetone, isopropyl alcohol and ethanol.",
"The reaction temperature may range from −5° C. to −20° C. Unsaturated fatty acids (UFA) are dissolved in the solvent while saturated fatty acids (SFA) precipitates under these conditions.",
"Filtration could be easily achieved by a filter press, a Sparkler filter, a centrifuge or similar equipment.",
"b) By crystallization of saturated fatty acids (SFA) using a detergent that coats the crystals, then increasing their specific weight.",
"Filtration could be achieved by previously described equipment.",
"c) By dry fractionation, based on differences between melting points of saturated (SFA) and unsaturated (UFA) free fatty acids.",
"The principal advantages of this technology are that there is no solvent required and the temperature range is warmer than previous cases (i.e.",
"over zero degrees).",
"Differential Scanning Calorimetry (DSC) was used in order to determine the best cooling profile.",
"FIG.",
"No1 shows the spectra for free fatty acids obtained after splitting of trap oils and greases.",
"Crystallization is carried out by a detailed program of cooling (i.e.",
"precision of 0.1° C.).",
"Details of this program are shown in FIG.",
"No2: Bleached Free Fatty Acids cooling curve.",
"It is important to note that unsaturated fatty acids (UFA) and saturated fatty acids (SFA) are mutual contaminants.",
"Poor filtration lets important quantities of UFA in SFA reducing the yield of UFA and decreasing the purity of SFA.",
"This problem could be successfully overcome by using a filter press under pressure.",
"Pressure is generated by squeezing the membrane, which Wraps the filter cloth.",
"A refrigerated liquid or gas could generate the pressure in order to keep the right temperature of crystallization.",
"Pressures could vary from 10 bars to 30 bars.",
"In step 6), unsaturated free fatty acids (UFA) are finally purified by another treatment of bleaching in similar conditions then that of free fatty acids (FFA).",
"In step 7), saturated free fatty acids (SFA) cake's is melted and send to be distillated.",
"Any distillation procedure (i.e.",
"falling film evaporation, wiped film evaporation, fractional distillation and molecular distillation) could be successfully achieved to separate stearic acid from palmitic acid due to the difference in their vapor pressures at high temperature (Table No4 and Table No5).",
"TABLE NO 4 Vapor Pressures of Palmitic and Stearic Acids.",
"Vapor Pressure (mm Hg) Temperature (° C.) Palmitic Stearic 200 6.15 2.60 230 22.53 10.56 260 67.01 41.15 TABLE NO 5 Boiling Points of Palmitic and Stearic Acids.",
"Vapor Pressure Boiling Point (C.) (mm Hg) 1 2 4 8 16 32 64 Palmitic Acid 165.9 178.0 191.2 205.7 221.5 239.1 258.6 Stearic Acid 182.5 195.0 208.6 223.6 240.0 285.3 278.6 Although the present invention has been explained hereinabove by way of a preferred embodiment thereof, it should be pointed out that any modifications to this preferred embodiment within the scope of the present invention is not deemed to alter or change the nature and scope of the present invention."
]
] | Patent_10275077 |
[
[
"Micro-arrayed organization of transcription factor target genes",
"The following invention outlines methodologies for the construction and utilization of transcription factor direct target gene microarrays of both DNA and corresponding protein/peptide target origin.",
"The technology entails the array/microarray annotation and organization of transcription factor direct loci and corresponding protein products identified through modified and improved versions of chromosomal immunoprecipitation (CHIP) and molecular cloning procedures.",
"It allows for the formulation of physiologically directed arrays which result in a thorough, focused characterization of the genetic and biochemical regulation occurring within a give population of cells or a given tissue.",
"Arrays and microarrays of direct targets for any given transcription factor created utilizing this technology are substantially more clinically relevant for purposes of medical diagnostics and patient prognostics than conventional microarrays due to the physiologically focused nature and the transcription factor targets.",
"In addition, the characterization and array organization of transcription factor target protein products and the assessment of their interactions with other proteins and/or small molecules is of critical importance for the purposes of understanding cellular and ultimately the design of therapeutics for human anomalies."
],
[
"1.A method according to the present invention which utilizes modified sequential chromosomal immunoprecipitation and cloning procedures for the discovery of transcription factor target genes from cells whereby said genes are organized into an array format.",
"2.A method according to claim 1 comprising the process of: a) cross-linking protein/DNA complexes in cells or tissues; b) immunoprecipitating said protein/DNA complexes with antibodies which recognize transcription factors; c) purifying DNA present within immunoprecipitated protein/DNA samples; d) organizing said purified DNA sequences into an array format.",
"3.A method according to claim 2 in which said purification of DNA present within immunoprecipitated protein/DNA samples includes amplification via inverse polymerase chain reaction (I-PCR) utilizing oligonucleotides corresponding to transcription factor binding sites to determine flanking nucleotide sequences present within discovered DNA fragments.",
"4.A method according to claim 2 in which said arrays consist of DNA templates bound to solic supports, for purposes of assessing the expression patterns or levels of transcription factor target genes.",
"5.A method according to claim 4 in which said transcription factor target genes consist of transcribed sequences, including coding sequences which correspond to amino acid composition.",
"6.A method according to claim 2 in which purified DNA fragments are utilized to cross hybridize against libraries of DNA sequences for the purposes of creating transcription factor target 7.An antibody according to claim 2 whereby said antibody allows for the purification of protein/protein and/or protein/DNA complexes from cells, for purposes of creating arrays and/or microarrays of transcription factor target genes.",
"8.A protein/DNA complex isolated from cells according to claim 2 whereby said protein DNA/complex results in the identification of transcription factor target genes, for purposes of constructing arrays and/or microarrays of said target genes.",
"9.DNA fragments isolated from protein/DNA complexes according to claim 8 whereby said DNA fragments encode transcription factor target genes, for purposes of constructing arrays and/or microarrays of said target genes.",
"10.Nucleotide sequences present in DNA fragments isolated according to methods described in claim 2 wherein said sequences represent transcription factor target genes and are utilized for purposes of constructing arrays of said sequences.",
"11.Arrays of transcription factor target gene sequences, for purposes of monitoring the expression patterns of transcription factor targets in given samples.",
"12.A method according to claim 2 which further comprises the process of translating isolated transcription factor target gene sequences for the purposes of constructing target protein arrays.",
"13.A method according to claim 12 in which said arrays are of a chemical/“nonliving” nature or biological/“living” nature.",
"14.Arrays of transcription factor target proteins as described in claim 13.15.A transcription factor target protein/protein interaction complex identified by arrays described in claim 14 in which said protein/protein complex represents the interaction between transcription factor target protein sequences and other protein sequences, for the purposes of characterizing transcription factor target protein interacting molecules.",
"16.A transcription factor target protein/small molecule complex identified by arrays described it claim 14 in which said protein/small molecule complex represents the interaction between transcription factor target protein sequences and small molecules, for the purposes of characterizing transcription factor target protein interacting molecules.",
"17.A transcription factor target protein/metal complex identified by arrays described in claim 14 in which said protein/metal complex represents the interaction between transcription factor target protein sequences and charged or uncharged metals, for the purposes of characterizing transcription factor target protein interacting molecules.",
"18.A transcription factor target protein/nucleotide sequence complex identified by arrays described in claim 14 in which said protein/nucleotide sequence complex represents the interaction between transcription factor target protein sequences and nucleotide sequences of DNA or RNA origin, for the purposes of characterizing transcription factor target protein interacting molecules.",
"19.Proteins which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays according to claim 14.20.Metals which are discovered as specifically interacting with transcription factor target proteii sequences through the use of arrays described by claim 14.21.Nucleotide sequences which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays described in claim 14.22.Simple sugars and oligosaccharides which are discovered as specifically interacting with transcription factor target protein sequences through the use of arrays described by claim 14.23.Therapies designed as a result of the knowledge obtained from the discovery of interactions between transcription factor target protein sequences and proteins, amino acid or peptide sequences, nucleotide sequences, small molecules, metals, simple sugars and oligosaccharides through the use of arrays according to claim 14."
],
[
"<SOH> 2.0 BACKGROUND OF THE INVENTION <EOH>Genetic activity, i.e.",
"the activation or repression of gene transcription, has long been directly correlated with gene function.",
"Transcriptional regulation is the first and perhaps most crucial mechanism by which cells regulate the functions of genes.",
"By providing or denying mRNA templates for translation it is possible to tightly control the intricate cellular mechanisms of determination, division, survival etc.",
"( FIG.",
"1 and for review see Moroy et al., 2000 , Cellular and Molecular Life Sciences, 57(6): 957-75).",
"Recently, a number of methods have been developed which allow for the rapid assessment of gene expression in a given sample and thus give insight as genetic profiles for various aspects of physiology and disease.",
"These include, but are not limited to, two dimensional arrays and microarrays of either cDNAs or oligonucleotides representing corresponding mRNAs on solid supports.",
"The arrayed aspect of the technology provides an organized, unbiased method for determining the quantitative and qualitative aspects of gene expression in a given sample population in a massive high-throughput format (a representative set of examples includes U.S. Pat.",
"Nos.",
"6,136,592, 6,100,030, 6,040,138 herein incorporated by reference Debouck et al., 1999 , Nature Genetics Supplement, 21: 48-50).",
"It is this macromolecular ability to monitor the expression patterns and levels of genes involved in physiology and disease which allows for many basic science as well as clinical applications such as the assessment of predisposition to particular disorders as well as the possibility of disease prevention or early treatment.",
"It is clear that array technology enables researchers to efficiently ascertain expression patterns and levels of a multitude of loci within a particular sample.",
"In addition, some effort has been directed towards the construction of microarrays which contain templates organized by physiology or functional entity such as cell cycle control or tissue specificity, yet these “focused arrays” are considerably lacking in gene content and limited in number.",
"In addition, it still remains that the majority of genetic microarrays consist of random sequences, the identity and composition of which are often even unknown.",
"Thus, for the most part, arrayed templates of either a nucleotide or peptide origin have yet to be developed such that the array of genes itself depicts something about physiology.",
"It is therefore imperative that more focused, biologically relevant arrays and microarrays of genes be created.",
"For example, arrays of genes known or hypothesized to be involved in a particular disease such as cancer, for example, would be of much more relevance clinically than arrayed organization of random gene sequences.",
"By clustering arrays and microarrays in the context of specific physiologic and disease categories, these arrays can then be more readily subjected to the appropriate sample populations for analysis.",
"This prevents the endless costly analysis of expression data which very well may not be relevant to the sample being studied.",
"Therefore, an initial establishment of clusters and “families” of genes predicted to play particular roles in physiology or disease, and subsequent organization of these clusters in an array and microarray format will allow for a new level of discrete and focused genetic profiling for basic science and medical diagnostics.",
"One method for clustering genes into particular physiologic and disease categories relies upon the exploitation of either the direct or indirect interaction between transcriptional regulators and terminal target genes (for review see Tjian and Maniatis, 1994 , Cell, 77: 5-8).",
"Many transcription factors have been extensively demonstrated to play specific roles in very “focused” areas of physiology and disease, primarily through the regulation of target genes.",
"It is possible to exploit this knowledge for the creation and production of functionally relevant arrays.",
"By establishing arrays and microarrays of transcription factor target loci it is possible to narrow the purpose of said arrays for the characterization of expression profiles for specific aspects of physiology.",
"In addition to transcription factor target genetic expression pattern profiling, it is clear that characterization of the biochemical interaction properties of transcription factor targets will enhance therapeutic discovery and development.",
"The ability to characterize protein/protein, chemical/protein, small molecule/protein and enzymatic reaction interactions in a high-throughput and saturable format is of unparalleled value for the eventual design of therapeutic intervention strategies for the treatment of disease.",
"In order to efficiently search for and analyze these types of interactions in a high-throughput yet sensitive format it is necessary to implement variations of array and microarray technology.",
"A number of groups have begun to focus upon the organization of proteins and/or peptide and amino acid sequences in array and microarray formats similar to that for nucleotides sequences.",
"Such an organization has been successfully implemented for the efficient identification of specific interactions between arrayed protein samples and other entities which include, but are not limited to, other proteins, enzymes, metals, sugars, oligosaccharides, chemical compounds, DNA and RNA molecules (a representative set of examples includes U.S. Pat.",
"Nos.",
"5,591,646, 6,156,511, 5,834,318 herein incorporated by reference; MacBeath et al., 2000 , Science, 289: 1760-1763 and for review see Emili et al., 2000 , Nature Biotechnology, 18: 393-397).",
"These arrays allow for the high-throughput sensitive and specific characterization of interactions between arrayed proteins and other molecules.",
"Yet in order to fully take advantage of protein array technology it is necessary to focus its application to discrete realms of physiology and disease.",
"By concentrating the identities of protein arrays on particular facets of biology a great deal of irrelevant biochemical screening and the costs associated with it can be eliminated.",
"It is the modification and narrowing of protein array and microarray technology in the context of transcription factor target proteins which is described in the present invention.",
"The creation and utilization of transcription factor target protein microarrays will allow for the high-throughput identification of small molecules, enzymes and other proteins which interact specifically with these targets.",
"Such characterizations will reveal novel enzymatic modification of protein targets as well as protein/protein, protein/DNA, protein/RNA and protein/small molecule interactions.",
"The resulting transcription factor target protein biochemical interaction data will enable researchers to more efficiently focus their efforts on specific aspects of human physiology and disease in order to optimize the design of novel therapeutic intervention strategies for particular human anomalies.",
"In order to create arrays and microarrays of transcription factor target genes and the corresponding target protein sequences, it is necessary to discover and isolate the target genes in a complete and saturable fashion, as the more target genes present in a defined array the more thorough and complete the assessment of the genetic profile for the sample being analyzed.",
"The chromosomal immunoprecipitation (ChIP) assay has been developed previously as a method for the analysis and characterization of transcription factor and/or regulatory protein interactions with known target sequences (Solomon et al., 1988 , Cell, 53: 937-947).",
"Recent advances in this technology now make it possible to identify and establish both direct and indirect relationships between transcriptional regulatory proteins and known as well as unknown target loci.",
"Optimized in a high-throughput format, it is now possible to manipulate regulatory protein/DNA interactions in order to “scan the genome” in search of genes involved in discrete, focused aspects of physiology and disease (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"By combining both modified chromosomal immunoprecipitation/target gene cloning methodologies and array/microarray technology, the presently described invention allows for creation of gene expression and protein interaction analysis tools such as expression and function-restricted arrays of particular focused physiologic relevance.",
"FIG.",
"2 illustrates the construction of transcription factor target nucleotide microarrays through an application of modified chromosomal immunoprecipitation procedures in combination with molecular cloning methodologies.",
"FIG.",
"4 diagrams methodology for the construction and implementation of transcription factor target protein “nonliving” arrays.",
"These arrays and microarrays eliminate random nucleotide and peptide sequence characterization and enhance the detailed analysis of physiologically directed expression and biochemical profiling.",
"Originally, in order to take advantage of the inherent ability of transcription factors to dictate the regulation of specific downstream target genes for purposes of target gene identification, technologies such as CHIP were developed to extract transcription factor/known target gene interactions from living cells and tissues (Solomon et al., 1988 , Cell, 53: 937-947).",
"This technology, however, was limited to the identification of only known transcription factor targets.",
"More recently, the ChIP methodology has been significantly improved upon and implemented for the efficient high-throughput identification and characterization of actively transcribed transcription factor target genes of both known and unknown origin (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Yet in order to fully take advantage of the knowledge of transcription factor target sequences for the purposes of therapeutic development it is apparent that efficient methodologies must be developed and employed which will reveal the genetic activity and biochemical nature of these target loci.",
"The herein described technology accomplishes these goals and further extends the value of transcription factor target gene identification at the biochemical level for purposes of therapeutic development."
],
[
"<SOH> 3.0 SUMMARY OF THE INVENTION <EOH>The application of array and microarray technologies for purposes of assessing genetic as well as biochemical interaction profiles of sample populations has been considerably limited by the construction of both nucleotide and peptide or protein arrays which do not represent discrete aspects of physiology and disease.",
"This lack of focus impairs the analysis of expression patterns by including a great deal of loci which are often not relevant to the particular sample being studied, thereby resulting in an unnecessary allocation of resources to nonrelevant gene expression and biochemical interaction analysis.",
"In addition, significant costs are associated with large-scale microarrays as well as misdirected analysis of valuable limited sample sources.",
"The presently described invention, based upon transcription factor function, circumvents these hindrances by allowing for the construction of physiologic and disease oriented arrays and microarrays.",
"By focusing the creation and implementation of arrays and microarrays on transcription factor target genes and the corresponding proteins, the presently described invention achieves significantly concentrated and discrete genetic and biochemical profiling.",
"Furthermore, the employment of protein arrays and microarrays for purposes of identifying protein/protein, protein/small molecule and enzymatic interactions is becoming increasing valuable for the high-throughput efficient analysis and characterization of potential avenues for therapeutic intervention.",
"It is the discrete organization and annotation of protein amino acid sequences in a format which allows for rapid assessment of interacting partners which drives the rapid accumulation of biochemical information.",
"Yet this organization is of limited value if the microarrayed proteins themselves are of limited utility with respect to the long-term goal of identifying therapeutics for the treatment of human anomalies.",
"The presently described invention lends significant improvement to protein array and microarray technologies by narrowing the arrayed material in a physiological context.",
"By arraying and microarraying proteins which are of known function and value due to their classification as specific transcription factor targets, it will be possible to considerably eliminate the analysis and characterization of irrelevant biochemical interactions.",
"Such narrowing of focus streamlines the drug discovery process, resulting in the requirement of fewer resources and a significant increase in the inherent value of the interaction data obtained.",
"Transcription factors such as p53, for example, are strategically chosen which have been previously demonstrated to play critical roles in certain aspects of disease and physiology ( FIG.",
"1 ).",
"In vivo cross-linkage of protein/DNA complexes is performed in cell lines expressing the factor of interest and immunoprecipitation of protein/chromosomal complexes is subsequently employed through the utilization of antibodies specific for the transcription factor being studied (Solomon et al., 1988 , Cell, 53: 937-947).",
"Cross-linkage is reversed and purified DNA fragments representing target genes for the factor of interest are subjected to gene sequence or corresponding protein microarray construction.",
"The transcribed downstream target sequences represent the functionality of the transcription factor in question as they directly carry out its function with respect to physiology.",
"The protein and peptide outputs for transcription factor target genes represent downstream biochemical effectors for transcription factor function and potentially encode therapeutic targets.",
"The aforementioned nucleotide and peptide or protein sequences are arrayed on solid supports such as nylon membrane, plastic or glass chips or even in vivo (see “living” arrays described below) and utilized to monitor the expression and interaction profiles of samples in question.",
"In order to successfully generate complex, saturable arrays and microarrays for particular aspects of physiology, the chromosomal immunoprecipitation assay has been modified and optimized for the high-throughput identification of both known and unknown transcription factor target loci ( FIG.",
"2 , FIG.",
"4 and PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Improvements include preimmunoprecipitation-immunoprecipitation (“preIP-IP”) utilizing antibodies specific for basal transcriptional machinery, which results in preisolation of only actively transcribed genes thus significantly reducing the acquisition of background random sequences.",
"Subsequent immunoprecipitation is conducted on isolated complexes with antibodies which recognize particular transcription factors involved in discrete aspects of physiology and disease.",
"In addition, sequences are isolated proximal to the transcriptional initiation site which often include 5′ untranslated and coding regions.",
"The ability to direct immunoprecipitation of protein/DNA complexes to only actively transcribed regions of the genome is accomplished in the present invention through the use of antibodies specific for the large subunit of RNA polymerase II, the central component of the basal transcriptional machinery (Chang et al., 1998 , Clinical Immunology and Immunopathology, 89(1): 71-8).",
"In addition, the use of antibodies conjugated to solid supports such as magnetic beads results in significant increases in yield and sensitivity, thus making high-throughput capability feasible (Dynal Corporation Technical Handbook, 1998, Biomagnetic Applications in Cellular Immunology).",
"These solid supports aid in the retrieval of protein/DNA complexes during initial and subsequent immunoprecipitation procedures by providing a matrix for retrieval of complexed material.",
"It is also stated that sequential immunoprecipitation may be performed in any order with the end result being decreased background random sequences and increased yield obtained.",
"Additionally, a further elimination of background random sequences is obtained through the employment of inverse polymerase chain reaction (1-PCR) utilizing oligonucleotides specific for the transcription factor binding site (Ochlnan et al., 1988 , Genetics, 120(3): 621-623; PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Acquisition of PCR products obtained by this methodology strongly infers direct target identity as products will only be obtained upon successful PCR extension from the inherent transcription factor binding sites present within immunoprecipitated fragments.",
"The combination of these novel technologies along with standard cloning procedures and the creation of arrays and microarrays of target sequences obtained allows for the discrete assessment of expression profiling for virtually any aspect of physiology or disease.",
"The proposed strategy would be indispensable for correct diagnostic tracing of disease progression and ultimately therapeutic intervention.",
"One embodiment of the present invention includes arrays and/or microarrays of transcription factor target genes, for the purposes of focusing genetic expression profiling experiments to particular specific entities of physiology and disease.",
"An additional embodiment of the present invention includes the methodology utilized to create the physiology, cellular morphology and disease oriented nucleotide arrays and microarrays.",
"Said methodology, described herein, includes chromosomal immunoprecipitation, double immunoprecipitation utilizing antibodies to the basal transcriptional machinery, solid phase separation technologies and inverse-PCR combined with standard molecular cloning methods.",
"Another embodiment of the present invention is the antibodies utilized to immunoprecipitate crosslinked protein/DNA complexes from intact cells and/or tissues for purposes of creating arrays of transcription factor target genes and ultimately transcription factor target proteins.",
"Yet another embodiment of the present invention includes antibodies conjugated to solid phase supports, such as but not limited to magnetic beads, for purposes of increasing the yield of DNA template obtained and/or reducing the background of nonspecific random sequences obtained, for the further purposes of creating arrays and microarrays of transcription factor target genes.",
"Another embodiment of the present invention includes protein/DNA complexes isolated by modified ChIP methodologies described herein, for purposes of creating arrays and microarrays of transcription factor target genes.",
"Still another embodiment of the present invention includes DNA fragments isolated by the methodology described herein, for the purposes of creating arrays and microarrays of transcription factor target genes.",
"An additional embodiment of the present invention includes the nucleotide sequences corresponding to the transcription factor target genes identified by the methodology described herein, for purposes of creating physiologically and disease focused arrays and microarrays of transcription factor target genes.",
"Still another embodiment of the present invention includes the genetic profile information gleaned from application of transcription factor target nucleotide arrays and microarrays.",
"It is this information which provides valuable insight with respect to particular realms of physiology and disease.",
"Yet another embodiment of the present invention is the application of transcription factor target gene sequence arrays and microarrays for purposes of medical diagnostics and patient prognostics.",
"Another embodiment of the present invention entails the peptide and amino acid sequences of the transcription factor target proteins which are organized and annotated in a microarrayed fashion.",
"It is these sequences which are analyzed for interactions with other proteins, nucleotide sequences and chemical small molecule entities.",
"Yet another embodiment of the present invention includes the methodology for constructing transcription factor target protein arrays.",
"It is the combination of modified chromosomal immunoprecipitation and molecular cloning and protein translation methods with biochemical array technology which results in the creation of valuable array reagents for therapeutic discovery.",
"An additional embodiment of the present invention includes “living”/biological arrays of transcription factor target proteins, for example, in the context of yeast colonies grown in a multiwel format which express the transcription factor target protein of interest.",
"Living arrays allow for the characterization of interactions with the protein of interest in a biological context in which other components or factors may be required and thus provided by the yeast machinery to catalyze interactions with arrayed transcription factor target proteins.",
"Yet another embodiment of the present invention includes “nonliving”/chemical arrays and microarrays of transcription factor target proteins, for example, in the context of amino acid sequences bound either covalently or noncovalently to membranes or glass microchips.",
"An additional embodiment of the present invention includes the proteins, metals, small molecules and nucleotide sequences which are tested for interaction specificities with transcription factor target protein arrays and microarrays.",
"Yet another embodiment of the present invention includes the knowledge obtained from protein microarray studies revealing specific interaction data on transcription factor target proteins and their interactions with other proteins, enzymes or small molecule chemicals.",
"It is the rapid accumulation of transcription factor target protein/protein and protein small molecule interaction data that will result in significant improvements in the efficiency and success of therapeutic development.",
"Still another embodiment of the present invention includes therapies developed as a result of knowledge obtained from the construction and implementation of transcription factor target protein arrays and microarrays."
],
[
"1.0 FIELD OF THE INVENTION The following invention describes the creation of array and microarray profiles of transcription factor targets for the purposes of physiologically focused medical diagnosis, patient prognosis and therapeutic development.",
"It is accomplished through the utilization of modified and improved versions of the chromosomal immunoprecipitation (ChIP) assay and specific cloning methods combined with nucleotide and peptide/protein microarray technology to generate microarrays of transcription factor target gone and peptide sequences.",
"These arrays allow for the efficient and saturable analysis of physiologically focused and restricted gene expression profiles and high-throughput biochemical screening of transcription factor drug target candidates for therapeutically relevant interacting molecules.",
"2.0 BACKGROUND OF THE INVENTION Genetic activity, i.e.",
"the activation or repression of gene transcription, has long been directly correlated with gene function.",
"Transcriptional regulation is the first and perhaps most crucial mechanism by which cells regulate the functions of genes.",
"By providing or denying mRNA templates for translation it is possible to tightly control the intricate cellular mechanisms of determination, division, survival etc.",
"(FIG.",
"1 and for review see Moroy et al., 2000, Cellular and Molecular Life Sciences, 57(6): 957-75).",
"Recently, a number of methods have been developed which allow for the rapid assessment of gene expression in a given sample and thus give insight as genetic profiles for various aspects of physiology and disease.",
"These include, but are not limited to, two dimensional arrays and microarrays of either cDNAs or oligonucleotides representing corresponding mRNAs on solid supports.",
"The arrayed aspect of the technology provides an organized, unbiased method for determining the quantitative and qualitative aspects of gene expression in a given sample population in a massive high-throughput format (a representative set of examples includes U.S. Pat.",
"Nos.",
"6,136,592, 6,100,030, 6,040,138 herein incorporated by reference Debouck et al., 1999, Nature Genetics Supplement, 21: 48-50).",
"It is this macromolecular ability to monitor the expression patterns and levels of genes involved in physiology and disease which allows for many basic science as well as clinical applications such as the assessment of predisposition to particular disorders as well as the possibility of disease prevention or early treatment.",
"It is clear that array technology enables researchers to efficiently ascertain expression patterns and levels of a multitude of loci within a particular sample.",
"In addition, some effort has been directed towards the construction of microarrays which contain templates organized by physiology or functional entity such as cell cycle control or tissue specificity, yet these “focused arrays” are considerably lacking in gene content and limited in number.",
"In addition, it still remains that the majority of genetic microarrays consist of random sequences, the identity and composition of which are often even unknown.",
"Thus, for the most part, arrayed templates of either a nucleotide or peptide origin have yet to be developed such that the array of genes itself depicts something about physiology.",
"It is therefore imperative that more focused, biologically relevant arrays and microarrays of genes be created.",
"For example, arrays of genes known or hypothesized to be involved in a particular disease such as cancer, for example, would be of much more relevance clinically than arrayed organization of random gene sequences.",
"By clustering arrays and microarrays in the context of specific physiologic and disease categories, these arrays can then be more readily subjected to the appropriate sample populations for analysis.",
"This prevents the endless costly analysis of expression data which very well may not be relevant to the sample being studied.",
"Therefore, an initial establishment of clusters and “families” of genes predicted to play particular roles in physiology or disease, and subsequent organization of these clusters in an array and microarray format will allow for a new level of discrete and focused genetic profiling for basic science and medical diagnostics.",
"One method for clustering genes into particular physiologic and disease categories relies upon the exploitation of either the direct or indirect interaction between transcriptional regulators and terminal target genes (for review see Tjian and Maniatis, 1994, Cell, 77: 5-8).",
"Many transcription factors have been extensively demonstrated to play specific roles in very “focused” areas of physiology and disease, primarily through the regulation of target genes.",
"It is possible to exploit this knowledge for the creation and production of functionally relevant arrays.",
"By establishing arrays and microarrays of transcription factor target loci it is possible to narrow the purpose of said arrays for the characterization of expression profiles for specific aspects of physiology.",
"In addition to transcription factor target genetic expression pattern profiling, it is clear that characterization of the biochemical interaction properties of transcription factor targets will enhance therapeutic discovery and development.",
"The ability to characterize protein/protein, chemical/protein, small molecule/protein and enzymatic reaction interactions in a high-throughput and saturable format is of unparalleled value for the eventual design of therapeutic intervention strategies for the treatment of disease.",
"In order to efficiently search for and analyze these types of interactions in a high-throughput yet sensitive format it is necessary to implement variations of array and microarray technology.",
"A number of groups have begun to focus upon the organization of proteins and/or peptide and amino acid sequences in array and microarray formats similar to that for nucleotides sequences.",
"Such an organization has been successfully implemented for the efficient identification of specific interactions between arrayed protein samples and other entities which include, but are not limited to, other proteins, enzymes, metals, sugars, oligosaccharides, chemical compounds, DNA and RNA molecules (a representative set of examples includes U.S. Pat.",
"Nos.",
"5,591,646, 6,156,511, 5,834,318 herein incorporated by reference; MacBeath et al., 2000, Science, 289: 1760-1763 and for review see Emili et al., 2000, Nature Biotechnology, 18: 393-397).",
"These arrays allow for the high-throughput sensitive and specific characterization of interactions between arrayed proteins and other molecules.",
"Yet in order to fully take advantage of protein array technology it is necessary to focus its application to discrete realms of physiology and disease.",
"By concentrating the identities of protein arrays on particular facets of biology a great deal of irrelevant biochemical screening and the costs associated with it can be eliminated.",
"It is the modification and narrowing of protein array and microarray technology in the context of transcription factor target proteins which is described in the present invention.",
"The creation and utilization of transcription factor target protein microarrays will allow for the high-throughput identification of small molecules, enzymes and other proteins which interact specifically with these targets.",
"Such characterizations will reveal novel enzymatic modification of protein targets as well as protein/protein, protein/DNA, protein/RNA and protein/small molecule interactions.",
"The resulting transcription factor target protein biochemical interaction data will enable researchers to more efficiently focus their efforts on specific aspects of human physiology and disease in order to optimize the design of novel therapeutic intervention strategies for particular human anomalies.",
"In order to create arrays and microarrays of transcription factor target genes and the corresponding target protein sequences, it is necessary to discover and isolate the target genes in a complete and saturable fashion, as the more target genes present in a defined array the more thorough and complete the assessment of the genetic profile for the sample being analyzed.",
"The chromosomal immunoprecipitation (ChIP) assay has been developed previously as a method for the analysis and characterization of transcription factor and/or regulatory protein interactions with known target sequences (Solomon et al., 1988, Cell, 53: 937-947).",
"Recent advances in this technology now make it possible to identify and establish both direct and indirect relationships between transcriptional regulatory proteins and known as well as unknown target loci.",
"Optimized in a high-throughput format, it is now possible to manipulate regulatory protein/DNA interactions in order to “scan the genome” in search of genes involved in discrete, focused aspects of physiology and disease (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"By combining both modified chromosomal immunoprecipitation/target gene cloning methodologies and array/microarray technology, the presently described invention allows for creation of gene expression and protein interaction analysis tools such as expression and function-restricted arrays of particular focused physiologic relevance.",
"FIG.",
"2 illustrates the construction of transcription factor target nucleotide microarrays through an application of modified chromosomal immunoprecipitation procedures in combination with molecular cloning methodologies.",
"FIG.",
"4 diagrams methodology for the construction and implementation of transcription factor target protein “nonliving” arrays.",
"These arrays and microarrays eliminate random nucleotide and peptide sequence characterization and enhance the detailed analysis of physiologically directed expression and biochemical profiling.",
"Originally, in order to take advantage of the inherent ability of transcription factors to dictate the regulation of specific downstream target genes for purposes of target gene identification, technologies such as CHIP were developed to extract transcription factor/known target gene interactions from living cells and tissues (Solomon et al., 1988, Cell, 53: 937-947).",
"This technology, however, was limited to the identification of only known transcription factor targets.",
"More recently, the ChIP methodology has been significantly improved upon and implemented for the efficient high-throughput identification and characterization of actively transcribed transcription factor target genes of both known and unknown origin (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Yet in order to fully take advantage of the knowledge of transcription factor target sequences for the purposes of therapeutic development it is apparent that efficient methodologies must be developed and employed which will reveal the genetic activity and biochemical nature of these target loci.",
"The herein described technology accomplishes these goals and further extends the value of transcription factor target gene identification at the biochemical level for purposes of therapeutic development.",
"3.0 SUMMARY OF THE INVENTION The application of array and microarray technologies for purposes of assessing genetic as well as biochemical interaction profiles of sample populations has been considerably limited by the construction of both nucleotide and peptide or protein arrays which do not represent discrete aspects of physiology and disease.",
"This lack of focus impairs the analysis of expression patterns by including a great deal of loci which are often not relevant to the particular sample being studied, thereby resulting in an unnecessary allocation of resources to nonrelevant gene expression and biochemical interaction analysis.",
"In addition, significant costs are associated with large-scale microarrays as well as misdirected analysis of valuable limited sample sources.",
"The presently described invention, based upon transcription factor function, circumvents these hindrances by allowing for the construction of physiologic and disease oriented arrays and microarrays.",
"By focusing the creation and implementation of arrays and microarrays on transcription factor target genes and the corresponding proteins, the presently described invention achieves significantly concentrated and discrete genetic and biochemical profiling.",
"Furthermore, the employment of protein arrays and microarrays for purposes of identifying protein/protein, protein/small molecule and enzymatic interactions is becoming increasing valuable for the high-throughput efficient analysis and characterization of potential avenues for therapeutic intervention.",
"It is the discrete organization and annotation of protein amino acid sequences in a format which allows for rapid assessment of interacting partners which drives the rapid accumulation of biochemical information.",
"Yet this organization is of limited value if the microarrayed proteins themselves are of limited utility with respect to the long-term goal of identifying therapeutics for the treatment of human anomalies.",
"The presently described invention lends significant improvement to protein array and microarray technologies by narrowing the arrayed material in a physiological context.",
"By arraying and microarraying proteins which are of known function and value due to their classification as specific transcription factor targets, it will be possible to considerably eliminate the analysis and characterization of irrelevant biochemical interactions.",
"Such narrowing of focus streamlines the drug discovery process, resulting in the requirement of fewer resources and a significant increase in the inherent value of the interaction data obtained.",
"Transcription factors such as p53, for example, are strategically chosen which have been previously demonstrated to play critical roles in certain aspects of disease and physiology (FIG.",
"1).",
"In vivo cross-linkage of protein/DNA complexes is performed in cell lines expressing the factor of interest and immunoprecipitation of protein/chromosomal complexes is subsequently employed through the utilization of antibodies specific for the transcription factor being studied (Solomon et al., 1988, Cell, 53: 937-947).",
"Cross-linkage is reversed and purified DNA fragments representing target genes for the factor of interest are subjected to gene sequence or corresponding protein microarray construction.",
"The transcribed downstream target sequences represent the functionality of the transcription factor in question as they directly carry out its function with respect to physiology.",
"The protein and peptide outputs for transcription factor target genes represent downstream biochemical effectors for transcription factor function and potentially encode therapeutic targets.",
"The aforementioned nucleotide and peptide or protein sequences are arrayed on solid supports such as nylon membrane, plastic or glass chips or even in vivo (see “living” arrays described below) and utilized to monitor the expression and interaction profiles of samples in question.",
"In order to successfully generate complex, saturable arrays and microarrays for particular aspects of physiology, the chromosomal immunoprecipitation assay has been modified and optimized for the high-throughput identification of both known and unknown transcription factor target loci (FIG.",
"2, FIG.",
"4 and PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Improvements include preimmunoprecipitation-immunoprecipitation (“preIP-IP”) utilizing antibodies specific for basal transcriptional machinery, which results in preisolation of only actively transcribed genes thus significantly reducing the acquisition of background random sequences.",
"Subsequent immunoprecipitation is conducted on isolated complexes with antibodies which recognize particular transcription factors involved in discrete aspects of physiology and disease.",
"In addition, sequences are isolated proximal to the transcriptional initiation site which often include 5′ untranslated and coding regions.",
"The ability to direct immunoprecipitation of protein/DNA complexes to only actively transcribed regions of the genome is accomplished in the present invention through the use of antibodies specific for the large subunit of RNA polymerase II, the central component of the basal transcriptional machinery (Chang et al., 1998, Clinical Immunology and Immunopathology, 89(1): 71-8).",
"In addition, the use of antibodies conjugated to solid supports such as magnetic beads results in significant increases in yield and sensitivity, thus making high-throughput capability feasible (Dynal Corporation Technical Handbook, 1998, Biomagnetic Applications in Cellular Immunology).",
"These solid supports aid in the retrieval of protein/DNA complexes during initial and subsequent immunoprecipitation procedures by providing a matrix for retrieval of complexed material.",
"It is also stated that sequential immunoprecipitation may be performed in any order with the end result being decreased background random sequences and increased yield obtained.",
"Additionally, a further elimination of background random sequences is obtained through the employment of inverse polymerase chain reaction (1-PCR) utilizing oligonucleotides specific for the transcription factor binding site (Ochlnan et al., 1988, Genetics, 120(3): 621-623; PCT Patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Acquisition of PCR products obtained by this methodology strongly infers direct target identity as products will only be obtained upon successful PCR extension from the inherent transcription factor binding sites present within immunoprecipitated fragments.",
"The combination of these novel technologies along with standard cloning procedures and the creation of arrays and microarrays of target sequences obtained allows for the discrete assessment of expression profiling for virtually any aspect of physiology or disease.",
"The proposed strategy would be indispensable for correct diagnostic tracing of disease progression and ultimately therapeutic intervention.",
"One embodiment of the present invention includes arrays and/or microarrays of transcription factor target genes, for the purposes of focusing genetic expression profiling experiments to particular specific entities of physiology and disease.",
"An additional embodiment of the present invention includes the methodology utilized to create the physiology, cellular morphology and disease oriented nucleotide arrays and microarrays.",
"Said methodology, described herein, includes chromosomal immunoprecipitation, double immunoprecipitation utilizing antibodies to the basal transcriptional machinery, solid phase separation technologies and inverse-PCR combined with standard molecular cloning methods.",
"Another embodiment of the present invention is the antibodies utilized to immunoprecipitate crosslinked protein/DNA complexes from intact cells and/or tissues for purposes of creating arrays of transcription factor target genes and ultimately transcription factor target proteins.",
"Yet another embodiment of the present invention includes antibodies conjugated to solid phase supports, such as but not limited to magnetic beads, for purposes of increasing the yield of DNA template obtained and/or reducing the background of nonspecific random sequences obtained, for the further purposes of creating arrays and microarrays of transcription factor target genes.",
"Another embodiment of the present invention includes protein/DNA complexes isolated by modified ChIP methodologies described herein, for purposes of creating arrays and microarrays of transcription factor target genes.",
"Still another embodiment of the present invention includes DNA fragments isolated by the methodology described herein, for the purposes of creating arrays and microarrays of transcription factor target genes.",
"An additional embodiment of the present invention includes the nucleotide sequences corresponding to the transcription factor target genes identified by the methodology described herein, for purposes of creating physiologically and disease focused arrays and microarrays of transcription factor target genes.",
"Still another embodiment of the present invention includes the genetic profile information gleaned from application of transcription factor target nucleotide arrays and microarrays.",
"It is this information which provides valuable insight with respect to particular realms of physiology and disease.",
"Yet another embodiment of the present invention is the application of transcription factor target gene sequence arrays and microarrays for purposes of medical diagnostics and patient prognostics.",
"Another embodiment of the present invention entails the peptide and amino acid sequences of the transcription factor target proteins which are organized and annotated in a microarrayed fashion.",
"It is these sequences which are analyzed for interactions with other proteins, nucleotide sequences and chemical small molecule entities.",
"Yet another embodiment of the present invention includes the methodology for constructing transcription factor target protein arrays.",
"It is the combination of modified chromosomal immunoprecipitation and molecular cloning and protein translation methods with biochemical array technology which results in the creation of valuable array reagents for therapeutic discovery.",
"An additional embodiment of the present invention includes “living”/biological arrays of transcription factor target proteins, for example, in the context of yeast colonies grown in a multiwel format which express the transcription factor target protein of interest.",
"Living arrays allow for the characterization of interactions with the protein of interest in a biological context in which other components or factors may be required and thus provided by the yeast machinery to catalyze interactions with arrayed transcription factor target proteins.",
"Yet another embodiment of the present invention includes “nonliving”/chemical arrays and microarrays of transcription factor target proteins, for example, in the context of amino acid sequences bound either covalently or noncovalently to membranes or glass microchips.",
"An additional embodiment of the present invention includes the proteins, metals, small molecules and nucleotide sequences which are tested for interaction specificities with transcription factor target protein arrays and microarrays.",
"Yet another embodiment of the present invention includes the knowledge obtained from protein microarray studies revealing specific interaction data on transcription factor target proteins and their interactions with other proteins, enzymes or small molecule chemicals.",
"It is the rapid accumulation of transcription factor target protein/protein and protein small molecule interaction data that will result in significant improvements in the efficiency and success of therapeutic development.",
"Still another embodiment of the present invention includes therapies developed as a result of knowledge obtained from the construction and implementation of transcription factor target protein arrays and microarrays.",
"4.0 DESCRIPTION OF THE FIGURES FIG.",
"1 Is a diagrammatic illustration of transcriptional regulation by the tumor suppressor protein p53.FIG.",
"2 Is an illustrative flowchart representing the manufacturing and construction of transcription factor target loci nucleotide microarrays for the purposes of medical diagnostics and patient prognostics (see text for details).",
"FIG.",
"3 Is a proposed example of an application of microarrayed p53 targets to the analysis of a particular sample as it progresses temporally from a normal to a tumorigenic cancerous phenotype and upon administration of different therapeutic strategies (see text for details).",
"FIG.",
"4 Is a diagrammatic illustration of the process of constructing and utilizing transcription factor target protein arrays and microarrays to determine target protein interacting molecules of either a chemical or biological nature (see text for details).",
"FIG.",
"5 Is a diagrammatic representation of the utilization of a “nonliving”/chemical transcription factor target protein microarray for the purposes of defining interacting molecules and the organization of data obtained into a database format (see text for details).",
"FIG.",
"6 Is a diagrammatic representation of the utilization of “living”/biological transcription facto target protein arrays for the purposes of defining interacting proteins, enzymes etc.",
"in the context of yeast (see text for details).",
"FIG.",
"7 Illustrates the implementation of transcription factor target protein arrays for the discovery and development of cancer therapeutics by focusing on the biochemical properties of targets for the transcription factor p53 (see text for details).",
"Table 1 Is an example of transcription factor target gene microarray expression pattern data accumulated in a numerical format (see text for details).",
"Table 2 Is an example of the combination of phenotypic and environmental influences on genetic expression patterns depicted in a microarrayed numerical format (see text for details).",
"5.0 DETAILED DESCRIPTION OF THE INVENTION 5.1 Expression Analysis: The Development of Nucleotide Microarrays Organized, large-scale analysis of expression patterns within given tissue or cell population samples has only recently become feasible.",
"The ability to monitor the expression patterns of large numbers of genes and thus obtain a “genetic profile” of virtually any particular sample at any given timepoint promises to reveal in great detail molecular clues to physiology and disease.",
"Indeed, known as “Transcriptomics,” this field is rapidly emerging as an essential and integral subdivision of the field of functional genomics (Drysdale et al., 2000, Yeast, 17(2):159-66).",
"A number of technologies have matured which allow for the organized annotation of genes for large-scale expression profiling purposes.",
"These currently include the use of photochemical or inkjet technologies to array either cDNA or oligonucleotide sequences on solid supports such as glass slides or nylon membranes (INSERT MICROARRAY PATENT REFS HERE DeRisi et al., Science, 278: 680-686).",
"It is predicted that eventually all genes from multiple organisms will be microarrayed for purposes of expression profiling of virtually any sample RNA population.",
"Indeed, the entire compilation of loci present in the yeast genome has already been organized into a microarray format and said arrays have been proven to reveal functional genomics information in a highly reproducible manner (Spellman et al., 1998, Cell, 9: 3273-3297).",
"The analysis of expression patterns and levels utilizing microarrays involves relatively straightforward recording of light emissions.",
"Nucleotide microarrays are analyzed primarily for changes in expression via altered light wavelengths upon binding of sample RNA to cDNA or oligonucleotide sequences.",
"The more bound RNA within a particular sequence slot present within the array, the brighter the emission of light and the greater the change in wavelength.",
"Expression levels can therefore be accurately monitored with extreme sensitivity.",
"In addition, given the micro aspect of the technology, relatively small sample populations can be analyzed for the actual character of the “transcriptome.” The application of nucleotide microarray technology for purposes of monitoring gene expression levels and patterns is clear.",
"From a basic science perspective, it is now possible to characterize changes in genetic expression patterns within a given tissue or cell line due to mutation or changes in environmental stimuli, for example.",
"From a medical perspective, disease diagnosis and prognosis will benefit enormously from microarray technology.",
"Monitoring gene expression patterns will result in the ability to diagnose predisposition to a certain disorder prior to its manifestation, and will allow doctors a head start on prevention and/or treatment.",
"Yet, as discussed, current nucleotide microarray technology fails to organize and annotate subsets of loci which are specific for particular realms of physiology and disease.",
"The presently described invention addresses this issue as well as the problems relating to it and provides a streamlined, high-throughput mechanism for the construction of physiologically specific microarrays of transcription factor target genes.",
"5.2 Protein Arrays and Microarrays More recently, array and microarray technology has been developed for the characterization of protein/protein and protein/small molecule interactions.",
"Specifically, methodologies have been developed which attach synthetic peptide and/or amino acid sequences corresponding to particular proteins to solid matrices with such sequences exposed on the surface of the matrix for purposes of accessibility by molecules of various origins which may or may not directly interact (MacBeath et al., 2000, Science, 289: 1760-1763).",
"These “nonliving”/chemical arrays provide high-throughput characterization of direct interactions between organized, annotated proteins and/or peptides and other proteins or small molecules including metals, oligosaccharides and nucleotide sequences (FIG.",
"5).",
"The technology is dependent upon the ability of these molecules to interact directly, however, without the requirement of other cofactors or modifications of the arrayed proteins which might be provided by living cells (Uetz et al., 2000, Nature, 403: 623-627).",
"“Living”/biological arrays have also been developed which provide the opportunity for the modification of either the arrayed proteins or putative interacting proteins by the eukaryotic cellular machinery.",
"Such modification or even the addition of other cellular components may be required for specific interaction between arrayed proteins and either small molecules or other proteins which are to be tested on the arrays.",
"Living arrays have been successfully formulated in the context of the yeast strain S. cerevisiae, although others including those of high eukaryotic or bacterial origin may be constructed.",
"In addition, however, protein arrays lack the focus necessary to efficiently scan for interacting molecules related to subsets of human physiology.",
"As an example, arrayed clones of yeast are propagated in minimal media such that DNA sequences encoding open reading frame/GALA activation domain fusion proteins are translated in each prospective yeast colony.",
"Interactions screens are performed by mating these arrayed yeast clones with another carrying ORF/GALA DNA binding domain fusion proteins.",
"Survival of these colonies in a minimal media environment is dependent upon the interaction of these proteins and subsequent recruitment to a GAL4 DNA binding site upstream of a minimal promoter driving synthesis of an essential amino acid which is lacking in the minimal media context (FIG.",
"6).",
"This strategy and others which include calorimetric assays in yeast have proven successful in identifying living arrayed protein interactions (Uetz et al., 2000, Nature, 403: 623-627).",
"5.3 Gene Expression and Function Over the past 10 years enormous efforts have been focused on the sequencing, either partial or full length, and annotation of libraries of actively transcribed genes from limitless sources originating from countless organisms (for review see Zweiger et al., 1997, Trends in Biotechnology, 17: 429436).",
"Recent advances in sequence database development have resulted in complex organization and annotation of known sequences into extensive gene families.",
"Often these families demonstrate considerable conservation in sequence, and surprisingly expression pattern identity between organisms as diverse as fly and man.",
"The gene encoding the transcription factor Nk×2.1 in humans, for example, is orthologous to the tinman locus in flies and each exibits similar roles in heart development for both fly and man (Chen et al., 1996, Developmental Genetics, 19(2): 119-30).",
"This is but one example whereby both sequence composition as well as expression pattern give insight as to genetic function.",
"The utility of studying gene expression patterns and correlating these patterns to genetic and/or genomic function has become standard.",
"Recent work in the analysis of yeast gene function suggests that the function of a particular locus or group of loci can be rapidly and accurately assessed by employing microarray technology to monitor changes in expression patterns of affected genes following mutation of particular loci.",
"In addition, the same group has demonstrated the applicability of microarray analysis of gene expression to the study of pharmacological target validation (Hughes et al., 2000, Cell, 102: 109-126).",
"Given these exciting results in yeast it is tempting to speculate how both nucleotide and protein/peptide array technology might be applied to the characterization of expression patterns in human cells and tissues.",
"Yet the enormous complexity of the human genome requires a much more directed and focused approach to microarray construction, implementation and analysis and poses unique problems and issues which the presently described invention seeks to overcome.",
"5.4 High-Throughput Identification of Transcription Factor Targets It is clear that transcription exemplifies function.",
"Through tight regulatory cascades transcription factors direct unique symphonies of gene expression which constantly change with respect to environmental and temporal cues.",
"A number of transcription factors have been characterized as functioning within a tight range with respect to physiology.",
"That is, transcription factors often focus function on specific physiologic entities, most often through the activation or repression of target gene expression (for a review focused on pituitary organogenesis see Rhodes et al., 1994, Current Opinions in Genetic Development, 4: 709-717).",
"Factors such as the estrogen receptor and the tumor suppressor p53, for example, control both cellular proliferation as well as programmed cell death and have been demonstrated to play crucial roles in the manifestation of breast cancer through the activation or repression of terminal target genes (FIG.",
"1; Tenbaum et al.",
"1997, International Journal of Biochemistry and Cell Biology, 29: 1325-1341; Levine et al., 1991, Nature, 351: 453-456).",
"Other factors play roles in regulating cellular fate through early steps in the determination of specific lineages during development.",
"An example of this is evident in the functional characterization of the transcription factor ikaros, which controls B and T cell development during hematopoiesis (Nichogiannopoulou et al., 1998 Seminars in Immunology, 10: 119-125).",
"Still other factors regulate the development and/or function of specific organs.",
"Similar to that mentioned for Nk×2.5 and tinman mentioned above, the GATA family of transcription factors has been shown to play a variety of roles in regulating cardiac specific gene activity both pre- and postnatally (Herzig et al., 1997, Proceedings of the National Academy of Sciences, 94: 7543-7548).",
"It is therefore evident that a dissection of the genetic hierarchies and ultimately an identification of target genes for these and other transcription factors as well as the biochemical interacting partners for these targets will yield valuable insight as to the genetic profile of particular discrete aspects of physiology.",
"In order to saturably identify and annotate in a microarray format transcription factor target genes and proteins of both known and unknown as well as direct and indirect origin, the presently described invention expands upon previously developed technology (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference) by organizing transcription factor target genes and the corresponding protein/peptide sequences into an annotated arrayed format for use in expression and biochemical profiling.",
"5.5 Construction of Transcription Factor Target Nucleotide Microarrays FIG.",
"2 illustrates the process for creation of transcription factor target nucleotide microarrays from manipulation of cell lines to final linkage of target sequences to two dimensional solid supports.",
"As initial technology for the identification of transcription factor targets is described in detail in a previous patent application (FIG.",
"2 and U.S. Patent Ser.",
"No.",
"60/225,225, filed Aug. 14, 2000 and herein incorporated by reference), it will only briefly be discussed herein.",
"The process for construction of target microarrays initiates with the growth and expansion of appropriate cell lines expressing the transcription factor of interest, either endogenously or ectopically.",
"Cell lines from which transcription factor target genes may be discovered via methodologies provided by the presently described invention include, but are in no way limited to 13C4 (mouse/mouse, hybrid, hybridoma), 143 B (human, bone, osteosarcoma), 2 BD4 E4 K99 (mouse/mouse, hybrid, hybridoma), 3 C9-D11-H11 (mouse/mouse, hybrid, hybridoma), 3 E 1 (mouse/mouse, hybrid, hybridoma), 34-5-8 S (mouse/mouse, hybrid, hybridoma), 3T3 (mouse, Swiss albino, embryo), 3T3 L1 (mouse, Swiss albino, embryo), 3T6 (mouse, Swiss albino, embryo), 5 C 9 (mouse/mouse, hybrid, hybridoma), 5G3 (hybrid, hybridoma), 6-23 (clone 6) (rat, thyroid, medullary, carcinoma), 7 D4 (mouse/rat, hybrid, hybridoma), 72 A1 (mouse/mouse, hybrid, hybridoma), 74-11-10 (mouse/mouse, hybrid, hybridoma), 74-124 (mouse/mouse, hybrid, hybridoma), 74-22-15 (mouse/mouse, hybrid, hybridoma), 74-9-3 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, E cell), 76-7-4 (mouse/mouse, hybrid, hybridoma), 7C2C5C12 (mouse/mouse, hybrid, B cells x myeloma, hybridoma), 9 BG 5 (mouse/mouse, hybrid, hybridoma), 94-3 (mouse/mouse, hybrid, hybridoma), A 172 (human, glioblastoma), A 375 (human, malignant melanoma), A 72 (dog, golden retriever, connective, not defined tumor), A-427 (human, Caucasian, lung, carcinoma), A-498 (human, kidney, carcinoma), A-704 (human, kidney, adenocarcinoma), A549 (human, lung, carcinoma), ACHN (human, Caucasian, kidney, adenocarcinoma), ACT 1 (mouse/mouse, hybrid, hybridoma), AE1 (mouse/mouse, hybrid, hybridoma), AE-2 (mouse/mouse, hybrid, hybridoma), Aedes albopictus (mosquito-Aedes albopictus, larvae), AGS (human, Caucasian, stomach, adenocarcinoma), AK-D (cat, lung, embryonic), Amdur II (human, Caucasian, skin, fibroblast, methylmalonicacidemia), AV 3 (human, amnion), B 95.8 (monkey, marmoset, leukocyte), B-63 (mouse, mammary gland, carcinoma), B2-1 (mouse, BALB/c, embryo), B50 (rat, nervous system, nervous tissue glial tumor), B69 (mouse/mouse, hybrid, hybridoma), B95a (monkey, marmoset), BAE (bovine, aorta), BALB 3T12-3 (mouse, BALB/c, embryo), BALB 3T3 clone A31 (mouse, BALB/c, embryo), BB (fish—Ictalurus nebulosus (bulhead brown catfish), trunk), BBM.1 clone E9 (mouse/mouse, hybrid, hybridoma), BC3H1 (mouse, brain, brain tumor), BCE C/D-1b (bovine, cornea), BeWo (human, placenta, choriocarcinoma), BF-2 (fish—bluegill fry, caudal trunk), BGM (monkey, African green, kidney), BEK 21 clone 13 (hamster, golden Syrian, kidney), BNL CL.2 (mouse, BALB/c, liver, embryonic), BNL SV A.8 (mouse, liver, embryonic), BS/BEK (bovine, kidney, embryonic), BSC-1 (monkey, African green, kidney), BT (bovine, turbinate), Bu (MR-31) (buffalo, lung), BUD-8 (human, Caucasian, skin, fibroblast), BXPC-3 (human, pancreas, adenocarcinoma), C 1271 (mouse, R1, mammary gland, mammary tumor), C2C12 (mouse, muscle), C32 (human, melanoma, amelanotic), C 6 (rat, glial tumor), Caco-2 (human, Caucasian, colon, adenocarcinoma), Caki-1 (human, Caucasian, kidney, carcinoma), Caki-2 (human, Caucasian kidney, carcinoma), CaLu-1 (human, Caucasian, lung, carcinoma, epidermoid), Calu-3 (human, Caucasian, lung, adenocarcinoma), CAPAN 1 (human, Caucasian, pancreas, adenocarcinoma), CAPAN 2 (human, Caucasian, pancreas, carcinoma), CAR (fish—goldfish, fin), CCF-STTG1 (human, Caucasian, astrocytoma, anaplastic, grade IV), CCRF S 180 II (mouse, CFW, sarcoma), CCRF-CEM (human, Caucasian, peripheral blood, leukemia, acute lymphoblastic), CCRF-SB (human, Caucasian, peripheral blood, leukemia, acute lymphoblastic), CEM/C2 (human, leukemia, T cell), Cf2Th (dog, thymus), Chang liver (human, liver), CHO K1 (hamster, Chinese, ovary), CHP 3 (human, Black, skin, fibroblast, galactosemia), CHP 4 (human, Black, skin, fibroblast, asymptomatic galactosemia), CHSE 214 (fish—salmon, embryo), Clone 1-5c WKD of Chang Conjunctiva (human, conjunctiva), Clone M-3 (mouse, (CxDBA) F1, skin, melanoma), CMT 93 (mouse, C57BL/ICRFat, rectum, carcinoma), COS-1 (monkey, African green, kidney), COS-7 (monkey, African green, kidney), CPA (bovine, endothelium, pulmonary artery), CPA 47 (bovine, endothelium, pulmonary artery), CPAE (bovine, endothelium, pulmonary artery), CRFK (cat, domestic, kidney), CR1-D11 (rat, NEDH, insulinoma), CSE 119 (fish—salmon, embryo), CV 1 (monkey, African green, kidney), CVC 7 (Agrothis segetum, hybrid, hybridoma), D 17 (dog, bone, sarcoma, osteogenic), Daudi (human, Black, lymphoma, Burkitt), DB 9 G.8 (mouse/mouse, hybrid, hybridoma), DB 1-Tes (dolphin, Delphinus bairdi, testis), DeDe (hamster, Chinese, lung), Detroit 510 (human, Caucasian, skin, fibroblast, galactosemia), Detroit 525 (human, Caucasian, skin, fibroblast, Turner syndrome), Detroit 529 (human, Caucasian, skin, fibroblast, trisomy 21/Down syndrome), Detroit 532 (human, Caucasian, foreskin, trisomy 21/Down syndrome), Detroit 539 (human, Caucasian, skin, fibroblast, trisomy 21/Down syndrome), Detroit 548 (human, Caucasian, skin, fibroblast, partial D trisomy), Detroit 550 (human, skin, fibroblast), Detroit 551 (human, Caucasian, skin, embryonic), Detroit 562 (human, Caucasian, pharynx, carcinoma), Detroit 573 (human, Caucasian, skin, fibroblast, B/D translocation), Detroit 6 (human, bone marrow), DK (dog, beagle, kidney), DON (hamster, Chinese, lung), DU 145 (human, Caucasian, prostate, carcinoma), Duck embryo (duck, Pekin, embryo), E.Derm (horse, dermis), EBTr (bovine, trachea, embryonic), ECTC (bovine, thyroid, embryonic), ECV304 (human, Asiatic, umbilical cord), EIAV 12E8.1 (mouse/mouse, hybrid, hybridoma), Ep 16 (mouse/mouse, hybrid, hybridoma), EPC (fish, carp epidermal, epithelioma), EREp (rabbit, skin, embryonic), ESK4 (pig, kidney, embryonic), FBHE (bovine, heart, embryonic), Fc 2 Lu (cat, lung, embryonic), Fc 3 Tg (cat, tongue, embryonic), FeLV 3281 (cat, lymphoma), FHM (fish—minnow, skin), FL (human, amnion), FRhK4 (monkey, rhesus, kidney, embryonic), G-7 (mouse, Swiss-Webster, muscle), G.8 (mouse, Swiss-Webster, muscle), GCT (human, lung, metastasis, histiocytoma), GH 1 (rat, Wistar-Furth, pituitary tumor), GH 3 (rat, Wistar-Furth, pituitary tumor), Girardi heart (human, heart), GK 1.5 (mouse/rat, hybrid, hybridoma) H 16-L104R 5 (mouse/mouse, hybrid, hybridoma), H 9 (human, leukemia, acute lymphoblastic), H 4-H-E (rat, liver, hepatoma), H4 (human, Caucasian, brain, nervous tissue glial tumor), H4-II-E-C3 (rat, AxC, liver, hepatoma), H4TG (rat, liver, hepatoma), H-9c2(2-1) (rat, BDIX, heart), Hak (hamster, Syrian, kidney), HCT 116 (human, colon, carcinoma), HCT-8 (human, intestine, ileocecal, adenocarcinoma), HEL 299 (human, Caucasian, lung, embryonic), HeLa (human, Black, cervix, carcinoma, epitheloid), HeLa 229 (human, Black, cervix, carcinoma, epitheloid), HeLa S 3 (human, Black, cervix, carcinoma, epitheloid), Hep 2 (human, Caucasian, larynx, carcinoma, epidermoid), Hep 3B2.1-7 (human, liver, carcinoma, hepatocellular), Hep G2 (human, Caucasian, liver, carcinoma, hepatocellular), Hepa 1-6 (mouse, liver, hepatoma), HFL (human, lung), HG 261 (human, Caucasian, sldn, fibroblast, Fanconi anemia), HGP 24 (human, gingival stroma), HL 60 (human, Caucasian, peripheral blood, leukemia), HOS (human, Caucasian, bone, osteosarcoma), HRT 18 (human, rectum-anus, adenocarcinoma), Hs 683 (human, neuroglia, glioma), Hs 863.T (human, bone, sarcoma, Ewing's), HS 883.T (human, bone, giant cell, sarcoma), HS 888 Lu (human: Caucasian, lung), Hs-27 (human, foreskin), HSDM1C1 (mouse, Swiss albino, fibrosarcoma), HT 1080 (human, Caucasian, acetabulum, fibrosarcoma), HT 1376 (human, Caucasian, bladder, carcinoma), HT-29 (human, Caucasian, colon, adenocarcinoma), HuTu 80 (human, adenocarcinoma), I 10 (mouse, BALB/cJ, testis, Leydig cells, testicular tumor), IB-RS-2 (pig, kidney), IBRS-2 D10 (pig, kidney), IEC-6 (rat, intestine, small), IM-9 (human, Caucasian, bone marrow, multiple myeloma), IMR 31 Bu (buffalo, lung), JMR 32 (human, Caucasian, neuroblastoma), IMR-90 (human, Caucasian, lung, embryonic), Intestine 407 (human, Caucasian, intestine, embryonic), J 111 (human, leukemia, monocytic), J 774A.",
"1 (mouse, BALB/c, monocyte-macrophage, not defined tumor), Jensen sarcoma (rat, sarcoma), JH 4 clone 1 (guinea pig, strain 13, lung), Jiyoye (human, Black, ascitic fluid, lymphoma, Burkitt), JM (human, leukemia, T cell), Jurka J6 (human, leukemia, T cell), K 562 (human, Caucasian, pleural effusion, leukemia, chronic myeloid), KATO III° (human, Mongoloid, stomach, carcinoma), KB (human, Caucasian, mouth, carcinoma, squamous cell), KHOS/NP (human, Caucasian, bone, osteosarcoma), KMP (mouse), L 1210 (mouse, ascitic fluid, leukemia, lymphocytic), L 132 (human, lung, embryonic), L 21.6 (mouse hybrid, hybridoma), L 243 (mouse/mouse, hybrid, hybridoma), L 5.1 (mouse/mouse, hybrid, hybridoma), L 929 (mouse, C3H (An, connective), L6 (rat, skeletal muscle), LC 540 (rat, Fisher, testis, Leydig cells, testicular tumor), LLC-MK2 (monkey, rhesus, kidney), LLC-PK1 (pig, kidney), LLC-RK1 (rabbit, New Zealand white, kidney), LLC-WRC 256 (rat, Walker, carcinoma), LM from NCTC clone 929 (mouse, C3H/An, connective), LM TK negative (mouse, C3H/An, connective), LNCaP.FGC (human, Caucasian, prostate, carcinoma), LS 180 (human, Caucasian, colon, adenocarcinoma), M 1 (mouse, SL, bone marrow, leukemia, myeloid), M-2E6 (mouse/mouse, hybrid, hybridoma), M2-1C6-4R3 (mouse/mouse, hybrid, hybridoma), MA 104 (monkey, African green, kidney, embryonic), mAB 35 (mouse/rat, hybrid, B cells x myeloma, hybridoma, B cell), MARC 145 (monkey, kidney), Mc Coy (mouse), MC/CAR (human, plasmacytoma, B cell), MCF 7 (human, Caucasian, breast, adenocarcinoma), MDBK (bovine, kidney), MDBK (13U 100) (bovine, kidney), MDCC MSB 1 (chicken, avian, spleen, lymphoma), MDCK (dog, cocker spaniel, kidney), MDOK (sheep, kidney), MDTC RP 19 (turkey, lymphocyte, Marek's disease), MEL Im (monkey, rhesus, mammary gland, mammary tumor), MG-63 (human, bone, osteosarcoma), MH 1 C 1 (rat, buffalo, liver, hepatoma), MH-S (mouse, lung), MIA PaCa-2 (human, Caucasian, pancreas, carcinoma), MiCl1 (mustela vison (mink), lung), MK-D6 (mouse/mouse, hybrid, hybridoma), MLA 144 (gibbon, lymphosarcoma), MOLT-3 (human, peripheral blood, leukemia, acute lymphoblastic T cell), MOLT4 (human, peripheral blood, leukemia), MPC-11 (mouse, BALB/c, myeloma), MPK (minipig, kidney), MRC 5 (human, lung, embryonic), MRSS-1 (mouse/mouse, hybrid, hybridoma, T cell), MS (monkey), Mv 1 Lu (mustela vison (mink), lung), MVPK-1 (pig, kidney), NA C 1300 clone (mouse, brain, neuroblastoma), Namalwa (human, Black, lymphoma, Burkitt), NCTC 2544 (human, skin, keratinocyte), NCTC clone 3526 (monkey, rhesus, kidney), Neuro-2a (mouse, albino, neuroblastoma), NIH:OVCAR-3 (human, Caucasian, adenocarcinoma, ovary), NOR 10 (mouse, muscle), NRK 49F (rat, kidney), NSO (mouse, BALB/c, myeloma), OA1 (sheep, brain), OHH1.K (deer, kidney), OKT 3 (mouse/mouse, hybrid, hybridoma), OKIT 4 (mouse/mouse, hybrid, hybridoma), OKT 8 (mouse/mouse, hybrid, hybridoma), P 3 HR 1 human, lymphoma, Burkitt), P3 88 D1 (mouse, DBA/2, monocyte-macrophage, lymphoma), P3 NS1 Ag4 (mouse, myeloma), P3NP/PFN (mouse/mouse, hybrid, hybridoma), P815 (mouse, mastocytoma), PANC-1 (human, Caucasian, pancreas, carcinoma), PC 61-5-3 (mouse/rat, hybrid, hybridoma), PC-12 (rat, adrenal medulla, pheochromocytoma), PD 5 (pig, kidney), PEG 1-6 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, B cell), PK 15 (pig, kidney), PLC/PRF/5 (human, liver, hepatoma, Alexander cells), Pt K1 (marsupial—potoroo, kidney), QT 35 (quail, Japanese, fibrosarcoma), QT 6 (quail, Japanese, fibrosarcoma), R 2 C (rat, Wistar-Furth, testis, Leydig cells, testicular tumor), R 9 ab (rabbit, New Zealand white, lung), R D (human, Caucasian, muscle, rhabdomyosarcoma, embryonal), R63 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, B cell), RAB-9 (rabbit, New Zealand white, skin, fibroblast), Raji (human, Black, lymphoma, Burkitt), RBL 1 (rat, leukemia, basophilic), RFL 6 (rat, Sprague-Dawley, lung), RK 13 (rabbit, kidney), RK 13/1 (rabbit, kidney), RPMI 1788 (human, Caucasian, peripheral blood), RPMI 1846 (hamster, golden Syrian, skin, melanoma, melanotic), RPMI 2650 (human, nasal septum, carcinoma, squamous cell), RPMI 8226 (human, peripheral blood, myeloma), RR 1022 (rat, Amsterdam, sarcoma), RTG 2 (fish—trout rainbow, gonad), RTO (fish—trout, rainbow, ovary), Saos-2 (human, Caucasian, bone, osteosarcoma), Sf 1 Ep (rabbit, domestic, epidermis), SIRC (rabbit, cornea), SK-LU-1 (human, Caucasian, lung, adenocarcinoma, grade EII), SK-MES-1 (human, lung, carcinoma, squamous cell), SK-NEP-1 (human, Caucasian, kidney, Wilms' tumor), SK-OV-3 (human, Caucasian, ovary, adenocarcinoma), SSE 5 (fish—trout, embryo), STO (mouse, SIM, embryo), SV-T2 (mouse, BALB/c, embryo), SW 13 (human, Caucasian, adrenal cortex, adenocarcinoma), T 98 G (human, Caucasian, glioblastoma), Th 1 Lu (bat, lung), TE 671 (human, Caucasian, medulloblastoma), TK TS 13 (hamster, Syrian, kidney), U 937 (human, Caucasian, pleural effusion, lymphoma, histiocytic), VERO (monkey, African green, kidney), VERO 76 (monkey, African green, kidney), VERO C 1008 (monkey, African green, kidney), WC 1 (fish, dermis, sarcoma), WF 2 (fish—Walley whole fry, fibroblast), WI 26 VA 4 (human, Caucasian, lung, embryonic), WI 38 (human, Caucasiar lung, embryonic), WI 38 VA 13 (human, Caucasian, lung, embryonic), WI-1003 (human, lung), WISH (human, amnion), WM 115 (human, skin, melanoma), XC (rat, Wistar, sarcoma), Y 1 (mouse, LAF1, adrenal cortex, adrenal tumor), ZR-75-1 (human, Caucasian, breast, carcinoma) and any other as yet undiscovered or uncharacterized cell lines through which the presently described invention may be implemented for the discovery of transcription factor target genes.",
"It is contemplated by the present invention that tissues of various sources may also be utilized for the purposes of constructing transcription factor target nucleotide and/or protein/peptide arrays.",
"Tissues include, but are not limited to heart, brain, spleen, lung, liver, muscle, kidney, testis, ovary, gut, hypothalamus, pituitary, tooth bud, mesoderm, ectoderm, endoderm, neural tube, somite, smooth muscle, cardiac muscle, skeletal muscle and all embryonic tissues from all possible organisms and all possible timepoints.",
"Intact cells or tissues are treated with protein/DNA cross-linkage reagents such as formaldehyde as previously described.",
"While the present invention employs formaldehyde as a chemical component for the cross-linking of protein/DNA complexes in living cells and tissues, it is in no way limited to this reagent for fixation.",
"Other chemicals may also be utilized to fix proteins to DNA (Benashski et al., Methods, 2000, 22: 365-371).",
"Some of these include, but are in no way limited to homobifunctional compounds difluoro-2,4-dinitrobenzene (DFDNB), dimethyl pimelimidate (DMP), disuccinimidyl suberate (DSS), thcarbodiimide reagent EDC, psoralens including 4,5′,8-trimethylpsoralen, photo-activatable azides such as 125I(S-[2-(4-azidosalicylamido)ethylthio]-2-thiopyridine) otherwise known as AET, (N-[4(p-axidosalicylamido)butyl]-3′[2′-pridyldithio]propionamide) also known as APDP, the chemical cross-linking reagent Ni(II)-NH2-Gly-Gly-His-COOH also known as Ni-GGH, sulfosuccinimidyl 2.",
"[(4-axidosalicyl) amino]ethyl]-1,3-dithiopropionate) also known as SASD, (N-14-(2-hydroxybenzoyl)-N-11 (4-azidobenzoyl)-9-oxo-8,11,14-triaza-4,5-ditheatetradecanoate) and any as yet uncharacterized or undiscovered reagents which result in the cross-linking of protein/DNA complexes in living cells and tissues.",
"Cellular extracts are purified and sonicated to yield the desired chromatin fragment size and said extracts are subjected to antibodies linked to solid phase supports such as M450 tosylactivated magnetic beads.",
"Other magnetic beads contemplated by the present invention and created by Dynal Corporation which may be utilized as a solid phase support for the chromosomal immunoprecipitation reaction described herein include Dynabeads M450 uncoated, Dynabeads M-280 Tosylactivated, Dynabeads M450 Sheep anti-Mouse IgG, Dynabeads M450 Goat anti-Mouse IgG, Dynabeads M450 Sheep anti-Rat IgG, Dynabeads M450 Rat anti-Mouse IgM, Dynabeads M-280 sheep anti-Mouse IgG, Dynabeads M-280 Sheep anti-Rabbit IgG, Dynabeads M450 sheep anti-Mouse IgG1, Dynabeads M-450 Rat anti-Mouse IgG1, Dynabeads M450 Rat anti-Mouse IgG2a, Dynabeads M-450 Rat anti-Mouse IgG2b, Dynabeads M450 Rat anti-Mouse IgG3.Other magnetic beads which are also contemplated by the present invention as providing utility for the purposes of sequential immunoprecipitation include streptavidin coated Dynabeads.",
"While the presently described invention employs magnetic beads as the solid phase to increase yield and recovery of protein/DNA complexes during sequential chromosomal immunoprecipitation, it is in no way the only solid phase support system which may be implemented successfully to increase yield and sensitivity.",
"Other solid phase supports contemplated by the present invention include, but are not limited to, sepharose, chitin, protein A cross-linked to agarose protein G cross-linked to agarose, agarose cross-linked to other proteins, ubiquitin cross-linked to agarose, thiophilic resin, protein G cross-linked to agarose, protein L cross-linked to agarose and an support material which allows for an increase in the efficiency of purification of protein/DNA complexes.",
"Antibodies specific for components of the basal transcriptional machinery and/or the transcription factor of interest recruit both the factor and bound potential target DNA sequences to the solid support matrix.",
"A series of washing steps removes nonspecific background bound sequences.",
"Cross-linkage is reversed and a heterogenous population of DNA templates putatively representing transcription factor target genes is retrieved.",
"The implementation of molecular biological procedures including inverse-PCR (Ochman et al., 1988, Genetics, 120(3): 621-623) and cDNA library screening results in the isolation of transcribed sequences for each target gene as well as confirmation of direct target gene identity.",
"Upon identification of transcription factor target loci, microarrays may subsequently be constructed which annotate and organize these target sequences into specific physiologically focused expression analysis tools based upon the original transcription factors immunoprecipitated in the modified sequential ChIP process.",
"Transcription factor target sequences are attached to solid supports such as nylon membranes, glass or plastic chips in the form of either cDNAs or oligonucleotides.",
"Although the presently described invention contemplates the use of nylon membranes as well as glass or plastic chips as solid phase supports it is in no way limited to these materials for the ultimate construction of transcription factor target nucleotide arrays.",
"Other solid supports include, but are in no way limited to nitrocellulose and metals of any kind.",
"A blueprint of each array documents the identity of each gene and its location relative to others on the two dimensional solid support.",
"Said arrays and microarrays are hence subjected to appropriate tissue and cell samples to produce sample expression profiles.",
"Hybridization of RNA or cDNA samples from test populations to the transcription factor target nucleotide arrays allows for sensitive expression profiling of particular realms of physiology.",
"By narrowing the focus of each array and microarray to transcription factor target genes, these arrays serve specific purposes with respect to physiology, morphology and disease, and eliminate many of the disadvantages of large-scale whole genome and/or unfocused array technology.",
"The creation of both nucleotide and peptide or protein arrays and microarrays can be performed for a variety of tissue and cell type-specific transcription factors for the purposes of physiologically focused gene expression analysis and biochemical interaction characterization.",
"While the presently described invention focuses on the discovery of both known and previously undiscovered target loci for the transcription factor p53 and the corresponding array construction for these targets, it is in no way limited in its utility for this particular transcription factor or the targets thereof.",
"Other transcription factors and corresponding targets of prokaryotic, eukaryotic and viral origin contemplated and covered by the present invention include, but are not limited to A2, AAF, abaA abd-A, Abd-B, ABF1, ABF-2, ABI4, Ac, ACE2, ACF, ADA2, ADA3, ADA-NF1, Adf-1, Adf-2a, Adf-2b, ADR1, AEF-1, AF-1, AF-2, AFLR, AFP1, AFX-1, AG, AG1, AG2, AG3, AGIE-BP1, AGL11, AGL12, AGL13, AGL14, AGL15-1, AGL15-2, AGL17, AGL2, AGL3, AGILA, AGL6, AGL8, AGL9, AhR, AIC3, AIC2, AIC3, AIC4, AIC5, AID2, AIIN3, ALF1B, ALL-1, alpha.",
"1, alpha2uNF1, alpha2uNF2, alph2uNF3, alpha-CP1, alpha-CP2a, alpha-CP2b, alpha-factor, alphaH0, alphaH2, alphaH3, alpha-IRP, alpha-PAL, alpha2uNF1, alpha2uNP3, alphaA-CRYBP1, alphaH2-alphaE3, alphaMHCBF1, Alx-3, ALx4, ALY, AMDA, AmdR, aM-2, AML1, AMLia, AMLlb, AMLlc, AMLIDeltaN, AML2, AML3, AMT1, AMY-1L, A-Myb, AN2, AnCF, ANF, ANF-2, ANR1, Antp, AP-1, AP-2, AP-2alphaisoform2, AP-2alphaisoform3, AP-2alphaisoform4, AP-3, AP3-1, AP3-2, AP4, AP-5, APC, APERALA1, APETALA3, AR, ARA, AREA, AREB6, ARG R1, ARG R11, armadillo, Arnt, ARP-1, ARP7, ARP9, ARR1, AS-C T3, AS321, ASF-1, ASH-1, ASH-3b, ASP, AT-13P2, ATBF1-A, ATBP, AT-BP1, AT-BP2, ATF, ATF-1, ATF-3, ATF-3deltaZ1P, ATF-adelta, ATF-like, Athb-1, Athb-2, Ato, Axial, AZF1, B factor, B″, BAF1, B-TFIID, band I factor, BAP, Barx-1, BAS, BBF1, BBF2a, BBF3, BBFa, Bcd, BCF1, Bcl-3, BCL-6, BD73, BDF1, beta-1, BETA1, BETA2, beta-catenin, beta-factor, BF-1, BF-2, BGP1, Binl, Blimp-1, BmFTZ-F1, B-Myb, B-Myc, BP1, BP2, B-Peru, BR-C Z1, BR-C Z2, BR-C Z4, Brachyury, BRF1, BrIA, Bm-3a, Brn4, Brn-5, BUF1, BUF2, BAF1, BAS1, BCFII, beta-factor, BETA3, BLyF, BP2, BR-C Z3, brachyuray, brahma, BRF1, Brn1, Brn2, Bm-3a, Brn-3b, Brn-4, Brn-5, Bro, Btd, BTEB, BTEB2, BUF, BUF1, BUF2, BUR6, byr3, BZIP910, BIP911, c-abl, c-Ets-1, c-Ets-2, c-Fos, c-Jun, c-Maf, c-myb, c-Myc, c-Qin, c-Re1, C/EBP, C/EBPalpha, C/EBPbeta, C/EBPdelta, C/EBPepsilon, C/EBPgamma, C1, CAC-binding protein, CACCC-binding factor, Cactus, Cad, CAD1, CAF17, CAL, CAP, CAR2, CArG box-binding protein, CAT8, CAUP, CBF1, CBF2, CBF3, CBF4, CBF5, CBF-A, CBF-B, CBF-C, CBP, CBTF, CCAAT-binding factor, CCBF, CCF, CCG1, CCK-1a, CCK-1b, CCR4, CD28RC, CDC10, Cdc68, CDF, cdk2, CDP, CDP2, Cdx-1, Cdx-2, Cdx-3, Cdx4, CEBF CEF1, ceh-1, ceh-10, ceh-12, ceh-13, ceh-14, ceh-16, CEH-18 and (all ceh related factors), CeMyoD, c-Ets-1, C-Ets-1A, c-Ets-1B, CF1, Cfla, CF2-I, CF2-11, CF2-III, CFF, CG-1, CHA4, CHOP-10, Chox-2.7, Chx10, CIN5, CIIIB1, c-Jun, CKB3, Clox, c-Maf, CMB1, CMB2, c-Myb, c-Myc, CNBP, Cnc, CoMP1, core-binding factor, CoS, COUP, COUP-TF, CP1, CP1A, CP1B, CP1C, CP2, CPBP, CPC1, CPE binding protein CPRF-1, CPRF-2, CPRF-3, CPM10, CPM5, CPM7, CPPI, CPRF-1, CPRF-2, CPRF-3, CPRF4a, CPRF-4b, all CREB related factors, CRE-BP1, CRE-BP2, CRE-BP3, CRE-BPa, CreA, CREB, CREB-2, CREBomega, CREMalpha, CREMbeta, CREMdelta, CREMepsilon, CREMgamma, CREMtaualpha, CRF, all CRM related factors, Croc, Crx, CRZ1, CSBP-1, CtBP, CTCF, CTF, CUM1, CUM10, CUP2, CUP9, CUS1, Cut, Cux, CWH-1, CWH-2, CWH-3, Cx, cyclin A, cyclin T, cyclin T1, cyclin T2, cyclin T2a, cyclin T2b, CYS3, D-MEF, Da, all DAL related factors, DAP, DAP1, DAT1, DAX1, DB1, DBF-A, DBF4, DBP, DBSF, dCREB, DDB, DDB-1, DDB-2, dDP, dE2F, DEAP3, DEF, DEFH2, Delilah, delta factor, deltaCREB, deltaE1, deltaEF1, deltaMax, DENF, DENF1, DENF2, DENF3, DEP, DEP2, DEP3, DEP4, DERmo-1, DF-1, DF-2, DF-3, Dfd, dFRA, DHR3, DHR38, DHR78, DHR96, dioxin receptor, dJRA D1, DII, all Dlx related factors, DM-SSRP1, DMLP1, Dof3, DP-1, DP-2, Dpn, Drl, all DREB related factors, DRF1, DRF2, DRTF, DSC1, DSIF, DSP1, DST1, DSXF, DSXM, DTF, E, E1A, E2, E2BP, E2F, E2F-BF, E2F-I, E4, E47, E4BP4, E4F, E4TF2, E7, E74, E75, EAP1, EAP2L, EAP2S, EAR2, EBF, EBF1, EBNA, EBP, EBP40, EC, EC5, ECF, ECF2, ECF3, ECH, ECM22, EcR, eE-TP EF-1A, EF-C, EF1, EFgamma, EGM1, EGM2, EGM3, Egr, EGR2, EGR3, eH-TF, E1a, EivF, EKLF, Elf-1, Elg, Elk-1, ELP, Elt-2, ErBP-1, embryo DNA binding protein, Emc, EMP, EMF2, EMF3, EMF4, Ems, Ernx, Emx-1, Emx-2, En, ENH-binding protein, ENKTF-1, epsilonF1, ER, Erbeta, EREBP-1, EREBP-2, EREBP-3, EREBP4, ERF1, Erg, Esc, Escl, esg, Esx-1a, Esx-1b, ETF, ETL, Eve, Evi, Evx, Exd, Ey, en-1, en-2, f(alpha-f(epsilon), F27E5.2, F2F, FACB, F-ACT1, factor 1, factor 2, factor 3, factor B 1, factor B2, factor delta, factor I, FAR, Fbfl, FBF-A1, FBP, FBP1, FBP11, FBP2, FBP6, FBP7, f-EBP, FHL1, FIM, FKBP59, Fkh, FKH1, Fkh-1, FKH2, Fkh-2, Fkh-3, Fkh4, Fkh-5, Fkh-6, FKHR, FKHRL1, FKHRL1P1, FKHRL1P2, FKHRP1, FlbD, FLC, FLF, Flh, Fli-1, FLO, FLO8, FLV-1, FOG, FosB, FosB/SF, Fra-1, Fra-2, Freac-1, Freac-10, Freac-2, Freac-3, Freac4, Freac-5, Freac-6, Freac-7, Freac-8, Freac-9, FRG Y1, FRG Y2, EWF, FTS, Ftz, FIZ-F1, FTZ-Flbeta, FZF1G factor, G factor, G/HBF-1, G10BP, G6 factor, GA-BF, GABP, GABP alpha, GABP-beta1, GABP-beta2, GAF, GAF1, GAF2, GAG2, GAL11, GAL4, GAL80, GammaCAAT, gammaCAC1, gammaCAC2, gamma-factor, gammaOBP, GAMYB, GAT1, GAT2, GAT3, GAT4, GATA-1, GATA-LA, GATA-1B, GATA-2, GATA-3, GATA4, GATA-5, GATA-5A, GATA-, GATA-6, GATA-6A, GATA-6B, GBF, GBF1, GBF12, GBF1A, GBF1B, GBF2, GBF2A, GBF2B, GBF3, GBF4, GBF9, GBP, GC1, GC2, GC3, GCF, GCM, GCMa, GCMb, GCN4 GCN5, GCNF, GCR1, GCR2, GE1, GEBF-I, GF1, GFI, Gfi-1, GFII, GHF3, GHF-5, GHF-7, GIS1, GKLF, GL1, Gl15, G12, Glass, GLI, GLI3, GLN3, GLO, GM-PBP-1, GP, GR, GR alpha, GR beta, GRF-1, Grg-4, Grg-5, GRIP1, Groucho, Gsb, GSBF1, Gsbn, Gsc, Gsc A, Gsc B, Gt, GT-1, GT-2, GT-IC, GT-IIA, GT-IIBalpha, GT-IIBbeta, GTS1, Gtx, GZF3, H16, H1TF1, H1TF2, H2B abp 1, H2RIIBP, H4TF-1, H4TF-2, HAC1, HAL9, HALF-I, HAP1, HAP2, HAP3, HAP4, HAP5, Hb, HB9, HBLF, HBP-1, HBP-1a, HBP-1a(1), HBP-1a(c14), HBP-1b, HIBP-1b(c1), HCM1, HDaxx, heat-induced factor, HEB, HEB1-p67, HEB1-p94, HEF-1B, HEF-1T, HEF4C, HEN1, HEN2, HeRunt-1, HES-1, HES-2, HES-3, HES-5, Hesxl, Hex, HFH-1, HFH-11A, HFH-11B, HFH-2, HFH-3, HFH-4, HFH-5, HFH-6, HFH-7, HPH-8, HIF-1, HIF-1alpha, HIF-1beta, HiNF-A, HiNF-B, HiNF-C, HiNF-D, HiNF-D3, HiNF-E, HiNF-M, HiNF-P, HIP1, HIR1, HIR2, H1R3, HIRA, HIV-EP2, HIlf, Hlf-alpha, Hlf-beta, HLX, Hlx, HMBP, HMG I, HMG I(Y), HiMG Y, HMGI-C, HMS1, HMS2, HNF-1, HNF-1A, HNF-1B, HNF-1C, HNF-3, HNF3(-like), HNF-3alpha, HNF-3B, HNF-3beta, HNF-3gamma, HNF-4, HNF4(D), HNF4alpha1, BNF4alpha2, HNF4alpha3, BNF-4alpha4, HNF4alpha7, HNF-4beta, HNF-4gamma, HNF-6, HNF-6alpha, HNF-6beta, hnRNP K, Hox11, HOXA1, HOXA10, HOXA10PL2, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXC10, HOXC11, HOXC12, HOXC13, HOXC4, HOXC5, HOXC6, HOXC6 (PR1), HOXC6 (PR11), HOXC8, HOXC9, HOXD1, HOXD10, HOXD11, HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, HP1 site factor, Hp55, Hp65, HrpF, HSE-binding protein, HSF, HSF1, HSF_, HSF24, HSF30, HSF8, hsp56, Hsp90, HST, HSTF, HY5, IBF, IBP-1, IBR, ICER, ICER-I, ICER-Igamma, ICER-II, ICER-Iigamma, ICP4, ICSBP, Id1, Id1.25, Id1H′, Id2 Id3, Id3/Heir-1, Id4, IDS1, IE1, IEBP1, IEFga, IF1, IF2, IFH1, IFNEX, IgPE-1, IgPE-2, IgPE-3, Ik-1, Ik-2, Ik-3, Ik4, Ik-5, Ik-6, Ik-7, Ik-8, IkappaB, IkappaB-alpha, IkappaB-beta, IkappaB-gamma IkappaB-gamma1, IkappaB-gamma2, IkappaBR, IK13, ILF, ILRF-A, IME1, IME4, IN02, IN04, INSAF, IPF1, I-POU, IRBP, IRE-ABP, IREBF-1, IRF-1, IRF-2, IRF-3, irIB-2a, Irx-3, ISGF-1, ISGF-3, ISGF-3alpha, ISGF-3gamma, Isl-1, ISRF, ISRFI, ITF, nT-1, ITF-2, IUF-1, Ixrl, JRF, Jun-D, JunB, JunD, K06B9.5, K07C11.1, kappaY factor, KAR4, KBF2, kBF-A, KBP-1, KCS1, KER1, -1, Kid-i, Kinl7, KN1, Kni, Knox3, KNRL, Koxl, Kr, Kreisler, KRF-1, Krox-20, Krox-24, Ku autoantigen, KUP, Lab, LAC9, LBP, LBP-1, LBP-1a, Lc, LCR-F1, LD, Ldbl, LEF-1, LEF-1B, LEF 1S, LEU3, LF-A1, LF-A2, LF-B2, LF-C, LFY, LG2, LH-2, Lhx-3, Lhx-3a, Lhx-3b, Lhx4, LHY, Lim-1, Lim-3, lin-1, lin-11, lin-14A, lin-14B1, lin-14B2, lin-29A, lin-29B, lin-31, lin-32, lin-39, LIP15, LIPl9, LIT-1, LKLF, Lmo1, Lmo2, Lmx-1, L-Myc1, L-Myc-1, L-Myc-1 (long form), L-Myc-1(short form), L-Myc-2, LR1, LSF, LSIRF-2, LUN, Lva, LVb-binding factor, LVc, LXRalpha LyF-1, Lyl-1, LYS14, Lz, M factor, M-Twist, M1, m3, Mab-18, MAC1, Mad, MAF, MafB, MafF, MafG, MafK, Mal63, MAPF1, MAPF2, MASH-1, MASH-2, mat-Mc, mat-Pc, MATal, MATalphal, MATalpha2, MATH-1, MATH-2, Max1, M factor, M1, m3, Mab-18 (284 AA), Mab-18 (296 AA), mab-5, MAC1, Madl, Mad3, Mad4, MADS1, MADS11, MADS16, MADS2, MADS24, MADS3, MADS4, MADS45, MADS5, MADS6, MADS7, MADS8, MADS9, MAF, MafB, MafF, MafG, MafK, MAL13, MAL23, MAL33, MAL63, MAPF1, MAPF2, MASH-1, MASH-2, Matl-Mc, MATal, MATalphal, MATalpha2, MATH-1, MATH-2, mat-Pc, Max, Max1, Max2, MAZ, MAZ1, MB67, MBF1, MBF-1, MBF2, MBF3, MBF-I, MBP1, MBP-1 (1), MBP-1 (2) MBP-2, MCBF, MCM1, MCM1+MATalpha1, MDBP, MDBP-2, MDS3, mec-3, MECA, MED11, MBD2, MED4, MED6, MED7, MED8, mediating factor, MEF1, MEF-2, MEF-2B, MEF-2B-1, MEF-2B-2, MEF-2B-3, MEF-2B4, MEF-2C, MEF-2C (433 AA form), MEF-2C (465 AA form), MEF-2C (473 AA form), MEF-2C/delta32 (441 AA form), MEF-2D, M-2D (506 AA form), MEF-2D (514 AA form), MEF-2D00, MEF-2DOB, MEF-2DA-0, MEF-2DA-B, MEF-2DA0, MEF 2DAB, Meis-1, Meis-1-1, Meis-1-2, Meis-1-3, Meis-1-4, Meis-1a, Meis-1b, Meis-2a, Meis-2b, Meis-2c, Meis-2d, Meis-3, Mesol, MET18, MET28, MET31, MET32, MET4, Mf2, MF3, MFH-1, Mfh-1, MGA1, Mhox, MHR1, M1, MIP1, MIF-1, MIG1, MIG2, Mix.1, Mix.2, Mix.3, Mix.4, Mixer, MIITA, Miz-1, MKR2, MLP, MM-1, MNBia, MNBlb, MNF1, MNR2, MOK-2, MOP3, MOT1, MOT3, MP4, MPBF, MR, MRF4, MRR, Msh, MSN1, MSN2, MSN4, Msx-1, Msx-2, MIB Zf, MTF1, MTF-1, MTH1, Mt11, mtTF1, M-Twist, muEBP-B, muEBP-C2, MUF1, MUF2, Mxi1, MYB A, MYB.PH1, MYB.PH2, MYB.PH3, MYB 1, Myb-1, all Myb related proteins, MYB-P1, MYBST1, myc-CF1, myc-PRF, MYC-RP, Myef-2, Myf-3, Myf-4, Myf-5, Myf-6, Myn, MyoD, Myogenin, MZF-1, Nabl, Nau, NBF, NC1, NCB2, NDT80, NELF, NeP1, NER1, Net, NeuroD, NF III-a, NF III-c, NF III-e, NF-1, NF-1/L, NF-1/Redl, NF-1A, NF-1A1, NF-1A1.1, NF-1A2, NF-1A3, NF-1A4, NF-1A5, NF-1B, NF-1B1, NF-1B2, NF-1B3, NF-1B4, NF-1C1, NF-1C2, NF-1C4, NF-1X NF-1X1, NF-1×2, NF-1×3, NF2d9, NF-4FA, NF-4FB, NP4FC, NF-A, NF-A3, NF-AB, NFalpha1, NFalpha2, NFalpha3, NFalpha4, NF-AT, NFAT-1, NF-AT3, NF-Atc, NF-ATc3, NF-Atp, NF-Atx, NF-BA1, NfbetaA, NF-CLEOa, NF-CLEOb, NF-D, NFdeltaE3A, NFdeltaE3B, NFdeltaE3C, NFdeltaE4A, NFdeltaE4B, NFdeltaE4C, Nfe, NF-E, NF-E1b, NF-E2, NP-E2 p45, NF-E3, NF-E4, NFE-6, NF-EM5, NF-Gma, NF-GMb, NF-H1, NF-H2, NF-H3, NFH3-1, NFH3-2, NF13-3, NPH3-4, NF-IL-2A, NP-IL-2B, NF-InsE1, NF-InsE2, NF-InsE3, NF-jun, NF-kappaB, NF-kappaB(-like), NF-kappaB 1, NF-kappaB I precursor, NF-kappaB2, NF-kappaB2 (p49), NF-kappaB2 precursor, NF kappaE1, NF-kappaE2, NF-kappaE3, NF-lambda2, NF-MHCIIA, NF-MHCIIB, NF-muE1, NP-muE2, NF-muE3, NF-muNR, NF-ODC1, NF-S, NP-TNF, NF-U1, NP-W1, NF-W2, NF-X, NF-X1, NF-X2NF-X3, NF-Xc, NF-Y, NF-Y′, NF-YA, NP-YB, NF-YC, NF-Zc, NF-Zz, NGFI-B, NGFI-C, NHP-1, NHP-2NHP3, NHP4, NHR1, NIP, NIRA, NIT2, NIT4, Nkx-2.1, Nkx-2.2, Nkx-2.5, NLS1, NMH7, NMHC5, Nmi, N-Myc, N-Mycl, N-Myc2, nob-1A, nob-1B, N-Oct-2alpha, N-Oct-2beta, N Oct-3, N-Oct-4, N-Oct-5a, N-Oct-5b, NOR1, NOT, NOT1, NOT2, NOT3, NOT5, NP-rn, NP-IV, NP-TCII, NP-Va, NPX1, NRD I, Nrf1, NRF-1, Nrf2, NRF-2NRF-2beta1, NRF-2gamma1, NRFA, NRG1, NRG2, NRL, NS-1, NSDD, NTF, NTF1, NUC-1, Nur77, NUT1, NUT2, OBF, OBF-1, OBF3.1, OBF3.2, OBF4, OBF5, OBP, OBPi, OC-2, OCA-B, OCSBF-1, OCSTF, Oct-1, Oct-10, Oct-11, Oct-1A, Oct-1B, Oct-1C, Oct-2, Oct-2.1, Oct-2.3, Oct-2.4, Oct-2.6, Oct-2.7, Oct-2.8, Oct-2B, Oct-2C, Oct4, Oct-4A, Oct-4B, Oct-5, Oct-6, Oct-7, Oct-8, Oct-9, Octa-factor, octamer-binding factor, oct-B2, oct-B3, Oct-R, Odd, ODR7, OG-12, OG-2, OG-9, OHP1, OHP2, Olf-1, OM1, ONR1, Opaque-2, OPM1, OSBZ8, Otd, Otx1, Otx2, Otx4, Ovo, OZF, P (long form), P (short form), P1, p107, p130, p28 modulator, p300, p38erg, p40x, p45, p49erg, p53 as, p55, p55erg, p58, p65delta p67, PAB1, PacC, PAF1, pag-3, PAGL1, pal-1, Pap1+, par-2, Paraxis, PARP, Pax-1, Pax-1/9, Pax-1/9 (AmphiPax-1), Pax-1/9-I, Pax-1/9-II, Pax-1/9-111, Pax-1/9-IV, Pax-1/9-V, Pax-1/9-VI, Pax-2, Pax-2.1, Pax-2.2, Pax-2/5/8, Pax-2a, Pax-2b, Pax-3, Pax-3A, Pax-3B, Pax-4, Pax-4a, Pax-4b, Pax-4c, Pax-4d, Pax-5, Pax-6, Pax-6 (Pax-QNR), Pax-6/Pd-5a, Pax-6 12.1, Pax-6 12.2, Pax-6 4.1, Pax-6 4.2, Pax-6 J2, Pax-7, Pax-8, Pax-8a, Pax-8b, Pax-8c, Pax-8d, Pax-8e, Pax-8f, Pax-8g, Pax-9, Pax-A, Pax-B, Pb, PBF, PBP, Pbx-1a, Pbx-1b, Pc, PC2, PC4, PC4 p9, PC5, Pcrl, PCRE1, PCT1, PDM-1, PDM-2, PDR1, PDR3, Pdx-1, PEA1, PEA2, PEA3, PEB1, PEBP2, PEBP2alpha, PEBP2alphaA/Osf2, PEBP2alphaA/til-1, PEBP2alphaA/til-1 (Y), PEBP2alphaA/til-1(U), PEBP2alphaA1, PEBP2alphaA2, PEBP2alphaB1, PEBP2alphaB2, PEBP2beta, PEBP2beta1, PEBP2beta2, PEBP2beta3, PEBP5, Pep-1, PERIANTIA, pes-lapes-1b, PF1, PF3, PGA4, PGD1, pha4, PHAN, PHD1, phiAP3, PHO2, PHO4, PHO80, Phox-2, php-3, P1, P11, P12, pie-1, PIHbox9, PIP2, Pit-1, Pit-1a, Pit-1b, Pit-1c, Pitx-3, PLE, PLE1/DEFH200, PLE/DEFH49, PLE/DEFH72, PLE/SQUA, PLZF, PNPI2, PO-B, pointedP1, pointedP2, Pontin52, pop-1POP2, POTM1-1, pou[c], Pou2, pox neuro, PP1, PP2, PPAR, PPARalpha, PPARbeta, PPARgamma, PPR1, PPUR, PPYR, PR PR A, PRb, Prd, PRDI-BF1, PRDI-BFc, PREB, Prop-1, protein a, protein b, protein c, protein d, PRP, PSE1, Psx-1, Psx-2, P-TEFb, PTF, PThL, PTF1-alpha, PTF1-beta, PTFalpha, PTFbeta, PTFdelta, PTFgamma, Ptx-1, Ptx-2, Ptx-2B, Pu box binding factor, Pu box binding factor (BJA-B), PU.1, Pu.1, PUB 1, PuF, PUF-I, Pur factor, Pur-1, PUT3, P-wr, PX, PZF1, qa-1F, QBP, QUT1, R, R1, R2, RAD1, Rad-1, RAD18, RAD2, RAF, RAP1, RAP2.5, RAR, RAR-alpha, RAR-alpha1, RAR-alpha2, RAR-beta, RAR-beta1, RAR-beta2, RAR-beta3, RAR-beta4, RAR-gamma, RAR-gamma1, RAR-gamma2, RAV1, RAV2, Rax, Rb, RBP60, RBP-Jkappa, Rc, RC1, RC2, RCS1, REB, REB1, Reb1p, Re1A, Re1B, repressor of CAR1 expression, REV-ErbAalpha, REX-1, RF1, RF2a, RFX, RFX1, RFX2, RFX3, RFX5, RF-Y, RGM1, RGR1, RGT1, RIC1, RIM1, RIP14, RITA 1, RLM1, RME1, RMS1, Ro, Roaz, ROM1, ROM2, RORalpha1, RORalpha2, RORalpha3, RORbeta, RORgamma, Rox, Roxl, ROX3, RPF1, RPGalpha, RPH1, RREB-1, RRF1, RRF2, RRF3 RRN10, RRN11, RRN3, RRN5, RRN6, RRN7, RRN9, RS2, RSC4, RSRFC4, RSRFC9, RSV-EF-11 RTF1, RTG1, RTG2, RTG3, Runt, RVF, Rx, Rx1, Rx2, Rx3, RXR-alpha, RXR-beta, RXR-beta1, RXR-beta2, RXR-gamma, S8, SAP1, SAP-1a, SAP-1b, SBF, SBF-1, Sc, SCBPalpha, SCBPbeta, SCBPgamma, SCD1/BP, SCM-inducible factor, Scr, S-CREM, S-CREMbeta, Sd, Sdc-1, SDS3, SEF1, SEF-1 (1), SEF-1 (2), SEF3, SEF4, SEM-4, SET1, SET2, SF1, SF-1, SF-2, SF-3, SF-A, SFL1, SGC1, SGF-1, SGF-2, SGF-3, SGF-4, Shn, SHP, SHP1, SHP2, SIF, SIG1, Sm, Sm-pllo, SfI-p15 SfI-p18, Sim1, Sim2, Six-1, Six-2, Six-3, Six-3alpha, Six-3beta, Six4, Six-4A, Six-4B, Six-4C, Six-5, Six-6, Skn-1, SKN7, SKO1, SLM1, SLM2, SLM3, SLM4, SLM5, Slp1, slp2, S-Myc, Sn, SN (sienna), Sna, SNF5, SNF6, SNP1, So, SOX-11, SOX-12, Sox-13, SOX-15, Sox-18, Sox-2, Sox-4, Sox-5, SOX-6, SOX-9, Sox-LZ, Sp1, Sp2, Sp3, Sp4, SPA, spE2F, Sph factor, Sp1-B, SpOtx, Sprin-1, SpRunt-1, SQUA, SRB10, SRBil, SRB2, SRB4, SRB5, SRB6, SRB7, SRB8, SRB9, SRD1, SR1 BP, SREBP-1, SREBP-1a, SREBP-1b, SREBP-1c, SREBP-2, SREP, SRE-ZBP, SRF, SRY, Sry h-1 Sry-beta, Sry-delta, ssDBP-1, ssDBP-2, SSRP1, Staf, Staf-50, STAT, STAT1, STATlalpha, STATibeta, STAT2, STAT3, STAT4, STAT5, STAT5A, STAT5B, STAT6, STC, STD1, Ste11, STE12, STE4, STF1, STF2, STKA, STM, STP1, Stral3, StuAp, su(f), Su(H), su(Hw), SUM-1, SUP, SVP, SVP46, SVI/SNF complex, SWIL, SWI2, SWI3, SWI4, SWI5, SWI6, SWP, T-Ag, t-Pou2, T3R, T3R-alpha, T3R-alpha1, T3R-alpha2, T3R-beta, T3R-beta1, T3R-beta2, TAB, T-Ag, TAG1, Tal-1, Tal-1beta, Tal-2, TAR factorTat, Tax, TCF, TCF-1TCF-1A, TCF-1B, TCF-1C, TCF-1D, TCF-1E, -1F, TCF-1G, TCF-2, TCF-2alpha, TCF-3, TCF-3B, TCF-3C, TCF-3D, TCF4, TCF-4(K), TCF-4B, TCF-4E, TCF-A, TCF-B, TCFbetal, TDEF, TEA1, TEC1, TEF, TEF 1, TEF-1, TEF2, TEF-2, Te1, TF68, TFE3, TFE3-L, TFp3-S, TFEB, TFEC, TFIIA, TFIIA (13.5 kDa subunit), Tf-LF1, Tf-LF2, TF-Vbeta, TGA, TGA1, TGAla, TGA2, TGA3, TGA6, TgF1, TGGCA-binding protein, TGT3, Th1, THM1, THM18, THM27, THRA1, TIF1, TIF2, TIN-1, TINY, TIP, tI-POU, TLE1, T11, Tlx, TM3, TM4, TM5, TM6, TM8, TMF, t-Pou2, TR2, TR2-11, TR2-9, TR3, TR4, Tra-1 (long form), Tra-1 (short form), TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREF2, TRF, TRF (2) Trident, TSAP, TSF3, Tsh, TIF-1, TTF-2, TTG1, Ttk 69K, Ttk 88K, TTP, Ttx, ttx-3, TUBF, Twi, TXREF, TyBF, UAY, UBF, UBF1, UBF2, UBP-1, Ubx, UCRB, UCRF-L, UEF-1, UEF-2, UEF-3, UEF4, UF1-H3beta, UFA, UFB, UFO, UGA3, UHF-1, UME6, unc-30, unc-37, unc4, Unc-86, URF, URSF, URTF, USF, USF2, vab-3, vab-7, vaccinia virus DNA-binding protein, Vav, Vax-1, Vax-2, VBP, VDR, v-ErbA, VETF, v-Ets, v-Fos, vHNF-1, vHNF-1A, vF-1B, vHNF-1C, VITF, v-Jun, v-Maf, Vmw65, v-Myb, v-Myb/v-Ets, V-Myc, v-Myc, Vpl, Vpr, v-Qin, v-Re1, VSF-1, WC1, WC2, Whn, WT1, WT1I, WZF1, X-box binding protein, X-Twist, X2BP, xaml, X-box binding protein, XBP-1, XBP-2, XBP-3, XF1, XF2, XFD-1, XFD-2, XFD-3, XFG20, XGRAF, Xiro1, Xiro2, Xiro3, xMEF-2, XPF-1, XrpFI, XW, XX, yan, YB-1, YB-3, Ybx-3, YEB3, YEBP, Y1, YNG2, YPF1, YY1, ZAP, ZEB, ZEML, ZEM2/3, Zen-1, Zen-2, Zeste, ZF1, ZF2, ZF5, Zfh-1, Zfh-2 Zfp-35, ZID, ZIP-1A, ZIP-2A, ZIP-2B, ZM1, ZM38, Zmhoxla, Zn-15, ZNF174, ZPT2-1, ZPT2-2, ZPT2-3, ZPT2-4, Zta.",
"In addition, any factors which retain the ability to regulate gene expression, either through activation or repression, and are as of yet previously undiscovered or uncharacterized are covered by the present invention.",
"5.6 Basic Biology Applications of Transcription Factor Target Gene Microarrays The study of gene regulation as it relates to cellular and even organismal biology is essential for the thorough understanding of events which occur at the molecular level to initiate and maintain biological processes which drive embryonic development or ensure survival.",
"By assessing the activation and/or repression of genetic loci known or predicted to plays roles in particular aspects of physiology, it is possible to correlate transcriptional regulatory mechanisms with specific phenotypes.",
"Nucleotide microarrays of transcription factor targets allow for the narrowed and focused assessment of expression profiles of genes relevant to the hypotheses being addressed.",
"Directing attention only to genes which are known or thought to play roles in a particular facet of biology saves much time and expense as needless irrelevant expression profiles are not pursued.",
"For example, the study of cell cycle control and cell division is at the forefront of cancer research and promises to ultimately provide avenues for treatment of this devastating disease.",
"A great deal of these studies focus on cell lines which progress temporally from a nontumorigenic state to a cancerous phenotype.",
"Microarray analysis of targets for transcription factors such as the tumor suppressor p53 (E1-Diery et al., 1993, Cell, 75: 817-825) and Rb (Iunaief et al., 1994, Cell, 79(1):119-30) utilizing these lines as RNA sources will undoubtedly reveal distinct genetic profiles for each stage of tumor progression.",
"A unique transcriptional profile or “transcriptome” may be obtained at different temporal points during progression of the tumorigenic phenotype.",
"Information gleaned from target nucleotide microarray studies of this nature not only provides unique fingerprints of cellular physiology but also reveals potential mechanisms that drive deviation from the normal cellular fate.",
"5.7 Medical Applications of Transcription Factor Target Gene Microarrays The presently described invention entails the creation of physiologically focused arrays and microarrays through the annotation and organization on solid phase supports of transcription factor target genes.",
"Transcription factors may be chosen which represent a particular clinical aspect of physiology, based upon previous research implicating these factors in said areas and the targets for these factors efficiently identified and arrayed.",
"The inherent ability of these factors to home in on target genes through either their DNA binding domains or through interactions with other proteins is exploited utilizing previously described technology (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"FIG.",
"3 is an illustrative example of the expressional characterization of a series of biopsied human tissue samples as disease progresses from no overt morphological alterations to a cancerous phenotype.",
"An expression profile of transcription factor target genes is taken at different temporal stages.",
"Therapeutic strategies may be implemented and subsequent microarray expression profiles analyzed to monitor the effectiveness of the therapy.",
"A reversion to profiles similar to those for early tumor progression or pre-tumorigenesis suggests effective treatment.",
"In the theoretical example of FIG.",
"3 therapeutic strategy B reverts tumor progression to near pretumorigenic stages.",
"It is contemplated and therefore covered by the present invention that virtually any type of cancer may be effectively monitored by transcription factor target nucleotide arrays and microarrays.",
"It is also contemplated and therefore covered by the present invention that maladies other that those related to a cancerous phenotype may also be monitored via transcription factor target nucleotide microarrays.",
"These include, but are in no way limited to inherited as well as sporadic conditions.",
"As mentioned above, not only are revealing expression profiles discerned from such transcription factor target nucleotide microarrays but potential points of therapeutic intervention or even therapeutic target discovery may be uncovered.",
"In addition, patient prognosis may be significantly improved with “standardized” expression profiles of transcription factor targets for both normal and diseased tissue at different stages of progression.",
"Finally, and perhaps the most intriguing aspect of the technology, is the ability to diagnose a disorder based upon gene expression patterns prior to the establishment of any overt symptoms.",
"Table 1 illustrates this point by providing a “transcriptome” of p53 targets for a number of tissue samples known to be isolated at different stages of cancer progression (T1 through T8).",
"Note that a unique expression level is annotated for each target gene a any given timepoint during tumor progression.",
"In addition, other stimuli such as environmental cue., and age may be correlated with a categorization of gene expression profiles.",
"Table 2 illustrates a compendium of alterations in target gene expression based upon internal and external influences.",
"Data accumulated from these profiles can undoubtedly yield significant insight into diagnostic applications as well as the development of preventative strategies.",
"5.8 Transcription Factor Target Protein Arrays The presently described invention details the construction and implementation of transcription factor target protein arrays and microarrays for the purposes of identifying interactions of these target proteins with chemical molecules, nucleotide sequences and other proteins of enzymatic or nonenzymatic origin.",
"FIG.",
"4 illustrates the scope of the process from the identification of transcription factor target genes to the characterization of target protein interacting molecules through the utilization of nonliving target protein arrays.",
"As described previously, transcription factor/DNA complexes are cross-linked in vivo via the addition of formaldehyde to cells in tissue culture or to isolated living tissues themselves.",
"In the presently described invention, antibody coated Dynabeads™ (Dynal Corporation) are added directly to cross-linked material and specific antibody/transcription factor/target gene complexes are immunoprecipitated, washed and DNA fragments representing target genes of interest subsequently isolated (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Pools of these fragments contain genomic sequences corresponding to actively transcribed regions of transcription factor target loci.",
"These sequences are screened against appropriate cDNA expression libraries to quickly and efficiently purify transcription factor target genes in the context of expression vectors for rapid production of the corresponding proteins.",
"cDNAs corresponding to transcription factor target genes are translated and transcription factor target protein products are subsequently arrayed into a format suitable for interaction screening.",
"These arrays are typically of a “living” or “nonliving” nature (see below).",
"Screens are implemented for the discovery of specific interactions between transcription factor target proteins and other proteins/enzymes, nucleotide sequences, metals or small molecule drugs.",
"It is contemplated and therefore covered by the present invention that any molecule identifies through the utilization of transcription factor target protein arrays may represent a potential avenue of therapy for particular aspects of human physiology and disease.",
"Interactions may be identified b) a number of methods but most often are revealed via fluorescent tags conjugated to the screen candidates of interest (MacBeath et al., 2000, Science, 289: 1760-1763).",
"Other biochemical detection methods include, but are in no way limited to radioactive hybridization, colorimetric detection and enzymatic activity such as that of horse radish peroxidase (HRP).",
"It should be noted that full-length transcription factor target protein sequences are not necessarily needed to produce interaction results, but rather target peptide or short amino acid sequences alone may be sufficient.",
"5.9 Advantages of the Presently Described Invention over Existing Technology While it is evident that nucleotide array and microarray characterization of gene expression patterns within specific sample populations is now a reality, a number of conceptual problems exist which must be overcome for the technology to become routine.",
"Reproducible data output is a primary concern.",
"The amount of sequence redundancy in the human genome is considerable.",
"The presently described invention aims to overcome this limitation by narrowing the scope of genes analyzed to only a subset of those contained in the genome.",
"A more limited and physiologically focused number of genes per microarray decreases redundancy and cross-hybridization issues and results in more accurate expression profiling.",
"As well, specificity in analysis will increase based upon smaller physiologically directed arrays.",
"More specific analyses means more comprehensive data accumulation during each round of expression characterization, thus eliminating errors introduced by large-scale characterization of irrelevant loci.",
"In addition, current microarrays lack the utility of control loci needed to ensure correct administration of experimental procedures.",
"The presently described invention eliminates this issue by providing numerous previously published known transcription factor target genes as controls for each physiologic and/or disease oriented nucleotide microarray.",
"These controls are not only expressed in the appropriate temporal and spatial manner, but often play functional roles related to the physiology being characterized.",
"For example, an array of target genes for the tumor suppressing transcription factor p53 (see FIG.",
"1) will contain a number of known targets, such as the WAF1 locus, which are shown to have functionality in regulating cell cycle and have affected expression patterns in tumorigenic tissue samples (E1-Diery et al., 1993, Cell, 75: 817-825).",
"The appropriate controlling of nucleotide microarray analysis will consistently reveal reproducible experimental results.",
"Sample availability also poses a unique problem to the microarray analysis of gene expression profiles.",
"The majority of DNA microarray experiments utilize sample RNA which has been harvested from at least 106-107 cells.",
"In some cases, especially those involving human tissue, sample size is small and therefore rate limiting.",
"Minute sample sizes may limit the number of microarray studies which may be performed as the larger the array of genetic loci the more sample required to get accurate readout and data acquisition.",
"This is especially true given the enormous complexity of the genes present within currently existing arrays and microarrays, most of which are likely to be irrelevant to the particular sample or aspect of physiology being studied.",
"By directing characterization of samples to microarrays which are focused on particular aspects of physiology an, disease, these arrays allow for the characterization of expression profiles for very limited sample sizes and increase the number of focused expression profiling experiments which may be undertaken.",
"In addition, given the large number of sequences which are annotated and linked to support material, the cost of construction of nonfocused nucleotide arrays and microarrays is quite significant.",
"The presently described invention circumvents this problem by focusing array construction and utilization only upon specific genes which play roles in particular aspects of physiology and disease.",
"While much progress has been made with respect to the high-throughput identification of biochemical interactions in both a biological and chemical context through the construction and use of protein arrays, several potential drawbacks of this technology limit its utility in the larger scope of optimizing the efficiency of biochemical interaction characterization and ultimately drug development (MacBeath et al., 2000, Science, 289: 1760-1763 and for review see Emili et al., 2000 Nature Biotechnology, 18: 393-397).",
"Perhaps the most relevant limitation of the above described methodologies is the shear magnitude of labor required to construct the arrays, either of a living or nonliving origin.",
"Given the estimated number of genes present in the human genome (at present 26,000) it would be extremely labor intensive and costly to organize the protein products of all such loci into biochemical arrays (Venter et al., 2001, Science, 291: 1304-1351).",
"In order to gain the maximum value and utility of protein arrays it is necessary to strategically choose the proteins which are to be organized and annotated in the array format.",
"Properly choosing which proteins or peptide sequences are to be included in each particular array will result in a focus on specific realms of physiology and even human disease, increasing the possibility of studying the appropriate interacting partners and ultimately developing therapeutics for the treatment of disease.",
"As transcription factors typically have been demonstrated time and again to control certain specific aspects of cellular and developmental biology, it is evident that the inherent ability of these factors to dictate discrete gene expression patterns allows for an excellent opportunity to define which gene products (proteins) mar be included in each array.",
"By organizing specific transcription factor target proteins into arrays the biochemical nature of entire realms of physiology and even disease can be studied thoroughly and efficiently.",
"6.0 EXAMPLES 6.1 Construction and Utilization of Transcription Factor Target Nucleotide Microarrays FIG.",
"2 is a flowchart representation of transcription factor target glass chip microarray construction.",
"Modified sequential chromosomal immunoprecipitation is performed on sonicated cross-linked chromatin isolated from cell lines and/or tissues (PCT patent application serial number PCT/US01/24823, filed Aug. 14, 2000 and herein incorporated by reference).",
"Upon reversal of cross-linkage precipitated DNA fragments containing putative transcription factor target genes are screened either via I-PCR or against cDNA libraries.",
"I-PCR results in the identification of promoter and enhancer elements specific for the transcription factor being studied and confirmation of direct target identity.",
"cDNA library screening reveals valuable 5′ untranslated and coding sequence information crucial to expression pattern characterizations.",
"Sequences are organized in a two-dimensional grid format for ease of target gene identification and analysis.",
"FIG.",
"3 illustrates the use of transcription factor target nucleotide microarrays for monitoring cancer patient prognosis during and prior to therapy.",
"Each square within the grid contains specific oligonucleotide sequences corresponding to p53 target genes and linked covalently to the solid support.",
"As RNA isolated from samples (or corresponding cDNA) is passed over the chip, evidence of target gene expression and quantitative analysis of levels is revealed by a change in light illumination for particular and specific target loci.",
"A temporal change in expression patterns is indicative of transcriptome alteration during tumor progression.",
"In addition, therapeutic effectiveness may be monitored through expression profiling as evidenced by changes in gene expression.",
"A reversion of patient transcriptome outputs to that of early tumor progression or even pretumorigenesis is indicative of effective therapeutic strategies.",
"In the illustrative example of FIG.",
"3 therapeutic strategy B reverts sample expression profiles to a pretumorigenic phenotype.",
"Table 1 is an example of temporal changes in gene expression patterns and levels as progression occurs from the normal to the tumorigenic phenotype.",
"Samples T1 through T7 represen controls for different known temporal stages of tumorigenesis (from early to late) while samples N1 through N3 are unknown samples.",
"Numbzrs listed linearly correlate with gene expression levels.",
"Note how certain transcription factor targets are activated while others are repressed upon phenotype manifestation.",
"From the data collected it is apparent that sample N1 correlates with an earlier manifestation of the disease as expression profiles are similar to that for sample T2.N2 exhibits a late stage expression profile resembling that of T5 and N3 shows no correlation to the disease phenotype.",
"Table 2 is a similar example of transcription factor target gene microarray expression classification upon issuance of external as well as internal influences.",
"These influences in this particular example include various environmental stimuli such as exposure to carcinogens as well as age.",
"Note how the expression profile of samples from patient A correlate with those in standard sample 1 while patient B samples exhibit similar expression profile to standard sample 3.6.2 Transcription Factor Target Protein Nonliving Arrays The ability to detect specific interactions of nucleotide sequences, small molecules, enzymes and other proteins with transcription factor target proteins allows for the ultimate design of therapeutics with higher efficacy and fewer side effects than those currently available.",
"Several different types of applications of transcription factor target microarray proteomics can be employed to achieve the desired results.",
"As mentioned above, there are primarily two types of array and microarray protein interaction screens which have been successfully utilized for the purposes of high-throughput interaction characterization (for review see Emili et al., 2000, Nature Biotechnology, 18: 393-397).",
"The presently described invention optimizes and focuses each of these methodologies for the analysis and characterization of various entities which may interact with transcription factor target proteins.",
"FIG.",
"5 is a diagrammatic flowchart of methodology employed for the utilization of “nonliving”/chemical transcription factor target protein microarrays.",
"Peptide sequences or bacterially expressed glutathione-S-transferase fusion proteins are immobilized on a solid phase support such as a nylon membrane or glass chip in a hydrated, folded state to preserve the naturally occurring 3-dimensional structure of the protein (Martzen et al., 1999, Science, 286: 1153-1155).",
"A number of assays may subsequently be implemented to determine the possibility of enzyme/substrate as well as simple protein/protein and protein/small molecule interactions.",
"Transcription factor target proteins present in the array may be tested as targets for enzymatic action by observing modification of the arrayed proteins upon incubation with the enzyme of interest.",
"Modifications such as phosphorylation or acetylation provide convenient tags which can be readily identified in vitro.",
"In addition, it is possible to characterize the interactions of transcription factor target proteins with those present in virtually any type of cell through the passage of whole cell lysates over the arrays.",
"Extensive washing and elution of bound proteins followed by mass spectrometry greatly enhances the scope of arrayed transcription factor target protein/protein interaction studies (Gygi et al., 1999, Nature Biotechnology, 17: 994-999; Neubauer et al., 1997, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 94: 385-390 and Lamond et al., 1997, Trends Cell Biol., 7: 139-142).",
"Finally, phage display methodologies complement transcription factor target protein array technology by allowing for the characterization of amplified libraries of proteins from virtually any source (Zozulya et al., 1999, Nature Biotechnol., 17: 1193-1198 and Hufton et al., 1999, J. Immunol.",
"Methods, 231: 39-51).",
"Bacteriophage samples expressing proteins on the surface of the phage are passed in contact with the transcription factor target protein array (FIG.",
"5).",
"Only specific interactions between the arrayed target proteins and those on the outer shell of the bacteriophage will allow for binding of the phage to specific targets within the array after rigorous washing.",
"These phage can be subsequently eluted from the protein array and the cDNA corresponding to the surface protein of interest can be purified and sequenced to reveal the protein's genetic identity and amino acid composition.",
"6.3 Transcription Factor Target Protein Living Arrays As mentioned above, it is also possible to construct biological/“living” arrays of transcription factor target proteins as described in yeast (Uetz et al., 2000, Nature, 403: 623-627).",
"The use of these transcription factor target protein arrays is illustrated diagrammatically in FIG.",
"6.Arrays of yeast colonies containing protein open reading frameIGAL4 activation domain fusions are mated with strains of yeast containing a single GAMA DNA binding domain fusion.",
"Upon nutritiona selection only yeast clones in which interaction between the two GAJA fusion proteins occurs will survive due to recruitment of the activation complex to a nutritional supplement/GAL4 DNA binding site locus engineered within the yeast genome.",
"Although GAJA interaction methodologies are described in the present invention, it is in no way limited to this particular transcription factor interaction and activation capacity.",
"Other transcription factors and their prospective binding sites may be utilized for the successful detection of protein/protein interactions and are therefore covered by the present invention.",
"Preparations of purified nucleotide sequences containing the cDNA encoding the interacting partner of interest are then performed from surviving yeast colonies.",
"DNA sequencing of these fragments will reveal the identity of the array tag containing the interaction partner.",
"The retrieval of information on protein/protein, protein/small molecule and enzyme/substrate interactions for transcription factor target proteins is of considerable value for the development of therapeutic agents.",
"Yet these data must be organized in a fashion that maximizes value and minimizes the complexity of the information at hand.",
"The presently described invention therefore describes the importing and organization of all data corresponding to the interactions of transcriptior factor targets into proteomics interaction databases which are easily searchable for the desired biochemical interaction information (FIGS.",
"5 and 6).",
"By implementing a vigorous bioinformatics platform to annotate and categorize these data researchers will have the opportunity to rapidly identify relevant transcription factor target protein interaction information for the ultimate design of therapeutics.",
"This type of annotation will speed the identification and exploitation of therapeutically relevant transcription factor target proteins.",
"6.4 Therapeutic Discovery Utilizing Transcription Factor Target Protein Arrays The biochemical data gleaned from the identification of interacting molecules with transcription factor target proteins is of unparalleled value for the development of agents of therapeutic intervention.",
"By focusing on the biochemical events downstream of a particular transcription factor it is possible to circumvent effects on undesired cellular cascades and design drugs which will exhibit higher efficacy and fewer side effects than those which are currently available.",
"FIG.",
"7 illustrates a theoretical example of the process for the identification of a therapeutic compound for the treatment of cancer through the implementation of transcription factor target protein array technology.",
"A transcription factor target protein array representing targets for the tumor suppressor p53 is tested against a fluorescent tag conjugated small molecule for interactions between the molecule and particular p53 target proteins.",
"Fluorescent light emission reveals a specific binding interaction between the small molecule and what is determined to be a G protein coupled receptor (GPCR) thought to inhibit tumorigenesis (for review see Gershengom et al.",
"2001, Endocrinology, 142: 2-10).",
"Tissue culture experiments are subsequently conducted to determine a putative negative or positive effect of the small molecule drug on the receptor's ability to transmit signals intracellularly to ultimately affect cellular proliferative and apoptotic events.",
"If the small molecule is determined to inhibit receptor function antagonists are designed hamper inactivation of the receptor thus driving constitutive receptor function.",
"If the small molecule is revealed to activate the receptor and thereby promote inhibition of tumorigenesis further analogs are developed to optimize interaction specificities and increase the activation state of the receptor.",
"In both cases it is possible to develop potential therapeutic agents superior to existing treatment strategies, the focus of which is directed at transcription factor target proteins in vivo.",
"7.0 REFERENCES Patent Documents Hudson et al., U.S. Pat.",
"No.",
"5,591,646, Issued January, 1997 Buettner et al., U.S. Pat.",
"No.",
"5,834,318, Issued November, 1998 Lockhart et al., U.S. Pat.",
"No.",
"6,040,138, Issued March, 2000 Burgess et al., PCT application #PCT/US01/24823, filed Aug. 14, 2000 McCasky et al., U.S. Pat.",
"No.",
"6,100,030, Issued August, 2000 Leighton et al., U.S. Pat.",
"No.",
"6,136,592, Issued October, 2000 Schatz et al., U.S. Pat.",
"No.",
"6,156,511, Issued December, 2000 Other References Benashski et al., Methods, 2000, 22: 365-371 Chang et al., 1998, Clinical Immunology and Immunopathology, 89(1): 71-8 Chen et al., 1996, Developmental Genetics, 19(2): 119-30 Debouck et al., 1999, Nature Genetics Supplement, 21: 48-50 DeRisi et al., Science, 278: 680-686 Drysdale et al., 2000, Yeast, 17(2):159-66 Dunaief et al., 1994, Cell, 79(1):119-30 E1-Diery et al., 1993, Cell, 75: 817-825 Emili et al., 2000, Nature Biotechnology, 18: 393-397 Gershengorn et al., 2001, Endocrinology, 142: 2-10 Gygi et al., 1999, Nature Biotechnology, 17: 994-999 Herzig et al., 1997, Proceedings of the National Academy of Sciences, 94: 7543-7548 Hufton et al., 1999, J. Immunol.",
"Methods, 231: 39-51 Hughes et al., 2000, Cell, 102: 109-126 Lamond et al., 1997, Trends Cell Biol., 7: 139-142 Levine et al., 1991, Nature, 351: 453456 MacBeath et al., 2000, Science, 289: 1760-1763 Martzen et al., 1999, Science, 286: 1153-1155 Moroy et al., 2000, Cellular and Molecular Life Sciences, 57(6): 957-75 Neubauer et al., 1997, Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 94: 385-390 Nichogiannopoulou et al., 1998 Seminars in Immunology, 10: 119-125 Ochman et al., 1988, Genetics, 120(3): 621-623 Rhodes et al., 1994, Current Opinions in Genetic Development, 4: 709-717 Solomon et al., 1988, Cell, 53: 937-947 Speliman et al., 1998, Cell, 9: 3273-3297 Tenbaum et al., 1997, International Journal of Biochemistry and Cell Biology, 29: 1325-1341 Tjian and Maniatis, 1994, Cell, 77: 5-8 Uetz et al., 2000, Nature, 403: 623-627 Venter et al., 2001, Science, 291: 1304-1351 Zozulya et al., 1999, Nature Biotechnol., 17: 1193-1198 Zweiger et al., 1997, Trends in Biotechnology, 17: 429436"
]
] | Patent_10275845 |
[
[
"Transcription factor target gene discovery",
"The ability to rapidly define transcription factor target genes allows for the study of genetic cascades involved in development, physiology and disease.",
"The presently descibed invention outlines the combination of novel chromosomal immunoprecipitation and molecular biology technologies fot the high-throughput in vico discovery and characterization of both known and unknown transcription factor target genes.",
"Through an application of solid phase support matrices and sequential chromosomal immunoprecipitation in combination with molecular cloning procedures this methodology allows for the rapid, stringent purification from cells and tissues of nucleotide sequences representing targets for regulation by transcription factors.",
"Implementation of the technology described herein will result in the efficient simultaneous characterization of both regulatory element and coding sequence target gene information and will be valuable for assessing genetic hierarchies and developing therapeutics."
],
[
"1.A method which utilizes chromosomal immunoprecipitation procedures for the discovery and characterization of transcription factor target genes.",
"2.A method according to claim 1 comprising a process of: a) attaching a protein binding entity to a support matrix; b) utilizing the support matrix/protein binding entity described in a) for the purposes of purifying protein/DNA complexes such as chromatin from cell extracts.",
"3.A method according to claim 2 wherein said protein binding entity is an antibody.",
"4.A method according to claim 1 in which said transcription factors are of a DNA binding nature.",
"5.A method according to claim 1 in which said transcription factors are recruited to DNA through contact with other proteins.",
"6.A method according to claim 1 in which said transcription factor target genes consist of coding sequences.",
"7.A method according to claim 1 in which said transcription factor target genes consist of noncoding sequences, including regulatory elements.",
"8.A method according to claim 7 in which said noncoding sequences are regulated by transcription factors.",
"9.A method according to claim 2 comprising a process of multiple sequential rounds of immunoprecipitation utilizing protein binding entities of differing origin which involves the process of: a) immunoprecipitation of cross-linked protein/DNA complexes utilizing protein binding entities specific for one protein followed by; b) a second round of immunoprecipitation of protein/DNA complexes utilizing complexes isolated by the first round as the substrate and protein binding entities specific for a different protein.",
"10.A method according to claim 9 which comprises more than two rounds of chromosomal immunoprecipitation.",
"11.A method according to claim 9 wherein said protein binding entity is specific for members of the basal transcriptional machinery.",
"12.A method which includes utilizing purified DNA fragments isolated by chromosomal immunoprecipitation procedures described in claim 1 to cross hybridize against libraries of nucleotide sequences.",
"13.A method which includes utilizing purified DNA fragments isolated by chromosomal immunoprecipitation procedures described in claim 1 to screen against arrays of nucleotide sequences.",
"14.A method according to claim 1 which includes the implementation of inverse PCR for the discovery of sequences directly bound by said transcription factors.",
"15.A method according to claim 1 which includes cloning of purified DNA fragments isolated by chromosomal immunoprecipitation into vectors for purposes of manipulation and sequence determination.",
"16.A protein/DNA complex isolated from cells according to methods described in claim 1.17.DNA fragments isolated from protein/DNA complexes described in claim 16.18.Nucleotide sequences present in DNA fragments described in claim 17 wherein said sequences represent noncoding sequences which may include 5 prime and 3 prime untranslated regions, introns, promoter, enhancer and/or silencer elements.",
"19.Nucleotide sequences present in DNA fragments described in claim 17 wherein said sequences represent coding sequences which correspond to specific amino acid sequences present in putative proteins.",
"20.Amino acid sequences encoded by nucleotide sequences described in claim 19.21.Proteins represented by amino acid sequences described in claim 20.22.A database of sequence information formed from isolated sequences by methods according to claim 1 which represents a cohesive organization of transcription factor target genes."
],
[
"<SOH> 2.0 BACKGROUND OF THE INVENTION <EOH>The specific regulation of transcription within the nucleus of the cell is one of the basic facets of the cellular machinery and is known to be the implicit foundation behind all cellular characteristics.",
"The ability to differentially regulate the activity of each of the estimated 26,000 genes depends upon the presence or absence of various transcriptional activator and/or repressor proteins (Venter et al., Science, 2001, 291(5507): 1304-1351).",
"FIG.",
"1 illustrates this concept for the steroid receptor class of transcription factors.",
"Steroid receptors, represented by the rectangles, dimerize and bind to target gene regulatory regions.",
"In the presence of a steroid ligand depicted by the ovals, target genes are activated.",
"In the absence of ligand the receptor is bound to corepressor machinery and target genes are inactive.",
"Interactions between these as well as other factors and their target loci have evolved over time into a complex series of temporal and biochemical events which governs transcription under tight regulatory constraints (for review see Semenza et al., Human Mutations, 1994, 3:180-199).",
"It is the interaction between these factors and sequence-specific regulatory elements that has shed insight into the mechanisms by which cells keep such entities as cell division, differentiation and immunomodulation in check By deciphering these genetic cascades and ultimately defining transcription factor targets it will be possible, for example, to determine just how tumor suppressing transcription factors such as p53 (Zambetti et al., Genes Dev., 6: 1143-1152, Zauberman et al., Oncogene , Jun.",
"15, 1995;10(12): 2361-6) and Rb (friend et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1987, 84: 9059-9063) exert their effects on both inhibiting cell division and promoting cell death.",
"Indeed, the value of transcription factor/regulatory interactions is evidenced by the wealth of patents recently issued in the United States relating directly to the factors themselves or technology pertaining to gene regulation (a representative set of examples includes U.S. Pat.",
"Nos.",
"5,53,036, 5,858,973, 5,863,757, 5,880,261 and 6,117,638 herein incorporated by reference).",
"The identification of transcription factor target loci requires an assessment of protein/DNA interactions in vivo.",
"The chromosomal immunoprecipitation (ChIP) assay has been demonstrated as a method which successfully allows for the purification of in vivo protein/protein interactions which occur in combination with DNA regulatory elements as well as direct protein/DNA interactions from cellular extracts of either cytoplasmic or nuclear origin (Solomon et al., Cell, 1988, 53: 937-947; de Belle et al., Biotechniques, 2000, 29(1): 170-175).",
"It is based upon the chemically catalyzed cross-linkage of biochemical interactions in living cells followed by purification of desired complexes from nonspecific contaminants.",
"To date, use of the ChIP assay has proven to be of value for the assessment of transcription factor complex recruitment to particular nucleotide sequences of known origin.",
"By determining the presence or absence of a particular transcription factor on a known DNA sequence or binding site present within a particular gene, for example, it is possible to establish whether specific known genes are targets for regulation by chosen factor.",
"However, in order to identify previously uncharacterized or undiscovered targets for potential regulation by a particular transcription factor a number of advances in the technology must be achieved.",
"For example, efficient recovery of quantities of DNA large enough to allow for cloning and sequencing of the potential transcription factor targets must occur.",
"In addition, an optimization for the opportunity to isolate transcribed portions of genes and eliminate noncoding genomic sequences which often do not reveal the identity of the target gene must be accomplished.",
"Finally, high-throughput organization of sequences obtained into a searchable database format should be undertaken to provide for maximum utility of the discovery transcription factor target genes.",
"Incorporation of the modified, significantly improved ChIP assay into the described present invention in combination with molecular cloning methods now allows for the high-throughput isolation and characterization of both known and unknown transcription factor target loci.",
"In addition, these modifications and improvements increase the sensitivity of target gene retrieval while simultaneously reducing background.",
"Solid phase technology has had a significant impact on the efficiency and sensitivity of protein complex purification.",
"Compounds such as sepharose and magnetic beads have allowed for extensive purification and characterization of protein/protein complexes from both in vitro and in vivo mixtures, without compromising the quantitative or qualitative aspects of samples obtained Dynal Corporation Technical Handbook, 1998, Sigma Corporation, cat.",
"#4B-200).",
"It's application for the purposes of identifying transcription factor target genes of unknown origin and in a high-throughput format, however, has yet to be implemented.",
"It is the use of solid phase technology in the presently described invention which significantly increases the sensitivity of obtaining real in vivo targets for transcription factors while reducing background false positive sequences obtained.",
"Through both a combination and modification of the above technologies as well as other molecular biology techniques such as exon scanning, inverse PCR and cDNA library screening the presently described invention allows for the extensive and exhaustive characterization of transcription factor target genes of both known and unknown origin and of a direct (the gene is bound by the factor) and indirect (interaction through other proteins) nature.",
"It is the implementation of chromosomal immunoprecipitation procedures improved via the use of-solid phase support and sequential immunoprecipitation for multiple proteins which permits the potential complete and thorough analysis of a great deal of the transcriptional cascades present in the nucleus of the cell.",
"The proposed technology described herein is applicable to a very limited quantity of cell or tissue samples, which makes it suitable for clinical analysis and comprehensive medical diagnostics.",
"The utilization of this technology will no doubt have a significant impact on the fields of therapeutics, medical diagnostics and basic research related to the realm of transcriptional regulation."
],
[
"<SOH> 3.0 SUMMARY OF THE INVENTION <EOH>The use of chromosomal immunoprecipitation (ChIP) for the identification of targets for transcriptional regulation by transcription factors has been limited by both the insensitivity of the technology to eliminate considerable nonspecific protein/DNA interactions as well as to the discovery and characterization of only previously identified nucleotide sequences.",
"The presently described invention overcomes the above limitations of chromosomal immunoprecipitation by employing a combination of novel sequential immunoprecipitation procedures utilizing antibodies to the basal transcriptional machinery, solid phase separation procedures and extensive cloning applications including a modified and significantly improved version of inverse PCR which allow for the discovery of target genes and their regulatory elements.",
"The combination of improved chromosomal immunoprecipitation procedures with expression profiling and cloning technologies described in the present invention has allowed for the discovery and characterization of both known and unknown target sequences for chosen transcription factors.",
"In addition, the presently described technology is highly automatable, allowing for extensive high-throughput analysis of virtually any genetic cascade.",
"One embodiment of the present invention is the formaldehyde fixation reaction process which cross-links DNA binding proteins with their prospective nucleotide binding sites present within close proximity or distal to target genes in living cells and or tissues.",
"This fixation reaction is designed and customized specifically for each particular cell line and/or tissue being studied.",
"An additional embodiment of the present invention is other chemical methods utilized for the purposes of fixing and/or cross-linking proteins to their prospective target nucleotide sequences in vivo directly through interaction with DNA or indirectly utilizing protein-protein contacts.",
"Another embodiment of the present invention is the cross-linked protein/target gene complex created by the formaldehyde crosslinkage reaction in vivo.",
"Said complex theoretically contains a mixture of protein/DNA complexes containing the desired transcription factor or regulatory protein directly or indirectly bound to its prospective target loci.",
"Another embodiment of the present invention is an antibody which is specific for Drosophila melanogaster or Sciara coprophila RNA Polymerase II protein large subunit.",
"The antibody may be of monoclonal or polyclonal origin and may recognize similar epitopes from different species.",
"Yet another embodiment of the present invention is an antibody which binds specifically to the mammalian transcription factor p53.Said antibody may be of monoclonal or polyclonal origin.",
"Still another embodiment of the present invention is an antibody-linked to magnetic beads which binds specifically to either Drosophila melanogaster or Sciara coprophila RNA polymerase II protein large subunit.",
"It is the solid-phase support linkage which enhances recovery and specificity of target chromatin upon immunoprecipitation.",
"Another embodiment of the present invention is an antibody which is linked to magnetic beads which binds specifically to the mammalian p53 protein.",
"Yet another embodiment of the present invention is the recovered fraction of the cross-linked, fixed chromatin protein/DNA complex.",
"Another embodiment of the present invention is the sonicated chemically cross-linked protein/DNA complex isolated after sonication but prior to immunoprecipitation.",
"Sonication allows for efficient immunoprecipitation of DNA fragment sizes small enough to be characterized in a high-throughput format via polymerase chain reaction (PCR) or other molecular biology techniques.",
"Still another embodiment of the present invention is the immunoprecipitated protein/DNA complex prior to release of the antibody and reversal of cross-linkage isolated utilizing antibodies which recognize either the Drosophila melanogaster or Sciara coprophila RNA polymerase II large subunit as well as the mammalian p53 protein.",
"An additional embodiment of the present invention is the sequential immunoprecipitation of cross-linked protein/DNA complexes from living cells and tissues utilizing antibodies to core transcriptional machinery factors first and to specific transcription factors second.",
"Sequential immunoprecipitation eliminates the majority of nontranscribed sequences and satellite DNA by focusing only upon transcribed and/or actively regulated genes.",
"It is primary immunoprecipitation with antibodies to proteins found in the basal transcriptional apparatus which results in increased sensitivity through a reduction in the amount of nontranscibed genomic DNA pulled down during subsequent immunoprecipitation reactions.",
"Theoretically only actively transcribed genetic sequences are present as templates for the second round of immunoprecipitation.",
"Secondary immunoprecipitation with antibodies to specific transcription factors is thereby significantly more efficient as is described herein and allows for the opportunity to characterize transcription factor function with respect to the regulation of gene activity.",
"These sequential rounds of immunoprecipitation may be performed in any order with the similar result of increased sensitivity for the discovery of transcription factor target genes and decreased background or nonspecific sequences obtained.",
"Additionally, solid phase chromosomal immunoprecipitation eliminates loss of cross-linked protein/DNA complex material initially precipitated from cellular extracts by providing a solid support and thereby enhances the potential ability to recover target DNA fragments and hence the nucleotide sequences corresponding to these fragments.",
"Excessive loss is prevented through clean, efficient recovery of antibody/protein/DNA complexes due to tight linkages between the solid phase (beads in the case of the present invention) and antibodies.",
"Yet another embodiment of the present invention is the utilization of polymerase chain reaction (PCR) to detect known target loci within the collection of pull-down fragments which putatively contains both known and unknown target genes.",
"It is the detection and monitoring of known controls which allows for a characterization of the efficiency of the system.",
"As well, an additional embodiment of the present invention is the utilization of inverse PCR (I-PCR) in combination with solid phase sequential chromosomal immunoprecipitation for purposes of defining only direct targets for regulation by specific transcription factors as well as for background reduction.",
"Specifically, oligonucleotides corresponding to transcription factor binding sites are used to PCR flanking sequences present in DNA fragment populations isolated by the technology described herein.",
"The application of this modified version of I-PCR to sequentially immunoprecipitated chromosomal templates hence results in the discovery and cloning of direct targets for regulation by the transcription factor in question.",
"Another embodiment of the present invention is the facilitated cloning of both known and unknown target genes from DNA fragments isolated by the presently described methods.",
"These potential targets, for transcription factors of DNA binding and nonDNA binding origin, are cloned through successive rounds of screening against cDNA libraries and genomic DNA libraries, ligation and transfer into bacteriophage and/or plasmid vectors, polymerase chain reaction including but not limited to I-PCR and DNA sequencing.",
"Yet another embodiment contemplated by the present invention is the screening of immunoprecipitated DNA fragments potentially containing target loci against libraries, arrays and/or microarrays of both known and unknown genes.",
"These libraries and arrays may be of either cDNA or oligonucleotide composition.",
"It is the screening of immunoprecipitated DNA fragments against these libraries, arrays and microarrays which facilitates the discovery of target genes for the transcription factor being studied.",
"Said screen allows for a rapid identification of coding sequences for transcription factor target loci present in the collection of DNA.",
"An additional embodiment of the present invention is the cloning of DNA fragment collections containing transcription factor target genes into bacteriophage arms and subsequent packaging into particles for the purposes of rapid conventional screening and sequencing.",
"These bacteriophage libraries may be screened with known DNA probes or other unknown probes for purposes of discovery of target loci.",
"Yet another embodiment of the present invention is the cloning of DNA fragment collections containing transcription factor target genes into exon scanning vectors which may be introduced into eukaryotic cells for purposes of rapidly identifying potential coding sequences within the collection of DNA fragments.",
"Another embodiment of the present invention includes the nucleotide sequences and corresponding amino acid sequences and protein products as determined to be targets for either direct or indirect transcriptional regulation.",
"An additional embodiment of the present invention is the organization of the nucleotide and corresponding amino acid sequences discovered into a database or databases for purposes of rapid search and characterization of these sequences for functional and possible therapeutic relevance."
],
[
"1.0 FIELD OF THE INVENTION The following invention describes the utilization of solid matrix binding technology in combination with sequential chromosomal immunoprecipitation and molecular cloning technologies to discover and characterize transcription factor target genes.",
"2.0 BACKGROUND OF THE INVENTION The specific regulation of transcription within the nucleus of the cell is one of the basic facets of the cellular machinery and is known to be the implicit foundation behind all cellular characteristics.",
"The ability to differentially regulate the activity of each of the estimated 26,000 genes depends upon the presence or absence of various transcriptional activator and/or repressor proteins (Venter et al., Science, 2001, 291(5507): 1304-1351).",
"FIG.",
"1 illustrates this concept for the steroid receptor class of transcription factors.",
"Steroid receptors, represented by the rectangles, dimerize and bind to target gene regulatory regions.",
"In the presence of a steroid ligand depicted by the ovals, target genes are activated.",
"In the absence of ligand the receptor is bound to corepressor machinery and target genes are inactive.",
"Interactions between these as well as other factors and their target loci have evolved over time into a complex series of temporal and biochemical events which governs transcription under tight regulatory constraints (for review see Semenza et al., Human Mutations, 1994, 3:180-199).",
"It is the interaction between these factors and sequence-specific regulatory elements that has shed insight into the mechanisms by which cells keep such entities as cell division, differentiation and immunomodulation in check By deciphering these genetic cascades and ultimately defining transcription factor targets it will be possible, for example, to determine just how tumor suppressing transcription factors such as p53 (Zambetti et al., Genes Dev., 6: 1143-1152, Zauberman et al., Oncogene, Jun.",
"15, 1995;10(12): 2361-6) and Rb (friend et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1987, 84: 9059-9063) exert their effects on both inhibiting cell division and promoting cell death.",
"Indeed, the value of transcription factor/regulatory interactions is evidenced by the wealth of patents recently issued in the United States relating directly to the factors themselves or technology pertaining to gene regulation (a representative set of examples includes U.S. Pat.",
"Nos.",
"5,53,036, 5,858,973, 5,863,757, 5,880,261 and 6,117,638 herein incorporated by reference).",
"The identification of transcription factor target loci requires an assessment of protein/DNA interactions in vivo.",
"The chromosomal immunoprecipitation (ChIP) assay has been demonstrated as a method which successfully allows for the purification of in vivo protein/protein interactions which occur in combination with DNA regulatory elements as well as direct protein/DNA interactions from cellular extracts of either cytoplasmic or nuclear origin (Solomon et al., Cell, 1988, 53: 937-947; de Belle et al., Biotechniques, 2000, 29(1): 170-175).",
"It is based upon the chemically catalyzed cross-linkage of biochemical interactions in living cells followed by purification of desired complexes from nonspecific contaminants.",
"To date, use of the ChIP assay has proven to be of value for the assessment of transcription factor complex recruitment to particular nucleotide sequences of known origin.",
"By determining the presence or absence of a particular transcription factor on a known DNA sequence or binding site present within a particular gene, for example, it is possible to establish whether specific known genes are targets for regulation by chosen factor.",
"However, in order to identify previously uncharacterized or undiscovered targets for potential regulation by a particular transcription factor a number of advances in the technology must be achieved.",
"For example, efficient recovery of quantities of DNA large enough to allow for cloning and sequencing of the potential transcription factor targets must occur.",
"In addition, an optimization for the opportunity to isolate transcribed portions of genes and eliminate noncoding genomic sequences which often do not reveal the identity of the target gene must be accomplished.",
"Finally, high-throughput organization of sequences obtained into a searchable database format should be undertaken to provide for maximum utility of the discovery transcription factor target genes.",
"Incorporation of the modified, significantly improved ChIP assay into the described present invention in combination with molecular cloning methods now allows for the high-throughput isolation and characterization of both known and unknown transcription factor target loci.",
"In addition, these modifications and improvements increase the sensitivity of target gene retrieval while simultaneously reducing background.",
"Solid phase technology has had a significant impact on the efficiency and sensitivity of protein complex purification.",
"Compounds such as sepharose and magnetic beads have allowed for extensive purification and characterization of protein/protein complexes from both in vitro and in vivo mixtures, without compromising the quantitative or qualitative aspects of samples obtained Dynal Corporation Technical Handbook, 1998, Sigma Corporation, cat.",
"#4B-200).",
"It's application for the purposes of identifying transcription factor target genes of unknown origin and in a high-throughput format, however, has yet to be implemented.",
"It is the use of solid phase technology in the presently described invention which significantly increases the sensitivity of obtaining real in vivo targets for transcription factors while reducing background false positive sequences obtained.",
"Through both a combination and modification of the above technologies as well as other molecular biology techniques such as exon scanning, inverse PCR and cDNA library screening the presently described invention allows for the extensive and exhaustive characterization of transcription factor target genes of both known and unknown origin and of a direct (the gene is bound by the factor) and indirect (interaction through other proteins) nature.",
"It is the implementation of chromosomal immunoprecipitation procedures improved via the use of-solid phase support and sequential immunoprecipitation for multiple proteins which permits the potential complete and thorough analysis of a great deal of the transcriptional cascades present in the nucleus of the cell.",
"The proposed technology described herein is applicable to a very limited quantity of cell or tissue samples, which makes it suitable for clinical analysis and comprehensive medical diagnostics.",
"The utilization of this technology will no doubt have a significant impact on the fields of therapeutics, medical diagnostics and basic research related to the realm of transcriptional regulation.",
"3.0 SUMMARY OF THE INVENTION The use of chromosomal immunoprecipitation (ChIP) for the identification of targets for transcriptional regulation by transcription factors has been limited by both the insensitivity of the technology to eliminate considerable nonspecific protein/DNA interactions as well as to the discovery and characterization of only previously identified nucleotide sequences.",
"The presently described invention overcomes the above limitations of chromosomal immunoprecipitation by employing a combination of novel sequential immunoprecipitation procedures utilizing antibodies to the basal transcriptional machinery, solid phase separation procedures and extensive cloning applications including a modified and significantly improved version of inverse PCR which allow for the discovery of target genes and their regulatory elements.",
"The combination of improved chromosomal immunoprecipitation procedures with expression profiling and cloning technologies described in the present invention has allowed for the discovery and characterization of both known and unknown target sequences for chosen transcription factors.",
"In addition, the presently described technology is highly automatable, allowing for extensive high-throughput analysis of virtually any genetic cascade.",
"One embodiment of the present invention is the formaldehyde fixation reaction process which cross-links DNA binding proteins with their prospective nucleotide binding sites present within close proximity or distal to target genes in living cells and or tissues.",
"This fixation reaction is designed and customized specifically for each particular cell line and/or tissue being studied.",
"An additional embodiment of the present invention is other chemical methods utilized for the purposes of fixing and/or cross-linking proteins to their prospective target nucleotide sequences in vivo directly through interaction with DNA or indirectly utilizing protein-protein contacts.",
"Another embodiment of the present invention is the cross-linked protein/target gene complex created by the formaldehyde crosslinkage reaction in vivo.",
"Said complex theoretically contains a mixture of protein/DNA complexes containing the desired transcription factor or regulatory protein directly or indirectly bound to its prospective target loci.",
"Another embodiment of the present invention is an antibody which is specific for Drosophila melanogaster or Sciara coprophila RNA Polymerase II protein large subunit.",
"The antibody may be of monoclonal or polyclonal origin and may recognize similar epitopes from different species.",
"Yet another embodiment of the present invention is an antibody which binds specifically to the mammalian transcription factor p53.Said antibody may be of monoclonal or polyclonal origin.",
"Still another embodiment of the present invention is an antibody-linked to magnetic beads which binds specifically to either Drosophila melanogaster or Sciara coprophila RNA polymerase II protein large subunit.",
"It is the solid-phase support linkage which enhances recovery and specificity of target chromatin upon immunoprecipitation.",
"Another embodiment of the present invention is an antibody which is linked to magnetic beads which binds specifically to the mammalian p53 protein.",
"Yet another embodiment of the present invention is the recovered fraction of the cross-linked, fixed chromatin protein/DNA complex.",
"Another embodiment of the present invention is the sonicated chemically cross-linked protein/DNA complex isolated after sonication but prior to immunoprecipitation.",
"Sonication allows for efficient immunoprecipitation of DNA fragment sizes small enough to be characterized in a high-throughput format via polymerase chain reaction (PCR) or other molecular biology techniques.",
"Still another embodiment of the present invention is the immunoprecipitated protein/DNA complex prior to release of the antibody and reversal of cross-linkage isolated utilizing antibodies which recognize either the Drosophila melanogaster or Sciara coprophila RNA polymerase II large subunit as well as the mammalian p53 protein.",
"An additional embodiment of the present invention is the sequential immunoprecipitation of cross-linked protein/DNA complexes from living cells and tissues utilizing antibodies to core transcriptional machinery factors first and to specific transcription factors second.",
"Sequential immunoprecipitation eliminates the majority of nontranscribed sequences and satellite DNA by focusing only upon transcribed and/or actively regulated genes.",
"It is primary immunoprecipitation with antibodies to proteins found in the basal transcriptional apparatus which results in increased sensitivity through a reduction in the amount of nontranscibed genomic DNA pulled down during subsequent immunoprecipitation reactions.",
"Theoretically only actively transcribed genetic sequences are present as templates for the second round of immunoprecipitation.",
"Secondary immunoprecipitation with antibodies to specific transcription factors is thereby significantly more efficient as is described herein and allows for the opportunity to characterize transcription factor function with respect to the regulation of gene activity.",
"These sequential rounds of immunoprecipitation may be performed in any order with the similar result of increased sensitivity for the discovery of transcription factor target genes and decreased background or nonspecific sequences obtained.",
"Additionally, solid phase chromosomal immunoprecipitation eliminates loss of cross-linked protein/DNA complex material initially precipitated from cellular extracts by providing a solid support and thereby enhances the potential ability to recover target DNA fragments and hence the nucleotide sequences corresponding to these fragments.",
"Excessive loss is prevented through clean, efficient recovery of antibody/protein/DNA complexes due to tight linkages between the solid phase (beads in the case of the present invention) and antibodies.",
"Yet another embodiment of the present invention is the utilization of polymerase chain reaction (PCR) to detect known target loci within the collection of pull-down fragments which putatively contains both known and unknown target genes.",
"It is the detection and monitoring of known controls which allows for a characterization of the efficiency of the system.",
"As well, an additional embodiment of the present invention is the utilization of inverse PCR (I-PCR) in combination with solid phase sequential chromosomal immunoprecipitation for purposes of defining only direct targets for regulation by specific transcription factors as well as for background reduction.",
"Specifically, oligonucleotides corresponding to transcription factor binding sites are used to PCR flanking sequences present in DNA fragment populations isolated by the technology described herein.",
"The application of this modified version of I-PCR to sequentially immunoprecipitated chromosomal templates hence results in the discovery and cloning of direct targets for regulation by the transcription factor in question.",
"Another embodiment of the present invention is the facilitated cloning of both known and unknown target genes from DNA fragments isolated by the presently described methods.",
"These potential targets, for transcription factors of DNA binding and nonDNA binding origin, are cloned through successive rounds of screening against cDNA libraries and genomic DNA libraries, ligation and transfer into bacteriophage and/or plasmid vectors, polymerase chain reaction including but not limited to I-PCR and DNA sequencing.",
"Yet another embodiment contemplated by the present invention is the screening of immunoprecipitated DNA fragments potentially containing target loci against libraries, arrays and/or microarrays of both known and unknown genes.",
"These libraries and arrays may be of either cDNA or oligonucleotide composition.",
"It is the screening of immunoprecipitated DNA fragments against these libraries, arrays and microarrays which facilitates the discovery of target genes for the transcription factor being studied.",
"Said screen allows for a rapid identification of coding sequences for transcription factor target loci present in the collection of DNA.",
"An additional embodiment of the present invention is the cloning of DNA fragment collections containing transcription factor target genes into bacteriophage arms and subsequent packaging into particles for the purposes of rapid conventional screening and sequencing.",
"These bacteriophage libraries may be screened with known DNA probes or other unknown probes for purposes of discovery of target loci.",
"Yet another embodiment of the present invention is the cloning of DNA fragment collections containing transcription factor target genes into exon scanning vectors which may be introduced into eukaryotic cells for purposes of rapidly identifying potential coding sequences within the collection of DNA fragments.",
"Another embodiment of the present invention includes the nucleotide sequences and corresponding amino acid sequences and protein products as determined to be targets for either direct or indirect transcriptional regulation.",
"An additional embodiment of the present invention is the organization of the nucleotide and corresponding amino acid sequences discovered into a database or databases for purposes of rapid search and characterization of these sequences for functional and possible therapeutic relevance.",
"4.0 DESCRIPTION OF THE FIGURES FIG.",
"1 Is a diagrammatic representation of transcriptional regulation by a steroid receptor transcription factor (see text for details).",
"FIG.",
"2 Is an illustration of the chemistry behind in vivo formaldehyde crosslinkage of nuclear protein/DNA interactions (see text for details).",
"FIG.",
"3 Is a diagrammatic illustration of the use of antibody-coated magnetic beads for the recovery of protein/DNA fragments (see text for details).",
"FIG.",
"4 Is a demonstration of the generation of “customizable” fragment sizes by adjustment of sonication conditions (see text for details).",
"FIG.",
"5 Is an outline of the technology described in the present invention for purposes of discovering transcription factor target genes (see text for details).",
"FIG.",
"6 Is a diagrammatic illustration of Exon Scanning.",
"FIG.",
"7A-D Is a demonstration of the utility of the described technology and invention through the analysis of RNA Polymerase II presence on the Sciara coprophila gene II/9-1 under different conditions (see text for details).",
"FIG.",
"8 Is a further demonstration of the utility of the described technology and invention and demonstrates p53 target gene identification after RNA Polymerase II large subunit “preIP/IP”, p53IP and stringent washing conditions (see text for details).",
"FIG.",
"9 Is a diagrammatic illustration of inverse PCR (I-PCR) applied towards DNA fragments isolated by methods described herein (see text for details).",
"Table 1 Is a listing of two target nucleotide sequences representing regulatory elements identified for the transcription factor p53 and the relative induction of transcription from these sequences linked to a minimal promoter in the presence of p53 (see text for details).",
"5.0 DETAILED DESCRIPTION OF THE INVENTION The presently described invention details a methodology for the rapid high-throughput identification of transcription factor target genes.",
"It is achieved through the implementation of solid phase sequential chromosomal immunoprecipitation utilizing antibodies to both tissue and cell-type restricted transcription factors and those of the basal core transcriptional machinery.",
"It is the application of this sequential immunoprecipitation which allows for efficient extraction of protein/DNA, RNA/DNA and RNA/DNA/protein complexes from living cells and or tissues.",
"Combined with the presently described standard as well as modified molecular cloning methodologies these techniques result in rapid and thorough identification and characterization of transcription factor target loci.",
"Particularly, implementation of solid phase sequential chromosomal immunoprecipitation in combination with modified inverse polymerase chain reaction, exon scanning and cloning strategies allows for the identification of direct transcription factor target loci.",
"Implementation of solid phase sequential chromosomal immunoprecipitation in combination with cDNA library and microarray hybridization technologies also allows for rapid identification of transcription factor target genes.",
"The utility of the presently described inventions lies in the rapid identification of transcription factor target genes of both a direct (i.e.",
"binds the factor) and indirect (factor is recruited to the gene through other proteins) nature from a living cell line or tissue.",
"Application of the presently described invention allows for the vast identification of target loci for virtually any transcription factor of either a DNA binding or nonDNA binding nature.",
"It is accomplished through a standard fixation of chromatin in living material, such as cells in tissue culture or isolated tissues, followed by successive immunoprecipitations of extracted protein/DNA complexes with antibodies specific to both transcription factors of interest as well as antibodies specific to the proteins of the core transcriptional machinery.",
"Typically, DNA isolated by these methodologies may then be subjected to various molecular biology procedures such as IPCR, cloning into exon-trapping vectors and/or screening against cDNA libraries or microarrays of known genes to determine the content of actively transcribed genes pulled down with antibodies against chosen transcription factors.",
"Antibodies contemplated by the present invention are utilized for the purposes of immunoprecipitating either DNA binding or nonDNA binding proteins and may be of monoclonal or polyclonal origin.",
"These antibodies described herein are designed against full length proteins as well as against particular epitope amino acid subsets present within those proteins.",
"The antibodies are of rabbit and goat origin, but may be produced through the immunization of any of a number of organisms typically used for research antibody production.",
"The solid phase technology contemplated by the present invention involves the use of magnetic beads.",
"These beads are conjugated to antibodies which specifically recognize particular proteins recovered from living cells and tissues.",
"The magnetic aspect of the bead allows for efficient separation of the bead/antibody/protein/DNA complex from nonspecific materials, including wash solutions, present in the reaction mixture.",
"Other solid phase technologies contemplated by the present invention include sepharose or other solid matrices linked to protein A, protein G or directly conjugated to antibodies which recognize specifically chosen proteins present within living cells/tissues.",
"For the purposes of the present invention the act of immunoprecipitating a protein/DNA complex will involve the utilization of an antibody of either polyclonal or monoclonal origin to directly and specifically recognize, bind and extract a protein/DNA complex from a bulk population of cross-linked protein/DNA complexes.",
"It is this immunoprecipative process which allows for the efficient isolation and ultimate characterization of transcription factor target genes.",
"Molecular biology procedures described in the present invention include use of the collection of DNA fragments potentially containing transcription factor target genes recovered after immunoprecipitation to screen cDNA and/or genomic libraries.",
"Additional molecular biology procedures include cloning the collection of DNA fragments potentially containing transcription factor target sequences into bacteriophage arms or plasmids for efficient screening and or sequencing.",
"For purposes of the present invention the term “gene” will refer to any and all regions of the genome of all organisms which code for proteins.",
"This definition will also include all control elements directly or indirectly associated with controlling the production of mRNA from the gene.",
"In addition, for the purposes of the present invention the term “control element” will refer to any regulatory element which dictates, controls or modulates the production of mRNA from the corresponding gene.",
"The production of mRNA is presumed to occur, at least in part, through the binding of transcription factors.",
"For the purposes of the present invention the term “transcription factor” will refer to any protein which binds directly or indirectly to a control element present within a gene and dictates, controls or modulates either the production or inhibition of production of mRNA from that particular gene.",
"As well, for the purposes of the present invention the term “transcriptional activator” will refer to any protein which binds either directly to a DNA control element or indirectly to a DNA control element through other proteins and activates or drives the production of mRNA from the gene corresponding to that particular control element.",
"For the purposes of the present invention the term “transcriptional repressor” will pertain to any protein which actively downregulates and thereby represses the production of mRNA from a gene to levels below those naturally occurring in an in vivo setting or to undetectable levels.",
"Also for the purposes of the present invention, the term “transcriptional modulator” will refer to any protein which dictates, controls or modulates the production of mRNA from a gene.",
"A gene will be delineated as active and therefore “expressed” when a nucleotide sequence referred to as an activating element is present within the gene or in close proximity to the gene and drives the production of detectable levels of mRNA, presumably through the actions of a transcriptional activating factor or transcriptional modulator.",
"A gene will be delineated as not expressed when mRNA cannot be detected, presumably due to the absence of control activating elements, due to the absence of transcriptional activators present on those elements or due to the presence of transcriptional repressors.",
"Finally, for the purposes of the present invention the term “active repression” will refer to the direct downregulation of a gene due to the presence of a silencing element within that gene or in close proximity to the gene, presumably through the binding at that particular silencing element or negative regulatory element of a transcriptional repressor.",
"5.1 Transcriptional Regulation and Human Physiology With the recent enormous influx of genomic information into the scientific community inevitably comes questions about genetic hierarchies and ultimately gene function.",
"How gene activity is regulated and in what context is as crucial to an understanding of our genetic makeup as the sequence itself.",
"More importantly, the question “What genes are expressed or repressed with respect to physiology?” represents an important concern regarding the discovery and characterization of drug targets.",
"It is clear that the regulation of transcription plays a critical role in a limitless array of physiological processes.",
"For example, a number of transcription factors have previously been implicated as either protooncogenes or tumor suppressors, thus affecting cancer progression by promotion or inhibition of cellular growth and apoptosis (for review see Levine et al., Nature, 1991, 351: 453-456).",
"The transcription factor p53 has been shown to play an indispensable role in the suppression of tumorigenesis and thus has become to be known as a tumor suppressor in its wild-type form (Seto et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1992, 89: 12028-12032).",
"The statistical predisposition to tumorigenesis correlating with mutations in p53 is staggering, with for example, approximately 75-80% of all colon carcinomas studied exhibiting a loss of both p53 alleles.",
"Such a preponderance for cancer upon inactivation of p53 DNA binding function strongly suggests that downstream targets for p53 transcriptional control may potentially play a role in tumor suppression and represent potential avenues of therapeutic intervention.",
"Indeed, several of the targets for direct regulation by p53 have been demonstrated to be involved in the arrest or downregulation of cell proliferation and/or cell death.",
"Examples of these include mdm2 (Oliner et al., Nature, 1993, 362: 857-860), p21/WAF1 (E1-Deiry et al., Cell, 1993, 75: 817-825), hsp70 (Maehara et al., Oncology, 2000, 58: 144-151), cyclin E (Smith et al., Exp.",
"Cell Res., 1997, 230: 61-68) and MDR1 (Achanzar et al., Toxicol.",
"Appl.",
"Pharmacol., 2000, 64: 291-330).",
"As p53 binding sites have been mapped in each of these loci, excellent internal controls exist for monitoring the sensitivity and background issues critical to the success of the technology described herein.",
"The p53 DNA recognition site consists of a dimer of two ten-mers which exists very rarely within the mammalian genome, occurring only around 300 times in a genome of three billion nucleotides (El-Deiry et al., Nat.",
"Genet., 1992, 1(1): 45-49).",
"This rare occurrence of the regulatory site for p53 provides a valuable assessment of the efficiency of the technology presented described technology.",
"Sequence information acquired from fragments immunoprecipitated can be scanned for the presence of this site and direct targets immediately identified while background is simultaneously assessed.",
"In addition to p53, other factors have also been implicated in the progression of cancer.",
"The female sex steroid hormone, estrogen, is required for the development and progression of human breast cancer.",
"To understand how endocrine therapy works and why tumors may become resistant to one therapy but not another, an understanding of the molecular mechanisms of estrogen receptor (ER) function and identification of molecular targets for ER are required.",
"The ER is a nuclear protein that functions as a transcription factor to regulate expression of estrogen responsive genes (Tenbaum et al., Int.",
"J. Biochem.",
"Cell Biol., 1997, 29: 1325-1341).",
"Some of these estrogen-regulated genes mediate growth and development of the mammary glands, and it is apparent that many are important for the effects of estrogen on tumor cell proliferation.",
"After estrogen or an estrogen analog binds to ER, dimerization of the receptor is induced which then allows binding of the complex to estrogen-responsive elements (ERE), a region in the promoter of estrogen target genes.",
"The binding of the ER dimer to this promoter region then facilitates transcription of that gene.",
"Most endocrine therapies for breast cancer inhibit tumor formation by depriving the cell of estrogen or by blocking its receptor.",
"Synthetic drugs like tamoxifen were first called antiestrogens because they bind ER and competitively block the effects of estrogen on tumor cell proliferation and on expression of certain genes.",
"However, it is not surprising that administration of this drug can have a spectrum of effects, depending on species, tissue, cell or gene context (Kazelenellenbogen et al., Breast Cancer Res.",
"Treat., 1997, 44: 23-38.).",
"In some cases, these “antiestrogens” can be estrogenic, stimulating transcription of genes which may change cellular morphology.",
"For instance, tamoxifen, which works as an antagonist to ER in breast cancer cells, can induce tumor development in the uterus (Deligdisch, L., Mod.",
"Pathol., 1993, 6(1): 94-106).",
"In other cases, sometimes in the same cell, they have predominant antiestrogenic activity.",
"These data provide the rationale for the identification of patterns for ER gene targets which can be activated and/or repressed upon the variety of drug treatments in different organs or tissues.",
"Those molecular targets could potentially be used as important tumor markers and/or could provide additional indispensable information on hormonal responsiveness and further therapeutic intervention.",
"Determination of cell fate and regulation of terminal differentiation by transcription factors represent major roles for these regulatory proteins in regulating physiology.",
"The ability to generate mature lymphocytic cells in tissue culture, for example, has been of intense interest for a number of years as the potential for replenishing low T and B cell counts in patients undergoing chemotherapy or are HIV positive, for example, becomes a reality.",
"The ikaros family of transcription factors has been shown to promote the differentiation of hematopoietic stem cells into the mature B and T cell lineages (Nichogiannopoulou et al., Semin.",
"Immunol., 1998, 10: 119-125).",
"Correspondingly, mice which possess a mutation in the conserved DNA binding domain of the ikaros locus fail to possess B and T lymphocytes as well as the earliest progenitors of these lineages (Winandy et al., Cell, 1995, 83: 289-299).",
"Thus, the ability to determine the downstream targets for ikaros allows for the potential to identify genes which promote hematopoietic stem cell differentiation and hence B and T cell production.",
"The DNA recognition sequence for the ikaros family has been previously characterized (Molhar et al., Mol.",
"Cell Biol., 1999, 14: 8292-8303), thus loci identified through the technology described herein as potential targets can be scanned for this recognition sequence as a confirmation of interaction.",
"Other more organogenic effects on human physiology are also controlled by transcription factors.",
"Cardiac hypertrophy, or enlargement of the heart, is the result of attempts by the cardiovascular system to compensate for progression of many forms of cardiac disease, including hypertension, mechanical load, heart attack (myocardial infarction) and others (for review see McKinsey et al., Curr.",
"Opin.",
"Genet.",
"Dev., 1999, 9: 267-274).",
"At the molecular-level, external stress factors such as hypertension and myocardial infarction result in a reactivation of the fetal cardiac genetic program, as well as a general physiological enlargement of the myocardium through increased myocardial cell size.",
"A number of transcription factors have been suggested to be involved in the initiation and maintenance of the reactivation of fetal cardiac genes.",
"GATA4, a member of the GATA family of transcription factors, is involved in the upregulation of several fetal cardiac (Herzig et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1997, 94: 7543-7348).",
"Studies of GATA4 and other factors involved in response to cardiac stress will reveal novel cascades of genes representing potential targets for therapeutic prevention and/or intervention of enlargement of the heart.",
"In addition, there are a number of human genetic disorders affecting both growth and reproductive capacity.",
"Several of these include mutations in transcription factors which have previously been shown to play vital roles in neuroendocrine organogenesis during embryonic development as well as appropriate functioning of this system in the adult (for review see Treier et al., Genes Dev., 1998, 12: 1691-1704).",
"Defects in human growth and fertility represent a major concern among the world population.",
"Many of the problems relating to these phenomena arise due to misregulation of genes which play crucial roles in the neuroendocrine system.",
"Progress in understanding this complex field has been aided by the fact that several murine animal models have been shown to exhibit phenotypes strikingly similar to that demonstrated by allelic mutations in humans.",
"For example, mutations in the human Prop-1 locus result in familial combined pituitary hormone deficiency, a finding quite similar to that found in the naturally occurring Ames mouse mutant (Wu et al., Nat.",
"Genet., 1998, 6: 1143-1152).",
"As well, both the Snell and Jackson dwarf mice have been shown to contain mutations within the Pit-1 locus (Rhodes et al., Curr.",
"Opin.",
"Genet.",
"Dev., 1994, 4: 709-717).",
"A number of human dwarfism cases which display similar pituitary lineage loss have now been demonstrated to carry mutations in the same locus (Wu et al., Nat.",
"Genet., 1998, 6: 143-1152).",
"Both the Prop-1 and Pit-1 genes are POU domain-containing homeobox transcription factors which act at distinct temporal and spatial points within the development of the pituitary gland.",
"Studies on the Ames dwarf have suggested that Prop-1 acts upstream of Pit-1 in the developmental regulatory cascade, putatively setting up a rudimentary organ from which Pit-1 is able to guide lineage determination and differentiation (Dasen et al., Cell, 1999,97: 587-598).",
"Indeed, Pit-1 has been shown by a number of groups to play an indispensable role in the survival and terminal differentiation of the somatotrope, lactotrope and thyrotrope pituitary cell lineages (Rhodes et al., Curr.",
"Opin.",
"Genet.",
"Dev., 1994, 4: 709-717).",
"In the absence of these cell populations, specifically that of the somatotrophic lineage, dwarfism and other growth defects occur (Treier et al., Genes Dev., 1998, 12: 1691-1704).",
"Many of the mutations which have been characterized in humans as well as other organisms for these factors lie in the DNA binding domain, which strongly suggests that an inability to effectively bind to and thus regulate downstream target genes is directly involved in the growth and fertility defects observed.",
"An application of the technology described herein to identify and characterize both direct and indirect targets for these factors will undoubtedly reveal novel pathways for potential therapeutic intervention for both growth and fertility defects in humans.",
"It is evident that the activation or repression of gene activity is essential for the appropriate development, growth and viability of an organism.",
"An understanding of the transcription factors described above as well as many others that govern these processes and specifically the identification of which target genes are controlled by these factors in both temporal and spatial manners during embryonic development and throughout adulthood is crucial to understanding various phenotypic characteristics.",
"Current technologies such as subtractive hybridization (Lockyer et al., Parasitology, 2000, 120: 399-407), differential display (Neilson et al., Genomics, 2000, 3: 13-24) and SAGE (Stephan et al., Mol.",
"Gen.",
"Metab., 2000, 70: 10-18), while effective at identifying target genes, are generally time consuming and do not implicitly arrive at direct transcription factor targets.",
"In addition, they require cell lines or tissues to differentially express the factor being studied, a task not often easily achieved.",
"Other methods such as protein/DNA affinity purification may deduce enhancer binding sites from the genome but lack the ability to reveal the gene regulated by the enhancer, thus requiring positional cloning of exonic coding sequences (Solomon et al., Cell, 1988, 53: 937-947).",
"Recent progress has been made, however, in the identification transcription factor targets and their corresponding coding sequences in vivo through the infection of living cells with modified retroviruses which seek out genomic transcription factor binding sites via integrase/transcription factor fusion proteins incorporated into the viral particle and “trap” exons (Burgess et al., U.S. Pat.",
"No.",
"6,139,833, Issued Oct. 31, 2000).",
"By studying the intricate outline of terminal target genes regulated by various transcription factors rather than the factors themselves, it will be possible not only to discover novel therapeutic targets, but also to efficiently focus drug delivery to discrete physiologic gene products thus enhancing effectiveness and reducing or eliminating side effects.",
"Moreover, the genes discovered as regulated by transcription factors may be used in a microarray format as phenotypical markers for medical diagnostics.",
"5.2 Modified-Chromosomal Immunoprecipitation In order to identify and characterize direct molecular targets for regulation by specific transcription factors, it is necessary to employ technologies which take advantage of the extremely specific protein/DNA contacts involved in gene regulation which are maintained within intact cells or tissues.",
"The Chromosomal Immunoprecipitation (ChIP) assay has been well established and may be successfully performed by those skilled in the art (Solomon et al., Cell, 1988, 53: 937-947; de Belle et al., Biotechniques, 2000, 29(19): 170-175).",
"It allows for manipulation of the above mentioned inherent physical interactions between proteins and DNA to delineate known downstream targets for virtually any transcription factor.",
"This method is based on the ability of formaldehyde or other chemicals to produce DNA/protein, RNA/protein and protein/protein cross-links at 2 angstrom resolution in vivo within intact cells or tissues.",
"Addition of formaldehyde to living cells results in formation of an extensively cross-linked network of biopolymers, thus preventing any large-scale redistribution of cellular components.",
"Formaldehyde does not react with free double-stranded DNA, avoiding kinetic constraints due to DNA damage.",
"In addition, formaldehyde crosslinks can be reversed under mild conditions so that DNA, RNA and protein complexes can be further analyzed separately.",
"FIG.",
"2 illustrates the chemistry behind the crosslinkage method.",
"The experimental design originates from the pioneering work of Alexander Varshavsky who developed the chromatin fixation, purification and immunoprecipitation scheme for analyzing the distribution of histones in the Drosophila heat-shock gene promoter (Solomon et al., Cell, 1988, 53: 937-947).",
"Upon reversal of crosslinkage and mechanical shearing of cellular DNA, protein/DNA interactions can be assessed by utilizing sequence information of known target loci in combination with the Polymerase Chain Reaction (PCR) (Innis et al., Academic Press, 1990; McPherson et al., IRL Press, 1991; Erlich, A. Stockton Press, 1989).",
"Recent work has demonstrated that the ChIP assay can be applied to the study of virtually any transcription factor which comes into contact, either directly or indirectly, with DNA (Scully et al., Science, 2000, 290(5494):1127-31; Jepsen et al., Cell, 2000, 102(6):753-63).",
"Living cells and/or isolated tissues are fixed with formaldehyde by adding cross-linking agent directly to the cell growth medium or tissue.",
"Although the presently described invention utilizes salivary glands from Sciara coprophila for RNA Polymerase II and Hela cells for p53 it is in no way limited to these particular tissues and cell types.",
"Other tissues from other organisms and species include, but are not limited to heart, brain, spleen, lung, liver, muscle, kidney, testis, ovary, gut, hypothalamus, pituitary, tooth bud, mesoderm, ectoderm, endoderm, neural tube, somite, smooth muscle, cardiac muscle, skeletal muscle and all embryonic tissues from all possible timepoints.",
"Cell lines from which transcription factor target genes may be discovered via methodologies provided by the presently described invention include, but are in no way limited to 13C4 (mouse/mouse, hybrid, hybridoma), 143 B (human, bone, osteosarcoma), 2 BD4 E4 K99 (mouse/mouse, hybrid, hybridoma), 3 C9-D11-H11 (mouse/mouse, hybrid, hybridoma), 3 E 1 (mouse/mouse, hybrid, hybridoma), 34-5-8 S (mouse/mouse, hybrid, hybridoma), 3T3 (mouse, Swiss albino, embryo), 3T3 L1 (mouse, Swiss albino, embryo), 3T6 (mouse, Swiss albino, embryo), 5 C 9 (mouse/mouse, hybrid, hybridoma), 5G3 (hybrid, hybridoma), 6-23 (clone 6) (rat, thyroid, medullary, carcinoma), 7 D4 (mouse/rat, hybrid, hybridoma), 72 A1 (mouse/mouse, hybrid, hybridoma), 74-11-10 (mouse/mouse, hybrid, hybridoma), 74-12-4 (mouse/mouse, hybrid, hybridoma), 74-22-15 (mouse/mouse, hybrid, hybridoma), 74-9-3 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, B cell), 76-74 (mouse/mouse, hybrid, hybridoma), 7C2C5C12 (mouse/mouse, hybrid, B cells x myeloma, hybridoma), 9 BG 5 (mouse/mouse, hybrid, hybridoma), 9-4-3 (mouse/mouse, hybrid, hybridoma), A 172 (human, glioblastoma), A 375 (human, malignant melanoma), A 72 (dog, golden retriever, connective, not defined tumor), A427 (human, Caucasian, lung, carcinoma), A498 (human, kidney, carcinoma), A-704 (human, kidney, adenocarcinoma), A549 (human, lung, carcinoma), ACHN (human, Caucasian, kidney, adenocarcinoma), ACT 1 (mouse/mouse, hybrid, hybridoma), AE-1 (mouse/mouse, hybrid, hybridoma), AE-2 (mouse/mouse, hybrid, hybridoma), Aedes albopictus (mosquito—Aedes albopictus, larvae), AGS (human, Caucasian, stomach, adenocarcinoma), AK-D (cat, lung, embryonic), Amdur II human, Caucasian, skin, fibroblast, methylmalonicacidemia), AV 3 (human, amnion), B 95.8 (monkey, marmoset, leukocyte), B-63 (mouse, mammary gland, carcinoma), B2-1 (mouse, BALB/c, embryo), B50 (rat, nervous system, nervous tissue glial tumor), B69 (mouse/mouse, hybrid, hybridoma), B95a (monkey, marmoset), BAE (bovine, aorta), BALB 3T12-3 (mouse, BALB/c, embryo), BALB 3T3 clone A31 (mouse, BALB/c, embryo), BB (fish—Ictalurus nebulosus (bullhead brown catfish), trunk), BBM.1 clone E9 (mouse/mouse, hybrid, hybridoma), BC3H1 (mouse, brain, brain tumor), BCE C/D-1b (bovine, cornea), BeWo (human, placenta, choriocarcinoma), BF-2 (fish—bluegill fry, caudal trunk), BGM (monkey, African green, kidney), BHK 21 clone 13 (hamster, golden Syrian, kidney), BNL CL.2 (mouse, BALB/c, liver, embryonic), BNL SV A.8 (mouse, liver, embryonic), BS/BEK (bovine, kidney, embryonic), BSC-1 (monkey, African green, kidney), BT (bovine, turbinate), Bu (IMR-31) (buffalo, lung), BUD-8 (human, Caucasian, skin, fibroblast), BXPC-3 (human, pancreas, adenocarcinoma), C 1271 (mouse, RIII, mammary gland, mammary tumor), C2C12 (mouse, muscle), C32 (human, melanoma, amelanotic), C6 (rat, glial tumor), Caco-2 (human, Caucasian, colon, adenocarcinoma), Caki-1 (human, Caucasian, kidney, carcinoma), Caki-2 (human, Caucasian, kidney, carcinoma), CaLu-1 (human, Caucasian, lung, carcinoma, epidermoid), Calu-3 (human, Caucasian, lung, adenocarcinoma), CAPAN 1 (human, Caucasian, pancreas, adenocarcinoma), CAPAN 2 (human, Caucasian, pancreas, carcinoma), CAR (fish—goldfish, fin), CCF-STTRG1 (human, Caucasian, astrocytoma, anaplastic, grade IV), CCRF S 180 II (mouse, CFW, sarcoma), CCRF-CEM (human, Caucasian, peripheral blood, leukemia, acute lymphoblastic), CCRF-SB (human, Caucasian, peripheral blood, leukemia, acute lymphoblastic), CEM/C2 (human, leukemia, T cell), Cf2Th (dog, thymus), Chang liver (human, liver), CHO K1 (hamster, Chinese, ovary), CHP 3 (human, Black, skin, fibroblast, galactosemia), CHP 4 (human, Black, skin, fibroblast, asymptomatic galactosemia), CHSE 214 (fish—salmon, embryo), Clone 1-5c4 WKD of Chang Conjunctiva (human, conjunctiva), Clone M-3 (mouse, (CxDBA) F1, skin, melanoma), CMT 93 (mouse, C57BL/ICRFat, rectum, carcinoma), COS-1 (monkey, African green, kidney), COS-7 (monkey, African green, kidney), CPA (bovine, endothelium, pulmonary artery), CPA 47 (bovine, endothelium, pulmonary artery), CPAE (bovine, endothelium, pulmonary artery), CRFK (cat, domestic, kidney), CRI-D11 (rat, NEDH, insulinoma), CSE 119 (fish—salmon, embryo), CV 1 (monkey, African green, kidney), CVC 7 (Agrothis segetum, hybrid, hybridoma), D 17 (dog, bone, sarcoma, osteogenic), Daudi (human, Black, lymphoma, Burkitt), DB 9 G.8 (mouse/mouse, hybrid, hybridoma), DB 1-Tes (dolphin, Delphinus bairdi, testis), DeDe (hamster, Chinese, lung), Detroit 510 (human, Caucasian, skin, fibroblast, galactosemia), Detroit 525 (human, Caucasian, skin, fibroblast, Turner syndrome), Detroit 529 (human, Caucasian, skin, fibroblast, trisomy 21/Down syndrome), Detroit 532 (human, Caucasian, foreskin, trisomy 21/Down syndrome), Detroit 539 (human, Caucasian, skin, fibroblast, trisomy 21/Down syndrome), Detroit 548 (human, Caucasian, skin, fibroblast, partial D trisomy), Detroit 550 (human, skin, fibroblast), Detroit 551 (human, Caucasian, skin, embryonic), Detroit 562 (human, Caucasian, pharynx, carcinoma), Detroit 573 (human, Caucasian, skin, fibroblast, B/D translocation), Detroit 6 (human, bone marrow), DK (dog, beagle, kidney), DON (hamster, Chinese, lung), DU 145 (human, Caucasian, prostate, carcinoma), Duck embryo (duck, Pekin, embryo), E.Derm (horse, dermis), EBTr (bovine, trachea, embryonic), ECTC (bovine, thyroid, embryonic), ECV304 (human, Asiatic, umbilical cord), E[AV 12E8.1 (mouse/mouse, hybrid, hybridoma), Ep 16 (mouse/mouse, hybrid, hybridoma), EPC (fish, carp epidermal, epithelioma), EREp (rabbit, skin, embryonic), ESK-4 (pig, kidney, embryonic), FBHE (bovine, heart, embryonic), Fc 2 Lu (cat, lung, embryonic), Fc 3 Tg (cat, tongue, embryonic), FeLV 3281 (cat, lymphoma), FHM (fish—minnow, skin), FL (human, amnion), FRhK-4 (monkey, rhesus, kidney, embryonic), G-7 (mouse, Swiss-Webster, muscle), G.8 (mouse, Swiss-Webster, muscle), GCT (human, lung, metastasis, histiocytoma), GH 1 (rat, Wistar-Furth, pituitary tumor), GH 3 (rat, Wistar-Furth, pituitary tumor), Girardi heart (human, heart), GK 1.5 (mouse/rat, hybrid, hybridoma), H 16-L10-4R 5 (mouse/mouse, hybrid, hybridoma), H 9 (human, leukemia, acute lymphoblastic), H-4-II-E (rat, liver, hepatoma), H4 (human, Caucasian, brain, nervous tissue glial tumor), H4-II-E-C3 (rat, AxC, liver, hepatoma), H4TG (rat, liver, hepatoma), H9c2(2-1) (rat, BDIX, heart), Hak (hamster, Syrian, kidney), HCT 116 (human, colon, carcinoma), HCT-8 (human, intestine, ileocecal, adenocarcinoma), HEL 299 (human, Caucasian, lung, embryonic), HeLa (human, Black, cervix, carcinoma, epitheloid), HeLa 229 (human, Black, cervix, carcinoma, epitheloid), HeLa S 3 (human, Black, cervix, carcinoma, epitheloid), Hep 2 (human, Caucasian, larynx, carcinoma, epidermoid), Hep 3B2.1-7 (human, liver, carcinoma, hepatocellular), Hep G2 (human, Caucasian, liver, carcinoma, hepatocellular), Hepa 1-6 (mouse, liver, hepatoma), HFL (human, lung), HG 261 (human, Caucasian, skin, fibroblast, Fanconi anemia), HGF 24 (human, gingival stroma), HL 60 (human, Caucasian, peripheral blood, leukemia), HOS (human, Caucasian, bone, osteosarcoma), HRT 18 (human, rectum-anus, adenocarcinoma), Hs 683 (human, neuroglia, glioma), Hs 863.T (human, bone, sarcoma, Ewing's), HS 883.T (human, bone, giant cell, sarcoma), HS 888 Lu (human, Caucasian, lung), Hs-27 (human, foreskin), HSDM1C1 (mouse, Swiss albino, fibrosarcoma), HT 1080 (human, Caucasian, acetabulum, fibrosarcoma), HT 1376 (human, Caucasian, bladder, carcinoma), HT-29 (human, Caucasian, colon, adenocarcinoma), HuTu 80 (human, adenocarcinoma), I 10 (mouse, BALB/cJ, testis, Leydig cells, testicular tumor), IB-RS-2 (pig, kidney), IBRS-2 D10 (pig, kidney), IEC-6 (rat, intestine, small), IM-9 (human, Caucasian, bone marrow, multiple myeloma), IMR 31 Bu (buffalo, lung), IMR 32 (human, Caucasian, neuroblastoma), IMR-90 (human, Caucasian, lung, embryonic), Intestine 407 (human, Caucasian, intestine, embryonic), J 111 (human, leukemia, monocytic), J 774A.",
"1 (mouse, BALB/c, monocyte-macrophage, not defined tumor), Jensen sarcoma (rat, sarcoma), JH 4 clone 1 (guinea pig, strain 13, lung), Jiyoye (human, Black, ascitic fluid, lymphoma, Burkitt), JM (human, leukemia, T cell), Jurkat J6 (human, leukemia, T cell), K 562 (human, Caucasian, pleural effusion, leukemia, chronic myeloid), KATO III° (human, Mongoloid, stomach, carcinoma), KB (human, Caucasian, mouth, carcinoma, squamous cell), KHOS/NP (human, Caucasian, bone, osteosarcoma), KMP (mouse), L 1210 (mouse, ascitic fluid, leukemia, lymphocytic), L 132 (human, lung, embryonic), L 21.6 (mouse, hybrid, hybridoma), L 243 (mouse/mouse, hybrid, hybridoma), L 5.1 (mouse/mouse, hybrid, hybridoma), L 929 (mouse, C3H/An, connective), L6 (rat, skeletal muscle), LC 540 (rat, Fisher, testis, Leydig cells, testicular tumor), LLC-MK2 (monkey, rhesus, kidney), LLC-PK1 (pig, kidney), LLC-RK1 (rabbit, New Zealand white, kidney), LLC-WRC 256 (rat, Walker, carcinoma), LM from NCTC clone 929 (mouse, C3H/An, connective), LM TK negative (mouse, C3H/An, connective), LNCaP.FGC (human, Caucasian, prostate, carcinoma), LS 180 (human, Caucasian, colon, adenocarcinoma), M 1 (mouse, SL, bone marrow, leukemia, myeloid), M-2E6 (mouse/mouse, hybrid, hybridoma), M2-1C6-4R3 (mouse/mouse, hybrid, hybridoma), MA 104 (monkey, African green, kidney, embryonic), mAB 35 (mouse/rat, hybrid, B cells x myeloma, hybridoma, B cell), MARC 145 (monkey, kidney), Mc Coy (mouse), MC/CAR (human, plasmacytoma, B cell), MCF 7 (human, Caucasian, breast, adenocarcinoma), MDBK (bovine, kidney), MDBK(BU 100) (bovine, kidney), MDCC MSB1 (chicken, avian, spleen, lymphoma), MDCK (dog, cocker spaniel, kidney), MDOK (sheep, kidney), MDTC RP 19 (turkey, lymphocyte, Marek's disease), MEL III (monkey, rhesus, mammary gland, mammary tumor), MG-63 (human, bone, osteosarcoma), MH 1 C 1 (rat, buffalo, liver, hepatoma), MH-S (mouse, lung), MIA PaCa-2 (human, Caucasian, pancreas, carcinoma), MiCl1 (mustela vison (mink), lung), MK-D6 (mouse/mouse, hybrid, hybridoma), MLA 144 (gibbon, lymphosarcoma), MOLT-3 (human, peripheral blood, leukemia, acute lymphoblastic T cell), MOLT-4 (human, peripheral blood, leukemia), MPC-11 (mouse, BALB/c, myeloma), MPK (minipig, kidney), MRC-5 (human, lung, embryonic), MRSS-1 (mouse/mouse, hybrid, hybridoma, B cell), MS (monkey), Mv 1 Lu (mustela vison (mink), lung), MVPK-1 (pig, kidney), NA C 1300 clone (mouse, brain, neuroblastoma), Namalwa (human, Black, lymphoma, Burkitt), NCTC 2544 (human, skin, keratinocyte), NCTC clone 3526 (monkey, rhesus, kidney), Neuro-2a (mouse, albino, neuroblastoma), NIH:OVCAR-3 (human, Caucasian, adenocarcinoma, ovary), NOR 10 (mouse, muscle), NRK 49F (rat, kidney), NSO (mouse, BALB/c, myeloma), OA1 (sheep, brain), OHH1.K (deer, kidney), OKT 3 (mouse/mouse, hybrid, hybridoma), OKT 4 (mouse/mouse, hybrid, hybridoma), OKT 8 (mouse/mouse, hybrid, hybridoma), P 3 HR 1 (human, lymphoma, Burkitt), P3 88 D1 (mouse, DBA/2, monocyte-macrophage, lymphoma), P3 NS1 Ag4 (mouse, myeloma), P3NP/PFN (mouse/mouse, hybrid, hybridoma), P815 (mouse, mastocytoma), PANC-1 (human, Caucasian, pancreas, carcinoma), PC 61-5-3 (mouse/rat, hybrid, hybridoma), PC-12 (rat, adrenal medulla, pheochromocytoma), PD 5 (pig, kidney), PEG 1-6 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, B cell), PK 15 (pig, kidney), PLC/PRF/5 (human, liver, hepatoma, Alexander cells), Pt K1 (marsupial—potoroo, kidney), QT35 (quail, Japanese, fibrosarcoma), QT 6 (quail, Japanese, fibrosarcoma), R 2 C (rat, Wistar-Furth, testis, Leydig cells, testicular tumor), R 9 ab (rabbit, New Zealand white, lung), R D (human, Caucasian, muscle, rhabdomyosarcoma, embryonal), R63 (mouse/mouse, hybrid, B cells x myeloma, hybridoma, B cell), RAB-9 (rabbit, New Zealand white, skin, fibroblast), Raji (human, Black, lymphoma, Burkitt), RBL 1 (rat, leukemia, basophilic), RFL 6 (rat, Sprague-Dawley, lung), RK 13 (rabbit, kidney), RK 13/1 (rabbit, kidney), RPMI 1788 (human, Caucasian, peripheral blood), RPMI 1846 (hamster, golden Syrian, skin, melanoma, melanotic), RPMI 2650 (human, nasal septum, carcinoma, squamous cell), RPMI 8226 (human, peripheral blood, myeloma), RR 1022 (rat, Amsterdam, sarcoma), RTG 2 (fish—trout, rainbow, gonad), RTO (fish—trout, rainbow, ovary), Saos-2 human, Caucasian, bone, osteosarcoma), Sf 1 Ep (rabbit, domestic, epidermis), SIRC (rabbit, cornea), SK-LU-1 (human, Caucasian, lung, adenocarcinoma, grade III), SK-MES-1 (human, lung, carcinoma, squamous cell), SK-NEP-1 (human, Caucasian, kidney, Wilms' tumor), SK-OV-3 (human, Caucasian, ovary, adenocarcinoma), SSE 5 (fish—trout, embryo), STO (mouse, SIM, embryo), SV-T2 (mouse, BALB/c, embryo), SW 13 (human, Caucasian, adrenal cortex, adenocarcinoma), T 98 G (human, Caucasian, glioblastoma), Tb 1 Lu (bat, lung), TE 671 (human, Caucasian, medulloblastoma), TK TS 13 (hamster, Syrian, kidney), U 937 (human, Caucasian, pleural effusion, lymphoma, histiocytic), VERO (monkey, African green, kidney), VERO 76 (monkey, African green, kidney), VERO C 1008 (monkey, African green, kidney), WC 1 (fish, dermis, sarcoma), WF 2 (fish—Walley whole fry, fibroblast), WI 26 VA 4 (human, Caucasian, lung, embryonic), WI 38 (human, Caucasian, lung, embryonic), WI 38 VA 13 (human, Caucasian, lung, embryonic), WI-1003 (human, lung), WISH (human, amnion), WM 115 (human, skin, melanoma), XC (rat, Wistar, sarcoma), Y 1 (mouse, LAF1, adrenal cortex, adrenal tumor), ZR-75-1 (human, Caucasian, breast, carcinoma) and any other as yet undiscovered or uncharacterized cell lines through which the presently described invention may be implemented for the discovery of transcription factor target genes.",
"Preliminary time course experiments spanning between 5 minutes and 1 hour of fixation are performed to yield the best combination of in vivo fixed chromatin, high DNA recovery, and small size of chromatin fragments.",
"For specific purposes, the cross-linking time can be considerably reduced or prolonged and must be optimized for the particular tissue or cell line and the transcription factors being studied.",
"FIG.",
"2 illustrates the chemical cross-linking of DNA and proteins by formaldehyde.",
"Formaldehyde (HCHO) is a very reactive dipolar compound in which the carbon atom is the nucleophilic center.",
"Amino and imino groups of the proteins (e.g.",
"the side chains of lysine and arginine) and of nucleic acids (e.g., cytosines) react with formaldehyde, leading to the formation of a Schiff base (reaction I).",
"This intermediate can react with a second amino group (reaction II) and condenses.",
"Cross-links may be reversed by heating in Tris-HCL—containing buffers.",
"This leads to a drop in pH and protonation of amino groups, thus forcing the equilibrium in the reverse direction.",
"In FIG.",
"2(A) illustrates formaldehyde-mediated cross-linking between the side chains of the lysines and (B) depicts cross-linking between cytosine and lysine.",
"While the present invention employs formaldehyde as a chemical component for the cross-linking of protein/DNA complexes in living cells and tissues, it is in no way limited to this reagent for fixation.",
"Other chemicals may also be utilized to fix proteins to DNA (Benashski et al., Methods, 2000, 22: 365-371).",
"Some of these include, but are in no way limited to homobifunctional compounds difluoro-2,4-nitrobenzene (DFDNB), dimethyl pimelimidate (DMP), disuccinimidyl suberate (DSS), thcarbodimide reagent EDC, psoralens including 4,5′, 8-trimethylpsoralen, photo-activatable azides such as 125I(S-[2-(4-azidosalicylamido)ethylthio]-2-thiopyridine) otherwise known as AET, (N-[4-axidosalicylamido)butyl]-3′[2′-pridyldithio]propionamide) also known as APDP, the chemical cross-linking reagent Ni(II)-NH2-Gly-Gly-His-COOH also known as Ni-GGH, sulfosuccinimidyl 2-[(4-axidosalicyl)amino]ethyl]-1,3-dithiopropionate) also known as SASD, (N-14-(2-hydroxybenzoyl)-N-11(4-azidobenzoyl)-9-oxo-8,11,14-triaza-4,5-ditheatetradecanoate) and any as yet uncharacterized or undiscovered reagents which result in the cross-linking of protein/DNA complexes in living cells and tissues.",
"Upon fixation of protein/DNA complexes in intact cells or tissues cellular lysis is accomplished through standard protocols which may be successfully implemented by those skilled in the art (Solomon et al, Cell, 1988, 53: 937-947; de Belle et al., Biotechniques, 2000, 29(1): 170-175).",
"For the purposes of chromosomal immunoprecipitation it is important that metal chelators such as EDTA and EGTA as well as protease inhibitors be added to the reaction to prevent degradation of protein/DNA complexes.",
"The mixture is subsequently mechanically lysed on ice via the its passage through a 26 G needle.",
"Typically 4 rounds of 25-30 needle passages per round are necessary for sufficient lysis and chromatin fractionation.",
"It is speculated that these parameters must be defined for each tissue or cell type.",
"Alternatively, samples may be lysed via the use of a Dounce homogenizer or the implementation of any mechanical stress which results in efficient breakage of cellular membranes and hence release of chromatin containing protein/DNA complexes.",
"Fixed, lysed cells or tissues are subsequently subjected to high resolution mechanical shearing, i.e.",
"sonication for the purposes of producing manageable DNA fragment sizes of the desired length.",
"In general, the size of the DNA fragments may be critical for high-resolution mapping studies as well as the identification of transcription initiation sites and/or exonic sequences, thus sonication provides a convenient method to “customize” fragment length as illustrated in FIG.",
"4.The sizes observed are a typical result obtained with Hela cells cross-linked for 30 minutes and sonicated by routine use of the Branson model 250 sonifer with microtip at constant power for various amounts of time.",
"While the presently described invention employs the use of a Branson model 250 sonifier/sonicator for the purposes of generating appropriate DNA fragment length from fixed lysed cells and/or tissues it is hypothesized that any mechanical instrument or enzymatic digestion capable of shearing or cutting soluble chromatin into lengths small enough to be manipulated via standard or modified molecular biology procedures for the purposes of discovering transcription factor targets may be utilized, These include, but are in no way limited to other sonicator models as well as restriction enzyme digestion by frequent as well as rare-cutting enzymes including, but in now way limited to, Acc I, Aci I, Acl I, Afe I, Afl II, Afl III Age I, Ahd I, Alu I, Alw I, AlwN I, Apa I, ApaL I, Apo I, Asc I, Ase I, Ava I, Ava II, Avr II, Bae I, BamH I, Ban I, Ban II, Bbs I, Bbv I, BbvC I, BceA I, Bcg I, BciV I, Bcl I, Bfa I, BfrB I, Bgl I, Bgl II, Blp I, Bmr I, Bpm I, BsaA I, BsaB I, BsaH I, Bsa I, BsaJ I, BsaW I, BsaX I, BseR I, Bsg I, BsiE I, BsiHKA I, BsiW I, Bsl I, BsmA I, BsmB I, BsmF I, Bsm I, BsoB I, Bsp1286 I, BspD I, BspE I, BspH I, BspM I, BsrB I, BsrD I, BsrF I, BsrG I, Bsr I, BssH II, BssK I, BssS I, BstAP I, BstB I, BstE II, BstF5 I, BstN I, BstU I, BstX I, BstY I, BstZ17 I, Bsu36 I, Btg I, Btr I, Bts I, Cac8 I, Cla I, Dde I, Dpn I, Dpn II, Dra I, Dra III, Drd I, Eae I, Eag I, Ear I, Eci I, EcoN I,EcoO109 I, EcoR I, EcoR V, Fau I, Fnu4H I, Fok I, Fse I, Fsp I, Hae II, Hae III, Hga I, Hha I, Hinc II, Hind III, Hinf I, HinP1 I, Hpa I, Hpa II, Hpy188 I, Hpy188 III, Hpy99 I, HpyCH4III, HpyCH4IV, HpyCH4V, Hph I, Kas I, Kpn I, Mbo I, Mbo II, Mfe I, Mlu I, Mly I, Mnl I, Msc I, Mse I, Msl I, MspA1 I, Msp I, Mwo I, Nae I, Nar I, Nci I, Nco I, Nde I, NgoM IV, Nhe I, Nla III, Nla IV, Not I, Nru I, Nsi I, Nsp I, Pac I, PaeR7 I, Pci I, Pf1F I, Pf1M I, Ple I, Pme I, Pml I, PpuM I, PshA I, Psi I, PspG I, PspOM I, Pst I, Pvu I, Pvu II, Rsa I, Rsr II, Sac I, Sac II, Sal I, Sap I, Sau3A I, Sau96 I, Sbf I, Sca I, Scrf I, SexA I, SfaN I, Sfc I, Sfi I, Sfo, SgrA I, Sma I, Sml I, SnaB I, Spe, Sph I, Ssp I, Stu I, Sty I Swa I, Taq I, Tfi I, Tli I, Tse I, Tsp45 I, Tsp509 I, TspR I, Tth111 I, Xba I, Xcm I, Xho I, Xma I, Xmn I and any other as yet uncharacterized or undiscovered restriction endonucleases which may be utilized to cut DNA for the purposes of implementing the presently described invention to discover transcription factor target genes of both known and unknown origin.",
"It is the ability to customize the length of said DNA fragments which allows for the cloning of transcription factor targets upon immunoprecipitation utilizing solid phase sequential immunoprecipitation.",
"After fixation and subsequent sonication, fixed chromatin fragments of defined length binding the protein (i.e.",
"transcription factor) of interest either directly or indirectly are purified by selective immunoprecipitation with antibodies specific to 1) proteins present within the core transcriptional machinery, an example of which is the large subunit (c) of RNA polymerase II and 2) the particular transcription factor for which target genes are being sought (see below for detailed description of this procedure).",
"As discussed below, it is the solid phase sequential immunoprecipitation procedure utilizing antibodies to both the core transcriptional machinery proteins as well as specific transcription factors which allows for the efficient cloning and characterization of coding sequences for transcription factor target genes.",
"5.3 Multiple Rounds of Chromosomal Immunoprecipitation Reduce Background While it is clear that it is possible to obtain known in vivo target loci for numerous transcription factors utilizing conventional chromosomal immunoprecipitation technologies, an inherent problem is the retrieval of nonspecific protein/DNA complexes.",
"These false positives are often the result of interactions between proteins and noncoding, inactive genomic DNA.",
"While often relevant, these interactions may be those which occur at great distances from the transcription initiation site and thus the identification of coding sequence for the target loci pertaining to these protein/DNA contacts becomes difficult.",
"The presently described technology circumvents the issues of nonspecificity and regulatory element distance from the transcription initiation site through an immunoprecipitation step utilizing antibodies to components of the basal transcriptional machinery.",
"As outlined in FIG.",
"5, chromatinized template is immunoprecipitated with antibodies specific for particular transcription factors.",
"In order to enrich for loci actively regulated by these factors, the presently described invention describes preincubation of chromatinized templates with monoclonal antibodies specific for the large subunit (c) of RNA polymerase II (Background reduction step 1).",
"This “preIP” immunoprecipitation enriches for genes actively transcribed by the Pol II transcription machinery thereby reducing the nonspecificity of the secondary immunoprecipitation and helps to overcome problems related to higher complexity of the genome by omitting noncoding regions and satellite DNA together with nontranscribed genes.",
"Said “preIP” is performed via the solid phase sequential chromosomal immunoprecipitation protocol described herein and may be successfully implemented by those known and skilled in the art.",
"FIG.",
"7 demonstrates the utility of the presently described invention as it pertains to chromosomal immunoprecipitation with antibodies specific for core chromatin proteins and antibodies specific for the large subunit of RNA polymerase II of Sciara coprophila (see Example 6.1 for details and Weeks et al., Genes Dev., 1993, 7: 2329-2344).",
"It illustrates the necessity of cross-linkage reversal as well as the customizable capability of sonication for the purposes of producing chromatin fragments which can be immunoprecipitated discretely with respect to core chromatin proteins or core transcriptional apparatus proteins.",
"IgG antibodies utilized and contemplated by the present invention include those specific for the Drosophila melanogaster RNA Polymerase II large subunit.",
"These antibodies also cross react with the large subunit of RNA Polymerase II from the fly species Sciara coprophila.",
"Species of origin for these antibodies is goat.",
"Termed gAP α-D1, the IgGs were affinity purified using a column carrying a fusion protein term D1, which contains residues A519-Gly992 of the IIc subunit.",
"As well as cross-reacting with Sciara coprophila RNA Polymerase II, the antibodies mildly cross react with the large subunit of yeast as well as mammalian RNA Polymerase II (Weeks et al., Genes & Development, 1993, 7: 2329-2344).",
"A 1:1000 dilution of the original stock solution of 22 ug IgG in 50 ul PBS was used.",
"In addition, a second set of antibodies affinity purified from rabbit immunosera, termed rAP α-PCTD, recognizes the hyperphosphorylated C-terminal domain of Drosophila RNA Polymerase II.",
"A dilution of 1:500 of an original stock solution of 0.054mg/ml in PBS/50% ethylene glycol was used.",
"A third set of antibodies utilized in the presently described invention, termed gAP α-CTD, specifically recognizes the unphosphorylated C-terminal domain of Drosophila RNA polymerase II large subunit.",
"A 1:2000 dilution of an original stock solution of 0.51mg/ml 2× PBS was used.",
"Other antibodies contemplated by the present invention include those designed to discrete regions of the RNA Polymerase II individual subunits including IIc.",
"These antibodies may be of either monoclonal or polyclonal origin.",
"Examples of these antibodies contemplated by the present invention include rabbit affinity purified polyclonal antibody specific for a peptide mapping within the tandem repeat domain of the large subunit of murine RNA Polymerase II.",
"An additional antibody contemplated by the present invention includes an affinity purified rabbit polyclonal antibody raised against a peptide mapping to the amino terminus of the large subunit of RNA Polymerase II.",
"Yet a third antibody contemplated by the present invention includes a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acid 1-224 of RNA Polymerase II of human origin (for review see Tjian, R. and Maniatis, T., Cell, 1994, 77: 5-8).",
"The presently described invention covers any antibodies designed to interact with or bind specifically the large subunit of RNA polymerase II.",
"The presently described invention is in no way limited to utilization of the above antibodies for purposes of first-round immunoprecipitation.",
"Additionally, antibodies to other proteins and subunits present within the core basal transcriptional machinery may be utilized.",
"It is contemplated by the present invention that sequential chromosomal immunoprecipitation utilizing antibodies to any protein present within the core transcriptional apparatus may substantially increase the ability to identify transcribed regions of transcription factor target loci (Kuras et al., Science, 2000, 19: 1244-1248).",
"Subunits of the core transcriptional apparatus, specifically that of the transcriptional initiation complex, for which chromosomal immunoprecipitation may be successfully carried out as discussed in the presently described invention include, but in no way are limited to species RNA polymerase IIA, RNA polymerase IIB and RNA polymerase IIc.",
"Other antibodies contemplated by the present invention may be designed to bind specifically to other core transcriptional apparatus proteins exclusive of the large subunit of RNA polymerase II (Nikolov et al., Proc.",
"Natl.",
"Acad, Sci.",
"USA, 1997, 94: 15-22; Hoffmann et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1997, 94: 8928-8935).",
"These include, but in no way are limited to TAF, TAF(I110), TAF(148), TAF(I63), TAF(II100), TAF(II110), TAF(II125), TAF(II135), TAF(II145), TAF(II150), TAF(II170), TAF(II18), TAF(II19), TAF(II20), TAF(II 25), TAF(II250), TAF(II25ODelta), TAF(II28), TAF(II30), TAF(II30alpha), TAF(II30beta), TAF(II31), TAP(II40), TAF(II47), TAF(II55), TAF(II60, TAF(II61), TAF(II67), TAF(II70-alpha), TAF(II70-beta), TAF(II70-gamma), TAF(II80), TAF-1, TAF-90, TAF-I, TAF-II, TAF-L, TBF1, TBP, TBP-1, TBP-2, TFIIA (32 kDa subunit, TFIIA-alpha/beta precursor (major), TFIlA-alpha/beta precursor (minor), TFIIA-gamma, TFIIA-L, TFIlA-S, TFIIB, TFIID, TFIIE, TFIIE-alpha, TFIIE-beta, TFIIF, TFIIF-alpha, TFIIF-beta, TFIIH, TFIIH core, TFIIH*,TFIIH-CAK, TFIIH-CCL1, TFIIH-cyclin H, TFIIH-ERCC2/CAK, TFIIH-KIN28, TFIIH-MAT1, TFIIH-MO15, TFIIH-p34, TFIIH-p37, TFIIH-p38, TFIIH-p44, TFIIH-p50, TFIIH-p55, TFIIH-p62, TFIIH-p73, TFIIH-p80, TFIIH-p85, TFIIH-p90, TFIIH-SSL2/RAD25, TFIIH-TFIIK, TFII-I, TFIIIA and any other as yet uncharacterized or undiscovered proteins which interact with the core transcriptional machinery for purposes of initiating transcriptional activation.",
"It should be noted that utilization of antibodies to these proteins may produce conflicting results as evidence exists that genes may be repressed even in the presence of core transcriptional machinery proteins, with the exception of the dephosphorylated form of the large subunit (c) of RNA polymerase II (Tjian and Maniatis, Cell, 1994, 77: 5-8).",
"As mentioned above, other as yet undiscovered and thus undescribed basal transcriptional apparatus proteins exclusive of the large subunit of RNA polymerase II are also contemplated by the present invention for the purposes of carrying out sequential immunoprecipitation to identify actively transcribed transcription factor target loci.",
"FIG.",
"8 demonstrates the utility of sequential immunoprecipitation for the purposes of identifying a known p53 target gene, p21.As is evidenced, very little quantitative PCR detection signal is lost due to sequential immunoprecipitation as compared to precipitation with antibodies only specific for the large subunit of RNA polymerase II (see the flowchart and lanes 1 through 4 which represent different stages of the sequential immunoprecipitation procedure for details).",
"As mentioned below, the presently described invention employs the use of a solid phase support, in this case magnetic beads, for increasing the yield of immunoprecipitated cross-linked chromatin during the implementation of sequential chromosomal immunoprecipitation.",
"In order to provide a cross-linked protein/DNA complex as a substrate for the second round of immunoprecipitation with an antibody specific for the particular transcription factor being studied, it is necessary to release previously bound solid phase support from the protein/DNA complex.",
"This is accomplished in the presently described invention via pH alteration.",
"By increasing the acidity of the complex mixture antibodies linked to the solid phase are denatured and bound cross-linked DNA/protein complexes are released.",
"In the experiment described the pH was adjusted from a neutral value of pH 7.6 to an acidity of 5.5 for the efficient denaturation of antibodies covalently linked to the solid phase.",
"pH alteration may be performed to successfully denature antibodies on the solid phase by those known and skilled in the art, but must be determined experimentally for each particular antibody and solid phase support utilized for sequential immunoprecipitation.",
"It is the denaturation of antibodies linked to solid phase which allows for the release of cross-linked pull-down DNA/protein complexes and the next round of chromosomal immunoprecipitation to be carried out and is hence covered by the present invention.",
"Other methods contemplated and covered by the present invention for the denaturation of antibodies bound to solid phase supports for the purposes of sequential immunoprecipitation include, but are in no way limited to enzymatic digestion including but not limited to proteolysis, temperature alteration, chemical, mechanical and UV dissociation.",
"In addition, it is contemplated by the present invention that the junction between the antibody and its solid support matrix may also be manipulated by the above methods for removal of chromatin template for the purposes of second round immunoprecipitation.",
"Table 1 delineates the identification of two previously uncharacterized target regulatory elements for the transcription factor p53 discovered through utilization of technology described by the present invention.",
"The nucleotide sequences listed demonstrate near consensus p53 binding sites and elicit a severalfold increase in stimulation in standard cotransfection induction experiments.",
"It is clear that by performing sequential immunoprecipitation utilizing antibodies specific to the large subunit (c) of RNA polymerase II only actively transcribed transcription factor target genes will be identified due to the required clearance of the promoter prior to large subunit attachment.",
"It is possible, however, to also discover and identify genes which are actively repressed by transcription factors which beacon repressor molecules that inhibit promoter clearance.",
"An example of such a repressor is NcoR (Heinzel et al., Nature, 1997, 387: 43-48).",
"It is contemplated in the presently described invention that utilization of antibodies specific for NcoR in combination with antibodies specific for factors which act to repress gene transcription that genes may be identified which are exclusively repressed for a variety of transcription factors.",
"Other repressor proteins thought to be recruited by DNA binding transcriptional repressors contemplated by the present invention and which may be utilized as targets for sequential immunoprecipitation include, but are in no way limited to SMRT, SunCoR, FunCoR, SIN1, Sin3A (1), Sin3A (2), Sin3A (3), Sin3B, HP1 and PcG (polycomb group proteins).",
"In addition, proteins which bind selectively to methylated DNA are speculated to be involved in mediating or playing a role in transcriptional repression and/or long-term silencing.",
"Thus these proteins serve as candidates for sequential immunoprecipitation to discover target genes actively repressed by certain transcription factors.",
"The proteins covered by the present invention for the purposes of identifying repressed or silenced transcription factor target genes include, but are in no way limited to the methyl DNA binding proteins MeCP1, MeCP2, MBD1, MBD2, MBD3 and MBD4.Other repressor proteins which have yet to be identified may also ultimately be targeted for sequential immunoprecipitation to define transcriptional repressor target genes.",
"In addition to the utilization of antibodies specific for the above mentioned and other proteins which may be recruited by specific transcription factors to actively repress genes, antibodies are also contemplated by the present invention which bind specifically proteins that cause modifications in the DNA and or core proteins in chromatin.",
"These modifications include, but are in no way limited to methylation of CpG islands, deacetylation and phosphorylation of histones.",
"Proteins involved in chromatin modification of this sort covered by the presently described invention include, but are in no way limited to HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8 and any other as yet undiscovered or uncharacterized proteins which effectively modify chromatin.",
"As evidenced above, it is the combination of immunoprecipitation with antibodies to tissue or cell type-specific factors, only one example of which is p53, with those specific for the core transcriptional machinery which allows for elimination of nontranscribed sequences and provides for optimal efficient recovery of transcription factor target genes.",
"Antibodies utilized by the present invention specific for the transcription factor p53 described in FIG.",
"8 are of polyclonal goat origin and recognize the full length protein (cat #sc6243, Santa Cruz Biotechnology, Inc.).",
"It is clear that sequential precipitation of cross-linked chromatin can be performed for a variety of tissue and cell type-specific transcription factors for the purposes of ultimately identifying both known and unknown target loci for these factors.",
"While the presently described invention demonstrates its applicability to the discovery of both known and previously undiscovered target loci for the transcription factor p53, it is in no way limited in its utility for this particular transcription factor.",
"Other transcription factors of prokaryotic, eukaryotic and viral origin contemplated and covered by the present invention include, but are not limited to A2, AAF, abaA abd-A, Abd-B, ABF1, ABF-2, ABI4, Ac, ACE2, ACF, ADA2, ADA3, ADA-NF1, Adf-1, Adf-2a, Adf-2b, ADR1, AEF3, AF-1, AF-2, AFLR, AFP1, AFX-1, AG, AG1, AG2, AG3, AGIE-BP1, AGL11, AGL12, AGL13, AGL14, AGL15-1, AGL15-2, AGL17, AGL2, AGL3, AGL4, AGL6, AGL8, AGL9, AhR, AIC3, AIC2, AIC3, AIC4, AIC5, AID2, AIIN3, ALF1B, ALL-1, alpha-1, alpha2uNF1, alpha2uNF2, alph2uNF3, alpha-CP1, alpha-CP2a, alpha-CP2b, alpha-factor, alphaH0, alphaH2, alphaH3, alpha-IRP, alpha-PAL, alpha2uNF1, alpha2uNF3, alphaA-CRYBP1, alphaH2-alphaH3, alphaMHCBF1, Alx-3, Alx-4, ALY, AMDA, AmdR, aMEF-2, AML1, AML1a, AML1b, AML1c, AML1DeltaN, AML2, AML3, AMT1, AMY-1L, A-Myb, AN2, AnCF,ANF, ANF-2, ANR1, Antp, AP-1, AP-2, AP-2alphaisoform2, AP-2alphaisoform3, AP-2alphaisoform4, AP-3, AP3-1, AP3-2, AP4, AP-5, APC, APETALA1, APETALA3, AR, ARA, AREA, AREB6, ARG RI, ARG RII, armadillo, Arnt, ARP-1, ARP7, ARP9, ARR1, AS-C T3, AS321, ASF-1, ASH-1, ASH-3b, ASP, AT-13P2, ATBF1-A, ATBP, AT-BP1, AT-BP2, ATF, ATF-1, ATF-3, ATF-3deltZIP, ATF-adelta, ATF-like, Athb-1, Athb2, Ato, Axial, AZF1, B factor, B″, BAF1, B-TFIID, band I factor, BAP, Barx-1, BAS, BBF1, BBF2a, BBF3, BBFa, Bcd, BCFI, Bcl-3, BCL-6, BD73, BDF1, beta-1, BETA1, BETA2, beta-catenin, beta-factor, BF-1, BF-2, BGP1, Bin1, Blimp-1, BmFTZ-F1, B-Myb, B-Myc, BP1, BP2, B-Peru, BR-C Z1, BR-C Z2, BR-C Z4, Brachyury, BRF1, Br1A, Brn-3a, Brn4, Brn-5, BUF1, BUF2, BAF1, BAS1, BCFII, beta-factor, BETA3, BLyF, BP2, BR-C Z3, brachyuray, brahma, BRF1, Brn1, Brn2, Brn-3a, Brn-3b, Brn4, Brn-5, Bro, Btd, BTEB, BTEB2, BUF, BUF1, BUF2, BUR6, byr3, BZIP910, BZIP911, c-abl, c-Ets-1, c-Ets-2, c-Fos, c-Jun, c-Maf, c-myb, c-Myc, c-Qin, c-Rel, C/EBP, C/EBPalpha, C/EBPbeta, C/EBPdelta, C/EBPepsilon, C/EBPgamma, C1, CAC-binding protein, CACCC-binding factor, Cactus, Cad, CAD1, CAF17, CAL, CAP, CAR2, CArG box-binding protein, CAT8, CAUP, CBF1, CBF2, CBF3, CBF4, CBF5, CBF-A, CBF-B, CBF-C, CBP, CBTF, CCAAT-binding factor, CCBF, CCF, CCG1, CCK-1a, CCK-1b, CCR4, CD28RC, CDC10, Cdc68, CDF, cdk2, CDP, CDP2, Cdx-1, Cdx-2, Cdx-3, Cdx4, CEBF, CEF1, ceh-1, ceh-10, ceh-12, ceh-13, ceh-14, ceh-16, CEH-18 and (all ceh related factors), CeMyoD, c-Ets-1, C-Ets-1A, c-Ets-1B, CF1, Cf1a, CF2-I, CF2-II, CF2-III, CFF, CG-1, CHA4, CHOP-10, Chox-2.7, Chx10, CIN5, CIIIB1, c-Jun, CKB3, Clox, c-Maf, CMB1, CMB2, c-Myb, c-Myc, CNBP, Cnc, CoMP1, core-binding factor, CoS, COUP, COUP-TF, CP1, CP1A, CP1B, CP1C, CP2, CPBP, CPC1, CPE binding protein CPRF-1, CPRF-2, CPRF-3, CPM10, CPM5, CPM7, CPPI, CPRF-1, CPRF-2, CPRF-3, CPRF4a, CPRF-4b, all CREB related factors, CRE-BP1, CRE-BP2, CRE-BP3, CRE-BPa, CreA, CREB, CREB-2, CREBomega, CREMalpha, CREMbeta, CREMdelta, CREMepsilon, CREMgamma, CREMtaualpha, CRF,all CRM related factors, Croc, Crx, CRZ1, CSBP-1, CtBP, CTCF, CTF, CUM1, CUM10, CUP2, CUP9, CUS1, Cut, Cux, CWH-1, CWH-2, CWH-3, Cx, cyclin A, cyclin T, cyclin T1, cyclin T2, cyclin T2a, cyclin T2b, CYS3, D-MEF2, Da, all DAL related factors, DAP, DAPI, DAT1, DAX1, DB1, DBP-A, DBF4, DBP, DBSF, dCREB, DDB, DDB-1, DDB-2, dDP, dE2F, DEAP3, DEF, DEFH2, Delilah, delta factor, deltaCREB, deltaE1, deltaEF1, deltaMax, DENF, DENF1, DENF2, DENF3, DEP, DEP2, DBP3, DEP4, DERmo-1, DF-1, DP-2, DF-3, Dfd, dFRA, DHR3, DHR38, DHR78, DHR96, dioxin receptor, dJRA, D1, DII, all Dlx related factors, DM-SSRP1, DMLP1, Dof3, DP-1, DP-2, Dpn, Dr1, all DREB related factors, DRF1, DRF2, DRTF, DSC1, DSIF, DSP1, DST1, DSXF, DSXM, DTF, E, E1A, E2, E2BP, E2F, E2F-BF, E2F-I, E4, E47, E4BP4, E4F, E4TP2, E7, E74, E75, EAP1, EAP2L, EAP2S, EAR2, EBF, EBF1, EBNA, EBP, EBP40, EC, EC5, ECF, ECF2, ECF3, ECH, ECM22, EcR, eE-TF, EF-1A, EF-C, EF1, EFgamma, EGM1, EGM2, EGM3, Egr, EGR2, EGR3, eH-TF, EIIa, EivF, EKLF, Elf-1, Elg, Elk-1, ELP, Elt-2, EmBP-1, embryo DNA binding protein, Emc, EMF, EMF2, EMP3, EMP4, Ems, Emx, Erx-1, Emx-2, En, ENH-binding protein, ENKTP-1, epsilonF1, ER, Erbeta, EREBP-1, EREBP-2, EREBP-3, EREBP4, ERF1, Erg, Esc, Esc1, esg, Esx-1a, Esx-1b, FM, ETL, Eve, Evi, Evx, Exd, Ey, en-1, en-2, f(alpha-f(epsilon), P27E5.2, F2F, FACB, F-ACT 1, factor 1, factor 2, factor 3, factor B1, factor B2, factor delta, factor I, FAR, Fbf1, FBF-A1, FBP, FBP1, FBP11, FBP2, FBP6, FBP7, f-EBP, FHL1, FIM, FKBP59, Fkh, FKH1, Fkh-1, FKH2, Fkh-2, Fkh-3, Fkh4, Fkh-5, Fkh-6, FKHR, FKHRL1, FKHRL1P1, FKHRL1P2, FKHRP1, FlbD, FLC, FLF, Flh, Fli-1, FLO, FLO8,FLV-1, FOG, FosB, FosB/SF, Fra-1, Fra-2, Freac-1, Freac-10, Freac-2, Freac-3, Freac4, Freac-5, Freac-6, Freac-7, Freac-8, Freac-9, FRG Y1, FRG Y2, FTF, FTS, Ftz, FTZ-F1, FTZ-F1beta, FZF1G factor, G factor, G/HBF-1, G10BP, G6 factor, GA-BP, GABP, GABP-alpha, GABP-beta1, GABP-beta2, GAF, GAF1, GAF2, GAG2, GAL11, GAL4, GAL80, GammaCAAT, gammaCAC1, gammaCAC2, gamma-factor, gammaOBP, GAMYB, GAT1, GAT2, GAT3, GAT4, GATA-1, GATA-1A, GATA-1B, GATA-2, GATA-3, GATA4, GATA-5, GATA-5A, GATA-, GATA-6, GATA-6A, GATA-6B, GBF, GBF1, GBF12, GBF1A, GBF1B, GBF2, GBF2A, GBF2B, GBF3, GBF4, GBF9, GBP, GC1, GC2, GC3, GCF, GCM, GCMa, GCMb, GCN4, GCN5, GCNF, GCR1, GCR2, GE1, GEBF-I, GF1, GFI, Gfi-1, GFII, GHF3, GHF-5, GHF-7, GIS1, GKLF, GL1, Gl15, G12, Glass, GLI, GLI3, GLN3, GLO, GM-PBP-1, GP, GR, GR alpha, GR beta, GRF-1, Grg-4, Grg-5, GRIP1, Groucho, Gsb, GSBF1, Gsbn, Gsc, Gsc A, Gsc B, Gt, GT-1, GT-2, GT-IC, GT-IIA, GT-IIBalpha, GT-IIBbeta, GTS1, Gtx, GZP3, H16, H1TF1, H1TF2, H2B abp 1, H2RIIBP, H4TF-1, H4TF-2, HAC1, HAL9, HALF-1, HAP1, HAP2, HAP3, HAP4, HAP5, Hb, HB9, HBLF, HBP-1, HBP-1a, HBP-1a(11), HBP-1a(c14), HBP-1b, HBP-1b(c1), HCM1, HDaxx, heat-induced factor, HE, HEB1-p67, HEB1-p94, HEF-1B, HEF-1T, HE-4C, HEN1, HEN2, HeRunt-1, HES-1, HES-2, HES-3, HES-5, Hesx1, Hex, HFH-1, HFH-11A, HFH-11B, HFH-2, HFH-3, HFH4, HFH-5, HFH-6, HFH-7, HFH-8, HIF-1, HIF-1alpha, HIF-1beta, HiNF-A, HiNF-B, HiNF-C, HiNF-D, HiNF-D3, HiNF-E, HiNF-M, HiNF-P, HIP1, HIR1, HIR2, HIR3, HIRA, HIV-EP2, Hlf, Hlf-alpha, Hlf-beta, HLX, Hlx, HMBP, HMG I, HMG I(Y), HMG Y, HMGI-C, HMS1, HMS2, HNF-1, HNF-1A, HNF-1B, HNF-1C, HNF-3, HNF-3(-like), HNF-3alpha, HNF-3B, HNF-3beta, HNF-3gamma, HNF-4, HNF-4(D), HNF-4alpha1, HNF-4alpha2, HNF-4alpha3, HNF-4alpha4, HNF-4alpha7, HNF-4beta, HNF-4gamma, HNF-6, HNF-6alpha, HNF-6beta, hnRNP K, Hox11, HOXA1, HOXA10, HOXA10 PL2, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8,HOXB9, HOXC10, HOXC11, HOXC12, HOXC13, HOXC4, HOXC5, HOXC6, HOXC6 (PRI), HOXC6 (PRII), HOXC8, HOXC9, HOXD1, HOXD10, HOXD11, HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, HP1 site factor, Hp55, Hp65, HrpF, HSE-binding protein, HSF, HSF1, HSF2, HSF24, HSF30, HSF8, hsp56, Hsp90, HST, HSTF, HY5, IBF, IBP-1, IBR, ICER, ICER-I, ICER-Igamma, ICER-II, ICER-Iigamma, ICP4, ICSBP, Id1, Id1.25, Id1H′, Id2, Id3, Id3/Heir-1, Id4, IDS1, IE1, IEBP1, IEFga, IF1, IF2, IFH1, IFNEX, IgPE-1, IgPE2, IgPE-3, Ik-1, Ik-2, Ik-3, Ik4, Ik-5, Ik-6, Ik-7, Ik-8, IkappaB, IkappaB-alpha, IkappaB-beta, IkappaB-gamma, IkappaB-gammal, IkappaB-gamma2, IkappaBR, IK13, ILF, ILRF-A, IME1, IME4, INO2, INO4, INSAF, IPF1, I-POU, IRBP, IRE-ABP, IREBF-1, IRF-1, IRF-2, IRF-3, irlB -2a, Irx-3, ISGP-1, ISGF-3, ISGF-3alpha, ISGF-3gamma, Is1-1, ISRF, ISRFI, ITF, ITF-1, ITF-2, IUF-1, Ixr1, JRF, Jun-D, JunB, JunD, K06B9.5, K07C11.1, kappaY factor, KAR4, KBF2, kBF-A, KBP-1, KCS1, KER1, −1, Kid-I, Kin17, KN1, Kni, Knox3, KNRL, Koxl, Kr, Kreisler, KRP-1, Krox-20, Krox-24, Ku autoantigen, KUP, Lab, LAC9, LBP, LBP-1, LBP-1a, Lc, LCR-F1, LD, Ldb1, LEF-1, LEF-1B, LEF-1S, LEU3, LF-A1, LF-A2, LF-B2, LF-C, LFY, LG2, LH-2, Lhx-3, Lhx-3a, Lhx-3b, Lhx-4, LHY, Lim-1, Lim-3, lin-1, lin-11, lin-14A, lin-14B1, lin-14B2, lin-29A, lin-29B, lin-31, lin-32, lin-39, LIP15, LIP19, LIT-1,LKLF, Lmo1, Lmo2, Lmx-1, L-Myc1, L-Myc-1, L-Myc-1(ong form), L-Myc-1(short form), L-Myc-2, LR1, LSF, LSIRF-2, LUN, Lva, LVb-binding factor, LVc, LXRalpha, LyF-1, Lyl-1, LYS14, Lz, M factor, M-Twist, M1, m3, Mab-18, MAC1, Mad, MAF, MafB, MafP, MafG, MafK, Mal63, MAPF1, MAPF2, MASH-1, MASH-2, mat-Mc, mat-Pc, MATal, MATalpha1, MATalpha2, MATH-1, MATH-2, Max1, M factor, M1, m3, Mab-18 (284 AA), Mab-18 (296 AA), mab-5, MAC1, Mad1, Mad3, Mad4, MADS1, MADS11, MADS16, MADS2, MADS24, MADS3, MADS4, MADS45, MADS5, MADS6, MADS7, MADS8, MADS9, MAF, MafB, MafF, MafG, MafK, MAL13, MAL23, MAL33, MAL63, MAPF1, MAPF2, MASH-1, MASH-2, Mat1-Mc, MATa1, MATalpha1, MATalpha2, MATH-1, MATH-2, mat-Pc, Max, Max1, Max2, MAZ, MAZi, MB67, MBF1, MBF-1, MBF2, MBF3, MBF-I, MBP1, MBP-1 (1), MBP-1 (2), MBP-2, MCBF, MCM1, MCM1+MATalpha1, MDBP, MDBP-2, MDS3, mec-3, MECA, MED11, MED2, MED4, MED6, MED7, MED8, mediating factor, MEF1, MEF-2, MEF-2B, MEF-2B-1, MEF-2B-2, MEF-2B-3, MEF-2B-4, MEF-2C, MEF-2C (433 AA form), MEF-2C (465 AA form), MEF-2C (473 AA form), MEF-2C/delta32 (441 AA form), MEF-2D, MEF-2D (506 AA form), MEF-2D (514 AA form), MEF-2D00, MEF-2D0B, MEF-2DA-0, MEF-2DA-B, MEF-2DA0, MEF-2DAB, Meis-1, Meis-1-1, Meis-1-2, Meis-1-3, Meis-1-4, Meis-1a, Meis-1b, Meis-2a, Meis-2b, Meis-2c, Meis-2d, Meis-3, Meso1, MET18, MET28, MET31, MET32, MET4, Mf2, MF3, MFH-1, Mfh-1, MGA1, Mhox, MHR1, Mi, MIBP1, MIF-1, MIG1, MIG2, Mix.1, Mix.2, Mix.3, Mix.4, Mixer, MA, Miz-1, MKR2, MLP, MM-1, MNB1a, MNB1b, MNF1, MNR2, MOK-2, MOP3, MOT1, MOT3, MP4, MPBF, MR, MRF4, MRR, Msh, MSN1, MSN2, MSN4, Msx-1, Msx-2, MTB-Zf, MTB1, MTF-1, MTH1, Mtll, mtTF1, M-Twist, muEBP-B, muEBP-C2, MUF1, MUP2, Mxi1, MYB A, MYB.PH1, MYB.PH2, MYB.PH3, MYB1, Myb1, all Myb related proteins, MYB-P1, MYBST1, myc-CF1, myc-PRF, MYC-RP, Myef-2, Myf-3, Myf-4, Myf-5, Myf-6, Myn, MyoD, Myogenin, MZF-1, Nab1, Nau, NBF, NC1, NCB2, NDT80, NELF, NeP1, NER1, Net, NeuroD, NF III-a, NP III-c, NF III-e, NF-1, NF-1/L, NF-1/Red1, NF-1A, NF-1A1, NF-1A1.1, NF-1A2, NF-1a3, NF-1A4, NF-1A5, NF-1B, NF-1B1, NF-1B2, NF-1B3, NP-1B4, NF-1C1, NF-1C2, NF-1C4, NF-1X, NF-1X1, NF-1X2, NF-1X3, NP2d9, NF4FA, NF-4FB, NF-4FC, NF-A, NF-A3, NF-AB, NFalpha1, NEalpha2, NPalpha3, NPalpha4, NF-AT, NFAT-1, NF-AT3, NF-Atc, NF-ATc3, NF-Atp, NF-Atx, NF-BA1, NfbetaA, NF-CLE0a, NF-CLE0b, NF-D, NFdeltaE3A, NFdeltaE3B, NFdeltaE3C, NFdeltaF4A, NFdeltaE4B, NFdeltaE4C, Nfe, NF-E, NF-E1b, NF-E2, NF-E2 p45, NF-E3, NF-E4, NFE-6, NF-EM5, NF-Gma, NF-GMb, NF-H1, NF-H2, NF-H3, NFH3-1, NFH3-2, NFH3-3, NFH3-4, NP-IL-2A, NF-IL-2B, NF-InsE1, NF-InsE2, NF-InsE3, NF-jun, NK-kappaB, NF-kappaB(-like), NF-kappaB1, NF-kappaB1 precursor, NF-kappaB2, NF-kappaB2 (p49), NF-kappaB2 precursor, NF-kappaE1, NF-kappaE2, NF-kappaE3, NF-lambda2, NF-MHCIIA, NF-MHCIIB, NF-muE1, NF-muE2, NF-muE3, NF-muNR, NF-ODC1, NF-S, NP-TNF, NF-U1, NF-W1, NF-W2, NF-X, NF-X1, NF-X2NF-X3, NF-Xc, NF-Y, NP-Y′, NF-YA, NF-YB, NF-YC, NF-Zc, NF-Zz, NGFI-B, NGFI-C, NHP-1, NBP-2NHP3, NHP4, NHR1, NIP, NIRA, NIT2, NIT4, Nkx-2.1, Nkx-2.2, Nkx-2.5, NLS1, NMH7, NMHC5, Nmi, N-Myc, N-Myc1, N-Myc2, nob-1A, nob-1B, N-Oct-2alpha, N-Oct-2beta, N-Oct-3, N-Oct4, N-Oct-5a, N-Oct-5b, NOR1, NOT, NOT1, NOT2, NOT3, NOT5, NP-III, NP-IV, NP-TCII, NP-Va, NPX1, NRD I, Nrf1, NRF-1, Nrf2, NRF-2NRF-2betal, NRF-2gamrnal, NRFA, NRG1, NRG2, NRL, NS-1, NSDD, NTF, NTF1, NUC-1, Nur77, NUT1, NUT2, OBF, OBF-1, OBF3.1, OBF3.2, OBF4, OBF5, OBP, OBP1, OC-2, OCA-B, OCSBF-1, OCSTF, Oct-1, Oct-10, Oct-11, Oct-1A, Oct-1B, Oct-1C, Oct-2, Oct-2.1, Oct-2.3, Oct-2.4, Oct-2.6, Oct-2.7, Oct-2.8, Oct-2B, Oct-2C, Oct4, Oct-4A, Oct4B, Oct-5, Oct-6, Oct-7, Oct-8, Oct-9, Octa-factor, octamer-binding factor oct-B2, oct-B3, Oct-R, Odd, ODR7, OG-12, OG-2, OG-9, OHP1, OHP2, Olf-1, OM1, ONR1, Opaque-2, OPM1, OSBZ8, Otd, Otx1, Otx2, Otx4, Ovo, OZP, P (long form), P (short form), P1, p107, p130, p28 modulator, p300, p38erg, p40x, p45, p49erg, p53as, p55, p55erg, p58, p65delta, p67, PAB1, PacC, PAF1, pag-3, PAGL1, pal-1, Pap1+, par-2, Parxis, PARP, Pax-1, Pax-1/9, Pax-1/9 (AmphiPax-1), Pax-1/9-I, Pax-1/9-II, Pax-1/9-III, Pax-1/9-IV, Pax-1/9-V, Pax-1/9-VI, Pax-2, Pax-2.1 , Pax-2.2, Pax-2/5/8, Pax-2a, Pax-2b, Pax-3, Pax-3A, Pax-3B, Pax4, Pax4a, Pax-4c, Pax-4d, Pax-5, Pax-6, Pax-6 (Pax-QNR), Pax-6/Pd-5a, Pax-6 12.1, Pax-6 12.2, Pax6 4.1, Pax-6 4.2, Pax6 J2, Pax-7, Pax-8, Pax-8a, Pax-8b, Pax-8c, Pax-8d, Pax-8e, Pax-8f, Pax-8g, Pax-9, Pax-A, Pax-B, Pb, PBF, PBP, Pbx-1a, Pbx-1b, Pc, PC2, PC4, PC4 p9, PC5, Pcr1, PCRE1, PCT1, PDM-1, PDM-2, PDR1, PDR3, Pdx-1, PEA1, PEA2, PEA3, PEB1, PEBP2, PEBP2alpha, PEBP2alphaA/Osf2, PEBP2alphaA/til-1, PEBP2alphaA/til-1 (Y), PEBP2alphaA/til-1(U), PEBP2alphaA1, PEBP2alphaA2, PEBP2alphaB1, PEBP2alphaB2, PEBP2beta, PEBP2beta1, PEBP2beta, PEBP2beta3, PEBP5, Pep-1, PERIANT, pes-1apes-1b, PF1, PF3, PGA4, PGD1, pha4, PHAN, PHD1, phiAP3, PHO2, PHO4, PHO80, Phox-2, php-3, PI, PI1, PI2, pie-1, PIHbox9, PIP2, Pit-1, Pit-1a, Pit-1b, Pit-1c, Pitx-3, PLE, PLE/DEPH200, PLE/DEFH49, PLE/DEFH72, PLEYSQUA, PLZF, PNPI2, PO-B, pointedP1, pointedP2, Pontin52, pop-1POP2, POTM1-1, pou[c], Pou2, pox neuro, PP1, PP2, PPAR, PPARalpha, PPARbeta, PPARgamma, PPR1, PPUR, PPYR, PR, PR A, PRb, Prd, PRDI-BF1, PRDI-BFc, PREB, Prop-1, protein a, protein b, protein c, protein d, PRP, PSE1, Psx-1, Psx-2, P-TEFb, PTF, PTP1, PF1-alpha, PTF1-beta, PTFalpha, PTFbeta, PTFdelta, PTFgamma, Ptx-1, Ptx-2, Ptx-2B, Pu box binding factor, Pu box binding factor (BJA-B), PU.1, Pu.1, PUB 1, PuF, PUF-I, Pur factor, Pur-1, PUT3, P-wr, PX, PZF1, qa-1F, QBP, QUT1, R, R1, R2, RAD1, Rad-1, RAD18, RAD2, RAF, RAP1, RAP2.5, RAR, RAR-alpha, RAR-alphal, RAR-alpha2, RAR-beta, RAR-beta1, RAR-beta2, RAR-beta3, RAR-beta4, RAR-gamma, RAR-gamma1, RAR-gamma2, RAV1, RAV2, Rax, Rb, RBP60, RBP-Jkappa, Rc, RC1, RC2, RCS1, REB, REB1, Reb1p, RelA, RelB, repressor of CAR1 expression, REV-ErbAalpha, REX-1, RF1, RF2a, RFX, RFX1, RFX2, RFX3, RFX5, RF-Y, RGM1, RGR1, RGT1, RIC1, RIM1, RIP14, RITA-1, RLM1, RME1, RMS1, Ro, Roaz, ROM1, ROM2, RORalpha1, RORalpha2, RORalpha3, RORbeta, RORgamma, Rox, Rox1, ROX3, RPF1, RPGalpha, RPH1, RREB-1, RRF1, RRF2, RRF3, RRN10, RRN11, RRN3, RRN5, RRN6, RRN7, RRN9, RS2, RSC4, RSRFC4, RSRFC9, RSV-EF-II, RTF1, RTG1, RTG2, RTG3, Runt, RVF, Rx, Rx1, Rx2, Rx3, RXR-alpha, RXR-beta, RXR-beta1, RXR-beta2, RXR-gamma, S8, SAP1, SAP-1a, SAP-1b, SBF, SBF-1, Sc, SCBPalpha, SCBPbeta, SCBPgamma, SCD1/BP, SCM-inducible factor, Scr, S-CREM, S-CREMbeta, Sd, Sdc-1, SDS3, SEP1, SEP-1 (1), SEP-1 (2), SEP3,SEF4, SEM-4, SET1,SET2, SF1, SF-1, SF-2, SF-3, SF-A, SFL1, SGC1, SGF-1, SGF-2, SGF-3, SGF4, Shn, SHP, SHP1, SHP2, SIF, SIG1, SIII, SIIII-p110, SIII-p15, SIII-p18, Sim1, Sim2, Six-1, Six-2, Six-3, Six-3alpha, Six-3beta, Six-4, Six-4A, Six-4B, Six-4C, Six-5, Six-6,Skn-1, SKN7, SKO1, SLM1, SLM2, SLM3, SLM4, SLM5, Slp1,slp2, S-Myc, Sn, SN (sienna), Sna, SNF5, SNF6, SNP1, So, SOX-11, SOX-12, Sox-13, SOX-15, Sox-18, Sox-2, Sox-4, Sox-5, SOX-6, SOX-9, Sox-LZ, Sp1, Sp2, Sp3, Sp4, SPA, spE2F, Sph factor, Spi-B, SpOtx, Sprm-1, SpRunt-1, SQUA, SRB10, SRB11, SRB2, SRB4, SRB5, SRB6, SRB7, SRB8, SRB9, SRD1, SRE BP, SREBP-1, SREBP-1a, SREBP-1b, SREBP-1c, SREBP-2, SREP, SRE-ZBP, SRF, SRY, Sry h-1, Sry-beta, Sry-delta, ssDBP-1, ssDBP-2, SSRP1, Staf, Staf-50, STAT, STAT1, STAT1alpha, STAT1beta, STAT2, STAT3, STAT4, STAT5, STAT5A, STATSB, STAT6, STC, SD1, Ste11, STF,12, STE4,STIF1, STEF2, STKA, STM, STP1, Stra13, StuAp, su(f), Su(H), su(Hw), SUM-1, SUP, SVP, SVP46, SWI/SNF complex, SWI1, SWI2, SWI3, SWI4, SWI5, SWI6, SWP,T-Ag, t-Pou2, T3R, T3R-alpha, T3R-alpha1, T3R-alpha2, T3R-beta, T3R-beta1, T3R-beta2, TAB, T-Ag, TAG1, Tal-1, Tal-1beta, Tal-2, TAR factorTat, Tax, TCF, TCF-1TCF-1A, TCF-1B, TCP-1C, TCF-1D, TCP-1E, -1F, TCF-1G, TCF-2, TCF-2alpha, TCF-3, TCP-3B, TCF-3C, TCF-3D, TCF4, TCF-4(K), TCF4B, TCF4E, TCF-A, TCF-B, TCFbeta1, TDEF, TEA1, TEC1, TEF, TEF 1, TEP-1, TEF2, TEF-2, Tel, TF68, TFE3, TFE3-L, TFE3-S, TFEB, TFEC, TFIIA, TFIIA (13.5 kDa subunit), Tf-LF1, Tf-LF2, TF-Vbeta, TGA, TGA1, TGA1a, TGA2, TGA3, TGA6, TgF1, TGGCA-binding protein, TGT3, Th1, THM1, THM18, THM27, THRA1, TIF1, TIF2, TIN-1, TINY, TIP, tI-POU, TLE1, Tll, Tlx, TM3, TM4, TM5, TM6, TM8, TMP, t-Pou2, TR2, TR2-11, TR2-9, TR3, TR4, Tra-1 (long form), Tra-1 (short form), TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREP2, TRF, TRF (2), Trident, TSAP, TSP3, Tsh,TTF-1, TTF-2, TTG1, Ttk 69K, Ttk 88K, TTP,Ttx, ttx-3, TUBF, Twi, TxREF, TyBF, UAY, UBF, UBF1, UBF2, UBP-1, Ubx, UCRB, UCRF-L, UEF-1, UEP-2, UEP-3, UEF-4, UF1-H3beta, UFA, UFB, UFO, UGA3, UHF-1, UME6, unc-30, unc-37, unc-4, Unc-86, URF, URSF, URTF, USF, USF2, vab-3, vab-7, vaccinia virus DNA-binding protein, Vav, Vax-1, Vax-2, VBP, VDR, v-ErbA, VETF, v-Ets, v-Fos, vHNF-1, vHNP-1A, vHNP-1B, vBNF-1C, VITF, v-Jun, v-Maf, Vmw65, v-Myb, v-Myb/v-Ets, V-Myc, v-Myc, Vp1, Vpr, v-Qin, v-Rel, VSF-1, WC1, WC2, Whn, WT1, WT1I, WZF1, X-box binding protein, X-Twist, X2BP, xam1, X-box binding protein, XBP-1, XBP-2, XBP-3, XF1, XF2, XFD-1, XFD-2, XFD-3, XFG20, XGRAF, Xirol, Xiro2, Xiro3, xMEF-2, XPF-1, XrpFI, XW, XX, yan, YB-1, YB-3, Ybx-3, YEB3, YEBP, Yi, YNG2, YPF1, YY1, ZAP, ZEB, ZEM1, ZEM2/3, Zen-1, Zen-2, Zeste, ZF1, ZF2, ZF5, Zfh-1, Zfh-2, Zfp-35, ZID, ZIP-1A, ZIP-2A, ZIP-2B, ZM1, ZM38, Zmhoxla, Zn-15, ZNF174, ZPT2-1, ZPT2-2, ZPT2-3, ZPT2-4, Zta.",
"In addition, any factors which retain the ability to regulate gene expression, either through activation or repression, and are as of yet previously undiscovered or as uncharacterized are covered by the present invention.",
"While the procedure of sequential immunoprecipitation of cross-linked protein/DNA complexes for purposes of detecting actively transcribed target genes in the presently described invention involves the sequential precipitation of protein/DNA complexes utilizing antibodies specific to the large subunit (c) of RNA polymerase II first and antibodies specific for the transcription factor of interest second, it is in no way limited to this particular order of immunoprecipitation.",
"It is contemplated by the present invention that the immunoprecipitation procedure may be reversed and thus performed with antibodies specific for the transcription factor of interest first and antibodies specific for the large subunit of RNA polymerase II second, although it is possible that a loss of target loci recovery may result due to initial precipitation of genes not activated by said transcription factor of interest.",
"It is also contemplated and covered by the presently described invention that sequential rounds of immunoprecipitation may be performed with antibodies specific to cell type and tissue restricted transcription factors for the purposes of identifying target genes for multiple factors.",
"For example, the technology described herein may be utilized to search for loci which are targets for regulation by both p53 and Rb, or by both Pit-1 and GATA2 (El-Diery et al., Cell, 1993, 75: 817-825; Dasen et al., Cell, 1999, 97: 587-598).",
"In addition, it is contemplated by the present invention that coimmunoprecipition utilizing antibodies specific for more than one transcription factor simultaneously may be successfully performed for the purposes of identifying target loci for two or more transcription factors.",
"5.4 Solid Phase Chromosomal Immunoprecipitation Increases both Yield and Sensitivity The presently described invention utilizes magnetic beads linked covalently to either monoclonal or polyclonal antibodies specific for discrete and particular transcription factors (Dynal Corporation).",
"It is clear that by implementing solid phase separation techniques for immunoprecipitation both the amount of material recovered as well as the specificity for real in vivo interactions is considerably enhanced.",
"This is due primarily to the increased ability to recover the protein/DNA complexes of limited quantity and implementation of additional washing procedures as compared to immunoprecipitation in the absence of using a solid phase base.",
"A diagrammatic illustration of the use of solid phase technology to increase yield and sensitivity is represented in FIG.",
"3.Cross-linked DNA/protein material is combined with magnetically charged Dynabeads upon which antibodies to the protein of interest have been conjugated.",
"Use of a magnet results in purification of protein/DNA complexes of interest.",
"Subsequent washing steps allow for the removal of the unbound cellular debris, proteins and DNA fragments.",
"Magnetic bead/protein/DNA complexes are subsequently subjected to further analysis as discussed below.",
"In the presently described invention linkage of antibodies to the solid phase support magnetic beads is accomplished via standard protocol (Dynal Corporation product information and specifications) and those known and skilled in the art are capable of establishing this linkage successfully.",
"Beads are washed briefly in phosphate buffered saline (PBS), pH 7.4.0.1-1.5 ug of antibody is added per ml of beads, the volume adjusted and the mixture incubated for 24 hours at 4 deg.",
"C. on a rotating shaker.",
"The beads are subsequently collected in test or eppendorph tubes via a magnet and the supernatant removed.",
"After two more rounds of washing in 10 mM Tris-HCl, pH 7.6 for an additional 16-24 hours the bead/antibody complex is ready for sequential immunoprecipitation of protein/DNA complexes.",
"The particular magnetic beads utilized as a solid phase supporting material in the presently described invention are Dynabeads M-450 Tosylactivated (Dynal Corporation).",
"Other magnetic beads contemplated by the present invention and created by Dynal Corporation which may be utilized as a solid phase support for the chromosomal immunoprecipitation reaction described herein include Dynabeads M-450 uncoated, Dynabeads M-280 Tosylactivated, Dynabeads M-450 Sheep anti-Mouse IgG, Dynabeads M-450 Goat anti-Mouse IgG, Dynabeads M-450 Sheep anti-Rat IgG, Dynabeads M-450 Rat anti-Mouse IgM, Dynabeads M-280 sheep anti-Mouse IgG, Dynabeads M-280 Sheep anti-Rabbit IgG, Dynabeads M-450 sheep anti-Mouse IgG1, Dynabeads M-450 Rat anti-Mouse IgG1, Dynabeads M-450 Rat anti-Mouse IgG2a, Dynabeads M-450 Rat anti-Mouse IgG2b, Dynabeads M-450 Rat anti-Mouse IgG3.Other magnetic beads which are also contemplated by the present invention as providing utility for the purposes of sequential immunoprecipitation include streptavidin coated Dynabeads.",
"While the presently described invention employs magnetic beads as the solid phase to increase yield and recovery of protein/DNA complexes during sequential chromosomal immunoprecipitation, it is in no way the only solid phase support system which may be implemented successfully to increase yield and sensitivity.",
"Other solid phase supports contemplated by the present invention include, but are not limited to, sepharose, chitin, protein A cross-linked to agarose, protein G cross-linked to agarose, agarose cross-linked to other proteins, ubiquitin cross-linked to agarose, thiophilic resin, protein G cross-linked to agarose, protein L cross-linked to agarose and any support material which allows for an increase in the efficiency of purification of protein/DNA complexes.",
"An alternative method of attaching antibodies to magnetic beads or other solid phase support material contemplated by the present invention is the procedure of chemical cross-linking.",
"Cross-linking of antibodies to beads may be performed by a variety of methods but may involve the utilization of a chemical reagent which facilitates the attachment of the antibody to the bead followed by several neutralization and washing steps to further prepare the antibody coated beads for sequential immunoprecipitation.",
"Yet another method of attaching antibodies to magnetic beads contemplated by the pent invention is the procedure of UV cross-linking.",
"A third method of attaching antibodies to magnetic beads contemplated by the present invention is the procedure of enzymatic cross-linking.",
"The presently described invention implements a solution of solid material in conjunction with antibody/protein/DNA complexes, yet other methodology, such as that which utilizes a column support fixture rather than a solution format may be successfully employed for purposes of solid phase sequential chromosomal immunoprecipitation.",
"In addition, support fixtures such as petry dishes, chemically coated test tubes or eppendorph tubes which may have the capability to bind antibody coated beads or other antibody coated solid phase support materials may also be employed by the present invention.",
"In the presently described invention the superparamagnetism of the beads allows for the use of a conventional magnet to separate bead/antibody/protein/DNA complexes from nonspecific interactions present with the reaction mixture.",
"The magnetic property of the bead is due to the presence of γFe2O3 and Fe3O4 found within the bead (Dynal Corporation product information and specifications), although it is also contemplated by the present invention that a number of other materials possessing magnetic properties may be sufficient to confer an ability for efficient separation of bead/antibody/protein/DNA complexes from nonspecific materials in the reaction mixture.",
"Upon sufficient isolation of protein/DNA complexes utilizing solid phase sequential immunoprecipitation technologies described in the present invention it is necessary to reverse cross-linkages so that DNA fragments containing transcription factor target genes may be precipitated and further manipulated through both standard and modified molecular biology procedures.",
"Those known and skilled in the art are capable of successfully reversing cross-linkages via conventional chromosomal immunoprecipitation protocols.",
"Reversal of cross-linkages is accomplished through an incubation of the isolated protein/DNA complexes at high temperatures, preferably 65 deg.",
"C. for 24 hours.",
"It is also contemplated by the present invention that alternative temperatures may be employed which effectively reverse cross-linkages in the protein/DNA complexes purified.",
"It is anticipated that temperatures higher or lower than 65 deg.",
"C. may also result in a reversal of cross-links.",
"It is speculated that any temperature above 37 deg.",
"C. may, to a certain degree, result in the reversal of chemically driven cross-links.",
"In addition, it is contemplated by the present invention that reversal of cross-linkages through chemical methods such as alkali treatment as well as UV or enzymatic manipulation may be implemented successfully and are covered by the presently described invention for the purposes of solid phase sequential immunoprecipitation and ultimately transcription factor target gene discovery.",
"Precipitation of DNA fragments containing potential transcription factor target loci is performed in the presently described invention through the use of a typical salt and ethanol mixture (Ausubel et al.",
"(editors), Current Protocols in Molecular Biology, 1994, Chapter 2, p 1-3).",
"Those known and skilled in the art of standard molecular biology procedures are capable of DNA fragment precipitation and collection.",
"It is contemplated that the salt may be omitted without a significant loss in sample recovery.",
"In addition, for the purposes of the presently described invention a coprecipitant is added which allows for visualization of the DNA pellet after precipitation and centrifugation.",
"The coprecipitant Pellet Paint® (Novagen Corporation) has been successfully employed in the present invention for purposes of precipitated DNA visualization and increased recovery (Lunyak et al., Innovations, 2001, 12: 4-5).",
"It is also contemplated by the presently described invention that other coprecipitants may be effectively used to prevent sample loss and increase yield.",
"Polyethylene glycol (PEG) and yeast RNA or any other coprecipitant which effectively acts as a carrier or allows for visualization of the DNA may also be used to accomplish increased yield and minimization of sample loss and are covered by the present invention.",
"5.5 Modified Inverse PCR in Combination with Sequential Solid Phase Chromosomal Immunoprecipitation Allows for the Discovery of Direct Transcription Factor Targets Since its inception by Kary Mullis almost 20 years ago, the polymerase chain reaction (PCR) has had a revolutionary impact on the study of DNA in general as well as gene expression in particular (Innis et al., Academic Press, 1990; McPherson et al., IRL Press, 1991; Erlich, A. Stockton Press, 1989; PCR is also illustrated in U.S. Pat.",
"Nos.",
"4,683,195, 4,683,202, 4,800,159, 4,965,188, 5,023,171, 5,066,584, 5,075,216, 5,091,310, 5,104,792 which are herein incorporated by reference).",
"One significant limitation of PCR is the requirement of the knowledge of nucleotide sequence in order to generate products of unknown sequence internal to those of known origin.",
"The inability to retrieve sequences external to known sequences was overcome by the development of inverse PCR (I-PCR) a method in which circularized DNA provided a template for the successful amplification of sequences of unknown nucleotide composition external to those known (Ochman et al., Genetics, 1988, 120(3): 621-623).",
"Thus it is now possible to retrieve nucleotide sequences adjacent to those of known composition for the purposes of assessing the identity of template DNA.",
"A modified version of the I-PCR technology is described in the present invention which takes advantage of the fact that for many transcription factors the binding site, or at least a consensus binding site, has been characterized through methods such as binding site selection (Ausubel et al.",
"(editors), Current Protocols in Molecular Biology, 1994, Chapter 12, p11/1-11/6).",
"By utilizing degenerate oligonucleotides specific for the binding site of a transcription factor in the format of inverse PCR it is possible to generate flanking sequence information which may aid in determining if the template in question is a target gene for the transcription factor being studied.",
"More specifically, it is now possible to determine if the template is a direct target for transcriptional regulation by the transcription factor being studied.",
"FIG.",
"9 illustrates the strategy behind the modified I-PCR technology described herein.",
"While a modified version of I-PCR is described in the present invention, other PCR technologies may be implemented for the rapid retrieval of transcription factor target genes from immunoprecipitated cross-linked protein/DNA complexes as well as DNA purified from these complexes upon reversal of cross-linkages.",
"Other PCR amplification technologies contemplated to be combined with solid phase sequential immunoprecipitation and therefore covered by the present invention include, but are in no way limited to RT-PCR, 5′ RACE (Rapid Amplification of cDNA Ends), 3′ RACE, nested PCR, degenerate oligonucleotide PCR, PCR using oligos coding for transcription factor binding sites in combination with oligos coding for sequences proximal to the transcriptional initiation site such as the TATAA box, and any PCR technology which aids the presently described invention for the purposes of identifying both known and unknown transcription factor target loci.",
"5.6 Combination of Solid Phase Technology, Sequential Chromosomal Immunoprecipitation, Modified I-PCR and other Molecular Cloning Methods for the Purposes of Transcription Factor Target Gene Discovery The sensitivity of the methodology relies heavily on the availability of high-quality monoclonal or polyclonal antibodies that can immunoprecipitate the antigen of interest in an efficient and specific manner.",
"The current technology described herein details a method which utilizes antibody coated magnetic beads in combination with the use of a coprecipitant for the precipitation of chosen antigens/DNA complexes with high efficiency and with virtually no background (FIG.",
"3).",
"Strategies implemented for the further reduction in background nonspecific binding are discussed below.",
"Originally, the ChIP technology was designed to characterize protein interactions with known DNA target sequences.",
"The methods described herein have been recently developed as to extend the ChIP assay to high-throughput cloning and analysis of both known and unknown sequences, many of which may represent potential transcription factor target loci.",
"FIG.",
"5 outlines methods developed and optimized for the efficient acquisition of target gene sequence information.",
"DNA cross-linked to binding proteins is isolated from tissue culture cells and sonicated to give the desired fragment length in preparation for immunoprecipitation.",
"Fragments may either be immunoprecipitated (IP'd) directly or “preIP/IP'd” utilizing an antibody to the large subunit of RNA polymerase II that allows for the isolation of only transcribed genes.",
"DNA fragments which have been sequentially immunoprecipitated are subsequently run through one or more of a series of sequence acquisition options.",
"Cloning of immunoprecipitated fragments into bacteriophage particles and/or exon scanning vectors allows for high-throughput retrieval of both coding and noncoding individual sequences.",
"FIG.",
"6 illustrates the process of exon scanning.",
"In addition, the employment of modified inverse PCR methodology described above reveals the possible presence of direct targets for regulation by the transcription factor being studied.",
"By assessing the presence of transcription factor binding sites present within the PCR'd and/or cloned fragments it is possible to draw conclusions as to the possibility of sequences obtained as representing real in vivo targets.",
"This observation is more applicable to factors which have large, invariant and therefore rare binding sites.",
"In addition, it is contemplated and therefore covered by the present invention that computer programs may be used to analyze sequences obtained in search of exons or other revealing attributes such as regulatory elements.",
"Finally, internal controls such as known genes, binding site information and gel electromobility shift assays (EMSA's) can be used for detailed assessment of signal-to-noise issues (Ausubel et al.",
"(editors), Current Protocols in Molecular Biology, 1996, Chapter 12, p/1-2/5).",
"Through implementation of the technology represented by the presently described invention it is possible to quickly and efficiently implement a saturation screen for target loci for virtually any transcription factor.",
"A demonstration of specific aspects of this technology is evidenced in FIG.",
"7 for the basal transcription factor RNA Polymerase II.",
"A series of positive internal controls such as immunoprecipitation with antibodies specific for histones as well as background minimization parameters are incorporated into the presently described technology which results in an unsurpassed level of “genome sifting” for genes regulated by transcription factors of either DNA binding or nonDNA binding origin.",
"Given the fact that background nonspecific chromatin immunoprecipitation could affect the ability to define a considerable percentage of the in vivo targets for the transcription factors being studied, an assessment of background levels is necessary.",
"Experimental procedures have been designed which will evaluate the “signal to noise” ratio for each factor.",
"This is possible by assessing within a defined population of immunoprecipitated chromatin the representation of known genes regulated by the chosen factor.",
"Therefore, for each factor an extensive Southern blot characterization of sample populations of fragments with known target loci as probes may be performed.",
"For example, p53 has previously been shown to be a transcriptional regulator of the p21 locus (El-Diery et al., Cell, 1993, 75: 817-825).",
"The binding site has been thoroughly mapped and sequenced.",
"Thus, it will be possible to utilize this locus as a probe to study in a given population of cloned fragments the percentage which hybridize to this particular probe.",
"Calculation of background can then be performed by assessing the percentage of the population which represents said known target and extrapolation from the predicted number of targets for p53.For example, by assuming a reasonable number of direct targets for p53 at between 30-50 (i.e.",
"for genes involved primarily in regulating proliferation, not apoptosis) it is possible to calculate the efficiency of the system.",
"5.7 The Creation of a Compendium of Transcription Factor Targets Application of the improved solid phase sequential chromosomal immunoprecipitation technologies combined with standard and modified molecular biology procedures described herein allows for the collection of DNA fragments putatively containing target loci for any of a multitude of both DNA binding and nonDNA binding transcription factors.",
"Said collection of DNA represents valuable nucleotide sequence information which may reveal novel gene cascades and ultimately therapeutic targets or strategies for the efficient design of therapeutics to treat a variety of human anomalies.",
"The genetic cascades and protein entities encoded by loci representing transcription factor target genes will undoubtedly reveal novel mechanisms of therapeutic intervention.",
"It is speculated that the collection of nucleotide sequence information obtained through implementation of the presently described invention or technologies described herein may be organized into a searchable database format.",
"This is particularly applicable with respect to each transcription factor or with respect to the discrete realms of human physiology and disease which are represented by the transcription factors for which target genes are discovered.",
"Database configuration of nucleotide sequence information for the purposes of therapeutic target discovery is not a new concept and has proven considerably beneficial to the scientific and medical communities (Celera Discovery System™, Celera Genomics, Inc.; Lifeseq™, Incyte Pharmaceuticals, Inc.; Deltabase™, Deltagen, Inc.) (Venter et al., Science, 2001, 291(5507): 1304-1351).",
"Thus the nucleotide sequences of either coding or noncoding origin (i.e.",
"regulatory elements) discovered through implementation of the technology described herein represent a valuable entity which may be mined for therapeutic utility via efficient computer algorithms.",
"Programming languages contemplated by the present invention which may be utilized to create searchable databases of transcription factor target genes include but are in no way limited to C, C+, C++, Visual C++, Basic, Visual Basic, Java, Visual Java, Perl and any other program which effectively annotates sequence information discovered by implementation of the technology described herein.",
"In addition, it is contemplated and therefore covered by the present invention that computer programs may be utilized to search or scan sequences obtained by technology described in the present invention for the purposes of discovery valuable coding sequence or regulatory information.",
"Use of programs such as, but not limited to BLAST, BLASTX, BLASTP, TBLASTN for such purposes are therefore contemplated and covered by the present invention (Altschul et al., J. Mol.",
"Biol., 215: 555-565; National Center for Biotechnology Information, Basic Local Alignment Search Tool computer algorithms and related variations; Ausubel et al.",
"(editors), Current Protocols in Molecular Biology, 1995, Chapter 19, p3/1-3/27).",
"Below are described several examples which illustrate application of the presently described invention for the purposes of discovering transcription factor target loci.",
"It should in no way be inferred that the below examples represent the only application of the described technology and hence the invention is not to be constrained by these particular examples.",
"6.0 EXAMPLES 6.1 Demonstration of Improved Chromosomal Immunoprecipitation For some transcription factor target genes DNA binding sites of either a direct or indirect nature may be located very proximal to the basal transcriptional machinery and transcriptional initiation site of target loci.",
"Other sites may be a distance of several kilobases from the promoter region and transcriptional initiation site.",
"Therefore the need for generating DNA fragment lengths of different sizes represents a crucial aspect of the described technology.",
"By varying fragment length it is possible to immunoprecipitate not only DNA molecules containing sites proximal but also distal to the transcriptional initiation region.",
"FIG.",
"4 illustrates the ability to “customize” DNA fragment length by varying sonication conditions.",
"DNA isolated from cells was sonicated under increasing temporal conditions, run on a 1.2% agarose gel in 0.5× TBE along with molecular weight markers and stained with ethidium bromide.",
"As the length of time for sonication is increased, it is evident that the fragment sizes of crosslinked DNA become smaller.",
"It is this customizable aspect of the described technology which makes is possible to isolate and characterize virtually any transcription factor target gene.",
"A basic demonstration of the application of the technology described herein focuses on the gene II/9-1 of Sciara coprophila (Gabrusewycz-Garcia, N. and Kleinfeld, R. G., Journal of Cell Biology, 1966, 29(2): 347-359).",
"FIG.",
"7 reveals the ability to immunoprecipitate target genes utilizing antibodies specific for the large subunit of RNA polymerase II.",
"In vivo CHIP assay reveals an engaged RNA Pol II at the Sciara coprophila gene 11(9-1 promoter during amplification stage of larval development.",
"25 salivary glands from either preamplification or amplification stages of larval development were dissected from larvae and incubated in Canon's medium containing 1.0% formaldehyde for 15 min at room temperature and for 30 min at 4 degrees C. In vivo fixed isolated chromosomal DNA's were recovered from tissues and digested with Hind III enzyme (Hind III restriction map of DNA Puff II/9A locus is given on the panel (A).",
"Released chromatin fragments were immunoprecipitated with antibodies raised against Drosophila melanogaster second large subunit of RNA Pol II (gAPD-1) (Weeks et al., Genes Dev., 7: 2329-2344) or with monoclonal anti-histone antibodies (Chemicon, Inc.) coated to Dynabeads (Dynal Corporation).",
"The specificity of these antibodies against Sciara coprophila protein extracts was analyzed by Western blot and shown on panel (A) Pellets were washed extensively and freed from cross-links by incubation of pelleted cross-linked protein/DNA complexes at 65 degrees C. overnight.",
"Purified DNA fragments were subjected to 30 cycles of PCR using a primer sets C and B and analyzed on a 2.5% agarose gel.",
"Primer set B was chosen as a control to demonstrate the specificity of immunoprecipitation with antibodies which recognize the large subunit of RNA polymerase II.",
"Given the fact that binding sites for set B are located 2.5 kb upstream of the geneII/9-1 promoter and sonication resulted in 300-500 bp fragments no ORI sequences should be detected.",
"(B) PCR analysis of input DNA's before immunoprecipitation.",
"Equivalent volumes of purified, in vivo formaldehyde-fixed, Hind III digested DNA samples from either preamplification stage (1) or amplification stage (2) without immunoprecipitation were freed of cross-links and analyzed by 30 cycles of PCR by primer set C. (C) ChIP of cross-linked DNA reveals an engaged RNA Pol II at the promoter of gene II/9-1 only during DNA amplification stage (8) but not at the preamplification stage of larval development (7).",
"At the same time formaldehyde cross-linked histones are detectable on a promoter containing DNA fragment during both preamplification and amplification stages of larval development (C).",
"Equal amounts of cross-linked, Hind III-digested DNA material were precipitated either with anti-histone antibodies (lanes 1,2,3,4), anti-Pol II antibodies (lanes 7, 8) or subjected to non-immune precipitation by magnetic beads as a control to monitor nonspecific precipitation of cross-linked complexes (5,6).",
"Samples in lanes 1,2,4,5,6,7,8 were freed of cross-links and 30 cycles of PCR with primer set C were done for each sample.",
"The absence of PCR product in lane 3 demonstrates the necessity of thermal reversal of the cross-links prior to PCR.",
"Lanes 5 and 6 show that no PCR products are detected in non-immune precipitants.",
"(D) Similar ChIP experiments with PCR analysis of a distinct genomic region after IP's were done in order to demonstrate completion of Hind III restriction digestion.",
"No products were obtained by PCR amplification with primer set B in the samples of anti-RNA Pol II IP for both stages.",
"The level of anti-histone pull down is the same (3, 4) as shown by primer set C. Multiple rounds of immunoprecipitation may result in the reduction of acquisition of significant amounts of DNA template due to loss upon repeated immunoprecipitation and washing.",
"Thus, it is necessary to test the recoverability of target genes both and after sequential immunoprecipitation.",
"FIG.",
"8 demonstrates both the efficiency and stringency of multiple immunoprecipitation rounds by assessing the quantitative presence of the p21 target gene for the transcription factor p53 both before and after sequential IP at very stages of the process (El-Deiry et al., Cell, 1993, 75: 817-825).",
"Hela cells were grown to 60% confluency on a 100 mm petry dish, irradiated at 0.5 Gry to stimulate a p53 dependent response and incubated for 6 hours at 37 deg.",
"C. and 7.2% CO2.Cells were cross-linked in 10% Fetal Bovine Serum Medium containing 1.0% formaldehyde for 30 minutes at 4 deg.",
"C. Cells were harvested, lysed and chromatin fragment length was customized to a length of 50-300 bp through implementation of microtip sonication via 9×15 second pulses of a Branson model 250 sonifier with a 5.0 minute incubation on ice between each 15 second pulse.",
"Samples of PCR template were taken at various points during the solid phase sequential immunoprecipitation procedure to assess the presence or absence of the p21 target gene.",
"p21 sequences were detected only in the sonicated sample prior to immunoprecipitation (sample #1) and in the fraction containing cross-linked protein/DNA adducts precipitated by both antibodies recognizing the large subunit of RNA polymerase II and holo p53 (sample #5).",
"Semi-quantitative PCR demonstrates that very little, if any, template is lost after double IP and the implementation of extensive washing conditions.",
"In addition, no detection of the p21 gene was observed in the supernatant after PolII large subunit immunoprecipitation (sample #2), the residual wash of PolII large subunit antibody conjugated beads after immunoprecipitation (sample #3) or the pelleted beads after pH adjustment and reversal of antibody/ligand binding (sample #4).",
"6.2 Demonstration of Novel p53 Target Gene Capture In order to demonstrate the utility of the presently described invention transcription factor target sequences were sought for the mammalian tumor suppressor p53 as mentioned above.",
"Specifically, Hela cells exhibiting 60% subconfluency on a 100 mm petry dish were subjected to 0.5 Gry and incubated for 6 hours at 37 deg.",
"C., 7.2% CO2.Irradiation of cells activates the p53 response to DNA damage and allows for a characterization of target gene activity.",
"Cells were subsequently cross-linked in 1.0% formaldehyde for 20 minutes, neutralized in 100 mM glycine and harvested for lysis.",
"After three rinses in PBS partial lysis was carried out in 200 ul 100 mM Hepes, pH 7.6, 2 mM EGTA, 2 mM EDTA, 2.0% Triton X-100 via 25 strokes through a 25 G needle.",
"After brief centrifugation and removal of supernatant lysis was continued by performing 25 strokes through a 25 G needle in the presence of 200 ul 100 mM Hepes, pH 7.6, 20 mM MgCl2, 3.0% sarcosyl, 150 mM NaCl.",
"After brief centrifugation and removal of supernatant lysis was completed by performing 25 strokes through a 25 G needle in the presence of 200 ul 100 mM Hepes, pH 7.6, 20 mM MgCl, 150 mM NaCl.",
"Upon removal of supernatant samples were dissolved in 100 ul 10 mM Tris, pH 7.6, 5 mM EDTA.",
"Chromatin fragment length was customized to a length of 50-300 bp through implementation of microtip sonication via 9×15 second pulses of a Branson model 250 sonifier with a 5.0 minute incubation on ice between each 15 second pulse.",
"FIG.",
"4 illustrates the fragment sizes obtained on a 1.2% agarose gel stained with Ethidium Bromide and analyzed under UV fluorescence.",
"Solid phase sequential chromosomal immunoprecipitation was performed with superparamagnetic tosylactivated Dynabeads (Dynal Corporation) linked to antibodies specific for the large subunit of RNA polymerase II and holo p53.Antibodies utilized in the current experiment were p53 (FL-393) (cat.",
"#6243, Santa Cruz Biotechnology, Inc.).",
"Fragments containing transcribed sequences were first isolated from sonicated chromatin samples through the use of beads coated with an antibody to the large subunit of RNA Polymerase II (cat.",
"#sc-9001, Santa Cruz Biotechnology, Inc.).",
"2 ul of antibody coated beads were incubated with 5 ul sonicated sample DNA in 10 ul PBS buffer overnight at 4.0 deg.",
"C. It was at this step that the pH was altered to a value of 5.5 to allow for the release of cross-linked protein/DNA adducts from the antibody-coated beads.",
"Subsequently immunoprecipitation using beads coated with antibodies to p53 was performed as described above.",
"After second round chromosomal immunoprecipitation bead/antibody/protein/DNA complexes were washed 3 times in 500 ul 10 mM Hepes, pH 7.6, 2 mM EGTA, 2 mM EDTA, 2% Triton X-100, 3.0% Empegen (cat.",
"#324690, Calbiochem Corporation).",
"A similar wash was repeated 3 times in the same buffer containing 1.0% Empegen.",
"A final wash was subsequently performed in a similar buffer without Empegen.",
"Proteinase K treatment was performed on samples for 1.0 hour at 50.0 deg.",
"C. by standard protocol.",
"DNA was precipitated via the addition of 250 ul 100% ethanol, 10 ul 2.5 M NaOAc and 2 ul Pellet Paint® coprecipitant (cat.",
"#70748-3, Novagen Corp.).",
"FIG.",
"9 illustrates the concept of modified inverse PCR (IPCR) for the purposes of defining transcription factor target loci in the context of sequential chromosomal immunoprecipitation.",
"PCR is possible through the addition of linkers bearing the restriction site and subsequent episomal circularization.",
"The success of the application of I-PCR itself suggests that the DNA fragments isolated may inherently be direct target genes of the transcription factor being studied, in this case p53.In the present example, degenerate oligonucleotide sequences corresponding to the p53 consensus DNA binding site (RRRCWWGYYYRRRCWWGYYY) linked to an EcoR1 restriction site were utilized to perform PCR and obtain flanking nucleotide sequence information (El-Diery et al., Nat Genet., 1992, 1(1): 45-49).",
"1.0 ug of sequentially immunoprecipitated DNA was blunt ended in the presence of Klenow fragment (cat.",
"#M-0212S, New England Biolabs) and 25 uM dNTP's for 1.0 hour at 37.0 deg.",
"C. After NucTrap™ (cat.",
"#400702, Stratagene Corporation) spin column purification EcoR1 linkers were ligated to the fragments at 4.0 deg.",
"C. overnight.",
"Fragments flanked by restriction sites were kinased in the presence of T4 polynucleotide kinase (cat.",
"#M0201, New England Biolabs) and 10 mM ATP and religated for circularization.",
"50 ng of circularized template DNA in combination with 100 ng each oligonucleotide was subjected to 25 rounds of PCR under the following conditions: 98.0 deg.",
"C. for 30 seconds, 55.0 deg.",
"C. for 30 seconds and 72.0 deg.",
"C. for 30 seconds.",
"PCR fragments were excised from a 1.2% agarose gel, blunted and shotgun subcloned into the Sma1 restriction site of pBluescript SK.",
"Plasmids containing fragments were sequenced via Sanger dideoxy sequencing methodology.",
"The presence of the EcoR1 linker sequence reveals the outermost flanks amplified by the PCR reaction.",
"Table 1 reveals two examples of nucleotide sequences obtained by procedures described herein.",
"Each sequence exhibits high sequence identity to the consensus binding site for p53 (bold letters denote nucleotides fitting the p53 binding site consensus).",
"Sequence A reveals similarity to nucleotide sequences present on Homo sapiens chromosome 17, GenBank accession #AC005562.Sequence B reveals homology to sequences present in Homo sapiens BAC clone RPII-557N21, GenBank accession #AC009242.Both genomic sequences obtained by I-PCR were subcloned upstream of a basal promoter linked to the luciferase reporter gene and cotransfected (20 ug each) with a eukaryotic expression vector containing a cDNA coding for human holop53 into Hela cells at 60% confluency.",
"Cells were subsequently harvested 24 hours after transfection for analysis of reporter gene induction.",
"Induction of transcription of the luciferase reporter was observed for both sequences as compared to basal levels (see Table 1) thus confirming the identification of novel enhancer elements regulatable by the transcription factor p53.The proximity of these regulatory elements with respect to transcribed sequences remains to be determined.",
"7.0 REFERENCES U.S. PATENT DOCUMENTS Mullis et al., U.S. Pat.",
"No.",
"4,683,195, Issued July, 1987 Mullis, U.S. Pat.",
"No.",
"4,683,202, Issued July, 1987 Mullis et al., U.S. Pat.",
"No.",
"4,800,159, Issued January, 1989 Mullis et al., U.S. Pat.",
"No.",
"4,965,188, Issued October, 1990 Ho et al., U.S. Pat.",
"No.",
"5,023,171, Issued June, 1991 Gyllensten et al., U.S. Pat.",
"No.",
"5,066,584, Issued November, 1991 Innis et al., U.S. Pat.",
"No.",
"5,075,216, Issued December 1991 Innis et al., U.S. Pat.",
"No.",
"5,091,310, Issued February, 1992 Silver et al., U.S. Pat.",
"No.",
"5,104,792, Issued February, 1992 Peterson et al., U.S. Pat.",
"No.",
"5,563,036, Issued October, 1996 Habener et al., U.S. Pat.",
"No.",
"5,858,973, Issued January, 1999 La Thangue et al., U.S. Pat.",
"No.",
"5,863,757, Issued January, 1999 Waeber et al., U.S. Pat.",
"No.",
"5,880,261, Issued March, 1999 Kushner et al., U.S. Pat.",
"No.",
"6,117,638, Issued September, 2000 Burgess et al., U.S. Pat.",
"No.",
"6,139,833, Issued October, 2000 OTHER REFERENCES Achanzar, W. E., Achanzar, K. B., Lewis, J. G., Webber, M. M., Waalkes, M. P. Cadmium induces c-myc, p53, and c-jun expression in normal human prostate epithelial cells as a prelude to apoptosis.",
"Toxicol.",
"Appl.",
"Pharmacol., 2000,64: 291-330.Altschul, S. F., Gish, W., Miller, W., Myers, E. W, Lipman, D. J., Basic local alignment search tool.",
"J. Mol.",
"Biol., 215: 555-565.Ausubel, F. M. et al.",
"(editors), Current Protocols in Molecular Biology, John Wiley & Sons and Green PublishingAssociates, Inc., New York, 1989, 1994, 1995, 1996.Benashski, S. E., King, S. M., Investigation of Protein-Protein Interactions within Flagellar Dynein Using Homobifunctional and Zero-Length Cross-linking Reagents.",
"Methods, 2000, 22: 365-371.Dasen, J. S., O'Connell, S. M., Flynn, S. E., Treier, M., Gleiberman, A. S., Szeto, D. P., Hooshmand, F., Aggarwal, A. K, Rosenfeld, M. G., Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types.",
"Cell, 1999, 97: 587-598.de Belle I, Mercola D, Adamson E D. Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking.",
"Biotechniques, July 2000;29(1):170-5.Deligdisch, L., Effects of hormone therapy on the endometrium.",
"Mod.",
"Pathol., 1993, 6(1): 94-106.Dynal Technical Handbook, Biomagnetic Applications in Cellular Immunology, 1998.El-Deiry, W. S., Kern, S. E., Pietenpol, J.",
"A., Kinzler, K. W., Vogelstein, B., Definition of a consensus binding site for p53.Nat.",
"Genet., 1992, 1(1): 45-49.El-Deiry, W. S., Tokino, T., Valculescu, V. E., Levy, D. B., Parson, R., Trent, J. M., Lin, D., Merser, W. E., Kinzler, K. W., Vogelstein, B. WAF1, a potential mediator of p53 tumor supression, Cell, 1993,75: 817-825.Erlich, A. PCR Technology: Principals and Applications of DNA Amplification, Stockton Press (1989).",
"Friend, S. H., Horowitz, J. M., Gerber, M. R., Wang, X. F., Bogenmann, E., Li, F. P., Weinberg, R. A., Deletions of a DNA sequence in retinoblastomas and mesenchymal tumors: Organization of the sequence and its encoded protein.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1987, 84: 9059-9063.Frohman, 1994, PCR Methods and Applications, 4:S40-S58.Gabrusewycz-Garcia, N. and Kleinfeld, R. G., A Study of the Nucleolar Material in Sciara Coprophila.",
"Journal of Cell Biology, 1966, 29(2): 347-359 Hecht, A., Strahl-Bolsinger, S., Grunstain, M., Mapping of DNA interaction sites of chromosomal proteins., Crosslinking studies in yeast.",
"Methods Mol.",
"Biol., 1999, 119: 469-479.Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K., Rosenfeld, M. G., A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression.",
"Nature, 1997, 387: 43-48.Herzig, T. C., Jobe, S. M., Aoki, H., Molkentin, J. D., Cowley, A. W. Jr., Izumo, S., Markham, B. E., Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1997, 94: 7543-7348.Hoffmann, A., Oelgeschlager, T., Roeder, R. G., Considerations of transcriptional control mechanisms: Do TIID-core promoter complexes recapitulate nucleosome-like functions?",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1997, 94: 8928-8935.Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990).",
"Jepsen, K., Hermanson, O., Onami, T. M., Gleiberman, A. S., Lunyak, V., McEvilly, R. J., Kurokawa, R., Kumar, V., Liu, F., Seto, E., Hedrick, S. M., Mandel, G., Glass, C. K., Rose, D. W., Rosenfeld, M. G. Combinatorial roles of the nuclear receptor corepressor in transcription and development.",
"Cell, 2000, 102(6):753-63.Kazelenellenbogen, B. S., Montano, M. M., Ekena, K., Herman, M. E., McInerney, E. M. William L. McGuire Memorial Lecture.",
"Antiestrogenes: mechanisms of action and resistance in breast cancer.",
"Breast Cancer Res.",
"Treat., 1997, 44: 23-38.Kuras, L., Kosa, P., Mencia, M., Struhl, K., TAF-containing and TAF-independent forms of transcriptionally active TBP in vivo., Science, 2000, 19: 1244-1248.Levine, A. J., Momand, J., Finlay, C. A.",
"The p53 tumor suppressor gene.",
"Nature, 1991, 351: 453-456.Lockyer, A. E., Jones, C. S., Noble, I. R., Rollinson, D., Use of differential display to detect changes in gene expression in the intermediate snail host Biomphalaria glabrata upon infection wuth Schistosoma mansoni., Parasitology, 2000, 120: 399-407.Lunyak, V., Baidya, R., Burgess, R., Enhanced sensitivity of ChIP through the application of Pellet Paint® Co-Precipitant, Innovations, 2001, 12, 45.Maehara, Y., Oki, E., Abe, T., Tokunaga, E., Shibahara, K., Kakeji, Y., Sugimachi, K., Overexpression of the heat shock protein HSP70 family and p53 protein and prognosis for patients with gastric cancer, Oncology, 2000, 58: 144-151.McKinsey, T A, Olson, E. N., Cardiac hypertrophy: sorting out the circuitry.",
"Curr.",
"Opin.",
"Genet.",
"Dev., 1999., 9: 267-274.McPherson, M. J. et al., PCR: A Practical Approach, IRL Press (1991).",
"Molhar, A., Georgopoulos, K., The Ikaros gene encodes a family of functionally deverse zink finger DNA-binding proteins.",
"Mol.",
"Cell Biol., 1999, 14: 8292-8303.Neilson, L., Andalibi, A., Kang, D., Coutifaris, C., Strauss, J. F., Stanton, J.",
"A., Green, D. P., Molecular phenotype of the human oocyte by PRC-SAGE.",
"Genomics, 2000, 63: 13-24.Nichogiannopoulou, A., Trevisian, M., Friedrich, C., Georgopoulos, K., Ikaros in hemopoietic lineage determination and homeostasis.",
"Semin.",
"Immunol., 1998, 10: 119-125.Nikolov, D. B., Burley, S. K., RNA polymerase II transcription initiation: A structural view.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1997, 94: 15-22.Ochman, H, Gerber A S, Hartl, D. L., Genetic applications of an inverse polymerase chain reaction.",
"Genetics, November 1988;120(3): 621-3.Oliner, J. D., Pietenpol, J.",
"A., Thiagalingam, S., Gyuris, J., Kinzler, K. W., Vogelstein, B., Oncoprotein MDM2 conceals the activation domain of tumor supressor p53.Nature, 1993, 362: 857-860.Rhodes, S. J., DiMattia, G. E., Rosenfeld, M. G., Transcriptional mechanisms in anterior pituitary cell differentiation., Curr.",
"Opin.",
"Genet.",
"Dev., 1994, 4: 709-717.Sambrook et al.",
"(1989) Molecular Cloning Vols.",
"I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Scully, K. M., Jacobson, E. M., Jepsen, K., Lunyak, V., Viadiu, H., Carriere, C., Rose, D. W., Hooshmand, F., Aggarwal, A. K., Rosenfeld, M. G. Allosteric effects of Pit-1 DNA sites on long-term repression in cell type specification.",
"Science, 2000, 290(5494): 1127-31.Semenza, G. L., Transcriptional Regulation of Gene Expression: Mechanisms and Pathophysiology, Human Mut., 1994, 3: 180-199.Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. J., Shenk, T. Wild-type p53 binds to the TATA—binding proteins and repress transcription.",
"Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA, 1992, 89: 12028-12032.Smith, M. L., Kontny, H. U., Bortnick, R., Fornace, A. J. Jr.,The p53-regulated cyclin G gene promotes cell growth: p53 downstream effectors cyclin G and Gadd45 exert different effects on cisplatin chemosensitivity, Exp.",
"Cell Res., 1997, 230: 61-68.Solomon, J., Larsen, P. L., Varshavsky, A., Mapping Protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene, Cell, 1988, 53: 937-947.Stephan, D. A., Chen, Y., Jiang, Y., Malechek, I., Gu, J.",
"Z., Robbins, C. M., Bithner, M. L., Morris, J.",
"A., Carstea, B., Meltzer, P. S., Adler, K., Garlick, B., Trent, J M., Ashlock, M. A., Positional cloning utilizing Genomic DNA microarrays: The Neimann-Pick Type C as a model system.",
"Mol.",
"Gen.",
"Metab., 2000, 70: 10-18.Tenbaum, S., Baniahmad, A.",
"Nuclear receptors: structure, function and involvement in disease.",
"Int.",
"J. Biochem.",
"Cell Biol., 1997, 29: 1325-1341.Tjian, R., Maniatis, T. Transcriptional activation: a complex puzzle with few easy pieces.",
"Cell, 1994, 77: 5-8.Treier, M., Gleiberman, A. S., O'Connell, S. M., Szeto, D. P., McMahon, J.",
"A., McMahon, A. P., Rosenfeld, M. G., Multistep signaling requirements for pituitary organogenesis in vivo.",
"Genes Dev., 1998, 12: 1691-1704.Venter et al., The sequence of the human genome.",
"Science, 2001, 291(5507): 1304-1351.Weeks, J. R., Hardin, S. E., Shen, J., Lee, J. M., Greenleaf, A. L., Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing, Genes Dev., 1993, 7: 2329-2344.Winandy, S., Wu, P., Georgopoulos, K., A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma, Cell, 1995, 83: 289-299.Wu, W., Cogan, J. D., Pfaffle, R. W., Dasen, J. S., Frisch, H., O'Connell, S. M., Flynn, S. E., Brown, M. R., Mullis, P. E., Parks, J. S., Phillips, J.",
"A.",
"3rd, Rosenfeld M G. Mutations in PROP1 cause familial combined pituitary hormone deficiency, Nat.",
"Genet., 1998,18: 147-9.Zambetti, G. P., Bargonetti, J., Walker, K., Prives, C., Levine, A. J. Wild-type p53 mediates positive regulation of the gene expression through a specific DNA sequence element., 1992, Genes Dev., 6: 1143-1152 Zauberman, A., Lupo, A., Oren, M., Identification of p53 target genes through inmmune selection of genomic DNA: the cyclin G gene contains two distinct p53 binding sites, Oncogene, Jun.",
"15, 1995;10(12): 2361-6."
]
] | Patent_10275846 |
[
[
"Process for the puritication of a salt of clavulanic acid",
"A process in which a salt of clavulanic acid, typically an amine salt or an alkali metal salt is exposed to conditions, particularly a pH of less than 6.0, which reduces the quantity of contaminating impurities.",
"The process may be a washing process, a recrystallisation process or a preparative process."
],
[
"1.A process comprising exposure of a salt of clavulanic acid to a liquid medium at a pH of less than 6.5.2.A process according to claim 1 wherein the pH is 3.5-5.5.3.A process according to claim 1 or 2 wherein the salt is washed with a wash medium at a pH of 6.0 or less.",
"4.A process according to claim 3 wherein the salt is an amine salt of clavulanic acid.",
"5.A process according to claim 4 wherein the amine salt is then subsequently converted into a final product pharmaceutically acceptable salt of clavulanic acid.",
"6.A process according to claim 3 wherein the salt which is washed is a metal salt of clavulanic acid.",
"7.A process according to any one of claims 3 to 6 wherein the wash medium is a mixture of water and a water-miscible organic solvent at a pH of 6.0 or less.",
"8.A process according to any one of claims 3 to 7 comprising a process in which a starting salt of clavulanic acid is provided containing one or more impurity, and the salt is washed with the wash medium, to thereby produce a product salt of clavulanic acid in which the level of the one or more impurity is lower.",
"9.A process according to any one of claims 3 to 8 comprising a process in which one or more impurity is removed from a starting salt of clavulanic acid, by washing of the starting salt of clavulanic acid with the wash medium.",
"10.A process according to claim 1 or 2 wherein the salt of clavulanic acid is recrystallised from a recrystallisation medium at pH of 6.0 or less.",
"11.A process according to claim 10 wherein the salt of clavulanic acid is an amine salt of clavulanic acid.",
"12.A process according to claim 11 wherein the amine salt is then subsequently converted into a final product pharmaceutically acceptable salt of clavulanic acid.",
"13.A process according to claim 10, 11 or 12 wherein the salt is dissolved in an aqueous medium at the pH 6.0 or less, or subsequently adjusted the pH 6.0 or less, then the salt is isolated from the aqueous solution by crystallisation by admixing the solution with a precipitating solvent.",
"14.A process according to any one of claims 10 to 13 comprising a process in which a starting salt of clavulanic acid is provided containing one or more impurity, and the salt is recrystallised from the recrystallisation medium, to thereby produce a product salt of clavulanic acid in which the level of the one or more impurity is lower.",
"15.A process according to any one of claims 10 to 14 comprising a process in which one or more impurity is removed from a starting salt of clavulanic acid, by recrystallisation of the starting salt of clavulanic acid from the recrystallisation medium.",
"16.A process according to claim 1 or 2, comprising a preparative process in which the salt of clavulanic acid is prepared in a liquid medium at a pH of 6.0 or lower.",
"17.A process according to claim 16 comprising preparation of an amine salt of clavulanic salt by reacting clavulanic acid with an amine in a liquid medium at a pH of 6.0 or lower.",
"18.A process according to claim 18 performed in a two phase system, being an organic solvent phase containing the clavulanic acid, and an aqueous phase into which the amine salt is extracted and which is at the pH of 6.0 or lower.",
"19.A process according to claim 1 or 2, comprising a preparative process in which a metal salt of clavulanic acid is formed as a product by a reaction between an amine salt of clavulanic acid and a metal salt precursor compound, in a liquid medium at the pH of 6.0 or lower.",
"20.A process according to claim 19 in which potassium clavulanate is prepared.",
"21.A process according to claim 19 or 20 performed with the amine salt in solution in a mixture of a water-miscible organic solvent and water either at the pH of 6.0 or less, or subsequently adjusted to this pH and the metal salt precursor is added to this solution of the amine salt.",
"22.A process according to any one of the preceding claims, being part of an overall process for preparation of potassium clavulanate from crude clavulanic acid as formed in a fermentation broth.",
"23.A process according to claim 22 involving the steps of (i) fermentation of a microorganism which produces an aqueous broth containing clavulanic acid, (ii) extraction of the clavulanic acid into an organic solvent, (iii) conversion of the clavulanic acid into an amine salt of clavulanic acid, (iv) exposure of the amine salt to a pH of 6.0 or lower, particularly 5.5 or lower, (v) conversion of the amine salt to potassium clavulanate.",
"24.A product amine salt of clavulanic acid being a product of a process according to any one of claims 1 to 18.25.Potassium clavulanate being a product of a process according to any one of claims 1 to 3 or 19 to 23.26.A process according to any one of claims 8, 9, 14 or 15 wherein one of the said one or more impurity is N-succinyl tyrosine.",
"27.A purification process in which a salt of clavulanic acid, containing or believed to be contaminated with N-succinyl tyrosine, is subjected to conditions which remove N-succinyl tyrosine.",
"28.A purification process according to claim 27 wherein the salt is exposed to a liquid medium containing a reagent which destroys N-succinyl tyrosine.",
"29.A purification process according to claim 27 wherein the salt is exposed to a reagent which destroys N-succinyl tyrosine, incorporated into or adsorbed onto a solid substrate.",
"30.A purification process in which a salt of clavulanic acid, containing or believed to be contaminated with N-succinyl tyrosine, is suspended or disssolved in a liquid medium and is exposed to a material which absorbs N-succinyl tyrosine and thereby removes N-succinyl tyrosine from the salt.",
"31.A preparative process in which a salt of clavulanic acid is prepared under conditions selected to minimise the formation of N-succinyl tyrosine and/or its retention in the product salt of clavulanic acid.",
"32.A process according to claim 31 wherein the salt of clavulanic acid is prepared in a liquid medium containing reagents which remove N-succinyl tyrosine, or at chemical or physical conditions which remove N-succinyl tyrosine."
],
[
"This invention relates to novel processes for the preparation of salts of clavulanic acid in a more pure state.",
"Clavulanic acid (Z)-(2R,5R)-3-(2-Hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid) is a β-lactamase inhibitor which is used commercially as a component of pharmaceutical formulations, usually in the form of its pharmaceutically acceptable salts, particularly potassium clavulanate.",
"Clavulanic acid is produced commercially by culture of the microorganism Streptomyces clavuligerus, for example as described in GB 1508977.Clavulanic acid may be extracted from the culture medium in various ways.",
"Normally the cells of the S. clavuligerus are first removed from the culture medium by such methods as filtration or centrifugation before such extraction procedures are commenced.",
"The clavulanic acid may be extracted from this clarified culture medium by solvent extraction from cold clarified culture medium adjusted to an acid pH.",
"Whole broth extraction is also feasible.",
"In the solvent extraction process the clavulanic acid is extracted into an organic solvent.",
"After separation of the phases clavulanic acid is found in solution in the organic phase.",
"The clavulanic acid may be back extracted from the organic phase into a new aqueous phase by making use of the greater water solubility of salts of clavulanic acid with organic amines, and isolating such an amine salt from the aqueous phase.",
"In such a process the amine salt is formed as an intermediate in the process of converting crude clavulanic acid into a pharmaceutically acceptable salt.",
"Such a process is described in for example EP-A-0 026 044, in which a solution of impure clavulanic acid in an organic solvent is contacted with t-butylamine to form the t-butylamine salt of clavulanic acid, which is then isolated.",
"Other similar processes are known which use other organic amines, such as tertiary octylamine (see EP-A-0 594 099 (Pharma Development)) diethylamine, tri-(lower alkyl) amines, dimethylaniline and NN′-diisopropyl-ethylenediamine.",
"WO-A-93/25557 (SmithKline Beecham) discloses a very extensive list of amines which can be used in this way.",
"WO-A-94/22873 (Gist Brocades) discloses use of various tertiary, tertiary diamines such as N,N,N′,N′-tetramethyl-1,2-diaminoethane, N,N,N′,N′-tetramethyl-1,6-diaminohexane, 1,2-dipiperidinoethane and dipiperidinomethane.",
"WO-A-96/20199 (Spurcourt) discloses use of diaminoethers such as bis (2-dimethylaminoethyl) ether.",
"GB-A-2298201 (Spurcourt) discloses use of various benzhydrylamines.",
"WO-A-96/33197 (LEK) discloses use of further amines including symmetrical N,N′-alkylethylene diamines, such as N,N′-diisopropyl-ethylenediamine, N,N′-diethylene diamine, N,N′-dibenzylethylene diamine and N,N,N′,N′-tetramethylene diamine.",
"WO-A-98/21212 (Gist-Brocades) for example discloses a process in which the amines N,N,N′,N′-tetramethylethylenediamine, 1,3-bis(di-methylamino)-2-propanol, benzhydrylamine and bis (2-(dimethylamino) ethyl) ether are used.",
"WO-A-98/23622 (Biochemie) discloses use of diisopropyl-ethylen-diamine.",
"After isolation the intermediate amine salt may be converted into a pharmaceutically useful salt of clavulanic acid, particularly an alkali metal salt especially potassium clavulanate, generally by reaction of the intermediate amine salt with a salt precursor compound such as potassium 2-ethylhexanoate.",
"The fermentation process by which clavulanic acid is prepared also produces side product impurities.",
"A number of such impurities have been identified as peaks in the HPLC trace of crude clavulanic acid, intermediate amine salts and pharmaceutically acceptable salts of clavulanic acid as prepared by conventional processes, but few have been chemically identified.",
"One such impurity has recently been identified by the present inventors as N-succinyl tyrosine.",
"It is desirable that as little of such impurities as possible are present in the final product pharmaceutically acceptable salt of clavulanic acid, and therefore that such impurities should be removed, either during the isolation of clavulanic acid or from the final product.",
"For example such impurities may be removed from the above-mentioned intermediate amine salt, during the step of conversion of the intermediate amine salt to the pharmaceutically acceptable salt, or from the final salt product.",
"Successful identification of this impurity as N-succinyl tyrosine has unexpectedly made available processes to prepare salts of clavulanic acid which are contaminated by less N-succinyl tyrosine than has hitherto been possible.",
"The process may be a purification process in which N-succinyl tyrosine is removed from a salt of clavulanic acid contaminated with it.",
"Therefore the invention provides a purification process in which a salt of clavulanic acid, contaminated with or believed to be contaminated with N-succinyl tyrosine, is subjected to conditions which are selected to remove N-succinyl tyrosine.",
"Such conditions may be chemical conditions, e.g.",
"treatment with one or more suitable reagent.",
"Suitably the salt of clavulanic acid, contaminated or believed to be contaminated with N-succinyl tyrosine, may be subjected to such conditions by exposure to a liquid medium, e.g.",
"an organic solvent or organic solvent-water mixture, containing such a reagent.",
"Such a process may involve washing of the salt of clavulanic acid in a solid state with such a liquid medium, or crystallisation of the salt of clavulanic acid from such a liquid medium.",
"Alternatively the salt of clavulanic acid, contaminated or believed to be contaminated with N-succinyl tyrosine, may be exposed to a suitable reagent in solid form.",
"Alternatively the purification process may be a process in which a salt of clavulanic acid, contaminated or believed to be contaminated with N-succinyl tyrosine, is suspended or dissolved in a liquid medium, e.g.",
"an organic solvent or organic solvent-water mixture, and is exposed to a material which is selected to absorb N-succinyl tyrosine and thereby removes N-succinyl tyrosine from the salt.",
"Alternatively the process may be a preparative process in which the salt of clavulanic acid is prepared under conditions selected to minimise the formation of N-succinyl tyrosine and/or its retention in the product salt of clavulanic acid.",
"For example the salt of clavulanic acid may be prepared in a liquid medium, e.g.",
"an organic solvent, an aqueous medium, or a mixture of an organic solvent, containing reagents which remove N-succinyl tyrosine, or subjected to chemical or physical conditions which remove N-succinyl tyrosine.",
"The salt of clavulanic acid purified or prepared in the above processes may be an amine salt of clavulanic acid, e.g.",
"with any of the amines referred to above, especially tertiary butylamine, and this amine salt may be subsequently be converted into a final product pharmaceutically acceptable salt of clavulanic acid, such as an alkali metal salt, e.g.",
"potassium clavulanate.",
"Alternatively the salt may be a metal salt of clavulanic acid, for example an alkali metal salt of clavulanic acid, particularly potassium clavulanate.",
"It has further unexpectedly been discovered that the level of impurities, such as the above-mentioned N-succinyl tyrosine, in a salt of clavulanic acid can reduced if the salt is exposed to a selected range of pH.",
"It is an object of this invention to use this discovery in the provision of improved processes for the preparation of such pharmaceutically acceptable salts.",
"According to this invention a process is provided comprising exposure of a salt of clavulanic acid to a liquid medium at a pH of less than 6.5, preferably 6.0 or less, more preferably pH 5.5 or less, more preferably pH 5.0 or less, more preferably being above pH 3.5, e.g.",
"pH 3.5-5.5, typically ca.",
"pH 4.5.The term “pH” as used herein includes the conventional usage of the term pH as the logarithm of the reciprocal of the hydrogen ion concentration.",
"Also the term pH as used herein includes the observed pH, i.e.",
"the pH as measured by exposing the medium to a conventional pH meter of known type, suitably calibrated by known methods.",
"Normally the media used in the process of this invention will contain some water.",
"Treatment of the salt in this way in the process of the invention can reduce the level of one or more impurities, particularly of N-succinyl tyrosine, in the salt, or in further salts of clavulanic acid, e.g.",
"a pharmaceutically acceptable salt, prepared from the salt.",
"In a first form of the process of this invention the salt may be washed with the medium, i.e.",
"a wash medium, at a pH of less than 6.5, preferably pH 5.5 or less.",
"The salt may be an amine salt of clavulanic acid, e.g.",
"with any of the amines referred to above, especially tertiary butylamine, and this amine salt may be washed with the wash medium, and then optionally the amine salt may subsequently be converted into a final product pharmaceutically acceptable salt of clavulanic acid, such as an alkali metal salt, e.g.",
"potassium clavulanate.",
"Alternatively the salt which is washed may be a metal salt of clavulanic acid, for example an alkali metal salt of clavulanic acid, particularly potassium clavulanate.",
"The wash medium may be an aqueous wash medium at such a pH, e.g.",
"acidified water or preferably a mixture of water and a water-miscible organic solvent such as a C1-7 alkyl alcohol, typically containing 0.5-20% v:v water, at such a pH.",
"Suitably the aqueous wash medium, e.g.",
"water or such a mixture of water and a water-miscible organic solvent may be acidified with a mineral acid, such as sulphuric, hydrochloric or nitric acid, or an organic e.g.",
"carboxylic acid such as a C1-7 alkanoic acid such as acetic acid.",
"This first form of the process may therefore be a process in which a starting salt of clavulanic acid is provided containing one or more impurity such as N-succinyl tyrosine, and the salt is washed with the wash medium, to thereby produce a product salt of clavulanic acid in which the level of the one or more impurity is lower.",
"This first form of the process may therefore be a process in which the one or more impurity such as N-succinyl tryosine is removed from a starting salt of clavulanic acid, by washing of the starting salt of clavulanic acid with the wash medium.",
"In second form of the process of this invention the salt may be recrystallised from the medium at pH of less than 6.5.For example an amine salt may be recrystallised from such a recrystallisation medium, then may subsequently be converted into a final product pharmaceutically acceptable salt of clavulanic acid, such as an alkali metal salt, e.g.",
"potassium clavulanate by known methods such as those referred to above, for example reaction with potassium 2-ethyl hexanoate.",
"Suitably in this recrystallisation process the salt, e.g.",
"an amine salt may be dissolved in an aqueous medium, e.g.",
"acidified water or a mixture of water and a water-miscible organic solvent such as a C1-7 alkyl alcohol.",
"Preferably the solution concentration is high, e.g.",
"e.g.",
"10-40% or more, but there appears to be no theoretical upper limit.",
"The aqueous medium may initially be at the pH less than 6.5, or the pH may be adjusted when the aqueous solution has been made up, e.g.",
"the aqueous solution may then be acidified with an acid, suitably a mineral acid, such as sulphuric, hydrochloric or nitric acid, or an organic acid e.g.",
"carboxylic acid such as a C1-7 alkanoic acid such as acetic acid.",
"A preferred pH is ca.",
"5.5 or lower.",
"The salt, e.g.",
"an amine salt, may then be isolated from the aqueous solution.",
"This may be achieved for example by crystallisation by admixing the solution, e.g.",
"an aqueous solution, with a precipitating solvent, such as a water miscible ketone.",
"Suitable ketones include aliphatic ketones, for example a di-C1-7 alkyl ketone, acetone being preferred.",
"Solvates of amine clavulanate salts with such ketones are known and an amine salt may precipitate as a ketone solvate.",
"For example the solution may be diluted with an excess, e.g.",
"a 5-50 times excess of the precipitating solvent e.g.",
"of a ketone.",
"Cooling of the diluted solution can help to improve the yield of the precipitated of crystals of the amine salt, e.g.",
"as a solvate, which may be isolated.",
"This second form of the process may therefore be a process in which a starting salt of clavulanic acid is provided containing one or more impurity such as N-succinyl tyrosine, and the salt is recrystallised in the recrystallisation medium, to thereby produce a product salt of clavulanic acid in which the level of the one or more impurity is lower.",
"This second form of the process may therefore be a process in which the one or more impurity is removed from a starting salt of clavulanic acid, by recrystallisation of the starting salt of clavulanic acid from the recrystallisation medium.",
"A third form of the process may be a preparative process in which the salt of clavulanic acid is prepared in a liquid medium at the pH of less than 6.5.For example this third form of the process may comprise a process in which an amine salt of clavulanic salt is prepared by reacting clavulanic acid with an amine in a liquid medium at a pH of less than 6.5.This third form of the process can be used to prepare amine salts of clavulanic acid containing a lower level of impurities such as N-succinyl tyrosine than occur in amine salts of clavulanic acid prepared by alternative or prior art methods.",
"In a preferred embodiment of this third form of the process the reaction is performed in a two phase system, being an organic solvent phase containing the clavulanic acid, and an aqueous phase into which the amine salt is extracted and which is at the pH of 6.5 or lower.",
"Suitably therefore the aqueous phase may comprise an aqueous solution or suspension of the amine, especially an aqueous solution or suspension of tertiary butylamine.",
"The organic solvent phase is preferably substantially imiscible with water, that is, although some mixing of the solvent with water may occur, over most of the phase diagram two phases are formed.",
"For example a two phase system may be established, comprising an organic solvent phase containing dissolved clavulanic acid, and a separate aqueous phase, and the organic amine may be introduced into this two phase system, e.g.",
"injected into the aqueous phase, with suitable mixing conditions e.g.",
"agitation or turbulence, so that the amine salt is extracted into the aqueous phase as it is formed.",
"Typically the organic phase may have a concentration of 5-100 g/L, for example 10-40 g/L in clavulanic acid, e.g.",
"ca.",
"30 g/L.",
"Suitable solvents for the organic phase include substantially water-immiscible C1-7 alkyl-C1-7 alkanoate esters such as ethyl acetate and tertiary butyl acetate, and di-C1-7 alkyl ketones such as methylisobutyl ketone.",
"A temperature of ca.",
"0-5° C. is preferred for this reaction.",
"Suitably the organic solvent phase may be the product of an extraction by the organic solvent of an optionally pre-purified, e.g.",
"filtered and carbon treated, fermentation broth in which clavulanic acid has been formed.",
"Suitably the aqueous phase can be provided by back-extraction from the organic solvent phase by a circulating extraction loop so that a high concentration of the amine salt can be built up in the aqueous phase.",
"The aqueous phase may be concentrated to a high concentration, e.g.",
"25 wt % or more e.g.",
"by circulation of the aqueous extraction loop for a suitable time.",
"Generally when clavulanic acid is extracted from an aqueous broth to form such an organic extraction phase the broth is acidified to pH below 2.0.Controlled addition of the organic amine into the above-described two phase system can be used to adjust the pH of the aqueous phase, which can be monitored on-line.",
"A preferred pH is again below 5.5.The amine salt may then be isolated from the aqueous phase.",
"This may be achieved for example by crystallisation by admixing the aqueous solution with a precipitating solvent, such as a water miscible ketone as described above, and this crystallisation can be performed in a manner analogous to the recrystallisation process as described above.",
"A fourth form of the process of the invention may be a preparative process in which a metal salt of clavulanic acid is formed as a product by a reaction between an amine salt of clavulanic acid and a metal salt precursor compound, in a liquid medium at the pH of less than 6.5.This fourth form of the process can be used to prepare metal salts of clavulanic acid containing a lower level of impurities such as N-succinyl tyrosine than occur in metal salts of clavulanic acid prepared by alternative or prior art methods.",
"Preferred product metal salts of clavulanic acid which may be prepared in this fourth form of the process are salts of alkali metals and alkaline earth metals, particularly potassium clavulanate.",
"This fourth form of the process of the invention appears to be suitable for use with all metal salt precursor compounds which can be converted into a pharmaceutically acceptable salt of clavulanic acid by reaction with an amine salt of clavulanic acid.",
"General classes of suitable metal salt precursor compounds include salts of alkali metal cations and alkaline earth metal cations with counter anions which include basic anions, such as hydrogen carbonate, carbonate or hydrogen phosphate, and in particular anions of weak organic carboxylic acids, such as alkanoic acids of formula R—CO2H where R is C1-20 alkyl, for example C1-8 alkyl, e.g.",
"salts of acetic, propionic and ethyl hexanoic acid, such as 2-ethyl hexanoic acid.",
"Some examples of precursor compounds in these general classes include sodium or potassium hydrogen carbonate, potassium hydrogen phosphate and calcium carbonate.",
"A preferred metal salt precursor compound for potassium clavulanate is potassium 2-ethylhexanoate.",
"The reaction of this fourth form of the process of this invention is preferably performed with the amine salt in solution or suspension in a mixture of a water-miscible organic solvent and water, for example a C1-8 alkyl alcohol or a mixture of such an alcohol with water, e.g.",
"an isopropanol/water mixture.",
"Suitable proportions for such a solvent:water mixture to suit particular requirements may be determined experimentally, e.g.",
"a solvent:water mixture containing 1-10% v:v water, e.g.",
"ca.",
"1-5% v:v water.",
"The amine salt may be dissolved in such a solvent which may be either at the pH of 6.0 or less, or which may be adjusted to the pH by addition of a suitable acid, e.g.",
"a mineral acid or organic acid as above.",
"The amine salt may suitably be present in a solution concentration 0.1-1.0M, e.g.",
"ca.",
"0.5 M in clavulanate moiety.",
"A preferred pH is 5.5 or below, e.g.",
"pH 5.5-5.0.Although mixing of the amine salt and precursor compound in any order is encompassed by the invention, the metal salt precursor is preferably added to a solution of the amine salt.",
"The precursor may be added as a solution, e.g.",
"in an alcohol, e.g.",
"isopropanol solution.",
"A suitable solution concentration for this precursor compound solution is 0.5-3.5M, e.g.",
"ca.",
"2M.",
"Slow addition of the precursor solution is preferred, preferably with stirring and preferably being chilled after the addition a temperature below ambient e.g.",
"0-5° C. The metal salt of clavulanic acid product may be formed as a precipitate from solution.",
"Precipitation of the product metal salt of clavulanic acid from solution may be encouraged by mixing the solution with a precipitating solvent, e.g.",
"isopropanol, as described above.",
"The solid product can be isolated from the reaction medium by for example filtration and washing of the product.",
"In each of the above processes the pH of the medium defined above can result in reduction of the quantity of one or more impurities such as N-succinyl tyrosine in the product relative to analogous processes at higher pH.",
"These processes of the invention may comprise a part of an overall process for preparation of potassium clavulanate from crude clavulanic acid e.g.",
"as formed in a fermentation broth, e.g.",
"in which an amine salt is used as an intermediate.",
"This overall process may involve the steps of (i) fermentation of a microorganism which produces an aqueous broth containing clavulanic acid, (ii) extraction of the clavulanic acid into an organic solvent, (iii) conversion of the clavulanic acid into an amine salt of clavulanic acid, (iv) exposure of the amine salt to a pH of 6.0 or lower, particularly 5.5 or lower, (v) conversion of the amine salt to potassium clavulanate.",
"The washing and/or recrystallisation processes of the invention may for example be applied to the salts of clavulanic acid prepared as products of either or both of steps (iii) and/or (v).",
"The preparative processes of the invention may for example be applied to either or both of preparation steps (iii) and/or (v), as described above.",
"In the processes of this invention which involve a pharmaceutically acceptable metal salt of clavulanic acid a preferred salt is potassium clavulanate.",
"In the processes of this invention which involve an amine salt of clavulanic acid a preferred amine salt is the tertiary butylamine salt of clavulanic acid.",
"This can be made, in a known (see for example EP-A-0 026 044, the content of which is included herein by reference) reaction, by reaction of tertiary butylamine with clavulanic acid, and easily isolated as an acetone solvate.",
"Both the tertiary butylamine salt and its acetone solvate can readily be converted into pharmaceutically acceptable salts of clavulanic acid, e.g.",
"potassium clavulanate, e.g.",
"as disclosed in EP 0026044A.",
"However the processes of the invention appear in principle to be applicable to all those amine salts which are known to be useable as intermediates in the conversion of clavulanic acid into a pharmaceutically acceptable salt such as potassium clavulanate.",
"Consequently these processes of the invention appears in principle to be effective with all such amines.",
"Examples of such amines, and their corresponding amine salts are disclosed for example in the publications referred to above, and include for example amine poly-, e.g.",
"di-, clavulanate salts if the amine has more than one amino-moiety.",
"Other suitable amine salts include those referred to in the publications mentioned above, e.g.",
"tertiary octylamine, diethylamine, tri-(lower alkyl) amines, dimethylaniline, NN′-diisopropyl-ethylenediamine, tertiary, tertiary diamines such as N,N,N′,N′-tetramethyl-1,2-diaminoethane, N,N,N′,N′-tetramethyl-1,6-diaminohexane, 1,2-dipiperidinoethane and dipiperidinomethane, diaminoethers such as bis (2-dimethylaminoethyl) ether, benzhydrylamines, N,N′-alkylethylene diamines, such as N,N′-diisopropylethylene diamine, N,N′-diethylene diamine, N,N′-dibenzylethylene diamine, N,N,N′,N′-tetramethylene diamine, N,N,N′,N′-tetramethylethylenediamine, 1,3-bis(di-methylamino)-2-propanol, benzhydrylamine and bis (2-(dimethylamino) ethyl) ether, and diisopropylethylendiamine.",
"The present invention further provides a product amine salt of clavulanic acid being a product of any of the above-described process, particularly tertiarybutylamine clavulanate.",
"The present invention further provides a product pharmaceutically acceptable metal salt of clavulanic acid being a product of any of the above-described process, particularly potassium clavulanate.",
"These products are characterised by impurity levels, particularly of N-succinyl tyrosine, which are less than those obtainable via known processes, particularly corresponding process which are carried out in a medium with a pH above that defined.",
"The invention will now be described by way of example only.",
"Examples 1, 2 and 3 are expressed in terms of the reduction of the total quantity of impurities in the product salt of clavulanic acid, and Examples 4, 5 and 6 are expressed in terms of reduction of the specific impurity N-succinyl tyrosine.",
"EXAMPLE 1 Acidification of Amine Salt During Amine Salt Crystallisation 30 g tertiary-butylamine (“t-BA”) clavulanate salt was dissolved in water to give a 30% pfa (i.e.",
"calculated as “pure free acid”, i.e.",
"clavulanic acid moiety by weight:volume) solution.",
"The pH of the solution was measured and acidified to pH 4.5 with approximately 1.1 ml of 50% sulphuric acid or concentrated nitric acid.",
"A 5× volume of acetone was added and the mixture stirred for 15 minutes.",
"Additional acetone was added over 20 minutes up to 35× the aqueous volume.",
"Throughout the foregoing the amine salt solution was maintained at ambient temperature.",
"The product t-BA salt crystallised and was chilled at 0-5° C. for 60 minutes and collected by filtration.",
"The product was washed with 600 ml of acetone, prior to drying for 12 hours in vacuo.",
"This procedure was repeated at a “natural” pH without acid addition, i.e.",
"ca.",
"pH 7.5.The t-BA salt obtained using these procedures was then converted into product potassium clavulanate using a known method, and levels of total impurities in the potassium clavulanate prepared using the samples of t-BA salt were measured as follows: Crystallisation pH % total impurities in potassium of t-BA solution clavulanate (wrt clav by HPLC) Natural (pH 7.0-7.5) 2.03 pH4.5 0.03 EXAMPLE 2 Preparation of Amine Clavulanate Salt from Clavulanic Acid and Amine Under Acid Conditions 100 L of clavulanic acid aqueous concentrate (ca.",
"20-40 g/L), from filtered concentrated fermentation broth was solvent extracted at pH 1.5 into methyl isobutyl ketone (“MIBK”).",
"The clavulanic acid rich MIBK was treated with carbon (17 L) and back extracted into water which was adjusted to pH 4.5 by controlled injection of a 50% MIBK solution of t-BA.",
"By controlling the t-BA addition rate the pH of the aqueous phase could be set, and pH 4.5 and pH 5.4 were used in separate experiments.",
"Once the aqueous back extraction solution had reached a suitable crystallisation concentration, a 100 ml aliquot was crystallised by addition of 35 volumes of acetone and the precipitated product isolated as in Example 1 above.",
"The subsequent crystallised amine was converted to potassium salt and analysed for total impurity levels.",
"This was then repeated with an aqueous phase pH of 5.4.By using the pH 4.5 the level of total impurities in the potassium clavulanate was reduced by 50% relative to the level at a pH of 5.4.EXAMPLE 3 Conversion of an Amine Salt to Potassium Clavulanate 25 g of t-BA clavulanate were dissolved in a mixture of isopropanol (81 ml) and distilled water (10 ml), and the pH was reduced to pH 6.8 with glacial acetic acid.",
"While stirring a further 145 ml of isopropanol was added and 59.5 ml potassium 2-ethylhexanoate (1.92N) dissolved in isopropanol added dropwise over 20 minutes.",
"The so-formed slurry was stirred for 120 minutes in an ice-bath at 0-5° C. and filtered.",
"The potassium clavulanate cake was washed with acetone (120 ml and 200 ml) and dried in vacuo with a nitrogen bleed.",
"Altering the volume of glacial acetic acid added varied the pH of the dissolution solution.",
"Results indicating the effect on the amount of total impurities in the potassium clavulanate (“Kclav”) and overall yield of potassium clavulanate are shown in the table below: % of total impurities Dissolution pH in Kclav (wrt clav) 6.8 0.87 6.5 0.92 6.0 0.63 5.5 0.57 5.0 0.13 5.0* 0.11 *The volume of water was reduced to compensate for the additional volume of acetic acid used.",
"The experiments described in Example 3 above were repeated using other acids than acetic acid to achieve the specified pH, for example hydrochloric acid was used with similar results.",
"EXAMPLE 4 Acidification of Amine Salt During Amine Salt Crystallisation 30 g tertiary-butylamine (“t-BA”) clavulanate salt was dissolved in water to give a 30% pfa (i.e.",
"calculated as “pure free acid”, i.e.",
"clavulanic acid moiety by weight:volume) solution.",
"The pH of the solution was measured and acidified to pH 4.5 with approximately 1.1 ml of 50% sulphuric acid or concentrated nitric acid.",
"A 5× volume of acetone was added and the mixture stirred for 15 minutes.",
"Additional acetone was added over 20 minutes up to 35× the aqueous volume.",
"Throughout the foregoing the amine salt solution was maintained at ambient temperature.",
"The product t-BA salt crystallised and was chilled at 0-5° C. for 60 minutes and collected by filtration.",
"The product was washed with 600 ml of acetone, prior to drying for 12 hours in vacuo.",
"This procedure was repeated at a “natural” pH without acid addition, i.e.",
"ca.",
"pH 8.5.The level of N-succinyl tyrosine in the t-BA salt obtained using these procedures was then measured as follows: Crystallisation pH % N-succinyl tyrosine in t-BA of t-BA solution clavulanate (wrt clav by HPLC) Natural (pH 8.5) 3.48 pH4.5 0.03 EXAMPLE 5 Preparation of Amine Clavulanate Salt from Clavulanic Acid and Amine Under Acid Conditions 100 L of clavulanic acid aqueous concentrate (ca.",
"2040 g/L), from filtered concentrated fermentation broth was solvent extracted at pH 1.5 into methyl isobutyl ketone (“MIBK”).",
"The clavulanic acid rich MIBK was treated with carbon (17 L) and back extracted into water which was adjusted to pH 4.5 by controlled injection of a 50% MIBK solution of t-BA.",
"By controlling the t-BA addition rate the pH of the aqueous phase could be set, and pH 4.5 and pH 5.4 were used in separate experiments.",
"Once the aqueous back extraction solution had reached a suitable crystallisation concentration, a 100 ml aliquot was crystallised by addition of 35 volumes of acetone and the precipitated product isolated as in Example 1 above.",
"The subsequent crystallised amine was converted to potassium salt and analysed for N-succinyl tyrosine levels.",
"This was then repeated with an aqueous phase pH of 5.4.By using the pH 4.5 the level of N-succinyl tyrosine impurity in the potassium clavulanate was reduced 26-fold relative to the level at a pH of 5.4.EXAMPLE 6 Conversion of an Amine Salt to Potassium Clavulanate 25 g of t-BA clavulanate were dissolved in a mixture of isopropanol (81 ml) and distilled water (10 ml), and the pH was reduced to pH 6.8 with glacial acetic acid.",
"While stirring a further 145 ml of isopropanol was added and 59.5 ml potassium 2-ethylhexanoate (1.92N) dissolved in isopropanol added dropwise over 20 minutes.",
"The so-formed slurry was stirred for 120 minutes in an ice-bath at 0-5° C. and filtered.",
"The potassium clavulanate cake was washed with acetone (120 ml and 200 ml) and dried in vacuo with a nitrogen bleed.",
"Altering the volume of glacial acetic acid added varied the pH of the dissolution solution.",
"Results indicating the effect on the amount of N-succinyl tyrosine impurity in the potassium clavulanate (“Kclav”) and overall yield of potassium clavulanate are shown in the table below: % of N-succinyl tyrosine Dissolution pH in Kclav (wrt clav) 6.8 0.62 6.5 0.65 6.0 0.44 5.5 0.40 5.0 0.04 5.0* 0.07 *The volume of water was reduced to compensate for the additional volume of acetic acid used.",
"The experiments described in Example 3 above were repeated using other acids than acetic acid to achieve the specified pH, for example hydrochloric acid was used with similar results."
]
] | Patent_10275852 |
[
[
"Eeg feedback controlled sound therapy for tinnitus",
"An automated method for treating tinnitus by habituation through use of neurological feedback, comprising the steps of connecting a subject through a set of attached headphones to an electronic sound player that is connected to a PC workstation presenting sound examples by software to the subject who can refine them by manipulating a series of controllers on the player, making an electronic recording of the sound in a digital music format, storing the recording in the computer, transferring a copy of the electronic sound file to the subject's electronic music player, generating an EEC signature of the subject's brain activity in response to the presented sound, sound using the customized sound to stimulate the auditory system while the brain activity is recorded, wherein the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately."
],
[
"1.An automated method for treating tinnitus by habituation to customized sound through use of neurological feedback, comprising the steps of: connecting a subject through headphones to an electronic sound player that is connected to a PC workstation; creating a customized sound profile for the subject's particular tinnitus by presenting a plurality of audible sound examples from tinnitus sound library software to the subject; allowing the subject to choose and refine the presented sounds that most closely resemble the tinnitus sound; recording the refined sound in a digital music format to create a custom sound profile; storing the custom sound profile recording in a computer; generating an EEG signature of the subject's brain activity in response to the presented sound by downloading a copy of the electronic sound file to the subject's electronic music player, presenting custom sound most closely matching the tinnitus to stimulate the auditory system, recording the subject's response to sounds adjacent to but not specifically corresponding to his tinnitus signature, recording the subject's brain activity during absence of sound stimuli uploading the EEG responses to sound stimuli and to the absence of sound into the computer to create the EEG signature; and exposing the subject to the custom sound for as long as practical each day wherein the EEG is actively monitored by the computer, which generates sound in response and periodically tests the signatures for tinnitus and silence and determines if the tinnitus is decreasing and the silence signal is strengthening, and wherein if these changes are not present, the computer slightly alters the sound stimuli and again checks for feedback, and wherein the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately to habituate the subject to his tinnitus.",
"2.A method for customized habituation treatment of tinnitus without masking sound or using subthreshold sound, comprising: matching narrowband sound frequency to a patient's perceived tinnitus; presenting the matched sound frequency to the patient wherein the presented matched sound activates the same population of neurons affected by tinnitus, and wherein habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible; and periodically updating frequency changes as required for maintaining maximum habituation.",
"3.An objective method for diagnosing tinnitus by detecting changes in the dynamic response characteristics of the auditory cortex induced by tinnitus, comprising: characterizing a subject's tinnitus perception by matching the pitch of his or her tinnitus to the frequericy of a pure sine tone generated by a function generator a programmable logarithmic amplifier that controlled in real time by a stimulus presentation and data collection program set the intensity of each stimulus; recording an auditory evoked potential to a variety of tone pitches, including the tinnitus pitch wherein the increased activation of the auditory cortex manifests itself as an increased slope of the AR; and calculating the slope of the AR response for tinnitus frequency tones wherein the observed increase in the slope of the AR in tinnitus is due to tinnitus-related activity present in the auditory cortex.",
"4.A method for treating tinnitus by habituation to its sound frequency, comprising the steps of: determining the “matching frequency” (pitch) of a subject's tinnitus; determining the hearing threshold for the tinnitus frequency; determining the “matching intensity” of the subject's tinnitus; determining the hearing threshold for the “off” frequencies; stimulating the auditory system in two series of tonal stimulation first at the tinnitus frequency, and second at the “off” frequency; collecting EEG data; and giving the subjects an electronic music player having the habituation stimulus downloaded wherein the subjects are asked to listen to it for as long as it is comfortable each day.",
"5.The method of claim 4, wherein the “matching frequency” is determined by 25 presenting the subjects with a continuous, audible tone varying in pitch, and asking them to indicate the frequency most closely matching the frequency of their tinnitus.",
"6.The method of claim 4, wherein to determine the threshold, subjects are presented with a continuous tone that gradually increases in volume, and are asked to indicate when they begin to hear the tone.",
"7.The method of claim 6, wherein the intensity at which the subjects begin to.",
"hear the tone is considered the hearing threshold.",
"8.The method of claim 4, wherein to determine the “matching intensity” subjects are presented with a continuous tone that increases or decreases in volume and are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus.",
"9.An EEG marker of tinnitus suitable for diagnosing the presence of tinnitus, comprising: a replica of a subject's sound experience; and a measure of the subject's EEG response to increasing intensity of the sound wherein the replica of sound is constructed from a patients subjective determination of sound most closely related to the annoying sound experienced by the patient, and the patient's EEG response is measured to determine peak amplitude of the N100 component."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention generally concerns the diagnosis and treatment of tinnitus.",
"The invention more specifically relates to the use of custom designed sound and feedback to determine the precise treatment sound matching the tinnitus, its neurophysiological effect, and monitoring the treatment effect by feedback from the brain.",
"2.Description of Related Art Commonly perceived as a “ringing in the ear,” tinnitus is a very frequent disorder of the auditory system, affecting about 17% of the general population and up to 33% in the elderly.",
"About a quarter of these people are sufficiently bothered by their tinnitus that they seek professional help [Jastreboff et al, 1996].",
"Tinnitus is a phantom perception and thus not associated with any auditory stimulus.",
"Until very recently, there were no objective measurements that could be related to tinnitus [Jastreboff et al, 1994], and diagnosis of tinnitus had to rely on various questionnaires, e.g.",
"[Wilson et al, 1991].",
"The fact that tinnitus is perceived as a sound, however, indicates that it is associated with aberrant neural activity in the auditory pathways.",
"Furthermore, the fact that tinnitus is associated with perception leads to the conclusion that central auditory structures such as the thalamus and auditory cortex must be involved.",
"Neural correlates of tinnitus have indeed been found in central auditory structures [Norena et al, 1 999; Mühlnickel et al, 1 998; Wallhäusser-Franke et al, 1996].",
"Previously, tinnitus had been viewed as being caused in the auditory periphery [Eggermont, 1990; Tonndorf, 1981; Salvi and Ahroon, 1983], and even though neuronal activity related to tinnitus has been found in the central auditory system rather than in the periphery, it remains possible that the chain of events that leads to the development of tinnitus may be set off by events taking place in the periphery."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>To address the beforementioned problem and the above solution the inventors disclose their invention as follows.",
"The invention contemplates an automated method for treating tinnitus by habituation to customized sound through use of neurological feedback.",
"The methodology comprises the following steps.",
"The tinnitus-suffering subject is connected through headphones to an electronic sound player that in turn is connected to a PC workstation.",
"A customized sound profile is created for the subject's particular tinnitus by presenting a plurality of audible sound examples from a tinnitus sound library in special software.",
"The subject is allowed to choose and refine the presented sounds that most closely resemble his or her tinnitus sound.",
"The refined sound is recorded in a digital music format to create a custom sound profile for that particular subject, and the custom sound profile recording is stored in a computer.",
"An EEG signature of the subject's brain activity in response to the presented sound is generated by downloading a copy of the electronic sound file to the subject's electronic music player, presenting custom sound most closely matching the tinnitus to stimulate the auditory system, and recording the subject's neurological response to sounds adjacent to but not specifically corresponding to his tinnitus signature.",
"The subject's brain activity during absence of sound stimuli is also recorded.",
"The EEG profile is uploaded into the computer to create the EEG signature.",
"When undergoing treatment, the EEG response is actively monitored by the computer, which generates sound in response.",
"The computer periodically tests the signatures for tinnitus and silence and determines if the tinnitus is decreasing and the silence signal is strengthening, and when if these desirable changes are not present, the computer slightly alters the sound stimuli and again checks for feedback.",
"Thus, the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately to habituate the subject to his tinnitus.",
"A method is also contemplated by this invention for customized habituation treatment of tinnitus without masking sound or using subthreshold sound.",
"This method involves matching narrowband sound frequency to a patient's perceived tinnitus and presenting the matched sound frequency to the subject, wherein the presented matched sound activates the same population of neurons affected by tinnitus, and wherein habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible.",
"Periodically, frequency changes are updated as required for maintaining maximum habituation.",
"An objective method for diagnosing tinnitus by detecting changes in the dynamic response characteristics of the auditory cortex induced by tinnitus is further contemplated.",
"This method comprises characterizing a subject's tinnitus perception by matching the pitch of his or her tinnitus to the frequency of a pure sine tone generated by a function generator, having a programmable logarithmic amplifier that is controlled in real time by a stimulus presentation and data collection program software to set the intensity of each stimulus.",
"An auditory evoked potential to a variety of tone pitches, including the tinnitus pitch is recorded, wherein the increased activation of the auditory cortex manifests itself as an increased slope of the AR.",
"The slope of the AR response for tinnitus frequency tones is calculated, wherein an observed increase in the slope of the AR in tinnitus indicates tinnitus-related activity present in the auditory cortex.",
"Another preferred method for treating tinnitus by habituation to its sound frequency comprises the steps of determining the “matching frequency” (pitch) of a subject's tinnitus, determining the hearing threshold for the tinnitus frequency; determining the “matching intensity” of the subject's tinnitus, and determining the hearing threshold for the “off” frequencies.",
"This is followed by stimulating the auditory system in two series of tonal stimulation; first at the tinnitus frequency, and second at the “off” frequency.",
"EEG data is collected and the subject is given an electronic music player having the habituation stimulus downloaded into it.",
"The subject is asked to listen to the player for as long as it is comfortable each day.",
"The “matching frequency” is determined by presenting the subjects with a continuous, audible tone varying in pitch, and asking them to indicate the frequency most closely matching the frequency of their tinnitus.",
"To determine the threshold, subjects are presented with a continuous tone that gradually increases in volume, and are asked to indicate when they begin to hear the tone.",
"The intensity at which the subjects begin to hear the tone is considered the hearing threshold.",
"“Matching intensity” is determined when subjects are presented with a continuous tone that increases or decreases in volume and are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus.",
"An EEG marker of tinnitus suitable for diagnosing the presence of tinnitus is also contemplated by this invention.",
"This marker comprises a replica of a subject's tinnitus sound experience and a measure of the subject's EEG response to increasing intensity of the sound.",
"The replica of sound is constructed from a subject's subjective determination of sound most closely related to the annoying sound experienced by him, and the patients EEG response is measured to determine peak amplitude of the N100 component.",
"These and other aspects and attributes of the present invention will become increasingly clear upon reference to the following drawings and accompanying specification."
],
[
"BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention generally concerns the diagnosis and treatment of tinnitus.",
"The invention more specifically relates to the use of custom designed sound and feedback to determine the precise treatment sound matching the tinnitus, its neurophysiological effect, and monitoring the treatment effect by feedback from the brain.",
"2.Description of Related Art Commonly perceived as a “ringing in the ear,” tinnitus is a very frequent disorder of the auditory system, affecting about 17% of the general population and up to 33% in the elderly.",
"About a quarter of these people are sufficiently bothered by their tinnitus that they seek professional help [Jastreboff et al, 1996].",
"Tinnitus is a phantom perception and thus not associated with any auditory stimulus.",
"Until very recently, there were no objective measurements that could be related to tinnitus [Jastreboff et al, 1994], and diagnosis of tinnitus had to rely on various questionnaires, e.g.",
"[Wilson et al, 1991].",
"The fact that tinnitus is perceived as a sound, however, indicates that it is associated with aberrant neural activity in the auditory pathways.",
"Furthermore, the fact that tinnitus is associated with perception leads to the conclusion that central auditory structures such as the thalamus and auditory cortex must be involved.",
"Neural correlates of tinnitus have indeed been found in central auditory structures [Norena et al, 1 999; Mühlnickel et al, 1 998; Wallhäusser-Franke et al, 1996].",
"Previously, tinnitus had been viewed as being caused in the auditory periphery [Eggermont, 1990; Tonndorf, 1981; Salvi and Ahroon, 1983], and even though neuronal activity related to tinnitus has been found in the central auditory system rather than in the periphery, it remains possible that the chain of events that leads to the development of tinnitus may be set off by events taking place in the periphery.",
"A Thalamic Model of Tinnitus The presence of tinnitus-related neural activity in the central auditory system agrees well with a recently proposed neurophysiological model of tinnitus [Jeanmonod et al, 1996] (FIG.",
"1).",
"According to the model, tinnitus arises in the thalamus.",
"Several parts of the thalamus interact to establish a reverberating loop.",
"Neuronal activity originating from this reverberating loop is transmitted to the auditory cortex, where it gives rise to the tinnitus perception.",
"This reverberating loop is established by disinhibition of neurons in the thalamus, which occurs when thalamic neurons receive inhibitory input causing a hyperpolarization.",
"These thalamic neurons are capable of generating action potentials, once the hyperpolarization has reached a threshold.",
"Thus, inhibitory input can cause these neurons to fire.",
"The action potentials generated by this mechanism are called low threshold spikes (LTS) and usually occur in rhythmic bursts.",
"Two structures in the thalamus receive afferent auditory input: the medial geniculate body (MGB) and the multimodal medial thalamus (MT).",
"Both these nuclei give excitatory input to the thalamic reticular nucleus (RT), and in turn receive inhibitory input from the RT.",
"When afferent input into the MGB is normal, an equilibrium of excitation and inhibition is present in both MT and MGB (FIG.",
"1A).",
"When the afferent auditory input into the thalamus fails, an imbalance of input is created.",
"The MGB will receive no input whereas the MT receives a slightly decreased input (FIG.",
"1B).",
"In this situation, the RT will still receive excitatory input from the MT, enough to generate the inhibitory output to both MT and MGB (FIG.",
"1C).",
"Since no excitation is present in the MGB, the inhibitory input from the RT will hyperpolarize the neurons in the MGB.",
"Neurons in the MGB will respond to this hyperpolarization with deinactivation leading to LTS bursts that are propagated to the auditory cortex (FIG.",
"1D).",
"Damage to the basilar membrane can set off the thalomocortical reverberating loop.",
"This model explains the appearance of tinnitus in conditions where the MGB is not receiving sensory input, e.g.",
"in silent environments, or when hearing thresholds are temporarily or permanently raised after noise exposure.",
"According to this model, once input into the MGB is restored, the tinnitus should vanish.",
"Clearly, this is not the case in those people who are so annoyed by their tinnitus that they seek professional help.",
"An additional mechanism must be in place to make tinnitus persist.",
"It has been noted that the presence of tinnitus cannot be determined from an audiogram, i.e.",
"tinnitus may be present even when the MGB is receiving normal sensory input.",
"The perception of tinnitus will only be stabilized when the person perceiving the phantom noise pays attention to it and associates it with unpleasant emotions [Jastreboff et al, 1996b; Langner and Wallhäusser-Franke, 1999](FIG.",
"2).",
"In this scenario, the person experiencing tinnitus is annoyed by it.",
"Consequently, he will direct his attention to the phantom sound, thereby activating the limbic system.",
"The limbic system is assumed to give rise to an increased generation of tinnitus-related activity.",
"The detection of tinnitus-related activity is facilitated by mechanisms of lateral inhibition in the central auditory system.",
"These will act to confine the phantom sound to regions representing distinct frequencies and increase the contrast between the tinnitus-related activity in these regions and the spontaneous activity in adjacent regions.",
"This will lead to increased tinnitus perception.",
"The increased perception can then lead to increased annoyance, completing a positive feedback cycle that will make tinnitus persistent.",
"The Auditory N100 As an Index of Cortical Responsiveness Event-related potential (ERP) studies have demonstrated that electrical activity time-locked to stimulus or response events and averaged over repeated trials reflects information processing in the cortex [Hillyard et al, 1978; Pritchard, 1981b; Duncan-Johnson and Donchin, 1977; Pineda et al, 1997; Pineda et al, 1998].",
"Considerable evidence to date indicates that long-latency potentials (approximately >100 ms poststimulus) appear primarily sensitive to cognitive variables that reflect task requirements and the psychological state of the subject.",
"These “endogenous” potentials most likely reflect non-obligatory activities invoked by the demands of the task.",
"One of the most widely studied endogenous components is the N100.Wolpaw and Penry [Wolpaw and Penry, 1975] were the first to propose that the N100 consisted of midline and temporal subcomponents.",
"Subsequent work has shown that midline components can be modeled with tangential generators on the superior temporal plane and pointing toward the midline of the scalp.",
"At least two midline components have been distinguished.",
"An early frontocentral peak (N100a) shows reliable tonotopic changes in distribution and sensitivity to attention [Woods, 1995].",
"A later midline component (N100b) shows the same distribution for tones of different frequency.",
"N100 components show increases in amplitude and decreases in latency with increasing stimulus intensity [Hillyard et al, 1978; Scherg and von Cramon, 1990; Mäkelä and Hari, 1990].",
"In some individuals, the increases in stimulus intensity often bring decreased amplitude and increased latency.",
"This tendency for N100 to increase or decrease in magnitude in response to stimuli of increasing intensity has been called the “augmenting/reducing” (AR) response.",
"These intensity-amplitude functions have been hypothesized to result from variations in the central modulation of sensory processing and/or the actions of nonspecific arousal systems [Zuckermann et al, 1974; von Knorring and Perris, 1981].",
"It has also been proposed that they.may reflect the “tuning” properties of a cortical gating mechanism that regulates sensory input [Buchsbaum and Silverman, 1968; Lukas and Siegel, 1977; Pritchard et al, 1985].",
"Some have related this mechanism to “attention” shifts and “overload” protection at high stimulus intensities.",
"A number of studies have shown that midline auditory N100 shows strong intensity dependence, while those recorded from temporal electrodes show weak intensity dependence [Pineda et al, 1991].",
"These differences suggest different N100 generators in the primary and secondary auditory areas [Pineda et al, 1991] [Connally, 1993], which is consistent with the multiple N100 generator hypothesis [Woods, 1995].",
"How Thalamo-Cortical Activity Affects the A/R-Response The rhythmic LTS bursting activity of thalamic neurons mentioned above is also observed in slow wave sleep [Pape and McCormick, 1989; Steriade and Llinas, 1988], where it is thought to be a gating mechanism blocking sensory input into the cortex [Pape and McCormick, 1989; Steriade and Llinas, 1988].",
"The argument has also been made that this mechanism is active not only during sleep but also during waking and may result in different attentional states.",
"The excitation of cortical tissue by tinnitus may compete with stimulus-induced activity.",
"The neural activity induced by the tinnitus may, therefore, be regarded as competition for cortical neuronal substrates.",
"This may lead to the reorganization of the auditory cortex [Mühlnickel et al, 1998].",
"That is, the processing of stimuli in the presence of tinnitus-related activity may lead to increases in the firing rate of neurons, the use of more neural substrate, or a combination of both.",
"It is hypothesized that these mechanisms for dealing with tinnitus-related activity in the auditory system lead to the increased intensity dependence of the auditory evoked potential that Inventors and others have observed [Norena et al, 1999].",
"The hypothesis that tinnitus-related neural activity is caused by oscillatory LTS activity in the thalamus is further supported by a combination of other findings.",
"First, application of serotonin in the lateral geniculate body (LGB) or MGB of the cat suppresses the hyperpolarization necessary to generate LTS-bursts [Pape and McCormick, 1989].",
"Second, the intensity dependence of the auditory evoked potential is strongly influenced by brain serotonergic activity [Juckel et al, 1999; Juckel et al, 1997; Hegerl et al, 1996].",
"Finally, these observations are linked by the fact that the thalamus contains a high density of binding sites for serotonergic drugs as well as serotonin uptake sites [Smith, 1999].",
"Elaboration on the Tinnitus Models Taken together the various models and evidence suggest that while tinnitus may be triggered by events in the periphery, the mechanisms that make tinnitus a persistent and annoying condition are located in the central auditory system.",
"Furthermore, it appears that people suffering from tinnitus unintentionally train themselves to have tinnitus by using negative reinforcement.",
"It has been shown that the cortical representation of tones associated with unpleasant sensations is enlarged [Gonzalez-Lima and Scheich, 1986] and/or changed to enhance the contrast between this particular tone and other tones of similar frequency [Ohl and Scheich, 1996].",
"The cortical representation of tones no longer associated with unpleasant sensations will return to a state where contrasts are no longer enlarged.",
"Moreover, responses to stimuli occurring while attention is directed at another task will decrease over time [Anderson and Oatman, 1980].",
"This suggests that if the association between tinnitus and unpleasant emotions can be broken, the aberrant neuronal activity in the central auditory system can be decreased by habituation.",
"Then tinnitus is treated like any other sound that does not carry relevant information: it is ignored.",
"The tinnitus retraining therapy (TRT) introduced by Jastreboff [Jastreboff et al, 1996a] makes use of the mechanism described above.",
"However, TRT as described by Jastreboff uses white or broadband noise as a habituating stimulus.",
"The rationale behind the use of white noise is to generate a decreased signal-to-noise ratio between the tinnitus-related neuronal activity and random background activity in the auditory system.",
"This would be achieved by introducing a quasi-random, stimulus-driven activity into all of the parallel tonotopic channels of the auditory system.",
"A precise computational model of tinnitus has been proposed by Langner et al [Langner and Wallhäusser-Franke, 1999] based on animal work.",
"This model assumes that the limbic system is necessary for stabilizing the tinnitus perception.",
"It also explains how a decreased auditory input resulting from a peripheral hearing deficit can give rise to a specific tinnitus pitch.",
"When the tinnitus sound is used as a habituation stimulus, Langners' model predicts the tinnitus would disappear.",
"Currently, there are sound therapies for tinnitus that use generic sounds.",
"These present therapies are only partially effective and require a long time for treatment.",
"It would, therefore, be desirable to obtain a brain signal feedback system, wherein one could rapidly suppress brain activity related to tinnitus and provide relief for this disorder.",
"SUMMARY OF THE INVENTION To address the beforementioned problem and the above solution the inventors disclose their invention as follows.",
"The invention contemplates an automated method for treating tinnitus by habituation to customized sound through use of neurological feedback.",
"The methodology comprises the following steps.",
"The tinnitus-suffering subject is connected through headphones to an electronic sound player that in turn is connected to a PC workstation.",
"A customized sound profile is created for the subject's particular tinnitus by presenting a plurality of audible sound examples from a tinnitus sound library in special software.",
"The subject is allowed to choose and refine the presented sounds that most closely resemble his or her tinnitus sound.",
"The refined sound is recorded in a digital music format to create a custom sound profile for that particular subject, and the custom sound profile recording is stored in a computer.",
"An EEG signature of the subject's brain activity in response to the presented sound is generated by downloading a copy of the electronic sound file to the subject's electronic music player, presenting custom sound most closely matching the tinnitus to stimulate the auditory system, and recording the subject's neurological response to sounds adjacent to but not specifically corresponding to his tinnitus signature.",
"The subject's brain activity during absence of sound stimuli is also recorded.",
"The EEG profile is uploaded into the computer to create the EEG signature.",
"When undergoing treatment, the EEG response is actively monitored by the computer, which generates sound in response.",
"The computer periodically tests the signatures for tinnitus and silence and determines if the tinnitus is decreasing and the silence signal is strengthening, and when if these desirable changes are not present, the computer slightly alters the sound stimuli and again checks for feedback.",
"Thus, the computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately to habituate the subject to his tinnitus.",
"A method is also contemplated by this invention for customized habituation treatment of tinnitus without masking sound or using subthreshold sound.",
"This method involves matching narrowband sound frequency to a patient's perceived tinnitus and presenting the matched sound frequency to the subject, wherein the presented matched sound activates the same population of neurons affected by tinnitus, and wherein habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible.",
"Periodically, frequency changes are updated as required for maintaining maximum habituation.",
"An objective method for diagnosing tinnitus by detecting changes in the dynamic response characteristics of the auditory cortex induced by tinnitus is further contemplated.",
"This method comprises characterizing a subject's tinnitus perception by matching the pitch of his or her tinnitus to the frequency of a pure sine tone generated by a function generator, having a programmable logarithmic amplifier that is controlled in real time by a stimulus presentation and data collection program software to set the intensity of each stimulus.",
"An auditory evoked potential to a variety of tone pitches, including the tinnitus pitch is recorded, wherein the increased activation of the auditory cortex manifests itself as an increased slope of the AR.",
"The slope of the AR response for tinnitus frequency tones is calculated, wherein an observed increase in the slope of the AR in tinnitus indicates tinnitus-related activity present in the auditory cortex.",
"Another preferred method for treating tinnitus by habituation to its sound frequency comprises the steps of determining the “matching frequency” (pitch) of a subject's tinnitus, determining the hearing threshold for the tinnitus frequency; determining the “matching intensity” of the subject's tinnitus, and determining the hearing threshold for the “off” frequencies.",
"This is followed by stimulating the auditory system in two series of tonal stimulation; first at the tinnitus frequency, and second at the “off” frequency.",
"EEG data is collected and the subject is given an electronic music player having the habituation stimulus downloaded into it.",
"The subject is asked to listen to the player for as long as it is comfortable each day.",
"The “matching frequency” is determined by presenting the subjects with a continuous, audible tone varying in pitch, and asking them to indicate the frequency most closely matching the frequency of their tinnitus.",
"To determine the threshold, subjects are presented with a continuous tone that gradually increases in volume, and are asked to indicate when they begin to hear the tone.",
"The intensity at which the subjects begin to hear the tone is considered the hearing threshold.",
"“Matching intensity” is determined when subjects are presented with a continuous tone that increases or decreases in volume and are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus.",
"An EEG marker of tinnitus suitable for diagnosing the presence of tinnitus is also contemplated by this invention.",
"This marker comprises a replica of a subject's tinnitus sound experience and a measure of the subject's EEG response to increasing intensity of the sound.",
"The replica of sound is constructed from a subject's subjective determination of sound most closely related to the annoying sound experienced by him, and the patients EEG response is measured to determine peak amplitude of the N100 component.",
"These and other aspects and attributes of the present invention will become increasingly clear upon reference to the following drawings and accompanying specification.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 shows the development of tinnitus (adapted from Jeanmonod et al 1996).",
"There is afferent auditory input to the medial geniculate body (MGB) and the multimodal medial thalamus (MT).",
"Both these nuclei give excitatory input to the thalamic reticular nucleus (RT), and in turn receive inhibitory input from the RT.",
"Further explanation is in the text.",
"FIG.",
"2 is an overview of the auditory system with regard to tinnitus generation and the Mechanisms stabilizing tinnitus.",
"Adapted from Jeanmonod 1996 and Langner 1999.Explanation is in the text.",
"FIG.",
"3 displays the auditory-evoked potential at the electrode position Fz, recorded at 30 dB (LOW), 42 dB (MED) and 54 dB (HIGH) above the subject's hearing threshold.",
"Note that the amplitude of the N100 component increases with increasing stimulus volume.",
"FIG.",
"4 shows the auditory-evoked potential at the electrode position Fz, recorded using various references.",
"The stimulus was a 1 kHz tone presented at 54 dB above the subject's hearing threshold.",
"The stimulus was presented 80 times in each condition.",
"FIG.",
"5 demonstrates that the intensity dependence of both amplitude (top) and latency (bottom) the N100 component of the auditory evoked potential in tinnitus subjects is changed by one hour of habituation training.",
"(N=4).",
"FIG.",
"6 reveals that neither the intensity dependence of amplitude (top) or latency (bottom) the N100 component of the auditory evoked potential in normal control subjects is affected by a one hour exposure to narrow band noise.",
"(N=2) FIG.",
"7 further shows that the intensity dependence of the N100 component of the auditory evoked potential varies with stimulus frequency.",
"Note that the A/R responses to 1 kHz tones are similar in normal control subjects and tinnitus subjects before habituation training (FIG.",
"5), whereas the AiR responses to 4 kHz tones in normal controls and the responses to the tinnitus frequency are in tinnitus subjects before habituation are very different.",
"(N=3) FIG.",
"8 is a spectrogram of a typical tinnitus habituation stimulus, showing two closely-spaced narrow band noises centered at 2800 and 3225 Hz, and a very narrow band noise centered at 7417 Hz which is almost 40 dB stronger than the first two.",
"FIG.",
"9 displays typical volume settings on the MPEG player used during habituation training.",
"FIG.",
"10 shows: at the Top, wavefonns of N100 responses to 1 kHz tones presented at 30 dB/SL, 48 dB/SL and 54 dB/SL; and at the Bottom, N100 Amplitudes plotted against stimulus intensity to show the intensity dependence function.",
"The slope of the linear regression was used as a measure of cortical responsiveness.",
"FIG.",
"11 is a comparison of the slopes of the intensity dependence between the tinnitus and control groups.",
"A responses to 1 kHz Tones; B responses to 2 kHz Tones; and C responses to tinnitus frequency/4 kHz Tones.",
"FIG.",
"12 shows an example of the N100 intensity dependence slope.",
"FIG.",
"13 is a representation of N100 intensity dependence slopes for the 1 kHz, 2 kHz, and tinnitus/4 kHz tone in tinnitus subjects and controls.",
"Only three midline sites (Fz, Cz, and Pz) are.",
"shown.",
"(p<0.05, Wilcoxon-Mann-Whitney U-Test).",
"DESCRIPTION OF THE PREFERRED EMBODIMENT Introduction Based on the aforementioned modes, Inventors devised a frequency-specific or custom-made habituation stimulus that sounds like the patient's tinnitus for habituation purposes.",
"Such a stimulus activates the same population of neurons affected by tinnitus.",
"It is Inventors' contention that habituation occurs when the tinnitus and the habituating stimulus sound are as much alike as possible.",
"The persistent activation of cortical tissue by tinnitus is assumed to compete for neural substrate with normal stimulus-induced activity in the primary auditory cortex.",
"In order to compensate, the processing of auditory stimuli in the presence of tinnitus-related activity may require an.",
"increase in firing rate of neurons, the use of more neural substrate, or a combination of both.",
"Inventors have hypothesized that these compensatory mechanisms lead to an increased intensity dependence in responses to external auditory events.",
"Since the magnitude of the largest possible response is limited, the presence of tinnitus may lead to a steeper gain function in the responsiveness of the primary auditory cortex and a decrease in the dynamic range of hearing sensitivity.",
"A correlate of this steeper gain function has been reported by Norena and colleagues [Norena, 1999], who observed an increased intensity dependence of the N100 auditory evoked potential in tinnitus subjects.",
"The N100 is known to increase in amplitude and decrease in latency with increasing stimulus intensity [Hillyard, 1978, Scherg, 1990, Mäkelä, 1990].",
"This intensity dependence of the N100 response is associated with the tangentially oriented dipole of the N100 component that is recorded primarily along midline sites.",
"According to studies of dipole source analysis [Verkindt, 1995, Scherg, 1991], this tangentially oriented dipole reflects mainly activity of the primary auditory cortex.",
"In contrast, the radially oriented dipole reflects activity of the secondary auditory cortex in the more lateral parts of the temporal lobe [Hegerl].",
"Since it is assumed that tinnitus-related activity involves the primary auditory cortex, the dependent measure for this study was the midline N100 component.",
"This component appears to be a good index for tinnitus because it reflects stimulus properties as well as attention and the psychological state of the subjects, both of which are presumed to contribute to tinnitus [Jastreboff, 1996].",
"Since tinnitus perception is subjective by its very nature, there have been no objective measurements that could be related to it, until very recently [Jastreboff 1994].",
"A diagnosis of tinnitus has had to rely on a variety of questionnaires [Kuk 1990, Wilson 1991].",
"On the other hand, changes in the dynamic response characteristics of the auditory cortex by tinnitus could be used as a basis for an objective diagnostic tool.",
"The N100 component of the auditory evoked potential has been shown to be an indicator of activity in the central auditory system [Pritchard, 1981a]; [Norena et al, 1999]; [Pineda et al, 1997; Pineda et al, 1998].",
"Because event-related potentials like the N100 are by definition evoked by physical stimuli, whereas tinnitus is not, event-related potentials can be used as more objective measures of tinnitus induced changes in the processing of tones rather than the tinnitus itself [Norena et al, 1999].",
"N100 shows reliable intensity dependence in both humans and non-human primates [Picton et al, 1976]; [Pineda et al, 1991].",
"That is, as sounds get louder, the magnitude of the response increases for many individuals, while it decreases for others.",
"This has been called an augmenting/reducing (AR) response and is assumed to index the “tuning” properties of a cortical gating mechanism that regulates sensory input [Buchsbaum and Silverman, 1968]; [Lukas and Siegel, 1977]; [Pritchard et al, 1985].",
"Inventors' research program had two main specific aims: 1.devise an objective method for the detection of tinnitus, and 2.assess the efficacy of customized acoustic habituation therapy.",
"Tinnitus and Lateral Inhibition EXAMPLE I Tinnitus Causes Specific Changes to the Augmenting/Reducing Response As Inventors have discovered in their own laboratory and has been shown elsewhere, tinnitus increases the slope of the AR response of the N100 component of the auditory evoked potential [Norena et al, 1999].",
"Furthermore, it has also been shown by means of magnetic source imaging that tinnitus induces tonotopically organized activity in the auditory cortex [Mühlnickel et al, 1998].",
"It was therefore hypothesized that the AR response at the specific frequencies at which tinnitus is perceived will have a steeper slope.",
"Tinnitus patients match the pitch of their tinnitus to the frequency of a pure tone.",
"The auditory evoked potential to a variety of tone pitches, including the tinnitus pitch, is 20 then be recorded from patients and normal controls.",
"The magnitude of N100 responses allows Inventors to calculate the slope of the AR response for tinnitus frequency tones, as well as for frequencies not related to tinnitus perception.",
"In normal controls, the tinnitus match is be substituted with a 4 kHz tone, since audiograms show a precise hearing threshold for this frequency and it is also near the 4.2 kHz average of tinnitus matching frequencies determined in Inventors' pilot studies.",
"Inventors propose that the observed increase in the slope of the AR in tinnitus is due to tinnitus-related activity present in the auditory cortex.",
"It is their contention that this activity interferes with stimulus driven activity in such a way that more cortical tissue has to be activated and that firing rates of individual cortical neurons have to be higher in order to allow for processing of peripheral stimuli.",
"This increased activation of the auditory cortex manifests itselfas an increased slope of the AR.",
"Plastic changes mediated by a positive feedback loop involving structures in the limbic system and mechanisms of lateral inhibition in the auditory pathway may lead to the maintenance of the tinnitus experience.",
"This line of argumentation links the thalamic model of Jeanmonod [Jeanmonod et al, 1996], which forms the basis for the proposal, and the changes in inventors' dependent variable.",
"The first step towards a characterization of the augmenting/reducing response was to develop a method that allowed Inventors to reliably record the N100 component and separate it from background noise.",
"The size of the N100 component is largely determined by the loudness of the stimulus.",
"Since Inventors anticipated working with subjects that have some degree of hearing loss, they chose to use stimulus volume settings that correspond to the subjects' individual hearing thresholds rather than use the same predetermined volume settings for all subjects.",
"This choice of volume settings should ensure that the perceived loudness of the stimuli is as similar as possible across subjects.",
"If the stimulus is presented at 30 dB or more above the subject's hearing threshold at the stimulus frequency, the signal-to-noise ratio is sufficient to reliably detect the N100 component and to demonstrate it's dependence on stimulus intensity (FIG.",
"3).",
"Another factor that affects the ability to detect the N100 component is the placement of the reference electrodes.",
"We, therefore, recorded the auditory evoked potentials comparing a number of different references, including a single mastoid, linked mastoid and the average reference method [Katznelson, 1981].",
"The results showed that the N100 component and the entire N100/P200 complex was seen most clearly using the linked mastoids as a reference.",
"The recordings made with the linked mastoids reference is also the only one in which the wave form of the N100/P200 complex closely resembles the waveforms found in previous publications, e.g.",
"[Picton et al, 1976] (FIG.",
"4).",
"Because the N100/P200 complex was present at various electrodes, re-referencing the single mastoid recordings to the average potential of all electrodes partially cancelled the N100/P200 signal, worsening the signal-to-noise ratio.",
"In another study, Inventors asked tinnitus subjects to match the pitch of their tinnitus to the frequency of a sine tone and recorded their auditory event-related potentials to tones of this frequency at 30 dB, 36 dB, 42 dB, 48 dB, and 54 dB above their individual hearing thresholds.",
"The N100 amplitude in response to these tinnitus frequency tones showed a much larger intensity dependence than to 1 kHz tones.",
"Likewise, N100 latencies increased with increasing stimulus volume (FIG.",
"5).",
"The responses to 2 kHz and 4 kHz (chosen to substitute for the tinnitus frequency) tones were recorded from normal listeners in two separate control experiments (FIG.",
"6 and FIG.",
"7).",
"In the control subjects, the responses to 1 kHz tones were much more strongly intensity dependent than responses to 4 kHz tones, while the N100 latency to the 4 kHz tones decreased with increasing stimulus volume.",
"Taken together these findings strongly suggest that tinnitus induces specific changes to the augmenting/reducing response.",
"Materials and Methods Subjects Eight subjects suffering from tinnitus (mean age: 46.8 yr.; S.D.=±11.7) and twelve control subjects (mean age: 39.1 yr.; S.D.=±10.7) were studied.",
"The hearing thresholds of all subjects were determined; using routine pure tone audiometry.",
"Patients suffering from tinnitus were referred from the Head and Neck Surgery Clinic at the UCSD Medical Center, while control subjects were recruited from the population of the UCSD campus.",
"An effort was made to match the age of the control subjects to the age of the tinnitus subjects.",
"Informed consent was obtained from all subjects.",
"The Institutional Review Board of the University of Califomia, San Diego approved the experimental procedures.",
"Tinnitus Matching Procedure A subject's tinnitus perception was characterized prior to every recording session by matching the pitch of their tinnitus to the frequency of a sine tone.",
"In our experience, this matching procedure has proven vulnerable to octave confusions, i.e.",
"some subjects matched their tinnitus pitch consistently at two different frequencies, depending on whether the matching procedure was begun at a frequency higher or lower than their tinnitus pitch.",
"These frequencies generally were one or two octaves apart.",
"If this was the case, we alternately presented both frequencies to the subject and asked which one was a better match.",
"EEG Experiments EEG was collected using standard methods.",
"Data were recorded from 15 electrode sites mounted on an elastic cap and located over the following scalp sites: F3, Fz, F4, C3, Cz, C4, P3, Pz, P4, T3, T4, T5, T6, O1, and O2 (according to the modified International 10-20 System).",
"Eye movement artifact, particularly blinks, was recorded from vertical and/or horizontal EOG electrodes.",
"In order to maintain compatibility with previous studie, all electrode sites were referred to linked mastoids.",
"Within each series, 80 stimuli of different intensities, for a total of 400 stimuli, were presented.",
"in random order and at intervals varying randomly between 1 and 3 sec.",
"The EEG was amplified by a factor of 10,000 and bandpass filtered between 0.01 to 100 Hz using 3 dB down filter skirts.",
"Analog signals were recorded and digitized at a sampling rate of 250 Hz.",
"The auditory stimuli were pure tones generated by a function generator.",
"A specially designed programmable logarithmic amplifier that was controlled in real time by a stimulus presentation and data collection program set the intensity of each stimulus.",
"Tone pips of 200 ms duration were presented at five different intensities (30 dB SL, 36 dB SL, 42 dB SL, 48 dB SL and 54 dB SL) through insert earphones (Eartone 3A transducers with Earlink foam eartips).",
"Inventors recorded auditory event-related potentials (ERP) in response to tones at three frequencies.",
"For the tinnitus subjects these frequencies were 1 kHz, 2 kHz and their tinnitus-match frequency.",
"In non-tinnitus subjects 4 kHz substituted for the tinnitus frequency.",
"Data Analysis The EEG data were analyzed using a two-stage approach.",
"In the first stage, traditional artifact rejection was employed to remove trials with amplifier blocking and those that contained eye movement artifacts.",
"The artifact-free EEG epochs for each intensity and condition were then averaged and the amplitudes of the N100 components measured.",
"Based on these data, intensity-amplitude functions were computed for all 15 electrode sites.",
"These were used to characterize an individual's responsiveness to specific tonal frequencies.",
"The peak amplitudes of the N100 component were measured and plotted against the stimulus intensity.",
"A linear regression was calculated, and its slope was used to characterize the intensity dependence of the N100 component recorded from the midline electrode sites Fz, Cz and Pz.",
"For each stimulus frequency, these intensity-amplitude functions were compared between the tinnitus and control groups using a 2-tailed Wilcoxon-Mann Whitney U-test (p<0.05).",
"Since three tests were performed for each stimulus condition, a correction for multiple comparisons based on the binomial distribution [Bortz, 1990] was performed by calculating α′: α ′ = ∑ i = n k ( k i ) α i · ( 1 - α ) k - i where k was the number of tests performed, n the number of tests that showed a significant result and a the significance level of the individual tests.",
"Differences in the intensity-amplitude functions were considered significant when α′ was less than the desired significance level, i.e.",
"α′<0.05.Results The results of the audiometric testing are shown in Table 1.An example of the N100 waveforms and its intensity-amplitude function is shown in FIG.",
"1.Comparison of the slopes of the intensity-amplitude functions between the tinnitus and control groups showed that the N100 responses from tinnitus patients to tones at their tinnitus frequency were slightly more intensity dependent (i.e.,.",
"steeper slopes) than those of non-tinnitus controls to 4 kHz tones (FIG.",
"2C).",
"In contrast, responses from the tinnitus group were significantly (p<0.05) less intensity dependent to 2 kHz tones than responses from the non-tinnitus control subjects (FIG.",
"2B).",
"The intensity dependence of responses to 1 kHz tones is.nearly identical in tinnitus and control subjects (FIG.",
"2A).",
"Taken together these findings strongly suggest that tinnitus induces specific changes to N100 intensity dependence.",
"Discussion The present results support the hypothesis that the presence of tinnitus related activity changes the intensity dependence of the N100 in a frequency TABLE 1 Hear- ing Right Left loss 1 kHz 2 kHz 4 kHz 1 kHz 2 kHz 4 kHz Tinnitus Wnl 7 6 4 8 7 3 Mild 1 1 1 0 1 5 Mod 0 1 3 0 0 0 Control Wnl 12 12 10 12 12 11 Mild 0 0 1 0 0 1 Mod 0 0 1 0 0 0 Abbreviations: Wnl: Within normal limits; Mod: Moderate specific manner.",
"The experimental data show statistically significant reductions in the intensity dependence of the N100 in response to 2 kHz tones and a non- significant increase in the intensity dependence of responses to the tinnitus frequency tones (4 kHz tones).",
"It is Inventors' contention that tinnitus related activity produces an increase in firing rate of neurons or activation of more neural substrate.",
"This, we believe, is reflected in the enhanced intensity dependence to.",
"tones at that frequency.",
"Furthermore, enhanced activation of this isofrequency region causes inhibition of neighboring regions via lateral inhibitory mechanisms.",
"This is reflected in the reduced intensity dependence to neighboring tones.",
"Inventors' working model of tinnitus is drawn from a recently proposed neurophysiological model of the disorder [Jeanmonod, 1996] in which tinnitus arises as a consequence of thalamocortical dysrhythmias.",
"More precisely, auditory nuclei in the thalamus interact to establish a reverberating loop in which neuronal activity originating from this reverberating loop gets transmitted to the auditory cortex, where it gives rise to the perception of tinnitus.",
"Such reverberating loops are established through disinhibition of cells in the thalamus, which occurs when thalamic relay cells are hyperpolarized by a lack of normal depolarizing sensory input.",
"The action potentials generated by this hyperpolarizing mechanism, or low threshold spikes (LTS), usually occur in rhythmic bursts.",
"A computational model of tinnitus recently proposed by Langner et al.",
"[Langner, 1999] accounts for how a decreased auditory input resulting from a peripheral hearing deficit can give rise to a specific tinnitus pitch.",
"According to this model, the detection of tinnitus-related activity is facilitated by mechanisms of lateral inhibition in the central auditory system.",
"These act to confine the neural activity causing the phantom perception to regions representing distinct frequencies and increase the contrast between the tinnitus-related activity and the spontaneous activity in adjacent regions.",
"Another indication that tinnitus related activity is confined to certain isofrequency regions of the auditory cortex comes from the fact that most tinnitus perceptions have distinct pitch.",
"Furthermore, this pitch is often related to the underlying pathology.",
"For example, noise-induced tinnitus tends to have a pitch near 4 kHz [Mitchell, 1984].",
"In cases where the tinnitus is perceived as tonal, tinnitus-related activity in the auditory cortex can be assumed to be limited to isofrequency regions that correspond to the tinnitus pitch.",
"Consequently, the changes in the intensity dependence of the midline auditory N100 response can be expected to be frequency specific.",
"Based on these models, a likely explanation for Inventors' findings is that tinnitus-related activity in the 4 kHz isofrequency region gives rise to lateral inhibition and thereby inhibits responses from the adjoining 2 kHz isofrequency region of the primary auditory cortex.",
"This produces decreased intensity dependence of the auditory evoked potential in response to 2 kHz tones.",
"This lateral inhibition effect must be limited in range such that responses to tones that are sufficiently different from the tinnitus frequency are not affected.",
"Indeed, the intensity dependence of responses to 1 kHz tones is nearly the same in tinnitus and control subjects.",
"This last finding contrasts with the increased intensity.",
"dependence of the N1/P2 component reported by Norena et al.",
"However, since the N1/P2 complex is thought to be generated by equivalent dipoles representing the primary and secondary auditory cortices, whereas the N100 observed in this study is thought to be generated by equivalent dipoles representing only the primary auditory cortex, the contribution of the secondary auditory cortex may contribute to the higher intensity dependence observed by Norena et al.",
"Another factor that can be expected to influence the intensity dependence of the N100 is hearing loss.",
"As shown in Table 1, about half of the tinnitus subjects had some hearing loss at their tinnitus frequency.",
"This hearing loss could lead to recruitment, i.e.",
"increased loudness growth in response to higher intensity stimuli.",
"Recruitment may be an alternative explanation for the increased intensity dependence of the N100 response at the tinnitus frequency.",
"However, hearing for the tinnitus subjects was less impaired at 2 kHz, i.e.",
"an octave lower than the tinnitus, so that hearing loss cannot account for the changed dynamics in N100 responses to these tones.",
"Furthermore, the effect of hearing loss, as described above would be to cause an increase of the N100 intensity dependence rather than the observed decrease.",
"Conclusions The present results suggest that tinnitus related activity in the primary auditory cortex changes the characteristics of the N100 component of the auditory evoked potential in a frequency specific manner.",
"In tinnitus subjects responses to tinnitus frequency tones are slightly more dependent on stimulus intensity than in controls, while responses to 2 kHz tones, i.e.",
"approximately one octave below the tinnitus frequency are significantly less dependent on stimulus intensity.",
"The lack of intensity dependence in responses to 2 kHz tones is most likely caused by lateral inhibition in the auditory cortex arising from the tinnitus related activity.",
"The observed changes in the dynamic properties of the N100 response is a way of demonstrating tinnitus related activity in the central neural system and may provide the basis for an objective.",
"tinnitus diagnostic tool.",
"EXAMPLE II Habituation Therapy Procedures Subject evaluation.",
"Two groups of subjects are selected and evaluated: normal controls and subjects with tinnitus.",
"They are evaluated initially to determine hearing thresholds, as well as tinnitus pitch and intensity levels.",
"The audiologist technician conduct s these procedures.",
"Subjects are initially screened at the Otolaryngology Clinic to ensure they have no serious physical or mental disorders, have no other auditory deficits, and do not take any medication or other substances, which may affect EEG recording.",
"Care is taken to ensure a balance between male and female subjects.",
"Group sizes according to study design.",
"Pure Tone Audiometry Procedure Prior to ERP experiments or habituation therapy, all subjects will have their audiogram taken using standard clinical procedures.",
"For tinnitus subjects the audiogram are repeated every 3 months during habituation therapy and after completing the habituation therapy.",
"Subjective Tinnitus Pitch/Intensity Determination A subject's perception of his or her tinnitus is characterized prior to every recording session by matching the pitch of their tinnitus to the frequency of a sine tone and the loudness of their tinnitus to the volume of a sine tone that matches the pitch of the tinnitus.",
"The following constitute typical instructions given to the subjects: “You are going to match a tone presented through the earphone or the speaker to the tone of your tinnitus for pitch/loudness.",
"Every time that this tone is presented, I want you to compare it to your tinnitus tone and report that the presented tone is either: “higher in pitch/loudness than your own tinnitus tone, equal to your own tinnitus tone, or lower in pitch/loudness to your own tinnitus tone” In Inventors' experience matching the pitch of the tinnitus to the frequency of a sine tone has proven to be vulnerable to octave confusions, i.e.",
"the tinnitus subjects match their tinnitus pitch consistently at two different frequencies, depending'on whether the matching procedure was begun at a frequency higher or lower than their tinnitus pitch.",
"These frequencies are usually roughly one or roughly two octaves apart.",
"If this is the case, Inventors present both tones to the subject and ask which one is a better match.",
"Tinnitus Masking Level Determination The tinnitus masking level is the level of the external stimulus tone that masks the tinnitus, according to the subject.",
"This should be a level just above the reported tinnitus intensity level.",
"Stimulus tones are initially presented at an intensity level below the determined intensity level of the tinnitus (this could even be at HL-5dB).",
"Subjects are given instructions similar to those described previously.",
"For example, “you are going to tell me when you can no longer hear your own tinnitus.",
"Every time that a stimulus tone is presented at a certain loudness, I want you to make a decision as to what you hear and report: “I hear my tinnitus only”.",
"“I hear both the stimulus tone and my tinnitus”.",
"“I hear the stimulus tone only”.",
"The masking level is the lowest intensity at which the subject reports that they only hear the stimulus tone.",
"The procedure is repeated once or twice for reliability.",
"EEG Procedures In order to detect tinnitus-related changes in the processing of auditory stimuli in the central auditory system, the auditory evoked potential is recorded.",
"This involves the recording and analysis of EEG and ERPs.",
"EEG is collected using standard methods.",
"Data are recorded from 15 electrode sites mounted on an elastic cap and located over a variety of scalp sites (F3, Fz, F4, C3, Cz, C4, P3, Pz, P4, T3, T4, T5, T6, O1, and O2 according to the modified International 10-20 System).",
"Eye movement artifacts, particularly blinks, are recorded from vertical and/or horizontal EOG electrodes.",
"In order to maintain backward compatibility with previous studies, e.g.",
"[Norena et al, 1999], and because Inventors' preliminary experiments show that the N100/P200 complex is best seen with referencing to linked mastoids, all electrode sites are referred to linked mastoids.",
"Within each series, 80 stimuli of different intensities, for a total of 400 stimuli, are presented in random order and at intervals varying randomly between 1 and 3 sec.",
"Inventors are aware that a better signal to noise ratio would be achieved with more stimulus presentations.",
"However, for the purposes of this study, their preliminary experiments show the signal-to-noise ratio to be sufficient (see FIG.",
"4).",
"The EEG is amplified by 10K and bandpass filtered between 0.01 to 100 Hz at 3 dB down.",
"Analog signals are recorded and digitized at a sampling rate of 250 Hz.",
"For stimulus presentation and data acquisition and analysis, the ADAPT scientific software ((c) A.Vankov, 1997) or Neuroscan software is used.",
"Both of these software packages permit the delivery of complex stimulus patterns and simultaneous data collection and analysis.",
"Auditory Stimulation The auditory stimulations are sine waves generated by a function generator (Goldstar FG2002C).",
"The intensity and the duration of each stimulus are set by a specially designed programmable.logarithmic amplifier that is controlled in real time by a stimulus presentation and data collection program running in ADAPT.",
"Auditory stimuli of five different intensities are presented through insert earphones (Eartone 3A transducers with Earl link foam eartips).",
"In one stimulation series, the tone frequency is the subjectively assessed frequency of the subject's tinnitus.",
"Three more stimulation series with stimulus frequencies at 1 kHz, 2 kHz and 8 kHz are carried out.",
"In all series, tones are of 200 ms duration and varying in intensity (+30 dB, +36 dB, +42 dB, +48 dB, and +54 dB above the subject's hearing threshold at that frequency).",
"For normal controls the auditory stimulation consists of series using I kHz, 2 kHz, 4 kHz, and 8 kHz, i.e.",
"the stimulation series using the tones of the tinnitus frequency is substituted by one using 4 kHz tones.",
"Experimental Paradigm On the day of testing, the sequence of the procedures is as follows: 1.In tinnitus subjects: Determine the “matching frequency” (pitch) of the subject's tinnitus.",
"Subjects are presented with a continuous, audible tone varying in pitch.",
"They are asked to indicate the frequency that most closely matches the frequency of their tinnitus.",
"Several runs are used to determine the mean of the reported frequencies.",
"Pitch matching is done in audiometrics.",
"2.In tinnitus subjects: Determine the hearing threshold for the tinnitus frequency.",
"Subjects are presented with a continuous tone that gradually increases in volume.",
"They are asked to indicate when they begin to hear the tone.",
"The intensity at which the subjects begin to hear the tone is considered the hearing threshold (0 db).",
"For EEG recording, tones at the tinnitus frequency are presented at volumes 30 dB, 36, dB, 42 dB, 48 dB, and 54 dB above this hearing threshold.",
"Several runs are used to determine the subject's hearing threshold.",
"3.In tinnitus subjects: Determine the “matching intensity” of the subject's tinnitus.",
"Subjects are presented with a continuous tone that increases or decreases in volume.",
"They are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus.",
"Several runs are used to determine the mean of the reported intensities.",
"Masking level is determined in audiometrics.",
"4.Determine the hearing threshold for the “off” frequencies.",
"The same procedure as in (2) is used, but with 1 kHz, 2 kHz, and 8 kHz tones (in control subjects: 1 kHz, 2 kHz, 4 kHz, and 8 kHz tones).",
"For EEG recording, these tones are presented at volumes 30 dB, 36, dB, 42 dB, 48 dB, and 54 dB above this hearing threshold.",
"Following the determination of the tinnitus pitch and intensity and subject's hearing threshold, one series of tonal stimulation and EEG collection takes place at each stimulus frequency.",
"The series are presented in random order determined with a dice.",
"EEG Data Analysis In one embodiment, the EEG data are analyzed using a two-stage approach.",
"In the first stage, they are artifact-rejected.",
"Initially, traditional artifact rejection is employed to remove trials with amplifier blocking and those that contain eye blinks.",
"In the second stage, the remaining single trials are concatenated and submitted to an ICA decomposition.",
"Components that account for eye, muscle, or movement artifacts are selected and those rows in the activation matrix are set to zero.",
"The data are then reconstructed without the artifacts (for a detailed description of the ICA algorithm and how it is applied to correct for EEG artifact, see [Makeig et al, 1997]; [Makeig et al, 1999]).",
"The artifact-free EEG epochs for each intensity and condition are averaged and the amplitudes of the N100 components measured.",
"Based on these data, intensity-amplitude functions are computed for all 15-electrode sites.",
"These are used to characterize an individual's responsiveness to specific tonal frequencies and as the measure of changes in that responsiveness.",
"EXAMPLE II EEG Index of Tinnitus Inventors determined whether N100 intensity dependence (i.e., the changes in amplitude in this brain signal as a finction of stimulus intensity) differs in tinnitus sufferers compared to non-tinnitus control subjects.",
"The experiment thus far has involved 24 tinnitus subjects and 14 control subjects.",
"Tinnitus subjects were initially asked to match the pitch of their internal tinnitus to the frequency of a sine tone.",
"Auditory ERPs in response to tones of up to four frequencies were recorded.",
"For the non-tinnitus control subjects, these frequencies were 1 kHz, 2 kHz, 4 kHz and 8 kHz.",
"In tinnitus subjects, their own tinnitus frequency was substituted for one of the frequencies used with the control subjects.",
"For all subjects, tone pips were presented at intensities of 30 dB SL, 36 dB SL, 42 dB SL, 48 dB SL and 54 dB SL.",
"The peak amplitudes of the N100 component were then measured and plotted as a function of stimulus intensity.",
"A linear regression was calculated, and its slope used to characterize the intensity dependence of the N100 component (see FIG.",
"12).",
"The resulting data from a subset of the tinnitus group, i.e., those subjects whose tinnitus pitch was near 4 kHz, show significantly higher intensity dependence of the N100 compared to controls (see FIG.",
"13).",
"Furthermore, comparison of the N100 intensity dependence of the tinnitus and control subjects showed that the N100 responses from tinnitus patients to 2 kHz tones was significantly less intensity dependent (i.e., smaller slopes) than those of non tinnitus controls (Wilcoxon-Mann-Whitney U-test, p<0.05).",
"In contrast, responses from the tinnitus group were slightly more intensity dependent to their tinnitus frequency tones (which had a mean of 4.2 kHz) than responses to the 4 kHz from the non-tinnitus control subjects.",
"Audiograms were taken from all of the tinnitus subjects and 11 of the control subjects.",
"Two of the tinnitus subjects had mild and one had moderate hearing loss at their tinnitus frequency.",
"The hearing loss observed at the tinnitus 15 frequency makes it possible that the stronger intensity dependence observed in the responses of tinnitus patients to tinnitus frequency tones may have been caused by recruitment (abnormal loudness growth) rather than tinnitus.",
"However, the weaker intensity dependence observed in responses to 2 kHz tones is opposite of the effect of recruitment, and thus not attributed to it.",
"Inventors attribute this effect on intensity dependence to the lateral inhibition caused by the tinnitus-related activity in the auditory cortex.",
"EXAMPLE III Customized Habituation Therapy with Habituation Stimuli Although their judgements are necessarily subjective, tinnitus subjects have evinced excellent consistency when it comes to matching habituation sounds with their tinnitus.",
"Before beginning the habituation therapy, Inventors required subjects to confirm that the habituation sound matched their tinnitus at two or more sessions separated by at least a week.",
"While there is some evidence that adjustments in the habituation stimulus may be desirable after habituation has progressed a few weeks, it seems reasonably clear, that this is due to changes in the characteristics of the tinnitus itself rather than a mismatch in the habituation sounds.",
"The frequency content of a typical habituation stimulus is shown in FIG.",
"8.Tinnitus retraining therapy requires habituation to constant noise exposure.",
"In order for habituation to occur, the habituation stimulus has to be audible, but must not be so loud as to mask the tinnitus [Jastreboff et al, 1996].",
"Therefore, the volume setting that a tinnitus subject uses on their noise generator gives an estimate of the loudness of the tinnitus.",
"The volume settings that Inventors' tinnitus subjects used during this experiment decreased over time, in one case so much that the subject exhausted the available volume settings and the stimulus had to be resynthesized at a lower intensity (FIG.",
"9).",
"Since the experimental recording procedure required a determination of the hearing threshold every time a recording of an auditory evoked potential was made, Inventors have records of the changes to the hearing thresholds during habituation therapy.",
"Hence, in a typical recording session, subjects had their hearing threshold determined and auditory evoked potentials recorded at their tinnitus frequency and at 1 kHz.",
"Subjects then listened to their customized habituation stimulus for an hour and the determination of the hearing thresholds and the recording of the auditory evoked potential was repeated.",
"Within a session, the hearing threshold at the tinnitus frequency was lowered by as much as 15 dB.",
"Over several weeks of habituation training, hearing thresholds at the tinnitus frequency were observed to decrease by as much as 25 dB (FIG.",
"10).",
"Habituation training caused changes in the intensity dependence of the N100 amplitude as well as N100 latencies oftinnitus subjects.",
"Before exposure to customized habituation stimulus the amplitude of the N100 component of the response to a tinnitus frequency tone was much more intensity dependent than the N100 component in response to a 1 kHz tone.",
"Likewise, the N100 latencies increased with increasing stimulus volume.",
"After subjects listened to the habituating stimulus for one hour, the intensity dependence of the response to the 1 kHz tone increased, and the latency of the N100 component of the response to the tinnitus frequency tone decreased with increasing stimulus volume (FIG.",
"5).",
"Normal hearing control subjects were exposed to a stimulus similar to the habituating one for the tinnitus patients.",
"The stimulus used in these control experiments was a narrow noise band with a center frequency of 4 kHz.",
"Neither the intensity dependence of N100 amplitude or latency were changed by the exposure to the control stimulus (FIG.",
"6).",
"Taken together, these findings suggest that the exposure to the habituating stimulus has an effect on tinnitus related activity in the auditory cortex.",
"of the tinnitus subjects.",
"A single one-hour exposure, however, is not enough to make the pattern of intensity dependence similar to the one observed in normal controls.",
"Habituation training in conjunction with directive counseling has been shown to be an effective treatment for tinnitus [Jastreboff et al, 1996b].",
"Typically, white noise is used as the habituating stimulus in tinnitus retraining therapy (TRT).",
"White noise evokes stimulus-induced neural activity in all of the parallel frequency-specific channels in the auditory system and is therefore likely to cause activity in those populations of neurons that also show tinnitus-induced activity.",
"For habituation training, it may be sufficient to excite a much smaller population of neurons, namely those that are contributing to the tinnitus.",
"A physical stimulus targeting these neurons would induce a similar perception as the tinnitus, i.e.",
"sound like the tinnitus.",
"Inventors propose to synthesize stimuli to match the patient's tinnitus and use this sound for habituation training.",
"By recording the patient's auditory evoked potentials, Inventors can quantitatively describe the changes in the electrophysiological markers induced by the habituation.",
"Further, Inventors will record the changes in audiometric tests as well as in subjective tinnitus measures.",
"Habituation therapy as proposed by Jastreboff [Jastreboff et al, 1996b] has been shown to achieve an 84% success rate in patients in terms of decreased annoyance induced by tinnitus and showing clear habituation of its perception.",
"Jastreboff claims that habituation therapy using white noise as a habituation stimulus causes the signal-to-noise ratio between the tinnitus related activity and spontaneous activity in the central auditory system to decrease.",
"At the same time, the central auditory system undergoes a habituation to the permanent presence of an auditory stimulus.",
"This habituation should lead to a decrease in tinnitus-related activity and consequently to decreased tinnitus perception and annoyance.",
"However, descriptions of tinnitus perception and Inventors' preliminary results indicate that the tinnitus perception and the perception caused by white noise are very different from each other.",
"When white noise is used for habituation therapy, two distinct continuous auditory perceptions are present.",
"One is the behaviorally irrelevant, stimulus-driven perception of the white noise, the other is the tinnitus, a highly annoying phantom perception.",
"In these conditions, habituation is likely to occur, but it is habituation to white noise rather than to the tinnitus perception.",
"Inventors' approach is designed to produce a habituation to the tinnitus sound, by making the perceptions of the tinnitus sound and the habituating stimulus as similar as they can possibly be.",
"This is done by using a customized sound that mimics the tinnitus perception as a habituation stimulus.",
"Thus, the perceptions experienced by Inventors' subjects are very similar, allowing the habituation to the customized habituation stimulus to extend to the tinnitus.",
"In this part of the study Inventors test whether this approach is an effective treatment of tinnitus, i.e.",
"causes tinnitus related annoyance to disappear, and whether the electrophysiological correlates of tinnitus described in Example I are changed as a consequence of this treatment.",
"Subjective Tinnitus Pitch/Intensity Determination The subjective experiences of tinnitus are characterized by Dr. Moore using a successive approximation technique.",
"Individual subjects are asked to verbally describe to a sound synthesis expert the “sounds” they experience using whatever vocabulary they possess for describing sounds.",
"Musical training and experience on the part of the subject are particularly valuable in this process, since they provide a useful language in which sound characteristics can be verbally communicated.",
"However, while musical training or experience is helpful, they are not necessary in order to achieve good and consistent tinnitus match.",
"In response to the subject's statements, sounds are synthesized using high-precision, general-purpose sound synthesis software.",
"At present Dr. Moore synthesizes the tinnitus sound according to the description provided by the subjects using the program “pcmusic” (see for example [Moore, 1990]).",
"The resulting sound is then played back to the subject, who responds with suggested adjustments, such as altering the pitch, number of components, balance or quality of the synthetic sound.",
"In response to these suggestions, the sound description file is modified, and the process is repeated until the subject reports that the synthesized sound matches well his or her subjective tinnitus experience.",
"The digitized sound is then downloaded into a small portable digital sound playback device (MPEG-player) for use during habituation.",
"The synthesized sound is completely documented by the description file together with the pcmusic program, as well as any number of post facto techniques, such as Fourier analysis.",
"The time needed for this successive approximation procedure can vary, depending on the subject's ability to describe their own subjective experience of tinnitus, and their ability to characterize differences between the tinnitus they experience and the sounds being synthesized by the computer.",
"Practical experience indicates that this can be done in about two or three 2-hour sessions devoted to accurately characterizing the subject's experience.",
"Tinnitus Masking Level Determination Determination of the tinnitus masking level is the same as in Example I. EEG Procedures EEG Procedures are the same as in Example I. Auditory Stimulation The auditory stimulation is similar to that used in Example I.",
"Since the 1 kHz and tinnitus frequency tone are presented before and after habituation (see ‘Experimental Paradigm’ below), the 2 kHz and 8 kHz tones are not presented.",
"Experimental Paradigm On the day of testing, the sequence of the procedures is as follows: Determine the “matchingfrequency” (pitch) of the subject's tinnitus.",
"Subjects are presented with a continuous, audible tone varying in pitch They are asked to indicate the frequency that most closely matches the frequency of their tinnitus.",
"Several runs are used to determine the mean of the reported frequencies.",
"Determine the Hearing Thresholdfor the Tinnitus Frequency Subjects are presented with a continuous tone that will gradually increase in volume.",
"They are asked to indicate when they begin to hear the tone.",
"The intensity at which the subjects begin to hear the tone is considered the hearing threshold (0 db).",
"For EEG recording, tones at the tinnitus frequency are presented at volumes 30 dB, 36, dB, 42 dB, 48 dB, and 54 dB above this hearing threshold.",
"Several runs are used.",
"to determine the subject's hearing threshold Determine the “matching intensity” of the Subject's Tinnitus Subjects are presented with a continuous tone that will increase or decrease in volume.",
"They are asked to indicate the moment when they perceive the tone as being of the same loudness as their tinnitus.",
"Several runs are used to determine the mean of the reported intensities.",
"Determine the hearing threshold for the “off” frequency.",
"The same.",
"procedure as above is used,.",
"but with a 1 kHi tone.",
"For EEG recording,.",
"1 kHz tones are presented at volumes 30 dB, 36, dB, 42 dB, 48 dB, and 54 dB above this hearing threshold.",
"Following the determination of the tinnitus pitch and intensity and subject's hearing threshold two series of tonal stimulation and EEG collection will take place.",
"The first series is at the tinnitus frequency, while the second one is at the “off” tonal frequency (1 kHz).",
"At the end of the tonal series, subjects are exposed to 1-hr of habituation with customized habituation sound.",
"The subjective “masking” threshold of the tinnitus is determined.",
"They will then listen to the sound at 6 dB below the masking threshold for approximately 1 hour.",
"The hearing.",
"threshold at the tinnitus frequency and at the “off” frequency is measured again at the end of the one-hour habituation.",
"At the end of the habituation period and after a 2-minute break, two more series of tonal stimulation and EEG collection, identical to the ones described above, are conducted.",
"Subjects are given the MP3 player with the habituation stimulus and asked to listen to it for as long as it is comfortable each day They are asked to return to the lab after one, four, and 12 weeks of habituation therapy for the same EEG evaluation.",
"Data Analysis The EEG data are analyzed using the procedures described in Example I.",
"When the slopes of the augmenting/reducing responses are calculated, Inventors look for changes within and between sessions.",
"These.",
"changes are correlated to the.",
"behavioral data obtained during the experimental sessions.",
"Habituation therapy using white noise leads to an improvement in 83% of cases [Jastreboffet al, 1996b].",
"Therefore, an important question to be addressed in this aim is not only whether successful tinnitus treatment returns behavioral and EEG indices to normal or whether one type of therapy is faster than another but whether these parameters have predictive value in terms of treatment efficacy.",
"This data analysis will also involve an analysis of all the data collected for Example I in order to develop a model that can make reasonable inferences and serve as a diagnostic tool to monitor habituation therapy.",
"If there is a specific pattern of alteration, clinicians will have a reliable tool to help assess the effectiveness of these acoustic therapies.",
"Subject Populations Approximately 200 normal, healthy, ethnically diverse adults (aged 18-30), both male and female, who are recruited from the general student population at UCSD are studied each year.",
"The population at UCSD is approximately 48% male and 52% female.",
"The breakdown in terms of ethnicity is Caucasian (39%), Asian (30%), Mexican-American (8%), Filipino (5%), others (18%).",
"Subjects are recruited through bulletin board ads, email, announcements in undergraduate courses, and personal references They are paid for their participation in the experiments on an hourly basis or compensated with credit toward undergraduate coursework.",
"They are asked to read and sign a consent form approved by the Institutional Review Board (IRB).",
"Subjects are made aware of the risks, which are minimal since all the hardware has been designed for this particular purpose, the stimuli are of low intensity and painless, and the subjects are not be placed under any stress.",
"Any discomfort due to the skin abrasion and electrode gel may remain for a short while after the experiment is completed EXAMPLE IV Altered Cortical Responses in Tinnitus Patients Following Habituation Therapy Inventors examined short and long term effects of habituation to a customized sound.",
"Habituation to a customized sound may have effects at both a behavioral and an electrophysiological level.",
"Patients descnbe the effect of listening to their customized sound as comforting, saying for example that it helps them fall asleep or make them feel in control of their tinnitus.",
"Patients give this feedback frequently within minutes after first turning on their MP3 players.",
"In order to substantiate these patient testimonials, patients were asked to complete the Tinnitus Handicap Questionnaire before and after the three week habituation period.",
"The questionnaire consists of 27 statements.",
"to which the patient responds by writing down a number between 0 and 100 next to each statement, where 100 means complete agreement and 0 complete disagreement with the statement.",
"The questionnaire is constructed in such a way that high scores indicate more severe tinnitus.",
"than low scores.",
"The questionnaire assesses the overall effect tinnitus has on the patient's life.",
"It contains three subscales that measure the effects tinnitus has on more specific aspects of a patients life, namely their mental health, their ability to hear and their attitude.",
"towards tinnitus.",
"So far three patients have.",
"completed the.",
"three week habituation protocol, giving the following scores: TABLE 2 Overall Mental Health Hearing Attitude before 11.2 17.9 5.0 5.0 after 5.6 5.7 1.7 5.0 before 50.0 67.9 29.2 37.5 after 37.0 53.2 23.3 27.5 before 48.8 68.6 23.3 40.0 after 48.0 64.3 37.5 25.0 Another way of assessing the effectiveness of the Customized Sound therapy is by observing.",
"the EEG correlate of tinnitus.",
"We undertook a series of EEG experiments in which we measured the change of the intensity dependence of the amplitude and latency ofthe auditory N100 response as a consequence ofone hour.",
"of habituation training.",
"We recorded the responses to tinnitus frequency tones and 1 kHz tones from tinnitus patients and control subjects before and after one hour of habituation.",
"Tinnitus patients used their customized sound for habituation, control subjects used narrowband noise with a center frequency of 4 kHz, and 4kHz tones were presented to substitute for the tinnitus frequency.",
"Due to the small number of subjects in this study the results of these experiments do not reach statistical significance.",
"However the provide an indication that the response pattern of tinnitus patients was changed by one hour of habituation (FIG.",
"5), while no such effect was observed in the control subjects (FIG.",
"6).",
"EXAMPLE V Automatic Feedback Habituation System A flow diagram representing the steps involved is seen in FIG.",
"11.From this diagram, the novelty of the invention is clearly apparent.",
"Subjects come to a PC workstation with electronic sound players.",
"Software designed by an electronic sound expert presents a series of tones to the subjects and refines them to match the subjects' tinnitus experience through a series of controllers that the subjects will manipulate themselves.",
"An electronic recording of the sound is then made in a digital music format, typically MP3 that is then stored on the computer.",
"A. copy of the electronic sound file is then transferred to the electronic music player.",
"This device is solid state electronics that stores and plays back music through a set of attached headphones.",
"After the customized sound generation, an EEG signature of a subject's brain activity is generated.",
"The customized sound is used to stimulate the auditory system while the brain activity is recorded.",
"The subject's response to sounds not corresponding to their tinnitus signature is recorded, as well as their brain activity during the absence of sound stimuli.",
"The EEG responses to sound stimuli and to the absence of sound are downloaded into the controlling computer.",
"While the EEG is actively being monitored by the computer, it generates the customized sound.",
"EEG signature for tinnitus is monitored as well as the signature for silence.",
"Periodically, the signatures for tinnitus and silence are tested for and the controlling computer determines if the tinnitus is going down and the silence signal is strengthening.",
"If these changes are not present, the computer slightly alters the sound stimuli and again checks for feedback that the appropriate changes are being seen.",
"The computer continuously monitors for the feedback signatures and drives the sound stimuli appropriately.",
"The method of using brain signals to drive the sound generation for suppression of tinnitus is unique.",
"Inventors assert that this method can likely be generalizable to a wide variety of medical conditions where input signals can be used to suppress brain activity and be maximized by brain signal feedback.",
"For example, brain signatures for motion sensation and absence of motion could be used with visual motion generation to reduce chronic vertigo.",
"Moreover, Parkinson's disease could suppressed by motor control stimuli and motor signal feedback, or depression could be suppressed by visual and auditory stimuli driven with EEG signature feedback.",
"The potential clinical application for this is the rapid treatment of tinnitus.",
"Currently there are sound therapies for tinnitus that use generic sounds.",
"The present therapies are only partially effective and require a long time.",
"With the brain signal feedback system, we are rapidly able to suppress brain activity related to tinnitus and provide relief for this disorder.",
"In accordance with these and other possible variations and adaptations of the present invention, the scope of the invention should be determined in accordance with the following claims, only, and not solely in accordance with that embodiment within which the invention has been taught.",
"REFERENCES Anderson, B. W. and Oatman, L. C. Auditory and Visual Evoked Potentials to Irrelevant Stimuli During Conditioning to a Visual Stimulus.",
"Neurol.Res.",
"1980; 1(3):281-90.Bortz, J., Liehnert, G. A., and Boehnke, K, Verteilungsfreie Methoden in der Biostatistik, Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong, Barcelona, Springer Verlag, (1990).",
"Buchsbaum, M. and Silverman, J.",
"Stimulus Intensity Control and the Cortical Evoked Response.",
"Psychosom.Med.",
"1968;30(1): 12-22.Connally, J. F. The Influence of Stimulus Intensity, Contralateral Masking and Handedness on the Temporal N1 and T Complex Components of the Auditory N1 Wave.",
"Electroenceph.Clin.Neurophysiol 1993; 86:58-68.Duncan-Johnson, C. C. and Donchin, E. On Quantifying Surprise: the Variation of Event-Related Potentials With Subjective Probability.",
"Psychophysiology 1977; 14(5):456-67.Eggermont, J. J.",
"On the Pathophysiology of Tinnitus; a Review and a Peripheral Model.",
"Hearing Research 1990; 48(1-2):111-23.Gonzalez-Lima, F. and Scheich, H. Neural Substrates for Tone-Conditioned Bradycardia Demonstrated With 2- Deoxyglucose.",
"II.",
"Auditory Cortex Plasticity.",
"Behav.Brain Res.",
"1986; 20(3):281-93.Hegerl, U., Juckel, G., and Möller, H. J.",
"Event Related Brain Potentials As Indicators of Neurochemical Dysfunctions in Psychiatric Patients.",
"von Knorring, L. and Perris, C. Biochemistry of the Augmenting-Reducing Response in Visual Evoked Potentials.",
"Neuropsychobiology 1981; 7(1):1-8.Wallhäusser-Franke, E., Braun, S., and Langner, G. Salicylate Alters 2-DG Uptake in the Auditory System: a Model for Tinnitus?",
"Neuroreport 7-8- 1996; 7(10): 1585-8.Wilson, P. H., Henry, J., Bowen, M., and Haralambous, G. Tinnitus Reaction Questionnaire: Psychometric Properties of a Measure of Distress Associated With Tinnitus.",
"Journal of Speech and Hearing Research 1991; 34(1): 197- 201.Wolpaw, J. R. and Penry, J. K. A Temporal Component of the Auditory Evoked Response.",
"Electroencephalogr.Clin.Neurophysiol.",
"1975; 39(6):609-20.Woods, D. L. The Component Structure of the N1 Wave of the Human Auditory Evoked Potential.",
"Electroencephalogr.Clin.Neurophysiol.Suppl 1995; 44:102-9.Zuckermann, M., Mrtaugh, D, and Siegel, J.",
"Sensation Seeking and Cortical Augmenting-Reducing.",
"Psychophysiology 1974; 11:535-42."
]
] | Patent_10276018 |
[
[
"Method for applying a layer containing at least polymeric material",
"In a method for applying a layer containing at least polymer material to a substrate, there is applied to the substrate a film of polymer particles dispersed in a non-reactive liquid.",
"By subjecting these at least polymer material-containing particles to an energy flow, which is locally at least substantially converted into heat, the particles will fuse with each other as a result of such a heat treatment.",
"In particular, the heat treatment is done with the aid of a laser device operative in the UV region, or a laser device operative outside this region, though then in combination with a heat transferring agent in the film."
],
[
"1.A method for applying a layer containing at least polymer material to a substrate, wherein the layer is obtained by applying, to the substrate, a film including at least polymer material-containing particles dispersed in a non-reactive liquid, and subjecting the particles to an energy flow which is locally substantially converted into heat, during which the particles fuse with each other.",
"2.A method according to claim 1, wherein the energy flow is provided with the aid of an Atomic Force Microscope thermal analyzer after evaporating the liquid in the film.",
"3.A method according to claim 1 wherein the energy flow is provided with the aid of a laser device.",
"4.A method according to claim 3 wherein the heat generated by the laser device is utilized first to remove the liquid from the film and then to fuse the polymer material-containing particles with each other.",
"5.A method according to claim 3 wherein, after the liquid has at least substantially disappeared from the film, the polymer material-containing particles fuse with each other with the aid of the laser device.",
"6.A method according to claim 1, wherein the film is formed by polymer material-containing particles dispersed in water.",
"7.A method according to claim 1, wherein the size of the dispersed particles is between about 5 and 30,000 nm.",
"8.A method according to claim 1, wherein polymer material-containing particles of different composition and/or size are present.",
"9.A method according to claim 1, wherein polymer material-containing particles have a multicomponent structure.",
"10.A method according to claim 1, wherein the film contains one or more further non-reactive components, from the group including: colors, pigments, and flowing property improving agents.",
"11.A method according to claim 1, wherein the film contains one or more reactive components, comprising crosslinking agents to be activated in a post-treatment.",
"12.A method according to claim 1, wherein a heat transferring agent is dispersed in the film.",
"13.A method according to claim 1, characterized in that the solids content in the wet film is less than 70 vol.",
"%.",
"14.A method according to claim 3 wherein the laser device is operative in the UV region is used.",
"15.A method according to claim 14, wherein the wavelength at which the laser device is operative is between 190 and 360 nm.",
"16.A method according to claim 14 wherein the laser devise is an excimer laser device.",
"17.A method according to claim 14 wherein the laser devise is an Nd:YAG laser device.",
"18.A method according to claim 14 wherein the laser devise is a solid state diode pumped laser.",
"19.A method according to claim 3 wherein an Nd:YAG laser device is used which is operative in the infrared region, and carbon black has been added to the film as heat transferring agent.",
"20.A method according to claim 14, wherein the laser device transmits a pulsed activation signal.",
"21.A method according to claim 14, wherein the laser device transmits a continuous activation signal.",
"23.A method according to claim 14, wherein the laser device, viewed over the width of the beam, has a “tophat” distribution intensity profile.",
"24.A method for manufacturing an object by building it up layer-by layer, wherein each of the layers to be successively applied onto each other is obtained by the use of the method according to claim 1.25.An apparatus for forming one or more layers containing at least polymer material to a substrate, wherein the apparatus is provided with a laser device and with a processing space having a substrate on which a layer, containing at least a polymer material, can be formed by the method defined in claim 1.26.An apparatus according to claim 25, wherein the laser device is provided with means for setting the intensity profile over the beam cross section.",
"27.An apparatus according to claim 25 wherein a mask is arranged in the optical path.",
"28.A coating obtained by the use of the method according to claim 1.30.A method according to claim 1, characterized in that the size of the dispersed particles is between about 100 and 1,000 nm.",
"31.A method according to claim 1, characterized in that the solids content in the wet film is less than 60 vol.",
"%.",
"32.A method according to claim 12 wherein the heat transferring agent is carbon black.",
"33.An apparatus according to claim 25 wherein a mask is arranged directly above the substrate."
],
[
"The present invention relates to a method for applying a layer containing at least polymer material to a substrate and to a method fork manufacturing an object by successively applying such layers onto each other.",
"It is known to manufacture such objects in a stereolithography process (SL).",
"In such a process, often use is made of UV radiation cation-curable epoxy resins or radical-curable acrylate resins.",
"Curing then takes place through irradiation from a laser of a particular wavelength.",
"By each time applying a next resin layer onto a preceding cured layer and selectively curing each next layer, an object can be obtained.",
"When the object has been completed, a post-treatment follows, often with organic solvents such as tripropylene glycol, monomethyl ether or 2-propanol, and post-curing in an oven.",
"In such a method, during curing and post-curing, shrinkage in the order of magnitude of 2 to 7% occurs.",
"The resultant faults (inaccuracies in form and dimensions) can cumulate strongly, especially when during curing already next layers are being applied.",
"Partly as a result of such shrinkage, the form and dimensions of an object to be manufactured have a limited resolution, in particular at best in the order of 25-50 μm.",
"In addition, resolution-limiting factors that apply are the minimum layer thickness and the homogeneity thereof, and the manner of crosslinking of the UV-crosslinkable stereolithography resins.",
"Further, drawbacks that apply are that acrylates during curing are sensitive to oxygen, while epoxy resins are hygroscopic.",
"Moreover, the resins to be used are relatively expensive.",
"In addition, there are other so-called rapid prototyping techniques known for manufacturing objects layer by layer, such as, for instance “laminated object manufacturing (LOM)”, “selective laser sintering (SLS)”.",
"These techniques also have all kinds of inherent disadvantages.",
"The technique mentioned first works with a fixed layer thickness and is suitable especially for relatively large, massive objects.",
"Hollow forms are difficult to realize.",
"When the latter technique is used, it is often rather cumbersome to provide sharp forms in the object, which means a reduced resolution, while further the process proceeds in a less well-defined manner; in particular, a temperature control cannot be realized with sufficient accuracy.",
"The object of the invention is to avoid the disadvantages of the known techniques as far as possible and to provide an entirely new method for applying a layer, in particular a relatively thin layer, comprising at least polymer material, which method is suitable in particular for forming relatively small objects.",
"According to the invention, to that end, the method such as it is indicated in the preamble is characterized in that a respective layer is obtained by applying to the substrate a film of at least polymer material-containing particles dispersed in a non-reactive liquid and by subjecting these particles to an energy flow which is converted in situ at least substantially into heat, during which heat treatment the particles fuse with each other.",
"The liquid does not serve as solvent here, but has the function of transport medium to enable the polymer material-containing particles to be applied uniformly to the substrate.",
"During the heat treatment, in principle no chemical reaction takes place, but only a direct, or indirect, thermal process, whereby evaporation of the liquid or a direct fusion of the dispersed particles can occur.",
"The heat treatment, whereby the polymer material-containing particles fuse, can be done with the aid of an AFM (“Atomic Force.",
"Microscope”) “thermal analyzer” after the liquid in the film has evaporated therefrom.",
"Depending on the volatility of the liquid and the desired process rate, a forced evaporation can take place with the aid of drying air.",
"The heat treatment can also be properly realized with the aid of a laser device.",
"In a first application, the energy generated by the laser device can be employed to bond together the polymer material-containing particles in a thermal manner (fusion).",
"In a general sense, it holds that when the energy treatment takes place utilizing a dispersion already dried without local heat treatment, this is designated as DLHT (dry layer heat treatment).",
"When a dispersion is involved which is dried forcibly during the heat treatment process, that is, the liquid is evaporated directly under the influence of the energy employed, this is called WLHT (wet layer heat treatment).",
"The result of the two methods depends on many factors, such as the layer thickness applied, the solids content in the layer applied, the composition of the transport medium, the particle size, the desired process rate, etc.",
"In a preferred embodiment, for ease of handling, the film is formed by polymer material-containing particles dispersed in water.",
"The size of the dispersed particles is between about 5 and 30,000 nm, and preferably between 100 and 1000 nm.",
"In case of an emulsion polymerization, the particles will typically be in the order of 30-1000 nm; in case of a suspension polymerization, they will typically be greater than about 1000 nm.",
"Above 30,000 nm, instead of the method according to the invention, selective laser sintering (SLS) is used.",
"According to the invention, both polymer particles of one single composition (homopolymer particles or copolymer particles) and of a different composition can be present.",
"In the latter case, it is also possible that the individual polymer particles are composed of different polymers.",
"Thus, the polymer particles can be built up from a core and a shell of different composition, for instance a core of an electrically conducting polymer and a shell of an electrically insulating polymer, or a core and a shell of different elastic properties.",
"In that case, depending on the heat treatment, either the shells alone, or both the shells and the cores can fuse with each other.",
"When also the cores fuse with each other, they can form particular patterns.",
"Through selective heat treatment, it is possible in this way, for instance, to incorporate electrical conductors into an otherwise electrically insulating plastic.",
"In addition to a makeup of polymer particles from a core and a shell of polymer material of different properties, it is also possible to take particles whose core consists of a non-polymer (for instance an organic (non-polymer), inorganic or metallic) phase which is enveloped with a polymeric phase.",
"In addition to the core-shell particle structure, other types of hybrid materials (composites) are useful, for instance particles having a raspberry, acorn or onion structure.",
"Naturally, all kinds of combinations of the above-mentioned polymer material-containing particles are possible.",
"Accordingly, the particles can differ both in composition and in size.",
"The film to be provided on the substrate can contain, in addition to the transport medium, and the polymers dispersed therein, one or more further non-reactive components, such as, for instance, colors, pigments, flow property improving or evaporation influencing substances.",
"It is also possible that the film contains one or more reactive components, such as, for instance, crosslinking agents to be activated in a post-treatment.",
"Further, under circumstances to be mentioned hereinafter, a heat transferring agent, such as carbon black, may be dispersed in the film.",
"It is also possible that ceramic particles have been added to the film, which ceramic particles are bonded to each other through the heat treatment and the fusion of the polymer material-containing particles, and, for instance, a composite pattern can be formed from which the polymeric material can be subsequently removed by firing.",
"To prevent coagulation in the film, the solids content in the film will be less than 70 vol.",
"% and preferably less than 60 vol.",
"%.",
"A minimum solids content is not defined, since a minimum layer thickness should always be achieved.",
"For carrying out the heat treatment, a laser device operative in the UV region can be used.",
"The wavelength at which the laser device is operative is then in particular between 190 and 400 nm, and is preferably between 240 and 310 nm.",
"Below 400 nm, the polymers useful for the method described here can directly absorb the energy supplied by the laser.",
"Moreover, such a laser device enables more accurate work: a good focusing on a relevant point on the film is rendered possible.",
"The wavelength is selected to be greater than 190 nm, because below that value it is no longer possible to work properly in air atmosphere; a vacuum or a gas atmosphere not reactive to the laser radiation is then highly desirable.",
"Further, the polymers can be damaged to a considerable extent by the high photon energy.",
"Further, above 190 nm; it is still, possible to work with standard optics.",
"Thus, working with, for instance, an F2 157 nm excimer laser device is more difficult and more expensive.",
"In view of the above, it is therefore preferred to work with a laser in the UV region.",
"Thus, for instance, an Argon-ion, Nd:YAG (3rd, 4th or 5th harmonic) or excimer laser device can be used.",
"In addition, it is also possible to use a laser device in the visible or infrared region, for instance an Nd:YAG laser device operative in the infrared region, in particular at a wavelength of about 1064 nm.",
"Because in that case the heat supplied by the laser cannot be absorbed by the polymers useful for the method described here, it will then be necessary to add to the film a heat transferring agent, such as carbon black, as already mentioned above.",
"For a laser device that is operative in the far infrared, such as a CO2 laser device, however, an addition of carbon black is not necessary, because there the laser energy can be absorbed directly by the polymers.",
"Further, other energy sources, such as, for instance, a UV lamp, can be used for drying the dispersion.",
"The laser device can transmit both a pulsed activation signal and a continuous activation signal.",
"For the practice of the method, use can then be made of a scanning technique, a masking technique or another imaging technique.",
"It is also possible, for instance, to split a laser beam into two the same way and having them meet at a point of the substrate, very locally a fusion can be effected.",
"When by the laser a laser beam is emitted with a normally used Gaussian intensity distribution over the beam width, the edges of the spot of the film layer to be activated will often be slightly thicker than the central portion of the spot.",
"To prevent this, the intensity of the beam energy over the cross section will be adapted to the operation.",
"This can be realized internally in the laser device or with the aid of other means, such as an external optical system.",
"The method as described so far is directed to the application of a single layer, that is, the application of a coating.",
"However, the invention also relates to a method for manufacturing an object, specifically by building it up layer by layer each of the layers to be successively applied onto each other being obtained by the practice of the above-described method.",
"The invention relates not only to a method but also to an apparatus for applying a layer containing at least polymer material to a substrate, or for manufacturing an object by building it up layer by layer.",
"To that end, the apparatus is provided with a laser device with optical components and with a processing space with a substrate on which a polymer layer, or several polymer layers to be successively applied onto each other, can be formed by the practice of the method described hereinabove.",
"This laser device can further be provided with means for setting the intensity profile over the beam cross section.",
"Also, a mask may be arranged in the optical path, in particular in combination with image reduction, or directly above the substrate (for a one-on-one image).",
"An automatic setting of the transmissivity of the mask is then possible; compared with the scanning with the laser beam, the laminated build-up of an object with the aid of grating, special lenses or other aids, such as all kinds of optical systems, then proceeds faster as a result.",
"Another possibility of selectively scanning larger surfaces is to make use of selective illumination.",
"The invention further relates to a coating, that is, a layer containing at least polymer material, and to an object formed by such layers, obtained by the use of the method described above.",
"The invention will now be further elucidated with reference to the accompanying drawing.",
"In the drawing: FIG.",
"1 shows a schematically represented set-up of an apparatus according to the invention; FIG.",
"2 shows the principle of a DLHT method according to the invention; FIGS.",
"3A-C show the principle of a WLHT method according to the invention; FIGS.",
"4A,B show a Gaussian distribution intensity profile over the laser beam cross section and a layer obtained with the aid thereof; and FIGS.",
"5A,B show a modified “tophat” intensity distribution over a laser beam cross section and a more homogeneous layer obtained with the aid thereof.",
"The set-up of an apparatus according to the invention as schematically represented in FIG.",
"1 comprises an excimer laser device 1, by means of which a laser beam 2 is obtained, which, via an optical system 1′ with various optical components, such as, for instance, a first and a second diaphragm 3 and 4, respectively, a mirror 5 and a lens 6, is directed into a space 7.In the space 7, a substrate 8 is present to which a layer, or several layers to be applied onto each other, can be applied according to the method of the invention.",
"On the substrate 8, with the aid of a doctor blade, a film 9 can be applied.",
"When using the method according to the invention, this film, in the examples described with reference to FIGS.",
"2 and 3A-C, consists of water having polymer particles dispersed therein.",
"When, as is indicated in FIGS.",
"3A-C, a WLHT method is pursued, the space 7 is filled with air of a high relative humidity.",
"Present on the bottom in the space 7, when applying the film 9, is an amount 10 of a solvent for the saturated vapor in the space 7.In a DLHT method, as is indicated in FIG.",
"2, a film 9 about 10 μm thick was applied to the substrate 8 with the aid of a doctor blade.",
"The solids content of the film was 16%.",
"The space 7 was filled with dry air.",
"After the water had evaporated virtually completely from the film 9, a layer of about 2 μm was left.",
"This layer was subjected to a heat treatment with the aid of an excimer laser.",
"The temperature in the film at the site of the spot exceeded the MFFT (“minimum film forming temperature”), in this case 35 to 40° C., except in the transition area formed as a result of heat diffusion, while the temperature outside the spot remained below this MFFT.",
"At the spot 11 irradiated by the laser beam, the polymer particles were fused with each other, and after about 1000 60 ns laser pulses a transparent layer of about 2 μm was formed.",
"The pulse frequency was found to be of little influence on the fusion process.",
"The pulse intensity was such that the above-mentioned MFFT-defined temperatures inside and outside the spot were maintained.",
"The unfused polymer particles around the spot 11 were subsequently rinsed away.",
"In a WLHT method, as is indicated in FIG.",
"3, a same film of about 10 μm was applied to the substrate 8.Now, however, the film was subjected directly to a heat treatment with the aid of the excimer laser, whereby first the water at the spot 11 evaporates, while the polymer particles descended, whereafter the polymer particles fused with each other again and a transparent layer of about 2 μm was formed.",
"In practice, the intensity profile over the cross section of the laser beam often has a Gaussian configuration; this is indicated in FIG.",
"4A.",
"The effect is that a layer is formed (see FIG.",
"4B) which is somewhat higher at the edges than in the middle.",
"To compensate this effect, a beam with a modified “tophat” distribution can be used, as indicated in FIG.",
"5A, so that a flat layer (see FIG.",
"6B) can be obtained.",
"In the following, various facets of the invention will be elucidated in and by more concrete examples.",
"1.Excimer Laser Use was made of a Lambda Physik EMG 1003i Excimer laser having a pulse energy of 200 mJ at a maximum, working at 248 nm (KrF gas mixture) with a pulse width of 60 ns.",
"The pulse frequency used is 50 Hz.",
"Imaging optics with a focal distance of 50 mm and an image distance of 75 mm were used, while the first pinhole for selecting a qualitatively good part of the laser beam (diameter: 1.5 mm) is at 5 cm from the laser, while the second pinhole (diameter: 1.5 mm) is at 100 cm behind the first pinhole (“filtering” of the most divergent parts), then a diaphragm and a 45°mirror to deflect the light from horizontal to vertical.",
"The substrate is on an Oriel XYZ-positioning table (see further FIG.",
"1).",
"2.Description of Emulsion Polymerization (JZ002) A 2 L double-walled, glass reactor equipped with a magnetic stirrer, a cooler and a thermocouple, was filled with 400 g butylmethacrylate, 0.4 g. tetrabromomethane and 1470 g demineralized water.",
"The mixture was stirred for 45 min.",
"(450 rpm) at room temperature, while nitrogen was passed through the mixture.",
"Next, the reactor was heated to 70° C., whereafter a degassed, aqueous ammonium persulfate solution (13.32 g in 41.4 g demineralized water) was added.",
"The stirring speed was lowered to 300 rpm immediately after addition of the initiator solution.",
"About 20 min.",
"after addition of the initiator, the stirring speed was further reduced to about 270 rpm.",
"After about 1 hour, the stirring speed was lowered once again, to 195 rpm.",
"2.5 hours after starting the polymerization, the heating was switched off and the dispersion slowly cooled to room temperature.",
"Thereupon the cooled dispersion was dialyzed in Visking dialysis tubes (31.70 mm diameter) in a bucket with flowing demineralized water.",
"The solids content of the dispersion was 18.4%.",
"The size of the dispersed polymer particles was determined with DCP (disk centrifuge photosedimentometry).",
"The number-average particle size, Dn, was 826 nm.",
"The weight-average particle size, Dw, was 827 nm.",
"The polydispersity of the system, expressed as Dw/Dn was 1.002.3.Description of Emulsion Polymerization (JZ057) A substantially equal procedure was followed as described with JZ002.With JZ057, 300 g butylmethacrylate, 0.3 g dodecyl thiol (instead of tetrabromomethane), 1.95 g sodium bicarbonate and 1.03 g sodium dodecyl sulfate and 1157 g demineralized water were used.",
"The reaction was started by addition of an ammonium persulfate solution (15.37 g in 70.29 g demineralized water).",
"The total polymerization time was 150 min.",
"The solids content of the dispersion was 16.1%.",
"The size of the dispersed polymer particles was determined with DCP (disk centrifuge photosedimentometry).",
"The number-average particle size, Dn, was 162 nm.",
"The weight-average particle size, Dw, was 164 nm.",
"The polydispersity of the system, expressed as Dw/Dn, was 1.008.A part of the dispersion was evaporated under vacuum with the aid of a rotation evaporator.",
"The solids percentage of the concentrated dispersion was 40.7%.",
"4.Applying Dispersion Layer With a pipette a few drops were applied onto a microscope glass (76×0.26 mm) and then spread out in the longitudinal direction of the glass plate with a 12 μm wire doctor blade.",
"5.A Thicker Layer was Applied by a Kind of Flow Coating Process.",
"A glass plate was held at an angle of about 40° and on top of the glass plate dispersion was squirted with a pipette.",
"6.WLHT According to the “Wet Layer Heat Treatment” method, an applied dispersion layer (JZ002) was laid onto the sample substrate (see Example 1: “description set-up”).",
"The applied dispersion layer was treated with a laser with the following characteristics: 50 Hz, 60 ns pulse width, 50 mm focal distance from the imaging lens, 75 mm image distance, first pinhole (diameter: 1.5 mm) at 5 cm from the laser, second pinhole (diameter: 1.5 mm) at 100 cm behind the first pinhole, then a diaphragm and a 45°mirror to deflect the light from horizontal to vertical.",
"50, 100, 250 and 2000 laser pulses were directed to different locations on the dispersion layer.",
"During the laser treatments, a crater formed in the wet dispersion layer which was not flooded after the treatment with surrounding liquid.",
"Thereafter the wet layer was dried in the air.",
"The locations treated could be well distinguished from the locations dried in the air.",
"The treated locations were completely transparent, while the surrounding layer was white.",
"7.First Variation on Example 6 Repetition of the example described under 6 “WLHT” with a focal distance of 50 mm gave spot formation only at 2000 laser pulses.",
"At fewer laser pulses, the surrounding liquid flowed over the treated spots again.",
"These locations were then not visible anymore after treatment.",
"8.Second Variation on Example 6 Repetition of the example described under 6 “WLHT”, wherein with a frequency of 50 Hz 2000 laser pulses were applied to a dispersion layer on a substrate moving at a speed of 0.1 mm/s for 50 s. After treatment, the dispersion was dried in the air; the result of this experiment was a transparent line in a white, dried dispersion.",
"9.Third Variation on Example 6 Repetition of the example described under 6 “WLHT”, using dispersion JZ057 (16%).",
"50, 100, 200, 500 and 1000 laser pulses were steered onto the wet dispersion layer.",
"The dispersion was dried in the air.",
"The locations with 50 and 100 pulses were not visible after laser treatment.",
"At the other locations, transparent spots could be clearly observed.",
"10.Variation on Example 9 Repetition of the example described under 9 “WLHT”, using dispersion JZ057 (41%).",
"11.DLHT According to the “Dry Layer Heat Treatment” method, a dispersion (JZ002) was provided on a microscope glass and subsequently dried in the air.",
"The dried film was white, which indicates that no film formation has taken place yet.",
"The microscope glass with the ‘dried’ dispersion layer was laid on the sample substrate (see Example 1: “description set-up”) and treated with a laser with the following characteristics: 50 Hz, 60 ns pulse width, 50 mm imaging lens, 75 mm focal distance, first pinhole (diameter: 1.5 mm) at 5 cm from the laser, second pinhole (diameter: 1.5 mm) at 100 cm behind the first pinhole, then a diaphragm and a 45°mirror to deflect the light from horizontal to vertical.",
"50, 100, 250 and 2000 laser pulses were controlled onto different locations on the dispersion layer.",
"The treated spots could be well distinguished with the aid of a microscope.",
"The treated locations were completely transparent, while the surrounding layer was white."
]
] | Patent_10276106 |
[
[
"Optical recording medium and its manufacturing method",
"A multi-layered film made of sequentially stacked first dielectric layer, recording layer, record-assist layer, second dielectric layer and first reflection layer on a disc substrate having a concavo-convex structure at least on one surface thereof.",
"An ultraviolet-setting resin layer is formed to cover the multi-layered film.",
"The record-assist layer is controlled in thickness to satisfy the relation d/4<a<3d, or more preferably the relation d/2≦a≦2d, between the thickness d of the recording layer and the thickness a of the record-assist layer.",
"The stacking order of the multi-layered film may be opposite.",
"In this case, a light-transmitting sheet is provided to cover the multi-layered film via a bonding layer."
],
[
"1.An optical recording medium comprising: a disc substrate having formed a concavo-convex structure formed at least on one surface thereof; and a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, wherein d/4<a<3d is satisfied where d is the thickness of the recording layer and a is the thickness of the auxiliary recording layer.",
"2.An optical recording medium according to claim 1 wherein said recording layer can record the information signals by reversible changes at least between two different states.",
"3.An optical recording medium according to claim 1 wherein said recording layer is made of a phase-changeable material capable of recording the information signals by phase changes between a crystal phase and an amorphous phase.",
"4.An optical recording medium according to claim 1 wherein the laser beam used for recording and/or reproducing information signals is irradiated to said recording layer from one side of the multi-layered film where the disc substrate exists.",
"5.An optical recording medium according to claim 4 wherein said multi-layered film includes a first dielectric layer, said recording layer, said record-assist layer, a second dielectric layer and a first reflection layer that are stacked sequentially from near the surface of the disc substrate having formed the concavo-convex structure.",
"6.An optical recording medium according to claim 4 wherein a layer of a synthetic resin is formed to cover the multi-layered film on the disc substrate.",
"7.An optical recording medium according to claim 1 wherein the laser beam used for recording and/or reproducing information signals is irradiated to the recording layer from one side of the disc substrate where the multi-layered film exists.",
"8.An optical recording medium according to claim 7 wherein said multi-layered film is made of a second reflection layer, third dielectric layer, said record-assist layer, said recording layer and a fourth dielectric layer that are stacked sequentially from near one surface of the disc substrate having said concavo-convex structure.",
"9.An optical recording medium according to claim 7 wherein a light-transmitting layer permitting the laser beam to pass through is formed to cover the multi-layered film on the disc substrate.",
"10.An optical recording medium according to claim 1 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.5≦Dl/Dg≦2.0.11.An optical recording medium according to claim 1 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.8≦Dl/Dg≦1.2.12.An optical recording medium according to claim 1 wherein an objective lens used for recording and/or reproducing the information signals has a numerical aperture not smaller than 0.45 and not larger than 0.60.13.An optical recording medium according to claim 1 wherein thickness of the recording layer is in the range not smaller than 5 nm and not larger than 50 nm.",
"14.An optical recording medium according to claim 1 wherein thickness of said recording layer is in the range not smaller than 10 nm and not larger than 40 nm.",
"15.An optical recording medium according to claim 1 wherein thickness of said record-assist layer is in the range not smaller than 3 nm and not larger than 100 nm.",
"16.An optical recording medium according to claim 1 wherein thickness of said record-assist layer is in the range not smaller than 5 nm and not larger than 60 nm.",
"17.An optical recording medium according to claim 1 wherein said recording layer is capable of recording the information signals in a write only mode.",
"18.An optical recording medium according to claim 1 wherein said recording layer is made of GeTe alloy or GeSbTe alloy.",
"19.An optical recording medium according to claim 1 wherein said record-assist layer is made of a material containing as its major component at least one compound selected from the group consisting of SnTe, SiN, SiC, SnSe, GeN, PbSe, PbTe, Bi2Te3 and Sb2Te3.20.An optical recording medium according to claim 1 wherein record marks can be recorded on both portions of the recording layer on top surfaces of ridges and portions of the recording layer on bottoms of the furrows of the concavo-convex structure of the disc substrate by said laser beam.",
"21.An optical recording medium according to claim 1 wherein said recording layer is located nearer to the irradiation side of the laser beam.",
"22.A method of manufacturing an optical recording medium, which forms a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, comprising: adjusting the thickness of the record-assist layer to satisfy d/4<a<3d where d is the thickness of the recording layer and a is the thickness of the record-assist layer.",
"23.A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of a material capable of recording the information signals by reversible changes at least between two different states 24.A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of a phase-changeable material capable of recording the information signals by phase changes between a crystal phase and an amorphous phase.",
"25.A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is configured to record the information signals in the recording layer by irradiation of the laser beam to the recording layer from one side of the multi-layered film where the disc substrate exists.",
"26.A method of manufacturing an optical recording medium according to claim 25 wherein said multi-layered film is made by stacking a first dielectric layer, said recording layer, said record-assist layer, a second dielectric layer and a first reflection layer that are stacked sequentially from near the surface of the disc substrate having formed the concavo-convex structure.",
"27.A method of manufacturing an optical recording medium according to claim 25 wherein a layer of a synthetic resin is formed to cover the multi-layered film after the multi-layered film is formed on the disc substrate.",
"28.A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is configured to record the information signals in the recording layer by irradiation of the laser beam to the recording layer from one side of the disc substrate where the multi-layered film exists.",
"29.A method of manufacturing an optical recording medium according to claim 28 wherein said multi-layered film is made by stacking a second reflection layer, third dielectric layer, said record-assist layer, said recording layer and a fourth dielectric layer sequentially from near one surface of the disc substrate having said concavo-convex structure.",
"30.A method of manufacturing an optical recording medium according to claim 28 wherein a light-transmitting layer permitting the laser beam to pass through is formed to cover the multi-layered film after the multi-layered film is formed on the disc substrate.",
"31.A method of manufacturing an optical recording medium according to claim 22 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.5≦Dl/Dg≦2.0.32.A method of manufacturing an optical recording medium according to claim 22 wherein, in a portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of a portion nearer to the injection side of the laser beam and width Dl of a level difference of a portion remoter from the injection side of the laser beam satisfy the ratio of 0.8≦Dl/Dg≦1.2.33.A method of manufacturing an optical recording medium according to claim 22 wherein an objective lens used for recording and/or reproducing the information signals has a numerical aperture not smaller than 0.45 and not larger than 0.60.34.A method of manufacturing an optical recording medium according to claim 22 wherein the recording layer is formed to have a thickness in the range not smaller than 5 nm and not larger than 50 nm.",
"35.A method of manufacturing an optical recording medium according to claim 22 wherein the recording layer is formed to have a thickness in the range not smaller than 10 nm and not larger than 40 nm.",
"36.A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is formed to have a thickness in the range not smaller than 3 nm and not larger than 100 nm.",
"37.A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is formed to have a thickness in the range not smaller than 5 nm and not larger than 60 nm.",
"38.A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is formed to be capable of recording the information signals in a write only mode.",
"39.A method of manufacturing an optical recording medium according to claim 22 wherein said recording layer is made of GeTe alloy or GeSbTe alloy.",
"40.A method of manufacturing an optical recording medium according to claim 22 wherein said record-assist layer is made of a material containing as its major component at least one compound selected from the group consisting of SiC, SiN, SnTe, SnSe, GeN, PbSe, PbTe, Bi2Te3 and Sb2Te3.41.A method of manufacturing an optical recording medium according to claim 22 wherein the optical recording medium is formed to be capable of recording record marks in top portions of ridges and bottom portions of furrows of the concavo-convex structure of the disc substrate."
],
[
"<SOH> BACKGROUND ART <EOH>Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.",
"The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.",
"In conventional optical discs, only lands or only grooves were used to record recording marks.",
"However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.",
"a land/groove recording technique, has been brought into use as a format of optical discs.",
"An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).",
"However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.",
"In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.",
"Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).",
"To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.",
"However, only change of the duty ratio cannot bring about ample freedom.",
"Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.",
"2000-215511, referred to as Literature 1).",
"However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.",
"This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.",
"Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.",
"It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability."
],
[
"<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.",
"1 is a cross-sectional view that shows an optical disc according to the first embodiment of the invention FIG.",
"2 is a graph that shows dependency or a carrier upon the thickness of a record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"3 is a graph that shows dependency of the signal amplitude upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"4 is a graph that shows dependency of a cross talk signal upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"5 is a cross-sectional view that shows an optical disc according to the second embodiment of the invention; FIG.",
"6 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; FIG.",
"7 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; and FIG.",
"8 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"TECHNICAL FIELD This invention relates to an optical recording medium and its manufacturing method especially suitable for application to an optical recording medium employing a land/groove recording technique.",
"BACKGROUND ART Recently, along with enhancement of recording density of optical recording mediums such as optical discs, development of the technique of effectively using the area on the recording surface of an optical disc has been pushed forward to enhance the recording density.",
"The recording surface of any disc substrate has concavo-convex called lands and grooves respectively.",
"In conventional optical discs, only lands or only grooves were used to record recording marks.",
"However, with the need of enhancing the recording density of optical discs, a technique for recording record marks on both lands and grooves, i.e.",
"a land/groove recording technique, has been brought into use as a format of optical discs.",
"An example of optical discs combined with the land/groove recording as their format is DVD-RAM (Digital Versatile Disc-Random Access Memory).",
"However, such optical discs employing the land/groove recording suffer a difference of the thermal property exerted to the recording layer because of the difference in physical configuration between lands and grooves when recording laser beams are irradiated to lands and grooves respectively.",
"In addition, lands and grooves form a periodical structure close to the wavelength of the laser beams.",
"Therefore, vector diffraction of light causes uneven distribution of irradiated laser beams between lands and grooves, and it has been difficult to record or reproduce record marks from both lands and grooves with a high C/N ratio (carrier-to-noise ratio).",
"To match lands and grooves in property, it is contemplated, for example, to change widths of lands and grooves and thereby change the duty ratio.",
"However, only change of the duty ratio cannot bring about ample freedom.",
"Under the circumstances, an optical disc was proposed, in which the recording layer on grooves is formed thicker than the recording layer on lands by selecting the sputtering condition, for example (Japanese Patent Laid-Open Publication No.",
"2000-215511, referred to as Literature 1).",
"However, in case of the optical disk disclosed in Literature 1, the condition for deposition of the recording layer is changed to form the recording layer on grooves thicker than the recording layer on lands.",
"This condition digresses the optimum condition for deposition of the recording layer and invites deterioration of signal characteristics of the recording layer.",
"Therefore, it has been demanded to develop a method capable of independently controlling the recording characteristics of lands and grooves.",
"It is therefore an object of the invention to provide an optical recording medium having a multi-layered film of a recording layer and a record-assist layer stacked adjacently on one surface of a disc substrate having formed a concavo-convex structure, and its manufacturing method, which enable freer designing while ensuring good signal characteristics in the recording layer on the concavo-convex structure and ensuring high reliability.",
"DISCLOSURE OF INVENTION The Inventor made vigorous researches to overcome the above-discussed problems the conventional techniques involved.",
"The researches are outlined below.",
"The recording principle of conventional optical mediums having a multi-layered film of a recording layer for recording information signals and a record-assist layer for supporting the recording layer is presumed to rely upon that, when the temperature is raised higher than the crystallization temperature lower than the melting point of the recording layer by laser beams irradiated to the recording layer, minute crystals produced in the crystallized auxiliary layer, and crystal growth of the recording layer occurs from the minute crystals as the seed crystals.",
"This principle has been brought about because record marks are different in amplitude between optical discs having the auxiliary layer and optical discs without the auxiliary layer.",
"The Inventor put forward the researches on the record-assist layer under the above presumption.",
"That is, the Inventor recorded 2T pulses on samples common in duty ratio but variously different in thickness of the record-assist layer, and measured dependency of a carrier upon the recording power in lands and grooves, respectively.",
"As a result, the Inventor has found that the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film and that there is almost no change of the carrier in lands even when the thickness of the record-assist layer is changed.",
"On the other hand, as to the dependency of the carrier upon the recording power in grooves, the Inventor has found that, although the dependency of the carrier upon the recording power in lands exhibits similar tendencies regardless of the thickness of the film, when the record-assist layer is changed thickness, the carrier rises as the thickness decreases.",
"This tendency was observed in the range close to the optimum value.",
"Accordingly, the Inventor got inspiration of using the phenomenon confirmed by the above-explained experiment.",
"That is, in case the duty ratio and the recording power are fixed, dependency of the carrier upon the thickness of the record-assist layer arises particularly in grooves, ad the dependency upon the thickness does not almost appear in lands.",
"From this fact, the Inventor had the idea that the carriers can be brought into agreement between lands and grooves without the need of changing the duty ratio.",
"The Inventor made further researches on changes of the carrier in grooves.",
"That is, the Inventor calculated how the change of amplitude of the recording layer of GeTe alloy depends upon the thickness of the record-assist layer by the effective Fresnel coefficient method.",
"From the result of the calculation, the Inventor got the knowledge that the amplitude increases as the thickness of the record-assist layer decreases.",
"With reference to the value when the thickness of the record-assist layer was 30 nm, when the thickness was decreased from 20 nm to 10 nm, the increase of the amplitude was approximately 1 dB according to the calculation by the effective Fresnel coefficient method.",
"Through the researches by the Inventor, the above-explained phenomenon can be assumed to occur because grooves are closer to the optical pickup than lands and less affected by track grooves, and therefore become more liable to reflect the amplitude changes in response to changes of the reflectance due to the difference of the thickness.",
"In regard to the carrier changes on the part of the grooves, the amplitude changes of the reflectance by the change of the film structure are considered to be dominant.",
"The present invention has been made based on the above-explained researches.",
"To accomplish the object, the first aspect of the invention is an optical recording medium comprising: a disc substrate having formed a concavo-convex structure formed at least on one surface thereof; and a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, wherein d/4<a<3d is satisfied where d is the thickness of the recording layer and a is the thickness of the auxiliary recording layer.",
"The second aspect of the invention is a method of manufacturing an optical recording medium, which forms a multi-layered film made by stacking at least a recording layer capable of recording information signals by irradiation of a laser beam and a record-assist layer adjacent to the recording layer on the surface of the disc substrate having the concavo-convex structure, comprising: adjusting the thickness of the record-assist layer to satisfy d/4<a<3d where d is the thickness of the recording layer and a is the thickness of the record-assist layer.",
"In the present invention, d/2≦a≦2d is preferably established when the thickness of the recording layer is d and the thickness of the record-assist layer is a.",
"In the present invention, the recording layer is typically configured to record the information signals by reversible changes at least between two different states, and it is preferably made of a phase-changeable material capable of recording the information signals by phase changes between a crystal phase and an amorphous phase.",
"In the present invention, the laser beam used for recording and/or reproducing information signals is typically irradiated to said recording layer from one side of the multi-layered film where the disc substrate exists.",
"In this case, the recording layer is preferably located nearer to the irradiation side of the laser beam.",
"More specifically, the multi-layered film is preferably made of a first dielectric layer, recording layer, record-assist layer, second dielectric layer and first reflection layer that are stacked sequentially from near the surface of the disc substrate having formed the concavo-convex structure.",
"Preferably, a layer of a synthetic resin such as a ultraviolet-setting resin (urethane-family, acrylic family, silicon family, polyester family, and so on) of a hot-melt adhesive agent is additionally formed on the top surface of the multi-layered film on the disc substrate (the surface opposite from the disc substrate) to cover the multi-layered film on the disc substrate for the purpose of protecting and reinforcing the multi-layered film.",
"Typically, the first dielectric layer and the second dielectric layer are made of zinc sulfide/silicon oxide mixture (ZnS—SiO2) having a diffractive index around 2.1 or a silicon nitride (SiN, Si3N4) having a diffractive index around 2.0.However, other materials are also usable.",
"In the resent invention, thickness of the first dielectric layer is adjusted not to exceed 200 nm within the range capable of forming the dielectric layer not to disconnect in form of islands.",
"In the present invention, the second dielectric layer is typically adjusted in thickness in the range not smaller than 1 nm and not smaller than 100 nm.",
"In the present invention, thickness of the first reflection layer is adjusted not to be smaller than 20 nm and not to be larger than 100 nm.",
"The first dielectric layer, recording layer, record-assist layer, second dielectric layer and first reflection layer are typically formed by sputtering, but any other film-making methods are also usable, such as electron beam vapor deposition, vacuum vapor deposition, epitaxial growth, chemical vapor deposition (CVD) and physical vapor deposition (PVD).",
"In the present invention, the laser beam used for recording and/or reproducing information signals is irradiated to the recording layer typically from one side of the disc substrate where the multi-layered film exists.",
"In the present invention, the multi-layered film is preferably made of a second reflection layer, third dielectric layer, said record-assist layer, said recording layer and a fourth dielectric layer that are stacked sequentially from near one surface of the disc substrate having said concavo-convex structure, and a light-transmitting layer permitting the laser beam to pass through is preferably formed to cover the multi-layered film on the disc substrate.",
"Although the third dielectric layer and the fourth dielectric layer are typically made of ZnS—SiO2 having a diffractive inde3x around 2.1, or SiN or Si3N4 having a diffractive index around 2.0, other materials can be used as well.",
"In the present invention, thickness of the third dielectric layer is typically adjusted in the range not thinner than 1 nm and not thicker then 100 nm.",
"In the present invention, thickness of the fourth dielectric layer is adjusted not to exceed 200 nm within the range capable of forming the dielectric layer not to disconnect in form of islands.",
"In the present invention, the second reflection layer is adjusted in thickness typically in the range not thinner than 20 nm and not thicker than 100 nm.",
"Although the second reflection layer, third dielectric layer, record-assist layer, recording layer and fourth dielectric layer are typically formed by sputtering, but any other film-making methods are also usable, such as electron beam vapor deposition, vacuum vapor deposition, epitaxial growth, chemical vapor deposition (CVD) and physical vapor deposition (PVD).",
"The light-transmitting layer formed to cover the multi-layered film on the disc substrate includes at least a light-transmitting sheet permitting light to pass through and an adhesive layer for bonding the light-transmitting sheet to the disc substrate.",
"The adhesive layer is an ultraviolet-setting resin or a pressure sensitive tackiness agent.",
"Instead of using the light-transmitting sheet, an ultraviolet-setting resin is usable to form the light-transmitting layer.",
"An example of optical discs configured for irradiation of a laser beam to the disc substrate from one side thereof having the multi-layered film is DVR (digital video recording system) having a thin light-transmitting layer.",
"This invention is applicable to optical discs such as DVR, including so-called DVR-red configured to record or reproduce information signals by using a semiconductor laser having an emission wavelength around 650 nm and so-called DVR-blue configured to record or reproduce information signals by using a semiconductor laser having an emission wavelength around 400 nm.",
"Such DVR is configured to record signals by using an objective lens that is typically enhanced in NA (Numerical Aperture) to around 0.85 by combining two lenses in series.",
"More specifically, it has the storage capacity around 22 GB per single plane.",
"Optical discs including DVR are preferably housed in cartridges, but application of the invention is not limited to cartridge-type optical discs.",
"In the present invention, if the multi-layered film formed on the disc substrate is covered with a thin light-transmitting layer, the light-transmitting layer is typically made of a non-magnetic material through which the laser beam from a GaN-family semiconductor laser (having an emission wavelength around 400 nm for blue emission), ZnSe-family semiconductor laser (having an emission wavelength around 500 nm for green emission) or AlGaInP-family semiconductor laser (having an emission wavelength around 635 to 680 nm for red emission) used at least for recording and reproducing information signals can pass.",
"More specifically, it is made of a light-transmitting thermoplastic resin such as polycarbonate.",
"In the present invention, in the portion of the disc substrate having the concavo-convex structure, width Dg of a level difference of concavo-convex in a portion nearer to the injection side of the laser beam and width Dl of a level difference of concavo-convex in a portion remoter from the injection side of the laser beam satisfy the ratio of 0.5≦Dl/Dg≦2.0, or more preferably the ratio of 0.8≦Dl/Dg≦1.2.In the present invention, numerical aperture of the objective lens used for recording and/or reproducing information signals is typically in the range not smaller than 0.45 and not larger than 0.60.In this range of the numerical aperture, the laser beam is irradiated from one side of the optical recording medium where the disc substrate exists.",
"To obtain good signals, maintain the optical contrast in a good condition and prevent thermal diffusion while preventing the recording layer from becoming discontinuous islands, thickness of the recording layer is typically adjusted in the range not thinner than 5 nm and not thicker than 50 nm, or more preferably, in the range not thinner than 10 nm and not thicker than 40 nm.",
"In the present invention, thickness of the record-assist layer is limited not to be thinner than 3 nm and not to be thicker than 100 nm, or more preferably in the range not thinner than 5 nm and not thicker than 60 nm.",
"In the present invention, the recording layer is typically configured to record information signals in a write only mode.",
"In this case, the recording layer is preferably made of a GeTe-family alloy or a GeSbTe-family alloy.",
"However, other materials are also usable for the recording layer, such as Ge—Te, Te—Sb, Te—Ge—Sb, Te—Ge—Sb—Pd, Te—Ge—Sb—Cr, Te—Ge—Sb—Bi, Te—Ge—Sn—O, Te—Ge—Sb—Se, Te—Ge—Sn—Au, In—Sb—Te, In—Sb—Se, Te—Ge—Sb—Sn, In—Sb—Te—Ag, In—Se and Te—Bi.",
"In the present invention, the optical recording medium is typically configured to record record marks with the laser beam in both the top portions of the ridges and the bottom portions of the furrows of the concavo-convex structure of the disc substrate.",
"That is, the optical recording medium according to the invention employs the land/groove recording technique.",
"In the present invention, in case the recording layer is made of a phase-changeable material for the purpose of improving the stability of record marks against reproduced light and simultaneously ensuring a good jitter property in high-density recording by mark-edge recording, the phase-changeable recording layer is typically made of a Te—Ge—Sb family alloy (which may contain other elements in addition to Te, Ge and Sb).",
"Its composition is in the range encircled by four points in the triangular graph of FIG.",
"6, which shows the composition of three components Te, Ge and Sb in coordinates (Te, Ge, Sb), namely, A(0.475, 0.05, 0.475), B(0.665, 0.05, 0.285), C(0.60, 0.40, 0) and D(0.40, 0.60, 0) (composition of the point A is Te47.5Ge5Sb47.5, composition of the point B is Te66.5Ge5Sb28.5, composition of the point C is Te60Ge40, and composition of the point D is Te40Ge60).",
"More preferably, the record-assist layer containing as its major component at least one compound selected from the group consisting of SiN, SiC, GeN, PbSe, PbTe, SnSe, SnTe, Bi2Te3 and Sb2Te3 is preferably formed immediately above and under the phase-changeable recording layer.",
"If the composition of the phase-changeable recording layer is outside the straight line connecting the point A and the point B on the triangular graph of FIG.",
"6 (where the content of Ge is less than 5 atomic %), then the crystallization temperature decreases, and portions without record marks (pre-recorded portions) become liable to be crystallized by reproduction reproduced light.",
"As a result, boundaries between record marks and pre-recorded portions become ambiguous, and the record marks may deteriorate.",
"Further, if the composition of the phase-changeable recording layer is outside the straight line connecting the point B and the point C and outside the straight line connecting the point D and the point A, then the jitter property deteriorates.",
"If the phase-changeable recording layer is inside the range surrounded by the points A, B, C and D but close to the straight line connecting the point A and the point B (where the content of Ge is 5 atomic % or more, but relatively small), then the difference in optical constant between the amorphous state and the crystal state becomes small.",
"This is not desirable from the viewpoint of the signal quality because the amplitude of the reproduction signal decreases.",
"In the present invention, more preferable composition range of the phase-changeable recording layer is the range where the coordinates (Te, Ge, Sb) are E(0.47, 0.30, 0.23), F(0.58, 0.30, 0.12), G(0.56, 0.44, 0) and H(0.44, 0.56, 0) on the triangular graph of FIG.",
"7 similar to FIG.",
"6.Composition of the point E is Te47Ge30Sb23, composition of the point F is Te58Ge30Sb12, composition of the point G is Te56Ge44, and composition of the point H is Te44Ge56.In the present invention, more preferable composition range of the phase-changeable recording layer is the range where the coordinates (Te, Ge, Sb) are J(0.47, 0.40, 0.13), K(0.55, 0.40, 0.05), L(0.52, 0.48, 0) and M(0.44, 0.56, 0) on the triangular graph of FIG.",
"8 similar to FIG.",
"6.Composition of the point J is Te47Ge40Sb13, composition of the point K is Te55Ge40Sb5, composition of the point L is Te52Ge48, and composition of the point M is Te44Ge56.In the present invention, the record-assist layer is preferably made of a material containing as its major component at least one compound selected from the group consisting of SnTe, SiN, SiC, SnSe, GeN, PbSe, PbTe, Bi2Te3 and Sb2Te3.The record-assist layer may be in a mixed crystal state mixing any one of those compounds as well.",
"From the viewpoint of the storage lifetime of the optical recording medium, SnTe is preferably selected from those compounds as the major component of the record-assist layer.",
"In the present invention, the record-assist layer contains one of above-specified compounds and may additionally contain other appropriate material.",
"In this case, the content of any of the above-specified compounds is adjusted to be 50 volume % or more.",
"If the content of the specific compound is less than 50 volume %, the phase-changeable recording layer may fail to crystallize well and may deteriorate the jitter property of the reproduced light.",
"The content of the specific compound in the record-assist layer is more preferably 70 volume % or more.",
"More specifically, usable materials of the record-assist layer are thorium phosphide (ThP), lanthanum sulfide (LaS), scandium antimony (ScSb), thorium selenium (ThSe), calcium selenium (CaSe), lead sulfide (PbS), scandium bismuth (ScBi), thorium arsenide (ThAs), BiSe, indium arsenide (InAs), yttrium tellurium (YTe), gallium antimony (GaSb), PbSe, tin antimony (SnSb), aluminum antimony (AlSb), copper iodide (CuI), strontium selenium (SrSe), SnTe, thorium antimony (ThSb), CaTe, barium sulfide (BaS), LaTe, PbTe, BiTe, SrTe, silver ionide (AgI), Sb2Te3, Bi2Se3, Bi2Te3 and so forth.",
"However, other materials are also usable where appropriate.",
"In the present invention, in case the multi-layered film on the disc substrate does not include the reflection layer and has the record-assist layer on one surface of the recording layer opposite from the disc substrate, a protective layer is preferably formed on one surface of the record-assist layer opposite from the surface in contact with the recording layer.",
"The protective layer is made of at least one material selected from metal or semi-metal oxides, carbides, nitrides, fluorides and sulfides.",
"More specifically, usable materials of the protective layer are oxides such as silicon oxides (SiO2, SiO), tantalum oxide (Ta2O5) and zirconium oxide (ZrO2), carbides such as silicon carbide (SiC) and titanium carbide (TiC) or simplex carbon (C), nitrides such as silicon nitride (Si3N4) aluminum nitride (AlN), sulfides such as zinc sulfide (ZnS), samarium sulfide (SmS) and strontium sulfide (SrS), and fluorides such as magnesium fluoride (MgF2).",
"Further, a mixture containing at least one or more kinds of those materials is preferably used as the material of the protective layer.",
"In the present invention, to ensure accurate control of the length of the record mark by mark-edge recording, the first reflection layer of the second reflection layer for reflecting light after passing the recording layer is preferably made of a material having thermal conductivity not lower than 50 W/m·K.",
"Examples of materials having thermal conductivity not lower than 50 W/m·K are metals selected from the group consisting of Al, Cr, Ni, Au, hafnium (Hf), palladium (Pd), tantalum (Ta), cobalt (Co), Mo, W, Cu and Ti, or alloys of those metals.",
"Among these materials, desirable materials for forming the reflection layer from various viewpoints are Al—Ti alloys, Al—Cr alloys, Al—Ta alloys, Al—Pd alloys, Al—Cu alloys, Ti—Al alloys, Ti—V alloys, Ti—Pd—Cu alloys, Ag alloys, and so on.",
"Composition of these alloys is determined in accordance with the required property.",
"Other materials are also usable if necessary.",
"According to the invention having the above-summarized configuration, since the thickness a of the record-assist layer is controlled to be larger than ¼ of the thickness d of the recording layer and smaller than three times of the thickness d, signal property can be changed by not only by changing the size of the concavo-convex but also by modifying the thickness of the record-assist layer.",
"Therefore, design freedom necessary for improvement of the signal property can be increased.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 is a cross-sectional view that shows an optical disc according to the first embodiment of the invention FIG.",
"2 is a graph that shows dependency or a carrier upon the thickness of a record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"3 is a graph that shows dependency of the signal amplitude upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"4 is a graph that shows dependency of a cross talk signal upon the thickness of the record-assist layer in the optical disc according to the first embodiment of the invention; FIG.",
"5 is a cross-sectional view that shows an optical disc according to the second embodiment of the invention; FIG.",
"6 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; FIG.",
"7 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention; and FIG.",
"8 is a triangular graph that shows suitable composition of a phase-changed recording layer in an optical recording medium according to the invention.",
"BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the invention will now be explained below with reference to the drawings.",
"First explained is an optical disc according to the first embodiment of the invention.",
"FIG.",
"1 shows the optical disc according to the first embodiment.",
"The optical disc according to the first embodiment is a WO (write once) type on which information signals are recorded in a WO mode.",
"As shown in FIG.",
"1, the optical disc 1 according to the first embodiment includes a multi-layered film made of a first dielectric layer 3, recording layer 4, record-assist layer 5, second dielectric layer 6 and first reflection layer 7 that are stacked on one major surface 2a of a disc substrate 2, and an ultraviolet-curing resin layer 8 formed to cover the multi-layered film.",
"The disc substrate 2 is made of a material that can transmit at least a laser beam used for recording and reproducing information signals.",
"Examples of materials usable for forming the disc substrate 2 are plastic materials such as polycarbonate-family resins, polyolefin resin and acrylic-family resins, and glass.",
"Among these materials, plastic materials are preferable from the economical viewpoint.",
"In the first embodiment, the disc substrate 2 is made of, for example, polycarbonate (PC).",
"The disc substrate 2 is sized 130 mm in diameter and approximately 1.2 mm in thickness, for example.",
"The major surface 2a of the disc substrate 2 has formed a track-like concavo-convex structure.",
"Ridges of the track-like concavo-convex structure are portions remoter from the laser beam injection side.",
"In the first embodiment, the ridges are called lands 2b.",
"The furrows are portions closer to the laser beam injection side.",
"In the first embodiment, the furrows are called grooves 2c.",
"Ratio between the width Dl of each land 2b and the width Dg of each groove 2, i.e.",
"the duty ratio (Dl/Dg), is selected from the range of 0.5 to 2.0.If the duty ratio is smaller than 0.5, then the width Dl of the land 2b is less than one half of the width Dg of the groove 2c, and it is difficult to control both the lands 2b and the grooves 2c to have a common signal property.",
"If the duty ratio is larger than 2.0, then the width Dg of the groove 2c is similarly narrowed to less than one half of the width Dl of the land 2b, and it is again difficult to control both the lands 2b and the grooves 2c to have a common signal property.",
"More preferable range of the duty ratio is 0.8 to 1.2.In an example of configurations and sizes of the disc substrate 2 according to the first embodiment, the track pitch (Tp) of the groove tracks is 0.77 μm, depth of the grooves 2c (depth of the furrows) is 70 nm, width Dl of the lands 2b is 0.82 μm, width Dg of the grooves 2c is 0.72 μm, and the duty ratio is (0.82/0.72=) 1.14.The first dielectric layer 3 is preferably made of a material exhibiting a low absorption power (absorptivity) to recording/reproducing laser beams, and more specifically, an extinction coefficient k not larger than 0.3.Taking the resistance to heat into consideration as well, SiN is one of preferable materials.",
"Thickness of the first dielectric layer 3 is adjusted not to exceed 200 nm.",
"In the first embodiment, it may be 10 nm thick, for example.",
"The recording layer 4 is made of GeTe alloy, for example.",
"If the thickness d of the recording layer 4 is smaller than 5 nm, the amplitude is optically too small.",
"If the thickness d is larger than 50 nm, then the recording layer degrades in heat diffusion effect, therefore becomes liable to hold heat therein, and will become incapable of recording record marks clearly.",
"Therefore, thickness d of the recording layer 4 is adjusted in the range of 5 nm≦d≦50 nm.",
"According to the knowledge of the Inventor obtained by an experiment of the signal property in the recording layer 4, thickness d of the recording layer is preferably adjusted in the range of 10 nm≦d≦40 nm, taking account of the range capable of obtaining good signals.",
"In the first embodiment, thickness of the recording layer 4 is around 20 nm, for example.",
"Material of the record-assist layer 5 contains as its major component at least one compound selected from the group consisting of SnTe, SiN, SiC, GeN, PbTe, SnSe, PbSe, Bi2Te3 and Sb2Te3.More specifically, it is a material containing any of the above-specified compounds by at least 50 volume %, or more preferably, by at least 70 volume %.",
"The record-assist layer 5 is preferably in uniform contact with the recording layer 4.In the first embodiment, the record-assist layer 5 is made of SnTe, for example.",
"Thickness a of the record-assist layer 5 is adjusted in the range larger than ¼ of the thickness d of the recording layer 4 and smaller than three times thereof.",
"That is, record-assist layer 5 is formed to satisfy the following relation between its thickness a and the thickness d of the recording layer 4, d/4<a<3d or more preferably to satisfy d/2≦a≦2d Details of the thickness of the record-assist layer 5 will be explained later.",
"The second dielectric layer 6 is preferably made of a material exhibiting a low absorption power to recording/reproducing laser beams, and more specifically, an extinction coefficient k not larger than 0.3.Taking the resistance to heat into consideration as well, ZnS—SiO2 (especially one having a mol ratio around 4:1) is one of preferable materials.",
"In the first embodiment, the first dielectric layer 3 and the second dielectric layer 6 are made of different materials.",
"However, they may be made of a common material.",
"The first reflection layer 7 is made of Al alloy, for example.",
"In the first embodiment, it is made of AlTi alloy.",
"If the first reflection layer 7 is thinner than 20 nm, heat generated in the recording layer 4 cannot diffuse sufficiently, and heat is not sufficiently cooled down.",
"If the reflection layer 7 is thicker than 100 nm, then its thermal property and optical property will be adversely affected, and a desired signal property will not be obtained.",
"Therefore, thickness of the first reflection layer 7 is adjusted in the range from 20 to 100 nm.",
"In the first embodiment, it is 60 nm thick, for example.",
"The ultraviolet-setting resin layer 8 is made of an ultraviolet-setting resin that is cured by irradiation of ultraviolet rays.",
"In the optical disc according to the first embodiment, having the above-explained configuration, a laser beam L1 is injected toward the multi-layered film including the sequentially stacked first dielectric layer 3, recording layer 4, record-assist layer 5, second dielectric layer 6 and first reflection layer 7 from one side thereof where the disc substrate 2 exists as shown in FIG.",
"1, and when it irradiates the recording layer 4, an information signal is recorded and/or reproduced.",
"Next explained is a manufacturing method of the optical disc 1 according to the first embodiment having the above-explained configuration.",
"First prepared is a clean disk substrate 2 having guide grooves (concavo-convex groove tracks) (for example, thickness: 1.2 mm, track pitch: 0.77 μm, width of lands: 0.82 μm, width of grooves: 0.72 μm, depth of grooves: 70 nm).",
"Subsequently, the disc substrate 2 is transported into a first sputtering chamber in which a Si target is set, and it is placed on a predetermined position.",
"After that, a film of SiN, for example, is formed on the major surface 2a of the disc substrate 2 by sputtering using nitrogen (N2) gas as the reactive gas.",
"As a result, the first dielectric layer 3 of SiN is deposited on the major surface 2a of the disc substrate 2.An example of the sputtering conditions is using an inactive gas such as argon (Ar) gas and a reactive gas such as N2 gas as the atmospheric gas, adjusting their flow rates to be Ar:N2=3:1, maintaining the pressure of the atmospheric gas in 0.6 Pa, and supplying the sputtering power of 2.5 kW.",
"After that, the disc substrate 2 having formed the first dielectric layer 3 is removed from the first sputtering chamber.",
"The disc substrate 2 is next introduced into a second sputtering chamber and placed on a predetermined position.",
"Thereafter, a film of GeTe, for example, is formed on the first dielectric layer 3 by sputtering.",
"As a result, the recording layer 4 of GeTe alloy is obtained.",
"An example of the puttering conditions used here is using an inactive gas such as Ar gas as the atmospheric gas, and adjusting the sputtering power to 0.3 kW and the pressure of the atmospheric gas to 0.4 Pa (3.0 mTorr).",
"Under these conditions, the recording layer 4 is formed by using a GeTe alloy target adjusted in composition such that the composition of the layer obtained becomes Ge50Te50, for example.",
"After that, the disc substrate 2 having formed the recording layer 4 as well is removed from the second sputtering chamber.",
"The disc substrate 2 having formed layers up to the recording layer 4 is next introduced into a third sputtering chamber and placed on a predetermined position.",
"After that, a SnTe alloy film is formed on the recording layer 4 by sputtering using a SnTe alloy target.",
"As a result, the record-assist layer 5 of SnTe alloy is formed evenly on the recording layer 4.An example of the sputtering conditions used here is using an inactive gas such as Ar gas as the atmospheric gas, adjusting the sputtering power to 0.3 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 2 having formed layers up to the record-assist layer 5 is removed from the third sputtering chamber.",
"The disc substrate having formed layers up to the record-assist layer is next introduced into a fourth sputtering chamber and placed on a predetermined position.",
"Thereafter, a ZnS—SiO2 film is formed on the record-assist layer 5 by sputtering using a ZnS—SiO2 target.",
"As a result, the second dielectric layer 6 of ZnS—SiO2 is deposited on the record-assist layer 5.An example of the sputtering conditions used here is using an inactive gas like Ar gas as the atmospheric gas and adjusting the sputtering power to 0.6 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 2 having formed layers up to the second dielectric layer 6 is removed from the fourth sputtering chamber.",
"The disc substrate 2 having formed layers up to the second dielectric layer 6 is next introduced into a fifth sputtering chamber and placed on a predetermined position.",
"Thereafter, an AlTi film is formed on the second dielectric layer 6 by sputtering using a target of AlTi alloy.",
"As a result, the first reflection layer 7 of AlTi alloy is deposited on the second dielectric layer 6.An example of the sputtering conditions used here is using an inactive gas like Ar gas as the atmospheric gas and adjusting the sputtering power to 2.5 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 2 having formed layers up to the first reflection layer 7 is removed from the fifth sputtering chamber.",
"After that, the ultraviolet-setting resin layer 8 is formed on the top surface of the first reflection layer 7 by spin coating or roll coating.",
"Through those steps, the WO type optical disc 1 according to the first embodiment, using GeTe, which is a phase-changeable material, as the recording layer 4 is completed.",
"As the optical disc 1 according to the first embodiment, which can be manufactured as explained above, the Inventor prepared optical discs 1 that are changed in thickness a of the record-assist layer 5 in the range of d/4≦a≦3d, namely as a=d/4, a=d/2, a=d, a=2d and a=3d, and measured their recording and reproducing characteristics by an evaluator.",
"That is, by evaluating recording/reproducing characteristics of find kinds of optical discs 1, the Inventor evaluated dependency of the carrier upon the thickness of the record-assist layer 5.The evaluator used for evaluation of those optical discs was adjusted to use laser beams having the wavelength of 660 nm and adjusted in numerical aperture of the objective lens to 0.575.A result of this evaluation is shown in FIG.",
"2.In the graph of FIG.",
"2, the abscissa indicates the thickness of the record-assist layer 5 and the ordinate indicates the carrier.",
"Evaluated values of lands are shown by ♦ and evaluated values of grooves by ▪.",
"The Inventor further carried out optical calculation about dependency of the laser beam amplitude upon the thickness of the record-assist layer 5.A result of the calculation is shown in FIG.",
"3.It is appreciated from FIG.",
"2 that, in grooves, the carrier gradually increases as the thickness a of the record-assist layer 5 decreases within the range from d/4 to 3d.",
"In contrast, in lands, the carrier changes less even when the thickness a of the record-assist layer 5 decreases within the range from d/4 to 3d.",
"That is, it is understood that the dependency upon the thickness of the record-assist layer 5 in changes of the carrier appears more clearly in grooves.",
"This means that, by intentionally changing the thickness of the record-assist layer 5, the signal characteristics in grooves can be changed while the signal characteristics in lands are maintained substantially constant.",
"It is also appreciated from FIG.",
"3 that the amplitude rapidly decreases as the thickness a of the record-assist layer 5 is once decreased and thereafter decreased to ¼ of the thickness d of the recording layer 4.This demonstrates that the control of the thickness of the record-assist layer 5 carried out for the purpose of assuring predetermined amplitude becomes very difficult.",
"On the other hand, in case the thickness a of the record-assist layer 5 is larger than ¼ of the thickness of the recording layer 4, changes of the amplitude are very moderate, and it will be easy to control the thickness of the record-assist layer 5.From this point of view, the thickness a of the record-assist layer 5 is preferably larger than ¼ of the thickness d of the recording layer 4.It is further appreciated from FIG.",
"2 that, when the thickness a of the record-assist layer 5 is decreased to d/4, both the value of the carrier in lands and the value of the carrier in grooves decreases.",
"Therefore, if the thickness a of the record-assist layer 5 is ¼ or less of the thickness d of the recording layer 4 (a≦d/4), both the signal property of lands and the signal property of grooves deteriorate.",
"That is, the thickness a of the record-assist layer 5 must be larger than ¼ of the thickness d of the recording layer 4 (d/4<a), and it is preferably adjusted to ½ or more of the thickness d of the recording layer 4 (d/2≦a).",
"Further, according to the knowledge of the Inventor obtained by the experiment, the record-assist layer 5 can be formed not to disconnect in form of islands on the recording layer 4 by adjusting the thickness a of the record-assist layer 5 to be larger than ¼ of the thickness d of the recording layer 4 (d/4<a).",
"Accordingly, it is possible to form the record-assist layer 5 evenly.",
"It is further appreciated from FIG.",
"3 that, as the thickness a of the record-assist layer 5 is larger, the amplitude gradually decreases.",
"Therefore, the thickness a of the record-assist layer 5 is preferably adjusted not to exceed three times of the thickness d of the recording layer 4.It is further appreciated from FIG.",
"2 that, when the thickness a of the record-assist layer 5 is increased to or beyond three times of the thickness d of the recording layer (3d≦a), both the value of the carrier in lands and the value of the carrier in grooves significantly decreases.",
"Therefore, if the thickness a of the record-assist layer 5 is three times or more of the thickness d of the recording layer (3d≦a), then both the signal property of lands and the signal property of grooves deteriorate and substantially result in the result of calculation shown in FIG.",
"3.That is, thickness a of the record-assist layer 5 must be limited below three times of the thickness d of the recording layer 4 (a<3d), and it is preferably adjusted not to exceed two times of the thickness d of the recording layer 4 (a≦2d).",
"For the purpose of investigating dependency of optical cross talk upon the thickness of the record-assist layer, the Inventor measured signal quantities of cross talk from lands to grooves and signal quantities of cross talk from grooves to lands upon recording 2T marks.",
"FIG.",
"4 shows the dependency of the cross talk signal quantity upon the dependency of the thickness of the record-assist layer 5.In FIG.",
"4, the ordinate shows cross talk in relative decibel for enabling comparison with the value of the carrier, and indicates cross talk from grooves to lands by ♦ and cross talk from lands to grooves by ▪.",
"It is appreciated from FIG.",
"4 that, as the thickness a of the record-assist layer 5 decreases from 3d to d/2, cross talk leaking from grooves to lands decreases even though the signal quantity in grooves increases (see FIG.",
"2).",
"On the other hand, cross talk leaking from lands to grooves increases only a little as the thickness a of the record-assist layer 5 decreases from 3d to d/2, and no remarkable increase is observed.",
"That is, even when the thickness of the record-assist layer 5 is changed, it is apparent that the carrier can be controlled in lands and grooves while suppressing the increase of the cross talk.",
"As explained heretofore, the optical disc according to the first embodiment, which has the multi-layered film including the first dielectric layer 3, recording layer 4, record-assist layer 5, second dielectric layer 6 and reflection layer 7 deposited on one major surface of the disc substrate 2 and employs the land/groove recording technique, can increase the signal amplitude especially in grooves 2c by adjustment of the thickness a of the record-assist layer to be larger than ¼ of the thickness d of the recording layer 4 and smaller than three times thereof, and can simultaneously keep both the signal property in lands 2b and the signal property in grooves 2c in a good condition by controlling the thickness of the record-assist layer 5 after optimizing the signal property in lands 2b.",
"Furthermore, since the signal amplitude in grooves 2c can be changed by while the signal amplitude in lands 2b is maintained constant, by intentional control of the thickness of the record-assist layer 5, both the signal amplitude in lands 2b and the signal amplitude in grooves 2c can be changed independently from each other.",
"This increases the design freedom and enables optimization of lands 2b and grooves 2c while maintaining the optimum deposition condition of the recording layer 4.Therefore, information signals can be reproduced from optical discs in a good condition of the signal property.",
"Thus a highly reliable optical disc can be obtained.",
"Next explained is an optical disc according to the second embodiment of the invention.",
"FIG.",
"5 shows the optical disc according to the second embodiment of the invention.",
"The optical disc according to the second embodiment is a programmable optical disc permitting rewriting of information signals.",
"As shown in FIG.",
"5, the optical disc 11 according to the second embodiment includes a multi-layered film made of a second reflection layer 13, third dielectric layer 14, record-assist layer 15, recording layer 16 and fourth dielectric layer 17 sequentially stacked on one major surface 12a of a disc substrate 12, and a light-transmitting layer 18 formed to cover the multi-layered film.",
"The disc substrate 12 is made of a low water-absorbing material such as polycarbonate (PC) or cycloolefin polymer (such as Zeonex (registered trademark)).",
"Thickness of the disc substrate 12 is, for example, in the range from 0.6 to 1.2 mm.",
"In the second embodiment, it is 1.1 mm thick, for example.",
"Diameter of the disc substrate 12 is 120 mm for example.",
"The optical disc according to the second embodiment is configured to record/reproduce information signals when a laser beam is irradiated to he disc substrate 12 from one side thereof where the thin light-transmitting layer 18 exists.",
"Therefore, the disc substrate 12 can be selected without taking it into consideration whether the material is permeable or not, and a substrate of a metal such as Al is also usable.",
"Alternatively, a glass substrate or a resin substrate of polyolefin, polyimide, polyamide, polyphenylene sulfide or polyethylene terephthalate is also usable as the disc substrate 12.The major surface 12a of the disc substrate 12 has formed a track-like concavo-convex structure.",
"Furrows of the track-like concavo-convex structure are portions remoter from the laser beam injection side.",
"In the second embodiment, the furrows are called lands 2b.",
"The ridges are portions closer to the laser beam injection side.",
"In the second embodiment, the ridges are called grooves 12c.",
"From the same reasons as those of the first embodiment, ratio between the width Dl of each land 2b and the width Dg of each groove 2, i.e.",
"the duty ratio (Dl/Dg), is selected from the range of 0.5 to 2.0, or more preferably from the range of 0.8 to 1.2.In an example of configurations and sizes of the disc substrate 12 according to the second embodiment, the track pitch (Tp) of the groove tracks is 0.77 μm, depth of the grooves (depth of the furrows) is 70 nm, width Dl of the lands 12b is 0.82 μm, width Dg of the grooves 12c is 0.72 μm, and the duty ratio is (0.82/0.72=) 1.14.The second reflection layer 13 is made of Al alloy, for example.",
"In the second embodiment, it is made of AlTi alloy.",
"From the same reasons as those of the first embodiment, thickness of the second reflection layer 13 is adjusted in the range from 20 to 100 nm.",
"For example, it may be 60 nm thick.",
"The third dielectric layer 14 is preferably made of a material exhibiting a low absorption power to recording/reproducing laser beams, and more specifically, an extinction coefficient k not larger than 0.3.Taking the resistance to heat into consideration as well, ZnS—SiO2 (especially one having a mol ratio around 4:1) is one of preferable materials.",
"Material of the record-assist layer 15 contains as its major component at least one compound selected from the group consisting of SnTe, SiN, SiC, GeN, PbTe, SnSe, PbSe, Bi2Te3 and Sb2Te3.More specifically, it is a material containing any of the above-specified compounds by at least 50 volume %, or more preferably, by at least 70 volume %.",
"The record-assist layer 15 is preferably in uniform contact with the recording layer 16.In the second embodiment, the record-assist layer 15 is made of SnTe, for example.",
"From the same reasons as those of the first embodiment, thickness a of the record-assist layer 15 is adjusted in the range larger than ¼ of the thickness d of the recording layer 16, explained later, and smaller than three times of same.",
"That is, record-assist layer 15 is formed to satisfy the following relation between its thickness a and the thickness d of the recording layer 16, d/4<a<3d or more preferably to satisfy d/2≦a≦2d from the viewpoint of improving the signal property.",
"The recording layer 16 is made of GeTe alloy, for example.",
"From the same reasons as those of the first embodiment, thickness d of the recording layer 4 is adjusted in the range of 5 nm≦d≦50 nm.",
"According to the knowledge of the Inventor obtained by an experiment of the signal property in the recording layer 16, thickness d of the recording layer is preferably adjusted in the range of 10 nm≦d≦40 nm, taking account of the range capable of obtaining good signals.",
"In the first embodiment, thickness of the recording layer 16 is around 20 nm, for example.",
"The fourth dielectric layer 17 is preferably made of a material exhibiting a low absorption power (absorptivity) to recording/reproducing laser beams, and more specifically, an extinction coefficient k not larger than 0.3.Taking the resistance to heat into consideration as well, SiN is one of preferable materials.",
"Thickness of the fourth dielectric layer 17 is adjusted not to exceed 200 nm.",
"In the second embodiment, it may be 10 nm thick, for example.",
"In the second embodiment, the third dielectric layer 14 and the fourth dielectric layer 17 are made of different materials.",
"However, they may be made of a common material.",
"In the optical disc according to the second embodiment, having the above-explained configuration, a laser beam L2 is injected toward the disc substrate 12 from one side thereof where the multi-layered film exists as shown in FIG.",
"5, and when it irradiates the recording layer 16 above the lands 12b and above the grooves 12c, information signals are recorded and/or reproduced.",
"Next explained is a manufacturing method of the optical disc 11 according to the second embodiment having the above-explained configuration.",
"First prepared is a clean disk substrate 12 having guide grooves (concavo-convex groove tracks) (for example, thickness: 1.1 mm, track pitch: 0.77 μm, width of lands: 0.82 μm, width of grooves: 0.72 μm, depth of grooves: 70 nm).",
"Subsequently, the disc substrate 12 having formed the concavo-convex groove tracks is transported into a first sputtering chamber, and placed in a predetermined position.",
"Thereafter, an AlTi film is formed at least on groove tracks with ridges and grooves by sputtering using a target of AlTi alloy.",
"As a result, the second reflection layer 13 of AlTi alloy is deposited on the disc substrate 12.An example of the sputtering conditions used here is using an inactive gas like Ar gas as the atmospheric gas and adjusting the sputtering power to 2.5 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 12 having formed the second reflection layer 13 is removed from the first sputtering chamber.",
"The disc substrate having formed the second reflection layer 13 is next introduced into a second sputtering chamber and placed on a predetermined position.",
"Thereafter, a ZnS—SiO2 film is formed on the second reflection layer 13 by sputtering using a ZnS—SiO2 target.",
"As a result, the third dielectric layer 14 of ZnS—SiO2 is deposited on the second reflection layer 13.An example of the sputtering conditions used here is using an inactive gas like Ar gas as the atmospheric gas and adjusting the sputtering power to 0.6 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 12 having formed layers up to the third dielectric layer 14 is removed from the second sputtering chamber.",
"The disc substrate 12 having formed layers up to the third dielectric layer 14 is next introduced into a third sputtering chamber and placed on a predetermined position.",
"After that, a SnTe alloy film is deposited on the third dielectric layer 14 by sputtering using a SnTe alloy target.",
"As a result, the record-assist layer 15 of SnTe alloy is formed evenly on the third dielectric layer 14.An example of the sputtering conditions used here is using an inactive gas such as Ar gas as the atmospheric gas, adjusting the sputtering power to 0.3 kW and the pressure of the atmospheric gas to 1.3×10−1 Pa (1.0 mTorr).",
"Thereafter, the disc substrate 12 having formed layers up to the record-assist layer 15 is removed from the third sputtering chamber.",
"The disc substrate 12 having formed layers up to the record-assist layer 15 is next introduced into a fourth sputtering chamber and placed on a predetermined position.",
"Thereafter, a film of GeSbTe, for example, is formed on the record-assist layer 15 by sputtering.",
"As a result, the recording layer 16 of GeSbTe alloy is obtained.",
"An example of the sputtering conditions used here is using an inactive gas such as Ar gas as the atmospheric gas, adjusting the sputtering power to 0.3 kW and the pressure of the atmospheric gas to 0.4 Pa (3.0 mTorr).",
"Thereafter, the disc substrate 12 having formed layers up to the recording layer 16 is removed from the fourth sputtering chamber.",
"The disc substrate 12 is next introduced into a fifth sputtering chamber and placed on a predetermined position.",
"After that, a film of SiN, for example, is formed on the second reflection layer 13 by sputtering using nitrogen (N2) gas as the reactive gas.",
"As a result, the third dielectric layer 14 of SiN is deposited on the second reflection layer 13.An example of the sputtering conditions is using an inactive gas such as argon (Ar) gas and a reactive gas such as N2 gas as the atmospheric gas, adjusting their flow rates to be Ar:N2=3:1, maintaining its pressure in 0.6 Pa, and supplying the sputtering power of 2.5 kW.",
"After that, the disc substrate 12 having formed the fourth dielectric layer 17 is removed from the fifth sputtering chamber.",
"Thereafter, a light-transmitting sheet 18b is bonded via a bonding layer 18a to the multi-layered film made of the second reflection layer 13, third dielectric layer 14, record-assist layer 15, recording layer 16 and fourth dielectric layer 17 by using a predetermined bonding machine (not shown).",
"As a result, the light-transmitting layer 18 is formed.",
"Through those steps, the optical disc 11 according to the second embodiment is completed.",
"With the optical disc 11 according to the second embodiment, the same effects as those of the first embodiment can be obtained by limiting the thickness a of the record-assist layer 15 within the range of d/4<a<3d relative to the thickness d of the recording layer 16, and it has been confirmed that the same effects as those of the optical disc 1 according to the first embodiment can be ensured also with the optical disc 11 having that configuration.",
"Although the invention has been explained by way of specific embodiments, the invention is not limited to those embodiments but can be modified in various modes based on the technical concept of the invention.",
"For example, numerical values and materials shown in the explanation of the embodiments are not but mere examples, and other numerical values and materials may be used if necessary.",
"For example, the first embodiment has been explained as applying the invention to a WO type optical disc having the recording layer 4 made of a phase-changeable material and configured to record in a WO manner.",
"However, application of the invention is not limited to WO type optical discs, but can be extended to other type optical discs such as programmable optical discs.",
"The second embodiment has been explained as using AlTi alloy as the material of the second reflection layer 13.However, material of the second reflection layer 13 may be selected from other Al alloys such as AlCu alloy, Al, silver (Ag), Ag alloys such as AgPdCu alloys, copper (Cu), Cu alloys, and so on.",
"Moreover, although the second embodiment has been explained as using the recording layer made of GeSbTe alloy, any other appropriate material such as GeTe alloy, GeInSbTe alloy, AgInSbTe alloy, or the like, can be used in accordance with the desired property.",
"Furthermore, although the second embodiment has been explained as employing 0.77 μm as the track pitch Tp of the concavo-convex tracks formed on one major surface 12a of the disc substrate 12, a different value may be employed where necessary.",
"In case a blue laser having the wavelength around 405 nm is used as the laser beam and NA is enhanced approximately to 0.85 by using two-group lenses, the track pitch Tp may be reduced to 0.74 μm or smaller, such as around 0.3 μm.",
"As described above, the invention enables freer design of optical recording mediums for obtaining more excellent signal characteristics and ensures optical mediums having excellent signal characteristics and high reliability by adjusting the thickness d of the recording layer and the thickness a of the record-assist layer to satisfy d/4<a<3d in optical recording mediums having the recording layer capable of recording information signals by irradiation of a laser beam and the multi-layered film stacking at least the recording layer and the record-assist layer at least on one surface of the disc substrate having formed concavo-convex."
]
] | Patent_10276126 |
[
[
"Bellows for consant-velocity joints",
"A convoluted boot for sealing an annular gap between two parts which are connected to one another in a rotationally fast way, which can be articulated relative to one another and which are axially displaceable relative to one another, especially of a constant velocity plunging joint, consisting of a low-strain polymer hard material, having a first larger collar 11 to be secured to a first component, a second smaller collar 12 to be secured to a second component and a plurality of annular fold units which extend between the first collar and the second collar and which, in the form of outer folds consisting of two annular flanks 31, 32, form a fold peak 21 between two fold valleys 22, in a first group A of at least three annular folds with a connection to the first collar 11, the diameters of the fold peaks 21 and fold valleys 22 decrease in the direction from the first collar to the second collar, in a second group B of annular folds with a connection to the second collar 12 of at least one fold, the diameters of the fold valleys and optionally of the fold peaks 21 are constant, the ratio of the diameter D1 of the fold peak 21 of the largest annular fold A1 of the first group A to the diameter D2 of the second collar 12 amounts to ≧2.5."
],
[
"1.A convoluted boot for sealing an annular gap between two parts which are connected to one another in a rotationally fast way, which can be articulated relative to one another and which are axially displaceable relative to one another, the boot comprising: a low-strain hard polymer material, having a first larger collar (11) to be secured to a first component, a second smaller collar (12) to be secured to a second component and a plurality of annular fold units which extend between the first collar and the second collar and which, in the form of outer folds comprising two annular flanks (31, 32), form a fold peak (21) between two fold valleys (22); wherein, in a first group (A) of at least three annular folds with a connection to the first collar (11), the diameters of the fold peaks (21) and fold valleys (22) decrease from the first collar to the second collar, and wherein in a second group (B) of at least one annular fold with a connection to the second collar (12), the diameters of the fold valleys are constant; and wherein the ratio of the diameter (D1) of the fold peak (21) of the largest annular fold (A1) of the first group (A) to the diameter (D2) of the second collar (12) is greater than or equal to 2.5 and the two annular flanks (31, 32) of each of the annular folds of the first group (A) form opposed angles with a radial plane (R), wherein a smaller angle (β) is formed by the annular flank (32) pointing towards the second collar (12) and wherein a larger angle (α) is formed by the annular flank pointing towards the first collar (11), the larger angle (α) being at least 25° greater than the smaller angle (β).",
"2.A convoluted boot according to claim 1, wherein the first group (A) comprises up to five annular folds.",
"3.A convoluted boot according to claim 1 wherein the second group (B) comprises up to eight annular folds.",
"4.A convoluted boot according to claim 1 wherein the annular flanks (31, 32) of each of the annular folds of the second group (B) form opposed angles with a radial plane, wherein the annular flank (32) pointing towards the second collar (12) forms first angle (β) and wherein the annular flank (31) pointing towards first collar (11) forms a second angle (α) being in a range from 5° smaller to 5° greater than the first angle (β).",
"5.A convoluted boot according to claim 1 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there are provided transition folds (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"6.A convoluted boot according to claim 5, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"7.A convoluted boot according to claim 1 wherein the convoluted boot comprises a thermoplastic elastomer (TPE).",
"8.A convoluted boot according to claim 7, wherein the TPE comprises a polyurethane (TPU).",
"9.A convoluted boot according to claim 7, wherein the TPE comprises a polyester (TPEE).",
"10.A convoluted boot according to claim 9, wherein the TPEE is a polyether ester or a polyester ester.",
"11.A convoluted boot according to claim 7, wherein the TPE comprises a polyamide (TPA).",
"12.A convoluted boot according to claim 7, wherein the TPE comprises a polyolefin (TPO).",
"13.A convoluted boot according to claim 12, wherein the TPO is a polypropylene or a polyethylene.",
"14.A convoluted boot according to claim 1 wherein the diameters of the fold peaks (21) of the second group (B) of annular folds are constant.",
"15.A convoluted boot according to claim 2 wherein the second group (B) comprises up to eight annular folds.",
"16.A convoluted boot according to claim 2 wherein the annular flanks (31, 32) of each of the annular folds of the second group (B) form opposed angles with a radial plane, wherein the annular flank (32) pointing towards the second collar (12) forms first angle (β) and wherein the annular flank (31) pointing towards first collar (11) forms a second angle (α) which differs from the first angle (β) not more than 5°.",
"17.A convoluted boot according to claim 3 wherein the annular flanks (31, 32) of each of the annular folds of the second group (B) form opposed angles with a radial plane, wherein the annular flank (32) pointing towards the second collar (12) forms first angle (β) and wherein the annular flank (31) pointing towards first collar (11) forms a second angle (α) which differs from the first angle (β) not more than 5°.",
"18.A convoluted boot according to claim 2 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there is provided at least one transition fold (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"19.A convoluted boot according to claim 3 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there is provided at least one transition fold (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"20.A convoluted boot according to claim 4 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there is provided at least one transition fold (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"21.A convoluted boot according to claim 16 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there is provided at least one transition fold (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"22.A convoluted boot according to claim 7 wherein, between the annular folds of the first group (A) and the annular folds of the second group (B), there is provided at least one transition fold (C) whose diameters deviate from one another at the fold valleys (22), wherein the diameter of the fold valley at the annular flank (31) pointing towards the first collar (11) is greater than the diameter of the fold valley at the annular flank (32) pointing towards the second collar (12).",
"23.A convoluted boot according to claim 18, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"24.A convoluted boot according to claim 19, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"25.A convoluted boot according to claim 20, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"26.A convoluted boot according to claim 21, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"27.A convoluted boot according to claim 22, wherein the annular flanks (31, 32) of the transition fold (C) form opposed angles with a radial plane (R), wherein the annular flank (32) pointing towards the second collar (12) forms a first angle (β) and that the annular flank (31) pointing towards the first collar (11) forms a second angle (α) which is defined by (β+25°)≧α≧(β+5°).",
"28.A convoluted boot according to claim 27 wherein the convoluted boot comprises a thermoplastic elastomer (TPE)."
],
[
"DESCRIPTION The invention relates to a convoluted boot for sealing an annular gap between two parts which are connected to one another in a rotationally fast way, which can be articulated relative to one another and which are axially displaceable relative to one another, especially of a constant velocity plunging joint, consisting of a low-strain hard polymer material, having a first larger collar to be secured to a first component, a second smaller collar to be secured to a second component and a plurality of annular fold units which extend between the first collar and the second collar and which, in the form of outer folds consisting of two annular flanks, form a fold peak between two fold valleys.",
"Because requirements become more and more stringent, convoluted boots of said type are produced from hard polymer materials to an increasing extent.",
"As compared to soft resilient materials used earlier, said hard polymer materials feature an improved resistance in mechanical and chemical respects, and in view of higher speeds and the need for a longer service life, it is inevitable that they are used.",
"One concern is their reduced flexibility which can be a problem at low temperatures.",
"In the case of constant velocity fixed joints which effect articulation only between two rotating components, convoluted boots made of said materials, even today, do meet the respective requirements in their entirety.",
"In the case of constant velocity plunging joints which, in addition to the angular movement, effect an axial displacement between the two rotating components, this means that the sealing convoluted boots are subject to friction contact between the annular flanks on the inside of the angle when the joint is in a telescoped and articulated condition and, when the joint is in the extended and articulated condition, the individual annular folds open up excessively widely on the outside of the angle, causing a collapsing of the annular folds or other irregularities.",
"At high speeds, in particular, this can result in the elasticity limit of the convoluted boot being exceeded and it can lead to boot damage.",
"It is therefore the object of the present invention to provide a convoluted boot of said type which is able to meet more stringent requirements in operation without suffering any damage and which is therefore particularly suitable for constant velocity plunging joints.",
"The objective is achieved by providing a convoluted boot of the initially mentioned type with the following characteristics: in a first group of at least three annular folds joining the first collar, the diameters of the fold peaks and fold valleys decrease from the first collar to the second collar, in a second group of annular folds joining the second collar of at least one fold, the diameters of the fold valleys and—if more than one present—of the fold peaks are constant, the ratio of the diameter D1 of the fold peak of the largest annular fold of the first group to the diameter D2 of the second collar amounts to ≧2.5.By providing two special groups of folds, the deformation of the convoluted boot when articulation and changes in axial length occur is allocated to said two groups in such a way that, in the first group consisting of folds of a decreasing size, deformation largely takes place in the form of articulation, whereas the second group consisting of at least one fold with a small diameter largely accommodates the change in axial length by being extended.",
"Convoluted boots with two different groups of folds are known from DE 33 09 386 C1, DE 43 23 686 C2, DE 196 01 096 A1 and DE 198 06 173 C1 for example.",
"However, in these cases, between the two groups of folds, there exists a region which is in constant contact with the inserted shaft.",
"From DE 38 73 496 T2, there is known a convoluted boot of said type with two different functional regions, having folds whose flanks extend parallel relative to one another and, on the outside, are connected to one another by a rounded annular bead.",
"The annular folds according to the present invention, however, when the two collars are coaxially aligned relative to one another in the fitted condition, are contact-free relative to the inserted shaft and the annular folds each, approximately, comprise the shape of a double cone.",
"The first group of folds can comprise up to five annular folds and the second group of folds can comprise up to eight annular folds.",
"The number of folds depends on the respective requirement profile.",
"It is obvious that with an increased number of annular folds, the need for a longer boot length also increases, but the angle work to be carried out by the individual folds generally decreases during articulation.",
"However, this does not affect the possibility of varying the shape of the individual folds within certain ranges, i.e.",
"that it is possible to provide more pointed and wider folds, but in principle, the folds of the first group are wider (have a larger volume) and the folds of the second group are more pointed (narrower).",
"According to a preferred embodiment it is proposed that the two annular flanks of each of the annular folds of the first group form opposed angles with a radial plane, wherein a smaller angle β is formed by the annular flank pointing towards the second collar and wherein a larger angle α with ≧(β+25°) is formed by the annular flank pointing towards the first collar.",
"Furthermore, it is proposed that the annular flanks of each of the annular folds of the second group form opposed angles with a radial plane, wherein the annular flank pointing towards the second collar forms an angle β and wherein the annular flank pointing towards the first collar forms an angle α which is defined by (β+5°) As the approximately conical annular flanks, in particular the annular flanks of the first group which point towards the first collar, can be curved so as to comprise a convex outside, it is specified hereby that the reference for the angle values has to be the conical face between the outer line of a fold valley and the outer line of a fold peak and that, if viewed in a longitudinal section, it has to be the straight line between the smallest outer radius of a fold valley and the greatest outer radius of a fold peak.",
"According to a further embodiment it is proposed that between the annular folds of the first group and the annular folds of the second group, there is provided a transition fold whose diameters at the fold valleys deviate from one another, wherein the diameter of the annular flank pointing towards the first collar is greater than the diameter of the annular flank pointing towards the second collar.",
"In particular, it is proposed that the annular flanks of the transition fold form opposed angles with a radial plane, wherein the annular flank pointing towards the second collar forms an angle β and that the annular flank pointing towards the first collar forms an angle α which is defined by (β+25°)≧α≧(β+5°).",
"Said transition fold is thus similar to the annular folds of the first group in that the diameter of the fold valleys decreases towards the second collar; however, as far as the angle configuration of the annular flanks is concerned, said transition fold is similar to the annular folds of the second group.",
"The purpose is to ensure that even at larger angular movements of the convoluted boot, there is preferably no friction contact between the fold valleys and the inserted shaft.",
"A preferred material for the convoluted boot is a thermoplastic elastomer (TPE); in particular, there are proposed materials based on polyurethane (TPU), on polyester (TPEE), in particular a polyether ester or a polyester ester, materials based on polyamide (TPA) or on polyolefin, in particular polypropylene or polyethylene.",
"A preferred embodiment of the convoluted boot in accordance with the invention will be described below with reference to the drawings wherein FIG.",
"1 shows an inventive convoluted boot in half a longitudinal section, with different individual characteristics having been drawn in.",
"FIG.",
"2 shows an illustration similar to that of FIG.",
"1 in which the dimensions specified by the invention are given.",
"FIG.",
"3 shows two annular folds in half a longitudinal section, giving the values of the angles.",
"FIG.",
"1 shows a convoluted boot comprising a first collar 11 with a larger diameter with an attaching region 11′, a second collar 12 with a smaller diameter with an attaching region 12′, as well as a total of seven complete annular folds.",
"A first group A comprises three annular folds A1, A2, A3, with the diameters of their fold valleys and fold peaks decreasing from fold to fold from the first collar 11 to the second collar 12.A second group B of two complete folds B1 and B2 and with a total of three fold peaks comprises fold valleys and fold peaks which comprise identical diameters.",
"A third transition fold C is positioned between the first group A and the second group B and comprises fold valleys with decreasing diameters from the first collar 11 to the second collar 12.At fold A1 it is indicated that a complete annular fold is regarded as an outer fold with a fold peak 21 between two fold valleys 22.In FIG.",
"2, any details identical to those shown in FIG.",
"1 have been given the same reference numbers.",
"To that extent, reference is made to the preceding description.",
"D1 refers to the outer diameter of the first fold A1 of the first group A and D2 refers to the outer diameter of the second collar 12.In accordance with the invention, the ratio of the two diameters is ≧2.5.FIG.",
"3 shows an individual fold, and in FIG.",
"3a, one fold, which can be fold A1 for example, comprises a first annular flank 31 and a second annular flank 32.A radial plane R is positioned normally on the longitudinal boot axis L. The opening angle of the first annular flank 31 relative to the radial plane R has been given the reference symbol a and the opening angle of the second annular flank 32 relative to the radial plane R has been given the reference symbol β.",
"FIG.",
"3b shows that at an annular flank 31 with a convex outer face or, optionally, with a concave outer face, the arm of the opening angle α deviating from the radial plane is formed by a straight line between the greatest circumferential line 41 of the fold peak and the smallest circumferential line 42 of the adjoining fold valley.",
"FIG.",
"3a shows that the arm of, the opening angle α deviating from the radial plane is formed by a strictly conical surface of the annular flank (32) itself."
]
] | Patent_10276208 |
[
[
"Method and apparatus of dsp resource allocation and use",
"Certain embodiments of the invention support the distribution and control of multiple instruction streams going to multiple steps of modules supporting arithmetic and memory, as well as method for numeric data processing optimizing non-additive functions and operations through a novel numeric representation and the use of novel ALU components to support numeric conversions and evaluations of non-additive functions."
],
[
"1.A circuit supporting partitioned operation of multiple concurrently presented instructions comprising: a first instruction wire bundle; a second instruction wire bundle; a clock wire bundle possessing a capture state; a partition control wire bundle possessing a partition control state; an arithmetic module circuit coupled to said partition control wire bundle and further comprised of a first memory circuit coupled to said partition control wire bundle; and a first arithmetic logic unit coupled to said partition control wire bundle; wherein said module circuit samples said first instruction wire bundle and said second instruction wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and wherein said arithmetic module circuit responds to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state; wherein said arithmetic module circuit response is further comprised of: said first memory circuit responds to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responds to at least one member of said sample instruction collection based upon said partition control state; wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein said first memory circuit is further comprised of at least two memory sub-circuits, each memory sub-circuit corresponding to a member of said ALU part collection; wherein each member of said part ALU collection and said corresponding memory sub-circuit are coupled to said partition control wire bundle; and wherein each member of said part ALU collection and said corresponding memory sub-circuit both respond to exactly one and the same member of said sample instruction collection based upon said partition control state.",
"2.The circuit of claim 1, wherein said first part ALU presents a first carry signal to said second part ALUs; wherein said second part ALU ignores said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.",
"3.The circuit of claim 1, further comprising: an input-output circuit coupled to said arithmetic module circuit, to said partition control wire bundle; wherein said input-output circuit responds to at least one member of said sample instruction collection based upon said partition control state.",
"4.The circuit of claim 3, wherein said input-output circuit presents a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; wherein said input-output circuit presents a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.",
"5.The circuit of claim 4, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein each of said second sub-circuits corresponding to said member of said ALU responds to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, said second sub-circuit further responds by approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.",
"6.The circuit of claim 5, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input; 7.The circuit of claim 6, wherein each member of said ALU part collection presents a result to said corresponding said second sub-circuit.",
"8.The circuit of claim 7, further comprising a second of said arithmetic module circuits coupled to said input-output circuit; wherein said input-output circuit presents said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein said input-output circuit presents said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"9.The circuit of claim 8, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"10.The circuit of claim 1, further comprising a first datapath instruction processor coupled to said first datapath instruction wire bundle presented to said module circuit; wherein said first datapath instruction processor asserts said first datapath instruction wire bundle; and a second datapath instruction processor coupled to said second datapath instruction wire bundle presented to said module circuit; wherein said second datapath instruction processor asserts said second datapath instruction wire bundle.",
"11.The circuit of claim 10, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said datapath instruction queue pointer responds to said clock wire bundle by asserting an asserted datapath instruction address; wherein said datapath instruction register responds to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and wherein said datapath instruction register further asserts said associated datapath instruction wire bundle based upon said datapath instruction capture state.",
"12.The circuit of claim 11, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of: an instruction store coupled to said datapath instruction address wire bundle and to said datapath instruction wire bundle; wherein said instruction store responds to said asserted datapath instruction address via said datapath instruction address wire bundle to asserting said datapath instruction wire bundle based upon said asserted datapath instruction address.",
"13.The circuit of claim 12, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.",
"14.A circuit implementation of claim 1, wherein a component list comprises said arithmetic module circuit, said first memory circuit coupled to said partition control wire bundle, and said first arithmetic logic unit; wherein each member of said component list is implemented with at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"15.The circuit implementation of claim 14, wherein the implementation of all members of said component list further includes only circuitry responding to said exactly one and the same member of said sample instruction collection based upon said partition control state.",
"16.A method of operating a module circuit containing at least an arithmetic module circuit further containing a first memory circuit and a first arithmetic logic unit using a clock wire bundle possessing a capture state and using a partition control wire bundle possessing a partition control state, said method comprising the steps of: said module circuit sampling a first instruction pair wire bundle and a second instruction pair wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and said arithmetic module circuit responding to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state; wherein the step of said arithmetic module circuit responding is further comprised of the steps of: said first memory circuit responding to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responding to at least one member of said sample instruction collection based upon said partition control state; wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein the step of said first arithmetic logic unit responding is further comprised of the steps of said first part ALU responding to exactly one member of said sample instruction collection based upon said partition control; and said second part ALU responding to exactly one member of said sample instruction collection based upon said partition control state; wherein said first memory circuit is comprised of at least two memory sub-circuits, each of said memory sub-circuits corresponding to a member of said ALU part collection; wherein the step of said first memory circuit responding is further comprised of, for each of said memory sub-circuits, the step of said memory sub-circuit responding to exactly one and the same member of said sample instruction collection as said corresponding member of said ALU part collection.",
"17.The method of claim 16, further comprising the step of said first part ALU presenting a first carry signal to said second part ALU; wherein the step of said second ALU responding is comprised of the steps of: said second part ALU ignoring said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.",
"18.The method of claim 16, further comprising the step of: an input-output circuit responding to at least one member of said sample instruction collection based upon said partition control state.",
"19.The method of claim 18, further comprising the steps of: said input-output circuit presenting a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; said input-output circuit presenting a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.",
"20.The method of claim 19, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein said method is further comprised, for each of said second sub-circuits corresponding to said member of said ALU part collection, of the step of: said second sub-circuit responding to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, the step of said second sub-circuit responding is further comprised of the step of: said second sub-circuit approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.",
"21.The method of claim 20, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input.",
"22.The method of claim 21, further comprising the steps of: for each member of said ALU part collection, said member presenting a result to said corresponding said second sub-circuit.",
"23.The method of claim 22, wherein said module circuit is further comprised of a second of said arithmetic module circuits coupled to said input-output circuit; wherein said method is further comprised of the step of: said input-output circuit presenting said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said input-output circuit presenting said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"24.The method of claim 23, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein said method is further comprised, for each member of said second circuit collection coupled to said internal wire bundle of the step of: said member asserting a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said member asserting a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"25.The method of claim 16, further comprising the steps of a first datapath instruction processor asserting said first datapath instruction wire bundle presented to said module circuit; and a second datapath instruction processor asserting said second datapath instruction wire bundle presented to said module circuit.",
"26.The method of claim 25, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said method is further comprised of the steps of said datapath instruction queue pointer responding to said clock wire bundle by asserting an asserted datapath instruction address; said datapath instruction register responding to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and said datapath instruction register asserting said associated datapath instruction wire bundle based upon said datapath instruction capture state.",
"27.The method of claim 26, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of an instruction store; wherein said method is further comprised of the steps of said instruction store responding to said asserted datapath instruction address to assert said datapath instruction wire bundle based upon said asserted datapath instruction address.",
"28.The method of claim 27, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.",
"29.A circuit implementation of the method of claim 16, wherein the steps of the method are each implemented within at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"30.A method of operating a module circuit containing at least an arithmetic module circuit further containing a first memory circuit and a first arithmetic logic unit using a clock wire bundle possessing a capture state and using a partition control wire bundle possessing a partition control state, said method comprising the steps of: said module circuit sampling a first instruction pair wire bundle and a second instruction pair wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and said arithmetic module circuit responding to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state.",
"31.The method of claim 30, wherein the step of said arithmetic module circuit responding is further comprised of the steps of: said first memory circuit responding to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responding to at least one member of said sample instruction collection based upon said partition control state.",
"32.The method of claim 31, wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein the step of said first arithmetic logic unit responding is further comprised of the steps of said first part ALU responding to exactly one member of said sample instruction collection based upon said partition control; and said second part ALU responding to exactly one member of said sample instruction collection based upon said partition control state; wherein said first memory circuit is comprised of at least two memory sub-circuits, each of said memory sub-circuits corresponding to a member of said ALU part collection; wherein the step of said first memory circuit responding is further comprised of for each of said memory sub-circuits, the step of said memory sub-circuit responding to exactly one and the same member of said sample instruction collection as said corresponding member of said ALU part collection.",
"33.The method of claim 32, further comprising the step of said first part ALU presenting a first carry signal to said second part ALU; wherein the step of said second ALU responding is comprised of the steps of: said second part ALU ignoring said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.",
"34.The method of claim 32, further comprising the step of: an input-output circuit responding to at least one member of said sample instruction collection based upon said partition control state.",
"35.The method of claim 34, further comprising the steps of: said input-output circuit presenting a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; said input-output circuit presenting a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.",
"36.The method of claim 35, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein said method is further comprised, for each of said second sub-circuits corresponding to said member of said ALU part collection, of the step of: said second sub-circuit responding to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, the step of said second sub-circuit responding is further comprised of the step of: said second sub-circuit approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.",
"37.The method of claim 36, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input.",
"38.The method of claim 37, further comprising the steps of: for each member of said ALU part collection, said member presenting a result to said corresponding said second sub-circuit.",
"39.The method of claim 38, wherein said module circuit is further comprised of a second of said arithmetic module circuits coupled to said input-output circuit; wherein said method is further comprised of the step of: said input-output circuit presenting said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said input-output circuit presenting said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"40.The method of claim 39, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein said method is further comprised, for each member of said second circuit collection coupled to said internal wire bundle of the step of: said member asserting a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; said member asserting a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"41.The method of claim 30, further comprising the steps of a first datapath instruction processor asserting said first datapath instruction wire bundle presented to said module circuit; and a second datapath instruction processor asserting said second datapath instruction wire bundle presented to said module circuit.",
"42.The method of claim 41, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said method is further comprised of the steps of said datapath instruction queue pointer responding to said clock wire bundle by asserting an asserted datapath instruction address; said datapath instruction register responding to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and said datapath instruction register asserting said associated datapath instruction wire bundle based upon said datapath instruction capture state.",
"43.The method of claim 42, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of an instruction store; wherein said method is further comprised of the steps of said instruction store responding to said asserted datapath instruction address to assert said datapath instruction wire bundle based upon said asserted datapath instruction address.",
"44.The method of claim 43, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle, and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.",
"45.A circuit implementation of the method of claim 30, wherein the steps of the method are each implemented within at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"46.A circuit supporting partitioned operation of multiple concurrently presented instructions comprising: a first instruction wire bundle; a second instruction wire bundle; a clock wire bundle possessing a capture state; a partition control wire bundle possessing a partition control state; an arithmetic module circuit coupled to said partition control wire bundle and further comprised of a first memory circuit coupled to said partition control wire bundle; and a first arithmetic logic unit coupled to said partition control wire bundle; wherein said module circuit samples said first instruction wire bundle and said second instruction wire bundle during said capture state on said clock wire bundle respectively generating a first sampled instruction state and a second sampled instruction state; and wherein said arithmetic module circuit responds to at least one member of a sample instruction collection based upon said partition control state; wherein said sample instruction collection is comprised of said first sampled instruction state and to said second sampled instruction state.",
"47.The circuit of claim 46, wherein said arithmetic module circuit response is further comprised of: said first memory circuit responds to at least one member of said sample instruction collection based upon said partition control state; and said first arithmetic logic unit responds to at least one member of said sample instruction collection based upon said partition control state.",
"48.The circuit of claim 47, wherein said first arithmetic logic unit is further comprised of an ALU part collection comprising a first part ALU and a second part ALU; wherein said first memory circuit is further comprised of at least two memory sub-circuits, each memory sub-circuit corresponding to a member of said ALU part collection; wherein each member of said part ALU collection and said corresponding memory sub-circuit are coupled to said partition control wire bundle; and wherein each member of said part ALU collection and said corresponding memory sub-circuit both respond to exactly one and the same member of said sample instruction collection based upon said partition control state.",
"49.The circuit of claim 48, wherein said partition control state includes to a carry partition control belonging to a partition control state collection comprising first carry control state and a second carry control state; wherein said first part ALU presents a first carry signal to said second part ALUs; wherein said second part ALU ignores said first carry signal whenever said exactly one sample instruction collection member for said first part ALU is different from said exactly one sample instruction collection member for said second part ALU.",
"50.The circuit of claim 48, further comprising: an input-output circuit coupled to said arithmetic module circuit, to said partition control wire bundle; wherein said input-output circuit responds to at least one member of said sample instruction collection based upon said partition control state.",
"51.The circuit of claim 50, wherein said input-output circuit presents a first input to a member of the collection comprising said first ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said first ALU part and said corresponding memory sub-circuit respond; wherein said input-output circuit presents a second input to a member of the collection comprising sad second ALU part and said corresponding memory sub-circuit based upon exactly one and the same member of said sample instruction collection to which said second ALU part and said corresponding memory sub-circuit respond.",
"52.The circuit of claim 51, wherein said arithmetic module circuit is further comprised of a second circuit coupled to said partition control wire bundle; wherein said second circuit is comprised of at least two second sub-circuits, each of said second sub-circuits corresponding to exactly one member of said ALU part collection; wherein each of said second sub-circuits corresponding to said member of said ALU responds to exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein for each of said second sub-circuits, said second sub-circuit further responds by approximately performing at least one member of a non-additive function collection comprising multiplication, division, square root, exponential base N, logarithm base N, sine, cosine, arcsine, arccosine, tangent, cotangent, secant, cosecant and polynomial functions based upon exactly one and the same member of said sample instruction collection to which said ALU part collection member responds; wherein said N is a positive number.",
"53.The circuit of claim 52, wherein an input collection is comprised of said first and said second input; wherein each member of said input collection is comprised of a log-input and a normal-input; 54.The circuit of claim 53, wherein each member of said ALU part collection presents a result to said corresponding said second sub-circuit.",
"55.The circuit of claim 54, further comprising a second of said arithmetic module circuits coupled to said input-output circuit; wherein said input-output circuit presents said first input to at least one member of the collection comprising said first ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein said input-output circuit presents said second input to at least one member of the collection comprising sad second ALU part of said second arithmetic module and said corresponding memory sub-circuit of said second arithmetic module based upon exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"56.The circuit of claim 55, wherein a second circuit collection is comprised of said second circuit of said arithmetic module circuit and said second circuit of said second arithmetic module circuit; wherein at least one member of said second circuit collection couples to an internal wire bundle comprised of a first sub-internal wire bundle and a second sub-internal wire bundle; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a first sub-internal wire state on said first sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said first ALU part of said arithmetic module circuit responds; wherein for each member of said second circuit collection coupled to said internal wire bundle asserts a second sub-internal wire state on said second sub-internal wire bundle based upon said exactly one and the same member of said sample instruction collection to which said second ALU part of said arithmetic module circuit responds.",
"57.The circuit of claim 46, further comprising a first datapath instruction processor coupled to said first datapath instruction wire bundle presented to said module circuit; wherein said first datapath instruction processor asserts said first datapath instruction wire bundle; and a second datapath instruction processor coupled to said second datapath instruction wire bundle presented to said module circuit; wherein said second datapath instruction processor asserts said second datapath instruction wire bundle.",
"58.The circuit of claim 57, wherein a datapath instruction processor collection is comprised of said first datapath instruction processor and said second datapath instruction processor; wherein said first datapath instruction wire bundle is an associated datapath instruction wire bundle of said first datapath instruction processor; wherein said second datapath instruction wire bundle is an associated datapath instruction wire bundle of said second datapath instruction processor; wherein at least one member of said datapath instruction processor collection is further comprised of: a datapath instruction pointer coupled to said clock wire bundle, and to a datapath instruction address wire bundle; and a datapath instruction register coupled to said clock wire bundle, to said asserted datapath instruction wire bundle, and to a datapath instruction wire bundle; wherein said clock wire bundle possesses a datapath instruction capture state; wherein said datapath instruction queue pointer responds to said clock wire bundle by asserting an asserted datapath instruction address; wherein said datapath instruction register responds to said clock wire bundle by capturing a datapath instruction capture state whenever said clock wire bundle is in said datapath instruction capture state; and wherein said datapath instruction register further asserts said associated datapath instruction wire bundle based upon said datapath instruction capture state.",
"59.The circuit of claim 58, wherein for at least one member of said datapath instruction processor collection comprising said datapath instruction pointer is further comprised of: an instruction store coupled to said datapath instruction address wire bundle and to said datapath instruction wire bundle; wherein said instruction store responds to said asserted datapath instruction address via said datapath instruction address wire bundle to asserting said datapath instruction wire bundle based upon said asserted datapath instruction address.",
"60.The circuit of claim 59, wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction processor is further comprised of a last instruction indicator coupled to a last instruction indicator wire bundle possessing a last instruction state and a non-last instruction state; wherein for at least one member of said datapath instruction processor collection comprising said instruction store, said datapath instruction pointer is further coupled to a new datapath instruction address wire bundle and to said last instruction indicator wire bundle; and wherein for at least one member of said datapath instruction processor collection comprising said instruction store is further comprised of: a branch-cache instruction processor coupled to said last instruction indicator and to said new datapath instruction address wire bundle; wherein said branch-cache instruction processor responds to said last instruction state via said last instruction indicator wire bundle by asserting a new datapath instruction address upon said new datapath instruction address wire bundle; and wherein said datapath instruction pointer responds to said last instruction indicator wire bundle by capturing said new datapath instruction address from said new datapath instruction address wire bundle.",
"61.A circuit implementation of claim 46, wherein a component list comprises said arithmetic module circuit, said first memory circuit coupled to said partition control wire bundle, and said first arithmetic logic unit; wherein each member of said component list is implemented with at least part of at least one member of the collection comprising a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array (FPGA); and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"62.The circuit implementation of claim 61,: wherein the implementation of all members of said component list further includes only circuitry responding to exactly one and the same member of said sample instruction collection based upon said partition control state.",
"63.A method of processing numeric data, comprising the step of: representing each member of a number collection by an integer part and a special part; log-converting a member of an input number collection to create a member of said number collection; and exp-converting a member of said number collection to create a member of an output number collection; wherein said special part representing said member of said number collection contains of exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity; wherein said special part of each member of said number collection further contains exactly one member of a second special value collection comprising a special-minus and a special-plus; wherein said integer part of each member of said number collection contains a sign and a magnitude; wherein, for each member of said number collection, said sign is a member of a sign collection consisting essentially of a positive-sign and a negative-sign; wherein said number collection comprises at least a first number and a second number; said method further comprising the steps of: performing all members of the arithmetic operation collection upon at least one of said members of said number collection; wherein said arithmetic operation collection is comprised of the steps of: adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result; wherein said number collection is further comprised of said add-result, said subtract-result, said exp-result and said log-result; wherein the step of adding is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; determining whether said special part of said second number contains said negative-infinity; and setting said special part of said add-result to contain said negative-infinity whenever said special part of at least one member of the collection said first number and said second number contains said negative-infinity; wherein the step of subtracting is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said subtract-result to contain said negative-infinity whenever said special part of said first number contains said negative-infinity; wherein the step of exponentiating is further comprised of the step of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said exp-result to contain said not-negative-infinity and setting said integer part to a zero-representation whenever said special part of said first number contains said negative-infinity; and setting said sign of said exp-result to essentially said negative-sign whenever said special part of said first number contains said special-minus; wherein the step of logarithming is further comprised of the steps of: determining whether said integer part of said first number is essentially equal to said zero-representation; setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number essentially equals said zero-representation; determining whether said sign part of said first number is essentially equal to said negative-sign; and setting said special part of said log-result to contain said special-minus whenever said sign part of said first number is essentially said negative-sign; wherein said input number collection is comprised of a first input number and a second input number.",
"wherein each member of said input number collection is comprised of an integer part; wherein the step log-converting said input number collection member is further comprised of the steps of: determining whether said integer part of said input number collection member is essentially equal to said zero-representation; and setting said special part of said number collection member to contain said negative-infinity whenever said integer part of said input number collection member essentially equals said zero-representation.",
"wherein said output number collection is comprised of a first output number and a second output number; wherein each of said output number collection members include a magnitude; wherein the step exp-converting is further comprised of the steps of: setting said magnitude of said output number collection member to said zero-representation whenever said special part of said number collection member contains said negative-infinity; and setting said sign of said output number collection member to said positive-sign whenever said special part of said number collection member contains said special-plus.",
"64.The method of claim 63, wherein said integer part of each of said number collection members is in a non-redundant numeric notation; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number equals said zero-representation.",
"65.The method of claim 63, wherein said integer part of each of said number collection members is in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number is a member of said zero-representation collection.",
"66.The method of claim 63, wherein said integer part of each of said number collection members is in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps; where the step determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: determining whether said sign part of said first number is equal to said negative-sign based upon performing at least one of the members of said negative-test collection.",
"67.The method of claim 63, wherein said integer part of each of said number collection members is in a non-redundant numeric notation possessing exactly one negative-test step; wherein the step of determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: performing said exactly one negative-test step based upon said first number.",
"68.The method of claim 63, wherein said integer part of each of said input number collection members contains a sign belonging to said sign collection and a magnitude; wherein the step of log-converting is further comprised of the steps of: determining whether said sign part of said input number collection member is essentially equal to said negative-sign; and setting said special part of said number collection member to contain said special-minus whenever said sign part of said input number collection member is essentially said negative-sign.",
"69.The method of claim 63, wherein each of said output number collection members include a sign belong to said sign collection; wherein the step exp-converting is further comprised of the step of: setting said sign of said output number collection member to said negative-sign whenever said special part of said number collection member contains said special-minus.",
"70.The method of claim 69, wherein each of said input number collection members is encoded as an N1 bit code; wherein said N1 is at least three; wherein said integer part of each of said number collection members is encoded as an N2 bit code; wherein N2 is greater than N1.71.A program system for processing numeric data, implementing the steps of claim 63, comprising program steps residing in a memory accessibly coupled to a computer, said program system comprising the program steps of: representing each member of a number collection by an integer part and a special part; log-converting a member of an input number collection to create a member of said number collection; exp-converting a member of said number collection to create a member of an output number collection; determining whether said special part of said first number contains said negative-infinity; adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result.",
"72.The program system of claim 71, wherein the program steps implementing the method are embodied in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon said computer.",
"73.A digital circuit generated from the program steps of claim 72.74.A circuit for processing numeric data, implementing the steps of claim 63, comprising: means for representing each member of a number collection by an integer part and a special part; means for determining whether said special part of said first number contains said negative-infinity; means for adding said first number to said second number to create an add-result; means for subtracting said first number by said second number to create a subtract-result; means for exponentiating said first number to create an exp-result; and means for logarithming said first number to create a log-result.",
"75.The circuit of claim 74, wherein at least one of the means of claim 74 is implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array; and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"76.The circuit of claim 74, wherein at least one of said input number collection members are implemented as a wire state collection received from a wire bundle coupled to said circuit.",
"77.The circuit of claim 74, wherein at least one of said output number collection members are implemented as a wire state asserted by said circuit onto a wire bundle.",
"78.A method of processing numeric data, comprising the step of: representing each member of a number collection by an integer part and a special part; wherein said special part representing said member of said number collection contains of exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity; wherein said number collection comprises at least a first number and a second number; said method further comprising the steps of: performing at least one member of the arithmetic operation collection upon at least one of said members of said number collection; wherein said arithmetic operation collection is comprised of the steps of: adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result; wherein said number collection is further comprised of said add-result, said subtract-result, said exp-result and said log-result; wherein the step of adding is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; determining whether said special part of said second number contains said negative-infinity; and setting said special part of said add-result to contain said negative-infinity whenever said special part of at least one member of the collection said first number and said second number contains said negative-infinity; wherein the step of subtracting is further comprised of the steps of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said subtract-result to contain said negative-infinity whenever said special part of said first number contains said negative-infinity; wherein the step of exponentiating is further comprised of the step of: determining whether said special part of said first number contains said negative-infinity; setting said special part of said exp-result to contain said not-negative-infinity and setting said integer part to a zero-representation whenever said special part of said first number contains said negative-infinity; wherein the step of logarithming is further comprised of the steps of: determining whether said integer part of said first number is essentially equal to said zero-representation; and setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number essentially equals said zero-representation.",
"79.The method of claim 78, wherein said integer part of each of said number collection members is in a non-redundant numeric notation; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number equals said zero-representation.",
"80.The method of claim 78, wherein said integer part of each of said number collection members is in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances; wherein the step of setting said special part of said log-result is further comprised of the step of: setting said special part of said log-result to contain said negative-infinity whenever said integer part of said first number is a member of said zero-representation collection.",
"81.The method of claim 78, wherein said integer part of each member of said number collection contains a sign and a magnitude; wherein, for each member of said number collection, said sign is a member of a sign collection consisting essentially of a positive-sign and a negative-sign; wherein said special part of each member of said number collection further contains exactly one member of a second special value collection comprising a special-minus and a special-plus; wherein the step of exponentiating is further comprised of the steps of: setting said sign of said exp-result to essentially said negative-sign whenever said special part of said first number contains said special-minus; wherein the step of logarithming is further comprised of the steps of: determining whether said sign part of said first number is essentially equal to said negative-sign; and setting said special part of said log-result to contain said special-minus whenever said sign part of said first number is essentially said negative-sign.",
"82.The method of claim 81, wherein said integer part of each of said number collection members is in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps; where the step determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: determining whether said sign part of said first number is equal to said negative-sign based upon performing at least one of the members of said negative-test collection.",
"83.The method of claim 81, wherein said integer part of each of said number collection members is in a non-redundant numeric notation possessing exactly one negative-test step; wherein the step of determining whether said sign part of said first number is essentially equal to said negative-sign is further comprised of the step of: performing said exactly one negative-test step based upon said first number.",
"84.The method of claim 81, further comprising the step of: log-converting a member of an input number collection to create a member of said number collection; wherein said input number collection is comprised of a first input number and a second input number.",
"85.The method of claim 84, wherein each member of said input number collection is comprised of an integer part; wherein the step log-converting said input number collection member is further comprised of the steps of: determining whether said integer part of said input number collection member is essentially equal to said zero-representation; and setting said special part of said number collection member to contain said negative-infinity whenever said integer part of said input number collection member essentially equals said zero-representation.",
"86.The method of claim 85, wherein said integer part of each of said input number collection members contains a sign belonging to said sign collection and a magnitude; wherein the step of log-converting is further comprised of the steps of: determining whether said sign part of said input number collection member is essentially equal to said negative-sign; and setting said special part of said number collection member to contain said special-minus whenever said sign part of said input number collection member is essentially said negative-sign.",
"87.The method of claim 86, further comprising the step of: exp-converting a member of said number collection to create a member of an output number collection; wherein said output number collection is comprised of a first output number and a second output number.",
"88.The method of claim 87, wherein each of said output number collection members include a magnitude; wherein the step exp-converting is further comprised of the step of: setting said magnitude of said output number collection member to said zero-representation whenever said special part of said number collection member contains said negative-infinity.",
"89.The method of claim 88, wherein each of said output number collection members include a sign belong to said sign collection; wherein the step exp-converting is further comprised of the steps of: setting said sign of said output number collection member to said negative-sign whenever said special part of said number collection member contains said special-minus; and setting said sign of said output number collection member to said positive-sign whenever said special part of said number collection member contains said special-plus.",
"90.The method of claim 89, wherein each of said input number collection members is encoded as an N1 bit code; wherein said N1 is at least three; wherein said integer part of each of said number collection members is encoded as an N2 bit code; wherein N2 is greater than N1.91.A program system for processing numeric data, implementing the steps of claim 78, comprising program steps residing in a memory accessibly coupled to a computer, said program system comprising the program steps of: representing each member of a number collection by an integer part and a special part; adding said first number to said second number to create an add-result; subtracting said first number by said second number to create a subtract-result; exponentiating said first number to create an exp-result; and logarithming said first number to create a log-result.",
"92.The program system of claim 78, wherein the program steps implementing the method are embodied in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon said computer.",
"93.A circuit generated from the program steps of claim 92.94.A circuit for processing numeric data, implementing the steps of claim 78, comprising: means for representing each member of a number collection by an integer part and a special part; means for adding said first number to said second number to create an add-result; means for subtracting said first number by said second number to create a subtract-result; means for exponentiating said first number to create an exp-result; and means for logarithming said first number to create a log-result.",
"95.The circuit of claim 94, wherein at least one of the means of claim 94 is implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection; wherein said programmable logic device collection comprises all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array; and wherein said fixed architecture device collection comprises all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"96.The circuit of claim 94, wherein at least one of said input number collection members are implemented as a wire state collection received from a wire bundle coupled to said circuit.",
"97.The circuit of claim 94, wherein at least one of said output number collection members are implemented as a wire state asserted by said circuit onto a wire bundle."
],
[
"<SOH> BACKGROUND OF INVENTION <EOH>Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.",
"These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.",
"Most of these response and transform functions are linear in nature.",
"Many of these functions operate on a finite grid of data, frequently known as a data window.",
"While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.",
"Many application systems need non-linear functions.",
"Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.",
"These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.",
"Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.",
"The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.",
"What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.",
"The above mentioned finite grids of data are often sampled in real-time by sensor devices.",
"Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.",
"Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.",
"Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.",
"The consequence of this is that bits go unused.",
"By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.",
"Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.",
"What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.",
"Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.",
"There is a central problem associated with these representations, the accumulation of arithmetic errors.",
"This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.",
"Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.",
"The consequence of this is that as calculations progress, additional precision is required, but not available.",
"What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.",
"By way of example, many real-time DSP applications possess the following common constraints.",
"High speed input sample rates, often on the order of 100K to 100 Million samples per second.",
"High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.",
"Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.",
"Many of these applications are developed against a further constraint of short time-to-market requirement.",
"Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.",
"Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.",
"Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.",
"Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.",
"Commercial examples of MIMD products include the high-end products of the ADSP product line.",
"Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.",
"There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.",
"The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.",
"Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.",
"This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.",
"Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.",
"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.",
"The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.",
"The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.",
"While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.",
"The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.",
"What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.",
"Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.",
"Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.",
"These computer have been largely constructed from arrays of FPGA's.",
"They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.",
"Such computer also tend to have problems with multiplication.",
"While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.",
"Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.",
"Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.",
"The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.",
"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.",
"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.",
"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.",
"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.",
"There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.",
"By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.",
"However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.",
"When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.",
"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.",
"Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).",
"However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.",
"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.",
"There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.",
"The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.",
"Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.",
"Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.",
"Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.",
"Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.",
"Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.",
"To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.",
"Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.",
"What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.",
"What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.",
"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.",
"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.",
"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.",
"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.",
"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.",
"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.",
"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods."
],
[
"<SOH> SUMMARY OF INVENTION <EOH>Certain embodiments of the invention solve all the above mentioned problems found in the prior art.",
"Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.",
"The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.",
"Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.",
"These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.",
"Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.",
"The logarithmic conversion of a zero yields a represented number with a minus-infinity.",
"The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.",
"Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.",
"Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.",
"Special-plus added to special-plus results in a special-plus.",
"When a special-minus number and a special-plus number are added, the result is a special-minus result.",
"This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.",
"In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.",
"A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.",
"Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.",
"This supports selecting input-output resources for optimal data bandwidth throughput.",
"They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.",
"The computation resource array may contain at least one instruction propagating resource.",
"The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.",
"The computation resource array may further contain at least one instruction processing resource.",
"Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.",
"The instruction processing resources may contain at least one instruction register.",
"The computation resource array may further contain at least one instruction fetching resource.",
"The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.",
"Certain embodiments of the invention include circuits generated by methods of this invention.",
"They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).",
"They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.",
"Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.",
"Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.",
"The shared bus wire bundle state is determined in part by the configuration register.",
"Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator."
],
[
"This application claims priority from the following provisional applications filed with the United States Patent and Trademark Office: Ser.",
"No.",
"60/204,113, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed May 15, 2000 by inventor, docket number ARITH001PR; Ser.",
"No.",
"60/215,894, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.",
"5, 2000 by inventor, docket number ARITH002PR; Ser.",
"No.",
"60/217,353, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Jul.",
"11, 2000 by inventor, docket number ARITH003PR; Ser.",
"No.",
"60/231,873, entitled “Method and apparatus of a digital arithmetic and memory circuit with coupled control system and arrays thereof”, filed Sep. 12, 2000 by inventor, docket number ARITH004PR; Ser.",
"No.",
"60/261,066, entitled “Method and apparatus of a DSP resource circuit”, filed Jan. 11, 2001 by inventor, docket number ARITH005PR; and Ser.",
"No.",
"60/282,093, entitled “Method and apparatus of a DSP resource circuit”, filed Apr.",
"6, 2001 by inventor, docket number ARITH006PR.",
"TECHNICAL FIELD This invention relates to digital signal processing engines, instruction processing mechanisms, arithmetic operational units of same, as well as circuitry generated based upon use of some or all of these elements.",
"BACKGROUND OF INVENTION Today, much of the growth in the planetary economy depends on rapid and reliable development of new products, many of which require Digital Signal Processing (DSP) hardware to solve the problems which attract customers to buy such products.",
"These problems are often solved by a wide variety of digital filtering techniques, often based upon Finite Impulse Response (FIR) filtering including various Discrete Fourier Transform based algorithms as well as Discrete Wavelet Transform algorithms.",
"Most of these response and transform functions are linear in nature.",
"Many of these functions operate on a finite grid of data, frequently known as a data window.",
"While existing hardware vehicles provide vehicles for such algorithms, there are several central problems which are difficult to solve with existing solutions.",
"Many application systems need non-linear functions.",
"Some commonly used arithmetic operations in engineering and applied science include but are not limited to the following: square roots, cube roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.",
"These commonly used arithmetic operations have found significant application in at least the areas of graphics models, statistical and probabilistic tools, dynamical systems, flow simulations, control systems, transistor and circuit modeling and other nonlinear models.",
"Additionally, there are large applications in the areas of multimedia and image processing including the requirements of filling an HDTV screen with an MPEG stream and various medical imaging applications.",
"The cellular radio industry possesses a number of base station related applications including 911 call location determination and signal separation in high capacity situations.",
"What is needed is arithmetic processing circuitry which can address at least these needs in a real-time fashion.",
"The above mentioned finite grids of data are often sampled in real-time by sensor devices.",
"Each data sample can vary from as small as 6 bits per data sample to 17 or more bits per data sample.",
"Usually, the data tends to be of fairly uniform size, the number of sample bits does not vary much, if at all, across a data grid.",
"Most DSP hardware has a fixed input data size, usually a multiple of 8 or 9 bits.",
"The consequence of this is that bits go unused.",
"By way of example, in a device supporting only 8 and 16 bit data input, a 9 bit sample requires all 16 bits be used, even though only slightly more than 50% of the input bits are actively being used.",
"Further, the data is often sampled at very high rates by multiple sensors, ranging from hundred of thousands of samples per second per sensor to a hundred million samples per second per sensor.",
"What is further needed is a high speed capability to efficiently accept and process widely varying data sample widths.",
"Today, arithmetic-based models are in widespread use employing various fixed point and floating point numeric representations.",
"There is a central problem associated with these representations, the accumulation of arithmetic errors.",
"This is an architectural result of the limited structural capabilities of contemporary arithmetic processors.",
"Such processors typically can only perform arithmetic of a fixed number of significant bits in a given instruction execution period.",
"The consequence of this is that as calculations progress, additional precision is required, but not available.",
"What is needed is arithmetic circuitry which can be readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.",
"By way of example, many real-time DSP applications possess the following common constraints.",
"High speed input sample rates, often on the order of 100K to 100 Million samples per second.",
"High output result rates, due to the prohibitive expense of storing these samples for longer than a few milliseconds to a few seconds.",
"Data sample sizes varying from 6 to 16 bits per sample, with a concomitant requirement to preserve or improve the signal to noise ratio from input sensors to internal use of these samples.",
"Many of these applications are developed against a further constraint of short time-to-market requirement.",
"Contemporary approaches to the performance problems of DSP include standard instruction processors, VLIW processors and reconfigurable computers.",
"Standard instruction processors can be further classified as embedded core DSP processors, Single Instruction Single Datapath (SISD) processors and Multiple Instruction Multiple Datapath (MIMD) processors.",
"Commercial examples of mbedded core DSP processors include the DSP group Oak and Pine processors.",
"Commercial examples of SISD processors include some of the components of the Analog Devices ADSP product line and products of the Texas Instrument 54XX DSP Family.",
"Commercial examples of MIMD products include the high-end products of the ADSP product line.",
"Commercial examples of VLIW processors can be found in the TI 60XX DSP product family.",
"There are at least two distinct performance bottlenecks which affect all or nearly all of the above mentioned approaches to arithmetic and instruction processing: the instruction fetch bottleneck and data access bottleneck.",
"The instruction fetch bottleneck is caused by the imbalance of memory access rate compared to instruction processing mechanisms.",
"Various approaches to solving this problem include adding cache memories, which then put the balance in favor of memories.",
"This leads to compensating by incorporating instruction decoders operating upon multiple instructions as found in super-scalar microprocessors.",
"Such circuitry increases the instruction processing capability of a single instruction path device, by greatly increasing the relative size of the instruction decoding mechanism to the arithmetic processors, as well as increasing the complexity of verifying instruction set execution compliance.",
"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.",
"The data access bottleneck often arises when memory is shared with other processes, such as the instruction fetching process mentioned above.",
"The standard approach to minimizing this problem is the use of either separate memories or providing caches, which in many cases are specifically dedicated to data memory operations.",
"While these approaches add to the availability of data for arithmetic processing, they do not address what the following major limitations found in all of the prior art.",
"The prior art does not provide the user with direct control over the input data width, the internal or intermediate precision width, nor the output data width.",
"What is needed is a way to provide the user of these circuits with direct control over input data with, internal or intermediate precision width and outut data width.",
"Today, VLIW architectures are available which show some flexibility, but are difficult to program due to complex, multiple-memory cycle instruction fetching mechanism, as well as having little or no flexibility regarding input data widths, internal/intermediate precision and output data widths.",
"Reconfigurable computers have been extensively researched since the 1990's, but have yet to have large scale commercial success.",
"These computer have been largely constructed from arrays of FPGA's.",
"They have tended to be very difficult to program, often requiring gate level or logic cell level programming, as opposed to support procedural computer language compilers.",
"Such computer also tend to have problems with multiplication.",
"While some FPGAs now contain cells supporting small multipliers, often 4 by 4 or 4 by 5 bit multipliers, when even a 16 by 16 bit multiplication is to be done, somewhere between 6 and 16 of these cells must be dedicated to that task.",
"Multipliers built this way do not lend themselves to ease of programming nor show themselves flexible in terms of changing input data width or output data width requirements.",
"Most people require less development time to create numeric applications using procedural programming languages such as C or Java than using assembly language, much less a gate or function cell level definition language.",
"The fact that it is possible to build a multiplier with an FPGA is not the problem system developers have to solve.",
"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.",
"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.",
"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.",
"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.",
"There are further problems in the organization of instruction processing mechanisms which significantly constrain performance due to the fixed configuration of internal operational resources.",
"By way of example, the number of arithmetic processing resources available to prepare for a branch decision is fixed.",
"However, high performance arithmetic-oriented applications often involve very large numbers of arithmetic operations being performed before any branching decisions need be made.",
"When branching is to be performed, a number of relatively short operation sequences are usually needed to determine the flow of execution and control.",
"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.",
"Today, multiple datapath architectures are either Single Instruction Multiple Datapath (SIMD) or Multiple Instruction Multiple Datapath (MIMD).",
"However, there are times when a system optimally acts in one fashion, and other times when it optimally would perform in the other fashion.",
"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.",
"There are additional problems facing the system designer intent upon making a new product: the system designer must often provide a complete systems solution, which often includes a package containing one or more printed circuits, which further contain integrated circuits performing numeric tasks within the package in normal operating modes.",
"The system designer needs to be able to test the printed circuits containing the integrated circuits in operation as early in the design process as possible.",
"Additionally, while there have been various attempts to use logarithmic numeric notations to perform arithmetic operations, none of the known approaches are readily extensible to varying precision widths.",
"Such notations tend to treat numbers as either floating point numbers possessing an exponent and mantissa, or as a fixed point number.",
"Both mechanisms have decided problems when applied to the varying needs of systems design, where the notation must be useful across a large collection of numeric ranges.",
"Floating point notations have fixed fields, which further tend to hide the most significant bit of the mantissa, rendering such a notation is inherently difficult to alter.",
"Both approaches to number notations lack any obvious way to convert 0 into a logarithm of 0.What is needed is a numeric notation readily supporting logarithmic numbers as well as being readily scalable to support differing amounts of precision in a real-time environment.",
"To summarize, what is needed is arithmetic processing circuitry addressing the need for advanced, often non-linear functions based upon much more than linear arithmetic operations in a real-time fashion.",
"Such operations include but are not limited to square roots, division, trigonometric functions, powers of numbers, polynomial functions, rational functions, exponential functions, logarithms, and determinants.",
"What is needed includes the ability to efficiently accept and process widely varying data sample widths at high speeds.",
"What is needed is arithmetic circuitry readily configured to provide a wide range of precision for any given instruction sequence in a given instruction execution period.",
"What is needed is a flexible instruction processing mechanism which can more efficiently utilize instruction memory bandwidth to drive the arithmetic processing circuitry.",
"What is needed is an instruction processing mechanism which can be optimally configured for both decision processes and computational sequences.",
"What is needed is an architecture supporting multiple datapaths in either an SIMD or MIMD mode, which can be rapidly reconfigured from one to the other.",
"What is needed is a mechanism providing the user with direct control over the input data width, the internal/intermediate precision width, and the output data width while providing a wide range of arithmetic operations in an efficient fashion.",
"What is further needed is a method of specifying such control and then generating efficient circuits satisfying those specifications.",
"What is further needed is an architecture which provides standard procedural language compiler support both for mechanisms supporting user control of input data width, internal/intermediate precision and output data width.",
"What is further needed is a target circuit compilation architecture providing automated support for procedural compilers specified and generated by such methods.",
"SUMMARY OF INVENTION Certain embodiments of the invention solve all the above mentioned problems found in the prior art.",
"Certain embodiments utilize partitionable datapath bit width units, which can be configured to provide a requested level of numeric precision.",
"The partitionable datapath bid width units include at least memory arrays and ALUs, which can collectively be configured to specific bit widths supporting the requested level of numeric precision in both a normal numeric realm and a logarithmic numeric domain.",
"Certain embodiments of the invention represent a collection of numbers as having at least a minus-infinity as a special part of each represented number.",
"These minus-infinity numbers act as annihilators in addition, so that minus-infinity plus anything else results in a minus-infinity in the special part of the represented result.",
"Thus, the fact that zero multiplying anything in the normal numeric realms translates upon taking logarithms of both numbers.",
"The logarithmic conversion of a zero yields a represented number with a minus-infinity.",
"The exponential conversion of a represented number with negative-infinity in its special part yields a 0 result.",
"Represented numbers may further include a special-plus or special-minus in their special parts, further supporting preservation of the input number sign upon into the represented number and conversion back to output numbers.",
"Note that this also requires that special-minus represented numbers, when added to special-minus represented numbers generate a represented number result with a special-plus.",
"Special-plus added to special-plus results in a special-plus.",
"When a special-minus number and a special-plus number are added, the result is a special-minus result.",
"This effects a logarithm of a first number added to the logarithm of a second number is essentially the same as the logarithm of the product of the first and second numbers.",
"In the logarithm domain, functions can be calculated which are very computationally expensive in the normal realm of numbers.",
"A level of efficiency previously unavailable in a programmable device of any kind is achieved by using at least some of the memories as table lookup mechanisms to approximately convert numbers between their logarithms, exponentials and other functions.",
"Certain embodiments of the invention include a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource comprising: selecting the input-output resources to create an input-output access collection comprised of at least an input-output access parameter; and selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.",
"This supports selecting input-output resources for optimal data bandwidth throughput.",
"They also support selecting datapath operational resources for optimal datapath resource allocation operating on data traversing the selected input-output resources.",
"The computation resource array may contain at least one instruction propagating resource.",
"The method may selecting the instruction propagating resources based upon the input-output access collection and the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.",
"The computation resource array may further contain at least one instruction processing resource.",
"Instruction processing resources may further be selected based upon the input-output access collection, datapath operational resource allocation and instruction propagating configuration.",
"The instruction processing resources may contain at least one instruction register.",
"The computation resource array may further contain at least one instruction fetching resource.",
"The method may include fetching using the instruction fetching resource to create a fetched instruction and loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.",
"Certain embodiments of the invention include circuits generated by methods of this invention.",
"They may be implemented using collections of one or more programmable logic devices, which may in turn include one or more programmable logic arrays and/or one or more Field Programmable Gate Arrays (FPGAs).",
"They may be implemented as part or all of an integrated circuit, having been specified using a method of this invention and/or simulated based upon their specification.",
"Their simulation may include implementations targeting logic hardware accelerators including programmable logic devices as execution elements.",
"Certain embodiments of the invention include an arithmetic module of multiple basic arithmetic circuits coupled to share several wire bundles including a first shared buss, a second shared buss and a third shared buss that synchronously execute either one or two instructions, depending upon a configuration register.",
"The shared bus wire bundle state is determined in part by the configuration register.",
"Each basic arithmetic circuit includes a basic arithmetic memory coupled with a basic arithmetic calculator.",
"BRIEF DESCRIPTION OF DRAWINGS FIG.",
"1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention; FIG.",
"2 shows a refinement of FIG.",
"1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400; FIG.",
"3 shows a refinement of FIG.",
"2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.",
"4 shows a refinement of FIG.",
"3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400; FIG.",
"5 shows a refinement of FIGS.",
"1, 2, 3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400; FIG.",
"6 shows a refinement of FIG.",
"5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100; FIG.",
"7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.",
"8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002; FIG.",
"9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result; FIG.",
"10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300; FIG.",
"11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIG.",
"11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8; FIGS.",
"12 to 16 show a configuration of instruction memories and MSRI[K] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples; FIG.",
"17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.",
"Note that in other embodiments of the invention, i could range over a different range; FIG.",
"17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.",
"FIGS.",
"18 to 20 show various configurations of PISRI's and their neighboring instruction memories; FIG.",
"21 shows a high level data flow diagram of a two dimensional array block 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported; FIG.",
"22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource and at least one datapath operational resource; FIG.",
"22B depicts a detail flowchart of user operation 2000 of FIG.",
"22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration; FIG.",
"23A depicts a detail flowchart of user operation 2000 of FIG.",
"22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration; FIG.",
"23B depicts a detail flowchart of user operation 2000 of FIG.",
"22A further performing the method of using/operating the array of computational resources; FIG.",
"24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500; FIG.",
"24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.",
"24A further comprised of instruction fetch mechanism 3800; FIG.",
"25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720; and FIG.",
"26 shows a detail block diagram of a branch processor 3700 extending the block diagram of FIG.",
"25 further comprised of cache manager 3740; FIG.",
"27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention; FIG.",
"28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.",
"27; FIG.",
"29 depicts a preferred version of ALU 1400, as used in FIG.",
"28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank; FIG.",
"30 depicts a flowchart of a method of processing numeric data, which may be variously embodied; FIG.",
"31 depicts a detail flowchart of operation 2222 of FIG.",
"30 further performing the arithmetic operation collection; FIG.",
"32A depicts a detail flowchart of operation 2272 of FIG.",
"31 further adding; FIG.",
"32B depicts a detail flowchart of operation 2282 of FIG.",
"31 further subtracting; FIG.",
"33A depicts a detail flowchart of operation 2292 of FIG.",
"31 further exponentiating; FIG.",
"33B depicts a detail flowchart of operation 2302 of FIG.",
"31 further logarithming; FIG.",
"34A depicts a detail flowchart of operation 2442 of FIG.",
"33B further setting the special part of the log-result; FIG.",
"34B depicts a detail flowchart of operation 2442 of FIG.",
"33B further setting the special part of the log-result; FIG.",
"35A depicts a detail flowchart of operation 2292 of FIG.",
"31 further exponentiating; FIG.",
"35B depicts a detail flowchart of operation 2302 of FIG.",
"31 further logarithming; FIG.",
"36A depicts a detail flowchart of operation 2532 of FIG.",
"35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.",
"36B depicts a detail flowchart of operation 2532 of FIG.",
"35B further determining whether the sign part of the first number is essentially equal to the negative-sign; FIG.",
"37A depicts a detail flowchart of operation 2232 of FIG.",
"30 further log-converting the input number collection member; FIG.",
"37B depicts a detail flowchart of operation 2232 of FIG.",
"30 further log-converting the input number collection member; FIG.",
"38A depicts a detail flowchart of operation 2242 of FIG.",
"30 further exp-converting; and FIG.",
"38B depicts a detail flowchart of operation 2242 of FIG.",
"30 further exp-converting.",
"DETAILED DESCRIPTION OF DRAWINGS As used herein a wire refers to a path connecting nodes of a circuit which carries a state between the connected nodes and/or refers to a resonant cavity propagating information in terms of state between the connected nodes.",
"A wire may be made out of metal, an optical chamber, or a tunnel path through a molecular substrate.",
"A wire bundle is a collection of at least one wire.",
"State as used herein refers to an element of a finite alphabet, which contains at least two symbols.",
"These two minimal symbols relate to ‘0’ and ‘1’ as used in Boolean logic.",
"FIG.",
"1 depicts a basic arithmetic processing unit 1400 comprised of a first memory circuit 1200 and a first ALU 1300 in accordance with certain embodiments of the invention.",
"The basic arithmetic unit 1400 includes a partitioning wire bundle 1002 presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1008 is presented to first memory circuit 1200 and first ALU 1300.Input wire bundle 1010 is presented only to first memory circuit 1200.First memory instruction wire bundle 1202 is presented to first memory circuit 1200.First memory circuit 1200 generates signals for first memory output wire bundle 1016 presented to first ALU 1300.First ALU instruction wire bundle 1302 is presented to first ALU circuit 1300.First ALU 1300 receives a carry-in wire bundle 1150, first ALU instruction wire bundle 1302 and generates a carry-out wire bundle 1152 and further generates signals for first ALU output wire bundle 1018.The basic arithmetic processing unit operates by receiving the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018, as well as first memory instruction wire bundle 1202, first ALU instruction wire bundle 1302 and carry-input wire bundle 1150.The received signal state of the partitioning wire bundle 1.002, further determines the operational partitioning of first memory circuit 1200 and first ALU circuit 1300 with regards to the signaling of input wire bundle 1008, input wire bundle 1010, first memory output wire bundle 1016 and first ALU output wire bundle 1018.First memory circuit 1200 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, input wire bundle 1010 and first memory instruction wire bundle 1202.Partitioning wire bundle 1002 signal state is used by first memory circuit 1200 to determine from the signal state of first memory instruction wire bundle 1202 at least one of first memory local instructions to be executed.",
"The first memory local instructions are executed by the first memory circuit 1200.First memory circuit 1200 asserts the signal state of first memory output wire bundle 1016.First ALU 1300 receives the signal state of partitioning wire bundle 1002, which determines the partitioning of the signaling of input wire bundle 1008, first memory output wire bundle 1016, first ALU instruction wire bundle 1302 and the effect of the signal state of carry-input wire bundle 1150.Partitioning wire bundle 1002 signal state is used by first ALU circuit 1300 to determine from the signal state of first ALU instruction wire bundle 1302 of at least one of first first ALU local instructions to be executed.",
"The first ALU local instructions are executed by the first ALU circuit 1300.First ALU circuit 1200 asserts the signal state of first ALU output wire bundle 1018.FIG.",
"2 shows a refinement of FIG.",
"1 further depicting an input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 of basic arithmetic unit 1400.Note that FIG.",
"2 operates in much the same fashion as FIG.",
"1, excepting Input-output circuit 1100 acts as an interface between at least three external data bus wire bundles, 1004 and 1006 controlled by instruction wire bundle 1102 as well as partitioning wire bundle 1002 and creating the state on wire bundles 1008, 1010 and 1012.FIG.",
"3 shows a refinement of FIG.",
"2 further depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and further coupled to a second memory circuit 1250 forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.",
"3 operates in a similar fashion to FIGS.",
"1 and 2 with regards to elements of similar reference numbers.",
"Second memory circuit 1250 may receive output 1018 from ALU 1300 as well as wire bundle 1014 from input-output circuit 1100 based upon partitioning wire bundle 1002.Second memory circuit 1250 may drive the state of wire bundle 1014.Second memory circuit 1250 may receive wire bundle 1012 from input-output circuit 1100.FIG.",
"4 shows a refinement of FIG.",
"3 depicting input-output circuit 1100 coupled to the first memory circuit 1200 and a first ALU 1300 and coupled to a second memory circuit 1400 with additional interconnections forming a basic arithmetic unit 1402, which is a refinement of basic arithmetic unit 1400.Note that FIG.",
"4 operates in a similar fashion to FIG.",
"1, 2, and 3 with regards to elements of similar reference numbers.",
"FIG.",
"5 shows a refinement of FIGS.",
"1, 2,3 and 4 depicting input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.",
"2, 3 and 4.FIG.",
"6 shows a refinement of FIG.",
"5 depicting a first input-output circuit 1100 coupled to multiple instances of basic arithmetic unit 1400 and a second instance of input-output circuit 1100.Basic arithmetic unit 1400 may at least be basic arithmetic unit 1402 as shown in FIGS.",
"2, 3 and 4.FIG.",
"7 shows a detail diagram of an ALU circuit 1300 in terms of four interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.FIG.",
"8 shows a detail diagram of first ALU circuit 1300 in terms of eight interconnected instances of a second ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.In FIGS.",
"8 and 9, each ALU circuit 1310 may support a consistent datapath bit width.",
"Each ALU circuit 1310 may further support a constant datapath bit width.",
"The constant datapath bit width may be at least one of 3, 4, and 5.In FIGS.",
"8 and 9, ALU circuit 1300 may contain instances of ALU circuit 1310 supporting carry propagation controlled by partitioning wire bundle 1002.The number of instances of ALU circuit 1310 is a power of two not necessarily shown in these Figures.",
"The number of instances of ALU circuit 1310 may not be a power of two.",
"The number of instances of ALU circuit 1310 may be 12.The constant bit width of ALU circuit 1310 may be 4 and the number of instances of ALU circuit 1310 belongs to a collection comprising 8, 12, 16, 24, 32, 48 and 64.The constant bit width of ALU circuit 1310 is 3 and the number of instances of ALU circuit 1310 belongs to a collection comprising 12, 16, 24, 32, 48 and 64.FIG.",
"9 depicts an arithmetic logic unit 1310 with multiple add-inputs presented to shifters and then added to generate an add-result.",
"Each shifter (S1, S2, S2, and S4)is controlled by add-instruction-components belonging to a collection comprising at least values representing same-sign, reverse-sign, do-not-use, shift-up and shift-down.",
"The effects of these values acting on the add-input are as follows: The add-input acted upon by the corresponding add-inst-component having same-sign generates the add-input.",
"The add-input acted upon by the corresponding add-inst-component having reverse-sign generates the negative of the add-input.",
"The add-input acted upon by the corresponding add-inst-component having do-not-use generates a zero.",
"The add-input acted upon by the corresponding add-inst-component having shift-up generates a positive power of two times the add-input.",
"The add-input acted upon by the corresponding add-inst-component having shift-down generates a negative power of two times the add-input.",
"The shift-instruction collection may further include shift-up-by-m and shift-down-by-m, where m is at least two.",
"Partitioning wire bundle 1002 is used to control carry propagation and shift bit propagation, but are not shown in this Figure to simplify the discussion.",
"The omission of partitioning wire bundle 1002 is not meant to limit nor require its presence for all embodiments of the invention.",
"Note that the logarithm of 0 will be negative-infinity, and that the square and square root of 0 are each 0.To preserve these facts in the logarithmic domain, shifting negative-infinity should produce negative infinity.",
"The add-inst controls for each shifter S1-S4 may further include shift-up-2 and shift-down-2, supporting shifting by 2 bits, as well as shift-up-3 and shift-down-3 supporting shifting by 3 bits.",
"Certain preferred embodiments of the invention employ a subset of all these add-inst controls including same-sign, reverse-sign, do-not-use, shift-up, shift-down, shift-up-2, shift-up-3, which may be coded as 2 bits designating same-sign, reverse-sign, do-not-use, shift-down; and 2 additional bits coding pass-through, shift-up, and shift-up-2.Note that as shown hereafter, when do-not-use is asserted, it does not matter what the other two bit field contains.",
"In general, when do-not-use is asserted, the contents of the other two bit field will be chosen to optimize at least one system characteristic, such as testability and/or logic complexity and/or signal propagation through the circuit, also known as the circuit's critical path delay.",
"The add-inst control signals may originate from the datapath instruction being presented for execution, or may alternatively be generated based upon part of a numeric data to perform a limited range of multiplications.",
"Consider the following table Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 do-not-use same-sign, shift-up-1 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => 4 same-sign, shift-up-2 do-not-use 101 => 5 = 4 + 1 same-sign, shift-up-2 same-sign, pass-through 110 => 6 = 4 + 2 same-sign, shift-up-2 same-sign, shift-up-1 111 => 7 = 8 − 1 reverse-sign, pass-through same-sign shift-up-3 Table One: a three bit multiplication based upon controlling a pair of shifter inputs.",
"Note that this further supports the circuitry shown in FIG.",
"9 as supporting the multiplication of 6 bits of an operand acting upon a second number, which may be further provided by a local memory.",
"Using this circuitry in such a fashion provides an interpolation capability supporting successive approximations, which may involve linear, multi-linear and through the feedback-cascading of partial results based upon this circuit, some forms of non-linear approximations to various non-linear functions including but not limited to various exponential functions, logarithms, trigonometric functions, among others.",
"An alternative approach to approximation interprets a three bit number as a signed number, leading to the following table: Multiplier Add-inst-1 Add-inst-2 000 => 0 do-not-use do-not-use 001 => 1 same-sign, pass-through do-not-use 010 => 2 same-sign, pass-through same-sign, pass-through 011 => 3 = 2 + 1 same-sign, pass-through same-sign, shift-up-1 100 => −4 reverse-sign, shift-up-1 reverse-sign, shift-up-1 101 => −3 = −4 + 1 reverse-sign, pass-through reverse-sign, shift-up-1 110 => −2 reverse-sign, pass-through reverse-sign, pass-through 111 => −1 reverse-sign, pass-through do-no-use Table Two: a signed three bit multiplication based upon controlling a pair of shifter inputs.",
"Note that these two tables may be concurrently employed in certain situations where a signed 6 bit numeric multiplication is desired.",
"The most significant 3 bits affect multiplication as shown in Table Two.",
"When the sign of the 6 bit quantity is negative, the least significant 3 bits may affect the multiplication as shown in Table One, with every instance of reverse-sign changed to same-sign, and every instance of same-sign changed to reverse-sign.",
"When the sign of the 6 bit quantity is positive, Table One may be used as shown.",
"These tables and discussions have been provided by way of example and are not meant to limit the scope of the claims.",
"As one of skill in the art will readily recognize, there are many alternative notations for the various operations presented herein which are essentially equivalent to the examples presented herein.",
"FIG.",
"10 shows Arithmetic Process Array 3000 comprising at least one Arithmetic Processor 3002, each coupled with 3104 Instruction Memory 3200 and with 3106 Instruction Memory 3300, in accordance with certain embodiments of the invention.",
"In certain embodiments of the invention, only one instruction memory 3200 is coupled to Arithmetic Processor Array 3000, feeding one instruction to each Arithmetic Processor 3002.FIGS.",
"11A to 16 refer to such embodiments of the invention.",
"Note that these embodiments of the invention do not require the partitioning wire bundle 3102, nor the partitioning wire bundle 1002.FIG.",
"11A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.",
"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.",
"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.",
"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIG.",
"11B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8.Note that in other embodiments of the invention, i could range over a different range.",
"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.",
"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.",
"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an MSRI[k] cell array where i ranges from 1 to k. FIGS.",
"12 to 16 show a configuration of instruction memories and MSRI[k] sufficient to perform a large number of realtime filtering and more sophisticated tasks upon an input stream of 8 or 9 bit samples.",
"Assume the horizontal datapath width of the ALU2 and memories in each MSRI is at least 3, preferably 4, bits.",
"When the horizontal datapath width of the ALU2 and memories in each MSRI is 4 bits, 10 bit samples could also be accepted.",
"The data stream enters MSRI[4] in FIG.",
"12 flowing to MSRI[5], then MSRI[6], MSRI[7], then MSRI[8] of FIG.",
"13 eventually leaving MSRI[231 having performed as a 12 pass arithmetic stream processor.",
"If each MSRI component performs a radix 4 FFT, then the system as a whole performs a 1K FFT by the time the data leaves MSRI[21], and a 4K FFT upon leaving MSRI[23].",
"The increase in depth of the MSRI's is to consistently extend the precision of the calculations to account for the accumulation of rounding errors.",
"In certain other embodiments of the invention, two instruction memories 3200 and 3300 are coupled to Arithmetic Processor Array 3000, feeding two instructions to each Arithmetic Processor 3002, the selection of which instruction is executed determined by the partitioning wire bundle 3102, which in turn drives the partitioning wire bundle 1002.FIGS.",
"17A to 20 refer to such embodiments of the invention.",
"FIG.",
"17A show a single column of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.",
"Note that in other embodiments of the invention, i could range over a different range.",
"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the firstmemories 1210 as second memories.",
"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.",
"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[k] cell array where i ranges from 1 to k. This circuit and the circuit of FIG.",
"17B use the partitioning wire bundle 1002 to control carry propagation and determination of which of the two instructions are executed in each horizontal strip of the PISRI.",
"FIG.",
"17B show four columns of coupled first memories 1210-i and ALU2 1310-i, where i=1 to 8, with two instructions memories 3200 and 3300 providing two instructions transmitted to each.",
"Note that in other embodiments of the invention, i could range over a different range.",
"Note also that in other embodiments of the invention, each ALU2 1310 unit could be coupled to the first memories 1210 as second memories.",
"Note that these circuits would be simpler, thus take up less circuit area and consume less power, but could not reconfigure the datapath bit-width, which would be determined by the number of cells (the range of i) and the datapath width of the horizontal strips.",
"The two dimensional strips containing memories 1210-i and ALU2 cells 1310-i are further integrated as an PISRI[K] cell array where i ranges from 1 to k. FIGS.",
"18 to 20 show various configurations of PISRI's and their neighboring instruction memories.",
"In FIG.",
"18, PSRI[11] can be configured to act as an MSRI[4] and MSRI[7], or as an MSRI[5] and MSRI[6].",
"The net effect can then be the processing flow of MSRI[4]==>MSRI[5]==>MSRI[6]==>MSRI[7].",
"Similarly, the PSRI[19]'s can be configured as ==>MSRI[9]==>MSRI[10]==>MSRI[11].",
"FIG.",
"19 shows a configuration of same size PISRI[8]'s which can be support dataflows as discussed above, and which provide a 32 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 4 bits.",
"Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.",
"FIG.",
"20 shows a configuration of same size PISRI[12]'s which can be support dataflows as discussed above, and which provide a 36 bit data interface to standard computer memories when the datapath bit width of the ALU2 1310 and memories 1210 cells is 3 bits.",
"Note that the partitioning wire bundle would also have an effect on the operation of the data interface to standard memories to preserve data alignments or to partition the external data memory into two address ranges, one for each partition.",
"FIG.",
"21 shows a high level data flow diagram of a two dimensional array blocks 1300 of ALU2 circuits 1310 and connecting datapaths between them by which data may be transported.",
"Data flow as depicted in FIG.",
"21 supports both nearest neighbor and global communication of array blocks in each of the two dimensions of the array.",
"Note that the vertical communications lines shown in FIG.",
"21 may be used to transfer data via an external data memory interface which is not shown.",
"Such an external data memory interface may receive addressing signal either from datapath element(s), and/or from one or more instruction processors.",
"FIG.",
"22A depicts a flowchart performing a method of using an array of computational resources containing at least one input-output resource, at least one datapath operational resource in accordance with certain embodiments of the invention.",
"User operation 2000 starts the usage of this flowchart.",
"Arrow 2002 directs the usage flow from user operation 2000 to user operation 2004.User operation 2004 performs selecting the input-output resources to create an input-output access collection comprised of at least one input-output access parameter Arrow 2006 directs usage from user operation 2004 to user operation 2008.User operation 2008 performs selecting the datapath operational resources based upon the input-output access collection to create a datapath operational resource allocation collection containing at least one datapath operation resource allocation.",
"Arrow 2010 directs usage from user operation 2008 to user operation 2012.User operation 2012 terminates the usage of this flowchart.",
"Note that in other embodiments of the invention, the flowchart of FIG.",
"22A may be used to depict a method of operating an array of computational resources containing at least one input-output resource, at least one datapath operational resource.",
"The array of computational resources may be implemented using one or more programmable logic devices, which may include or more programmable logic arrays and/or one or more Filed Programmable Gate Arrays (FPGAs).",
"Certain further embodiments of the invention include the array of computation resources further containing at least one instruction propagating resource.",
"FIG.",
"22B depicts a detail flowchart of user operation 2000 of FIG.",
"22A further performing selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration, in accordance with certain embodiments of the invention.",
"Arrow 2030 directs the usage flow from starting user operation 2000 to user operation 2032.User operation 2032 performs selecting the instruction propagating resources based upon the input-output access collection and based upon the datapath operational resource allocation collection to create an instruction propagating configuration collection containing at least one instruction propagating configuration.",
"Arrow 2034 directs usage from user operation 2032 to user operation 2036.User operation 2036 terminates the usage of this flowchart.",
"FIG.",
"23A depicts a detail flowchart of user operation 2000 of FIG.",
"22A performing selecting the instruction processing resources based upon the input-output access collection, the datapath operational resource allocation collection, and the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration in accordance with certain embodiments of the invention.",
"Arrow 2050 directs the usage flow from starting user operation 2000 to user operation 2052.User operation 2052 performs selecting the instruction processing resources based upon the input-output access collection and based upon the datapath operational resource allocation collection and based upon the instruction propagating configuration collection to create an instruction processing configuration collection containing at least one instruction processing configuration.",
"Arrow 2054 directs usage from user operation 2052 to user operation 2056.User operation 2056 terminates the usage of this flowchart.",
"Certain further embodiments of the invention include the array of computation resources further containing at least one instruction fetching resource and the instruction processing resources containing at least one instruction register.",
"FIG.",
"23B depicts a detail flowchart of user operation 2000 of FIG.",
"22A further performing the method of using/operating the array of computational resources in accordance with certain embodiments of the invention.",
"Arrow 2070 directs the usage flow from starting user operation 2000 to user operation 2072.User operation 2072 performs fetching using the instruction fetching resource to create a fetched instruction.",
"Arrow 2074 directs usage from user operation 2072 to user operation 2076.User operation 2076 performs loading the fetched instruction into the instruction register to create an instruction register state based upon the fetched instruction.",
"Arrow 2078 directs usage from user operation 2076 to user operation 2080.User operation 2080 terminates the usage of this flowchart.",
"FIG.",
"24A shows a detail block diagram of an instruction memory 3200 comprised of instruction register 3600, branch processor 3700 and instruction memory array 3500, in accordance with certain embodiments of the invention.",
"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.",
"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.",
"24B shows a detail block diagram of an instruction memory 3200 extending the block diagram of FIG.",
"24A further comprised of instruction fetch mechanism 3800, in accordance with certain embodiments of the invention.",
"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.",
"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.FIG.",
"25 shows a detail block diagram of an branch processor 3700 comprised of branch sequence register 3710, program counter 3730 and branch address look-up table 3720, in accordance with certain embodiments of the invention.",
"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.",
"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.",
"Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.",
"FIG.",
"26 shows a detail block diagram of an branch processor 3700 extending the block diagram of FIG.",
"25 further comprised of cache manager 3740, in accordance with certain embodiments of the invention.",
"Branch processor 3700 in certain further embodiments of the invention includes a branch return stack.",
"In certain further embodiments of the invention, the branch return stack can be unloaded and reloaded via arrow 3702.Branch Address Loop-Up Table 3720 may include an interpreter address look-up table supporting an interpretive language, in certain further embodiments of the invention.",
"Such interpretive languages may include but are not limited to JAVA, FORTH and Smalltalk.",
"FIG.",
"27 depicts a high level system block diagram of a DSP Resource Circuit in accordance with certain embodiments of the invention.",
"The DSP Resource Circuit is comprised a Datapath Resource Array 5000.The Datapath Resource Array 5000 is coupled to at least one of the following: the Digital Device Interface 5300 and the System and Control Interface 5400.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5312,5314 and/or 5316 with Digital Device Interface 5300.In certain applications, coupling 5312 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.",
"Coupling 5314 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5316 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.",
"Note that the couplings 5302, 5304, and 5306 preferably respectively relate to the external communications associated with couplings 5312, 5314, and 5316 in such applications.",
"In other applications, one input-output processor may strictly receive data via coupling 5316 which is generated based upon an external input stream via coupling 5306.Additionally, an input-output processor may strictly output data via coupling 5312 which is used to generate an external data stream presented via coupling 5302.In certain applications, each of these coupling may be preferably split into two such couplings, each under the control of a separate input-output processor.",
"The Datapath Resource Array 5000 may also be coupled to a Local Memory Interface 5500.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5512,5514 and/or 5516 to Local Memory Interface 5500.Preferably, coupling 5512 communicates memory access request information including but not limited to address information, and where appropriate, memory access length.",
"Coupling 5514 preferably communicates data received from the Datapath Resource Array 5000 for storage elsewhere, as well as data being sent to the Datapath Resource Array 5000.Coupling 5516 may be used to convey status information, which may include but is not limited to at least one of the following: memory latency-wait states, which may be current or projected, as well as error status information including but not limited to checksum errors and other error detection related information.",
"Note that preferably, couplings 5502, 5504, and 5506 respectively relate to the external communications associated with couplings 5512, 5514, and 5516.When applicable, Datapath Resource Array 5000 is coupled by at least one of 5402 and/or 5404 to System and Control Interface 5400.Each or either of couplings 5402 and 5404 may be comprised of a collection of couplings such as discussed above in FIG.",
"27 with unidirectional couplings and possibly strictly bi-directional couplings.",
"In certain applications, coupling 5402 may preferably convey system control and status information related via 5406 with an external system environment.",
"In certain applications, coupling 5404 may convey data communicated via 5406 with the external system environment.",
"Such data may be provided during system initialization time for conveyance not only into internal memory within the Datapath Resource Array 5000.Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Local Memory Interface 5500 for storage in local memory.",
"Coupling 5404 may also be used during system initialization for further data conveyance through Datapath Resource Array 5000 to Digital Device Interface 5300 for use elsewhere.",
"FIG.",
"28 depicts a simplified floor plan of a layout of the DSP Resource Circuit of FIG.",
"27.Datapath Resource Array 5000 is comprised of at least one instruction processor 3800 and an array of DSP resources 1400.By way of example, FIG.",
"28 depicts an array of 8 rows and 8 columns of DSP resource 1400 instances.",
"Instruction processor 3800 may be closely coupled with an instruction memory array 3500.In certain applications, Datapath Resource Array 5000 is preferably comprised of two instruction processors 3800-1 and 3800-2.At this level of abstraction the partition control wire bundle is not visible.",
"However, it is assumed to support partitioning the array of DSP resources into two horizontal regions.",
"By way of example, the top three rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 1 3800-1.The remaining bottom 5 rows of the array of DSP resources may be partitioned to act based upon an instruction state communicated from instruction processor 2 3800-2.In certain applications a further partitioning of instruction processing resources may be preferred.",
"An instruction processor 3800 may be partitioned into two instruction processing streams, each with independent branch mechanism.",
"The first instruction processing stream may be partitioned to control the instruction state asserted for the three left-most columns of the array of DSP resources.",
"The second instruction processing stream would control the remaining 5 columns of the array of DSP resources.",
"Note that in certain applications, partitioning may support more than two instruction processing streams being sent from an instruction processor.",
"For simplicity of discussion, no more than two instruction streams will be discussed hereafter.",
"This is not meant to limit the scope of the claims.",
"By way of example, the applications and configurations of FIGS.",
"12 to 20 may be implemented by circuitry illustrated in FIG.",
"28.Digital Device Interface 5300 is comprised of at least one at least one input-output instruction processor 1130-1 controlling an input-output processor 1120-1.Digital Device Interface 5300 may be further comprised of a second input-output instruction processor 1130-2 controlling input-output processor 1120-2.In certain applications, at least one input-output instruction processor 1130 may be coupled to an input-output instruction memory 1140.Input-output processors preferably possess couplings to all the rows of their associated array of DSP resources 1400.Input-output processors preferably configure communication to the array of DSP resources based upon the partition state information which configures the rows of the array of DSP resources to communicate together.",
"By way of example, the first input-output processor 1120-1 may communicate with the top three rows of the array of DSP resources, when they are so partitioned.",
"The second input-output processor 1120-2 may then preferably communicate with the remaining 5 bottom rows.",
"Note that the rows of DSP resources may be partitioned into more than two communicating components.",
"Similarly, the Digital Device Interface 5300 may comprise more than two input-output instruction processors controlling more than two input-output processors.",
"Each input-output processor may include at least one of the following: a data memory, ALU, and specialized logical functions such as bit packing-unpacking circuits.",
"Note that the circuitry of FIGS.",
"27 and 28 may further reside in a package where at least one of the interfaces couples to analog circuitry including but not limited to at least one of the following: A/D converters, D/A converters, frequency synthesizers, threshold detectors, and amplifiers.",
"Note that implementations of multiple instances of the circuitry of FIG.",
"28 may include situations where one input-output processor of a first instance may not couple to elements of another array of DSP resources 1400 within another instance.",
"System and Control Interface 5400 may be comprised of at least one input-output processor 1120-3 controlled by input-output instruction processor 1130-3.The preceding discussion regarding the Digital Device Interface is applicable to System and Control Interface 5400, and will not be repeated for reasons of brevity.",
"However, this is not meant limit the scope of the claims.",
"A common branching mechanism is preferably employed through the instruction processors discussed in FIG.",
"28.This branching mechanism can be embodied to support caching and accessing of memory through a local memory interface 5500, or may be embodied without support of external memory.",
"The overall instruction processing principles embodied in this invention include the following design/architectural goals: Input-output and datapath configurations dominate the embodied architectures, not the other way around.",
"The hardware supports software debugging and test.",
"The embodied architectures can support multiple systems levels of instruction fetching.",
"They can support both SIMD and MIMD, as well as SISD and MISD processing applications.",
"Implementations support separate references to data and instructions.",
"There are several consequences to separate data and instruction references.",
"The runtime environments of procedural languages such C, C++, PASCAL, FORTRAN and JAVA.",
"By employing an invariant instruction set across a variety of datapath size ranges, a single program can handle a wide range of input data sizes with the arithmetical precision preserved by construction.",
"Another consequence is the requirement of parameter passing to functions and subroutines by reference only.",
"Consider for a moment the runtime stack frame requirements of the C programming language.",
"C's runtime stack frame contains the following components: Branch related pointers, loop counters, data address references and data values, all of which may have differing data widths.",
"By partitioning the stack frame into multiple stack frame into separate stack frames to handle differing data widths, the communication and manipulation of data to “fit” onto a single stack frame, or “fit” back into the processing element where it is needed is minimized.",
"There are further architectural preferences which ease the task of compilation of procedural language programs as well as improve the reliability of these translations.",
"FIG.",
"29 depicts a preferred version of ALU 1400, as used in FIG.",
"28 and earlier Figures supporting configurations of its internal memories and system input 1002 emulating a multiplier-accumulator with local register bank.",
"Arithmetic processor 1400 preferably contains 3 ALUs, 1300-1, 1300-2 and 1300-3, with two memories 1200-1 and 1200-2 respectively feeding 1016 and 1018 the first two ALUs 1300-1 and 1300-2.The third ALU 1300-3 is feed by at least ALU 1300-2 and wire bundle 1002.System input 1002 preferably contains representations of normal numbers and numbers in a logarithmic domain.",
"The logarithmic domain will be discussed in detail shortly.",
"The normal number representation be stored in memory 1200-1.ALU 1300-2 may further be implemented in a fashion as shown in FIG.",
"9, with shift controls being provided from part of the numeric signaling received from wire bundle 1018, as well as at least part of the addressing being provided by parts of the numeric signals received from wire bundle 1018.The results of the two ALU 1300-1 and 1300-2 operations are sent 1018 and 1036 to ALU 1300-3, where the results are combined to form an efficient and very accurate approximation of a sum of numerical products.",
"As mentioned earlier, there are some fundamental problems with multipliers.",
"Most importantly, they tend to grow with the product of the input precision.",
"While this may be somewhat constrained by output precision requirements, it remains a fundamental problem.",
"An approach that solve this problem will now be described.",
"Consider the action of representing each member of a number collection by an integer part and a special part, where the special part contains exactly one member of a first special value collection comprising negative-infinity and not-negative-infinity.",
"FIG.",
"30 depicts a flowchart of a method of processing numeric data, which may be variously embodied.",
"Arrow 2210 directs the flow of execution from starting operation 2200 to operation 2212.Operation 2212 performs representing each member of a number collection by an integerpart and a special part.",
"Arrow 2214 directs execution from operation 2212 to operation 226.Operation 226 terminates the operations of this flowchart.",
"Arrow 2220 directs the flow of execution from starting operation 2200 to operation 2222.Operation 2222 performs performing at least one member of the arithmetic operation collection upon at least one of the members of the number collection.",
"Arrow 2224 directs execution from operation 2222 to operation 226.Operation 226 terminates the operations of this flowchart.",
"Certain embodiments of the invention may perform all members of the arithmetic operation collection upon the relevant member of the number collection.",
"Certain embodiments of the invention may further include one or both of the following operational steps.",
"Arrow 2230 directs the flow of execution from starting operation 2200 to operation 2232.Operation 2232 performs log-converting a member of an input number collection to create a member of the number collection.",
"Arrow 2234 directs execution from operation 2232 to operation 226.Operation 226 terminates the operations of this flowchart.",
"Arrow 2240 directs the flow of execution from starting operation 2200 to operation 2242.Operation 2242 performs exp-converting a member of the number collection to create a member of an output number collection.",
"Arrow 2244 directs execution from operation 2242 to operation 2216.Operation 2216 terminates the operations of this flowchart.",
"FIG.",
"31 depicts a detail flowchart of operation 2222 of FIG.",
"30 further performing the arithmetic operation collection.",
"Arrow 2270 directs the flow of execution from starting operation 2222 to operation 2272.Operation 2272 performs adding the first number to the second number to create an add-result.",
"Arrow 2274 directs execution from operation 2272 to operation 2276.Operation 2276 terminates the operations of this flowchart.",
"Arrow 2280 directs the flow of execution from starting operation 2222 to operation 2282.Operation 2282 performs subtracting the first number by the second number to create a subtract-result.",
"Arrow 2284 directs execution from operation 2282 to operation 2276.Operation 2276 terminates the operations of this flowchart.",
"Arrow 2290 directs the flow of execution from starting operation 2222 to operation 2292.Operation 2292 performs exponentiating the first number to create an exp-result.",
"Arrow 2294 directs execution from operation 2292 to operation 2276.Operation 2276 terminates the operations of this flowchart.",
"Arrow 2300 directs the flow of execution from starting operation 2222 to operation 2302.Operation 2302 performs logarithming the first number to create a log-result.",
"Arrow 2304 directs execution from operation 2302 to operation 2276.Operation 2276 terminates the operations of this flowchart.",
"Note that the number collection is further comprised of the add-result, the subtract-result, the exp-result and the log-result.",
"FIG.",
"32A depicts a detail flowchart of operation 2272 of FIG.",
"31 further adding.",
"Arrow 2330 directs the flow of execution from starting operation 2272 to operation 2332.Operation 2332 performs determining whether the special part of the first number contains the negative-infinity.",
"Arrow 2334 directs execution from operation 2332 to operation 2336.Operation 2336 terminates the operations of this flowchart.",
"Arrow 2340 directs the flow of execution from starting operation 2272 to operation 2342.Operation 2342 performs determining whether the special part of the second number contains the negative-infinity.",
"Arrow 2344 directs execution from operation 2342 to operation 2336.Operation 2336 terminates the operations of this flowchart.",
"Arrow 2350 directs the flow of execution from starting operation 2272 to operation 2352.Operation 2352 performs setting the special part of the add-result to contain the negative-infinity whenever the special part of at least one member of the collection the first number and the second number contains the negative-infinity.",
"Arrow 2354 directs execution from operation 2352 to operation 2336.Operation 2336 terminates the operations of this flowchart.",
"FIG.",
"32B depicts a detail flowchart of operation 2282 of FIG.",
"31 further subtracting.",
"Arrow 2370 directs the flow of execution from starting operation 2282 to operation 2372.Operation 2372 performs determining whether the special part of the first number contains the negative-infinity.",
"Arrow 2374 directs execution from operation 2372 to operation 2376.Operation 2376 terminates the operations of this flowchart.",
"Arrow 2380 directs the flow of execution from starting operation 2282 to operation 2382.Operation 2382 performs setting the special part of the subtract-result to contain the negative-infinity whenever the special part of the first number contains the negative-infinity.",
"Arrow 2384 directs execution from operation 2382 to operation 2376.Operation 2376 terminates the operations of this flowchart.",
"FIG.",
"33A depicts a detail flowchart of operation 2292 of FIG.",
"31 further exponentiating.",
"Arrow 2410 directs the flow of execution from starting operation 292 to operation 2412.Operation 2412 performs determining whether the special part of the first number contains the negative-infinity.",
"Arrow 2414 directs execution from operation 2412 to operation 2416.Operation 2416 terminates the operations of this flowchart.",
"Arrow 2420 directs the flow of execution from starting operation 292 to operation 2422.Operation 2422 performs setting the special part of the exp-result to contain the not-negative-infinity and setting the integer part to a zero-representation whenever the special part of the first number contains the negative-infinity.",
"Arrow 2424 directs execution from operation 2422 to operation 2416.Operation 2416 terminates the operations of this flowchart.",
"FIG.",
"33B depicts a detail flowchart of operation 2302 of FIG.",
"31 further logarithming.",
"Arrow 2430 directs the flow of execution from starting operation 2302 to operation 2432.Operation 2432 performs determining whether the integer part of the first number is essentially equal to the zero-representation.",
"Arrow 2434 directs execution from operation 2432 to operation 2436.Operation 2436 terminates the operations of this flowchart.",
"Arrow 2440 directs the flow of execution from starting operation 2302 to operation 2442.Operation 2442 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number essentially equals the zero-representation.",
"Arrow 2444 directs execution from operation 2442 to operation 2436.Operation 2436 terminates the operations of this flowchart.",
"Note that the integer part of each of the number collection members may be in a non-redundant numeric notation.",
"FIG.",
"34A depicts a detail flowchart of operation 2442 of FIG.",
"33B further setting the special part of the log-result.",
"Arrow 2470 directs the flow of execution from starting operation 2442 to operation 2472.Operation 2472 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number equals the zero-representation.",
"Arrow 2474 directs execution from operation 2472 to operation 2476.Operation 2476 terminates the operations of this flowchart.",
"Alternatively, the integer part of each of the number collection members may be in a redundant numeric notation possessing a zero-representation collection comprising at least two zero-representation instances.",
"FIG.",
"34B depicts a detail flowchart of operation 2442 of FIG.",
"33B further setting the special part of the log-result.",
"Arrow 2490 directs the flow of execution from starting operation 2442 to operation 2492.Operation 2492 performs setting the special part of the log-result to contain the negative-infinity whenever the integer part of the first number is a member of the zero-representation collection.",
"Arrow 2494 directs execution from operation 2492 to operation 2496.Operation 2496 terminates the operations of this flowchart.",
"Note that the integer part of each member of the number collection may contains a sign and a magnitude.",
"Further, for each member of the number collection, the sign may be a member of a sign collection consisting essentially of a positive-sign and a negative-sign.",
"The special part of each member of the number collection may further contain exactly one member of a second special value collection comprising a special-minus and a special-plus.",
"FIG.",
"35A depicts a detail flowchart of operation 2292 of FIG.",
"31 further exponentiating.",
"Arrow 2510 directs the flow of execution from starting operation 2292 to operation 2512.Operation 2512 performs setting the sign of the exp-result to essentially the negative-sign whenever the special part of the first number contains the special-minus.",
"Arrow 2514 directs execution from operation 2512 to operation 2516.Operation 2516 terminates the operations of this flowchart.",
"FIG.",
"35B depicts a detail flowchart of operation 2302 of FIG.",
"31 further logarithming.",
"Arrow 2530 directs the flow of execution from starting operation 2302 to operation 2532.Operation 2532 performs determining whether the sign part of the first number is essentially equal to the negative-sign.",
"Arrow 2534 directs execution from operation 2532 to operation 2536.Operation 2536 terminates the operations of this flowchart.",
"Arrow 2540 directs the flow of execution from starting operation 2302 to operation 2542.Operation 2542 performs setting the special part of the log-result to contain the special-minus whenever the sign part of the first number is essentially the negative-sign.",
"Arrow 2544 directs execution from operation 2542 to operation 2536.Operation 2536 terminates the operations of this flowchart.",
"The integer part of each of the number collection members may be in a redundant numeric notation supporting determination of negativity by a negative-test collection comprising at least two negative-test steps.",
"FIG.",
"36A depicts a detail flowchart of operation 2532 of FIG.",
"35B further determining whether the sign part of the first number is essentially equal to the negative-sign.",
"Arrow 2570 directs the flow of execution from starting operation 2532 to operation 2572.Operation 2572 performs determining whether the sign part of the first number is equal to the negative-sign based upon performing at least one of the members of the negative-test collection.",
"Arrow 2574 directs execution from operation 2572 to operation 2576.Operation 2576 terminates the operations of this flowchart.",
"Alternatively, the integer part of each of the number collection members is in a non-redundant numeric notation possessing exactly one negative-test step.",
"FIG.",
"36B depicts a detail flowchart of operation 2532 of FIG.",
"35B further determining whether the sign part of the first number is essentially equal to the negative-sign.",
"Arrow 2590 directs the flow of execution from starting operation 2532 to operation 2592.Operation 2592 performs performing the exactly one negative-test step based upon the first number.",
"Arrow 2594 directs execution from operation 2592 to operation 2596.Operation 2596 terminates the operations of this flowchart.",
"Note that each member of the input number collection may be comprised of an integer part.",
"FIG.",
"37A depicts a detail flowchart of operation 2232 of FIG.",
"30 further log-converting the input number collection member.",
"Arrow 2600 directs the flow of execution from starting operation 2232 to operation 2602.Operation 2602 performs determining whether the integer part of the input number collection member is essentially equal to the zero-representation.",
"Arrow 2604 directs execution from operation 2602 to operation 2606.Operation 2606 terminates the operations of this flowchart.",
"Arrow 2610 directs the flow of execution from starting operation 2232 to operation 2612.Operation 2612 performs setting the special part of the number collection member to contain the negative-infinity whenever the integer part of the input number collection member essentially equals the zero-representation.",
"Arrow 2614 directs execution from operation 2612 to operation 2606.Operation 2606 terminates the operations of this flowchart.",
"Note that the integer part of each of the input number collection members may contain a sign belonging to the sign collection and a magnitude.",
"FIG.",
"37B depicts a detail flowchart of operation 2232 of FIG.",
"30 further log-converting the input number collection member.",
"Arrow 2630 directs the flow of execution from starting operation 2232 to operation 2632.Operation 2632 performs determining whether the sign part of the input number collection member is essentially equal to the negative-sign.",
"Arrow 2634 directs execution from operation 2632 to operation 2636.Operation 2636 terminates the operations of this flowchart.",
"Arrow 2640 directs the flow of execution from starting operation 2232 to operation 2642.Operation 2642 performs setting the special part of the number collection member to contain the special-minus whenever the sign part of the input number collection member is essentially the negative-sign.",
"Arrow 2644 directs execution from operation 2642 to operation 2636.Operation 2636 terminates the operations of this flowchart.",
"Note that each of the output number collection members may include a magnitude.",
"FIG.",
"38A depicts a detail flowchart of operation 2242 of FIG.",
"30 further exp-converting.",
"Arrow 2650 directs the flow of execution from starting operation 2242 to operation 2652.Operation 2652 performs setting the magnitude of the output number collection member to the zero-representation whenever the special part of the number collection member contains the negative-infinity.",
"Arrow 2654 directs execution from operation 2652 to operation 2656.Operation 2656 terminates the operations of this flowchart.",
"Also, each of the output number collection members may include a sign belonging to the sign collection.",
"FIG.",
"38B depicts a detail flowchart of operation 2242 of FIG.",
"30 further exp-converting.",
"Arrow 2670 directs the flow of execution from starting operation 2242 to operation 2672.Operation 2672 performs setting the sign of the output number collection member to the negative-sign whenever the special part of the number collection member contains the special-minus.",
"Arrow 2674 directs execution from operation 2672 to operation 2676.Operation 2676 terminates the operations of this flowchart.",
"Arrow 2680 directs the flow of execution from starting operation 2242 to operation 2682.Operation 2682 performs setting the sign of the output number collection member to the positive-sign whenever the special part of the number collection member contains the special-plus.",
"Arrow 2684 directs execution from operation 2682 to operation 2686.Operation 2686 terminates the operations of this flowchart.",
"Note that each of the input number collection members may be encoded as an N1 bit code; wherein the N1 is at least three.",
"The integer part of each of the number collection members may be encoded as an N2 bit code; wherein N2 is greater than N1.This method of numeric processing may be implemented as a program system comprised of program steps implementing the steps of the method.",
"The program steps may reside in a memory accessibly coupled to a computer, which executes these program steps.",
"The program system may further be implemented as program steps in at least one member of the language collection comprising C, C++, JAVA, FORTRAN, PASCAL, VERILOG, VHDL, assembly language and executable code for at least one computational engine implemented upon the computer.",
"The invention includes circuitry generated from those program steps.",
"The invention also includes circuitry implemented within at least one circuit component belonging to a programmable logic device collection and a fixed architecture device collection.",
"Where the programmable logic device collection refers to all integrated circuits at least partially embodying at least one programmable logic array and all integrated circuits at least partially embodying a Field Programmable Gate Array.",
"The fixed architecture device collection refers to all integrated circuits generated using gate array templates, fuse programmable integrated circuits, standard cell libraries, memory generators, and custom layout technologies.",
"The preceding embodiments of the invention have been provided by way of example and are not meant to constrain the scope of the following claims."
]
] | Patent_10276414 |
[
[
"Centrifugal pump, in particular a spirally-housed centrifugal pump for a heat exchange medium",
"In a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), parts of the casing cover (18) are a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction.",
"The bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a), and the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56)."
],
[
"1.A centrifugal pump, in particular a spiral-casing centrifugal pump, for a heat carrier medium, comprising a spiral casing (14) having an impeller (40), a casing cover (18) and an associated casing attachment portion (34) for a pump shaft (35, 36, 36a) provided with a shaft bearing (76, 76a) near the spiral casing (14) and a shaft seal (56) disposed at a spacing in relation to the shaft bearing in a sealing chamber (48), characterised in that a bearing cup (28) accommodating the shaft bearing (76, 76a), a tubular portion (32) surrounding the pump shaft (35, 36, 36a) as a heat barrier and support ribs (56) or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover (18).",
"2.A centrifugal pump as set forth in claim 1 characterised in that the bearing cup (28) is held at an axial spacing towards the impeller (40) by the tubular portion (32) surrounding the pump shaft (35, 36, 36a).",
"3.A centrifugal pump as set-forth in claim 1 or claim 2 characterised in that the casing attachment portion (34) attached to the casing cover (18) or an edge ring (30) carried by the support ribs (26) thereof delimits the sealing chamber (48) for the shaft seal (56).",
"4.A centrifugal pump as set forth in claim 3 characterised in that at their ends remote from the base region (20) of the casing cover (18) the support ribs (26) are connected by the edge ring (30) surrounding the longitudinal axis (A) of the pump.",
"5.A centrifugal pump as set forth in one of claims 1 through 4 characterised in that the support ribs (26) are inclined at an angle (w) to the longitudinal axis (A) of the pump from the base region (20) of the casing cover (18) towards the shaft seal (56).",
"6.A centrifugal pump as set forth in claim 5 characterised by an angle (w) of between 30° and 60°.",
"7.A centrifugal pump as set forth in one of claims 1 through 6 characterised in that the cross-sectional height (h) of the support ribs (26) which preferably extend in axial planes (E) passing radially through the longitudinal axis (A) of the pump increases towards the sliding ring seal (56).",
"8.A centrifugal pump as set forth in one of claims 1 through 7 characterised in that the sealing chamber (48) adjoins the shaft bearing (76, 76a).",
"9.A centrifugal pump as set forth in one of claims 1 through 8 characterised in that at least one abutment step (28) directed away from the spiral casing (24) for a further pump portion (96) is arranged at the casing cover (18).",
"10.A centrifugal pump as set forth in claim 9 characterised in that the abutment step is a front ring (22) formed on the casing cover (18).",
"11.A centrifugal pump as set forth in claim 9 or claim 10 characterised in that the abutment step or the front ring (22) projects from an annular flange (21) which surrounds the support ribs (26) and which is disposed at the transition thereof to a base region (20) of the casing cover (18).",
"12.A centrifugal pump comprising a sealing ring (59) of the shaft seal which is in the form of a sliding ring seal (56), the sealing ring extending peripherally at a counterpart ring (60) provided at the housing side, as set forth in one of claims 1 through 11, characterised in that the counterpart ring (60) for the sealing ring (59) is arranged in a shaped stub portion (64) of the casing attachment portion (34), the stub portion projecting axially into the sealing chamber (48), and associated with the stub portion is at least one passage (58) inclined with respect to the longitudinal axis (A) of the pump, with a closure screw (52) closing the passage.",
"13.A centrifugal pump as set forth in claim 12 characterised by an angle (z) between the longitudinal axis (A) of the pump and the longitudinal axis (B) of the passage (53) of about 45°.",
"14.A centrifugal pump as set forth in claim 12 or claim 13 characterised by an axial length (f) of the stub portion (64) which corresponds approximately to half the axial length (e1) of the sealing chamber (48).",
"15.A centrifugal pump as set forth in one of claims 1 through 14 characterised in that the shaft bearing is in the form of a plain bearing (76) with a ratio of the length (g) of the bearing bush (77) to the diameter (d) of the pump shaft (35, 36, 36a) of between about 1.5 and 2.0, preferably about 1.8.16.A centrifugal pump as set forth in claim 15 characterised in that the bearing bush (77) of the plain bearing (76, 76a) is adapted to be dismantleable.",
"17.A centrifugal pump as set forth in claim 15 or claim 16 characterised in that the plain bearing (76, 76a) or its bearing bush (77) is externally embraced approximately at its longitudinal center by a clamping or tolerance ring (80) which engages into the bearing cup (28).",
"18.A centrifugal pump as set forth in claim 17 characterised in that the width (n) of the clamping or tolerance ring (80) corresponds to a fraction of the length (g) of the bearing bush (77).",
"19.A centrifugal pump as set forth in claim 18 characterised in that the width (n) of the external clamping or tolerance ring (80) corresponds approximately to between a fifth and a sixth of the length (g) of the bearing bush (77).",
"20.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76a) includes an inner bearing sleeve (82) surrounded by a bearing bush (77) at the bearing cup side (FIGS.",
"6 and 7).",
"21.A centrifugal pump as set forth in one of claims 15 through 20 characterised in that within the plain bearing (76a) the pump shaft (35, 36a) is embraced by at least one tolerance ring (80i) which engages into the pump shaft.",
"22.A centrifugal pump as set forth in one of claims 17 through 21 characterised in that provided on respective sides of the outer tolerance ring (80), viewed in longitudinal section of the centrifugal pump (10a), is a respective inward tolerance ring (80i) which engages into the pump shaft (35, 36a).",
"23.A centrifugal pump as set forth in one of claims 15 through 19 characterised in that the plain bearing (76) includes a bearing bush (77) at the bearing cup side, with a slidable internal peripheral surface portion (78) embracing the pump shaft (36).",
"24.A centrifugal pump as set forth in claim 23 characterised by an internal peripheral surface portion (28) of the plain bearing (76) comprising carbon (FIGS.",
"4 and 5).",
"25.A centrifugal pump as set forth in claim 23 or claim 24 characterised by a hardened pump shaft (36) or a corresponding shaft portion which is associated with the bearing bush (77).",
"26.A centrifugal pump as set forth in one of claims 1 through 25 characterised in that the sliding ring seal (56) embraces a shaft sleeve (89) at the shaft side, wherein at least one O-ring (88) is provided between the shaft sleeve (89) and the pump shaft (36, 36a).",
"27.A centrifugal pump as set forth in one of claims 1 through 26 characterised in that a rearward shaft bearing (68) for the pump shaft (36, 36a) is arranged in the casing attachment portion (34) at an axial spacing relative to the sliding ring seal (56) outside the sealing chamber (48).",
"28.A centrifugal pump as set forth in claim 27 characterised in that at least one hollow space (66) is arranged between the sealing chamber (48) and the shaft bearing (68) in the casing attachment portion (34).",
"29.A centrifugal pump as set forth in one of claims 1 through 28 characterised in that the pump shaft (36a) is in the form of a plug-in stub shaft with clamping ring (92).",
"30.A centrifugal pump as set forth in one of claims 8 through 29 characterised in that the spiral casing (14) and the bearing carrier casing (16) are embraced by a spacer (96) which butts against the abutment step or steps or the front ring (22) respectively and it is attached to a motor (100)."
],
[
"The invention concerns a centrifugal pump—in particular a spiral-casing centrifugal pump—for a heat carrier medium, comprising a spiral casing having an impeller, a casing cover and an associated casing attachment portion for a pump shaft provided with a shaft bearing near the spiral casing and a shaft seal disposed at a spacing in relation to the shaft bearing in a sealing chamber.",
"Such centrifugal pumps are described by way of example in a prospectus from the present applicants in relation to the range NHT for heat carrier purposes up to 350° C. A spiral casing for an impeller is connected to a bearing casing by means of a throttle section having external ribs.",
"The impeller is fixed at one end of a pump shaft which extends in the throttle section.",
"Arranged between the throttle section and the impeller is a plain bearing of silicon carbide and arranged downstream of the throttle section is a sliding ring seal which is followed by a rolling bearing.",
"This arrangement involves a horizontal spiral-casing centrifugal pump of a process type of structure which is single-stage in terms of intake flow.",
"The pump-side silicon carbide plain bearing is lubricated by the fluid being delivered, while used at the drive side is a grease-lubricated grooved-type ball bearing.",
"In consideration of that state of the art, the inventor set himself the aim of providing a centrifugal pump of the kind set forth in the opening part of this specification, which is simple to manufacture and which can be inexpensively and particularly well used.",
"That object is attained by the teaching of the independent claim; the appendant claims set forth advantageous developments.",
"In addition the scope of the invention embraces all combinations comprising at least two of the features disclosed in the description, the drawing and/or the claims.",
"In accordance with the invention a bearing cup accommodating the shaft bearing, a tubular portion surrounding the pump shaft as a heat barrier and so-called support ribs or the like profile elements arranged at a spacing relative to each other in the peripheral direction are parts of the casing cover.",
"Those features permit the pump to be of a simple structure with only two pressure-bearing parts for the bearing carrier casing—comprising the casing cover and the casing attachment portion—as well as a particularly high level of stability for the bearing carrier casing.",
"In addition the bearing cup is to be held at an axial spacing towards the impeller by the tubular portion which surrounds the pump shaft.",
"The sealing chamber for the shaft seal is advantageously delimited by the casing attachment portion which is preferably joined to the casing cover or an edge ring which is carried by the support ribs thereof and which surrounds the longitudinal axis of the pump; the edge ring is connected to and preferably integrally formed on the support ribs at their ends remote from the base region of the casing cover.",
"Disposed between the spiral casing and the above-mentioned sealing chamber is a latticework-like or trussing-like rib arrangement comprising those support ribs which are to be inclined towards the longitudinal axis from the spiral casing at a preferred angle of between 40° and 45° and—at a radial spacing relative to each other—surround the bearing cup and the heat barrier and form jointly therewith the casing cover.",
"For that purpose it has proven to be desirable for the cross-sectional height of the support ribs which preferably extend in axial planes which pass radially through the longitudinal axis of the pump to be caused to increase towards the sliding ring seal in order to enhance the level of stability and minimise heat losses.",
"In accordance with a further feature of the invention the sealing chamber is to adjoin the shaft bearing.",
"Inter alia that ensures an adequate supply of lubricating medium to the bearing.",
"In addition the bearing contributes to circulation of the medium in the sealing chamber for flushing and cooling the seal.",
"In order to enlarge the use of the centrifugal pump according to the invention and the combination options thereof, it is possible to arrange on the casing cover at least one abutment stage or step directed away from the spiral casing, for a further part of the pump; that abutment step should preferably be a front ring which is formed on the casing cover.",
"An associated pump part can moreover be a spacer which extends around the bearing carrier casing and which abuts against the abutment step or steps or the front ring and to which a motor is attached.",
"It has proven to be desirable for the abutment step or the front ring to be arranged to project from an annular flange which extends around the support ribs and which is mounted to the transition thereof to a base region of the casing cover.",
"The scope of the invention includes a centrifugal pump with a sealing ring of the shaft seal—in the form of a sliding ring seal—, the sealing ring extending therearound on a counterpart ring provided at the casing side; the counterpart ring is arranged for the sealing ring in a shaped stub portion of the casing attachment portion, the stub portion projecting axially into the sealing chamber.",
"At last one opening or passage which is inclined with respect to the longitudinal axis of the pump and with a closure screw for closing it is to be associated with the stub portion—which is of an axial length approximately corresponding to half the axial length of the sealing chamber—; the longitudinal axis of the passage advantageously delimits an angle of about 45° with the longitudinal axis of the pump.",
"Thus the sliding ring seal is advanced within the sealing chamber in order to make available sufficient space for collecting air or outgassings of the delivery medium—in particular when the arrangement is positioned vertically.",
"The described arrangement of the passage with the closure screw easily permits complete venting of the sealing chamber both when the pump is positioned horizontally and also vertically.",
"The frictional heat of the sliding ring seal is desirably dissipated to the surroundings by virtue of a large surface area of the surrounding sealing chamber and a large volume of fluid in the sealing chamber.",
"In accordance with the invention the shaft bearing is in the form of a plain bearing with a ratio of the length of the bearing bush to the diameter of the pump shaft of between about 1.5 and 2.0, preferably about 1.8.In addition the bearing bush of the plain bearing should be designed to be dismantled.",
"Advantageously the plain bearing, as viewed from the impeller, is disposed behind the heat barrier, which contributes to simplicity of possible fixing and also contributes to the heat barrier being of a desirable design configuration.",
"In order to achieve a low level of heat conduction from the spiral casing to the sealing chamber, a small cross-section and a long length of the tubular portion, which is in the form of the heat barrier, of the casing cover has proven to be desirable.",
"That can be implemented particularly well with the specified bearing arrangement.",
"The positioning of the impeller-side bearing behind the heat barrier means that the delivery medium which serves as a lubricating agent is at a lower temperature at that location than in the pump casing.",
"As a result it is of higher viscosity, which is advantageous for the lubricating capability.",
"In addition the vapor pressure of the medium at that location is markedly lower than in the pump casing, thereby minimising the risk of the bearing running dry.",
"It has proven to be desirable for the plain bearing or the bearing bush thereof, for fixing purposes, to be surrounded approximately at the longitudinal center thereof on the outside by a clamping or tolerance ring which engages into the bearing cup.",
"The width of the clamping or tolerance ring should correspond to a fraction of the length of the bearing bush, for example between a fifth and a sixth of the length of the bearing bush.",
"That provides for easy (dis-)mantling as well as compensation for alignment errors by virtue of tilting mobility.",
"There are a number of design configurations of the plain bearing, in accordance with the invention.",
"In one case the plain bearing includes an inner bearing sleeve which is surrounded by a bearing bush at the bearing cup side.",
"In that case the pump shaft is surrounded within the plain bearing by at least one tolerance ring which engages into the pump shaft.",
"In particular an inward tolerance ring should be provided on each of the two sides of the outer tolerance ring which engages into the bearing cup—as viewed in longitudinal section of the centrifugal pump—, the inward tolerance ring engaging into the pump shaft.",
"The bearing sleeve and the bearing bush are preferably made from silicon carbide.",
"Another design configuration in accordance with the invention involves a plain bearing with a bearing bush at the bearing cup side, the bearing bush including a slidable internal peripheral surface portion which extends around the pump shaft.",
"In that case the dismantleable plain bearing bush is preferably provided with an inner carbon coating.",
"The bush runs on a hardened shaft or a hardened shaft portion.",
"Fixing is effected by a centrally disposed outer tolerance ring, as already mentioned.",
"In accordance with a further feature of the invention the sliding ring seal extends around a shaft sleeve at the shaft side, wherein at least one O-ring is provided between the shaft sleeve and the pump shaft.",
"That arrangement, in a construction which is relieved of load, permits assembly of the sliding ring seal from the impeller side, which is desirable in particular when the shaft is in the form of a plug-in shaft.",
"In accordance with another feature of the invention arranged in the casing attachment portion at an axial spacing relative to the sliding ring seal outside the sealing chamber is a rearward shaft bearing for the pump shaft; at least one hollow space can also be provided in the casing attachment portion between the sealing chamber and the shaft bearing.",
"In a particular configuration the pump shaft is in the form of a plug-in shaft with clamping ring.",
"By virtue of the described different design configurations of the plain bearing, in combination with the three types of structure: pump with main plate and coupling; pump of a block-type structure; and pump of an in-line type of structure there are at least six structural variants for the centrifugal pump according to the invention.",
"Further advantages, features and details of the invention will be apparent from the description hereinafter of preferred embodiments and with reference to the drawing in which: FIGS.",
"1 and 8 each show a perspective view of different embodiments of a centrifugal pump according to the invention for heat carrier media, FIG.",
"2 shows an end view of the FIG.",
"1 structure, FIG.",
"3 shows a partial longitudinal section through FIG.",
"2 along line III-III therein, FIG.",
"4 shows a view in longitudinal section on an enlarged scale through the pump in FIG.",
"2 taken along line IV-IV therein, FIG.",
"5 shows a view on an enlarged scale of a part of the FIG.",
"4 structure, FIG.",
"6 shows a longitudinal section corresponding to FIG.",
"4 through another embodiment of the pump shown in FIG.",
"1, FIG.",
"7 shows a view on an enlarged scale of a portion of the FIG.",
"6 structure, FIG.",
"9 shows an end view of the FIG.",
"8 structure, FIG.",
"10 shows a perspective view of a casing cover of the pump, FIG.",
"11 shows a view on an enlarged scale through the pump of FIGS.",
"8 and 9 taken along line XI-XI in FIG.",
"9, FIG.",
"12 shows a partial longitudinal section on an enlarged scale in relation to FIG.",
"11 taken along line XII-XII in FIG.",
"9, FIGS.",
"13 and 14 show different perspective views of a part of a further embodiment of a centrifugal pump according to the invention, FIG.",
"15 shows an end view of the structure shown in FIGS.",
"13 and 14, FIG.",
"16 shows a partial longitudinal section through the pump of FIGS.",
"13 through 15 taken along line XVI-XVI in FIG.",
"15, and FIG.",
"17 shows a partial section on an enlarged scale in relation to FIG.",
"16 through that pump taken along line XVII-XVII in FIG.",
"15.A spiral-casing centrifugal pump 10, on a volute or spiral casing 14 provided with connecting flanges 12, 12a,—with the interposition of seals (not shown)—has a bearing carrier casing 16 which includes a casing cover 18 with a casing attachment portion 34 fitted thereto.",
"The casing cover 18 is provided with an annular flange 21 which projects from a dish-like base region 20 and through which pass connecting screws 24, on a front ring 22 which is formed on the annular flange 21 and which forms an abutment step; the connecting screws 24 are fitted into sleeve ribs 15 which are formed in axis-parallel relationship externally on a peripheral wall 13 of the spiral casing 14, the peripheral wall engaging over the dish wall 19, and the screws 24 centeringly connect the casing cover 18 to the spiral casing 14.Formed on the casing cover 18 or the base region 20 thereof respectively are support ribs 26 of substantially constant thickness b, which are determined by axial planes E—passing radially through the longitudinal axis A of the pump—and which are inclined at an angle w of about 40° towards the longitudinal axis A of the pump.",
"The cross-sectional height h of the support ribs 26 steadily increases in the longitudinal extent or in the axial direction away from the casing cover 18.The support ribs 26 are cast at the other end to a bearing cup 28 axially associated with the longitudinal axis A of the pump and an edge ring 30 which surrounds the bearing cup.",
"An axial tubular portion 32 which is integral with the casing cover 18 connects the bearing cup 28 thereto, in centered relationship.",
"The bearing cup 28, with its edge ring 30, and the support rings 26—of which there are four here—are also part of the casing cover 18 whose length a, approximately corresponds to a third of the pump length a.",
"Passing through the casing cover 18 and the rearward casing attachment portion 34—that is to say the bearing carrier casing 16—is a pump shaft 36 of a diameter d, while an impeller 40 is held on the front stepped end 37 thereof by a nut 38.The impeller 40 rotates in the spiral casing 14 which includes an intake or suction chamber 42, whose inlet in the region of the axial connecting flange 12 is identified by reference 43 and whose radial outlet in the connecting flange 12a is identified by reference 44.Disposed near the impeller 40 in the casing cover 18 is a stuffing-box packing 46 at the periphery of the cylindrical tubular portion 32.The internal space 29 of the bearing cup 28 is connected to the cylindrical tubular portion 32 which extends around the pump shaft 36 to form a heat barrier.",
"If for example a flow agent of low viscosity at about 350° C. passes through the inlet 43, flow agent of higher viscosity at 130° C. is disposed in the internal space of the bearing cup.",
"The internal space 29 of the bearing cup 28, which adjoins the cylindrical tube portion 32, communicates with a sealing chamber 48 which is arranged in the bearing carrier casing 16 or in the casing attachment portion 34 and which is surrounded by an annular wall 50 and which is provided with at least one closure screw 52 together with a screwthreaded opening or passage 53 which accommodates the screw and which is inclined at an angle z of 45° relative to the longitudinal axis A of the pump, and with blade-like surface elements 55 which are inclined relative to each other and which each have a respective edge 54 parallel to the axis, the axial length e1 thereof approximately corresponding to half the length e of the casing attachment portion 34.An outlet screw 52b towards the bottom is arranged in a radial opening or passage in the region of the chamber which is towards the bottom.",
"In addition FIG.",
"2 shows a lateral closure screw 52a which is required for venting when the pump 10 is used in a vertical position.",
"A rear axial sliding ring seal 56—which is not relieved of load—can be seen in the sealing chamber 48.Projecting from the annular wall 50 are four radial lugs 51 which are connected to corresponding counterpart lugs 51a of the edge ring 30 by screws 24a.",
"In embodiments which are not shown here—in particular of large dimensioning—an annular flange can also be provided, instead of the above-mentioned radial lugs 51.In order to increase the surface area of the outside of the annular wall, axially extending surface portions can be arranged on the outside of the annular wall.",
"The sliding ring seal 56 includes a coil spring 58—which is supported against a ring step or a ring 38 which is pushed on to the pump shaft 36—and which loads a rotating sliding ring 59 which with a stationary counterpart ring 60 forms a sealing gap.",
"The counterpart ring 60 is disposed in the tubular space 62 of an axial shaped stub portion 64 of an axial length f which here corresponds approximately to half the sealing chamber length e1.Arranged subsequently to the annular end part 65 of the stub portion 64 in the casing attachment portion 34 is a dish-like hollow space 66.Between the latter and the adjacent stepped shaft end 37e the pump shaft 36 is surrounded by a grooved-type ball bearing 68 with spacer disks 69 and a securing ring 70; that unit is covered by a bearing cover 72 of the casing attachment portion 34.Projecting radially beneath the latter is a plate-like support leg 74 which is fixed by screws 24b, comprising an angle plate member, in opposite relationship to which, in the region of the spiral casing 14, are two supports 74a which project in parallel in eccentric relationship.",
"The supports 74a and the support leg 74 are connected at the other end to a base plate (not shown).",
"Arranged in the internal space 29 in the bearing cup 28 in FIGS.",
"3 through 5 is a front hydrodynamic plain bearing 76 comprising a bearing bush 77 of a length g with an internal peripheral surface portion 78 of carbon material; the plain bearing 78, at its longitudinal center, is surrounded by a clamping or tolerance ring 80 of a width n, the ring 80 being provided in the bearing cup 28—that is to say at the housing side—; the ring 80 is of an elastic nature and force-lockingly connects the plain bearing 76 to the bearing cup.",
"The plain bearing 76a of the pump 10a as shown in FIGS.",
"6 and 7 has within the bearing bush 77 a coaxial bearing sleeve 82 and—besides the above-described outer tolerance ring 80 at the longitudinal center—two inner tolerance rings 80i of a width i which are disposed at the shaft side and which—viewed in longitudinal section—flank the outer tolerance ring 80 at the pump shaft 36 at a radial spacing on both sides.",
"The width n of the outer tolerance ring 80 and the width i of the inner tolerance rings 80i are relatively short in relation to the length g of the bearing bush, for example the latter corresponds approximately to between five and six times the width i and n respectively.",
"Reference 84 in FIGS.",
"6 and 7 shows a nut which is disposed between the front plain bearing 76a and the rear sliding ring seal 56—which is here relieved of load—for the purposes of fixing the bearing sleeve 82.The sliding ring components for carrying the radial forces which occur preferably comprise silicon carbide in the illustrated embodiment.",
"The two above-discussed bearing types 76, 76a can also be used in the centrifugal pump 11 shown in FIGS.",
"8 through 12 in which the pump shaft 35 is in the form of a plug-in or stub shaft and is provided with an O-ring 88 within a shaft sleeve 86 of the sliding ring seal 56a, the shaft sleeve 86 surrounding the pump shaft.",
"The O-ring 88 is disposed between the pump shaft 35 and a shaft sleeve 89 embracing it.",
"At the rearward end of the bearing carrier casing 16 the pump shaft 35 includes a clamping ring 92 which carries a fan wheel 90 and which is surrounded towards the front by a protective grid 93 as protection to prevent articles from being poked in.",
"The rearward shaft bearing—which is disposed in the motor—cannot be seen in the drawing.",
"The impeller 90 is surrounded by a wall ring 94—with apertured plate members 95—of a spacer 96 with endward flange rings 97, 97a; they are connected by longitudinal ribs 98 which are parallel to the axis.",
"The flange ring 97 which is at the right in the drawing is screwed to a parallel flange ring 99 of a motor 100, the flange ring 99 bearing against the spiral casing 14; the motor 100 is fixed by means of the spacer 96 to the pump 11.In the configuration of a centrifugal pump 11a with a vertical shaft 35 as shown in FIG.",
"13 ff, the inlet 43 and the outlet 44 of the spiral casing 14 are on a straight diametral line Q.",
"This pump 11a can also be provided with both of the described bearing forms 76, 76a."
]
] | Patent_10276581 |
[
[
"Allocating access across shared communication medium",
"A method of providing network access across a shared communications medium between competing users (86) includes the step of allocating network access for each user for a future time interval (102).",
"Features include forecasting network access (1100) of the users in a future time interval, and prioritizing the users for allocating network access to the users.",
"The network access allocations represent network access allowances available to the users during the future time interval, and further may represent network access usage.",
"Classes of users can be allocated network access first (1114), and then each user allocated network access from the class allocation."
],
[
"1.A method of providing network access across a shared communications medium between at least two competing users, comprising the steps of: (a) prior to a first time interval, determining for each user a respective first network access allowance representing a respective first maximum level of network access available to the user during the first time interval, but not necessarily representing the level of network access that will be utilized by the user during the first time interval; (b) during the first time interval, providing network access to each user such that the respective first network access allowance for each user is not exceeded; (c) prior to a second consecutive time interval, determining for each user a respective second network access allowance representing a respective second maximum level of network access available to the user during the second time interval, but not necessarily representing the level of network access that will be utilized by the user during the second time interval, at least one respective second network access allowance for a user differing from such user's respective first network access allowance; and (d) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.",
"2.The method of claim 1, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"3.The method of claim 1, wherein the first and second time intervals are consecutive time intervals.",
"4.The method of claim 1, wherein each of the first time interval and the second time interval has a period of between one minute and sixty minutes.",
"5.The method of claim 1, wherein the second network access allowance for each respective user differs from the first network access allowance for such user.",
"6.The method of claim 1, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.",
"7.The method of claim 6, wherein the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.",
"8.The method of claim 7, wherein the first allocation policy differs from the second allocation policy.",
"9.The method of claim 7, further comprising requesting a minimum level of network access for the first time interval for a user, and wherein said allocating network access to such user for the first time interval comprises setting the level of network access allocated to such user to a level equal to or greater than the requested minimum level.",
"10.The method of claim 9, wherein said allocating network access to such user for the second time interval comprises setting the level of network access allocated to such user to a level less than the requested minimum level for the first time interval.",
"11.The method of claim 1, further comprising the step of monitoring network access usage by each user.",
"12.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted from and to each user during a past time interval.",
"13.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a past time interval.",
"14.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.",
"15.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a past time interval.",
"16.The method of claim 11, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a time interval.",
"17.The method of claim 1, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.",
"18.The method of claim 1, wherein the Shared Access Carrier Network comprises a wireless network.",
"19.The method of claim 1, further comprising prioritizing the users for determining network access allowances.",
"20.The method of claim 19, wherein said prioritizing is based on fairness considerations.",
"21.The method of claim 20, wherein the users are prioritized based on user throughput during a past time interval, with a user with lesser throughput receiving priority over a user with greater throughput.",
"22.The method of claim 20, wherein the users are prioritized based on data loss for each user during a past time interval, with a user with greater data loss having priority over a user with lesser data loss.",
"23.The method of claim 20, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"24.The method of claim 20, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.",
"25.The method of claim 20, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"26.The method of claim 19, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.",
"27.The method of claim 26, further comprising the step of monitoring network access usage by each user.",
"28.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"29.The method of claim 27, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"30.The method of claim 27, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.",
"31.The method of claim 27, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"32.The method of claim 27, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"33.The method of claim 27, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises setting the respective levels of network access equal to each user's specified respective minimum level of network access.",
"34.The method of claim 11, further comprising the step of forecasting network access usage by each user during a future time interval based on said step of monitoring network access usage by each user.",
"35.The method of claim 34, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"36.The method of claim 34, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.",
"37.The method of claim 34, wherein the first network access allowances for the users are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.",
"38.The method of claim 37, wherein said step of allocating network access comprises setting the respective levels of network access of the users proportional to each user's forecasted network access usage.",
"39.The method of claim 37, further comprising the step of prioritizing the users for allocating network access.",
"40.The method of claim 39, wherein said prioritizing is based on each user's forecasted network access usage.",
"41.The method of claim 40, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"42.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"43.The method of claim 40, wherein said step of allocating network access comprises setting respective user levels of network access equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"44.A method of providing during two consecutive time intervals network access across a shared communications medium between two competing users, comprising the step of varying between the two time intervals a respective network access allowance for each user, each respective network access allowance representing a maximum level of network access available to the respective user during a particular time interval, but not necessarily representing the level of network access that is utilized by the user during the particular time interval.",
"45.A method of providing network access across a shared communications medium in a downstream direction towards competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; and (b) based on said monitored network access usage, allocating network access to each user on a per user basis for a future time interval.",
"46.The method of claim 45, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"47.The method of claim 45, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.",
"48.The method of claim 47, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.",
"49.The method of claim 45, wherein said step of monitoring comprises monitoring bandwidth that is consumed by each user in a downstream direction at time intervals of fifteen minutes to sixty minutes.",
"50.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"51.The method of claim 45, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"52.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of logical data units transmitted to each user during a time interval.",
"53.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted to each user during a time interval.",
"54.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"55.The method of claim 45, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"56.The method of claim 45, wherein said step of allocating network access comprises allocating network access equally to the users.",
"57.The method of claim 45, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"58.The method of claim 57, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"59.The method of claim 57, wherein the Shared Access Carrier Network comprises a wireless network.",
"60.The method of claim 45, further comprising prioritizing the users for allocating network access.",
"61.The method of claim 60, wherein said prioritizing is based on fairness considerations.",
"62.The method of claim 61, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput receiving priority over a user with greater throughput.",
"63.The method of claim 61, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss having priority over a user with lesser data loss.",
"64.The method of claim 61, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"65.The method of claim 61, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.",
"66.The method of claim 61, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"67.The method of claim 60, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.",
"68.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"69.The method of claim 67, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"70.The method of claim 67, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.",
"71.The method of claim 67, wherein SLAs provide respective fees for network access usage, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"72.The method of claim 67, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"73.The method of claim 67, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.",
"74.The method of claim 45, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.",
"75.The method of claim 74, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"76.The method of claim 74, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.",
"77.The method of claim 74, wherein said step of allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.",
"78.The method of claim 74, further comprising the step of prioritizing the users for allocating network access.",
"79.The method of claim 78, wherein said prioritizing is based on each user's forecasted network access usage.",
"80.The method of claim 78, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"81.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"82.The method of claim 78, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"83.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) forecasting network access usage by each user during a future time interval based on said monitored network access usage by each user; and (c) based on said forecasted network access usage, allocating network access to each user for the future time interval.",
"84.The method of claim 83, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"85.The method of claim 83, wherein said step of forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.",
"86.The method of claim 83, wherein said step of allocating network access comprises allocating network access to the users proportionally based on each user's forecasted network access usage.",
"87.The method of claim 83, further comprising the step of prioritizing the users for allocating network access.",
"88.The method of claim 87, wherein said prioritizing is based on each user's forecasted network access usage.",
"89.The method of claim 87, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"90.The method of claim 87, wherein said prioritizing is based on fairness considerations.",
"91.The method of claim 87, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.",
"92.The method of claim 87, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.",
"93.The method of claim 87, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"94.The method of claim 87, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.",
"95.The method of claim 87, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"96.The method of claim 87, wherein said step of prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.",
"97.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"98.The method of claim 96, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"99.The method of claim 96, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usages both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access would have been utilized for such users.",
"100.The method of claim 96, wherein SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"101.The method of claim 96, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"102.The method of claim 96, wherein SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.",
"103.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"104.The method of claim 87, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"105.The method of claim 83, wherein each network access allocation represents a bandwidth allowance of a respective user during the future time interval.",
"106.The method of claim 83, wherein each network access allocation represents bandwidth utilized by each user during the future time interval.",
"107.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) of each user that will be transmitted over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.",
"108.A method of providing network access across a shared communications medium of a Cable Network between competing users, comprising the steps of: (a) monitoring network access usage requested by each user for a time interval; (b) based on said monitoring, forecasting the number of logical data units (LDUs) that will be requested by each user over a future time interval; and (c) based on said forecasting, allocating network access available to each user for the future time interval.",
"109.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"110.The method of claim 109, wherein said step of allocating further comprises allocating any surplus network access remaining equally to the users.",
"111.The method of claim 109, wherein said step of allocating further comprises allocating in decreasing user priority any surplus network access to the users proportionally to the user's forecasted network access usage.",
"112.The method of claim 109, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"113.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.",
"114.The method of claim 109, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.",
"115.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"116.The method of claim 109, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"117.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"118.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"119.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"120.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"121.The method of claim 109, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.",
"122.The method of claim 109, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"123.The method of claim 122, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"124.The method of claim 122, wherein the Shared Access Carrier Network comprises a wireless network.",
"125.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on said monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total available network access to the total forecasted network access usage of all the users, and subject to a respective, predetermined maximum value and subject to availability.",
"126.The method of claim 125, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"127.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.",
"128.The method of claim 125, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.",
"129.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"130.The method of claim 125, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"131.The method of claim 125, wherein said step of monitoring network access includes collecting data representative of the logical data units transmitted from and to each user during a time interval.",
"132.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"133.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"134.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"135.The method of claim 125, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.",
"136.The method of claim 125, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"137.The method of claim 126, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"138.The method of claim 126, wherein the Shared Access Carrier Network comprises a wireless network.",
"139.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"140.The method of claim 139, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"141.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.",
"142.The method of claim 139, wherein said step of monitoring comprises monitoring network access usage by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.",
"143.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"144.The method of claim 139, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"145.The method of claim 139, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"146.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"147.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"148.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"149.The method of claim 139, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.",
"150.The method of claim 139, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"151.The method of claim 150, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"152.The method of claim 150, wherein the Shared Access Carrier Network comprises a wireless network.",
"153.A method of providing network access across a shared communications medium between competing users, comprising the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on said monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum specified value and subject to availability.",
"154.The method of claim 153, wherein any additional network access remaining after said allocation is earmarked for the users such that each user's allocation equals the user's forecasted network access usage, subject to a respective, predetermined maximum allowed level of network access and subject to availability.",
"155.The method of claim 154, wherein any surplus network access remaining after said distribution is further earmarked in equal amounts for the users, subject to a respective, predetermined maximum allowed level of network access.",
"156.The method of claim 154, further comprising allocating to the users network access only in an amount equal to the forecasted network access usage multiplied by the ratio of total network access available for allocation to the total forecasted network access usage of the users, but only when the total forecasted network access usage of the users exceeds the total network access available for allocation.",
"157.The method of claim 153, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"158.The method of claim 153, wherein said step of monitoring comprises monitoring network access usage by the users in an upstream direction at time intervals of one minute to fifteen minutes.",
"159.The method of claim 153, wherein said step of monitoring comprises monitoring network access by the users in a downstream direction at time intervals of fifteen minutes to sixty minutes.",
"160.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"161.The method of claim 153, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"162.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"163.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"164.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are dropped during a time interval.",
"165.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of each user that are dropped during a time interval.",
"166.The method of claim 153, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of each user that are requested to be transmitted in the upstream direction during a time interval.",
"167.The method of claim 153, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"168.The method of claim 167, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"169.The method of claim 167, wherein the Shared Access Carrier Network comprises a wireless network.",
"170.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing said monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.",
"171.The method of claim 170, wherein the threshold value represents a respective maximum level of network access for each user.",
"172.The method of claim 170, wherein the threshold value represents a respective maximum burstable level of network access with target probability for each user.",
"173.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via email.",
"174.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via instant messaging.",
"175.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.",
"176.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.",
"177.The method of claim 170, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.",
"178.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent basis.",
"179.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user with a maximum burstable level of network access with target probability.",
"180.The method of claim 170, further comprising charging the user a fee for the modification of the SLA.",
"181.The method of claim 170, wherein the modification of the user's SLA includes guaranteeing a level of network access to the user on a temporary basis.",
"182.The method of claim 170, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"183.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in an upstream direction of communication across the shared communications medium at time intervals of one minute to fifteen minutes.",
"184.The method of claim 170, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each user in a downstream direction of communication across the shared communications medium at time intervals of fifteen minutes to sixty minutes.",
"185.The method of claim 170, wherein said step of monitoring network access includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"186.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"187.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"188.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"189.The method of claim 170, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.",
"190.The method of claim 170, wherein the shared communications medium is part of a Shared Access Carrier Network.",
"191.The method of claim 190, wherein the Shared Access Carrier Network comprises a Cable Network and the shared communications medium comprises a coaxial cable.",
"192.The method of claim 190, wherein the Shared Access Carrier Network comprises a wireless network.",
"193.The method of claim 170, further comprising, based on said monitored network access usage, allocating network access to each user for a future time interval.",
"194.The method of claim 193, wherein said step of allocating network access comprises allocating network access equally to the users.",
"195.The method of claim 193, further comprising prioritizing the users for allocating network access.",
"196.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for the users, and said step of prioritizing includes comparing said monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"197.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and said step of prioritizing includes comparing said monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when said monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"198.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and said step of prioritizing includes comparing said monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.",
"199.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide a respective fee for network access usage by users, and said step of prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"200.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and said step of prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"201.The method of claim 195, wherein said step of prioritizing is based on the SLAs of the users, wherein the SLAs specify respective minimum levels of network access for users, and said step of allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.",
"202.The method of claim 195, wherein said prioritizing is based on fairness considerations.",
"203.The method of claim 202, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.",
"204.The method of claim 202, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.",
"205.The method of claim 202, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"206.The method of claim 202, wherein the users are prioritized based on both user throughput and data loss during a time interval.",
"207.The method of claim 202, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"208.The method of claim 193, further comprising the step of forecasting network access usage by each user during the future time interval based on said step of monitoring network access usage by each user.",
"209.The method of claim 208, wherein said step of forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"210.The method of claim 208, wherein said step of forecasting comprises applying an adaptive-response-rate signal exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of the users.",
"211.The method of claim 208, wherein said step of allocating comprises allocating network access to the users proportionally based on each user's forecasted network access usage.",
"212.The method of claim 208, further comprising the step of prioritizing the users for allocating network access.",
"213.The method of claim 212, wherein said prioritizing is based on each user's forecasted network access usage.",
"214.The method of claim 212, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"215.The method of claim 212, wherein said step of allocating comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"216.The method of claim 212, wherein said step of allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"217.A method of providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users, comprising the steps of: (a) monitoring network access usage by each user for respective predetermined past time intervals; (b) identifying a recurrent period of high network access usage of a user based on said monitoring; and (c) soliciting a user to modify the user's SLA to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.",
"218.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via email.",
"219.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via instant messaging.",
"220.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via redirection of a web browser of the user to a solicitation web page.",
"221.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via generation and mailing of literature.",
"222.The method of claim 217, wherein said step of soliciting a user comprises contacting the user via a telephonic communication.",
"223.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for all future recurrent periods of high network access usage.",
"224.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user for a predetermined number of future recurrent periods of high network access usage.",
"225.The method of claim 217, wherein the modification of the user's SLA includes guaranteeing a minimum level of network access to the user with a maximum burstable level of network access with target probability for the future period of high network access usage.",
"226.The method of claim 217, wherein the recurrent time period of high network access usage comprises a particular time of day.",
"227.The method of claim 217, further comprising charging the user a fee for modification of the user's SLA.",
"228.A method of providing network access across a DOCSIS 1.0 compliant cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user a respective first bandwidth allowance for a first future time interval; (b) generating a first set of cable modem configuration files, each of which limits bandwidth consumption by a cable modem (CM) of a respective user to that user's first bandwidth allowance; (c) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; (d) sending a command either to each user's CM or to a cable modem termination system (CMTS) to which each user's CM is connected to cause each CM to acquire its new respective configuration file of the first set for the first time interval; (e) determining for each user a respective second bandwidth allowance for a second future time interval, a second bandwidth allowance of at least one of the users differing from that user's first bandwidth allowance; and (f) for each user having a second bandwidth allowance different from that user's first bandwidth allowance, (i) generating a second cable modem configuration file which limits bandwidth consumption of that user's CM to a value representative of that user's second bandwidth allowance; (ii) sending the second configuration file for that user to the TFTP Server of the DOC Network; and (iii) sending a command either to that user's CM or to the CMTS to which that user's CM is connected to cause the CM to acquire the second configuration file for that user for the second time interval.",
"229.The method of claim 228, further comprising the steps of monitoring bandwidth of each user for a time interval and, based on said monitored bandwidth, determining each user's second bandwidth allowance.",
"230.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes prioritizing each user and allocating bandwidth to each user according to such priority.",
"231.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth with respective values specified by service level agreements (SLAs) of the users.",
"232.The method of claim 230, wherein said step of prioritizing includes comparing said monitored bandwidth of each user with an established minimum quality of service value.",
"233.The method of claim 229, wherein said step of determining each user's second bandwidth allowance includes forecasting each user's bandwidth for the second future time interval.",
"234.The method of claim 233, wherein said step of determining each user's bandwidth allowance includes prioritizing the users for allocation of bandwidth based on said forecasting.",
"235.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the upstream direction during time intervals of one minute to fifteen minutes.",
"236.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is requested by each CM in the upstream direction during time intervals of one minute to fifteen minutes.",
"237.The method of claim 229, wherein said step of monitoring comprises monitoring the bandwidth that is consumed by each CM in the downstream direction during time intervals of fifteen minutes to sixty minutes.",
"238.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"239.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"240.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"241.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"242.The method of claim 229, wherein said step of monitoring includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in the upstream direction during a time interval.",
"243.The method of claim 228, wherein the first time interval and the second time interval are consecutive time intervals.",
"244.The method of claim 228, wherein each of the first time interval and the second time interval has a period of one to sixty minutes.",
"245.The method of claim 228, wherein for each user, the first bandwidth allowance of that user differs from the second bandwidth allowance of that user.",
"246.The method of claim 228, wherein the first bandwidth allowance for each user differs from the first bandwidth allowance of all other users.",
"247.The method of claim 228, wherein the second bandwidth allowance for each user differs from the second bandwidth allowance of all other users.",
"248.The method of claim 228, wherein the first bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the first time interval in accordance with a first selected allocation policy.",
"249.The method of claim 248, wherein the second bandwidth allowance of each user is determined by allocating bandwidth to each user on a per user basis for the second time interval in accordance with a second selected allocation policy.",
"250.The method of claim 249, wherein the first and second allocation policies differ.",
"251.The method of claim 249, wherein the first and second allocation policies are the same.",
"252.A method of continuously providing network access across a cable network of a data-over-cable (DOC) network to at least two users competing for bandwidth, comprising the steps of: (a) determining for each user during present time intervals bandwidth allowances of such user for respective future time intervals, each bandwidth allowance of such user for a future time interval representing a maximum level of bandwidth consumption for such user during the future time interval, but not necessarily representing the amount of bandwidth that will be consumed by such user during such future time interval, the bandwidth allowances of each user varying as between time intervals; and (b) for each user, limiting during each respective future time interval bandwidth consumption of that user's cable modem (CM) to that user's respective bandwidth allowance for such time interval by implementing a new cable modem configuration file in the CM.",
"253.The method of claim 252, wherein the present and future time intervals have a uniform period of one minute to sixty minutes.",
"254.The method of claim 252, wherein each user's bandwidth allowance for each future time interval is determined by allocating bandwidth to the users on a per user basis for such future time interval in accordance with a selected allocation.",
"255.A Carrier Network for providing communication between multiple users and a Service Provider, comprising: (a) computer network equipment defining an Intermediate Network and a shared communications medium, said Intermediate Network and said shared communications medium extending between the users and the Service Provider for conveying data therebetween, said shared communications medium joining the users with said Intermediate Network such that data of each user is conveyed over said shared communications medium to and from the Intermediate Network whereby the users compete for network access; and (b) software for managing network access of the users across said shared communications medium, said software including computer-executable instructions that performs the steps of: (i) monitoring the network access usage by each user for a time interval; and (ii) based on the monitored network access usage, setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.",
"256.The Carrier Network of claim 255, wherein said network equipment futher defines a Cable Network and said shared communications medium comprises a coaxial cable of said Cable Network.",
"257.The Carrier Network of claim 256, wherein said software comprises software modules distributed within computer readable media of said network equipment.",
"258.The Carrier Network of claim 256, wherein said software comprises software modules stored in a computer readable medium of a hardware component.",
"259.The Carrier Network of claim 258, wherein said hardware component is physically located with said network equipment.",
"260.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a regional data center of said DOC Network.",
"261.The Carrier Network of claim 259, wherein said Carrier Network comprises a Data-Over-Cable (DOC) Network and said hardware component is located within a headend of said DOC Network.",
"262.The Carrier Network of claim 258, wherein said hardware component is physically located remotely from said network equipment.",
"263.The Carrier Network of claim 262, wherein said hardware component is directly connected with said network equipment for communication therebetween.",
"264.The Carrier Network of claim 262, wherein said hardware component is indirectly connected with said network equipment through the Internet for communication therebetween.",
"265.The Carrier Network of claim 256, wherein said software modules include: (i) a first software component that manages a database for storage and retrieval of data; (ii) a second software component that collects data representative of network access usage from said network equipment, and that sends data representative of network access usage to said first component for storage thereof; and (iii) a third component that retrieves data representative of network access usage stored in said first component and sets, based at least partially thereon, network access allowances for the users for the future time interval.",
"266.The Carrier Network of claim 265, further includes a fourth component having a graphical user interface for sending data to said first component for storage and for retrieval of data stored by said first component.",
"267.The Carrier Network of claim 255, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"268.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in an upstream direction across said shared communications medium in time intervals of one minute to fifteen minutes.",
"269.The Carrier Network of claim 267, wherein the step of monitoring comprises monitoring bandwidth that is consumed by each user in the downstream direction across said shared communications medium in time intervals of fifteen minutes to sixty minutes.",
"270.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval are equal in length.",
"271.The Carrier Network of claim 255, wherein the time interval for which network access usage is monitored and the future time interval each is approximately one minute to sixty minutes in length.",
"272.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units transmitted from and to each user during a time interval.",
"273.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets transmitted from and to each user during a time interval.",
"274.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a time interval.",
"275.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of bytes and data packets of the user that are dropped during a time interval.",
"276.The Carrier Network of claim 255, wherein the step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction across said shared communications medium during a time interval.",
"277.The Carrier Network of claim 255, wherein said step of setting a network access allowance for each user comprises allocating network access equally to the users and setting the network access allowance for each user equal to that user's allocated network access.",
"278.The Carrier Network of claim 277, wherein said software further includes computer-executable instructions that perform the additional step of prioritizing the users for allocating network access.",
"279.The Carrier Network of claim 278, wherein the prioritizing is based on fairness considerations.",
"280.The Carrier Network of claim 279, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.",
"281.The Carrier Network of claim 279, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.",
"282.The Carrier Network of claim 279, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"283.The Carrier Network of claim 279, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.",
"284.The Carrier Network of claim 279, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"285.The Carrier Network of claim 278, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.",
"286.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing each user's monitored network access usage with the specified respective minimum levels of network access, and awarding priority to a user when said respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"287.The Carrier Network of claim 285, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"288.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.",
"289.The Carrier Network of claim 285, wherein SLAs provide respective fees for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"290.The Carrier Network of claim 285, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"291.The Carrier Network of claim 285, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.",
"292.The Carrier Network of claim 285, wherein said software further includes computer-executable instructions that perform the step of forecasting network access usage by each user during the future time interval based on the monitoring of network access usage by each user.",
"293.The Carrier Network of claim 292, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"294.The Carrier Network of claim 292, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.",
"295.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to users proportional to each user's forecasted network access usage.",
"296.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions that perform the step of prioritizing the users for allocating network access.",
"297.The Carrier Network of claim 296, wherein prioritizing is based on each user's forecasted network access usage.",
"298.The Carrier Network of claim 296, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"299.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"300.The Carrier Network of claim 292, wherein said step of setting a network access allowance for each user comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"301.A computer-readable medium having computer-executable instructions that manage network access across a shared communications medium between competing users of a Carrier Network, said instructions performing the steps of: (a) monitoring the network access usage by each user for a time interval; and (b) setting a network access allowance for each user representing a level of network access made available for utilization by the user during a future time interval, but not necessarily representing the level of network access that will be utilized by such user during the future time interval.",
"302.The computer-readable medium of claim 301, wherein said step of setting a network access allowance for each user comprises allocating network access, and wherein said software further includes computer-executable instructions performing the step of prioritizing the users for allocating network access.",
"303.The computer-readable medium of claim 302, wherein the prioritizing is based on fairness considerations.",
"304.The computer-readable medium of claim 302, wherein the users are prioritized based on user throughput during a time interval, with a user with lesser throughput rate receiving priority over a user with greater throughput rate.",
"305.The computer-readable medium of claim 302, wherein the users are prioritized based on data loss for each user during a time interval, with a user with greater data loss rate having priority over a user with lesser data loss rate.",
"306.The computer-readable medium of claim 302, wherein the users are prioritized based on network access usage for a particular time of day, with a user with lesser network access usage for the particular time of day receiving priority over a user with greater network access usage for the particular time of day.",
"307.The computer-readable medium of claim 302, wherein the users are prioritized based on both user throughput and data loss of the user during a time interval.",
"308.The computer-readable medium of claim 302, wherein users are prioritized based on an established minimum quality of service (QoS) standard.",
"309.The computer-readable medium of claim 302, wherein prioritizing is based on service level agreements (SLAs) of the users regarding the provision of network access.",
"310.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for the users with the specified respective minimum levels of network access, and awarding priority to a user when the respective monitored network access usage for such user falls below the user's specified respective minimum level of network access.",
"311.The computer-readable medium of claim 309, wherein SLAs specify respective time-of-day (TOD) minimum levels of network access for users, and prioritizing includes comparing the monitored network access usages for such users during the specified respective TOD with the specified respective TOD minimum levels of network access, and awarding priority to a user when the monitored network access usage during the specified respective TOD for such user falls below the user's specified respective TOD minimum level of network access.",
"312.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access up to a maximum burstable levels with target probability for users, and prioritizing includes comparing the monitored network access usage both with the respective minimum levels of network access for such users and with the respective maximum burstable levels of network access for such users, and comparing the instances the respective maximum levels of network access were obtained for such users out of all instances the respective maximum levels of network access were requested for such users.",
"313.The computer-readable medium of claim 309, wherein SLAs provide a respective fee for network access usage, and prioritizing comprises sorting such users based on each user's respective fee in decreasing order, with a user with a higher fee receiving priority over a user with a lesser fee.",
"314.The computer-readable medium of claim 309, wherein SLAs provide respective credits for levels of network access below respective guaranteed levels for users, and prioritizing comprises sorting such users based on each user's respective credit in decreasing order, with a user with a higher credit receiving priority over a user with a lower credit.",
"315.The computer-readable medium of claim 309, wherein SLAs specify respective minimum levels of network access for users, and allocating network access comprises allocating network access to such users equal to each user's specified respective minimum level of network access.",
"316.The computer-readable medium of claim 309, further comprising computer-executable instructions performing the step of forecasting network access usage by each user during the future time interval based the monitoring of network access usage by each user.",
"317.The computer-readable medium of claim 316, wherein forecasting comprises predicting future network access usage of each user based upon monitored past network access usage patterns of each user.",
"318.The computer-readable medium of claim 316, wherein forecasting comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to the monitored network access usages of the users.",
"319.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to users proportional to each user's forecasted network access usage.",
"320.The computer-readable medium of claim 316, further comprising computer-executable instructions performing the step of prioritizing the users for allocating network access.",
"321.The computer-readable medium of claim 320, wherein prioritizing is based on each user's forecasted network access usage.",
"322.The computer-readable medium of claim 320, wherein said users are prioritized in increasing order of each user's forecasted network access usage, with a user with a lesser forecasted network access usage receiving priority over a user with a greater forecasted network access usage.",
"323.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access equally to the users.",
"324.The computer-readable medium of claim 316, wherein allocating network access comprises allocating network access to the users equal to each user's forecasted network access usage, and then allocating any remaining network access to the users proportionally based on each user's forecasted network access usage.",
"325.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users; and (d) allocating bandwidth to each user sequentially in decreasing order of user priority.",
"326.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth requested for each user.",
"327.The computerized method of claim 325, wherein the bandwidth that is monitored is the bandwidth consumption of each user.",
"328.The computerized method of claim 327, wherein the bandwidth that is forecasted is the bandwidth consumption of each user.",
"329.The computerized method of claim 328, wherein users are prioritized based on each user's forecasted future bandwidth consumption in increasing order, whereby a user with a lesser forecasted bandwidth consumption receives a higher priority than a user with a greater forecasted bandwidth consumption.",
"330.The computerized method of claim 328, wherein each user's allocation of bandwidth for the future time interval is set to equal each user's forecasted bandwidth consumption subject to a respective, predetermined maximum value and subject to bandwidth availability 331.The computerized method of claim 330, further comprising distributing equally to the users any unallocated bandwidth.",
"332.The computerized method of claim 330, further comprising distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidth consumptions.",
"333.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise the number of logical data units transmitted from and to each user during the past time interval.",
"334.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes transmitted from and to each user during the past time interval.",
"335.The computerized method of claim 330, wherein the data representative of past bandwidth consumption comprise data representative of the number of data packets transmitted from and to each user during the past time interval.",
"336.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.",
"337.The computerized method of claim 336, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.",
"338.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.",
"339.The computerized method of claim 336, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.",
"340.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee charged to each user in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.",
"341.The computerized method of claim 340, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.",
"342.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.",
"343.The computerized method of claim 340, wherein the data representative of past bandwidth comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.",
"344.A computerized method of allocating among a plurality of users bandwidth for conveying information across a shared communications medium, comprising the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective bandwidth levels specified to the users; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.",
"345.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.",
"346.The computerized method of claim 344, wherein any unallocated bandwidth remaining is distributed equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.",
"347.The computerized method of claim 344, further comprising setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.",
"348.The computerized method of claim 344, wherein the data representative of past bandwidth comprise the number of logical data units transmitted from and to each user during the past time interval.",
"349.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of bytes and data packets transmitted from and to each user during the past time interval.",
"350.The computerized method of claim 344, wherein the data representative of past bandwidth consumption comprise data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during the past time interval.",
"351.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.",
"352.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing equally to the users any unallocated bandwidth.",
"353.The computer-readable medium of claim 351, wherein said instructions further perform the step of distributing to the users any unallocated bandwidth remaining in amounts proportional to the users' forecasted bandwidths.",
"354.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available to the users' total forecasted bandwidth, and subject to a respective, predetermined maximum value and subject to bandwidth availability.",
"355.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth by each user during a time interval, and data representative of respective fees that are charged to the users for bandwidth; (b) forecasting future bandwidth by each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective fee in decreasing order, whereby a user with a higher fee receives a higher priority than a user with a lesser fee; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.",
"356.A computer-readable medium having computer-executable instructions that allocates among a plurality of users bandwidth for conveying information across a shared communications medium, said instructions performing the steps of: (a) receiving data representative of past bandwidth of each user during a time interval, and data representative of respective credits that are applied to accounts of the users for bandwidth shortfalls below respective levels of network access specified to the users; (b) forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; (c) prioritizing users based on the respective credit in decreasing order, whereby a user with a higher credit receives a higher priority than a user with a lesser credit; and (d) sequentially for each user in decreasing order of user priority, setting each user's allocation of bandwidth for the future time interval equal to each user's forecasted bandwidth subject to a respective, predetermined maximum specified value and subject to availability.",
"357.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth to the users sequentially in decreasing user priority such that each user's allocation equals the user's forecasted bandwidth, subject to a respective, predetermined maximum allowed value of bandwidth and subject to availability.",
"358.The computer-readable medium of claim 356, wherein said instructions further perform the step of distributing any unallocated bandwidth equally to the users subject to a respective, predetermined maximum allowed value of bandwidth.",
"359.The computer-readable medium of claim 356, wherein said instructions further perform the step of setting each user's allocation of bandwidth for the future time interval only in an amount equal to the forecasted bandwidth of the user multiplied by the ratio of total bandwidth available for allocation to the total forecasted bandwidth of the users, but only when the total forecasted bandwidth exceeds the total bandwidth available for allocation.",
"360.A method of providing network access across a shared communications medium between at least four competing users, with at least a first pair of users being grouped within a first class and at least a second pair of different users being grouped within a second class, comprising the steps of: (a) determining class and user allowances of network access for a first time interval by allocating network access to each user class for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval, (b) providing network access to each user during the first time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' network allowance; (c) determining class and user allowances of network access for a second time interval by allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval, the allocated network access for the second time interval for at least one user differing from that user's allocated network access for the first time interval; and (d) providing network access to each user during the second time interval such that no user receives more network access than that user's allowance and no class receives more collective network access than that class' allowance.",
"361.The method of claim 360, wherein for at least one class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.",
"362.The method of claim 360, wherein for each class the allocated network access for the first time interval for each user differs from the allocated network access for the second time interval for that user.",
"363.The method of claim 360, wherein the collective network access allocated to each class for the first time interval differs from the collective network access allocated to each class for the second time interval.",
"364.The method of claim 360, further comprising requesting a minimum level of network access for a user for utilization during the first future time interval, and wherein said allocating network access to such user for the first future time interval comprises setting the level of network access allocated to such user to an amount equal to or greater than the requested minimum level.",
"365.The method of claim 364, wherein said allocating network access to such user for the second future time interval comprises setting the level of network access allocated to such user to an amount less than the requested minimum level for the first time interval.",
"366.The method of claim 360, wherein each of the first future time interval and the second future time interval has a period of between one minute and sixty minutes.",
"367.The method of claim 360, wherein network access comprises bandwidth across the shared communications medium for consumption by each user in conveying data of the user.",
"368.The method of claim 360, further comprising the step of monitoring network access usage by each user.",
"369.The method of claim 368, further comprising the step of tracking network access usage for each user class.",
"370.The method of claim 368, wherein said step of monitoring network access usage by each user includes collecting data representative of logical data units transmitted from and to each user during a past time interval.",
"371.The method of claim 368, further comprising tracking logical data units transmitted from and to each user class during a past time interval.",
"372.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are dropped during a past time interval.",
"373.The method of claim 368, further comprising tracking the number of logical data units that are dropped for each user class during a past time interval.",
"374.The method of claim 368, wherein said step of monitoring network access usage includes collecting data representative of the number of logical data units of the user that are requested to be transmitted in an upstream direction during a past time interval.",
"375.The method of claim 368, further comprising tracking the number of logical data units of each user class that are requested to be transmitted in an upstream direction during a past time interval.",
"376.The method of claim 360, wherein the shared communications medium is part of a Cable Network and the shared communications medium comprises a coaxial cable.",
"377.The method of claim 360, wherein the shared communications medium is part of a Shared Access Carrier Network comprises a wireless network.",
"378.The method of claim 360, further comprising prioritizing the user classes for allocating network access.",
"379.The method of claim 378, further comprising prioritizing the users for allocating, network access.",
"380.The method of claim 378, wherein said prioritizing of the user classes is based on fairness considerations.",
"381.The method of claim 380, wherein the user classes are prioritized based on collective user throughput during a past time interval, with a user class with lesser collective user throughput receiving priority over a user class with greater collective user throughput.",
"382.The method of claim 380, wherein the user classes are prioritized based on collective data loss for each user class during a past time interval, with a user class with greater collective data loss having priority over a user class with lesser collective data loss.",
"383.The method of claim 380, wherein the user classes are prioritized based on collective network access usage for a particular time of day, with a user class with lesser collective network access usage for the particular time of day receiving priority over a user class with greater collective network access usage for the particular time of day.",
"384.The method of claim 380, wherein the user classes are prioritized based on both collective user throughput for the class and collective data loss of the users for the class during a time interval.",
"385.The method of claim 380, wherein user classes are prioritized based on an established minimum quality of service (QoS) standard.",
"386.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) for at least two user classes regarding the provision of network access to each respective class.",
"387.The method of claim 386, further comprising the step of tracking network access usage by each user class.",
"388.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for the user classes with the specified respective minimum levels of collective network access therefor, and awarding priority to a user class when said monitored network access usage for such user class falls below the specified respective minimum level of collective network access for such class.",
"389.The method of claim 387, wherein each CSLA specifies a respective time-of-day (TOD) minimum level of collective network access for the respective users therein, and said step of prioritizing includes comparing said monitored network access usages for such user classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a user class when said monitored network access usage during the specified respective TOD for such user class falls below the specified respective TOD minimum level of collective network access of such user class.",
"390.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access up to a maximum burstable level with target probability for the respective users therein, and said step of prioritizing includes comparing for each user class said monitored network access usage both with the respective minimum level of collective network access and with the respective maximum burstable level of collective network access, and comparing the instances the respective maximum level of network access were obtained out of all instances the respective maximum level of network access could have been utilized.",
"391.The method of claim 387, wherein each CSLA provides a respective fee for collective network access usage, and said step of prioritizing comprises sorting such user classes based on each class' respective fee in decreasing order, with a user class with a higher fee receiving priority over a user class with a lesser fee.",
"392.The method of claim 387, wherein each CSLA provides a respective credit for a level of collective network access falling below a respective guaranteed level for the user class, and said step of prioritizing comprises sorting such user classes based on each class' respective credit in decreasing order, with a user class with a higher credit receiving priority over a user class with a lower credit.",
"393.The method of claim 387, wherein each CSLA specifies a respective minimum level of collective network access for the user class, and said step of allocating network access comprises setting the respective level of collective network access of the user class equal to the class' specified respective minimum level of collective network access.",
"394.The method of claim 368, further comprising the step of forecasting collective network access usage by each user class during a future time interval based on said step of monitoring network access usage by each user.",
"395.The method of claim 394, wherein said step of forecasting comprises summing within each class a forecasted network access usage of each user.",
"396.The method of claim 394, wherein said step of forecasting network access usage of each user comprises applying an adaptive-response-rate single exponential smoothing function and a Holt-Winters' seasonal exponential smoothing function to said monitored network access usages of each user.",
"397.The method of claim 395, wherein said step of allocating network access to each user class comprises setting the respective levels of network access of the user classes proportional to each class' forecasted network access usage.",
"398.The method of claim 395, further comprising the step of prioritizing the user classes for allocating network access.",
"399.The method of claim 398, wherein said prioritizing is based on each class' forecasted network access usage.",
"400.The method of claim 398, wherein said user classes are prioritized in increasing order of each class' forecasted network access usage, with a user class with a lesser forecasted network access usage receiving priority over a user class with a greater forecasted network access usage.",
"401.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportional to the number of users within each class.",
"402.The method of claim 398, wherein said step of allocating network access comprises setting the respective levels of network access equal to each class' forecasted network access usage, and then allocating any remaining network access to the user classes proportionally based on each class' forecasted network access usage.",
"403.The method of claim 378, wherein said step of prioritizing is based on class service level agreements (CSLAs) regarding the provision of collective network access.",
"404.The method of claim 403, further comprising the step of monitoring collective network access usage of each class.",
"405.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes with the specified respective minimum levels of collective network access, and awarding priority to a class when said respective monitored network access usage for such class falls below the class' specified respective minimum level of collective network access.",
"406.The method of claim 404, wherein CSLAs specify respective time-of-day (TOD) minimum levels of collective network access for classes, and said step of prioritizing includes comparing said monitored network access usages for such classes during the specified respective TOD with the specified respective TOD minimum levels of collective network access, and awarding priority to a class when said monitored network access usage during the specified respective TOD for such class falls below the class' specified respective TOD minimum level of collective network access.",
"407.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access up to a maximum burstable levels with target probability for classes, and said step of prioritizing includes comparing said monitored network access usage for each such class both with the respective minimum levels of collective network access and with the respective maximum burstable levels of collective network access, and comparing the instances the respective maximum levels of collective network access were obtained for each such class out of all instances the respective maximum levels of collective network access could have been utilized.",
"408.The method of claim 404, wherein CSLAs provide respective fees for collective network access usage of classes, and said step of prioritizing comprises sorting such classes based on each class' respective fee in decreasing order, with a class with a higher fee receiving priority over a class with a lower fee.",
"409.The method of claim 404, wherein CSLAs provide respective credits for levels of collective network access below respective guaranteed levels for classes, and said step of prioritizing comprises sorting such classes based on each class' respective credit in decreasing order, with a class with a higher credit receiving priority over a class with a lower credit.",
"410.The method of claim 404, wherein CSLAs specify respective minimum levels of collective network access for classes, and said step of allocating network access comprises setting the allocations of collective network access for such classes equal to each class' specified respective minimum level of collective network access"
],
[
"<SOH> BACKGROUND OF THE PRESENT INVENTION <EOH>As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.",
"The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).",
"The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.",
"The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.",
"For example, a Carrier or a related entity may serve as an Internet service provider (ISP).",
"Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.",
"One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.",
"Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).",
"For example, with regard to DOC Networks, and with reference to FIG.",
"1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30 , which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.",
"All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34 .",
"Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.",
"Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.",
"The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.",
"Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.",
"This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.",
"1 .",
"The DOC Network 40 also includes multiple headends 36 , 64 , 66 .",
"In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.",
"Examples of a dedicated connection are shown for comparison in FIG.",
"1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76 .",
"Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.",
"Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.",
"A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.",
"A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.",
"Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.",
"Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.",
"It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.",
"Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.",
"For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.",
"Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.",
"Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.",
"A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.",
"Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.",
"Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.",
"Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.",
"The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.",
"In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.",
"In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.",
"Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.",
"Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.",
"Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.",
"A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.",
"A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.",
"More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.",
"At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.",
"While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.",
"Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.",
"Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.",
"Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.",
"Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.",
"Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium."
],
[
"<SOH> SUMMARY OF THE PRESENT INVENTION <EOH>Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.",
"First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.",
"In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.",
"Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.",
"The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.",
"The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.",
"Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.",
"Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.",
"In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.",
"Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.",
"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.",
"With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.",
"Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.",
"In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.",
"In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.",
"Another additional feature of the present invention includes the prioritizing of the users for allocating network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.",
"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.",
"Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.",
"In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.",
"Another feature of the present invention includes the prioritizing of the users for allocating network access.",
"A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.",
"A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.",
"Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.",
"The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.",
"Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.",
"Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.",
"The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.",
"The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.",
"The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.",
"The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.",
"A charge preferably is applied to the user for the modification.",
"A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.",
"Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.",
"A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.",
"Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.",
"Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.",
"The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.",
"The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.",
"The Carrier Network also includes software that manages the network access of the users.",
"In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.",
"In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.",
"The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.",
"One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.",
"Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.",
"The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.",
"A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.",
"Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.",
"In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.",
"The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.",
"Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.",
"A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.",
"Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.",
"The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.",
"Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.",
"In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.",
"The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.",
"Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.",
"The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.",
"As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.",
"As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.",
"The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.",
"The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.",
"Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.",
"In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.",
"Accordingly, all users of a shared communications network can be classified.",
"In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.",
"Another feature includes the step of prioritizing the users for the allocation of network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.",
"Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted."
],
[
"FIELD OF THE PRESENT INVENTION The present invention generally relates to allocating access across a shared communications medium and, in particular, to allocating bandwidth used to convey data of competing users across a shared communications medium of a Carrier Network.",
"BACKGROUND OF THE PRESENT INVENTION As used herein, a “Carrier Network” generally refers to a computer network through which users (such as homes and businesses) communicate with various service providers.",
"The Carrier Network extends from the location of each user to an intermediate switched/routed network (hereinafter “Intermediate Network”).",
"The service providers, in turn, are connected to the Intermediate Network, either directly or indirectly via the Internet, for communications with the users.",
"The Carrier Network is maintained by a “Carrier,” which also may serve as a service provider for certain services.",
"For example, a Carrier or a related entity may serve as an Internet service provider (ISP).",
"Two prevalent types of Carrier Networks include a “Shared Access Carrier Network,” in which data of multiple users are conveyed together over a shared communications medium between the users and the Intermediate Network, and a “Dedicated Connection Carrier Network,” in which data of each user are conveyed alone between the user and the Intermediate Network and are not combined with data of other users.",
"One of the most prevalent Shared Access Carrier Networks today is found in the Data-Over-Cable (DOC) Network, which includes the traditional network constructed from coaxial cable and the hybrid fiber coaxial (HFC) network constructed with both fiber optical cabling and coaxial cable.",
"Other Shared Access Carrier Networks include wireless and digital subscriber line (XDSL) networks (the xDSL lines typically being aggregated onto an oversubscribed backhaul trunk into the Intermediate Network, with the trunk defining the shared communications medium).",
"For example, with regard to DOC Networks, and with reference to FIG.",
"1 wherein a conventional DOC Network 40 is illustrated, data packets are transmitted in a downstream direction from a cable modem termination system (CMTS) 30, which is located in a headend 36 (or distribution hub) of a Carrier, over a coaxial cable 32 to respective cable modems (CMs) 34 of users.",
"All of the CMs 34 are attached by the coaxial cable 32 to the CMTS 30 in an inverted tree configuration, and each CM 34 connected to the coaxial cable 32 listens to all broadcasts from the CMTS 30 transmitted through the coaxial cable 32 for data packets addressed to it, and ignores all other data packets addressed to other CMs 34.Theoretically, a CM 34 is capable of receiving data in the downstream direction over a 6 MHz channel with a maximum connection speed of 30-40 Mbps.",
"Data packets also are transmitted in the upstream direction over a 2 MHz channel by the CMs 34 to the CMTS 30 typically using time division multiplexing (TDM) and at a maximum connection speed of 1.5-10 Mbps.",
"The headend 36 in the DOC Network 40 includes a plurality of CMTSs, with each CMTS supporting multiple groups of CMs each connected together by a respective coaxial cable.",
"Each such group of CMs connected to a CMTS defines a Shared Access Carrier Network, with the coaxial cable in each representing the shared communications medium.",
"This arrangement of a group of CMs connected to a CMTS by a coaxial cable is referred to herein as a “Cable Network.” Accordingly, the DOC Network 40 includes a plurality of Cable Networks 38 originating from CMTSs at the headend 36 of the Carrier, with a particular Cable Network 38 being illustrated in an expanded view in FIG.",
"1.The DOC Network 40 also includes multiple headends 36,64,66.In contrast to the Shared Access Carrier Network, a user in the Dedicated Connection Carrier Network establishes a dedicated connection directly with the Intermediate Network for the transfer of data directly therebetween, and no data of other users travel over the dedicated connection.",
"Examples of a dedicated connection are shown for comparison in FIG.",
"1 and include a connection established by a telephony modem 74 and a connection established by an ISDN modem 76.Both downstream and upstream connection speeds in a Dedicated Connection Carrier Network range from a maximum of 53 kbps in a telephony modem connection to a maximum of 128 kbps in a basic rate interface ISDN connection.",
"Connection speeds and, more importantly, throughput rate—the amount of data actually transmitted successfully in a given time interval—are important in minimizing downtime that users spend waiting for HTML documents to download from the Web.",
"A Shared Access Carrier Network is considered superior to a comparable Dedicated Connection Carrier Network because the maximum instantaneous connection speed offered by the Shared Access Carrier Network is greater.",
"A Shared Access Carrier Network is considered “comparable” to a Dedicated Connection Carrier Network where the entire bandwidth over a shared communications medium of the Shared Access Carrier Network equals an aggregate bandwidth that is divided between and dedicated to users in a Dedicated Connection Carrier Network.",
"Accordingly, Shared Access Carrier Networks are able to offer significantly faster downloads of web documents, emails, and file transfers that are not considered available in Dedicated Connection Carrier Networks.",
"Furthermore, new multimedia applications and Internet services, such as voice and video communications via the Internet, now are offered which require even greater throughput rates for acceptable levels of service than that of the traditional Internet services, i.e., throughput rates greater than that required for acceptable text-based Web browsing, file transferring, and email communication.",
"It is believed that these new multimedia applications and Internet services cannot adequately be provided for over Dedicated Connection Carrier Networks and that, consequently, Shared Access Carrier Networks ultimately will prevail as the predominant type of Carrier Network for Internet access by users.",
"Of course, the actual throughput rates experienced by a particular user rarely, if ever, will equate to the maximum connection speeds of which the Shared Access Carrier Network is capable because of the shared nature of the communications medium.",
"For example, in a Cable Network the total bandwidths available over the shared cable in the downstream and upstream directions, which determine the respective maximum connection speeds, must be shared among all of the users communicating at a given time.",
"Thus, rarely will a single user have available for use a large portion of the entire bandwidth in a particular direction.",
"Further, as a Carrier adds users to the Cable Network, the actual downstream and upstream bandwidths available to the user—and thus throughput rates of the user—generally will decrease.",
"A Carrier therefore must be careful to draw a balance between the number of users connected to a Cable Network and the performance users experience communicating over the network.",
"Unfortunately, Shared Access Carrier Networks that have been established were designed to provide the traditional Internet services, and not the new multimedia applications and Internet services that require higher throughput rates for acceptable levels of service.",
"Consequently, each balance previously struck by Carriers in establishing Shared Access Carrier Networks was based on considerations of the throughput rates required for the traditional Internet services, and user throughput rates currently experienced by users in such networks are believed to fall short of acceptable quality of service (QoS) standards believed required in a Carrier Network for the new multimedia applications and Internet services.",
"Additionally, with regard to new Shared Access Carrier Networks that are being established, considerations of the new multimedia applications and Internet services tend to reduce the number of users that a Carrier now can reasonably expect to connect to the shared communications medium before degrading the performance levels of the new multimedia applications and Internet services.",
"The balance is being shifted towards less users per shared access medium in exchange for higher throughput rates and, thus, higher QoS standards.",
"In an attempt to avoid reducing the number of users, it has been proposed, at least in DOC Networks, to discriminate between the traditional Internet services and the new multimedia applications and Internet services with regard to priority of data packet transmissions.",
"In particular, the generally accepted standard in the United States governing communication protocols over cable is DOCSIS version 1.0, which was ratified by the International Telecommunication Union in March of 1998.DOCSIS stands for “Data Over Cable Service Interface Specifications.” When DOCSIS 1.0 was developed, it was generally believed that, in view of the “fast” connection speeds of Cable Networks, the provision of bandwidth on a best effort basis would be sufficient to meet all user requirements.",
"Accordingly, each user subscribed to receive network access pursuant to a service level agreement (SLA) which provided for network access (or bandwidth in Cable Networks) only on a best effort basis.",
"Now, in an effort to address the foreseen ever-increasing demand for higher throughput rates, DOCISIS version 1.1 has been proposed, in accordance with which each data packet transmitted over a DOC Network now must include a classification designation for prioritization purposes by network equipment.",
"Subsequently, data packets representing voice or video, for example, now can be identified and given priority transmission over data packets representing email, file transfers, and text-based Web documents.",
"A benefit of such flow classification is that, while overall bandwidth generally available to a user may otherwise remain unchanged, throughput rates of data for voice and video now may be provided at a higher rate than throughput rates of data for the traditional Internet services, thereby increasing the performance of voice and video applications and services while at least maintaining the traditional number of users connected to a Cable Network.",
"A disadvantage of the revisions to DOCSIS 1.1 is that the revisions do not enhance established Cable Networks constructed with only DOCSIS 1.0 compliant equipment, as such equipment does not support the added functionality of DOCSIS 1.1 so as to distinguish between data packets.",
"More broadly, another disadvantage of the classification of data packets into Internet Protocol (IP) flows based on the services represented by the data packets is that such classification discriminates against users who do not utilize multimedia applications and services receiving the prioritized transmissions.",
"At least for some extensive users of the traditional Internet services, some degradation in performance may be noticed by lower classification of their data packets, particularly if the user engages in, for example, web hosting.",
"While the transmissions of data packets for documents, files, and emails are not as time-sensitive as data packets for voice and video, increased data packet latency for documents, files, and emails, even if incrementally small, nevertheless will result in service degradation for large or numerous documents, files, and emails.",
"Accordingly, a need exists for a method and apparatus that will accommodate differing demands for network access by users competing for such access across a shared communications medium of a Shared Access Carrier Network, whether new or established, and irrespective of data packet classifications.",
"Furthermore, as Shared Access Carrier Networks emerge as the favored type of network, it is believed that open access to such networks by different competing service providers will become an important commercial and legislative issue.",
"Moreover, as more and more service providers seek to provide users with services over Shared Access Carrier Networks, it is believed that users of such service providers will receive inadequate bandwidth over the Shared Access Carrier Networks to meet minimum standards of quality, especially in Cable Networks where bandwidth is provided on a best efforts basis.",
"Accordingly, it is believed that an additional need exists for a method by which a service provider competing for users of a shared communications medium can seek protection against bandwidth starvation of the users of the shared communications medium that are its customers.",
"Conversely, it is also believed that a need exists for a method that will accommodate differing demands for network access by users competing for such access across the shared communications medium.",
"SUMMARY OF THE PRESENT INVENTION Briefly summarized, the present invention relates to a method of providing network access across a shared communications medium between competing users, and include eight different aspects.",
"First Aspect of the Present Invention In the first aspect of the present invention, the method broadly includes the steps of: (a) prior to first and second time intervals, respectively determining for each user first and second network access allowances; (b) during the first time interval, providing network access to each user such that the respective first network access allowance is not exceeded; and (c) during the second time interval, providing network access to each user such that the respective second network access allowance for each user is not exceeded.",
"In accordance with the present invention, the “network access allowance” represents a respective maximum level of network access that can be made available to the user during a particular time interval, and does not represent necessarily the level of network access that will be utilized by the user during such time interval.",
"Furthermore, at least one respective second network access allowance for a user differs from such user's respective first network access allowance.",
"The first network access allowances for the users preferably are determined by allocating network access to each user on a per user basis for the first time interval in accordance with a first allocation policy.",
"The first network access allowance for each respective user then is equated to the network access allocated to that user under the first allocation policy.",
"Likewise, the second network access allowances for the users are determined by allocating network access to each user on a per user basis for the second time interval in accordance with a second allocation policy that may or may not differ from the first allocation policy.",
"Again, the second network access allowance for each respective user then is equated to the network access allocated to that user under the second allocation policy.",
"In additional features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.",
"Another additional feature of the present invention includes the step of prioritizing the users for the allocation of network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for determining bandwidth allowances during the future time interval.",
"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.",
"With regard to at least two users, the favored user allocations, and thus the user allowances, differ as between future time intervals.",
"Second Aspect of the Present Invention In the second aspect of the present invention, the method broadly includes the steps of monitoring network access usage of each user for a past time interval and, based on the monitored network access usage, allocating network access to each user for a future time interval.",
"In a feature of the present invention, the network access that is monitored and allocated is in the downstream direction towards the users.",
"In a separate and independent feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.",
"Another additional feature of the present invention includes the prioritizing of the users for allocating network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network; based on the monitored bandwidth consumption, the forecasting of bandwidth consumption of each user in a future time interval; the prioritization of users; and the subsequent allocation of bandwidth to each user in decreasing priority for the future time interval.",
"Users are prioritized based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.",
"Third Aspect of the Present Invention In the third aspect of the present invention, the method broadly includes the steps of monitoring network access usage by each user for a past time interval and, based on the monitored network access usage, allocating network access for each user for a future time interval.",
"In a feature of the present invention, network access usage of each user is forecast for a future time interval based on the monitoring, and allocations are made based on the forecasting.",
"Another feature of the present invention includes the prioritizing of the users for allocating network access.",
"A preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; (c) prioritizing users based on each user's forecasted network access usage in increasing order, whereby a user with a lesser forecasted network access usage receives a higher priority than a user with a greater forecasted network access usage; and (d) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"Another preferred method of providing network access to the users includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) based on the monitoring, forecasting network access usage by each user over a future time interval; and (c) allocating network access available to each user during the future time interval, with each user's allocation of network access being equal to each user's forecasted network access usage multiplied by a ratio of the total network access available for allocation to the total forecasted network access usage of all users, and subject to a respective, predetermined maximum value and subject to availability.",
"A third preferred method of providing network access to the users includes the steps of: (a) charging each user a respective fee for network access usage; (b) monitoring network access usage by each user during a time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum value and subject to availability.",
"Yet a fourth preferred method of providing network access to the users includes the steps of: (a) applying respective credits to users for network access shortfalls below respective levels of network access specified to the users; (b) monitoring network access usage by each user during a past time interval; (c) based on the monitoring, forecasting network access usage by each user over a future time interval; (d) prioritizing users based on each user's respective credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit; and (e) allocating network access available to each user during the future time interval in decreasing order of user priority, with each user's allocation of network access being equal to each user's forecasted network access usage subject to a respective, predetermined maximum guaranteed value and subject to availability.",
"Fourth Aspect of the Present Invention In the fourth aspect of the present invention, the method includes providing network access across a shared communications medium between competing users pursuant to service level agreements (SLAs) of the users.",
"The method broadly includes the steps of: (a) monitoring network access usage by each user during a time interval; (b) comparing the monitored network access usage by each user with a predetermined threshold value; and (c) soliciting a user to modify the user's SLA if the user's monitored network access usage varies from the predetermined value by a predetermined tolerance.",
"Features of the present invention include the additional steps of allocating network access to each user for a future time interval, and forecasting network access usage by users in the future time interval.",
"Another additional feature of the present invention includes the step of prioritizing the users for allocating network access to the users.",
"The network access usage includes the user throughput rate, bandwidth consumption, and/or bandwidth requested for a predetermined period of time.",
"The threshold value preferably represents a respective maximum level of network access (whether maximum allowed or maximum guaranteed) for each user or a respective maximum burstable level of network access with target probability for each user.",
"The solicitation is conducted via email, instant messaging, redirection of a web browser of the user to a solicitation web page, generation and mailing of literature, telephonic communication, or other communication means.",
"The solicited modification of the user's SLA includes guaranteeing a level of network access to the user on a permanent or on a temporary basis.",
"A charge preferably is applied to the user for the modification.",
"A preferred method of the present invention includes the identification of a recurrent period of high network access usage by a user based on the monitoring, and in response thereto, the solicitation of the user to modify the user's SLA in order to guarantee a minimum level of network access during an anticipated future recurrent period of high network access usage.",
"Fifth Aspect of the Present Invention In the fifth aspect of the present invention, the method includes incorporating allocations of network access into a DOCSIS 1.0 compliant cable network wherein users compete for bandwidth.",
"A preferred method includes the steps of: (a) generating cable modem configuration files, each of which limits bandwidth consumption by a cable modem of a user to a value representative of that user's allowance of bandwidth; (b) sending the configuration files to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network; and (c) sending a command either to each user's cable modem, or to a cable modem termination system to which each user's cable modem is connected, to cause the cable modem to acquire its new respective configuration file.",
"Features of the present invention include the additional steps of prioritizing users for allocation of bandwidth, and forecasting bandwidth of each user in a future time interval.",
"Sixth Aspect of the Present Invention In the sixth aspect of the present invention, a Carrier Network provides communications between multiple users and Service Providers.",
"The Carrier Network includes computer network equipment defining a Cable Network and an Intermediate Network which, combined, extends between the users and the Service Providers.",
"The Cable Network includes a shared communications medium joining the users with the Intermediate Network, and users compete for network access across the shared communications medium in conveying data.",
"The Carrier Network also includes software that manages the network access of the users.",
"In accordance with the present invention, the software includes computer-executable instructions performing the steps of monitoring network access usage by each user for a time interval and, based thereon, allocating network access to the users for a future time interval.",
"In additional features of the present invention, the software includes computer-executable instructions performing the additional steps of forecasting network access usage by each user during the future time interval, and prioritizing the users for allocation of network access In additional features of the present invention, the software is distributed within network equipment of the Carrier Network or, alternatively, is stored in hardware physically located at a remote location from the Carrier Network but linked with the Carrier Network for communications therebetween.",
"The hardware remotely located is directly linked with the Carrier Network or is linked indirectly through the Internet.",
"One of many preferred embodiments of the present invention includes prioritization of users based on one or more various prioritization policies, including the prioritization of users based upon fairness considerations, such as user throughput during a particular time interval, user data loss for a particular time interval, network access usage for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users based upon provisions found in each user's respective service level agreement (SLA), and the prioritization of users based upon each user's forecasted network access usage for a future time interval.",
"Seventh Aspect of the Present Invention In the seventh aspect of the present invention, the present invention relates to a method of (and to a computer-readable medium with computer-executable instructions for performing the method of) providing network access across a shared communications medium between competing users.",
"The method broadly includes the steps of receiving data representative of past bandwidth of each user during a time interval; forecasting future bandwidth of each user over a future time interval based on the data representative of the past bandwidth; and setting each user's allocation of bandwidth for the future time interval.",
"A feature of the present invention includes the step of prioritizing the users for allocating bandwidth to the users.",
"Preferably, each user's bandwidth allocation represents that user's limit on bandwidth consumption for the future time interval (“bandwidth allowance”), and does not necessarily represent that user's bandwidth consumption during the future time interval.",
"In a first preferred method, the users are prioritized based on each user's forecasted future bandwidth in increasing order, whereby a user with a lesser forecasted bandwidth receives a higher priority than a user with a greater forecasted bandwidth.",
"The users are then allocated sequentially in decreasing order of user priority, with each user's allocation being set to equal the user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to bandwidth availability.",
"Another preferred method includes allocating bandwidth that is to be made available to each user during the future time interval, with each user's allocation of bandwidth being set to equal each user's forecasted bandwidth multiplied by a ratio of the total bandwidth available for allocation to the total forecasted bandwidth of all users, and subject to a respective, predetermined maximum value and subject to availability.",
"A third preferred method includes receiving data representative of a respective fee that is charged to each user for bandwidth, prioritizing users based on each user's fee in decreasing order, whereby a user having a greater fee receives a higher priority than a user having a lesser fee, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.",
"Yet a fourth preferred method includes receiving data representative of a respective credit that is applied to each user's account for bandwidth shortfalls below a specified bandwidth value, prioritizing users based on each user's credit in decreasing order, whereby a user having a greater credit receives a higher priority than a user having a lesser credit, and setting in decreasing user priority each user's allocation of bandwidth equal to each user's forecasted bandwidth subject to a respective, predetermined maximum value and subject to availability.",
"The bandwidth monitored and forecast includes bandwidth that is consumed or, alternatively, bandwidth that is requested for consumption.",
"Eighth Aspect of the Present Invention Finally, in the eighth aspect of the present invention, the present invention relates to a method of providing network access across a shared communications medium between competing users.",
"In particular, the present invention includes the steps of allocating network access to at least two “user classes” for a first future time interval and, for each user class, allocating network access to each user within the class for the first time interval.",
"The present invention further includes the additional step of allocating network access to each user class for a second future time interval succeeding the first time interval and, for each user class, allocating network access to each user for the second time interval.",
"Each user receives a first determined allowance of network access for utilization during the first time interval equal to that user's allocation for the first time interval, and a second determined allowance of network access for utilization during the second time interval equal to that user's allocation for the second time interval.",
"The user allocations—and hence the user allowances—preferably differ as between the first and second time intervals.",
"As used herein, a “bandwidth allowance” represents a respective maximum level of network access that will be made available to a user class or to a user during a particular time interval, and does not necessarily represent the level of network access that will be utilized by the user class or user during such time interval.",
"As used herein, a “user class” is intended to refer to a grouping of users who compete for access across a shared communications medium and who have some characteristic in common.",
"The characteristic may, for example, be that the users are customers who receive Internet service over the shared communications medium from the same service provider.",
"The characteristic also may, for example, be that the users each subscribe to receive a particular level of network access across the shared communications medium, or that the users receive the same level of a particular service that is provided across the shared communications medium.",
"Furthermore, a user class is a grouping of users to which, collectively, a determined amount of bandwidth is allocated as opposed to other user classes.",
"In this regard, users that are not classified are considered to be part of a default user class having in common that fact that no other classification applies to them.",
"Accordingly, all users of a shared communications network can be classified.",
"In features of the present invention, the method includes the steps of monitoring network access usage by the users, and forecasting network access usage of each user in a future time interval.",
"Another feature includes the step of prioritizing the users for the allocation of network access.",
"One of many preferred embodiments of the present invention includes the monitoring of bandwidth consumption of each user across the shared communications medium of a Cable Network and tracking the collective bandwidth consumption of each user class; based on the monitored bandwidth consumptions, the forecasting of bandwidth consumption of each user in a future time interval and the calculation based thereon of the collective bandwidth consumption of each user class; the prioritization of users and the prioritization of user classes; and the subsequent allocation of bandwidth to each user class, and then to each user, in decreasing order of priority for determining bandwidth allowances during the future time interval.",
"Users and user classes are prioritized based on one or more various prioritization policies, including fairness considerations such as individual or collective user throughput during a particular time interval, individual or collective user data loss for a particular time interval, individual or collective user bandwidth consumption for a particular time-of-day, an established minimum quality of service (QoS) standard, or combination thereof.",
"Other prioritization policies include the prioritization of users and user classes based upon provisions found in each user's respective service level agreement (SLA) or provisions found in each class' service level agreement (CSLA), and the prioritization of users based upon each user's forecasted bandwidth consumption for the future time interval and the prioritization of user classes based upon each class' collective forecasted bandwidth consumption.",
"In alternative embodiments, the bandwidth that is requested, rather than the bandwidth that is consumed, is monitored and forecasted.",
"BRIEF DESCRIPTION OF THE DRAWINGS Further features and benefits of the present invention will be apparent from a detailed description of preferred embodiments thereof taken in conjunction with the following drawings, wherein like elements are referred to with like reference numbers, and wherein: FIG.",
"1 illustrates a conventional DOC Network; FIG.",
"2 illustrates a first DOC Network of the present invention; FIG.",
"3 illustrates a second DOC Network of the present invention; FIG.",
"4 illustrates a third DOC Network of the present invention; FIG.",
"5 illustrates a fourth DOC Network of the present invention; FIG.",
"6 illustrates a system architecture of software components that perform preferred methods of the present invention in the DOC Networks of FIGS.",
"2-5; FIG.",
"7 illustrates a flowchart of the steps of a preferred routine for forecasting bandwidth of each user for a future time interval; FIG.",
"8 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the ARRSES Function of the preferred routine of FIG.",
"7; FIG.",
"9 illustrates a flowchart of the steps of generating a forecasted bandwidth for a user in accordance with the HW Function of the preferred routine of FIG.",
"7; FIG.",
"10 illustrates a graph of user throughput rates versus user data loss rates for two users relative to a target minimum QoS standard; FIG.",
"11 illustrates a flowchart of a first preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.",
"12 illustrates a flowchart of a second preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.",
"13 illustrates a flowchart of a third preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.",
"14 illustrates a flowchart of a fourth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.",
"15a and 15b illustrate a flowchart of a fifth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIGS.",
"16a and 16b illustrate a flowchart of a sixth preferred method of prioritizing classes and allocating collective bandwidth to each class; FIG.",
"17 illustrates a flowchart of a first preferred method of prioritizing users and allocating bandwidth to each user within a class; FIG.",
"18 illustrates a flowchart of a second preferred method of prioritizing users and allocating bandwidth within a class; FIG.",
"19 illustrates a flowchart of a third preferred method of prioritizing users and allocating bandwidth within a class; FIG.",
"20 illustrates a flowchart of a fourth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.",
"21a and 21b illustrate a flowchart of a fifth preferred method of prioritizing users and allocating bandwidth within a class; FIGS.",
"22a and 22b illustrate a flowchart of a sixth preferred method of prioritizing users and allocating bandwidth within a class; FIG.",
"23 illustrates a flowchart of a preferred method of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network; FIG.",
"24 illustrates the allocation of bandwidth to users within a class during a first time interval; FIG.",
"25 illustrates the allocation of bandwidth to the users of FIG.",
"24 during a second time interval; and FIG.",
"26 illustrates a flowchart of a preferred method of soliciting a user to modify the user's SLA based on monitored network access usage of the user.",
"DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In the following detailed description, numerous specific details are set forth with regard to preferred embodiments of the present invention in order to provide a thorough understanding of the present invention; however, it will be apparent to ordinary artisans that the present invention may be practiced without all of these specific details.",
"Well-known structures and devices also are shown in block diagram form, the specific details of which are not considered a necessary part of the present invention.",
"Furthermore, as will become apparent to ordinary artisans, the present invention may be embodied in or performed by hardware, firmware, or software, or various combinations thereof.",
"As described above, a conventional DOC Network 40 is shown in FIG.",
"1 and includes a plurality of Cable Networks 38, with a particular Cable Network 38 being illustrated in an expanded view and comprising a group of CMs 34, each connected to a computer 44 representing a user.",
"Additionally, as used herein, “user” includes not only a person who interacts with a computer 44, but any additional persons who also interact with the same computer 44, as well as any group of persons all of whom interact with computers attached either to the same CM 34 or to the same computer 44 which, itself, is attached to a CM 34.While not shown, such additional arrangements are well known in the art.",
"The CMs 34 are connected by a coaxial cable 32 with a CMTS 30 and, specifically, to a card 31 mounted within the CMTS 30.Each of the CMTSs of the DOC Network 40 preferably includes a plurality of cards, with each card supporting a group of CMs connected thereto in an inverted tree configuration to define a Cable Network 38.Furthermore, each CMTS conventionally supports up to 1,500 users, although recent CMTSs have been introduced that support up to 15,000 users.",
"Each Cable Network 38 defines a Shared Access Carrier Network, wherein data of respective users in each are conveyed together through a shared coaxial cable.",
"For instance, data packets (or frames) addressed to at least one of the computers 44 are transmitted by the CMTS 30 downstream over the coaxial cable 32 to all of the CMs 34 within a 6 MHz data channel.",
"Conversely, data packets intended for delivery to the CMTS 30 and beyond are transmitted by a CM 34 upstream to the CMTS 30 over the coaxial cable 32 within a 2 MHz channel.",
"The Cable Network 38 shown in expanded view in FIG.",
"1 is a traditional all coaxial cable network.",
"The other Cable Networks 38 collectively include both traditional all coaxial cable networks as well as HFC networks.",
"The CMTS 30 transmits and receives data packets between the Cable Networks 38 and an Intermediate Network 46, which begins with a router 48 in the headend 36, and includes switched and routed network equipment at a Regional Data Center 50 that provides connectivity to service providers 52,54,56,58, either directly or through the Internet 60.In this regard, during user communications the router 48 conveys data packets from the CMTS 30 to the Regional Data Center 50 of the DOC Network 40 and, conversely, routes data packets received from the Regional Data Center 50 to the appropriate CMTS for delivery to a particular user.",
"Data packets that are conveyed to the Regional Data Center 50, in turn, are directed on to an appropriate service provider 52,54 directly connected to the Regional Data Center 50, or to an appropriate service provider 56,58 indirectly connected to the Regional Data Center 50 via the Internet 60.Alternatively, data packets from users are conveyed to a server of an application server group 62 of the Regional Data Center 50, which includes, for example, servers supporting Web hosting, news, chat, SMTP, POP3, Proxy, cache and content replication, and streaming media.",
"The Cable Networks 38 stemming from headend 36 are maintained by a Carrier which also may maintain the Regional Data Center 50 as well as serve as a service provider.",
"Moreover, the Carrier may maintain the Cable Networks of additional headends 64,66, or of only one or more of the headends 64,66.In any event, the Cable Networks that are maintained by the Carrier are administered on a daily basis through an element management system (EMS) 68.The EMS 68 comprises an operations system designed specifically to configure and manage CMTSs and associated CMs, and includes a CM database 70.Operational tasks performed by the EMS 68 include provisioning, day-to-day administration, and testing of various components of each CMTS.",
"The EMS 68 typically is located at a central network operations center of the Carrier, but may be collocated at the headend 36 of the Carrier as shown in FIG.",
"1.The DOC Network 40 is managed through a control plane server group 72 typically located at the Regional Data Center 50.The control plane server group 72 includes the usual servers necessary to run the DOC Network 40, such as user authorization and accounting servers, log control servers (Syslog), IP address assignment and administration servers (DHCP, TFTP), domain name servers (DNS), and DOCSIS control servers.",
"For purposes of comparison, two dedicated connections also are shown in FIG.",
"1, wherein a telephony modem 74 and an ISDN modem 76 are connected directly to the Intermediate Network 46 at the Regional Data Center 50.As will be immediately apparent, data conveyed over each dedicated connection is between a single user and the Intermediate Network 46, and is not combined with data of other users over a shared communications medium as in each Cable Network 38.As is common in conventional Cable Networks 38 such as those shown in the DOC Network 40 of FIG.",
"1, when a CM comes online the CM is assigned a configuration file which, inter alia, sets a constant limit on the bandwidth that can be utilized in the downstream direction by the CM during any particular interval of time, and sets a constant limit on the bandwidth that can be utilized in the upstream direction by the CM during any particular interval of time.",
"The configuration file also includes other parameters, such as the IP address for the CM.",
"The configuration file for each CM conventionally is obtained by the CM when first brought online, or when the CM is reset.",
"The upstream and downstream bandwidth limits are predetermined by the Carrier or other appropriate entity, the determination of which is based on the expected number of users to be serviced by the particular Cable Network 38 to which the CM belongs.",
"With particular regard to data transmissions in the downstream direction, when the bandwidth limit is reached in receiving data within a particular time interval, the CM transmits a signal to the router 48 to cease further data forwarding for the remainder of the time interval.",
"Thereafter, whereas any data received by a CMTS is relayed on to the CM as the data is received, any additional data received by the router 48 during the remainder of this time interval is stored for later transmission in a buffer up to a threshold limit and, thereafter, any further data received within the time interval is dropped.",
"With regard to data transmissions in the upstream direction, when the CM registers with the CMTS following receipt by the CM of its configuration file, the CM informs the CMTS of the constant bandwidth limit to be applied to upstream transmissions from the CM.",
"Then, actual requests for bandwidth (i.e., requests for timeslots) for transmission of data in the upstream direction are submitted regularly by each CM to the CMTS.",
"In response to the submissions, the CMTS schedules timeslots in a particular time interval to the CMs for exclusive transmission of data within each timeslot by a respective CM.",
"However, the CMTS does not grant an amount of bandwidth (by assigning too many timeslots) to a particular CM that would exceed the constant bandwidth limit for the particular CM.",
"The timeslots are assigned to requesting CMs based on an established assignment policy.",
"For example, timeslots may be assigned by the CMTS on a first-in-first-out basis, or timeslots may be assigned equally to the CMs that request bandwidth within a particular window of time.",
"The requesting CMs also may be prioritized by the CMTS for assignment of the timeslots.",
"Preferred embodiments 78,80,82,84 of a DOC Network in accordance with the present invention are shown, respectively, in FIGS.",
"2-5, wherein each includes a “network access manager” 86 in accordance with the present invention.",
"In FIG.",
"2 the network access manager 86 is located in the headend 36 of the DOC Network 78, in FIG.",
"3 the network access manager 86 is located at the Regional Data Center 50 of the DOC Network 80, and in FIGS.",
"4-5 the network access manager 86 is remotely located, but is disposed for communication with the respective DOC Network 82,84, either directly as shown in the DOC Network 82 of FIG.",
"4, or indirectly via the Internet 60 as shown in the DOC Network 84 of FIG.",
"5.The network access manager 86 preferably comprises a hardware component having software modules for performing methods in accordance with the present invention.",
"For commercial purposes, especially in enhancing existing DOC Networks, preferably the network access manager 86 is self-contained and need only be connected in communication with the DOC Network to operate correctly.",
"In a DOC Network that is being upgraded or established, preferably the software modules are distributed within the DOC Network itself and may or may not include any additional hardware components such as the network access manager 86.For example, the software modules may be incorporated into the EMS, CMTS, and control plane server group of a DOC Network, thereby avoiding the expense of additional computer hardware components.",
"In order to accommodate deployment and implementation of the present invention, the software modules preferably are designed as peers within a messaging infrastructure and, in particular, within a CORBA infrastructure 87, the system architecture of which is shown in FIG.",
"6.Due to the interoperability of the peers to the CORBA infrastructure 87, the separate modules readily call upon each other as described in detail below without regard to differences in location between the modules.",
"Nevertheless, for ease of deployment, the network access manager 86 is best suited for deployment and implementation of the present invention in established DOC Networks, whether situated within the Intermediate Network as in FIGS.",
"2-3, or remotely situated as in FIGS.",
"4-5.The software modules include a Data Collector 88, a Database Manager 90, Bandwidth Allocator 92, and GUI & Report Generating Engine 94.The Data Collector 88 and Bandwidth Allocator 92 each includes an external system interface layer 96,98, respectively, that enables it to communicate with network equipment of a DOC Network.",
"In the system architecture of preferred embodiments, the Data Collector 88 communicates with each CMTS and CMs of each Cable Network for which network access is managed by the network access manager 86, and the Bandwidth Allocator 92 communicates with the control plane server group 72 of the DOC Network as well as with the CMTS and CMs.",
"If a DOC Network is DOCSIS 1.0 compliant, then each external system interface layer 96,98 is a DOCSIS external system interface layer.",
"If a DOC Network uses proprietary interface specifications, then each external system interface layer 96,98 is designed based on the proprietary interface specifications.",
"In either case, however, the Data Collector 88 and Bandwidth Allocator 92 generally need not be modified; only the external systems interface layers 96,98 thereof need be changed based on the particularities of the DOC Network.",
"Each of the Data Collector 88 and Bandwidth Allocator 92 also includes a scheduling element 100,102, respectively, that schedules the timing of actions and communications thereof with the network equipment of a DOC Network.",
"The GUI & Report Generating Engine 94 communicates with an Administrator 106 of the network access manager 86, preferably through a web server, whereby the Administrator 106 sets up and configures the network access manager 86 and accesses reports generated by the network access manager 86, such as graphs of bandwidth consumption and bandwidth requested per time interval for a user.",
"The Administrator 106 may be the Carrier, a service provider, or some other entity, such as the entity managing the Regional Data Center 50 or a third-party responsible for maintenance of the network access manager 86.The Database Manager 90 stores configuration and setup information received from the GUI & Report Generating Engine 94, as well as information processed by the Data Collector 88.The Database Manager 90 also provides information to the Bandwidth Allocator 92 and GUI & Report Generating Engine 94 as requested via the CORBA infrastructure 87.Having now described in detail the structure of preferred DOC Networks 78,80,82,84, preferred methods of the present invention will be described with reference thereto.",
"In accordance with preferred methods of the present invention, network access usages of each user in the upstream and downstream directions are monitored through the Data Collector 88.Specifically, the Data Collector 88 issues queries to the CMTS and CM to which counter values of logical data units (LDUs) are returned for a user.",
"Preferably, counter values are returned for the number of bytes and the number of data packets that are transmitted in both the upstream and downstream directions, the number of bytes and the number of data packets that are dropped in both the upstream and downstream directions, the number of bytes and the number of packets that are requested to be transmitted in the upstream direction, and the time for which the counter values are returned.",
"Accordingly, as used herein the phrase “monitoring network access usage” is intended to refer to the collection of data representative of at least one of: (i) the number of LDUs that are transmitted in a particular direction across a shared communications medium; (ii) the number of LDUs that are dropped in transmitting in a particular direction across a shared communications medium; and (iii) the number of LDUs that are requested to be transmitted in a particular direction across a shared communications medium.",
"In a DOCSIS compliant DOC Network, the information is collected from the CMTS and CMs of a Cable Network via the simple network management protocol (SNMP).",
"The counter values for bytes and data packets that are transmitted and that are dropped in the upstream direction from each CM, and the number of bytes and data packets that are requested to be transmitted in the upstream direction from each CM, are recorded by the CMTS in accordance with a management information base (MIB) of a DOCSIS compliant CMTS.",
"Likewise, the counter values for bytes and data packets that are transmitted and that are dropped in the downstream direction from the CMTS to a CM are recorded by the CM in accordance with a MIB of a DOCSIS compliant CM.",
"Both bytes and data packets are monitored since each data packet may vary in the number of bytes it contains.",
"The scheduling element 100 of the Data Collector 88 initiates the data collection from each CMTS and from the CMs connected thereto, preferably at different predetermined time intervals.",
"For example, the data collection from a CMTS preferably occurs at five minute intervals and data collection from the CMs connected thereto preferably occurs at thirty minute intervals.",
"The data collection from the CMs preferably is less often than the data collection from the CMTS in order to minimize consumption of bandwidth across the Cable Network that otherwise would be allocated to users.",
"When the counter values and time thereof are returned to the Data Collector 88, the Data Collector 88 calculates the change over time for each counter value to arrive at the average rates of bytes and data packets that are successfully transmitted, the average rates of bytes and data packets that are requested to be transmitted, and the average rates of bytes and data packets that are dropped.",
"The respective rates and time intervals for the rates (as well as the counter values and time stamp data) are then communicated to the Database Manager 90, which stores the information in a user statistics table (“stats”) for later use by the Bandwidth Allocator 92 and GUI & Report Generating Engine 94.The Bandwidth Allocator 92 continually determines the network access—or bandwidth in a Cable Network—that may be utilized by each user class, and by each user within each class, over succeeding time intervals.",
"Each allowance is determined by first allocating bandwidth to the user classes, and then allocating bandwidth to the users in each class, in accordance with one or more selected allocation policies.",
"Furthermore, as set forth above, each allowance is an amount of bandwidth up to which a user class or user may consume, but is not necessarily the amount of bandwidth that a user class or user will consume; it is an upper limit on such amount.",
"For example, with reference to FIG.",
"24, a selected allocation policy has resulted in the allocation of bandwidth to the users of the shared communications medium 2450 for a time interval extending from t0 to (t0+dt), User 2 and User K each is allocated a single bandwidth unit (b/w unit 3 and b/w unit X, respectively), while User 1 and User 3 each is allocated two bandwidth units (b/w unit 1 and b/w unit 2 to User 1, and b/w unit 4 and b/w unit 5 to User 3).",
"As shown in FIG.",
"25, in the next time interval extending from (t0+dt) to (t0+2dt), User 1, User 3, and User K each is allocated a single bandwidth unit (b/w unit 1, b/w unit 5, and b/w unit X, respectively), while User 2 is allocated three bandwidth units (b/w unit 2, b/w unit 3, and b/w unit 4).",
"In this example, all users are grouped within the same class, and the bandwidth units in this example broadly represent network access to the communication member 2400 that is shared between the users across the shared communications medium 2450.In accordance with the present invention, respective user bandwidth allowances for each time interval are equated with these user allocations of bandwidth, whereby no user receives more bandwidth in a time interval than that user's respective bandwidth allowance for that time interval.",
"Furthermore, it is important to distinguish what a user actually may be “allocated” in the context of the bandwidth that is actually utilized or consumed by such user, as opposed to bandwidth allocations to a user in accordance with the present invention.",
"The bandwidth allocation in accordance with the present invention represents a limit on the amount of bandwidth that can be allocated to a user for a time interval—and hence is equated with a bandwidth allowance; it does not represent per se the amount of bandwidth that the user actually will utilize in the time interval.",
"In determining network access allocations (and thus allowances) in the preferred embodiments herein described, the Bandwidth Allocator 92 preferably performs three routines, including: the prediction of bandwidth of each user class, and each user within each class, in a predetermined future interval of time (“First Routine”); the prioritization of user classes, and users within each class, for allocation of bandwidth (“Second Routine”); and the actual allocation of bandwidth for each user class, and each user within each class, for determining the bandwidth allowances for the future time interval (“Third Routine”).",
"The First Routine preferably is performed utilizing statistical analysis of past bandwidth consumption of each user or, alternatively, past bandwidth requested for each user, and the forecasted bandwidth includes the bandwidth expected to be consumed by each user or, alternatively, the bandwidth expected to be requested by each user.",
"Any function, method, or algorithm that generates an estimate of a future sample based on previously encountered samples may be used and many are well known in the art of statistical analysis as is evident from SPYROS MAKRIDAKIS ET AL., FORECASTING METHODS AND APPLICATIONS (3d.",
"Ed.",
"John Wiley & Sons 1998), which is hereby incorporated by reference.",
"With regard to user classes, preferably a collective forecasted bandwidth for each class is determined by summing the forecasted bandwidth of all users within the class.",
"The preferred algorithm for predicting each user's forecasted bandwidth includes the combined use of an adaptive-response-rate single exponential smoothing function (ARRSES Function) and a Holt-Winters' seasonal exponential smoothing function (HW Function).",
"These two functions are utilized according to the forecast generation flowchart of FIG.",
"7.The input includes a list of active users and the applicable time intervals for bandwidth allocation.",
"The First Routine 700 begins by identification (Step 702) of the users of the Cable Network to which bandwidth is to be allocated in the Third Routine.",
"Then, for each user, bandwidth for a succeeding time interval is predicted according to either the ARRSES Function or HW Function by first determining (Step 704) whether the user previously has been assigned a forecast function.",
"If not, then in Step 706 the ARRSES Function is assigned to the user and the ARRSES Function is used to generate and record the forecasted bandwidth for the succeeding time interval.",
"On the other hand, if it is determined in Step 704 that a forecast function is assigned, but it is determined in Step 707 that the forecast function is not the HW Function, then a determination is made (Step 708) whether to check for a seasonal cycle of the user.",
"This determination in Step 708 is made by checking the elapsed time since the last seasonal check was made, with a seasonal check being made after a predetermined period of time elapses.",
"If the determination in Step 708 is affirmative, then a seasonal identifier algorithm is executed (Step 710), in which an autocorrelation function and a seasonal identifier function are performed.",
"The autocorrelation function is well known in the art of statistical analysis, and is used to identify elements in a time series which are influential on a current observation of that same series.",
"Based on the output of the autocorrelation function, the seasonal identifier function identifies possible seasonal cycles of the time series by identifying local maxima of the results of the autocorrelation function.",
"Based on the results of the seasonal identifier function, a determination is made (Step 712) whether an actual seasonal pattern exists.",
"If a seasonal pattern is not found, or if it is not yet time to check for a seasonal cycle, then a forecast is generated and recorded (Step 714) using the ARRSES Function.",
"If a seasonal pattern is found, then the HW Function is assigned (Step 716) to the user, the HW Function is initialized (Step 718), and the first forecast is generated and recorded (Step 720) using the HW Function.",
"If it is determined in Step 707 that the current function assigned to the user already is the HW Function, then the determination is made (Step 722) whether the last forecasted bandwidth was acceptable.",
"This determination is made by comparing whether the forecasted bandwidth was within 10% of the actual bandwidth consumed or requested.",
"If this determination in Step 722 is negative, then the ARRSES Function is assigned to the user and the new forecast is generated and recorded in accordance with the ARRSES Function (Step 706).",
"If the last forecast is determined (Step 722) to have been acceptable, then a determination is made (Step 724) whether the seasonal cycle has ended.",
"If the seasonal cycle has ended, then the HW Function is reinitialized (Step 726), and the first forecast of the next seasonal cycle is generated and recorded (Step 728) via the HW Function.",
"If the seasonal cycle has not expired, then the next forecast is generated and recorded (Step 730) in accordance with the HW Function.",
"Following each of Step 706, Step 714, Step 728, and Step 730, the Bandwidth Allocator 92 determines (Step 732) whether the forecasting has been completed for all users and, if not, then repeats (Step 738) a forecast loop for a remaining user.",
"If it is determined in Step 732 that all users have been evaluated, then the forecasts are communicated (Step 736) to the Database Manager 90 and the forecasting routine ends.",
"A forecast of bandwidth for a user in a future time interval is generated in accordance with the ARRSES Function via the following formulas: FN+1=FN+αN(BN−FN) αN+1=|SEN/SAEN| SEN+1=SEN+β(BN+1−FN+1−SEN) SAEN=β|(BN−FN)|+(1−β)SAEN−1 wherein, F is the bandwidth that is expected to be consumed by a user for a time interval (or the bandwidth that is expected to be requested by a user); B is the bandwidth that is actually consumed by a user for the time interval (or the bandwidth that is actually requested by a user); N is the present time interval; N−1 is the previous (immediate past) time interval; N+1 is the next (immediate future) time interval; and β is a selected parameter affecting the responsiveness to change of the ARRSES Function when the bandwidth of a user changes between time intervals.",
"Bandwidth is predicted both for the 6 MHz channel in the downstream direction as well as the 2 MHz channel in the upstream direction.",
"Preferably each time interval is thirty minutes in length, but preferably may range from fifteen minutes to sixty minutes in length when bandwidth is forecast in the downstream direction.",
"Preferably each time interval is five minutes in length, but preferably may range from one minute to fifteen minutes in length when bandwidth is forecast in the upstream direction.",
"The steps in generating a forecast in accordance with the ARRSES Function are set forth in FIG.",
"8, and include the calculation (Step 802) of a forecast error, the calculation (Step 804) of a smoothed error, the calculation (Step 806) of a smoothed absolute error, the calculation (Step 808) of alpha, and the calculation (Step 810) of the new forecast.",
"A forecast of bandwidth of a user for a future time interval is generated in accordance the HW Function via the following formulas: Ls=1/s(Y1+Y2+ .",
".",
".",
"+Ys) bs=1/S[(Ys+1−Y1)/s+(Ys+2−Y2)/s+ .",
".",
".",
"+(Y2s−Ys)/s] S1=Y1/Ls,S2=Y2/Ls, .",
".",
".",
"Ss=Ys/Ls Lt=α(Yt/St−s)+(1−α)(Lt−1+bt−1) bt=β(Lt−Lt−1)+(1−β)bt−1 St=γYt/Lt+(1−γ)St−s Ft+m=(Lt+btm)St−s+m wherein, Li=an average level of bandwidth after time interval i, bi=the trend after time interval i, si=the seasonal influence at time interval i, s=length of seasonal cycle (in number of time intervals), Yi=monitored bandwidth consumed or requested in time interval i t=time of initialization, m=the number of time intervals into the future for which a forecast is made, and α, β, and γ are parameters of the forecast method whose values are determined by doing a grid search over the domain of possible values of these parameters in an attempt to minimize the mean-squared-error of the forecast method, each of α, β, and γ falling between 0 and 1.The steps in generating a forecast in accordance with the HW Function are set forth in FIG.",
"9, and include the initialization of the HW Function by determining Ls, bs, and S1, S2 .",
".",
".",
", Ss in Step 902, if appropriate; the determination of the intermediate values of Lt, bt, and St in Step 904; and the determination of the forecast in Step 906, all in accordance with the above formulas.",
"The Second Routine performed by the Bandwidth Allocator 92 comprises the prioritizing of user classes, and of users within each class, to determine respective orders of allocations.",
"Prioritization is performed in accordance with one or more of various possible prioritization policies for users and for user classes.",
"With regard to users within each class, the prioritization policies may depend upon, for example, (i) each user's SLA, (ii) each user's forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.",
"User SLAs that at least partially affect prioritization policies include those that specify, for example: (i) a guaranteed minimum level of bandwidth; (ii) a time-of-day (TOD) minimum level of bandwidth; or (iii) a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability.",
"Equivalently, such provisions also may be found in a CSLA for a class of which the user is a member.",
"Under a SLA or CSLA providing for a guaranteed minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for use at all times.",
"Accordingly, if the available bandwidth to such a user otherwise would fall below the minimum guaranteed level, then such a user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.",
"Similarly, under a SLA or CSLA providing for a TOD minimum level of bandwidth for a user, a user will have a guaranteed minimum level of bandwidth for a particular TOD.",
"If the available bandwidth to such a user otherwise would fall below the minimum guaranteed level during the particular TOD, then such user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.",
"Finally, under a SLA or CSLA providing for a guaranteed minimum level of bandwidth up to a maximum burstable level of bandwidth with target probability for a user, a user will have a guaranteed minimum level of bandwidth at all times and, in addition thereto, probably will have additional bandwidth up to a maximum level at any given time in accordance with the target probability.",
"Accordingly, if the bandwidth available to such user otherwise would fall below the minimum guaranteed level, then the user is given priority over all other users whose guaranteed minimum levels of bandwidth (if applicable) have been satisfied.",
"The user also is given priority over such other users in allocating additional bandwidth as needed up to the maximum level in accordance with the target probability.",
"Other SLA or CSLA provisions not relating to guaranteed levels of bandwidth also may affect a prioritization policy for users.",
"Thus, for example, a SLA or CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon bandwidth consumption by a user for a particular amount of time, and the fee may be different as between users.",
"Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid.",
"Similarly, a SLA or CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier to an account based upon a bandwidth shortfall to a user for a particular amount of time when a guaranteed level of bandwidth for the user is not met.",
"Moreover, the credit may be different as between users.",
"Under these circumstances, prioritization may be determined so as to minimize the collective credits that a Carrier must apply.",
"An example of prioritization based upon the forecasted bandwidth of each user includes giving priority to a first user over all other users, each of whom have a forecasted bandwidth that is greater than that of the first user.",
"Prioritization may also be performed based on unilateral fairness considerations, especially when SLAs or CSLAs do not guarantee minimum levels of bandwidth for individual users, or when users otherwise would share equally in priority.",
"Thus, users may be prioritized based on, for example: (i) the throughput of each of the users for a given time interval, with priority going to the user with the lesser throughput; (ii) data packets dropped over a given time interval, with priority going to the user with the greater data loss; and (iii) throughput experienced during a particular time of day or day of the week with priority going to the user with the lesser throughput for the particular time of day or day of the week.",
"An example of fairness considerations that may be utilized in determining priority is illustrated in FIG.",
"10, wherein user throughput for a time interval is graphed against user data packets dropped in the time interval for Users A and B.",
"A target QoS standard for minimum throughput and maximum packet loss rates are established by the Carrier, whereby in the illustrated example each user is prioritized based on the user's absolute distance from the target QoS standard.",
"Thus, under this policy, User A experiencing higher throughput rate and a lower packet loss rate, and thus having a shorter distance from the standard, is prioritized lower than User B having a lower throughput rate and higher data loss rate.",
"With regard to user classes, prioritization policies are similar to those of the users and include, for example, (i) each CSLA, (ii) each class' collective forecasted bandwidth, (iii) fairness considerations, or (iv) any combination thereof.",
"CSLAs that at least partially affect prioritization policies for user classes include those that specify, for example: (i) a guaranteed minimum level of collective bandwidth for the user class; (ii) a time-of-day (TOD) minimum level of collective bandwidth for the user class; or (iii) a guaranteed minimum level of collective bandwidth up to a maximum burstable level of collective bandwidth with target probability for the user class.",
"Other CSLA provisions not relating to guaranteed levels of collective bandwidth also may affect a prioritization policy.",
"Thus, for example, each CSLA may specify a fee (in dollars per unit time per unit bandwidth) that is paid based upon collective bandwidth consumption by the users of a class for a particular amount of time, and the fee may be different as between different classes of users.",
"Under these circumstances, prioritization may be determined so as to maximize fee revenues that are paid to a Carrier.",
"Similarly, each CSLA may specify a credit (in dollars per unit time per unit bandwidth) that is applied by the Carrier based upon a collective bandwidth shortfall to the users of the class for a particular amount of time when a guaranteed level of collective bandwidth is not met.",
"Moreover, the credit may be different as between user classes.",
"Under these circumstances, prioritization may be determined so as to minimize the total credits that a Carrier may have to apply.",
"An example of prioritization based upon the collective forecasted bandwidth of each user class includes giving priority to a first user class over all other user classes, each of which has a respective collective forecasted bandwidth that is greater than that of the first user class.",
"Prioritization may also be performed based on unilateral fairness considerations, especially when CSLAs do not guarantee minimum levels of collective bandwidth, or when classes otherwise would share equally in priority.",
"Thus, user classes may be prioritized based on, for example: (i) the collective throughput of the users of a class for a given time interval, with priority going to the class with the lesser collective throughput; (ii) the collective data packets of a user class that are dropped over a given time interval, with priority going to the user class with the greater collective data loss; and (iii) the collective throughput of the users of a class experienced during a particular time of day or day of the week, with priority going to the user class with the lesser collective throughput for the particular time of day or day of the week.",
"The Third Routine performed by the Bandwidth Allocator 92 is the allocation of bandwidth to the user classes, and then to the users within each class, in accordance with one or more allocation policies as desired.",
"Examples of allocation policies for users include: (i) the equal distribution of all available bandwidth to all users; (ii) the distribution of all available bandwidth to all users proportional to each user's respective forecasted bandwidth; (iii) the distribution of bandwidth to each user equal to the user's respective forecasted bandwidth, with any surplus bandwidth being distributed to the users either equally or proportionally based upon the user's respective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user based upon the minimum of the user's guaranteed bandwidth or the forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users.",
"Likewise, examples of allocation policies for user classes include: (i) the distribution of all available bandwidth by the Bandwidth Allocator 92 to all user classes proportional to the number of active users in each class; (ii) the distribution of all available bandwidth to all user classes proportional to each class' respective collective forecasted bandwidth; (iii) the distribution of bandwidth to each user class equal to the class' respective collective forecasted bandwidth, with any surplus bandwidth being distributed to the user classes either equally or proportionally based upon the class' respective collective forecasted bandwidth; and (iv) the initial distribution of bandwidth to each user class based upon the minimum of the class' guaranteed collective bandwidth or the collective forecasted bandwidth and, thereafter, incremental allocations of remaining bandwidth to all of the users classes.",
"Examples of alternate preferred methods of prioritizing user classes, and then allocating bandwidth to the classes, will now be described in detail, each of which utilizes one or more of the aforementioned user class prioritization and allocation policies.",
"Alternative preferred methods of prioritizing users within each class, and then allocating bandwidth to the users in each class, are set forth thereafter.",
"In either case, the preferred methods of prioritizing and allocating are initiated pursuant to the scheduling module 102 of the Bandwidth Allocator 92, which operates independently of the scheduling module 100 of the Data Collector 88.With regard to prioritization of and allocation to user classes, a first preferred method 1100 is illustrated in FIG.",
"11 and begins with the retrieval (Step 1102) of the collective forecasted bandwidth from the Database Manager 90 for all active user classes.",
"Whether a user class is active is determined by past collective bandwidth consumption of the class (or, alternatively, collective requested bandwidth for the users of the class), as revealed by the user stats maintained by the Database Manager 90.All user classes are then prioritized (Step 1104) based on each class' collective forecast in increasing order, whereby a class having a lesser collective forecasted bandwidth will be prioritized over a class having larger collective forecasted bandwidth.",
"A “surplus” is then set (Step 1106) to the total bandwidth available for allocation to the classes in the particular direction of communication over the shared communications medium at issue, and the total bandwidth available is then allocated (Step 1108) to each user class in an amount equaling the collective forecasted bandwidth subject to a respective maximum collective bandwidth value of the user class.",
"Preferably the maximum collective bandwidth value is determined either in the appropriate CSLA or by the Carrier, Administrator 106, or other entity.",
"Allocation of bandwidth to a user class additionally is subject to the actual availability of bandwidth following previous allocations thereof to user classes with equal or higher priority.",
"Following allocations to all user classes, any bandwidth determined (Step 1110) to be remaining is then allocated (Step 1112) to the classes in amount proportional to the number of active users in each class, subject of course to the respective maximum collective bandwidth value of the class.",
"The resulting class allocations are then recorded in the Database Manager 90 (Step 1114) as the bandwidth allowances for the classes.",
"The method 1200 illustrated in FIG.",
"12 is the same as that of FIG.",
"11, except that surplus bandwidth, if any, is allocated (Step 1102) proportional to the collective forecasted bandwidths of the user classes, again subject to the respective maximum collective bandwidth value of each user class.",
"The preferred method 1300 illustrated in FIG.",
"13 does not prioritize the user classes for purposes of allocation but, instead, treats all classes equally.",
"The method 1300 begins with the retrieval (Step 1302) of the collective forecasted bandwidth of each user class from the Database Manager 90.The surplus is then set to the total bandwidth available in the particular direction of communication, and the sum of all the collective forecasts is calculated (Step 1304).",
"The available bandwidth then is allocated (Step 1306) to all classes proportional to the class' collective forecasted bandwidth, again subject to the respective maximum collective bandwidth value for each class.",
"The resulting class allocations then are recorded in the Database Manage 90 (Step 1308) as the bandwidth allowances for the classes.",
"The preferred method 1400 illustrated in FIG.",
"14 seeks to maximize revenues from fees (F) that are paid for class bandwidth consumption.",
"The method 1400 begins with the retrieval (Step 1402) of the collective forecast for each user class as well as a fee that is paid for the collective bandwidth of the class.",
"The classes are then sorted (Step 1404) based on these fees in decreasing order, with the class with the highest fee receiving the highest priority.",
"Next the surplus is set (Step 1406) to the total bandwidth available for allocation to the classes in the particular direction of communication.",
"Bandwidth then is allocated (Step 1408) to the classes as available from highest to lowest priority in an amount equal to the class' collective forecasted bandwidth, subject to the respective maximum collective bandwidth value for the class.",
"Both preferred method 1500 of FIGS.",
"15a and 15b, and preferred method 1600 of FIGS.",
"16a and 16b differ from the other methods 1100,1200,1300,1400 in that these two methods allocate bandwidth to the user classes in multiple allocation rounds.",
"Method 1500 begins in FIG.",
"15a with the retrieval (Step 1502) of the collective forecasted bandwidths of the classes as well as a credit (C) that applies if a respective class does not receive up to a guaranteed maximum level of collective bandwidth.",
"The classes are then prioritized (Step 1504) based on each class' respective credit in decreasing order, with those classes having higher credits being given priority over classes with lesser credits.",
"Next, the surplus is set (Step 1506) to the total bandwidth available to the classes in the particular direction of communication.",
"Bandwidth then is allocated (Step 1508) as available in a first round to the classes from highest to lowest priority.",
"The allocation for each class in the first round is equal to the minimum of the collective forecasted bandwidth or the maximum collective bandwidth that is guaranteed, subject to the respective maximum collective bandwidth value for the class.",
"If any additional bandwidth is determined (Step 1510) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 1514).",
"Bandwidth then is allocated (Step 1516) as available to each class in the same class order.",
"Assuming sufficient bandwidth remains available, the allocation in the second round brings each class' allocation up to the class' collective forecasted bandwidth subject to the class' respective maximum collective bandwidth value.",
"Following the second allocation round, a determination is made (Step 1518) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 1522) to the classes proportional to each class' collective forecasted bandwidth, and subject to each class' respective maximum collective bandwidth value.",
"The resulting class allocations are then recorded (Step 1524) in the Database Manager 90 as the bandwidth allowances of the classes.",
"If it is determined that no bandwidth remains available in either of Step 1510 or Step 1518, then the class allocations are completed and are recorded in the Database Manager 90 in Steps 1512,1524, respectively.",
"Method 1600 of FIGS.",
"16a and 16b differs from that of FIGS.",
"15a and 15b only in that the sum of the collective forecasted bandwidths for all classes is calculated (Step 1602) and a determination is made (Step 1604) whether the sum exceeds the total bandwidth available for allocation to the classes.",
"If the sum exceeds the total available bandwidth, then bandwidth is allocated (Step 1606) to each class in an amount equal to the collective forecasted bandwidth of the class, subject to the class' maximum guaranteed collective bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.",
"Thus, for example, if the sum of all collective forecasted bandwidths exceeds the total available bandwidth for allocation in an amount equal to 20% of all collective forecasted bandwidths, then each class is allocated bandwidth in an amount equal to the class' collective forecasted bandwidth (subject to the class' maximum guaranteed collective bandwidth), then less 20% thereof.",
"The information including fees, credits, guaranteed collective bandwidths, and respective maximum collective bandwidth values in the aforementioned preferred methods, is obtained from each CSLA and/or is predetermined by the Administrator 106, Carrier, or other entity.",
"Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a CSLA table for each class as well as information regarding users associated therewith, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of class and user records in a timely fashion.",
"Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once class allocations have been determined, the Database Manager 90 is updated with the new class allocations.",
"Then, for each class, allocations of bandwidth are made to the users in the class.",
"Furthermore, allocations within each class may be made by different methods.",
"A first preferred method 1700 of prioritizing users and allocating bandwidth (whether upstream or downstream) by the Bandwidth Allocator 92 is illustrated in FIG.",
"17 and begins with the retrieval (Step 1702) of the forecasted bandwidth from the Database Manager 90 for all active users.",
"Whether a user is active is determined by past bandwidth consumption of the user (or, alternatively, requested bandwidth for the user), as revealed by the user stats maintained by the Database Manager 90.All users are then prioritized (Step 1704) based on each user's forecast in increasing order, whereby users having lesser forecasted bandwidths will be prioritized over users having larger forecasted bandwidths.",
"The “surplus” is then set (Step 1706) to the total allocated bandwidth of the class (i.e., the class' collective bandwidth allowance) in the particular direction of communication, and the entire bandwidth allowance of the class is then allocated (Step 1708) to each user in an amount equaling the forecasted bandwidth of the user subject to a respective maximum bandwidth value of the user.",
"Preferably the respective maximum bandwidth value is determined either in the user's SLA, the respective CSLA of the class, or by the Carrier, Administrator 106, or other entity.",
"Allocation of bandwidth to a user additionally is subject to the actual availability of bandwidth following previous allocations thereof to users with equal or higher priority.",
"Following allocations to all users, any bandwidth determined (Step 1710) to be remaining out of the total class allowance is then allocated equally (Step 1712) to the users subject to the respective maximum bandwidth value for each user.",
"The new user allocations are then incorporated (Step 1714) into the DOC Network as the bandwidth allowances of the users.",
"The method 1800 illustrated in FIG.",
"18 is the same as that of FIG.",
"17, except that surplus bandwidth in the class, if any, is allocated (Step 1802) proportional to the forecasted bandwidths of the users in the class, again subject to each user's respective maximum bandwidth value.",
"The preferred method 1900 illustrated in FIG.",
"19 does not prioritize the users for purposes of allocation but, instead, treats all users equally.",
"The method 1900 begins with the retrieval (Step 1902) of the forecasted bandwidth for each user in the class from the Database Manager 90.The surplus is then set to the total allocated bandwidth of the class in the particular direction of communication, and the sum of all forecasts of the users in the class is calculated (Step 1904).",
"The total allocated bandwidth of the class then is allocated (Step 1906) to all users in the class proportional to the user's forecasted bandwidth, again subject to each user's respective maximum bandwidth value.",
"The user allocations then are incorporated into the DOC Network (Step 1908) as the bandwidth allowances of the users.",
"The preferred method 2000 illustrated in FIG.",
"20 seeks to maximize revenues from fees (F) that are paid for bandwidth consumption by the users.",
"The method 2000 begins with the retrieval (Step 2002) of the forecast for each user as well as a fee that is paid for bandwidth by each user.",
"The users are then sorted (Step 2004) based on user fees in decreasing order, with the user paying the most for bandwidth receiving the highest priority.",
"Next, the surplus is set (Step 2006) to the total allocated bandwidth of the class in the particular direction of communication.",
"Bandwidth then is allocated (Step 2008) to the users in the class as available from highest to lowest priority in an amount equal to each user's forecasted bandwidth, and subject to the user's respective maximum bandwidth value.",
"Both preferred method 2100 of FIGS.",
"21a and 21b, and preferred method 2200 of FIGS.",
"22a and 22b differ from the other methods 1700,1800,1900,2000 in that these two methods allocate bandwidth to the users in multiple allocation rounds.",
"Method 2100 begins in FIG.",
"21a with the retrieval (Step 2102) of the forecasted bandwidths of the users as well as a credit (C) that applies if a respective user does not receive up to a guaranteed maximum level of bandwidth.",
"The users are then prioritized (Step 2104) based on each user's respective credit in decreasing order, with those users having higher credits being given priority over users with lesser credits.",
"Next, the surplus is set (Step 2106) to the total allocated bandwidth of the class in the particular direction of communication.",
"Bandwidth then is allocated (Step 2108) as available in a first round to the users from highest to lowest priority.",
"The allocation in the first round for each user is equal to the minimum of the forecasted bandwidth or the maximum bandwidth that is guaranteed, subject to the user's respective maximum bandwidth value.",
"If any additional bandwidth is determined (Step 2110) to remain after the first allocation round, then the surplus is set to the additional bandwidth (Step 2114).",
"Bandwidth then is allocated (Step 2116) as available to each user in the same user order.",
"Assuming sufficient bandwidth remains available, the allocation in the second round brings the user's allocation up to the user's forecasted bandwidth subject to the user's respective maximum bandwidth value.",
"Following the second allocation round, a determination is made (Step 2118) whether any remaining bandwidth exists and, if so, then the remaining bandwidth is allocated (Step 2122) equally to the users, subject to each user's respective maximum bandwidth value.",
"The user allocations are then incorporated (Step 2124) into the DOC Network as the users' bandwidth allowances.",
"If it is determined that no bandwidth remains available in either of Step 2110 or Step 2118, then the user allocations are completed and are incorporated into DOC Network in Steps 2112,2124, respectively, as the users' bandwidth allowances.",
"Method 2200 of FIGS.",
"22a and 22b differs from that of FIGS.",
"21a and 21b only in that the sum of the forecasted bandwidths for all users is calculated (Step 2202) and a determination is made (Step 2204) whether the sum exceeds the total allocated bandwidth of the class.",
"If the sum exceeds the total allocated bandwidth of the class, then the bandwidth is allocated (Step 2206) to each user in an amount equal to the forecasted bandwidth, subject to the user's maximum guaranteed bandwidth, and less an amount thereof proportional to the total bandwidth shortfall.",
"Thus, for example, if the sum of all forecasted bandwidths exceeds the total allocated bandwidth of the class in an amount equal to 20% of the sum of all the forecasted bandwidths, then each user is allocated bandwidth in an amount equal to the user's forecasted bandwidth (subject to the user's maximum guaranteed bandwidth), then less 20% thereof.",
"The applicable class bandwidth allowances used in the aforementioned methods are obtained from the Database Manager 90.The information, including fees, credits, guaranteed user bandwidths, and maximum bandwidth values in the aforementioned methods, is obtained from each user's SLA or from any applicable CSLA, and/or is predetermined by the Administrator 106, Carrier, or other entity.",
"Moreover, this information is retrieved by the Bandwidth Allocator 92 from the Database Manager 90, which includes and maintains a user SLA table as well as a user billing table, as updated from time-to-time by the Administrator 106.Specifically, the information is configured and maintained through GUIs provided as part of the GUI & Report Generating Engine 94, and is preferably accessed by the Administrator 106 either directly or indirectly through the Internet 60.Alternatively, information is retrieved by the Bandwidth Allocator 92 from an external database maintained by the Administrator, Carrier, or other entity through an application program interface (API) incorporated into the external system interface layer 98 of the Bandwidth Allocator 92.The use of an external database is preferred not only for the CSLAs, but also for the SLAs and user billing tables, as it eliminates any duplicative maintenance of information otherwise maintained by the Database Manager 90 which must be synchronized with the external database, including periodic updating of user records in a timely fashion.",
"Regardless of the particular method or policies utilized by the Bandwidth Allocator 92, once user allocations have been determined under the aforementioned allocation policies, the respective DOC Network is updated with the resulting user allocations as the bandwidth allowances for the users for a particular time interval.",
"Each user is then allocated bandwidth during the particular time interval in an amount that is less than, or equal to, that user's bandwidth allowance.",
"Similarly, the collective bandwidth consumptions of a class by users therein is limited by that class' bandwidth allowance.",
"Preferably, the DOC Network is updated at periodic intervals of between one to fifteen minutes and, preferably every five minutes.",
"Furthermore, the periodic interval preferably corresponds to the scheduling of the Bandwidth Allocator 92 with regard to upstream transmissions.",
"With particular reference to FIG.",
"23, a preferred method 2300 of updating a DOC Network for a DOCSIS 1.0 compliant Cable Network with the user allowances is illustrated.",
"The DOC Network is updated by incorporating (Step 2302) the user allocations as bandwidth allowances (i.e., bandwidth limits) into CM configuration files (MD-5 files) for the CMs of the respective users.",
"As set forth above, each CM configuration file contains instructions for a respective CM that limits the actual bandwidth consumed by the CM in the upstream direction and in the downstream direction.",
"The CM configuration files are then sent (Step 2304) by the Bandwidth Allocator 92 to a Trivial File Transfer Protocol (TFTP) Server of the DOC Network, which maintains CM configuration files for the CMs of the Cable Network.",
"A command is also sent (Step 2306) to either of the CMs or the CMTS of the respective Cable Network causing the CMs to acquire and implement the CM configuration files maintained on the TFTP Server.",
"In addition to maintaining information regarding CSLAs, class allocations, SLAs, and user billing data in the Database Manager 90, the GUI & Report Generating Engine 94 further enables the Administrator 106 to analyze the user stats updated by the Data Collector 88, including the generation of reports and graphs regarding, for example, network access usage of the users over time as well as user throughput rates vs. data loss rates similar to that shown in FIG.",
"10.It additionally should be noted that a user may or may not be permitted to be grouped in one or more classes in accordance with the present invention.",
"If it is desired that classes be mutually exclusive, then some policy should be established for determining which class with which a user is associated as between competing classes.",
"If it is desired that classes not be mutually exclusive, then users falling within two or more classes will be allocated bandwidth within each class to the extent that no conflict arises as between the classes, and subject to any maximum allowed aggregated user bandwidth for all classes that may be established.",
"As now will readily be seen, the preferred methods and preferred networks of the present invention described in detail herein enable a Carrier to accommodate bandwidth concerns of service providers competing for the business of users of a shared communications medium in a Shared Access Carrier Network.",
"In particular, CSLAs now can be constructed in accordance with the present invention whereby a service provider is guaranteed some collective level of network access for the users of the shared communications medium that are customers of the service provider.",
"Furthermore, the provision of bandwidth to users who are customers of competing service providers can now be based on fairness considerations, even if one of the service providers is related to the Carrier.",
"In addition thereto, the differing demands for instantaneous throughput by users competing for access across the shared communications medium now can be accommodated in accordance with the present invention.",
"Indeed, a Carrier now is able to continuously vary bandwidth consumption limits for each user on an individual basis and for small time intervals, either in accordance with fairness considerations, forecasted network access usage of the users, or under contractual provisions governing network access.",
"It also will now be evident that the present invention gives rise to new business models that may be implemented by service providers for providing network access to users thereof and, in particular, to new ways of selling network access, which is also considered part of the present invention.",
"For example, in accordance with the present invention, network access now can be “wholesaled” to service providers by considering the users of the service provider a class and allocating bulk network access to such class pursuant to a CSLA between the Carrier and the service provider.",
"Through a CSLA, a Carrier can offer to the service provider a guaranteed minimum level of network access for the class that is constant throughout the day or week, or a guaranteed minimum level of network access that varies depending upon considerations such as the time of day or the day of week.",
"A Carrier also now can offer a guaranteed minimum level of network access to the class with a guaranteed maximum level of network access provided as needed in accordance with a target probability.",
"The service providers, in turn, then can offer different SLAs to the users that are its customers, essentially selling network access at the retail level.",
"Accordingly, service providers can be assured of levels of network access for the users that are their customers, and users can be assured of appropriate levels of network access to meet their individual demands.",
"Moreover, Carriers and/or service providers now can differentiate between users in charging for network access, thereby allowing Carriers and/or service providers to differentiate revenue streams for maximization of revenues.",
"The present invention also enables Carriers and/or service providers to offer “dynamic SLAs” to users.",
"The term “dynamic SLA” refers to a SLA that can be modified by a user as the user's demand for network access significantly changes, whether such modification is permanent or temporary.",
"In this regard, and in accordance with a preferred method 2600 of the present invention as illustrated in FIG.",
"26, the “network access retailer” (the entity selling the network access to the user) monitors (Step 2602) network access usage by users of a Shared Access Carrier Network and determines (Step 2604), for each user based on network access usage, whether a SLA provision other than those found in the user's current SLA would better meet the user's needs.",
"This determination is made by comparing the user's throughput, bandwidth consumption, and/or bandwidth requested for a predetermined period of time against a set of threshold values, including any guaranteed level of network access provided for in the user's SLA as well as any minimum QoS standard that are deemed necessary for user satisfaction by the network access retailer or other appropriate entity.",
"Thus, if the user's level of throughput, bandwidth consumption, and/or bandwidth requested for the predetermined time interval differs by a predetermined tolerance from a minimum threshold value, then the user is identified (Step 2606) as a “candidate” for modifying the SLA.",
"A similar process alternatively is used, wherein the user's forecasted bandwidth is compared to the threshold values and, if the difference exceeds a predetermined tolerance, then the user is deemed a candidate for modifying the user's SLA.",
"Once users have been identified as candidates, the candidates are filtered by screening (Step 2608) the candidates against a list of users for which solicitations are not to be made.",
"Those candidates passing the screening are then invited (Step 2610) to modify their respective SLAs.",
"The solicitation of the user preferably is performed via email, instant messaging, redirection of the user's web browser to a solicitation web page, generation and mailing of solicitation literature via U.S. mail, telemarketing, or other means of communication.",
"The solicitation includes an invitation for the user to modify the user's SLA by increasing for a fee the minimum level of network access guaranteed to the user.",
"The solicitation preferably also includes an invitation to make the modification permanent, or to make the modification only temporary and for a specific period of time.",
"Thus, for example, if a user is identified as having a high usage pattern at recurrent periods of time (such as every Saturday night when a particular webcast is viewed, or when an Internet game is played), then the user automatically is solicited with an invitation via instant messaging on the following Saturday night to increase the user's guaranteed network access for that night, for a predetermined number of following Saturday nights, and/or for every Saturday night.",
"Acceptance of the invitation by each user results in the modification (Step 2612) of the user's SLA for the appropriate period of time by increasing the level of network access the user is guaranteed (and/or the user's respective maximum bandwidth value, depending upon the policies used).",
"The solicited modification to the user's SLA is updated in the SLA database, which is then used during user prioritization and allocation of bandwidth by the Bandwidth Allocator 92.The resulting higher bandwidth allowance should enhance the user's experience and overall satisfaction with the Carrier Network.",
"In particular, the higher bandwidth (greater network access) should enhance the viewing of the webcast or the playing of the Internet game.",
"On the other hand, SLAs for which users decline solicitations are not modified.",
"Furthermore, if deemed appropriate, users declining a solicitation are recorded in the list against which candidates are screened.",
"Preferably, the Bandwidth Allocator 92 analyzes the user stats maintained by the Database Manager 90, identifies those users that are candidates for SLA modification, and initiates the solicitation of such candidates.",
"Information for each user's SLA for comparison with the user's stats automatically is obtained either from the Database Manager 90, or from an external database maintained by the network access retailer or other appropriate entity.",
"Furthermore, the Bandwidth Allocator 92 preferably performs this analysis for solicitation on a regularly scheduled basis.",
"In addition to such solicitations, a user of course may request a change in the level of network access guaranteed without having to receive first a solicitation.",
"Furthermore, the user may request that the change be for a temporary period of time such that, for example, the change is reversed after only a few hours, which would cover a viewing of a particular webcast or the playing of a particular Internet game beginning at the time of the request.",
"In view of the foregoing detailed description of the preferred embodiments and methods of the present invention, it readily will be understood by those persons skilled in the art that the present invention is susceptible of broad utility and application.",
"Many embodiments and adaptations of the present invention other than those herein described, as well as many variations, modifications, and equivalent arrangements, will be apparent from or reasonably suggested by the present invention and the foregoing description thereof, without departing from the substance or scope of the present invention.",
"Accordingly, while the present invention has been described herein in detail in relation to preferred embodiments, it is to be understood that this disclosure only is illustrative and exemplary of the present invention and is made merely for purposes of providing a full and enabling disclosure of the invention.",
"The foregoing disclosure is not intended nor is to be construed to limit the present invention or otherwise to exclude any such other embodiments, adaptations, variations, modifications and equivalent arrangements, the present invention being limited only by the claims appended hereto and the equivalents thereof.",
"Thus, for example, it will be apparent that, while preferred embodiments of the present invention have been described in the context of DOC Networks (including either a network of all coaxial cable, or a HFC network), the present invention nevertheless relates to any other network (whether wireline or wireless) wherein competing users share access across a shared communications medium including, for example, home networks and small networks in mass transit vehicles."
]
] | Patent_10276654 |
[
[
"Novel nucleic acids and polypeptides",
"The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof."
],
[
"1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, and complementary sequences thereof.",
"2.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.",
"3.An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 wherein said polynucleotide is DNA.",
"5.An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.",
"6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to comprise the polynucleotide of claim 1.9.A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.",
"10.An isolated polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide encoded by any one of the polynucleotides of claim 1; and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-8051.11.A composition comprising the polypeptide of claim 10 and a carrier.",
"12.An antibody directed against the polypeptide of claim 10.13.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.",
"14.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.",
"15.The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.",
"16.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.",
"17.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.",
"18.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.",
"19.A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-8051, a mature protein coding portion of SEQ ID NO: 1-8051, an active domain of SEQ ID NO: 1-8051, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID NO: 1-8051, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).",
"20.An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8052-16102, the mature protein portion thereof, or the active domain thereof.",
"21.The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.",
"22.A collection of polynucleotides, wherein the collection comprises the sequence information of at least one of SEQ ID NO: 1-8051.23.The collection of claim 22, wherein the collection is provided on a nucleic acid array.",
"24.The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.",
"25.The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.",
"26.The collection of claim 22, wherein the collection is provided in a computer-readable format.",
"27.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.",
"28.A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier."
],
[
"<SOH> 2.BACKGROUND <EOH>Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.",
"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).",
"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.",
"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences."
],
[
"<SOH> 3.SUMMARY OF THE INVENTION <EOH>The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.",
"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.",
"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.",
"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.",
"These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.",
"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.",
"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.",
"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.",
"The identifying sequence can be 100 base pairs in length.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.",
"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.",
"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.",
"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.",
"The collection can also be provided in a computer-readable format.",
"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.",
"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.",
"In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.",
"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.",
"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.",
"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.",
"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.",
"host cells) of the invention.",
"The invention also provides compositions comprising a polypeptide of the invention.",
"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.",
"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.",
"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.",
"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.",
"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.",
"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.",
"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.",
"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.",
"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.",
"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.",
"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.",
"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.",
"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.",
"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.",
"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.",
"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.",
"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.",
"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.",
"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.",
"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.",
"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.",
"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.",
"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.",
"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.",
"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).",
"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.",
"2.BACKGROUND Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemokines, and interleukins) has matured rapidly over the past decade.",
"The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning).",
"More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.",
"Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.",
"3.SUMMARY OF THE INVENTION The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.",
"The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.",
"The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases.",
"The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.",
"These nucleic acid sequences are designated as SEQ ID NO: 1-8051.The polypeptides sequences are designated SEQ ID NO: 8052-16102.The nucleic acids and polypeptides are provided in the Sequence Listing.",
"In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases.",
"In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.",
"The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-8051 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-8051.A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-8051 or a degenerate variant or fragment thereof.",
"The identifying sequence can be 100 base pairs in length.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-8051.A collection as used in this application can be a collection of only one polynucleotide.",
"The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array.",
"In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment.",
"The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.",
"The collection can also be provided in a computer-readable format.",
"This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors.",
"Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art.",
"In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-8051 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-8051; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-8051.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing (e.g., SEQ ID NO: 8052-16102); (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g.",
"orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.",
"The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-8051; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.",
"Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated.",
"The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.",
"host cells) of the invention.",
"The invention also provides compositions comprising a polypeptide of the invention.",
"Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The invention also provides host cells transformed or transfected with a polynucleotide of the invention.",
"The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells.",
"Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.",
"Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.",
"These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like.",
"For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.",
"In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.",
"The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.",
"For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.",
"Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.",
"The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.",
"Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.",
"In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.",
"The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample.",
"Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.",
"The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected.",
"The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.",
"The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.",
"Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.",
"The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.",
"Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.",
"Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.",
"The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound that binds to a polypeptide of the invention is identified.",
"The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies.",
"In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products.",
"Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.",
"The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in the sequence listing).",
"If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.",
"4.DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.",
"The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.",
"According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.",
"Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.",
"The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.",
"The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing.",
"For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.",
"Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules.",
"The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.",
"The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.",
"The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.",
"The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.",
"PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.",
"Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.",
"The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.",
"As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.",
"EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).",
"One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.",
"The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.",
"These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.",
"In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U).",
"It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil).",
"Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.",
"The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.",
"The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.",
"Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.",
"Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.",
"A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.",
"Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NO: 1-8051.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.",
"(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).",
"They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.",
"Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.",
"The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-8051.The sequence information can be a segment of any one of SEQ ID NO: 1-8051 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-8051.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.",
"Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes.",
"Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.",
"The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.",
"Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.",
"The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).",
"The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.",
"The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.",
"The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.",
"The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.",
"For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.",
"While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.",
"repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.",
"The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.",
"A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.",
"The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.",
"A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.",
"The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.",
"Preferably the peptide is from about 5 to about 200 amino acids.",
"To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.",
"The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.",
"The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.",
"The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence.",
"The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence.",
"The peptide may have been produced by processing in the cell which removes any leader/signal sequence.",
"The mature protein portion may or may not include an initial methionine residue.",
"The methionine residue may be removed from the protein during processing in the cell.",
"The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.",
"The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.",
"The term “variant”(or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.",
"Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.",
"Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.",
"Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.",
"Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.",
"Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.",
"“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.",
"For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.",
"“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.",
"The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.",
"Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.",
"Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.",
"For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.",
"Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.",
"For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.",
"The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.",
"In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).",
"The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.",
"In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.",
"The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.",
"The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.",
"“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.",
"As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.",
"Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.",
"The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.",
"An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.",
"Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.",
"Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.",
"This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.",
"The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.",
"Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.",
"This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.",
"Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.",
"The cells can be prokaryotic or eukaryotic.",
"The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.",
"“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.",
"“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.",
"“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.",
"Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.",
"Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.",
"(1998) Annu.",
"Rev.",
"Immunol.",
"16:27-55) Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.",
"Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.",
"The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.",
"Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).",
"Other exemplary hybridization conditions are described herein in the examples.",
"In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).",
"As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.",
"Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).",
"Such a sequence is said to have 65% sequence identity to the listed sequence.",
"In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).",
"Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.",
"Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.",
"Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% identity, more preferably at least about 98% sequence identity, and most preferably at least about 99% sequence identity.",
"For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.",
"For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.",
"Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.",
"(1990) Methods Enzymol.",
"183:626-645).",
"Identity between sequences can also be determined by other methods known in the art, e.g.",
"by varying hybridization conditions.",
"The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.",
"The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.",
"The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.",
"The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.",
"As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.",
"UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.",
"The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.",
"The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.",
"As described above, a UMF will increase the frequency of uptake of a linked marker sequence.",
"Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.",
"4.2 Nucleic Acids of the Invention Nucleotide sequences of the invention are set forth in the Sequence Listing.",
"The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-8051; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 8052-16102; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polypeptides of any one of SEQ ID NO: 8052-16102.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-8051; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 8052-16102.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.",
"The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.",
"The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.",
"The present invention also provides genes corresponding to the cDNA sequences disclosed herein.",
"The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.",
"Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.",
"Further 5′ and 3′ sequence can be obtained using methods known in the art.",
"For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-8051 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-8051 or a portion thereof as a probe.",
"Alternatively, the polynucleotides of SEQ ID NO: 1-8051 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.",
"The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.",
"The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.",
"The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.",
"Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99%, sequence identity to a polynucleotide recited above.",
"Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-8051, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.",
"Fragments of, e.g.",
"15, 17, or 20 nucleotides or more that are selective for (i.e.",
"specifically hybridize to any one of the polynucleotides of the invention) are contemplated.",
"Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.",
"The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.",
"Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-8051, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-8051 with a sequence from another isolate of the same species.",
"Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.",
"In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.",
"The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NO: 1-8051 can be obtained by searching a database using an algorithm or a program.",
"Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol.",
"Evol.",
"36 290-300 (1993) and Altschul S. F. et al.",
"J. Mol.",
"Biol.",
"21:403-410 (1990)).",
"Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.",
"Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.",
"Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.",
"The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.",
"The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.",
"These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.",
"There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.",
"Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.",
"These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).",
"Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.",
"Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.",
"Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.",
"Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.",
"Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.",
"In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.",
"This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.",
"In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).",
"A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.",
"10:6487-6500 (1982).",
"PCR may also be used to create amino acid sequence variants of the novel nucleic acids.",
"When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.",
"PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.",
"The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.",
"A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.",
"Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.",
"Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.",
"Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.",
"The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.",
"The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.",
"Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.",
"In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-8051, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.",
"Also included are the cDNA inserts of any of the clones identified herein.",
"A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.",
"(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).",
"Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.",
"Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.",
"In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.",
"Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.",
"A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.",
"The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof or any other polynucleotides of the invention.",
"In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-8051 or a fragment thereof is inserted, in a forward or reverse orientation.",
"In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.",
"Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.",
"The following vectors are provided by way of example.",
"Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).",
"Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).",
"The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.",
"19, 4485-4490 (1991), in order to produce the protein recombinantly.",
"Many suitable expression control sequences are known in the art.",
"General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).",
"As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.",
"Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.",
"Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.",
"Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.",
"Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.",
"Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.",
"Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.",
"The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.",
"Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.",
"Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.",
"The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.",
"Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.",
"As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).",
"Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).",
"These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.",
"Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.",
"Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.",
"Polynucleotides of the invention can also be used to induce immune responses.",
"For example, as described in Fan et al., Nat.",
"Biotech.",
"17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.",
"The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.",
"4.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-8051, or fragments, analogs or derivatives thereof.",
"An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.",
"In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.",
"Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 8052-16102 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-8051 are additionally provided.",
"In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.",
"The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.",
"In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.",
"The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).",
"Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-8051), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.",
"The antisense nucleic acid molecule can be complementary to the entire coding region of a mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of a mRNA.",
"For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a mRNA.",
"An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.",
"An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.",
"For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.",
"Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.",
"Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).",
"The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.",
"The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.",
"An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.",
"Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.",
"For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.",
"The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.",
"To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.",
"In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule.",
"An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al.",
"(1987) Nucleic Acids Res 15: 6625-6641).",
"The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.",
"(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.",
"(1987) FEBS Lett 215: 327-330).",
"4.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.",
"Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as a mRNA, to which they have a complementary region.",
"Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave a mRNA transcripts to thereby inhibit translation of a mRNA.",
"A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-8051).",
"For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an mRNA of SEQ ID NO: 1-8051 (see, e.g., Cech et al.",
"U.S. Pat.",
"No.",
"4,987,071; and Cech et al.",
"U.S. Pat.",
"No.",
"5,116,742).",
"Alternatively, polynucleotides of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.",
"See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.",
"See generally, Helene.",
"(1991) Anticancer Drug Des.",
"6: 569-84; Helene.",
"et al.",
"(1992) Ann.",
"N.Y. Acad.",
"Sci.",
"660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.",
"For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.",
"(1996) Bioorg Med Chem 4: 5-23).",
"As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.",
"DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.",
"The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.",
"The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.",
"(1996) above; Perry-O'Keefe et al.",
"(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.",
"For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.",
"PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.",
"(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.",
"(1996), above; Perry-O'Keefe (1996), above).",
"In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.",
"For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.",
"Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.",
"PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).",
"The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.",
"(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.",
"(1989) Nucl Acid Res 17: 5973-88).",
"PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.",
"(1996) above).",
"Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.",
"See, Petersen et al.",
"(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.",
"Natl.",
"Acad.",
"Sci.",
"U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.",
"Natl.",
"Acad.",
"Sci.",
"84:648-652; PCT Publication No.",
"W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.",
"W089/10134).",
"In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.",
"Res.",
"5:539-549).",
"To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.",
"4.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.",
"For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.",
"The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.",
"Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide.",
"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels.",
"The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences.",
"See, for example, PCT International Publication No.",
"WO94/12650, PCT International Publication No.",
"WO92/20808, and PCT International Publication No.",
"WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.",
"If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.",
"The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.",
"Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).",
"The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.",
"Any host/vector system can be used to express one or more of the ORFs of the present invention.",
"These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.",
"The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.",
"Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.",
"Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.",
"Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.",
"Various mammalian cell culture systems can also be employed to express recombinant protein.",
"Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).",
"Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.",
"Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.",
"DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.",
"Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.",
"Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.",
"Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.",
"Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.",
"Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.",
"Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.",
"Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.",
"If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.",
"Such covalent attachments may be accomplished using known chemical or enzymatic methods.",
"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.",
"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.",
"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.",
"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.",
"These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.",
"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.",
"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.",
"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.",
"Here, the naturally occurring sequences are deleted and new sequences are added.",
"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.",
"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.",
"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.",
"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.",
"No.",
"5,272,071 to Chappel; U.S. Pat.",
"No.",
"5,578,461 to Sherwin et al.",
"; International Application No.",
"PCT/US92/09627 (WO93/09222) by Selden et al.",
"; and International Application No.",
"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.",
"4.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 8052-16102 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-8051 or the corresponding full length or mature protein.",
"Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NO: 1-8051 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.",
"The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 8052-16102 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.",
"Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 8052-16102.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.",
"Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.",
"Chem.",
"Soc.",
"114, 9245-9253 (1992), both of which are incorporated herein by reference.",
"Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.",
"The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.",
"The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.",
"The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.",
"The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.",
"Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided.",
"In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.",
"Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.",
"The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.",
"By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.",
"Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.",
"A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.",
"At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.",
"The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.",
"This technique is particularly useful in producing small peptides and fragments of larger polypeptides.",
"Fragments are useful, for example, in generating antibodies against the native polypeptide.",
"Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.",
"The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.",
"As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.",
"One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.",
"The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.",
"For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.",
"The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.",
"Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.",
"In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.",
"One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.",
"These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.",
"See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.",
"Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.",
"The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.",
"These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.",
"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.",
"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.",
"In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.",
"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 8052-16102.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.",
"The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.",
"For example, modifications in the peptide or DNA sequence can be made by those skilled in the art using known techniques.",
"Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.",
"For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.",
"Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.",
"No.",
"4,518,584).",
"Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.",
"Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.",
"This type of analysis determines the importance of the substituted amino acid(s) in biological activity.",
"Regions of the protein that are important for protein function may be determined by the eMATRIX program.",
"Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.",
"Such modifications are encompassed by the present invention.",
"The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.",
"Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.",
"1555 (1987), incorporated herein by reference.",
"As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.",
"The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.",
"The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.",
"Alternatively, the protein of the invention may also be expressed in a form that will facilitate purification.",
"For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His-tag.",
"Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.",
"), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.",
"The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.",
"One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).",
"Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.",
"Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.",
"The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).",
"This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted.",
"Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.",
"Such analogs may exhibit improved properties such as activity and/or stability.",
"Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells.",
"Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids.",
"Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.",
"4.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.",
"Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.",
"Biol.",
"215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.",
"vol.",
"25, pp.",
"3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.",
"Biol., Vol.",
"6, pp.",
"219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol.",
"4, pp.",
"202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol.",
"26 (1), pp.",
"320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol.",
"Biol, 157, pp.",
"105-31 (1982), incorporated herein by reference).",
"The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.",
"NCB NLM NIH Bethesda, Md.",
"20894; Altschul, S., et al., J. Mol.",
"Biol.",
"215:403-410 (1990).",
"4.7 Chimeric and Fusion Proteins The invention also provides chimeric or fusion proteins.",
"As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.",
"Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.",
"In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.",
"In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.",
"Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.",
"The polypeptide can be fused to the N-terminus or C-terminus.",
"For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.",
"In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.",
"In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprises one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.",
"The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.",
"The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.",
"Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e,g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival.",
"Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.",
"A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.",
"For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.",
"In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.",
"Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.",
"(eds.)",
"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).",
"Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).",
"A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.",
"4.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.",
"The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.",
"Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).",
"See, for example, Anderson, Nature, supplement to vol.",
"392, no.",
"6679, pp.",
"25-20 (1998).",
"For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).",
"Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).",
"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.",
"Treated cells can then be introduced in vivo for therapeutic purposes.",
"Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.",
"It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.",
"Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.",
"Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.",
"The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.",
"These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.",
"Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.",
"Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.",
"The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.",
"See, for example, PCT International Publication No.",
"WO 94/12650, PCT International Publication No.",
"WO 92/20808, and PCT International Publication No.",
"WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.",
"If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.",
"In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.",
"As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.",
"Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.",
"Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.",
"These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.",
"The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.",
"Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.",
"Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.",
"Here, the naturally occurring sequences are deleted and new sequences are added.",
"In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.",
"The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.",
"Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.",
"The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.",
"No.",
"5,272,071 to Chappel; U.S. Pat.",
"No.",
"5,578,461 to Sherwin et al.",
"; International Application No.",
"PCT/US92/09627 (WO93/09222) by Selden et al.",
"; and International Application No.",
"PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.",
"4.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].",
"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.",
"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.",
"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.",
"No.",
"5,557,032, incorporated herein by reference.",
"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.",
"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.",
"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.",
"No.",
"5,489,743 and PCT Publication No.",
"WO94/28122, incorporated herein by reference.",
"Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.",
"Inactivation can be carried out using homologous recombination methods described above.",
"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.",
"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.",
"The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide.",
"Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.",
"In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].",
"Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.",
"Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.",
"Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.",
"No.",
"5,557,032, incorporated herein by reference.",
"Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.",
"Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.",
"Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.",
"No.",
"5,489,743 and PCT Publication No.",
"WO94/28122, incorporated herein by reference.",
"Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.",
"Inactivation can be carried out using homologous recombination methods described above.",
"Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.",
"The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.",
"4.10 Uses and Biological Activity The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.",
"Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).",
"The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.",
"Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.",
"Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.",
"The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.",
"4.10.1 Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.",
"The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.",
"Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.",
"The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.",
"Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.",
"Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.",
"Methods for performing the uses listed above are well known to those skilled in the art.",
"References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.4.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.",
"Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.",
"In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.",
"In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.",
"4.10.3 Cytokine and Cell Proliferation/Differentiation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.",
"The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.",
"Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.",
"137:3494-3500, 1986; Bertagnolli et al., J. Immunol.",
"145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.",
"149:3778-3783, 1992; Bowman et al., I. Immunol.",
"152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.",
"J. E. e.a.",
"Coligan eds.",
"Vol 1 pp.",
"3.12.1-3.12.14, John Wiley and Sons, Toronto.",
"1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.",
"J. E. e.a.",
"Coligan eds.",
"Vol 1 pp.",
"6.8.1-6.8.8, John Wiley and Sons, Toronto.",
"1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.",
"J. E. e.a.",
"Coligan eds.",
"Vol 1 pp.",
"6.3.1-6.3.12, John Wiley and Sons, Toronto.",
"1991; deVries et al., J. Exp.",
"Med.",
"173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6-Nordan, R. In Current Protocols in Immunology.",
"J. E. Coligan eds.",
"Vol 1 pp.",
"6.6.1-6.6.5, John Wiley and Sons, Toronto.",
"1991; Smith et al., Proc.",
"Natl.",
"Aced.",
"Sci.",
"U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.",
"In Current Protocols in Immunology.",
"J. E. Coligan eds.",
"Vol 1 pp.",
"6.15.1 John Wiley and Sons, Toronto.",
"1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.",
"In Current Protocols in Immunology.",
"J. E. Coligan eds.",
"Vol 1 pp.",
"6.13.1, John Wiley and Sons, Toronto.",
"1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 77:6091-6095, 1980; Weinberger et al., Eur.",
"J. Immuni.",
"11:405-411, 1981; Takai et al., J. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988.4.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.",
"Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.",
"The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.",
"It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein I-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).",
"Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.",
"Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations.",
"This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.",
"Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.",
"Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.",
"No.",
"5,690,926).",
"Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.",
"This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.",
"These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.",
"These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.",
"Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.",
"For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.",
"The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.",
"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.",
"In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.",
"Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.",
"A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.",
"The selectable marker allows only cells of the desired type to survive.",
"For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.",
"Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.",
"Lanza et al., Academic Press (1997)).",
"Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.",
"In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.",
"Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.",
"Proc.",
"Natl.",
"Acad.",
"Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.",
"The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.",
"as described by Bernstein et al., Blood, 77: 2316-2321 (1991).",
"4.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.",
"Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.",
"in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.",
"Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.",
"Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.",
"Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.",
"In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.",
"In Culture of Hematopoietic Cells.",
"R. I. Freshney, et al.",
"eds.",
"Vol pp.",
"139-162, Wiley-Liss, Inc., New York, N.Y. 1994.4.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.",
"A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.",
"Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.",
"De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.",
"A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.",
"Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)",
"mediated by inflammatory processes may also be possible using the composition of the invention.",
"Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.",
"Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.",
"Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.",
"De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.",
"The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.",
"The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.",
"The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.",
"The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.",
"for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.",
"More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.",
"Further conditions that may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.",
"Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.",
"Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.",
"Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.",
"Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.",
"A polypeptide of the present invention may also exhibit angiogenic activity.",
"A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.",
"A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.",
"Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.",
"WO95/16035 (bone, cartilage, tendon); International Patent Publication No.",
"WO95/05846 (nerve, neuronal); International Patent Publication No.",
"WO91/07491 (skin, endothelium).",
"Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps.",
"71-112 (Maibach, H. I. and Rovee, D. T., eds.",
"), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz J.",
"Invest.",
"Dermatol 71:382-84 (1978).",
"4.10.7 Immune Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such activities.",
"A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.",
"These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.",
"More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.",
"and various fungal infections such as candidiasis.",
"Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.",
"include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.",
"Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.",
"Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.",
"The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch.",
"Toxocol.",
"73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.",
"Environ.",
"Health 53: 563-79).",
"Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.",
"Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.",
"The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.",
"Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.",
"Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.",
"Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.",
"Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).",
"For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.",
"Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.",
"The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.",
"Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.",
"Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.",
"To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.",
"The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.",
"Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.",
"Natl.",
"Acad.",
"Sci USA, 89:11102-11105 (1992).",
"In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.",
"846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.",
"Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.",
"Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self-tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.",
"Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.",
"Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.",
"Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.",
"The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.",
"Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.",
"840-856).",
"Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.",
"Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.",
"For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.",
"Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.",
"Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.",
"The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.",
"A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.",
"In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.",
"Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.",
"Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.",
"Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.",
"The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.",
"128:1968-1974, 1982; Handa et al., J. Immunol.",
"135:1564-1572, 1985; Takai et al., I. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.",
"153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.",
"144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.",
"J. E. e.a.",
"Coligan eds.",
"Vol 1 pp.",
"3.8.1-3.8.16, John Wiley and Sons, Toronto.",
"1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.",
"137:3494-3500, 1986; Takai et al., J. Immunol.",
"140:508-512, 1988; Bertagnolli et al., J. Immunol.",
"149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.",
"134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.",
"Nat.",
"Acad.",
"Sci.",
"USA 88:7548-7551, 1991.4.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.",
"A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.",
"Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).",
"Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.",
"Administration of sufficient amounts of other inhibins can induce infertility in these mammals.",
"Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.",
"See, for example, U.S. Pat.",
"No.",
"4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.",
"The activity of a polypeptide of the invention may, among other means, be measured by the following methods.",
"Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 83:3091-3095, 1986.4.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.",
"Chemotactic or chemokinetic compositions (e.g.",
"proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.",
"For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.",
"A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.",
"Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.",
"Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.",
"Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.",
"Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.",
"J. Clin.",
"Invest.",
"95:1370-1376, 1995; Lind et al.",
"APMIS 103:140-146, 1995; Muller et al Eur.",
"J. Immunol.",
"25:1744-1748; Gruber et al.",
"J. of Immunol.",
"152:5860-5867, 1994; Johnston et al.",
"J. of Immunol.",
"153:1762-1768, 1994.4.10.10 Hemostatic and Thrombolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.",
"Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.",
"A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).",
"Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.",
"Pharmacol.",
"26:131-140, 1986; Burdick et al., Thrombosis Res.",
"45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.4.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.",
"Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.",
"For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.",
"Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.",
"Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.",
"Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.",
"Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.",
"Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.",
"Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.",
"reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.",
"The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.",
"An anticancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.",
"The use of anti-cancer cocktails as a cancer treatment is routine.",
"Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.",
"In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.",
"There are hereditary conditions and/or environmental situations (e.g.",
"exposure to carcinogens) known in the art that predispose an individual to developing cancers.",
"Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.",
"In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.",
"These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.",
"Can.",
"Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.",
"J. Dev.",
"Biol., 40: 1189-97 (1999) and Li et al., Clin.",
"Exp.",
"Metastasis, 17:423-9 (1999), respectively.",
"Suitable tumor cells lines are available, e.g.",
"from American Type Tissue Culture Collection catalogs.",
"4.10.12 Receptor/Ligand Activity A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.",
"A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.",
"Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.",
"Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.",
"A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.",
"The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology; Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.",
"Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 84:6864-6868, 1987; Bierer et al., J. Exp.",
"Med.",
"168:1145-1156, 1988; Rosenstein et al., J. Exp.",
"Med.",
"169:149-160 1989; Stoltenborg et al., J. Immunol.",
"Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).",
"Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.",
"Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.",
"The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or toxin molecules by conventional methods.",
"(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.",
"182 (1990) Academic Press, Inc. San Diego).",
"Examples of radioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.",
"Examples of toxins include, but are not limited, to ricin.",
"4.10.13 Drug Screening This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.",
"The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.",
"One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.",
"Drugs are screened against such transformed cells in competitive binding assays.",
"Such cells, either in viable or fixed form, can be used for standard binding assays.",
"One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.",
"Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.",
"Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.",
"The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.",
"Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.",
"For a review, see Science 282:63-68 (1998).",
"Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.",
"Of particular interest are peptide and oligonucleotide combinatorial libraries.",
"Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.",
"For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.",
"Opin.",
"Biotechnol.",
"8:701-707 (1997).",
"For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol.",
"Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1 (1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).",
"Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.",
"The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.",
"In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.",
"The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.",
"The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.",
"Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.",
"4.10.14 Assay for Receptor Activity The invention also provides methods to detect specific binding of a polypeptide e.g.",
"a ligand or a receptor.",
"The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.",
"For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.",
"As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.",
"There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.",
"Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.",
"The responses of the two cell populations to the addition of ligands(s) are then compared.",
"Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).",
"As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.",
"The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.",
"For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.",
"The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.",
"Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.",
"phosphorylation.",
"Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.",
"4.10.15 Anti-Inflammatory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.",
"The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.",
"Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.",
"Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.",
"4.10.16 Leukemias Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.",
"Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).",
"4.10.17 Nervous System Disorders Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.",
"Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.",
"Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.",
"For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.",
"Such effects may be measured by any method known in the art.",
"In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.",
"(1990, J. Neurosci.",
"10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.",
"(1980, Exp.",
"Neurol.",
"70:65-82) or Brown et al.",
"(1981, Ann.",
"Rev.",
"Neurosci.",
"4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.",
"In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).",
"4.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.",
"4.10.19 Identification of Polymorphisms The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.",
"Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.",
"For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.",
"Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.",
"For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.",
"Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).",
"In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.",
"Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.",
"The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.",
"In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.",
"Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.",
"4.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis are determined in an experimental animal model system.",
"The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int.",
"Arch.",
"Allergy Appl.",
"Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).",
"The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.",
"The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.",
"The control consists of administering PBS only.",
"The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.",
"An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.",
"4.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.",
"Examples of therapeutic applications include, but are not limited to, those exemplified herein.",
"4.11.1 Example One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.",
"While the mode of administration is not particularly important, parenteral administration is preferred.",
"An exemplary mode of administration is to deliver an intravenous bolus.",
"The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician.",
"It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.",
"Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.",
"For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.",
"Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.",
"The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.",
"The preparation of such solutions is within the skill of the art.",
"4.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.",
"Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.",
"The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).",
"The characteristics of the carrier will depend on the route of administration.",
"The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.",
"In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.",
"These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.",
"The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.",
"Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.",
"Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).",
"A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.",
"As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.",
"As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).",
"Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.",
"A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.",
"When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.",
"When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.",
"In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.",
"Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.",
"When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.",
"If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.",
"4.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.",
"Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.",
"Intravenous administration to the patient is preferred.",
"Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.",
"In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.",
"Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.",
"The liposomes will be targeted to and taken up selectively by the afflicted tissue.",
"The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.",
"The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.",
"Preferably for wound treatment, one administers the therapeutic compound directly to the site.",
"Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.",
"Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.",
"4.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.",
"These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.",
"Proper formulation is dependent upon the route of administration chosen.",
"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.",
"When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.",
"The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.",
"When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.",
"The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.",
"When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active-ingredient of the present invention.",
"When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.",
"The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.",
"A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.",
"The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.",
"For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.",
"For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.",
"Such penetrants are generally known in the art.",
"For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.",
"Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.",
"Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.",
"Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).",
"If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.",
"Dragee cores are provided with suitable coatings.",
"For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.",
"Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.",
"Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.",
"The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.",
"In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.",
"In addition, stabilizers may be added.",
"All formulations for oral administration should be in dosages suitable for such administration.",
"For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.",
"For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.",
"In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.",
"Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.",
"The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.",
"Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.",
"The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.",
"Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.",
"Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.",
"Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.",
"Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.",
"Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.",
"Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.",
"The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.",
"In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.",
"Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.",
"Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.",
"A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.",
"The co-solvent system may be the VPD co-solvent system.",
"VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.",
"The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.",
"This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.",
"Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.",
"Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g.",
"polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.",
"Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.",
"Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.",
"Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.",
"Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.",
"Various types of sustained-release materials have been established and are well known by those skilled in the art.",
"Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.",
"Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.",
"The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.",
"Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.",
"Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.",
"Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.",
"The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens.",
"The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.",
"B-lymphocytes will respond to antigen through their surface immunoglobulin receptor.",
"T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.",
"MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.",
"The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.",
"Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.",
"The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.",
"Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.",
"Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.",
"Nos.",
"4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.",
"The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.",
"Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.",
"Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.",
"Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.",
"It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.",
"For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.",
"When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.",
"Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.",
"Topical administration may be suitable for wound healing and tissue repair.",
"Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.",
"Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.",
"Such matrices may be formed of materials presently in use for other implanted medical applications.",
"The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.",
"The particular application of the compositions will define the appropriate formulation.",
"Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.",
"Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.",
"Further matrices are comprised of pure proteins or extracellular matrix components.",
"Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.",
"Matrices may be comprised of combinations of any of the above-mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.",
"The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.",
"Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.",
"In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.",
"A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).",
"Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).",
"The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.",
"In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.",
"These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).",
"The therapeutic compositions are also presently valuable for veterinary applications.",
"Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention.",
"The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.",
"The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.",
"For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.",
"Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.",
"Polynucleotides of the present invention can also be used for gene therapy.",
"Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.",
"Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).",
"Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.",
"Treated cells can then be introduced in vivo for therapeutic purposes.",
"4.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.",
"More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.",
"Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.",
"For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.",
"For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.",
"For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.",
"Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).",
"The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.",
"The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.",
"The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.",
"The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.",
"The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.",
"See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.",
"1 p. 1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).",
"The MEC will vary for each compound but can be estimated from in vitro data.",
"Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.",
"However, HPLC assays or bioassays can be used to determine plasma concentrations.",
"Dosage intervals can also be determined using MEC value.",
"Compounds should be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.",
"In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.",
"An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.",
"Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.",
"The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.",
"4.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.",
"The pack may, for example, comprise metal or plastic foil, such as a blister pack.",
"The pack or dispenser device may be accompanied by instructions for administration.",
"Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.",
"4.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.",
"The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.",
"Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library.",
"In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.",
"Certain classes have subclasses as well, such as IgG1, IgG2, and others.",
"Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.",
"Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.",
"An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.",
"The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.",
"An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of any of the full length proteins of the invention, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.",
"Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.",
"Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.",
"In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region on the surface of the protein of the invention, e.g., a hydrophilic region.",
"A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.",
"As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.",
"See, e.g., Hopp and Woods, 1981, Proc.",
"Nat.",
"Acad.",
"Sci USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.",
"Biol.",
"157: 105-142, each of which is incorporated herein by reference in its entirety.",
"Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.",
"A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.",
"Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).",
"Some of these antibodies are discussed below.",
"5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.",
"An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.",
"Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.",
"Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.",
"The preparation can further include an adjuvant.",
"Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.",
"), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.",
"Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).",
"The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.",
"Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.",
"Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.",
"14, No.",
"8 (Apr.",
"17, 2000), pp.",
"25-28).",
"5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.",
"In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.",
"MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.",
"Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).",
"In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.",
"Alternatively, the lymphocytes can be immunized in vitro.",
"The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.",
"Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.",
"The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.",
"59-103).",
"Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.",
"Usually, rat or mouse myeloma cell lines are employed.",
"The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.",
"For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.",
"Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.",
"More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.",
"Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.",
"51-63).",
"The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.",
"Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).",
"Such techniques and assays are known in the art.",
"The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.",
"Biochem., 107:220 (1980).",
"Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.",
"After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.",
"Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.",
"Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.",
"The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.",
"The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.",
"No.",
"4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).",
"The hybridoma cells of the invention serve as a preferred source of such DNA.",
"Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.",
"The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.",
"No.",
"4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.",
"Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.",
"5.13.2 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.",
"These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.",
"Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.",
"Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.",
"(See also U.S. Pat.",
"No.",
"5,225,539.)",
"In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.",
"Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.",
"In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.",
"The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.",
"Op.",
"Struct.",
"Biol., 2:593-596 (1992)).",
"5.133 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.",
"Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.",
"Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.",
"77-96).",
"Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.",
"77-96).",
"In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.",
"Biol., 227:381 (1991); Marks et al., J. Mol.",
"Biol., 222:581 (1991)).",
"Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.",
"Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.",
"This approach is described, for example, in U.S. Pat.",
"Nos.",
"5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et at.",
"(Bio/Technology 10, 779-783 (1992)); Lonberg et al.",
"(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.",
"Rev.",
"Immunol.",
"13 65-93 (1995)).",
"Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.",
"(See PCT publication WO94/02602).",
"The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.",
"The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.",
"An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.",
"The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.",
"The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.",
"Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.",
"An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.",
"No.",
"5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.",
"A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.",
"No.",
"5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.",
"The hybrid cell expresses an antibody containing the heavy chain and the light chain.",
"In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.4 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.",
"No.",
"4,946,778).",
"In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.",
"Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.",
"5.13.5 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.",
"In the present case, one of the binding specificities is for an antigenic protein of the invention.",
"The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.",
"Methods for making bispecific antibodies are known in the art.",
"Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).",
"Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.",
"The purification of the correct molecule is usually accomplished by affinity chromatography steps.",
"Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.",
"The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.",
"It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.",
"DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.",
"For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).",
"According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.",
"The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.",
"In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g tyrosine or tryptophan).",
"Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.",
"alanine or threonine).",
"This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.",
"Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.",
"F(ab′)2 bispecific antibodies).",
"Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.",
"For example, bispecific antibodies can be prepared using chemical linkage.",
"Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.",
"These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.",
"The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.",
"One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.",
"The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.",
"Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.",
"Shalaby et al., J. Exp.",
"Med.",
"175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.",
"Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.",
"The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.",
"Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.",
"For example, bispecific antibodies have been produced using leucine zippers.",
"Kostelny et al., J. Immunol.",
"148(5):1547-1553 (1992).",
"The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.",
"The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.",
"This method can also be utilized for the production of antibody homodimers.",
"The “diabody” technology described by Hollinger et al., Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.",
"The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.",
"Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.",
"Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.",
"See, Gruber et al., J. Immunol.",
"152:5368 (1994).",
"Antibodies with more than two valencies are contemplated.",
"For example, trispecific antibodies can be prepared.",
"Tutt et al., J. Immunol.",
"147:60 (1991).",
"Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.",
"Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.",
"CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.",
"Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.",
"These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.",
"Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).",
"5.13.6 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.",
"Heteroconjugate antibodies are composed of two covalently joined antibodies.",
"Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.",
"No.",
"4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).",
"It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.",
"For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.",
"Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.",
"No.",
"4,676,980.5.13.7 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.",
"For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.",
"The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).",
"See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.",
"Immunol., 148: 2918-2922 (1992).",
"Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.",
"Cancer Research, 53: 2560-2565 (1993).",
"Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.",
"See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).",
"5.13.8 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).",
"Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.",
"Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.",
"A variety of radionuclides are available for the production of radioconjugated antibodies.",
"Examples include 212Bi, 131I, 131In, 90Y, and 186Re.",
"Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).",
"For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).",
"Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.",
"See WO94/1 1026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.",
"4.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.",
"As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.",
"Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.",
"A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.",
"As used herein, “recorded” refers to a process for storing information on computer readable medium.",
"A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.",
"A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.",
"The choice of the data storage structure will generally be based on the means chosen to access the stored information.",
"In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.",
"The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.",
"A skilled artisan can readily adapt any number of data processor structuring formats (e.g.",
"text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.",
"By providing any of the nucleotide sequences SEQ ID NO: 1-8051 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NO: 1-8051 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.",
"Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.",
"The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.",
"Biol.",
"215:403-410 (1990)) and BLAZE (Brutlag et al., Comp.",
"Chem.",
"17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.",
"Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.",
"As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.",
"The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.",
"A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.",
"As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.",
"As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.",
"As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.",
"Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.",
"A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.",
"Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).",
"A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.",
"As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.",
"A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.",
"The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues.",
"However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.",
"As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.",
"There are a variety of target motifs known in the art.",
"Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.",
"Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).",
"4.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.",
"Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.",
"Acids Res.",
"6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem.",
"56:560, (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).",
"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.",
"Both techniques have been demonstrated to be effective in model systems.",
"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.",
"4.16 Diagnostic Assays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.",
"In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.",
"Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.",
"In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.",
"In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.",
"Conditions for incubating a nucleic acid probe or antibody with a test sample vary.",
"Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.",
"One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.",
"Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.",
"1 (1982), Vol.",
"2 (1983), Vol.",
"3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).",
"The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.",
"The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.",
"Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.",
"In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.",
"Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.",
"In detail, a compartment kit includes any kit in which reagents are contained in separate containers.",
"Such containers include small glass containers, plastic containers or strips of plastic or paper.",
"Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.",
"Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.",
"), and containers which contain the reagents used to detect the bound antibody or probe.",
"Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.",
"One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.",
"4.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).",
"See, e.g., Kunkel et al., U.S. Pat.",
"No.",
"5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.",
"4.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NO: 1-8051, or bind to a specific domain of the polypeptide encoded by the nucleic acid.",
"In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.",
"In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.",
"Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.",
"Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.",
"Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).",
"Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).",
"Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.",
"The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.",
"The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.",
"For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.",
"Alternatively, agents may be rationally selected or designed.",
"As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.",
"For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W.H.",
"Freeman, NY (1992), pp.",
"289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.",
"In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.",
"As described above, such agents can be randomly screened or rationally designed/selected.",
"Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.",
"One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.",
"Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.",
"Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.",
"Acids Res.",
"6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.",
"56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).",
"Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.",
"Both techniques have been demonstrated to be effective in model systems.",
"Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.",
"Agents that bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.",
"Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.",
"4.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.",
"The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-8051.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-8051 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.",
"Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.",
"PCR as described in U.S. Pat.",
"Nos.",
"4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.",
"Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.",
"The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.",
"Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.",
"Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.",
"The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.",
"The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.",
"These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.",
"The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.",
"Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981 f).",
"Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.",
"The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.",
"4.20 Preparation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.",
"Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.",
"One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.",
"Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.",
"Microbiol.",
"28 (6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol.",
"Cell Probes 3 (2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.",
"Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.",
"For example, Broude et al.",
"(1994) Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA 91 (8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.",
"Streptavidin-coated beads may be purchased from Dynal, Oslo.",
"Of course, this same linking chemistry is applicable to coating any surface with streptavidin.",
"Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).",
"Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.",
"Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.",
"CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.",
"CovaLink Modules may be purchased from Nunc Laboratories.",
"DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal.",
"Biochem.",
"198 (1) 13842).",
"The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991).",
"In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res.",
"11 (8) 6513-29).",
"This is beneficial as immobilization using only a single covalent bond is preferred.",
"The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.",
"To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.",
"It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.",
"More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.",
"at 95° C. and cooling on ice for 10 min.",
"Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIM7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.",
"Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIM7, is made fresh and 25 ul added per well.",
"The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).",
"It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.",
"This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.",
"The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.",
"Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.",
"An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.",
"For example, addressable laser-activated photodeprotection may be Fodor et al.",
"(1991) Science 251 (4995) 767-73, incorporated herein by reference.",
"Probes may also be immobilized on nylon supports as described by Van Ness et at (1991) Nucleic Acids Res.",
"19 (12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal.",
"Biochem.",
"169 (1) 104-8; all references being specifically incorporated herein.",
"To link an oligonucleotide to a nylon support, as described by Van Ness et al.",
"(1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.",
"One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91 (11) 5022-6, incorporated herein by reference).",
"These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).",
"These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.",
"A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.",
"4.21 Preparation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.",
"For example, Sambrook et al.",
"(1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).",
"DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.",
"Samples may be prepared or dispensed in multiwell plates.",
"About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.",
"The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.",
"(1989), shearing by ultrasound and NaOH treatment.",
"Low pressure shearing is also appropriate, as described by Schriefer et al.",
"(1990) Nucleic Acids Res.",
"18 (24) 7455-6, incorporated herein by reference).",
"In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.",
"A lever device allows controlled application of low to intermediate pressures to the cell.",
"The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.",
"One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.",
"(1992) Nucleic Acids Res.",
"20 (14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.",
"The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.",
"Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs).",
"Fitzgerald et al.",
"(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.",
"Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.",
"As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.",
"Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.",
"This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.",
"Phosphate groups must also be removed from genomic DNA by methods known in the art.",
"4.22 Preparation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.",
"Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.",
"By offset printing, a density of dots higher than the density of the wells is achieved.",
"One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.",
"By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.",
"Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.",
"Each of the subarrays may represent replica spotting of the same samples.",
"In one example, a selected gene segment may be amplified from 64 patients.",
"For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).",
"A plate for each of the 64 patients is prepared.",
"By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.",
"Subarrays may contain 64 samples, one from each patient.",
"Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.",
"Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.",
"a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.",
"A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.",
"The present invention is illustrated in the following examples.",
"Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.",
"Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.",
"The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.",
"Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.",
"Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.",
"All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.",
"5.0 EXAMPLES 5.1 Example 1 Novel Nucleic Acid Sequences Obtained From Various Libraries A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques.",
"The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts.",
"Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences.",
"The clones were clustered into groups of similar or identical sequences.",
"Representative clones were selected for sequencing.",
"In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol.",
"PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.",
"Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.",
"In some cases RACE (Rapid Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.",
"5.2 Example 2 Novel Contigs The novel contigs of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases.",
"The sequences for the resulting nucleic acid contigs are designated as SEQ ID NO: 1-8051 and are provided in the attached Sequence Listing.",
"The contigs were assembled using an EST sequence as a seed.",
"Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 115, gb pri 115, and UniGene version 103, and exons from public domain genomic sequences predicted by GenScan) that belong to this assemblage.",
"The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.",
"Further, the inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.",
"The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ ID NO: 1-8051) of the present invention are incorporated in the attached Sequence Listing.",
"A subset of the predicted polypeptide sequences contain an unknown amino acid; a stop codon; a possible nucleotide deletion; or a possible nucleotide insertion.",
"These sequences have also been shown in their entirety in Table 2.Table 2 also shows the corresponding start and stop nucleotide locations to each of SEQ ID NO: 1-8051.Table 2 also indicates the method by which the polypeptide was predicted.",
"Method A refers to a polypeptide obtained by using a software program called FASTY (available from http://fasta.bioch.virginia,edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W. R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).",
"Method B refers to a polypeptide obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C. Burge and S. Karlin, J. Mol.",
"Biol., 268:78-94 (1997), incorporated herein by reference).",
"Method C refers to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.",
"The nearest neighbor results for SEQ ID NO: 1-8051 were obtained by a BLASTX version 2.0al 19 MP-WashU search against Genpept release 123 and Geneseq release 200110 (Derwent), using BLAST algorithm.",
"The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-8051.The nearest neighbor results for SEQ ID NO: 1-8051, having identifiable function(s) are incorporated in the attached Sequence Listing.",
"Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.",
"Biol., Vol.",
"6 pp.",
"219-235 (1999) herein incorporated by reference), all the polypeptide sequences were examined to determine whether they had identifiable signature regions.",
"The attached Sequence Listing provides the results obtained by eMatrix analysis for each polypeptide as follows: the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.",
"Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol.",
"26 (1) pp.",
"320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains.",
"The attached Sequence Listing provides the results obtained by pFam analysis for each polypeptide, namely: the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.",
"Tables 1 and 2 follow.",
"Table 1 shows the various tissue sources of SEQ ID NO: 1-8051.Table 2 shows the start and stop nucleotides for the translated amino acid sequence for which each assemblage encodes.",
"Table 2 also provides a correlation between the amino acid sequences set forth in the Sequence Listing, the nucleotide sequences set forth in the Sequence Listing and the SEQ ID NO: in U.S. Ser.",
"No.",
"09/577,408.LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00001 Please refer to the end of the specification for access instructions.",
"LENGTHY TABLE REFERENCED HERE US20070060743A1-20070315-T00002 Please refer to the end of the specification for access instructions.",
"LENGTHY TABLE The patent application contains a lengthy table section.",
"A copy of the table is available in electronic form from the USPTO web site ().",
"An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3)."
]
] | Patent_10276817 |
[
[
"Automatic scanning system, shoppers scanpass system",
"The checkout system described herein is to show a system how to eliminate the lines at the regiters, to eliminate the removal of items from shopping cart in order to “ring” them at the checkout counters.",
"As clients lowering their goods into shopping carts, an auto scanner mounted at the top of the cart shall trigger a beam to scan the items which are lower into the cart.",
"Once client completes his shopping he than read the total amount of sale which can be verified at the top of the shopping cart.",
"The cart with all the intended purchased items is wheeled to a checkout areas, in which the total amount is paid either by cash or credit cards."
],
[
"1.Checkout system eliminating the lines at the register, so to eliminate the removal of items from shopping carts in order to “ring” them at the checkout counters As clients lowering their goods into shopping carts, an automatic scanner mounted at the top of the carts shall trigger a beam to scan the items which are lowered into the carts."
],
[
"Systematic design for most retail establishments which “ring” their items thru a checkout counter, where clients/shoppers can conduct the process of shopping without the standard waiting on lines in front of cash registers.",
"DESCRIPTION Items meanning any commodity which can be handled mostly by hand, as a self service method of purchase, within retail establishment such as supermarkets, retail chains stores, retail chain outlets of any type of marketing in a self-service system, can benefit greatly by using this system.",
"Any items which are for the most part are picked up from the selling offering area and are lower into shopping wagons by clients who wheel the wanted goods to the cash registers counters.",
"Shopping cart.",
"Specifically designed as carriers and for an automatic instant scanning for all items which are lowered into them.",
"An automatically scanning devise installed at the top brim, frame of shopping cart that electronically sensetive to the coded pricing tags attached to every item.",
"Once an item which carried the coded price tag is passed thru the top frame of the shopping cart and lower onto cart will instantally trigger the pricing scanner installed at their top Wagons O Matic Pricing Scanner.",
"Wagons, Carts are made of a type of materials, components and designed specifically to be used as a carriers and scanners.",
"The designed wagons/carts will described in a separate chapter.",
"Shoppers will have the ability to monitor each item by the unit pricing as well as the price itself thru a scanner window installed at the top of each shopping cart.",
"In the event of a reversed scanning such as a removal of an item from cart, the scanner installed at the top frame of cart will automatically deduct the amount from the total desired purchase.",
"(By pressing a botton which says CANCEL).",
"A processing checkout booths shall be constructed at the location designated to receive all carts filled with goods which are wheeled into specifically designed Cashiers scanning booths.",
"Each booth equipped with a remote scanner shall beam directly into the “total price” window located at the top of each shopping cart.",
"A total amount of sale shall be displayed and announced for each individual shopping cart.",
"Scanning booths shall be equipped with slots machines mounted on their walls to recieve payment for each purchase by cash, credit cards or store cards.",
"Customers shall be instructed to insert cards at appropriate slot machines.",
"Once payments accepted a gate/door will be opened for each shopping cart to be wheeled out into a packing booths or a similar packing counter.",
"At each gate/door a push button shall activate a trigger for the total sale reciept.",
"Each time door/gate is opened a cc tv system will monitor that individual shopping cart.",
"All Check—points of entries and exits shall be monitored by a cc tv systems.",
"An optional checkout cashier counters, “the old system” will still be operational for those who wish to be informed about various store applications as well as the privilege to converse with cashier in general.",
"The advantage use of the SCANPASS system is to reduce the long lines in front of each cash registers.",
"Shoppers will not leave unwanted items by registers.",
"There will be a dramatic reduction in shoplifting, probably not at all.",
"If store employees are engaged in theft, than any items not accounted for, unscanned will have to exit the back doors, an area which can be easily monitored at all hours.",
"Produce and related loose food items shall be packed and wrapped accordingly meaning by wanted desired volume, with a protected see thru wrap, bag which eliminate the direct contact of skin.",
"Each wrapped produced, food bag will carry a scanning tag.",
"and be protected against an outside contact of foreign bacterias, as well as human breath, speach, cough, hair, etc.",
"No item could be carried out of the store undetected unpaid or unaccounted for, since the scanned tag attached will always trigger the alarm systematic devise on the way out.",
"And if any attempt shall be made to carry the item out of the store thru the SCANPASS exit (gate), then simply the gate will not open.",
"Type of “labels” attached to all products will be of a design that applicable to the size and shape of the item, and be constructed as a wrap around ribbon with a thin cord inserted or attached to it, visible from all directions for scanning detections and if item relatively large in size than 2 or three scanning labels shall be attached.",
"Once all shopping carts are moved into the bagging counters which shall be supported by store employees or upon shoppers choice to bag their items themselves, A conveyor belt shall be constructed at that area for all carts to be wheeled on and eventually be carried to the the front store entrance.",
"SCANPASS System An Automatic SCANPASS System.",
"Specifically designed/Systematic design for retail establishments which “ring” their items thru a checkout counter, where clients/shoppers conduct the process of shopping without the standard of waiting on lines in front of the cash registers.",
"and use the specifically special designed built shopping cart/wagon equipped with an automatic scanner at the top of its frame to scan/trigger all items which are entering, lowering into wagons/carts.",
"SCANPASS To be registered as a trade mark.",
"And said title shall be recognized as a title for the system described herein on page 5 and on exhibit E. Wagon O Matic System Wagon O Matic Pricing Scanner.",
"Specifically designed shopping wagon equipped with an automatic scanner/trigger, mounted at the top of its frame to scan/trigger all item which are lowered into the wagon.",
"Wagon O Matic To be registered as a trade mark.",
"and said title shall be recognized as a title for the system described herein on page 6 and on exhibit F."
]
] | Patent_10292688 |
[
[
"Thermoplastic Hydraulic Composition, Formed Article Prepared From The Composition By Hydration- Hardening And Method For Preparing The Hydration- Hardened Former Article",
"The object of the present invention is to produce general machine parts, OA machine part, and the like which can be produced by injection molding and which have excellent mechanical properties, thermal resistance, dimension stability and processability.",
"The thermoplastic hydraulic composition of the present invention comprises 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.",
"It is characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.",
"It is characterized in that the hydraulic composition preferably is a mixed powder which comprises from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less."
],
[
"1.A thermoplastic hydraulic composition that is formed into a given shape and then is hardened by removal of at least a part of a thermoplastic resin from the inside, followed by the introduction of moisture, which is characterized by comprising 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition.",
"2.The thermoplastic hydraulic composition according to claim 1 further comprising at least one of from 1 to 200 parts by weight of a fiber and from 0.5 to 10.0 parts by weight of a releasing agent added.",
"3.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic composition is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.",
"4.The thermoplastic hydraulic composition according to claim 1, wherein the hydraulic powder is at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium alumino-ferrite, calcium phosphate, hemihydrate or anhydride gypsum and a powder of calcium oxide possessing a self-hardening property.",
"5.The thermoplastic hydraulic composition according to claim 1, wherein the non-hydraulic powder is at least one powder selected from a potassium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a flyash powder, a silica powder, a clay powder and a silica fume powder.",
"6.The thermoplastic hydraulic composition according to claim 1, wherein the thermoplastic resin is constituted of from 50 to 90 wt % of a thermoplastic low molecular compound and 50 to 10 wt % of a thermoplastic high molecular compound.",
"7.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic low molecular compound is selected from low melting point compounds of paraffin wax, montan wax, carnauba wax, a fatty acid ester, glycerite and modified wax.",
"8.The thermoplastic hydraulic composition according to claim 6, wherein the thermoplastic high molecular compound is a single substance or a copolymer of two or more substances selected from polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.",
"9.A hydration-hardened formed article characterized by being obtained by forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape, subsequently removing the thermoplastic resin from the inside of the resulting formed article, and further introducing moisture into the formed article to harden it.",
"10.A hydration-hardened formed article characterized by being obtained by forming the hydraulic composition according to claim 1 into a given shape, subsequently removing the thermoplastic resin from the resulting formed article completely or removing only a thermoplastic low molecular compound, and further introducing moisture into the inside of the formed article to harden it.",
"11.A method for producing a hydrogen-hardened formed article, the method being characterized in that it comprises forming a thermoplastic hydraulic composition comprising 100 parts by weight of a thermoplastic resin and from 500 to 1000 parts by weight of a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.",
"12.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic resin from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic high molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.",
"13.The method for producing a hydration-hardened formed article according to claim 11, wherein the degreasing step is carried out at a temperature of from 400 to 500° C. for a period of 12 hours or longer.",
"14.A method for producing a hydration-hardened formed article, the method being characterized in that it comprises forming the hydraulic composition according to claim 1 into a given shape to obtain an unhardened formed article, subsequently removing only a thermoplastic low molecular compound from the inside of the formed article by degreasing the unhardened formed article at a temperature not lower than the melting point of the thermoplastic low molecular compound, and then hydration-hardening the unhardened formed article by curing it to introduce moisture thereinto.",
"15.The method for producing a hydration-hardened formed article according to claim 14, wherein the degreasing step is carried out at a temperature of 270° C. or lower for a period of 12 hours or shorter.",
"16.The method for producing a hydration-hardened formed article according to claim 11, wherein said curing step is carried out at a temperature of 80° C. or higher by any one of normal pressure steam curing, high pressure steam curing and hot water curing, or in combination of the normal pressure steam curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing."
],
[
"<SOH> BACKGROUND ART <EOH>As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.",
"However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.",
"Hei-10-177099, Hei-10-177100 and Hei-11-233636.)",
"However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.",
"The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions."
],
[
"TECHNICAL FIELD The present invention relates to a thermoplastic hydraulic composition that allows an injection molding technique mainly employed for production of plastic molded articles and ceramic molded articles to be applicable even for hydraulic compositions, and to formed articles prepared from the hydraulic compositions by hydration-hardening.",
"The present invention relates more particularly to provide thermoplastic hydraulic compositions, formed articles thereof having excellent mechanical properties, thermal resistance, dimensional stability and processability, as well as being capable of being applied for, e.g., general machine parts and OA machine parts.",
"BACKGROUND ART As a material for machine parts, metallic materials have conventionally been used widely due to their superior material characteristics.",
"However, a recent technical progress has diversified needs for machine parts and machine parts in which non-metallic materials such as sintered ceramics or plastics have come to be used for making up for drawbacks of metallic materials The present inventors made a variety of investigations in order to comply with such demands and found that a hydraulic composition comprising a combination of a hydraulic powder, a non-hydraulic powder with an average particle diameter at least one order smaller than that of the hydraulic powder, a workability improver and a formability improver can be applied for machine parts such as paper sheet-feeding rollers through its pressure forming or extrusion forming; they filed patent applications directed thereto (Japanese Patent Application Nos.",
"Hei-10-177099, Hei-10-177100 and Hei-11-233636.)",
"However, these hydraulic compositions and methods for forming the same can be easily applied for machine parts of simple shapes but are difficult to be applied for machine parts of complex shapes.",
"The fact of being restricted in shape which can be produced problematically results directly in the restriction on the application range of the hydraulic compositions.",
"DISCLOSURE OF THE INVENTION The object of the present invention is to provide a thermoplastic hydraulic composition and a formed article prepared from the composition by hydration-hardening that make it possible to produce machine parts with a shape more complex than those obtained by conventional pressing or extrusion forming by allowing the above-mentioned hydraulic composition for machine parts to have a composition such that the resulting hydraulic composition can be injection molded and forming it.",
"The present inventors made intensive studies to achieve the above object and found that a thermoplastic hydraulic composition containing a thermoplastic resin, a hydraulic powder, a fiber and a releasing agent respectively in given amounts is of excellent injection moldability and that a molded article obtained by injection molding the thermoplastic hydraulic composition and hardening it by curing is excellent in mechanical properties, thermal properties and formability, thus achieving reproduction of a complex shape.",
"Specifically, the thermoplastic hydraulic composition of the present invention is characterized by containing 100 parts by weight of two types of thermoplastic resins differing in melting point and from 500 to 1000 parts by weight of a hydraulic composition, and is preferably characterized by further comprising from 1 to 200 parts by weight of a fiber and/or from 0.5 to 10.0 parts by weight of a releasing agent added.",
"The foregoing hydraulic composition is preferably a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder.",
"BEST MODE FOR CARRYING OUT OF THE INVENTION A more detailed description on the thermoplastic hydraulic composition of the present invention will be made below.",
"(1) Hydraulic Composition (1-1) In the thermoplastic hydraulic composition of the present invention, it is preferable that the hydraulic composition is used in an amount of from 500 to 1000 parts by weight, particularly from 650 to 750 parts by weight, based on 100 parts by weight of the two types of thermoplastic resins differing in melting point.",
"If less than 500 parts by weight, the strength of formed articles will be reduced, whereas when exceeding 1000 parts by weight, the amount of the thermoplastic resin incorporated will be reduced, resulting in deterioration of formability to cause a problem.",
"By the hydraulic composition is meant a powder that is hardened by water and preferably comprises at least one powder selected from portland cement, calcium silicate, calcium aluminate, calcium fluoroaluminate, calcium sulphoaluminate, calcium aluminoferrite, calcium phosphate, hemihydrate or anhydride gypsum, and a powder of calcium oxide possessing a self-hardening property.",
"The hydraulic powder preferably has an average particle diameter of from about 10 to about 40 μm, and preferably has a specific surface area by blaine of 2500 cm2/g or more from the viewpoint of securing a high strength of formed articles.",
"(1-2) The aforementioned hydraulic composition preferably is a mixed powder comprising from 50 to 90 wt % of a hydraulic powder and from 10 to 50 wt % of a non-hydraulic powder having an average particle diameter of 1/10 that of the hydraulic powder or less.",
"The aforementioned non-hydraulic powder indicates a powder that is incapable of hardening by itself even by its contact with water and includes a powder such that some of the components thereof are eluted in an alkaline or acid state or under a high-pressure steam atmosphere and then react with other eluted components to form a product.",
"Preferred as the non-hydraulic powder is at least one powder selected from a calcium hydroxide powder, a dihydrate gypsum powder, a calcium carbonate powder, a slag powder, a fly ash powder, a silica powder, a clay powder and a silica fume powder.",
"The non-hydraulic powder has an average particle diameter at least one order, preferably at least two order, smaller than that of the hydraulic powder, but no lower limit is set unless the effect of the present invention is damaged.",
"(2) The thermoplastic resin is a resin which can obtain a plasticity to a degree such that it can be formed by heating and is a resin which can be used for extrusion forming and injection molding.",
"In the present invention, a hydraulic composition is melt kneaded together with the thermoplastic resin at a temperature not lower than the softening point of the thermoplastic resin to be formed into a pellet form, which is used as a raw material for the subsequent injection molding.",
"The pellet-formed raw material is melted and kneaded again within a heating cylinder mounted inside an injection molding machine and is filled into a mold by the injection device.",
"The mixture of the hydraulic composition and the thermoplastic resin filled into the mold is formed into a formed article through cooling and hardening of the thermoplastic resin, which can be removed from the mold.",
"Hydraulic compositions are generally provided with a flowability by water, but it takes a long time for them to be released from a mold.",
"Therefore, forming methods such as injection molding can not be applied for them.",
"Moreover, if the hydraulic compositions come into contact with water, a hydration reaction will proceed and therefore articles defectively formed can not be recycled.",
"However, a mixture of the aforementioned thermoplastic resin and the hydraulic composition can afford a shape to the hydraulic composition without use of water and can realize releasing from a mold in a short time.",
"In addition, since no water is used in a stage of forming, no hydration reaction of the hydraulic composition is started and therefore the mixture can be recycled multiple times if it has not been cured yet.",
"Since the hydraulic composition of the present invention uses no water during its forming, it is necessary to supply moisture after the forming.",
"The spaces between particles of the hydraulic composition in a formed article after injection molding are filled with a thermoplastic resin.",
"Therefore, if the formed article is left in such a condition, the supply of water to the inside of the formed article will be inhibited, resulting in an insufficient hydration reaction of the formed article.",
"For this reason, it is necessary to remove a part or the whole of the resin from the inside of the formed article to form a channel for water supply.",
"When removing the resin from the inside of a formed article, it is desirable to use two types of resins differing in melting point when considering minimizing the dimensional change of the formed article.",
"Use of two types of resins differing in melting point makes it possible to injection mold the thermoplastic hydraulic composition and to obtain a formed article with a complex shape because a low molecular resin melts at a relatively low temperature and enables the hydraulic composition to fluidize easily.",
"Such a low molecular resin starts to decompose at a relatively low temperature (about 100° C. in the case of some resins), but a high molecular resin starts to decompose at a relatively high temperature (about 200° C. in the case of some resins).",
"Accordingly, if the removal of resin components at an intermediate temperature, only the low molecular resin is removed from the inside of the formed article and the vacant spaces resulting from the removal of the low molecular resin becomes channels for moisture supply, which facilitates a hydration reaction in the inside of the formed article.",
"On the other hand, the high molecular resin remaining in the inside of the formed article instead of being decomposed exists in the inside of the formed article to inhibit, e.g., dimensional change of the formed article.",
"In addition, when the resin components are removed at a temperature not lower than the melting point of the high molecular resin, all of the low molecular resin and the high molecular resin are removed and a completely inorganic hardened article will be obtained.",
"In such a case, since the low molecular resin has a melting point different than that of the high molecular resin, the low molecular resin is removed first and then the high molecular resin is removed.",
"Such removal of the resin components with a time lag can inhibit dimensional change of formed articles.",
"The thermoplastic low molecular compound with a lower melting point preferably has a molecular weight of 200 to several thousands.",
"The thermoplastic high molecular compound with a higher melting point preferably has a molecular weight of 10000 or more.",
"It is desirable to set upper limits based on appropriate selection from the viewpoint of kneadability and the like, because the increase of molecular weight have a great influence to kneadability.",
"Regarding blending of the thermoplastic resin, it is preferable to blend a low molecular compound in an amount of from 50 to 90 wt %, as described above, and most preferably from 55 to 65 wt %.",
"The amount of the thermoplastic high molecular compound is preferably from 50 to 10 wt %, and more preferably from 45 to 35 wt %.",
"If the amount of the thermoplastic low molecular compound is less than 50 wt %, the channel for moisture supply is reduced and hydration proceeds insufficiently.",
"On the other hand, if it is more than 90 wt %, the dimensional change during removal from a mold will adversely become large.",
"(2-1) As the aforementioned thermoplastic low molecular compound, it is preferable to use a single substance or two or more substances selected from low melting point compounds including paraffin wax, montan wax, carnauba wax, fatty acid esters, glycerite, and modified wax.",
"(2-2) As the thermoplastic high molecular compound, used are a single substance or two or more substances of polystyrene, an ethylene-vinyl acetate copolymer, an ethylene-ethyl acrylate copolymer, polypropylene, an acrylonitrile-butadiene-styrene copolymer, an acrylonitrile-styrene copolymer, methyl methacrylate, a vinyl chloride-vinyl chloride acetate copolymer, a vinyl chloride-vinyl acetate-maleic acid copolymer, a vinyl chloride-vinyl acetate copolymer, a vinyl chloride-vinylidene chloride copolymer, a vinyl chloride-acrylonitrile copolymer, an ethylene-vinyl chloride copolymer, a propylene-vinyl chloride copolymer, vinyl acetate, polyvinyl alcohol, polyamide, polyacetal, polyester, polycarbonate, polysulfone, polyether imide, polyamidoimide, and polyphenylene sulfide.",
"(3) The fiber is preferably added in an amount of from 1 to 200 parts by weight, and more preferably from 100 to 170 parts by weight based on 100 parts by weight of the thermoplastic resin.",
"When no fiber is added or when the amount of fiber added is less than 40 parts by weight, it may be impossible to improve the low impact strength and low tensile strength, which are inherent to hydraulic compositions.",
"If more than 200 parts by weight, it will have much influence to flowability to result in poor formability.",
"As the fiber, fibers of glass, carbon, aramid, polyamide, boron and the like and potassium titanate whisker can be used.",
"The length of the fiber is preferably from 0.1 to 6 mm, and more preferably from 3 to 6 mm.",
"The diameter of the fiber is preferably from 5 to 30 μm.",
"(4) The releasing agent is added preferably in an amount of from 0.5 to 10.0 parts by weight, and more preferably in an amount of from 2.0 to 3.0 parts by weight.",
"If less than 0.5 part by weight, the releasability from a mold will be poor.",
"If exceeding 10.0 parts by weight, the hydration reaction of the hydraulic composition may be inhibited.",
"As the releasing agent, stearic acid, stearyl alcohol, ethylene bisstearic acid amide, glycerol triester, glycerol monoester and the like can be used.",
"As other additives, alkylphenols, 2.6-tertialbutylparacresol, bisphenol A and the like for inhibiting oxidation of the thermoplastic resin can be used.",
"In addition, salicylic acid esters, benzene acid esters or the like may optionally be added as an ultraviolet absorber.",
"(5) Method for Producing Hardened Articles The hydration-hardened formed article of the present invention is a product produced by forming a thermoplastic hydraulic composition comprising a thermoplastic resin and a hydraulic composition into a given shape to obtain an unhardened formed article, subsequently heating it at a temperature not lower than the melting point of the thermoplastic resin to remove the thermoplastic resin from the inside of the formed article (this operation is also called degreasing), and curing the resulting formed article to introduce moisture, thereby hydration-hardening the formed article.",
"The thermoplastic resin may be made up of one compound, but it is preferable to use one made up of two types of substances including a thermoplastic low molecular compound and a thermoplastic high molecular compound as described previously.",
"When the thermoplastic resin is constituted of such two types of compounds, preferred is degreasing at a temperature between the melting point of the thermoplastic low molecular compound having a lower melting point and the melting point of the thermoplastic high molecular compound having a higher melting point (270° C. or less).",
"This will remove only the thermoplastic low molecular compound from the inside of a formed article.",
"Introduction of moisture using the vacant spaces resulting from the degreasing as channels for moisture supply will increase the hydration reaction rate to cause hydration hardening.",
"Specifically, injection molding, extrusion forming, pressure forming and the like can be employed as a forming method.",
"Furthermore, regarding a degreasing method, when DEP and an ethylene-vinyl acetate copolymer are used as a low molecular compound and a high molecular compound, respectively, decomposition of DEP starts at 100° C. and ends at 190° C.; whereas the ethylene-vinyl acetate copolymer starts to decompose at 210° C. Accordingly, it is possible to selectively remove only DEP by heating formed articles at 200° C. When curing at a temperature of from 400 to 500° C., it is possible to remove these thermoplastic resins completely.",
"The time required for resin removal can be set optionally depending, for example, on the kind, incorporation amount, pressure and temperature of the resin.",
"When the combination of the resins described in the above example is degreased at 200° C. under atmospheric pressure, the degreasing ratio will reach about 70% in 3 hours and about 100% in 12 hours.",
"Further, curing may be carried out by a curing method such as a normal pressure steam curing, a high pressure steam curing and a hot water curing conducted alone at a temperature not lower than 80° C., or in combination of the normal pressure curing and the hot water curing, or in combination of the high pressure steam curing and the hot water curing.",
"As a degreasing method, methods other than heating can be applied and are exemplified by solvent extraction and a removal method using reduced pressure.",
"The following is a description on embodiments of the present invention.",
"EXPERIMENTAL EXAMPLE 1 A thermoplastic hydraulic composition was prepared by blending an ethylene-vinyl acetate copolymer resin (EVA, molecular weight: 30000 to 50000) as a thermoplastic high molecular compound, carnauba wax (molecular weight: 300 to 500) as a thermoplastic low molecular compound and stearic acid as a releasing agent, respectively in the ratios given in Table 1, to a powder resulting from mixing of portland cement (average particle diameter 20 μm) as a hydraulic powder and fly ash and silica powder as non-hydraulic powders, and then was kneaded with a hot roll at 140° C. for 45 minutes to yield pellets.",
"Subsequently, an injection molded article 120 mm long, 10 mm wide and 3 mm thick was obtained by use of the pellets.",
"The resulting unhardened molded article was degreased through a degreasing step by heating (1): at 200° C. for 12 hours, 2) at 500° C. for 12 hours) and then a molded article was produced by autoclave curing (175° C., 7 hours, 8.8 atm).",
"The molded article was tested for its flexural strength, deflection temperature under load, and coefficient of linear expansion and was compared.",
"The results are shown in Table 1.<Test Method> HDT Test (Test of Deflection Temperature under Load) .",
".",
".",
"According to JIS K 7191-2, Method A A specimen 120 mm long, 10 mm wide and 3 mm thick was prepared.",
"The specimen was held between supporting points with an interval of 100 mm and the temperature thereof was raised at a constant rate while a flexural stress of 1.8 MPa was applied downward to its center.",
"The temperature at which the deflection reached a standard deflection was used as the deflection temperature under load.",
"Coefficient of linear expansion .",
".",
".",
"According to ASTM D-648 A φ3×20 mm specimen was prepared and the coefficient of linear expansion was measured in the temperature range of from 30 to 80° C. using a push-rod type measuring device.",
"TABLE 1 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 1 2 3 4 5 1 2 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 37.0 40.4 105 110 22.5 ** 8.6 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 80 80 38 39 150 ** 250 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.",
"The results shown above reveal that all the molded articles of the present invention are superior in mechanical strength in terms of flexural strength to those of the Comparative Examples.",
"In addition, it is shown that the molded articles of the present invention have higher deflection temperatures under load and therefore are superior also in thermal resistance.",
"Moreover, the fact that the molded articles of the present invention have smaller coefficients of linear expansion shows that those molded articles are superior also in dimension stability.",
"On the other hand, the molded articles of Comparative Examples are inferior in all of these properties to those of the present invention.",
"In Comparative Example 2, cracks were formed during curing.",
"EXPERIMENTAL EXAMPLE 2 Using the same materials as those used in Experimental Example 1, a thermoplastic hydraulic composition with the composition shown in Table 2 was kneaded while being heated to 150° C. with a hot roll and formed into a pellet form.",
"Subsequently, the resultant was formed into a 120 mm long, 10 mm wide, 3 mm thick specimen using an injection machine and then the unhardened molded article was degreased through a degreasing step by heating ((1): at 200° C. for 12 hours, (2) at 500° C. for 12 hours).",
"A molded article thereafter was produced by normal pressure steam curing (100° C., 7 hours) and then compared its flexural strength, deflection temperature under load, and coefficient of linear expansion in the same way as Experimental Example 1.The results are shown in Table 2.TABLE 2 Unit: part(s) by weight Compara- Compara- tive tive Example Example Example Example Example Example Example 6 7 8 9 10 3 4 Portland cement 80 80 80 80 80 80 80 Fly ash (average 10 10 10 10 10 10 10 particle diameter 1 to 2 μm) Silica powder 10 10 10 10 10 10 10 Carnauba wax 7.5 8.5 7.5 8.5 4.0 9.0 0 Ethylene-vinyl 3.5 3.5 3.5 3.5 6.0 0 9.0 acetate copolymer Stearic acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Carbon fiber 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Degreasing (1) (1) (2) (2) (1) (1) (1) method Flexural strength* 31.2 32.3 70.5 80.4 19.6 ** 6.0 (N/mm2) HDT(° C.) 180 178 >280 >280 95 ** 50 Coefficient of 100 100 50 48 179 ** 254 linear expansion (×10−7) *According to JIS K 7171 Bending test **Cracks were formed during curing.",
"The results shown above reveal that all the formed articles of the present invention are superior in mechanical strength measured as flexural strength to those of Comparative Examples.",
"Further, it is also shown that the formed articles of the present invention have higher deflection temperature under load and are superior in thermal resistance.",
"Furthermore, the fact that those formed article have small coefficients of linear expansion shows that those formed articles are also superior in dimensional stability.",
"On the other hand, the formed articles of the Comparative Examples are inferior in all of such properties to the formed articles of the present invention.",
"In Comparative Example 3, cracks were formed during curing.",
"As described above, the thermoplastic hydraulic composition of the present invention can be injection molded without addition of water.",
"When this composition is cured, general machine parts, OA machine parts and the like with excellent mechanical properties, thermal resistance, dimensional stability and processability can be produced.",
"By blending two types of thermoplastic resins differing in melting point and removing only a thermoplastic low molecular compound having a low melting point followed by curing, it is possible to promote the hydration reaction to the inside of formed articles and simultaneously to harden the formed articles while maintaining their shapes by the action of the remaining high molecular compounds having high melting points.",
"In such a manner, machine parts excellent in mechanical characteristics, thermal resistance, dimensional stability or the like can be produced.",
"Furthermore, curing after complete removal of the thermoplastic resin at higher temperatures makes it possible to further improve such properties of formed articles."
]
] | Patent_10296092 |
[
[
"Combinations of enzyme inhibitor-containing preparations and the use thereof",
"The invention comprises a process wherein the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ) and of T cells as well is inhibited by the simultaneous and combined inhibition of the enzyme activity (I) of alanyl aminopeptidase and of dipeptidyl peptidase IV; (II) of dipeptidyl peptidase IV and of the angiotensin-converting enzyme; (III) of dipeptidyl peptidase IV and of prolyl oligopeptidase; and (IV) of dipeptidyl peptidase IV and of X-Pro-aminopeptidase to an extent which cannot be achieved by an application of a single one of said enzyme inhibitors, even at a higher dosage.",
"Although the said inhibitors exercise an influence on the very same process finally, i.e.",
"the DNA synthesis and, thus, the proliferation of immune cells, this effect is not complete and is not long lasting when a single inhibitor is applied.",
"From the functional overlap of enzymatic activities results an additive/superadditive inhibitory effect on the DNA synthesis and proliferation by the simultaneous inhibition of more than one of the above enzymes, as our data show.",
"Our invention shows that the simultaneous application of substances inhibiting the above enzymes or of corresponding preparations and administration forms, respectively, is well suitable for a therapy of autoimmune diseases and chronic diseases with an inflammatory genesis as well as for a treatment of rejection episodes after a transplantation."
],
[
"1.Use of inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having the same substrate specificity (DP IV-analogous enzyme activity) in combination with inhibitors of alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro-aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and/or of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) for a superadditive inhibition of an activation, DNA synthesis and proliferation of human T lymphocytes and mononuclear cells.",
"2.Use according to claim 1, wherein the inhibitors of DP IV are preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivative), corresponding derivatives, preferably dipeptide phosphonic acid diaryl estates, dipeptide boronic acids (e.g.",
"Pro-boro-Pro) and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably NE-4-nitrobenzyloxycarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure.",
"3.Use according to claim 1, wherein amino acid amides, e.g., NE-4-nitrobenzyloxy-carbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative, are preferably employed as the DP IV inhibitor.",
"4.Use according to claim 1, wherein Actinonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phosphinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, preferably act as inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-L-proline, the corresponding L-proline methyl esters, (2S,3R)-HAMH/(2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof preferably act as inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof preferably act as inhibitors of ACE; Postatin, Eurystatin A or B, Na-protected peptide aldehydes, preferably benzyloxy-carbonyl-L-prolyl-L-prolinal or benzyloxycarbonyl-L-thioprolyl-L-thioprolinal, Na-protected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyano-pyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof preferably act as inhibitors of POP (PEP).",
"5.Use of inhibitor combinations of claim 1 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.",
"6.Use of inhibitor combinations of claim 1 for a suppression of transplant rejection and for a therapy of tumor diseases.",
"7.Pharmaceutical preparations, comprising inhibitors of dipeptidyl peptidase IV (DP IV) as well as of enzymes having DP IV-analogous enzyme activity in combination with inhibitors of any of the enzymes alanyl aminopeptidase (aminopeptidase N, APN) as well as of enzymes having the same substrate specificity (APN-analogous enzyme activity), of X-Pro aminopeptidase (aminopeptidase P, APP), of the angiotensin-converting enzyme (ACE) and of prolyl oligopeptidase (POP, prolyl endopeptidase, PEP) and in combination with per se known carrier, additive and/or auxiliary substances.",
"8.Pharmaceutical preparation according to claim 7, comprising preferably Xaa-Pro-dipeptides (Xaa=α-amino acid or side chain-protected derivatives) corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and salts thereof, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (Xaa=α-amino acid, n=0-10), corresponding derivatives and salts thereof or amino acid (Xaa)-amides, corresponding derivatives and salts thereof, wherein Xaa is an α-amino acid or a side chain-protected derivative, respectively, preferably Ne-4-nitrobenzyloxcarbonyl L-lysine, -L-proline, -L-tryptophane, -L-isoleucine, -L-valine, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine and derivatives thereof act as amide structure, as the inhibitors of DP IV.",
"9.Pharmaceutical preparation according to claim 7, comprising preferably amino acid amides, e.g.",
"Ne-4-nitrobenzyloxcarbonyl-L-lysine-thiazolidid, -pyrrolidid and -piperidid as well as the corresponding 2-cyanothiazolidid, 2-cyanopyrrolidid and 2-cyanopiperidid derivative as the inhibitors of DP IV.",
"10.Pharmaceutical preparation according to claim 7, comprising, as the inhibitors of APN, APP, ACE and POP (PEP), preferably Actnonin, Leuhistin, Phebestin, Amastin, Probestin, β-aminothiols, α-amino phosphinic acids, α-amino phoshinic acid derivatives, preferably D-Phe-ψ-[PO(OH)—CH2]-Phe-Phe and salts thereof, as the inhibitors of APN; Apstatin, (2S,3R)-HAMH-L-proline, (2S,3R)-HAPB-pyrrolidides, -thiazolidides (HAMH=3-amino-2-hydroxy-5-methyl hexanoyl, HAPB=3-amino-2-hydroxy-4-phenyl butanoyl) and salts thereof as the inhibitors of APP; Captopril, Enalapril, Lisinopril, Cilazopril and salts thereof as inhibitors of ACE; Postatin, Eurystatin A or B, Naprotected peptide aldehydes, preferably benzyloxycarbonyl-L-prolyl-L-prolinal or benzyloxcarbonyl-L-thioprolyl-L-thioprolinal, Naprotected amino acid (Xaa)-pyrrolidides or -thiazolidides (Xaa=α-amino acid, preferably L-alanine, L-valine, L-isoleucine) as well as the corresponding 2-cyanopyrrolidid or 2-cyanothiazolidid derivatives, substrate-analogous Na-protected peptide phosphonic acid diaryl esters or peptide diazo methyl ketones or peptide ammonium methyl ketones and salts thereof as inhibitors of POP (PEP).",
"11.Pharmaceutical preparation according to claim 7 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.",
"12.Pharmaceutical preparation according to claim 7 for a systemic use of the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramusclar, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.",
"13.Pharmaceutical preparation according to claim 7 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.",
"14.Use of inhibitor combinations of claim 2 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.",
"15.Use of inhibitor combinations of claim 2 for a suppression of transplant rejection and for a therapy of tumor diseases.",
"16.Use of inhibitor combinations of claim 3 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.",
"17.Use of inhibitor combinations of claim 3 for a suppression of transplant rejection and for a therapy of tumor diseases.",
"18.Use of inhibitor combinations of claim 4 for a prevention and therapy of autoimmune diseases, preferably of Rheumatoid Arthritis, Lupus Erythematodes, Multiple Sclerosis, IDDM, Morbus Crohn, Colitis Ulcerosa, Psoriasis, Neurodermitis, Glomerulonephritis, Interstitial Nephritis, Vasculitis, autoimmune diseases of the thyroid gland or autoimmune hemolytic anemia as well as of other chronic diseases with an inflammatory genesis as, for example, allergies and arteriosclerosis.",
"19.Use of inhibitor combinations of claim 4 for a suppression of transplant rejection and for a therapy of tumor diseases.",
"20.Pharmaceutical preparation according to claim 8 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.",
"21.Pharmaceutical preparation according to claim 8 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.",
"22.Pharmaceutical preparation according to claim 8 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.",
"23.Pharmaceutical preparation according to claim 9 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.",
"24.Pharmaceutical preparation according to claim 9 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.",
"25.Pharmaceutical preparation according to claim 9 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.",
"26.Pharmaceutical preparation according to claim 10 comprising two or more of the inhibitors of DP IV or of enzymes having a DP IV-analogous enzyme activity, of APN or of enzymes having an APN-analogous enzyme activity, of ACE, of POP (PEP) and of XPNPEP2 in a spaced apart formulation in combination with per se known carrier, auxiliary and/or additive substances for a simultaneous or a directly consecutive administration with the aim of a combined action.",
"27.Pharmaceutical preparation according to claim 10 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, vaginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.",
"28.Pharmaceutical preparation according to claim 10 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications.",
"29.Pharmaceutical preparation according to claim 11 for a systemic use for the oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular, rectal, virginal, sublingual application, together with per se known carrier, auxiliary and/or additive substances.",
"30.Pharmaceutical preparation according to claim 11 for a topical use in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles including instillative applications."
],
[
"The present invention describes the combined inhibition of the activity of the enzymes Aminopeptidase N (APN; E.C.",
"3.4.11.2.; CD13), Dipeptidyl peptidase IV (DP IV; E.C.",
"3.4.14.5; CD26), Prolyl oligopeptidase (POP; Prolyl endopeptidase; PEP; E.C.",
"3.4.21.26), the membrane-adherent Aminopeptidase P (X-Pro-Aminopeptidase; APP; XPNPEP2; E.C.",
"3.4.11.9) and Angiotensin-converting Enzyme (Angiotensin-konvertierendes Enzym; ACE; CD156; E.C.",
"3.4.15.1;CD156) by a simultaneous application of respective specific inhibitors of the above-referenced enzymes on the basis of amino acid derivatives, peptides or peptide derivatives, by which the activation, the DNA synthesis and, thus, the proliferation of immune cells is suppressed.",
"It is applicable to all diseases showing autoimmune pathogenesis that the disease is based on, or consists of, an activation and proliferation of immune cells, in particular of autoreactive T cells.",
"The very same mechanisms effect the acute or chronic rejection episodes after a transplantation of organs.",
"It was shown that membrane-adherent peptidases as, for example, DP IV or APN play a key role in the process of activation and clonal expansion of immune cells, particularly of T lymphocytes [B. Fleischer: “CD26, a surface protease involved in T cell activation”, Immunology Today 1994, 15, 180-184; U. Lendeckel et al.",
": “Role of alanyl peptidase in growth and function of human T cells”, International Journal of Molecular Medicine 1999, 4, 17-27; D. Riemann et al.",
": “CD13—not just a marker in leukemia typing”, Immunology Today 1999, 20, 83-88].",
"Several functions of mitogene-stimulated mononuclear cells (mitogen-stimulierte mononukleäre Zellen; MNZ) or of enriched T lymphocytes as, for example, the DNA synthesis, production and secretion of immune-stimulating cytokines (IL2, IL6, IL12, IFN-γ) and helper functions for B cells (synthesis of IgG and IgM) may be inhibited in the presence of specific inhibitors DP IV and APN [E. Schön et al,: “The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes”, Biomed.",
"Biochim Acta 1985, 2, K9-K15; E. Schön et al.",
": “The role of dipeptidyl peptidase IV in humane T lymphocyte activation, inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro”, Eur.",
"J. Immunol.",
"1987, 17, 1821-1826; D. Reinhold et al.",
": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360; U. Lendeckel et al.",
": “Induction of the membrane alanyl peptidase gene and surface expression in human T cells by mitogenic activation”, Biochem.",
"J.",
"1996, 319, 817-823; T. Kähne et al.",
": “Dipeptidyl peptidase IV: A cell surface peptidase involved in regulating T cell growth (Review)”, Int.",
"J. Mol.",
"Med.",
"1999, 4, 3-15; U. Lendeckel et al.",
": “Role of alanyl aminopeptidase in growth and function of human T cells (Review)”, Int.",
"J. Mol.",
"Med.",
"1999, 4, 17-27].",
"It is already known that the treatment of autoimmune diseases and rejection reactions after transplantations can be effected by an inhibition of dipeptidyl peptidase IV localized on immune cells by means of synthetic inhibitors.",
"Reference is made, for example, to EP 764 151 A1, WO 09529691, EP 731 789 A1, EP 528 858).",
"With respect to the enzymes aminopeptidase N, angiotensin-converting enzyme, X-Pro-aminopeptidase and prolyl oligopeptidase, such effects are not known up to now.",
"The invention is based on the surprising finding that the simultaneous inhibition of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, as well as (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase inhibits the DNA synthesis and, thus, the proliferation of mononuclear cells (MNZ), and of T cells as well, to an extent which cannot be achieved by the application of a single one of these enzyme inhibitors, even at a higher dosage.",
"Although the above-mentioned inhibitors finally exhibit an effect to the same process, i.e.",
"the DNA synthesis and, thus, the proliferation of immune cells, said effect is marked to a weaker extent and is not long lasting in the case of an application of single inhibitors.",
"Due to the functional overlap of the enzymatic activities of said enzymes, there can be observed a superadditive inhibitory effect on the DNA synthesis and proliferation resulting from the simultaneous inhibition of two or more of these enzymes, as can be concluded from our data.",
"Our invention shows that the simultaneous application of inhibitory substances of the above-mentioned enzymes or, respectively, of corresponding preparations and administration forms is well suitable for the therapy of autoimmune diseases and inflammatory diseases as well as for the treatment of rejection reactions after a transplantation.",
"In detail, the invention is based on the finding that the DNA synthesis of mononuclear cells (MNZ) and T cells is inhibited, in a superadditive way, by the simultaneous administration of inhibitors of the enzymatic activities of (I) dipeptidyl peptidase IV and aminopeptidase N, (II) dipeptidyl peptidase IV and angiotensin-converting enzyme, (III) dipeptidyl peptidase IV and prolyl oligopeptidase, (IV) dipeptidyl peptidase IV and X-Pro-aminopeptidase.",
"The application of enzyme inhibitors is a new method and complementary therapy form for the treatment of the above-mentioned diseases.",
"The inhibitors of dipeptidyl peptidase IV, of aminopeptidase N, of prolyl oligopeptidase, of the angiotensin-converting enzyme and of X-Pro-aminopeptidase applied in accordance with the invention may be employed in pharmaceutically applicable formulation complexes as inhibitors, substrates, pseudo-substrates, peptides and peptide derivatives having inhibitory effect and as antibodies of said enzymes as well.",
"Preferred effectors for DP IV are, for example, Xaa-Pro-dipeptides, their corresponding derivatives, preferably dipeptide phosphonic acid diaryl esters and their salts, Xaa-Xaa-(Trp)-Pro-(Xaa)n-peptides (n=0-10), their corresponding derivatives and their salts and amino acid (Xaa)-amides, their corresponding derivatives and salts, wherein Xaa is an α-amino acid/imino acid or an α-amino acid/imino acid derivative, respectively, preferably Nε-4-nitrobenzylcarbonyl-L-lysin, -L-prolin, -L-tryptophan, -L-isoleucin, -L-valin, and cyclic amines as, for example, pyrrolidine, piperidine, thiazolidine, and their derivatives serve as the amide structure.",
"Such compounds and their preparation were described in a prior patent (K. Neubert et al., DD 296 075 A5).",
"The inhibitors are administered simultaneously with known carrier substances.",
"The administration is conducted, on one hand, as a topical application in the form of, for example, creams, ointments, pastes, gels, solutions, sprays, liposomes, shaked mixtures, hydrocolloid dressings, and other dermatological bases/vehicles, respectively, including instillative applications and, on the other hand, systemic applications for an oral, transdermal, intravenous, subcutaneous, intracutaneous, intramuscular administration in suitable recipes and in a suitable galenic application form, respectively.",
"WORKING EXAMPLES Example 1 Inhibition of the DNA Synthesis of Human T Lymphocytes by Incubation with Synthetic Inhibitors of DP IV and of APN Our investigations showed that the DNA synthesis of human peripheric T lymphocytes is inhibited in a superadditive manner by the simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (Actinonin).",
"The T cells were incubated for 72 h in the presence of said inhibitors and subsequently, the DNA synthesis rate was determined via a measurement of the 3[H]-thymidin incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al,: “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"1 shows the dose-dependent superadditive inhibition of the DNA synthesis.",
"FIG.",
"1 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and aminopeptidase N (actinonine) on the DNA synthesis of human T lymphocytes.",
"Human peripheric T cells were incubated over 3 days with the concentrations of the inhibitors shown.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine inserted into the DNA was measured after further 6 hours.",
"Example 2 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of APN Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of APN (actinonine).",
"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al.",
": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"2 shows the dose-dependent, superadditive inhibition of the DNA synthesis.",
"FIG.",
"2 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and APN (actinonine) on the DNA synthesis of human mononuclear cells (MNZ).",
"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.",
"Example 3 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-thiazolidid).",
"The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al.",
": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"3 shows the dose-dependent, superadditive inhibition of the DNA synthesis.",
"FIG.",
"3 show a the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human peripheric T lymphocytes.",
"Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.",
"Example 4 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of POP Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of prolyl oligopeptidase (Boc-Ala-Thiazolidid).",
"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al.",
": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"4 shows the dose-dependent, superadditive inhibition of the DNA synthesis.",
"FIG.",
"4 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of prolyl oligopeptidase (Boc-Ala-Thia) on the DNA synthesis of human mononuclear cells (MNZ).",
"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.",
"Example 5 Inhibition of the DNA Synthesis of Human T Lymphocytes by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human T lymphocytes is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).",
"The T cells were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al.",
": “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"5 shows the dose-dependent, superadditive inhibition of the DNA synthesis.",
"FIG.",
"5 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) X and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human peripheric T lymphocytes.",
"Human T cells were incubated for three days with the concentrations of inhibitors mentioned above.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.",
"Example 6 Inhibition of the DNA Synthesis of Human Peripheric Mononuclear Cells by an Incubation with Synthetic Inhibitors of DP IV and of ACE Our experiments showed that the DNA synthesis of human peripheric mononuclear cells (MNZ) is inhibited in a superadditive manner by a simultaneous administration of inhibitors of DP IV (Lys[Z(NO2)]-thiazolidid=I49) and of the angiotensin-converting enzyme (Captopril).",
"The MNZ were incubated for 72 h in the presence of said inhibitors, and the DNA synthesis rate was determined subsequently by the measurement of the 3[H]-thymidine incorporation, as was described by Reinhold et al.",
"[D. Reinhold et al.",
"; “Inhibitors of dipeptidyl peptidase IV induce secretion of transforming growth factor β1 in PWM-stimulated PBMNC and T cells”, Immunology 1997, 91, 354-360].",
"FIG.",
"6 shows the dose-dependent, superadditive inhibition of the DNA synthesis.",
"FIG.",
"6 shows the synergistic and dose-dependent effect of inhibitors of DP IV (I49) and of the angiotensin-converting enzyme (Captopril) on the DNA synthesis of human mononuclear cells (MNZ).",
"Human MNZ were incubated for three days with the concentrations of inhibitors mentioned above.",
"Subsequently, 3[H]-methyl thymidine was added to the culture medium, and the amount of 3[H]-thymidine incorporated into the DNA was measured after further 6 hours.",
"Example 7 Inhibition of the Proliferation of Human Peripheric Mononuclear Cells (MNZ) by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin) FIG.",
"7: The MNZ were incubated for a time period of 72 h without addition (control), with an addition of the mitogenic lectin phytohemagglutinin (PHA), and with the addition of PHA and the inhibitors mentioned above, respectively.",
"Subsequently, the number amount of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.",
"Example 8 Inhibition of the Proliferation of the Human T Cell Line KARPAS-299 by a Single and Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of APN (Actinonin and Probestin) FIG.",
"8: The KARPAS-299 cells were incubated without addition (control) and in the presence of single inhibitors mentioned above as well as in the presence of combinations thereof, respectively.",
"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.",
"Example 9 Inhibition of the Proliferation of Activated Human Peripheric T Cells by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]-thiazolidid) and of APN (Actinonin and Probestin) FIG.",
"9: The T cells, with the exception of the untreated control, were activated by an addition of phytohemagglutinine and phorbol-12-myristate-13-acetate to the culture medium and were incubated for a time period of 72 h in the presence of the above-mentioned inhibitors single and in combination.",
"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer.",
"Example 10 Inhibition of the Proliferation of PHA-Activated Human Mononuclear Cells (MNZ) by a Single or Simultaneous Administration of Inhibitory Substances of DP IV (I49=Lys[Z(NO2)]thiazolidid) and of X-Pro-Aminopeptidase (APP) (Apstatin) FIG.",
"10: The mononuclear cells (MNZ) were incubated for a time period of 72 h in the presence of the inhibitors mentioned above single and in combination.",
"Subsequently, the number of metabolically active cells was determined by using the commercially available WST-1 cell proliferation assay (Takara Inc.) in accordance with the provisions of the manufacturer."
]
] | Patent_10296102 |
[
[
"Induction heating of rail welds",
"An apparatus (10) and method for preheating welds uses a centered induction plate (12) having preferably a plurality of induction coils (30, 32) to impart the generation of heat in the materials to be welded (14), being interactively controlled by at least a temperature sensor (90, 92) and power supply control loop (16) so that even preheating can be obtained for a selected length of time given the parameters of the weld desired."
],
[
"1.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating element affixed to the first side of the tool and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensor positioned proximate the first workpiece and the first heating element; a second induction heating element affixed to the second side of the tool and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensor positioned proximate the first workpiece and the second heating element; a controller coupled to the first and second temperature sensors, the controller activating the first and second heating elements independently to achieve a predetermined welding temperature in both first and second heating zones.",
"2.The induction heating system of claim 1, further comprising a first power supply coupled to the first heating element, and a second power supply coupled to the second heating element, wherein both first and second power supplies are coupled to the controller.",
"3.The induction heating system of claim 1, wherein the first and second induction heating elements are ferrite core heating elements.",
"4.The induction heating system of claim 1, wherein the temperature sensors comprise thermocouple temperature sensors.",
"5.The induction heating system of claim 1, wherein the temperature sensors comprise pyrometer temperature sensors.",
"6.The induction heating system of claim 1, wherein the controller provides a discernible indication when the workpieces have reached the predetermined welding temperature.",
"7.The induction heating system of claim 6, wherein the discernible indication is a visual indication.",
"8.The induction heating system of claim 1, wherein the controller provides positioning information to a positioning robot that adjusts relative position of the tool until the predetermined welding temperature is achieved.",
"9.The induction heating system of claim 1, wherein cooling fluid is provided proximate each of the heating elements.",
"10.The induction heating system of claim 9, wherein the controller controls cooling fluid activation.",
"11.The induction heating system of claim 1, wherein there are three or more heating elements.",
"12.The induction heating system of claim 1, wherein there are three or more heating zones.",
"13.The induction heating system of claim 1, wherein the first and second workpieces are railroad rails.",
"14.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the work-pieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; an induction heating element affixed to the tool and disposed proximate the first and second workpieces; a heating zone defined by a temperature sensor positioned proximate the first workpiece and the heating element; a controller coupled to the temperature sensor, the controller activating the heating element to achieve a predetermined welding temperature in the heating zone.",
"15.An induction heating system for preheating first and second workpieces to be welded together comprising: a tool disposed in a gap defined by facing ends of the workpieces, the tool further including mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the tool longitudinally with respect to the workpieces for proper proximity to the workpieces; a plurality of induction heating elements affixed to the tool and disposed proximate the first and second workpieces; as plurality of heating zones defined by multiple temperature sensors positioned proximate the first workpiece and the heating elements; a controller coupled to the temperature sensors, the controller activating the heating elements to achieve a predetermined welding temperature in the heating zones.",
"16.An induction heating system for preheating first and second workpieces to be welded together comprising: mounting means having first and second opposing sides, and disposed in a gap defined by facing ends of the workpieces, wherein the mounting means further further includes mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; a first induction heating means affixed to the first side of the mounting means and disposed proximate the first and second workpieces; a first heating zone defined by a first temperature sensing means positioned proximate the first workpiece and the first heating means; a second induction heating means affixed to the second side of the mounting means and disposed proximate the first and second workpieces; a second heating zone defined by a second temperature sensing means positioned proximate the first workpiece and the second heating means; a controller means coupled to the first and second temperature sensing means, the controller means activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones.",
"17.The induction heating system of claim 16, wherein the mounting means comprises a tool having first and second opposing sides.",
"18.The induction heating system of claim 16, further comprising a first power supply means coupled to the first heating means, and a second power supply means coupled to the second heating means, wherein both first and second power supplies means are coupled to the controller means.",
"19.The induction heating system of claim 16, wherein the first and second induction heating means comprise ferrite core heating elements.",
"20.The induction heating system of claim 16, wherein the temperature sensing means comprise thermocouple temperature sensors.",
"21.The induction heating system of claim 16, wherein the temperature sensing means comprise pyrometer temperature sensors.",
"22.The induction heating system of claim 16, wherein the controller means provides a discernible indication when the workpieces have reached the predetermined welding temperature.",
"23.The induction heating system of claim 22, wherein the discernible indication is a visual indication.",
"24.The induction heating system of claim 16, wherein the controller means provides positioning information to a positioning means that adjusts relative position of the tool until the predetermined welding temperature is achieved.",
"25.The induction heating system of claim 16, wherein cooling fluid is provided proximate each of the heating means.",
"26.The induction heating system of claim 25, wherein the controller means controls cooling fluid activation.",
"27.The induction heating system of claim 16, wherein there are three or more heating means.",
"28.The induction heating system of claim 16, wherein there are three or more heating zones.",
"29.The induction heating system of claim 16, wherein the first and second workpieces are railroad rails.",
"30.A method for preheating first and second workpieces to be welded together, the method comprising the steps of: (a) disposing, within a gap defined by facing ends of the workpieces, mounting means having first and second opposing sides; (b) providing mechanical centering assemblies disposed adjacent the facing ends of the workpieces, wherein the mechanical centering assemblies position the mounting means longitudinally with respect to the workpieces for proper proximity to the workpieces; (c) disposing a first induction heating means affixed to the first side of the mounting means proximate the first and second workpieces; (d) defining a first heating zone by positioning a first temperature sensing means proximate the first workpiece and the first heating means; (e) disposing a second induction heating means affixed to the second side of the mounting means proximate the first and second workpieces; (f) defining a second heating zone by positioning a second temperature sensing means proximate the first workpiece and the second heating means; and (g) activating the first and second heating means independently to achieve a predetermined welding temperature in both first and second heating zones."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.",
"The descriptions herein of rail welds should be considered with this more expansive use in mind.",
"Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.",
"This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.",
"This eliminates the human element, resulting in an automated high quality weld preheat method.",
"The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.",
"One advantage in this regard is the ability to manipulate the heat gradient.",
"Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.",
"The invention provides consistent heat through a wide range of ambient temperatures.",
"Another advantage is that of use in different rail geometry, rail chemistries and welding methods.",
"In addition to heating, an analogous plate or array can be used to control cooling after welding.",
"In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.",
"Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.",
"An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.",
"While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.",
"2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.",
"The use of items such as gas or other torches, gas burners or electrically powered devices.",
"Field welding in the past commonly preheated with torches and gas burners.",
"Such methods introduce human intervention positioning, timing or estimating heat input and temperature.",
"Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.",
"Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.",
"1 is a schematic view showing the layout of the components of the invention.",
"FIG.",
"2 is an elevational view showing the induction plate of the invention.",
"FIG.",
"2A is an elevational view showing an induction coil with ferrite core.",
"FIG.",
"3 is a wiring diagram showing the control wiring of the invention.",
"FIG.",
"4 is a wiring diagram showing the power supply wiring to control separate heating zones.",
"FIG.",
"5 is a side elevational view showing the induction plate of the invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"CLAIM OF PRIORITY Priority is claimed based upon Provisional Patent Application Ser.",
"No.",
"60/209,030, filed Jun.",
"2, 2000, which is incorporated by reference as if fully set forth herein.",
"STATEMENT OF INVENTORSHIP Assignee, Holland Company, believes that the subject matter was the invention of Richard F. Miller.",
"FIELD OF THE INVENTION Some welding techniques require precise consistent and controlled heating, which is difficult or impossible to obtain with torches, gas burners or other electric devices.",
"Instead, this is achievable through the use of induction heating wherein a plate or other locating and holding device is placed in a gap between work pieces to be welded, the plate containing an array of induction heating elements which, when energized, produce shaped, varying electromagnetic fields which link with and induce a voltage in the work pieces which in turn results in eddy current flows and subsequent power losses, as well as hysteresis losses, so that the workpiece temperature is raised to a desired, uniform level prior to welding.",
"BACKGROUND OF THE INVENTION 1.Summary of Invention The preferred embodiment is adapted to use in welding railroad rails, however, other difficult to weld work pieces could be advantageously preheated with the invention.",
"The descriptions herein of rail welds should be considered with this more expansive use in mind.",
"Induction type heat allows for precise heating at ideal locations and can be used to control heat gradients.",
"This control is possible though use of a feedback system, controller, coil arrangement and a positioning mechanism.",
"This eliminates the human element, resulting in an automated high quality weld preheat method.",
"The use of an automated electrically powered and computer controlled induction heating system using the induction heating coils and heating plate with sensitive temperature control and feedback interfaced within the power supply enables higher quality and more consistent welds of difficult to weld pieces such as railroad rails and similar high strength and complex shaped generally ferric items.",
"One advantage in this regard is the ability to manipulate the heat gradient.",
"Also included in advantages over prior art methods are the facts that no consumables are required, there is no need for gases or fuel on board or during transportation and cleanliness—in that there are no combustion byproducts.",
"The invention provides consistent heat through a wide range of ambient temperatures.",
"Another advantage is that of use in different rail geometry, rail chemistries and welding methods.",
"In addition to heating, an analogous plate or array can be used to control cooling after welding.",
"In the preferred and alternative embodiments, the invention envisions the use of independent, single or multiple coils and/or power units.",
"Independent, single or multiple coils and/or power units enable the precise location of heating, subdivides locations of heating and provides flexibility in the control of heating areas.",
"An added benefit of using an independent preheating unit, as compared to including the welder or portions of the welder's power supply or the like is that of efficiencies gains due to multi tasking during welding process.",
"While preheating is occurring, the welder itself can be independently set up for welding operations, or, indeed, one rail may be welded while the adjacent rail is preheated, should the rail gaps be proximate the rail welder's cable runs.",
"2.Description of Related Art While preheating of metal pieces for welding as a general concept is well known, heretofore generally manual application of heat has been used.",
"The use of items such as gas or other torches, gas burners or electrically powered devices.",
"Field welding in the past commonly preheated with torches and gas burners.",
"Such methods introduce human intervention positioning, timing or estimating heat input and temperature.",
"Combustion variables including fuel, air, pressure, position and shape of a flame relative to rails, ignition steps, initial temperature of the workpieces and even weather contribute to imprecision in temperature control in the prior art.",
"Resistance electrical devices have power and conductivity variables including both electrical and thermal limitations that also contribute to imprecision in temperature control.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIG.",
"1 is a schematic view showing the layout of the components of the invention.",
"FIG.",
"2 is an elevational view showing the induction plate of the invention.",
"FIG.",
"2A is an elevational view showing an induction coil with ferrite core.",
"FIG.",
"3 is a wiring diagram showing the control wiring of the invention.",
"FIG.",
"4 is a wiring diagram showing the power supply wiring to control separate heating zones.",
"FIG.",
"5 is a side elevational view showing the induction plate of the invention.",
"DETAILED DESCRIPTION OF THE INVENTION Weld quality and consistency can be improved with precise control of heating (temperatures, location zones and heat gradients).",
"Heating becomes more critical when dealing with certain alloying, geometry or ambient temperatures.",
"This is more of an art gained through experience or a routine that must be followed carefully to attempt to produce consistent, quality welds.",
"An induction heating system 10 uses a tool or plate 12 to heat a railroad rail 14.Power control 16 is operatively connected to a pair of power supplies 18,20.Power supplies 18,20 are in turn operatively connected to a heat zone control unit 22 using control output connections 24, 26.Cables (not shown) interconnect power supplies 18,20 to plate 12, generally, and to heating elements 30, 32 specifically through connectors 34, 36.FIG.",
"2A shows the heating element 32 in more detail.",
"Each element 30, 32 is generally broken out into a separate heating module 38, 40 using the elements 30, 32 and other features more fully described below to provide a unitary heating module 38, 40.The heating system 10 is a 5 Kw, 25 Khz induction heating system broken out in the form of two 2.5 Kw modules 38, 40.While this is the preferred capacity of the system, different welding operations could advantageously be accommodated through the use of a heating system with a different heating capacity.",
"These are capable of independent heating of two heat zones, 42, 44.Each module 38, 40 is connected to a heating element 30, 32 with ferrite cores 46, 48 in a common housing in plate 12 capable of being inserted in an approximate one inch gap between rails 14.It will be understood that the rails 14 are shown in section and in the field there will be to rails 14 with a gap between them intended to be welded.",
"For preheating of normal section railroad rails 14 for gas shielded arc welding, the preferred heat zones 42, 44 consist of two heating locations 50, 52 generally near the bottom flanges 54, 56.The preferred embodiment will be further described below after general description of the field that requires induction preheating.",
"Gas shielded arc welding without the preheating taught by the invention in this application has been practiced under controlled laboratory and/or workshop conditions but is believed unsuitable for use in the field.",
"One method used under controlled conditions is generally taught by U.S. Pat.",
"Nos.",
"5,773,779 and 5,877,468, which are incorporated by reference as if fully set forth herein.",
"It is believed that one reason the method described in these two patents is inoperative in field conditions is inadequate control of preheating.",
"Delivery of gas shielded arc welding equipment and the alignment and restraint of railroad rails and the deployment of a weld containment unit for application of weld beads is taught in published International Application No.",
"WO 99/31322 published 16 Dec. 1998 entitled “Mail Welding Apparatus Incorporating Rail Restraining Device, Weld Containment Device and Weld Delivery Unit.” The teachings of this application are also incorporated by reference.",
"It is believed that the apparatus and method taught herein are essential in effective practice of gas shielded arc welding of rails.",
"Additionally, other welding methods are believed to be capable of enhancement through the use of the invention taught here.",
"Other weld methods, such as thermite, on site foundry and even certain flux-based arc welding may prove suitable for high strength welds of complex shapes with adequate and well controlled preheating.",
"While the preferred embodiment of induction heating for rail welding using gas shielded arc welding anticipates using two heating modules 38, 40, other uses could use fewer, more or differently arranged heating modules.",
"Thus, for certain rail welding or joining methods it may prove advantageous to heat the entire rail section simultaneously.",
"The invention is not limited to rail welding using two heating modules.",
"Plate 12 is formed to fully support and contain heating elements 30, 32.Accordingly plate 12 has a body portion 60 ending in a protective ceramic cover 62 which fully covers ferrite cores 46, 48 and the corresponding conductors of elements 30, 32.Side edges 64, 66 of plate 12 are fitted with centering bar assemblies 68, 70.Assemblies 68, 70 use centering adjustment mechanism 72 to adjust bars 74, 76 outwardly or inwardly to fit the rail gap.",
"It will be understood that assemblies 68, 70 are symmetric and accordingly only one assembly 70 is shown and illustrated in FIG.",
"5.Precise cutting of rails in the field is quite difficult, thus there is often variation in the size of gaps and the orientation of their faces.",
"The adjustable and expandable centering bar assembles enabling side to side and top to bottom centering are important in aligning plate 12 as close to the center of the gap as practicable to maximize the uniform heating of the rail ends.",
"In this manner the assemblies 68, 70 are aligned for maximum effectiveness and uniformity, being centered between faces that may themselves be non-parallel due to the difficulty of cutting in the field.",
"Each heating element 30, 32 is fitted with a respective pair of water/power connections 80, 82 illustrated in FIG.",
"2A.",
"These enable both the electrical power connection necessary to energize the ferrite core 46, 48 and provide conduits for the transmission of cooling fluid to dissipate the heat radiantly transmitted from the preheated rails 14 to the plate 12.Spring clips 84, 86 are used to retain elements 30, 32 in place in plate 12.Temperature measuring devices such as spring loaded thermocouples 90, 92 are used as an integral part of both plate 12 and heat zone control unit 22.Thermocouples 90, 92 are operatively connected to heat zone control unit 22 which is in turn used to control power supplies 18, 20.Power supplies 18, 20 receive signals from unit 22 interconnected through connections 24, 26 which provide power on or power off control signals depending on the heat measured at thermocouples 90, 92.It will be observed, particularly from FIG.",
"1 and 2 that thermocouples 90 and 92 are positioned proximate ferrite cores 48, thereby providing an accurate temperature reading from rails 14 which are heated as a result of the energizing of elements 30, 32 and particularly cores 46, 48.The general principles of induction heating will be recognized, namely the providing of a sufficiently large energy output at elements 30, 32 and cores 46, 48 will create heat in adjacent steel rails 14.In this manner heat zones 42, 44 are interactively controlled so that a controlled heat results although a variety of factors, whether an imprecise gap, power, magnetic fluctuations, unequal temperature differential or even environmental factors such as a crosswind, impact the actual temperature and heat distribution in the rails.",
"As noted above while locating heat zones 42, 44 near the rail flanges is advantageous for gas shielded arc welding other types of welding may require the use of different heat zones such as heating the entire rail section including flanges, web and head, or for the welding of different shapes such as I-beams and the like.",
"In these other uses and applications, the number of heat zones and their orientation can be controlled consistent with the principles of this invention.",
"The use of one or more coil elements 30, 32 gives flexibility in applying the two preferred heat zones 46, 48.In other, particularly non-rail welding applications, a single heat zone or multiple heat zones could be used.",
"A plurality of coil elements, two or more, also provide flexibility in temperature differentials that may be required by particular metallurgical or welding considerations.",
"The invention enables precision controlling of the heat gradient in the pieces to be welded.",
"The interactive control between elements 30, 32 and thermocouples 90, 92 in zones 42, 44 enables the ability to manipulate the effects of applied heat to the particular metallurgy of the rails 14 and weld material and method used.",
"Control unit 22 can also be interconnected to corresponding additional fixtures or controls.",
"The invention contemplates a feedback system that enables, but is not limited to input from a robot or positioner or a controller/computer that calculates heat soak/rail temperature for particular conditions.",
"With this data compared to the induction heat inputs directly supplied by unit 22 and temperature measurement enabled by thermocouples 90, 92, control unit can be modified for particular time and energy parameters, given known metallurgical and welding requirements.",
"This full feedback system maximizes the quality control of the welding process so that it will be repeatable and monitored.",
"The full feedback system also records actual temperatures and adjusts automatically.",
"The fill feedback system is further programmable for various materials, conditions and methods.",
"With greater and better data regarding when the weld pieces reach correct temperature(s) in singular and/or multiple zones fully integrated with weld delivery controls, the user is provided a seamless system with no additional mechanisms, components required, such as the prior art burners or torches, and welding materials requiring multiple unrelated and uncontrolled steps.",
"Finally, the fully integrated system can be manipulated by robot for deployment, movements during heating process and retractment.",
"In operation, the steps of the invention are premised on the step in which each heat zone is monitored by a temperature measuring device which checks temperature on one side of the zone.",
"Even heating is achieved on either side of the zone because the inductor is centered between the rails with a mechanical centering device, which independently and exactly centers the inductor relative to side one and side two.",
"By so doing each side is brought up to a preferably preheat temperature even though the rails may have a variable gap.",
"It will be noted that the temperature may, of course, vary based on the materials welded and the method of welding used.",
"Rail ends are preheated by induction heating for preparation of welding.",
"The ideal temperature and heat gradient is controlled by a feedback system.",
"The feedback system uses temperature-measuring devices like thermocouple's, pyrometers, and other heat sensors with or without a controlling device.",
"During the pre-heating, different zone(s) of rail ends can be simultaneously heated independently of each other.",
"Frequency, proximity and number of cycles allow for control of the heat gradient.",
"This is complemented though coil designs in the tool and/or power inputs.",
"This tool fits between the rail ends in the gap and can be manipulated by a robotic arm or manually.",
"The gaps between rails are approximately ¼″ and up.",
"Material and mechanical designs of this tool enhance durability and efficiencies.",
"Process requirements are monitored and recorded for quality control.",
"Parameter measurements give a go/no go signal to proceed with welding or intervene with corrections to meet parameters.",
"In addition post-heating enjoys many of the same benefits.",
"The complete system is mobile and portable.",
"In operation, each zone begins heating simultaneously.",
"Should one zone reach temperature prior to the other the heater output is reduced so as to maintain at temperature until the second zone also achieves required temperature.",
"Only at this time does the controller send a signal to the weld controller indicating that welding can commence."
]
] | Patent_10296635 |
[
[
"Carboxylate salts in heat-storage applications",
"The use of an alkali metal salt or alkaline earth metal salt or of a bring solution of a C1-C16 carboxylic acid, or a mixture thereof, as a medium for the storage and use of thermal energy.",
"The salts or solution can be used in a heat exchange fluid or a lubricant or hydraulic fluid or soap."
],
[
"1-13.",
"(canceled) 14.A method for the storage and use of thermal energy comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.",
"15.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C3-C18 carboxylic acid or hydrated salts of a C3 to C7 carboxylic acid with a source of thermal energy.",
"16.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C2 carboxylic acids and a salt of one or more C3-C5 carboxylic acids.",
"17.The method of claim 1 wherein said mixture comprises a salt of one or more C1-C5 carboxylic acids and one or more C6-C18 carboxylic acids.",
"18.The method of claim 4 wherein said mixture comprises a salt of one or more C3-C5 carboxylic acids and one or more C6-C16 carboxylic acids.",
"19.The method of claim 1 wherein the temperature range of the thermal energy source is 20 to 180 degrees C. 20.The method of claim 1 further comprising contacting one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.",
"21.The method of claim 1 further comprising contacting one or a mixture of hydrated alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1 carboxylic acid with a source of thermal energy.",
"22.A method for improving the thermal properties of a fluid comprising dispersing in said fluid one or a mixture of anhydrous alkali metal salts, alkali metal earth salts, amine salts or ammonium salts of a C1-C18 carboxylic acid or hydrated salts of a C1 to C7 carboxylic acid with a source of thermal energy.",
"23.The method of claim 9 wherein the fluid is a water soluble alcohol freezing point depressant selected from the group consisting of ethylene glycol, propylene glycol, ethanol and methanol.",
"24.The method of claim 9 wherein the fluid is a lubricant or hydraulic fluid selected from the group consisting of mineral oil, synthetic oil, mineral soap, synthetic soap, mineral grease and synthetic grease."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Thermal energy originating from any energy source is reusable if it can be stored.",
"Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.",
"in refineries and steam generation plants).",
"Energy generated in peak load time can be managed and stored for later use.",
"Examples are solar heating and electrical heating on low tariff hours.",
"The problem of cold car engine start in wintertime is well known.",
"Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.",
"Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.",
"Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.",
"However these solutions put an extra burden on the vehicle's electrical power system.",
"Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.",
"Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.",
"Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.",
"An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.",
"The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.",
"The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates."
],
[
"This invention relates to the application of carboxylate salts for storing thermal energy.",
"Melted salts are used for heat-storage because salts absorb heat during the transition from the solid to the liquid phase.",
"This heat is stored in latent form as long as the liquid state persists and released again during the transition from the liquid to the solid phase when the liquid salt solidifies.",
"BACKGROUND OF THE INVENTION Thermal energy originating from any energy source is reusable if it can be stored.",
"Examples of reusable energy are excess heat from stationary and automotive internal combustion engines, heat generated by electrical motors and generators, process heat and condensation heat (e.g.",
"in refineries and steam generation plants).",
"Energy generated in peak load time can be managed and stored for later use.",
"Examples are solar heating and electrical heating on low tariff hours.",
"The problem of cold car engine start in wintertime is well known.",
"Frost and damp on windscreen and windows, difficult engine start, cold in the passenger compartment.",
"Car manufacturers are aware of this problem and make every possible effort to improve the driver's comfort under such circumstances.",
"Electrical heating of windshield, rear windows, steering wheel and passenger seats are offered as comfort options.",
"However these solutions put an extra burden on the vehicle's electrical power system.",
"Engine manufacturers are looking for solutions that make preferably use of excess heat generated by the engine that can be controllably released to the environment.",
"Heat-storage salts or functional fluids containing heat-storage salts may find new applications in emerging technologies.",
"Heat-storage salts could for instance be applied to maintain fuel cells at constant temperatures.",
"An aspect of this invention is that in automotive and heavy-duty engine applications, excess engine heat can be stored in carboxylic salts or in carboxylic salt solutions integrated into the engine heat-exchange system.",
"The stored heat can be used to rapidly heat critical engine components, engine fluids and exhaust gas catalyst Heating of these critical components before engine start helps avoids the discomfort, high fuel consumption, high exhaust emissions and increased engine wear linked to cold engine start.",
"The heat stored in carboxylic salts or in carboxylic salts solutions can also be used to heat the passenger compartment to improve driver and passenger comfort in cold climates.",
"PRIOR ART Hydrated fluoride-, chloride-, sulfate- and nitrate salts or salt combinations have been described as heat-storage media.",
"U.S. Pat.",
"No.",
"4,104,185 describes a heat accumulator in which the heat-energy storage medium consists essentially of a potassium fluoride/water solution having a fluoride content between 44 and 48% by weight.",
"U.S. Pat.",
"No.",
"5,567,346 relates to a latent heat storage material composition comprising 65 to 85 wt % of sodium sulfate decahydrate, 1 to 20 wt % of ammonium chloride and 1 to 20 wt % of sodium bromide, and optionally 1 to 20 wt % of ammonium sulfate.",
"U.S. Pat.",
"No.",
"5,728,316 relates to heat-storage salt mixtures composed of magnesium nitrate hexahydrate and lithium nitrate in mass ratio 86-81:14-19.U.S.",
"Pat.",
"No.",
"5,755,988 relates to a process for moderating the thermal energy content of closed container comprising mixtures of organic acids.",
"Co-assigned EP 0,229,440, EP 0,251,480, EP 0,308,037 and EP 0,564,721 describe the use of carboxylate salts as corrosion inhibitors in aqueous heat exchange fluids or corrosion-inhibited antifreeze formulations.",
"EPA No.",
"99930566.1 describes aqueous solutions of carboxylates that provide frost and corrosion protection.",
"Aqueous solutions of low carbon (C1-C2) carboxylic acid salts, in combination with higher carbon (C3-C5) carboxylic acid salts, were found to provide eutectic freezing protection.",
"Improved corrosion protection was found by adding one or more than one C6-C16 carboxylic acids.",
"The advantage of these carboxylic salts based cooling fluids over ethylene glycol- or propylene glycol cooling fluids is improved heat-transfer due to a higher specific heat and improved fluidity resulting from the higher water content at the same frost protection.",
"It is another objective of this invention to add heat-storage capacity to the above heat-exchange fluids and other functional fluids and soaps like lubricants and greases.",
"It is an object of the present invention to provide heat storage salt combinations that are less toxic and less burdensome to the environment than the fluoride-, chloride-, sulfate- and nitrate salts or the acidic salt combinations used in prior art Another object of the invention is to provide heat storage salt combinations that are less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.",
"FIELD OF THE INVENTION One aspect of the invention relates to the application of alkali metal salts or alkali earth metal salts of carboxylic acids, and combinations of such salts as latent heat-storage media The carboxylate heat-storage salts of this invention are less toxic and more environmentally friendly than the fluoride-, chloride-, sulfate- and nitrate salts or salt combinations used in prior art.",
"They are also less corrosive to the metals and materials used in heat transfer- and heat-storage equipment.",
"They are similar to the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.",
"They are also compatible with the carboxylates (formates and/or acetates) used as freezing point depressant in aqueous heat-exchange fluids.",
"In heat storage applications it is important to find media with melting temperatures that are in line with the temperature operating range of the heat source and which have high latent heat capacity.",
"It is another aspect of this invention that mixtures of carboxylic salts can be tuned to provide melting temperatures that fit the application temperatures.",
"Similarly, combinations with high heat capacity can be selected to optimize storage capacity.",
"This can be done by mixing different salts of the same carboxylate (for instance the potassium, lithium and/or sodium salt of the same carboxylate) or by mixing the salts of different carboxylates.",
"In heat storage applications it is also important that the heat storage salts can withstand unchanged and unlimited cycles of heat storage and heat release.",
"Hydrated heat-storage salts are particularly susceptible.",
"Loss of water from hydrolyzed crystals will introduce anhydrous crystalline structures with different melting temperatures and different latent heat capacities that may no longer be suitable for the application.",
"Dehydration at temperatures above the melting temperature of a hydrated salt can be avoided by using hermetically sealed containers and limiting the free space where water can condense without contact with the heat-storage salts.",
"These measures limit to some extent the use of hydrated salts in heat-storage applications.",
"It is another aspect of this invention to disperse carboxylate salts with heat-storage capacity in the heat-transfer fluid.",
"Heat-storage salts can be selected that have limited solubility in the heat-transfer fluid of choice.",
"The total amount of heat-storage salts added to the solution can be tuned to the heat capacity required in a particular system.",
"As the melting temperature of the dispersed heat-storage salts is reached, the salts will start to melt and extract heat from the fluid by phase transmission.",
"The fluid temperature can only rise again when all the heat-storage salts are in molten state.",
"In the case where hydrated heat-storage salts are used, the use of an aqueous heat-exchange fluid in which the salts are dispersed ensures hydration.",
"Heat-storage salts can be selected that have densities that are close in solid and liquid phase so that there is no risk of damage to the container or system due to expansion upon phase transition.",
"In many heat-exchange applications, however, a fluid phase will be preferred to allow easy transport of heat.",
"Dual heat-exchange systems can of course be used, in which the primary system contains the heat-storage salts, and the secondary system contains the heat-transport fluid.",
"It is another aspect of the invention to improve the heat capacity of a heat-exchange fluid by dispersion of heat-storage particles in existing heat exchange fluids or other functional fluids or soaps.",
"Examples are: 1 Heat-exchange fluids based on water soluble alcohol freezing point depressants such as ethylene glycol, propylene glycol, ethanol or methanol.",
"2 Heat-exchange fluids based on aqueous solutions of low carbon (C1-C2) carboxylic acid salts (formates, acetates) or mixtures thereof.",
"3 Heat-exchange fluids, lubricants or hydraulic fluids based on mineral- or synthetic oil, mineral and synthetic soaps or greases.",
"Suspended particles provide heat-storage capacity in the bulk of the existing exchange medium, lubricant or grease.",
"The alkali metal salts of carboxylic acids have low toxicity, are biodegradable and are not corrosive towards many materials.",
"An additional advantage of alkali metal carboxylates is that they are similar and/or compatible with the carboxylates used as freezing point depressant and with the carboxylates used as corrosion inhibitors in aqueous and glycol based heat-exchange fluids.",
"EXAMPLES The invention will be more specifically described by way of reference to the following examples.",
"A number of formulations were evaluated, by subjecting known quantities of salts to controlled heating and cooling cycles between 20° C. and 180° C. EXAMPLE COMPOSITION Comparative A Magnesium Chloride Hexahydrate Comparative B Magnesium Nitrate Hexahydrate Invention 1 Potassium Octanoate Invention 2 Potassium Heptanoate Invention 3 Potassium Octanoate (90%)/Potassium Heptanoate (10%) Invention 4 Potassium Propionate Invention 5 Sodium Propionate (30%)/Potassium Formate (70%) Invention 6 Potassium Octanoate (70%)/Potassium Heptanoate (30%) Invention 7 Brine solution of 80 w/w % Potassium Propionate Invention 8 Sodium Propionate (20%)/Potassium Formate (20%)/ Potassium Heptanoate (10%)/Water (50%) FIGURES The Figures show heat transformation curves for the formulations of the examples.",
"They are more specifically described hereinafter.",
"DISCLOSURE OF THE INVENTION Application of Carboxylate Salts in Heat-Storage Applications.",
"One aspect of the invention is that alkali metal salts and alkali earth metal salts of carboxylic acids have been found to have heat-storage capacities which allow these salts to be used in heat-storage applications.",
"To evaluate the heat-storage capacities, salts were subjected to controlled heating and cooling cycles over a preset temperature range.",
"For instance, to evaluate possible automotive applications, known quantities of the salts were subjected to controlled heating and cooling cycles between 20° C. and 180° C. When, upon heating, the melting point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is melted.",
"By measuring the temperature differential between salt and a reference recipient subjected to the same temperature cycles, the melting point can be determined.",
"By integrating the temperature differential over time, the latent heat capacity of the sample can be measured.",
"Similarly, when, upon cooling the solidification point is reached, the temperature measured within the salt will tend to remain constant until all of the salt is solidified.",
"Again, by integrating the temperature differential overtime, the latent heat capacity of the sample can be estimated (differential scanning calorimetric technique).",
"By repeating the temperature cycles, the stability of the heat-storage salt can be evaluated.",
"Literature provides information on the melting point and heat capacity of some known heat-storage salts.",
"For instance, magnesium chloride hexahydrate (comparative example A) has been reported to have a melting point of 117° C. and a latent heat capacity of 165 KJ/kg.",
"FIG.",
"1 shows experimental curves for magnesium chloride hexahydrate.",
"The temperature cycle has been repeated five times.",
"FIG.",
"2 shows the temperature differentials versus time.",
"In FIG.",
"3, the temperature differentials are plotted in function of temperature.",
"From these curves it can be derived that the melting point is indeed 117° C. Under-cooling upon solidification is shown.",
"Repeatability of the melting point in successive temperature cycles or series is good.",
"Some reduction in heat capacity is however noted, likely the result of partial dehydration of the salt.",
"More extreme shifts in melting point(s) and heat-storage capacity are shown in FIG.",
"4 for magnesium nitrate hexahydrate (comparative example B).",
"In this experiment heat-storage capacity is lost in the second and third temperature cycle (series 2 and 3).",
"Additional temperature cycles for the sample of magnesium nitrate hexahydrate are shown in FIG.",
"5.Dehydration of the salt apparently causes further changes and shifts in melting and solidification points towards higher temperatures.",
"Carboxylate Salts Provide Stable Heat-Storage Properties.",
"Surprisingly, a much more stable behavior is found for alkali metal salts of carboxylic acids that are also employed as corrosion inhibitors.",
"For example, FIG.",
"6 shows five successive temperature cycles for potassium octanoate (invention example 1).",
"The melting point of the salt is 57° C. An additional example using potassium heptanoate is shown in FIG.",
"7 (invention example 2).",
"The melting point for potassium heptanoate is 61° C. The Melting Point of Carboxylate Salts can be Tuned for a Specific Heat-Storage Application.",
"The melting point can be tuned for a specific application by the selection and mixing ratio of the alkali metal carboxylates.",
"For example, a mixture of potassium octanoate (90%) and potassium heptanoate (10%) (invention example 3 shown in FIG.",
"8) was found to have a melting temperature of about 48° C., particularly suited for heat-storage at lower temperatures.",
"In aqueous solutes these carboxylate salts or salt combinations show excellent corrosion protection properties.",
"In addition, they are similar and thus fully compatible with the carboxylates used as corrosion inhibitors in ethylene glycol and propylene glycol heat-exchange fluids and water treatment chemicals.",
"The low carbon (C1-C2) carboxylic acid alkali metal salts and the medium carbon (C3-C5) carboxylic acid alkali metal salts, or combinations of the two can be used as heat-storage salts.",
"For example, FIG.",
"9 (invention example 4) shows consecutive heating and cooling cycles for potassium propionate, with melting temperature of 79° C. A mixture of 30% sodium propionate and 70% potassium formate—FIG.",
"10 (invention example 5)—was found to have a melting temperature of 167° C. Heat-Storage Properties of Wetted or Hydrated Carboxylate Salts are Easily Restored.",
"It was found that, starting from hydrated or wetted carboxylate salts, stable heat-storage properties can easily be obtained by one or more temperature cycles in which the water is evaporated.",
"This is illustrated in FIG.",
"11 (invention example 6) for a mixture of the potassium octanoate (70%) and potassium heptanoate (30%), with a melting temperature of about 42° C.—water is boiled off from a wet sample in the first heatig cycle and latent heat can already be recovered in the first cooling cycle at a solidification temperature of about 45° C. This allows heat-exchange applications in which water is evaporated or added to the heat-storage salts, for instance to remove excess heat efficiently.",
"Brine Solutions of Carboxylate Salts have Heat-Storage Capacity.",
"Brine solutions of carboxylate salts can also be used as heat-storage medium.",
"For example, FIGS.",
"12 to 14 (invention example 7) shows the different curves for consecutive heating and cooling cycles for a brine solution of 80 w/w % of potassium propionate.",
"Contrary to the salts, the aqueous brine solution was contained in a closed container, not allowing evaporation of water.",
"In the experiment, phase transition on the lower temperature range was apparently not completed when the heating cycle was re-started, due to the high heat-storage capacity of the medium.",
"Silicone oil was used as reference fluid.",
"Dispersed Carboxylates Salts Provide Heat-Storage Capacity to Fluids or Soaps.",
"It is another aspect of this invention to disperse the hydrated salts with heat-storage capacity in the heat-transfer fluid.",
"For example, FIG.",
"15 (invention example 8) shows the consecutive heating and cooling cycles for a mixture of 20% sodium propionate and 20% potassium formate and 10% potassium heptanoate with 50% water in comparison with a brine solution without the addition of the potassium heptanoate.",
"The effect of the heptanoate additions is clearly seen.",
"This is even more evident from the curves in FIG.",
"16, showing the differential temperatures in function of time.",
"FIG.",
"17 shows the effect of the potassium heptanoate.",
"The effect of solidification at about 73° C. which is also observed for pure potassium heptanoate (FIG.",
"7) is clearly seen.",
"Dispersion of carboxylate heat-storage salts in other fluids will have similar effects.",
"This will particularly be the case for glycol based heat exchange fluids.",
"Many carboxylate salts have limited solubility in glycol and water and can thus be dispersed in such fluids to add heat-storage capacity.",
"Similarly, this is possible in other functional products such as lubricants or hydraulic fluids based on mineral- or synthetic oil, and in mineral-and synthetic soaps or greases."
]
] | Patent_10296826 |
[
[
"Stabiliser for radiopharmaceuticals",
"The present invention provides an improved stabiliser for radiopharmaceuticals which inhibits impurities from being produced by two kinds of decomposition mechanisms and exhibits such an effect that the shelf life of a radiopharmaceutical after its preparation is prolonged as compared with conventional ones.",
"The improvement comprises a combination of an amino-substituted aromatic carboxylic acid or its salt, ester or amide in combination with a diphosphonic acid or its salt."
],
[
"1.A stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical which is susceptible to either reductive degradation or radiolysis; (ii) a first stabiliser for said radiopharmaceutical which includes an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a second stabiliser for said radiopharmaceutical which includes a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"2.The composition of claim 1 wherein the amino-ubstituted aromatic carboxylic acid or its salt, ester or amide is selected from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 3, 5-diaminobenzoic acid, 4-amino-salicylic acid, and salts, esters and amides thereof.",
"3.The composition of claim 1, wherein the diphosphonic acid or its salt is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof.",
"4.The composition of claim 1, wherein the amino-substituted aromatic carboxylic acid or salt, ester or amide thereof is sodium 4-aminobenzoate, and the diphosphonic acid is methylenediphosphonic acid.",
"5.The composition of claim 1, wherein the radiopharmaceutical includes a γ-ray emitter or a β-ray emitter.",
"6.The composition of claim 5, wherein the radiopharmaceutical includes Tc-99m, Re-186 or Re-188.7.The composition of claim 1, wherein the radiopharmaceutical includes a metal complex of a radionuclide with a ligand.",
"8.The composition of claim 7, wherein the ligand is an amine oxime.",
"9.The composition of claim 8, wherein the amine oxime is selected from the group consisting of d,l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.",
"10.A non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition of claim 1 which comprises: (i) a first stabiliser which comprises an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; and (ii) a second stabiliser which comprises a diphosphonic acid or salt thereof, wherein the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"11.The kit of claim 10, which includes 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.",
"12.The kit of claim 10, further comprising a tin (II) reductant, wherein the molar ratio of the diphosphonic acid or its salt to tin (II) is in the range 1 to 10.13.The kit of claim 10, further comprising an amine oxime ligand.",
"14.The kit of claim 13 wherein the amine oxime is selected from the group consisting of d, l-hexamethylpropyleneamine oxime and 4,9-diaza-3, 3, 10, 10-tetramethyldodecane-2, 11-dione dioxime.",
"15.The kit of claim 10, wherein the first and second stabilzers are lyophilised.",
"16.In a method of preparing a radiopharmaceutical composition which includes adding stabilizers to a radiopharmaceutical, wherein the radiopharmaceutical is not a metal complex of diphosphonic acid, the improvement comprising using a diphosphonic acid or a salt thereof as a stabiliser.",
"17.Cancelled 18.The method of claim 16, wherein the radiopharmaceutical comprises Tc-99m.",
"19.The method of claim 16, wherein the diphosphonic acid is selected from the group consisting of methylenediphosphonic acid, hydroxymethanediphosphonic acid, hydroxyethanediphosphonic acid, ethylenediaminetetraphosphonic acid, and salts thereof."
],
[
"<SOH> BACKGROUND TO THE INVENTION <EOH>Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.",
"Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.",
"In the case of Tc-99m, the latter is referred to as post-reconstitution instability.",
"U.S. Pat.",
"No.",
"4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99m Tc-complexes of diphosphonic acids.",
"Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99m Tc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.",
"99m Tc-HMPAO is particularly unstable with respect to post-reconstitution stability.",
"99m Tc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.",
"The function of the stannous ion is to reduce the 99m Tc-pertechnetate ( 99m TcO 4 − ), ie.",
"technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99m Tc-HMPAO metal complex.",
"The radiochemical purity (Rcp) of 99m Tc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99m Tc labelling.",
"After Tc-99m radiolabelling, the Rcp of 99m Tc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99m Tc complex of unknown structure derived from 99m Tc-HMPAO, 99m Tc-pertechnetate ( 99m TcO 4 − ) and reduced-hydrolysed-technetium [ 99m Tc].",
"Of these impurities, both the secondary complex and 99m Tc-pertechnetate are decomposition products of 99m Tc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.",
"Med.",
"29, 1568-1576, 1988).",
"The secondary complex is believed to be produced when the lipophilic 99m Tc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.",
"stannous) remaining from the pertechnetate reduction step.",
"On the other hand, the 99m Tc-pertechnetate impurity is produced when 99m Tc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.",
"radiolysis of the solvent.",
"Accordingly, in order to inhibit the production of both the 99m Tc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.",
"Thus, Nucl.",
"Med.",
"Biol.",
"7, 675-680 (1989); Eur.",
"J. Nucl.",
"Med.",
"16, 541 (1990); Eur.",
"J. Nucl.",
"Med.",
"20, 661-666 (1993) and Eur.",
"J. Nucl.",
"Med.",
"22, 1163-1172 (1995) all report attempts to stabilise 99m Tc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.",
"In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99m Tc-HMPAO to at least 80% at 4 hours post reconstitution.",
"Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99m Tc-HMPAO at 6 hours post reconstitution to at least 80%.",
"The stabilisation mechanisms of 99m Tc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.",
"Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99m Tc-HMPAO.",
"However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99m Tc-blue or cobalt chloride is used as a stabiliser for 99m Tc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99m Tc-pertechnetate.",
"Accordingly, any kit for the preparation of 99m Tc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).",
"One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.",
"The other is a vial containing the stabiliser (methylene blue or cobalt chloride).",
"Thus, the most successful prior art methods of stabilising 99m Tc-HMPAO to date all require the use of 2-vial kits.",
"When the kit for preparing 99m Tc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99m Tc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.",
"methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.",
"It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.",
"Too short a time, and 99m Tc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.",
"Too long a time, and the stabilising effect is delayed.",
"In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.",
"The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.",
"In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.",
"Moreover, when methylene blue is added to 99m Tc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.",
"There is therefore a need for a single vial kit for the preparation of 99m Tc-HMPAO which has both shelf-life and post-radiolabelling stability.",
"The present invention provides a kit which solves this problem, and is straightforward to use.",
"The Present Invention.",
"The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid."
],
[
"FIELD OF THE INVENTION The present invention relates to a stabilised radiopharmaceutical composition, together with non-radioactive kits for the preparation of the stabilised radiopharmaceutical composition.",
"BACKGROUND TO THE INVENTION Some radiopharmaceuticals undergo decomposition due to either radiolysis or redox reactions, and hence exhibit undesirable instability.",
"Non-radioactive kits for the preparation of radiopharmaceuticals, especially Tc-99m radiopharmaceuticals, may suffer from two types of instability: (i) shelf-life instability of the non-radioactive composition over time, (ii) instability of the radiopharmaceutical post-formation.",
"In the case of Tc-99m, the latter is referred to as post-reconstitution instability.",
"U.S. Pat.",
"No.",
"4,451,451 discloses that para-aminobenzoic acid (pABA) and analogues are useful stabilisers for technetium non-radioactive kits, including kits for the preparation of 99mTc-complexes of diphosphonic acids.",
"Tc-99m-hexamethylpropyleneamine oxime (referred to hereinafter as 99mTc-HMPAO), is a radiopharmaceutical commercially available as a regional cerebral blood flow imaging agent.",
"99mTc-HMPAO is particularly unstable with respect to post-reconstitution stability.",
"99mTc-HMPAO is usually prepared from a lyophilised, non-radioactive kit which contains HMPAO and stannous ion.",
"The function of the stannous ion is to reduce the 99mTc-pertechnetate (99mTcO4−), ie.",
"technetium in oxidation state Tc(VII), to the Tc(V) oxidation state of the 99mTc-HMPAO metal complex.",
"The radiochemical purity (Rcp) of 99mTc-HMPAO one hour after Tc-99m labelling is only about 80%, so that it must be used within 30 minutes of 99mTc labelling.",
"After Tc-99m radiolabelling, the Rcp of 99mTc-HMPAO decreases with time due to the growth of three different radioactive impurities, namely: a hydrophilic secondary 99mTc complex of unknown structure derived from 99mTc-HMPAO, 99mTc-pertechnetate (99mTcO4−) and reduced-hydrolysed-technetium [99mTc].",
"Of these impurities, both the secondary complex and 99mTc-pertechnetate are decomposition products of 99mTc-HMPAO; however, it is reported that the decomposition mechanisms are different (J. Nucl.",
"Med.",
"29, 1568-1576, 1988).",
"The secondary complex is believed to be produced when the lipophilic 99mTc-HMPAO complex is exposed to excess unoxidised tin(II) (ie.",
"stannous) remaining from the pertechnetate reduction step.",
"On the other hand, the 99mTc-pertechnetate impurity is produced when 99mTc-HMPAO and the secondary complex are oxidised by the free radicals produced in solution by the action of radiation, ie.",
"radiolysis of the solvent.",
"Accordingly, in order to inhibit the production of both the 99mTc-pertechnetate and secondary complex impurities, the addition of stabilisers has been disclosed.",
"Thus, Nucl.",
"Med.",
"Biol.",
"7, 675-680 (1989); Eur.",
"J. Nucl.",
"Med.",
"16, 541 (1990); Eur.",
"J. Nucl.",
"Med.",
"20, 661-666 (1993) and Eur.",
"J. Nucl.",
"Med.",
"22, 1163-1172 (1995) all report attempts to stabilise 99mTc-HMPAO by the addition of either: gentisic acid, sodium decahydroxypyrophosphate, methylene blue, cobalt chloride or the like.",
"In particular, the post-radiolabelling addition of methylene blue improves the Rcp of 99mTc-HMPAO to at least 80% at 4 hours post reconstitution.",
"Similarly, the post-radiolabelling addition of cobalt chloride has been found to improve the Rcp of 99mTc-HMPAO at 6 hours post reconstitution to at least 80%.",
"The stabilisation mechanisms of 99mTc-HMPAO by methylene blue and cobalt chloride are believed to be essentially the same.",
"Both are in redox equilibrium in solution, and oxidise excess tin(II), thus stabilising 99mTc-HMPAO.",
"However, when the reducing tin(II) and methylene blue or cobalt chloride coexist in solution before the Tc-99m radiolabelling step, the tin(II) reductant is completely oxidised, so that the Tc-99m labelling becomes impossible because there is no longer any reducing agent present to reduce the Tc(VII) 99mTc-blue or cobalt chloride is used as a stabiliser for 99mTc-HMPAO, it must be added after the Tc-99m radiolabelling step and cannot be pre-mixed with the ligand (HMPAO) and 99mTc-pertechnetate.",
"Accordingly, any kit for the preparation of 99mTc-HMPAO employing such stabilisers, must be composed of two vials (referred to hereinafter as a 2-vial kit).",
"One vial is a freeze-dried vial containing the HMPAO ligand together with the tin(II) reductant and other excipients.",
"The other is a vial containing the stabiliser (methylene blue or cobalt chloride).",
"Thus, the most successful prior art methods of stabilising 99mTc-HMPAO to date all require the use of 2-vial kits.",
"When the kit for preparing 99mTc-HMPAO is a 2-vial kit, the radiolabelling operation is more complicated than for a single vial kit and comprises two steps: (1) 99mTc-pertechnetate solution is added to the vial containing the HMPAO ligand and the resulting mixture is mixed by shaking; (2) a stabiliser solution from the second vial (eg.",
"methylene blue or cobalt chloride) is added to the reconstituted mixture from step (1) in the first vial.",
"It is necessary that the time between the first and second steps is controlled so as to be as close as possible to two minutes.",
"Too short a time, and 99mTc-HMPAO complex formation may be incomplete and hence addition of the stabiliser may adversely affect the Rcp by oxidising the stannous ion before the reduction of the pertechnetate starting material is complete.",
"Too long a time, and the stabilising effect is delayed.",
"In such a procedure it is also necessary that care be taken with respect to the amounts of the solutions added.",
"The operator must also take due care to ensure that the vials are not inadvertently mixed up at any stage.",
"In addition, there is an increased risk of radiation dose to the operator due to the increased number of manipulations.",
"Moreover, when methylene blue is added to 99mTc-HMPAO, a precipitate is produced, so that a filtration step becomes necessary, and thus the procedure becomes more complicated.",
"There is therefore a need for a single vial kit for the preparation of 99mTc-HMPAO which has both shelf-life and post-radiolabelling stability.",
"The present invention provides a kit which solves this problem, and is straightforward to use.",
"The Present Invention.",
"The present invention relates to a stabiliser for radiopharmaceuticals which comprises a combination of an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof, with a diphosphonic acid or a salt thereof with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides: a stabilised radiopharmaceutical composition which comprises: (i) a radiopharmaceutical; (ii) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (iii) a diphosphonic acid or salt thereof, is with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"By the term ‘amino-substituted aromatic carboxylic acid’ is meant a compound in which at least one hydrogen atom on the aromatic ring of an aromatic carboxylic acid is substituted with at least one amino group.",
"The aromatic group is preferably benzene.",
"Preferred amino-substituted aromatic carboxylic acids are: 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid (pABA), 3,5-diaminobenzoic acid and 4-aminosalicylic acid.",
"4-aminobenzoic acid (pABA) is especially preferred.",
"The salt of the amino-substituted aromatic carboxylic acid is suitably a salt with a biocompatible cation.",
"By the term “biocompatible cation” is meant a positively charged counterion which forms a salt with an ionised, negatively charged group (here a carboxylate group), where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body.",
"Examples of suitable biocompatible cations include: the alkali metals (eg.",
"sodium or potassium); the alkaline earth metals (eg.",
"calcium, magnesium and barium); and the ammonium ion.",
"A preferred biocompatible cation is sodium.",
"Preferred salts of the present invention include: sodium 2-aminobenzoate, sodium 3-aminobenzoate, sodium 4-aminobenzoate (NapABA), sodium 3,5-diaminobenzoate, sodium 4-aminosalicylate, potassium 2-aminobenzoate, potassium 3-aminobenzoate, potassium 4-aminobenzoate, potassium 3,5-diaminobenzoate and potassium 4-aminosalicylate.",
"Sodium 4-aminobenzoate (NapABA) is especially preferred.",
"Suitable esters of the aromatic carboxylic acid include methyl, ethyl or propyl esters.",
"Preferred esters are: methyl 2-aminobenzoate, methyl 3-aminobenzoate, methyl 4-aminobenzoate, methyl 3,5-diaminobenzoate, methyl 4-aminosalicylate, ethyl 2-aminobenzoate, ethyl 3-aminobenzoate, ethyl 4-aminobenzoate, ethyl 3,5-diaminobenzoate, ethyl 4-aminosalicylate, propyl 2-aminobenzoate, propyl 3-aminobenzoate, propyl 4-aminobenzoate, propyl 3,5-diaminobenzoate and propyl 4-aminosalicylate.",
"Suitable amides of the amino-substituted aromatic carboxylic acid are amides formed by derivatising the carboxyl group of the aromatic carboxylic acid with either ammonia or a compound having at least one amino group, and include such compounds as 2-aminobenzamide, 3-aminobenzamide and 4-aminobenzamide.",
"The diphosphonic acid of the present invention is suitably a 1,1- or a 1,2-diphosphonic acid, or a diphosphonic acid derivative of an amine such as ethylenediaminetetraphosphonic acid (EDTMP).",
"1,1-diphosphonic acids are preferred, such as methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP).",
"Methylenediphosphonic acid (MDP) and hydroxymethanediphosphonic acid (HMDP) are especially preferred.",
"Suitable salts of the diphosphonic acid are with a ‘biocompatible cation’ as defined above.",
"Preferred such diphosphonate salts include: sodium methylenediphosphonate, sodium hydroxymethanediphosphonate, sodium hydroxyethanediphosphonate, sodium ethylenediamine-tetraphosphonate, ammonium methylenediphosphonate, ammonium hydroxymethanediphosphonate, ammonium hydroxyethanediphosphonate and ammonium ethylenediamine-tetraphosphonate.",
"The combination of compounds in the stabiliser for radiopharmaceuticals of the present invention is preferably a combination of sodium 4-aminobenzoate (NapABA) with methylenediphosphonic acid (MDP) or hydroxymethanediphosphonic acid (HMDP).",
"Without wishing to be bound by theory, it is believed that the stabilisers of the present invention act as follows: The amino-substituted aromatic carboxylic acid or its salt, ester or amide has a reducing ability and removes any oxidising free radicals which result from the radiolysis of the solution, and hence inhibits the oxidative destruction of the radiopharmaceutical by free radical attack.",
"On the other hand, when excess tin(II) reductant remains after completion of the radiolabelling, the diphosphonic acid or its salt inhibits the excess stannous ion from reductively degrading the radiopharmaceutical.",
"It is a surprising finding of the present invention that such stabilisers can be present in a single step radiopharmaceutical preparation without either: (i) preventing or hindering the stannous reduction of the radiopharmaceutical precursor, with consequent adverse effect on Rcp; (ii) complexing with the radiometal to form undesirable radioactive impurities (eg.",
"diphosphonic acid complexes of the radionuclide).",
"Accordingly, the amount of the diphosphonic acid or its salt to be added to the radiopharmaceutical depends upon the amount of the reducing tin(II) contained in the radiopharmaceutical preparation or kit.",
"The amount of the diphosphonic acid or its salt effective to stabilise the radiopharmaceutical is 1 to 10 moles per mole of stannous, preferably 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.",
"The radionuclide of the radiopharmaceutical of the present invention is a γ-ray or β-ray emitter, preferably Tc-99m, Re-186 or Re-188, most preferably Tc-99m.",
"γ-ray emitters are mainly used for radiodiagnosis and β-ray emitters are mainly used for radiotherapy.",
"When a radiodiagnostic radiopharmaceutical of this invention is intracorporeally administered to a human being, the level of radioactivity used is in the range of from 370 to 1,480 MBq, preferably from 370 to 1,110 MBq.",
"When a radiotherapeutic agent of the radiopharmaceuticals of this invention is intracorporeally administered to a human being, the radioactivity level is in the range of from 37 to 18,500 MBq, preferably from 370 to 7,400 MBq.",
"The radiopharmaceutical comprises an active ingredient which is susceptible to either degradation by the reducing action of the reductant (present to help effect labelling of the radionuclide), or radiolysis.",
"By using the stabiliser composition of the present invention to stabilise such active ingredients, it is possible to prolong the useful lifetime post-radiolabelling to at least twice that of the prior art.",
"The stabilisers of the present invention are particularly useful when the active ingredient comprises a ligand having a tetradentate diaminedioxime donor set, especially d,l-hexamethylpropyleneamine oxime or HMPAO (exametazime), 4,9-diaza-3,3,10,10-tetramethyldodecane-2,11-dione dioxime (PnAO), and similar compounds.",
"In a second aspect, the present invention provides a non-radioactive kit for the preparation of the stabilised radiopharmaceutical composition described above which comprises: (i) an amino-substituted aromatic carboxylic acid or a salt, ester or amide thereof; (ii) a diphosphonic acid or salt thereof, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"Thus, the stabiliser for radiopharmaceuticals of the present invention can be pre-mixed with the ligand prior to radiolabelling, so that the formulation of a single vial, non-radioactive kit for the preparation of a stabilised radiopharmaceutical (which is not a radionuclide complex of the diphosphonic acid stabilizer) is possible.",
"This simplifies the labelling procedure of the prior art 2-vial kit, shortens the labelling time and diminishes the risk of operator exposure to harmful radiation.",
"The single vial kit of the present invention also has an extended stability post radioactive preparation, which in turn extends the useful lifetime for the user of the kit, eg.",
"a clinician.",
"Suitable non-radioactive kits of the present invention comprise 0.01 to 10 mg of an amino-substituted aromatic carboxylic acid or its salt, ester or amide, and 0.01 to 1 mg of a diphosphonic acid or its salt.",
"When the kit contains stannous, the amount of the diphosphonic acid or its salt effective to stabilise is 1 to 10 moles per mole of stannous.",
"Preferably, the ratio of diphosphonic acid or its salt is 2 to 8 moles per mole of stannous, most preferably 4 to 6 moles per mole of stannous.",
"In a third aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid suitable diphosphonic acid salts are those with a biocompatible cation as described above.",
"Preferably the radiopharmaceutical also comprises stannous in the formulation.",
"Most preferably, the radiopharmaceutical comprises 99mTc.",
"Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.",
"In a fourth aspect, the present invention discloses the use of a diphosphonic acid or a salt thereof as a stabiliser for non-radioactive kits for the preparation of radiopharmaceuticals, with the proviso that the radiopharmaceutical is not a metal complex of the diphosphonic acid.",
"Suitable diphosphonic acid salts are those with a biocompatible cation as described above.",
"Preferably the non-radioactive kit further comprises stannous in the formulation.",
"Most preferably, the non-radioactive kit is for the preparation of a 99mTc radiopharmaceutical.",
"Preferably, the diphosphonic acid or salt thereof used as a stabiliser is methylenediphosphonic acid (MDP), hydroxymethanediphosphonic acid (HMDP), hydroxyethanediphosphonic acid (HEDP), ethylenediaminetetraphosphonic acid (EDTMP), and salts thereof.",
"DESCRIPTION OF THE DRAWINGS FIG.",
"1 compares the change in Rcp of 99mTc-HMPAO prepared from Composition III, Composition III+lactose and a commercially available kit for preparing 99mTc-HMPAO, each of which had been stored for 6 months prior to reconstitution.",
"The present invention is explained in more detail with reference to the following Examples: Example 1 is a comparative example for a two step process (including prior art stabilisers such as cobalt chloride).",
"An Rcp of 99mTc-HMPAO of at least 80% 3 hours after the addition is judged as effective stabilisation.",
"However, as is clear from Table 1, the only single stabiliser which meets this criterion is cobalt chloride.",
"Example 2 shows that 99mTc-HMPAO can be effectively stabilised in a single-step process, using a mixture of two compounds as stabilisers, and that the stability is significantly increased over that achieved using the 2-step process of Example 1.Example 3 provides further data on optimising the combinations of MDP/NapABA and HMDP/NapABA in a single step process.",
"The molar ratio of Sn2+ to MDP/HMDP was kept constant (1:5), and the amounts added were varied.",
"Example 4 shows that lyophilised non-radioactive kits for the preparation of 99mTc radiopharmaceuticals can be prepared using the compositions of the present invention.",
"Lactose (a known cryoprotectant excipient in freeze-dried formulations), is shown to be useful to preserve stannous levels for extended shelf-life periods.",
"Example 5 shows that the combination HMDP/NapABA is also effective as a stabiliser for 99mTc-HMPAO.",
"Example 6 provides data on the stannous or tin(II) content of the freeze-dried kits, as a function of shelf-life storage in the dark at 4° C., and shows that the amount of reducing tin(II) in Composition III+lactose is higher than that in Composition III.",
"Moreover, the amount of the reducing tin(II) in Composition III shows some tendency to decrease with increasing storage time.",
"Accordingly, when the freeze-dried kit is stored for a prolonged period of time, lactose may be used in the kit composition to maintain the amount of the tin(II) reductant constant.",
"Example 7 compares the Rcp after 6-months storage of the freeze-dried kits of the present invention, with the Rcp for 99mTc-HMPAO prepared from a commercially available kit.",
"The Rcp decreases as a function of time post-reconstitution for the commercial kit, but for Composition III and Composition III+lactose, the Rcp's at 6 hours post-reconstitution were at least 80%.",
"This shows that, whilst the stannous levels without lactose do show some modest decrease (Example 5), the decrease does not impair the performance of the kit.",
"Example 8 shows that freeze-dried kits containing the stabiliser composition HMDP/NapABA have an effective shelf-life.",
"Example 9 compares the biodistribution in rats of 99mTc-HMPAO prepared using kit Composition III and kit Composition III+lactose of the present invention, with a commercial 99mTc-HMPAO kit preparation.",
"No significant difference in uptake in target organs was found.",
"This includes the brain, which is the diagnostic imaging application of the present commercial 99mTc-HMPAO agent.",
"These results show that the added compounds (MDP, NapABA and lactose), which are not present in the commercially available kit, do not adversely affect the biological behaviour of the 99mTc radiopharmaceutical.",
"Example 10 describes various lyophilisation methods suitable for freeze-dried kit preparation of formulations of the present invention.",
"Example 11 compares the Rcp of the various freeze-dried kits of Example 10.Example 1 The Rcp of 99mTc-HMPAO Using a Single Stabiliser: Two Step Process (Comparative Example) The radiochemical purity (Rcp) of 99mTc-HMPAO prepared using a commercially available kit (CERETEC™) for preparing 99mTc-HMPAO is 80% at one hour post-reconstitution, and the Rcp decreases thereafter with time.",
"On the other hand, in the case of 99mTc-HMPAO stabilised with cobalt chloride, the Rcp 6 hours after radiolabelling is 80%.",
"The following known medically acceptable compounds and additives were studied: ascorbic acid, sodium ascorbate, gentisic acid, gentisic acid ethanolamide, methylenediphosphonic acid (MDP), succinic acid, 4-aminobenzoic acid (pABA) and sodium 4-aminobenzoate (NapABA).",
"To each of ten vials each containing 0.5 mg of HMPAO and 4.0 μg of Sn2+ (stannous), was added sodium pertechnetate (1.48 GBq in 5 ml of saline) to carry out the radiolabelling.",
"To each of 9 of the ten reconstituted vials was individually added the compound shown in Table 1 at a time of 2 minutes post-radiolabelling.",
"Nothing was added to the remaining vial.",
"At 1 minute and 3 hours after the addition, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).",
"The results are given in Table 1: TABLE 1 Radiochemical purity (Rcp) of 99mTc-HMPAO after the addition of a single stabiliser (%, n = 3).",
"Name of Additive Rcp 1 min from Rcp 3 hours from (amount) addition (%) addition (%) No additive 87 ± 4 38 ± 15 Cobalt chloride 94 ± 3 89 ± 2 (200 μg) Ascorbic acid 83* 41* (4.5 μg) Sodium ascorbate 90* 51* (10 mg) Gentisic acid 87 ± 2 77 ± 3 30 μg) Gentisic acid ethanolamide 61 ± 3 29 ± 13 (30 μg) Methylene diphosphonic acid 79* 30* (10 μg) 4-Aminobenzoic acid 80 ± 8 67 ± 5 (30 μg) Sodium4-amino-benzoate 69 ± 5 39 ± 11 (30 μg) Succinic acid 51* 14* (30 μg) Note: *n = 1 Example 2 Examination of Stabilisation of 99mTc-HMPAO by a Combination of Two Compounds: Single Step Process The combinations studied were: (i) ascorbic acid and hydroxymethanediphosphonic acid (HMDP); (ii) sodium 4-aminobenzoate (NapABA) and methylenediphosphonic acid (MDP).",
"A composition comprising 0.5 mg of HMPAO, 4.0 μg of Sn2+, 1.0 μg of HMDP and 0.5 μg of ascorbic acid (Composition A), and a composition comprising 0.5 mg of HMPAO, 5.4 μg of Sn2+, 40.5 μg of MDP and 0.5 mg of NapABA (Composition B) were prepared.",
"To each of A and B was added sodium pertechnetate (1.48 GBq in 5 ml) to carry out the labelling.",
"After 3 hours, an aliquot was taken from each, and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).",
"The Rcp of 99mTc-HMPAO 3 hours after Tc-99m labelling was about 62% in the case of Composition A and about 80% in the case of Composition B.",
"Example 3 Optimisation of Stabiliser Composition: Single Step Process As shown in Table 2, six different sample compositions were prepared and stored in the dark at 4° C. One vial of each formulation was taken out at each time point (1, 7, 31 and 32-days storage), and sodium pertechnetate (1.48 GBq in 5 ml) was added.",
"At 6 hours post addition of pertechnetate, an aliquot was taken from each vial and the Rcp measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline, filter paper/50% aqueous acetonitrile).",
"The results are shown in Table 2: TABLE 2 Sample Compositions and Rcp of 99mTc-HMPAO 6 hours post-radiolabelling (n = 1, *n = 3).",
"Composition I II III IV V VI Preparation HMPAO 0.5 0.5 0.5 0.5 0.5 0.5 Conditions (mg) NapABA 0.5 0.1 0.1 0.1 0.5 — Sn2+ 5.4 5.4 8.1 8.1 8.1 8.1 (μg) MDP 40.5 40.5 61.0 61.0 — — (μg) HMPD — — — — 66.0 66.0 (μg) Rcp (%) - 1 day 86.4 79.5 82.2 73.8 84.8 ± 2.7* 77.3 ± 5.5* after storage 7 days 80.8 80.9 82.1 81.1 — — time 32 days 79.0 81.8 84.3 66.5 — — 31 days — — — — 83.5 ± 2.2* 72.0 ± 0.2* Note: *n = 3.Compositions II, III and V show that, even in the case of samples stored for 32 days in the dark at 4° C., the Rcp at 6 hours post-reconstitution was at least 80%.",
"Compositions III and V, which exhibited a higher Rcp, 5 were therefore used as the basis for subsequent tests.",
"Example 4 Stabilisation of 99mTc-HMPAO by a Combination of MDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition III of Example 3, and a composition prepared by adding lactose to Composition III (referred to hereinafter as Composition III+lactose) were used to prepare four lots of freeze-dried kit in each case.",
"The amounts of compounds shown below correspond to the scale for a production batch of about 100 freeze-dried kit vials.",
"First, anhydrous stannous chloride (25.8 mg) and MDP (122 mg) were dissolved in 0.1 M hydrochloric acid (1,000 ml).",
"The resulting solution is referred to hereinafter as Solution 1.d,l-HMPAO (100 mg) and NapABA (100 mg) were dissolved in Solution 1 (100 ml), to give a solution referred to hereinafter as Solution 2.Solution 2 was divided into two 50 ml portions, one of which was adjusted to a pH of 8.5-9.0 with sodium hydroxide and thereafter the total volume was adjusted to 10.0 ml with water by use of a measuring cylinder.",
"In the other 50 ml portion was dissolved 300 mg of lactose monohydrate, the resulting solution was adjusted to a pH of 8.5 to 9.0 with sodium hydroxide, and thereafter, the total volume was adjusted to 100 ml with water by use of a measuring cylinder.",
"1.0 ml of each of the solutions obtained was placed in each vial, which was then frozen at −50 to −78° C. and then freeze-dried for about 24 hours.",
"After completion of the freeze-drying, the vials were sealed and the freeze-dried kit was stored in the dark at 4° C. The same procedure was repeated four times to prepare four separate lots of each of, Composition III and Composition III+lactose.",
"Lot Nos.",
"ID-01 to ID-04 were of Composition III and Lot Nos.",
"ID-05 to ID-08 were of Composition III+lactose.",
"The production conditions and results of each lot are shown in Table 3.TABLE 3 Production conditions and results of each lot Freezing Number of pH before temperature Vials Lot No.",
"freezing (° C.) Produced Property# ID-01 9.0 −50 99 Fair ID-02 9.0 −78 90 Bad ID-03 8.5 −78 39 Bad ID-04 9.0 −50 99 Fair ID-05 9.0 −50 91 Good ID-06 9.0 −78 92 Good ID-07 8.5 −78 94 Good ID-08 8.5 −50 91 Good #where ’property' refers to the behaviour during lyophilisation: Good = the cake in the freeze-dried kits kept well-formed; Fair = the cake in the freeze-dried kits was partly broken, but there was no loss of powder from the vials during lyophilisation; Bad = the cake in the freeze-dried kits was completely broken, and some of the powder was lost from the vials during lyophilisation.",
"In all the lots other than ID-03 (which is of Composition III), at least 90 vials were produced.",
"Among the four lots of Composition III, only ID-03 was adjusted to a pH of 8.5 before freezing.",
"Moreover, in the case of Composition III+lactose, even when the pH was adjusted to 8.5 before freezing (ID-07, ID-08), at least 90 vials could be produced.",
"Accordingly, when a freeze-dried kit is produced, it is desirable to adjust the pH to 9.0 in the case of lactose-free Composition III.",
"Example 5 Stabilisation of 99mTc-HMPAO by a Combination of HMDP and NapABA: Single Step Process using a Freeze-Dried Kit Composition V of Example 3, and a composition prepared by adding lactose to Composition V (referred to hereinafter as Composition V+lactose) were used to prepare one lot of freeze-dried kits in each case.",
"The freeze-dried kits were prepared in much the same manner as that of Example 4, except that Solution 1 was slightly different—in this case anhydrous stannous chloride (25.8 mg) and HMDP-2Na (132.0 mg) were dissolved in 0.1M hydrochloric acid (1,000 mL).",
"Lot Nos.",
"ID-09 was of Composition V and Lot Nos.",
"ID-10 was of Composition V+lactose.",
"The production conditions and results of each lot are shown in Table 4.TABLE 4 Production conditions and results of each lot.",
"Freezing Number of pH before Temperature Vials freezing (° C.) Produced Property# ID-09 8.8 −50° C. 49 Fair ID-10 8.8 −50° C. 45 Good #defined in Example 4.Example 6 Determination of the Amount of tin(II) in the Freeze-Dried Kit as a Function of Storage Time Vials of Composition III (ID-01, O2, 03 and 04), Composition III+lactose (ID-05, 06, 07 and 08), Composition V (ID-09) and Composition V+lactose (ID-10) which had been stored in the dark at 4° C. for periods of 1 day, 3-months and 6-months, were allowed to warm to room temperature.",
"The amount of tin(II) was then determined by an absorbance method.",
"The results obtained are shown in Table 5.TABLE 5 Amounts of reducing tin(II) determined (μg/vial, n = 2).",
"1-day storage 3-month storage 6-month storage Composition III 9.5 ± 0.2 9.1 ± 0.5 8.7 ± 0.5 Composition III + 10.3 ± 0.5 10.5 ± 0.5 10.8 ± 0.4 lactose Composition V 10.1 ± 0.0 — — Composition V + 10.3 ± 0.3 — — lactose Note Each value was an average value of 4 lots.",
"Example 7 Radiochemical Purity of Freeze-Dried Kits Containing MDP and NapABA Studied over a 6-Month Shelf-Life Storage Period Four lots of each of Composition III and Composition III+lactose produced in Example 4 were subjected to examination of radiochemical purity after 6 -months storage in the dark at 4° C. Vials of freeze-dried Composition III (ID-01, O2, 03 and 04) and Composition III+lactose (ID-OS, 06, 07 and 08) were stored for 6 months in the dark at 4° C. After a storage period of 1 day, 1, 3 and 6 months, the vials were allowed to warm to room temperature, and Tc-99m labelling was carried out.",
"Thus, to one vial of each lot was added sodium pertechnetate (1.48 GBq in 5 ml).",
"Aliquots were taken after times of 2 minutes, one hour, 3 hours and 6 hours and the Rcp was measured by a combination of three chromatographic systems (stationary phase/developing solvent: silica gel/methyl ethyl ketone, silica gel/saline and filter paper/50% aqueous acetonitrile).",
"The results obtained at 6 hours are shown in Table 6.When Composition III and Composition III+lactose were radiolabelled after 6-month shelf-life storage, simultaneously, sodium pertechnetate (1.48 GBq/5 ml) was added to a commercially available kit for preparing 99mTc-HMPAO (containing 0.5 mg of HMPAO and 4.0 μg of Sn2+) as a control.",
"An aliquot thereof was taken after times of one minute, one hour, 3 hours and 6 hours, and then the Rcp was measured in the same manner.",
"The results obtained are shown in FIG.",
"1.TABLE 6 Rcp of 99mTc-HMPAO at 6 hr post reconstitution by a combination of MDP and NapABA as a function of storage time.",
"STORAGE PERIOD 1-day 1-month 3-months 6-months Rcp Composition III 86.4 ± 2.2 87.4 ± 1.5 86.5± 2.7 88.4 ± 3.4 (%) Composition III + 86.2 ± 2.3 86.2 ± 2.4 87.9 ± 2.6 85.6 ± 2.7 Lactose Example 8 Radiochemical Purity of Freeze-Dried Kits Containing HMDP and NapABA for 1-Month Shelf-Life Storage One lot of each of Composition V and Composition V+lactose produced in Example 5 were subjected to examination of radiochemical purity for 1-month storage in the dark at 4° C. The Rcp was measured in the same manner.",
"The results obtained are shown in Table 7.TABLE 7 Rcp of 99mTc-HMPAO at 6 hr post-reconstitution stabilised by a combination of HMDP and NapABA as a function of storage time.",
"Storage Time 1-day 1-month Rcp Composition V 84.8 ± 2.7 83.5 ± 2.2 (%) Composition V + 87.3 ± 2.8 86.0 ± 1.0 Lactose Example 9 Biodistribution in the Rat To one vial of each of Composition III (ID-O2) and Composition III+lactose (ID-06) obtained in Example 4 was added sodium pertechnetate in a proportion of 1.48 GBq/5 ml to carry out the radiolabelling.",
"After 2 hours, 3.0 to 3.7 MBq of the reconstituted solution was administered to a female Sprague-Dawley strain rat (body weight 140-170 g) which had been previously anaesthetised with sodium thiopentobarbital in the tail vein.",
"At one hour post-administration, the animal was sacrificed and the radioactivity in each organ was measured using a NaI single channel analyser.",
"Separately, sodium pertechnetate was added in a proportion of 1.48 GBq/5 ml to the commercially available kit for preparing 99mTc-HMPAO.",
"In the same manner, at 15 minutes post-reconstitution, 3.0-3.7 MBq of the solution was administered to the same kind of rat, and the biodistribution of radioactivity in each organ measured.",
"The results obtained are shown in Table 8: TABLE 8 Biodistribution of Commercial 99mTc-HMPAO, Composition III (ID-02) and Composition III + lactose (ID-06) in the female rat at one hour post administration (upper row: % ID/organ, lower row: % ID/g, n = 5).",
"Composition III + Commercial Composition lactose 99mTc-HMPAO III (ID-02) (ID-06) Brain 1.66 ± 0.13 1.65 ± 0.10 1.73 ± 0.15 0.96 ± 0.07 0.96 ± 0.15 1.01 ± 0.07 Blood 15.38 ± 0.72 14.41 ± 1.68 14.07 ± 1.51 1.56 ± 0.04 1.64 ± 0.06 1.45 ± 0.18 Heart 0.81 ± 0.04 0.85 ± 0.03 0.90 ± 0.10 1.61 ± 0.08 1.75 ± 0.10 1.72 ± 0.03 Lung 3.41 ± 0.20 3.15 ± 0.18 3.39 ± 0.38 4.05 ± 0.28 4.26 ± 0.29 4.14 ± 0.36 Liver 5.91 ± 0.20 6.49 ± 0.48 7.68 ± 0.38 1.10 ± 0.07 1.36 ± 0.13 1.44 ± 0.14 Spleen 0.78 ± 0.10 0.79 ± 0.18 0.86 ± 0.12 2.17 ± 0.12 2.24 ± 0.12 2.25 ± 0.21 Kidney 4.27 ± 0.27 5.04 ± 0.48 4.81 ± 0.31 4.39 ± 0.16 4.78 ± 0.64 4.46 ± 0.21 Stomach 1.41 ± 0.54 2.15 ± 0.46 1.70 ± 0.79 0.49 ± 0.17 1.24 ± 0.43 0.93 ± 0.54 Small 17.27 ± 0.85 17.61 ± 0.87 20.23 ± 1.50 Intestine 2.86 ± 0.17 3.47 ± 0.33 3.94 ± 0.46 Large 2.08 ± 0.25 2.14 ± 0.34 2.05 ± 0.24 Intestine 0.35 ± 0.03 0.40 ± 0.04 0.34 ± 0.03 Skin 0.78 ± 0.61 0.68 ± 0.55 0.51 ± 0.34 0.39 ± 0.04 0.37 ± 0.09 0.31 ± 0.09 Muscle 0.91 ± 0.49 0.67 ± 0.41 0.49 ± 0.21 0.28 ± 0.03 0.29 ± 0.03 0.16 ± 0.02 Urine 12.36 ± 1.18 11.78 ± 2.18 12.07 ± 1.11 Carcass 36.96 ± 0.83 37.76 ± 2.18 33.95 ± 1.37 0.34 ± 0.02 0.37 ± 0.02 0.31 ± 0.01 Example 10 Methods of Freeze-Dried Kit Preparation The freeze-dried kit (Composition V+lactose of Example 5) was prepared by two methods.",
"The first method (ID-11) was similar to that of Example 5, but with a modified Solution 2—thus, d,l-HMPAO (250 mg),NapABA (250 mg) and lactose monohydrate (1.5 g) were dissolved in Solution 1 (250 mL).",
"The solution was added to water (250 mL) and adjusted to pH 8.9.The production conditions and results for ID-11 are shown in Table 9.The second method (ID-12) is described below.",
"Thus, lactose monohydrate (3.03 g), NapABA (505 mg), d,l-HMPAO (505 mg), HMDP (66.6 mg) and anhydrous stannous chloride (13.1 mg) were dissolved in 0.1M hydrochloric acid (1000 mL) in the order given.",
"The solution was then adjusted to pH 9.4.The production conditions and results for ID-12 are shown in Table 9.TABLE 9 Production conditions and results of each lot Freezing Number pH before Temperature of Vials freezing ° ( C.) Produced Property# ID-11 8.9 −40° C. 290 Good ID-12 9.4 −40° C. 240 Good #as defined in Example 4.Example 11 RCP of Freeze-Dried Kits Prepared by Different Methods The freeze-dried kits in Example 10 were used to examine the RCP.",
"The RCP was measured in the same manner as in Example 7.The results obtained are shown in Table 10.TABLE 10 Rcp of 99mTc-HMPAO at the time after reconstitution 2 min 1 hour 3 hours 6 hours ID-11 90.2 ± 0.4 89.3 ± 1.8 85.9 ± 1.7 85.7 ± 1.7 ID-12 87.1 ± 2.1 81.4 ± 2.4 70.6 ± 1.7 60.7 ± 4.0"
]
] | Patent_10296952 |
[
[
"METHODS FOR MEDIATING GENE SUPPRESSION",
"The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.",
"The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.",
"It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs."
],
[
"1.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"2.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"3.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"4.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"5.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"6.A method according to any one of claims 1 to 5, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.",
"7.A method according to any one of claims 1 to 6, wherein the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.",
"8.A method according to any one of claims 1 to 7, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.",
"9.A method according to any one of claims 1 to 8, wherein the factor is selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"10.A method according to any one of claims 1 to 9, wherein the RNAi factor is an res sequence.",
"11.A method according to claim 10, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"12.A method according to claim 11, wherein the res sequence is obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.13.A method according to claim 11, wherein the res sequence is represented by Seq ID Nos 1 to 4.14.A method according to any one of claims 1 to 13, wherein the cell is a eukaryotic cell.",
"15.A method according to 14, wherein the eukaryotic cell is a mammalian.",
"16.A method according to claim 1 or claim 2, wherein the cell is a Schizosaccharomyces pombe cell.",
"17.A method according to any one of claims 1 to 16, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.",
"18.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"19.A pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"20.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"21.A cell according to claim 20, wherein the cell is a eukaryotic cell.",
"22.A cell according to claim 20 or claim 21, wherein the cell is a Schizosaccharomyces pombe cell.",
"23.A method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.",
"24.A method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"25.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"26.A method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"27.A method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"28.A method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"29.A method according to any one of claims 23 to 28, wherein the target nucleic acid is an exogenous nucleic acid or a part thereof.",
"30.A method according to any one of claims 23 to 29, wherein the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.",
"31.A method according to any one of claims 23 to 30, wherein the cell is a eukaryotic cell.",
"32.A method according to 31, wherein the eukaryotic cell is a mammalian.",
"33.A method according to claim 23 or claim 24, wherein the cell is a Schizosaccharomyces pombe cell.",
"34.A method according to any one of claims 23 to 33, wherein the antisense nucleic acid corresponds to a part of the target nucleic acid.",
"35.A cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"36.A cell according to claim 35, wherein the cell is a eukaryotic cell.",
"37.A cell according to claim 35 or claim 36, wherein the cell is a Schizosaccharomyces pombe cell.",
"38.Method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.",
"39.A factor identified by the method of claim 38.40.A factor according to claim 39, wherein the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.",
"41.A factor according to claim 39 or claim 40, wherein the factor is selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"42.A factor according to any one of claims 39 to 41, wherein the factor is an res sequence.",
"43.A factor according to claim 42, wherein the factor is selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"44.An RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.45.An RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.46.A Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.",
"Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.",
"It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.",
"One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.",
"The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.",
"Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.",
"The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.",
"These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.",
"The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.",
"The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.",
"Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.",
"The second strategy for finding key players in RNAi has involved the use of cell free assays.",
"These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.",
"However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.",
"Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.",
"This would enable the identification of factors which can enhance or reduce the gene silencing effect.",
"It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.",
"Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.",
"This is indicative of an RNAi-like mechanism of gene suppression.",
"It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.",
"This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.",
"The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.",
"The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.",
"According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.",
"Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.",
"It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.",
"Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.",
"More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"Even more preferred is an RNAi factor which is an res sequence.",
"Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.",
"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.",
"According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.",
"As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.",
"According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.",
"Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.",
"Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.",
"Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.",
"However it will be understood that the entire native sequence of the target nucleic acid may be employed.",
"Conveniently, the target nucleic acid may be coupled to a selectable marker.",
"The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.",
"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.",
"According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.",
"According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.",
"According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.",
"The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.",
"More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"Even more preferred is a factor having an res sequence.",
"Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.",
"The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.",
"The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.",
"This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).",
"The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).",
"It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.",
"The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.",
"It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.",
"The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.",
"The effective concentration range can be determined using routine methodology known in the art.",
"Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.",
"Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.",
"The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics."
],
[
"TECHNICAL FIELD The present invention is concerned with methods for enhancing gene suppression in cells and in particular it is concerned with improved methods for enhancing RNAi-mediated gene silencing by manipulation of factors associated with RNAi.",
"The present invention is also concerned with methods for identifying factors which down-regulate as well as those which up-regulate RNAi.",
"It is also concerned with genetic constructs useful for enhancing or modulating gene silencing and cells harbouring such constructs.",
"BACKGROUND OF THE INVENTION The use of double-stranded RNA (dsRNA) to specifically interfere with gene expression has received considerable attention because of its demonstrated potency in a range of organisms, including some which have so far been genetically intractable.",
"Termed RNA interference (RNAi), it has been implicated in viral defense, control of transpositional elements, genetic imprinting and endogenous gene regulation.",
"It has been hypothesised to be the central mechanism in post-transcriptional gene silencing (PTGS), co-suppression, quelling, and antisense RNA-mediated gene suppression.",
"One model that has been proposed is that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and then dissociated and free to attack homologous mRNA by RNA nuclease-mediated degradation.",
"The application of this technique will greatly facilitate the dissection of gene function and the validation of genes involved in disease states.",
"Recently at least two different strategies have been undertaken to identify the cellular proteins composing a proposed multi-protein complex involved in the recognition of dsRNA and the activation of dsRNA-mediated gene interference.",
"The first involves the use of classical chemical mutagenesis or insertional mutagenesis to isolate mutants completely defective for RNAi and cloning of the relevant genes using complementation.",
"These studies have been carried out in genetically tractable organisms such as plants, worms and fungi.",
"The genetic screens described involve the use of RNAi systems in which the degree of suppression is complete.",
"The mutagenesis produces mutants in which the RNAi effect is completely reversed indicating the loss of a cellular factor (function) required for the RNAi effect.",
"Thus these genetic screens would most likely miss factors that have subtle effects or rate limiting or rate determining roles in RNAi.",
"The second strategy for finding key players in RNAi has involved the use of cell free assays.",
"These in vitro reconstitution assays, on the other hand, identify cellular factors that impact on RNAi outside of the cellular context and therefore the cellular role of these factors must always be tested.",
"However, the major disadvantages of these strategies are that genes will not be identified if they are essential to the organism, nor will they directly identify gene activities which will enhance RNAi when overexpressed.",
"Thus there is a need for models which can demonstrate a range of RNAi efficacies, with both increasing and decreasing quantitative activities being selectable.",
"This would enable the identification of factors which can enhance or reduce the gene silencing effect.",
"It is therefore an object of the present invention to overcome or at least ameliorate one or more disadvantages of the prior art, or provide a useful alternative.",
"SUMMARY OF THE INVENTION Through the use of a fission yeast model for the study of dsRNA-mediated gene silencing and in the search for factors involved in this process, it was surprisingly found that the natural level of RNAi activity can be enhanced by manipulating factors associated with RNAi activity or efficacy.",
"Thus, it has been found that by increasing the steady-state levels of a target nucleic acid sequence in the presence of the same pool of the corresponding antisense sequence, or a part thereof, the antisense-mediated suppression was not only maintained, but enhanced.",
"This is indicative of an RNAi-like mechanism of gene suppression.",
"It has also been found that overexpression of certain sequences, named herein RNAi enhancing sequences (res), (also referred to herein as anti-sense enhancing sequences—aes), also had the ability to enhance RNAi.",
"This ability of RNAi activity to be enhanced in S. pombe provides a model system which enables the analysis of RNAi processes and the identification and study of factors which either up-regulate or down-regulate its activity.",
"The system has been further used to identify RNAi enhancing gene sequences which increase PTGS efficacy when their resulting protein activities are augmented in vivo.",
"The model used to exemplify the present invention and the methods described are also applicable to treatment of disorders in which gene expression requires more efficient modulation or silencing.",
"According to a first aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) elevating in the cell the level of an RNAi factor, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to a second aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of target nucleic acid expression, which method comprises elevating the level of an RNAi factor in a cell that expresses said target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to a third aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"According to a fourth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) elevating the level of an RNAi factor in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"According to a fifth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) elevating the level of an RNAi factor in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.",
"Preferably the level of the RNAi factor is elevated by introducing into the cell additional copies of, or agents which give rise to, the RNAi factor.",
"It will be understood therefore that up-regulating the expression of an endogenous RNAi factor will also achieve the same result and is contemplated herein as part of the invention.",
"Preferably the factor is selected from the group consisting of a gene, cDNA, RNA or a protein.",
"More preferred is a factor selected from the group consisting of a transcriptional activator of the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"Even more preferred is an RNAi factor which is an res sequence.",
"Also for preference the factor can be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"Further preferred factors are represented by the res sequences which are obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.Preferably the res sequence is represented by any one of Seq ID Nos 1 to 4.The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.",
"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.",
"According to a sixth aspect there is provided a pharmaceutical composition for use in performing the method of any one of the previous aspects comprising (i) an expressible nucleic acid encoding, or capable of increasing or decreasing the expression of, an RNAi factor; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"According to a seventh aspect there is provided a pharmaceutical composition for use in performing the method of any one of claims 2 to 17 comprising (i) an nucleic acid which is the target nucleic acid or a part thereof, or an expressible nucleic acid encoding a factor capable of elevating the intracellular level of the target nucleic acid; (ii) a nucleic acid encoding a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid; and (iii) a pharmaceutically acceptable carrier, wherein the nucleic acids of (i) and (ii) may be situated on the same or different molecules.",
"According to an eighth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses a target nucleic acid and (ii) comprises an elevated level of an RNAi factor, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"For preference the cell is a eukaryotic cell but more preferred is a mammalian cell.",
"As indicated above, in certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"According to a ninth aspect there is provided a method for inhibiting the expression of a target nucleic acid in a cell, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the cell, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of said target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of said target nucleic acid.",
"According to a tenth aspect there is provided a method of increasing cellular susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which method comprises augmenting the level of the target nucleic acid or a part thereof in a cell expressing the target nucleic acid, with the proviso that the cell is to have prior, concurrently or subsequently introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"According to an eleventh aspect there is provided a method for treating a subject suffering from a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of said target nucleic acid or a part thereof in the subject's cells where the target nucleic acid is expressed, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid, thereby treating the subject.",
"According to a twelfth aspect there is provided a method for inhibiting in a subject the onset of a disorder whose alleviation is mediated by inhibiting the expression of a target nucleic acid, which method comprises the steps of (i) augmenting the level of the target nucleic acid or a part thereof in the subject's cells where the target nucleic acid would be expressed if the subject were suffering from the disorder, and (ii) prior, concurrently with or subsequent to performing step (i), introducing into such cells a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid, under conditions permitting an increase in the degree to which the anti-sense nucleic acid would inhibit expression of the target nucleic acid were such expression to occur, thereby inhibiting in the subject the onset of the disorder.",
"According to a thirteenth aspect there is provided a method of determining whether inhibiting the expression of a particular target nucleic acid or the activity of its product may alleviate a disorder, which method comprises the steps of (i) augmenting the level of the target nucleic acid in a cell whose phenotype correlates with that of a cell from a subject having the disorder; (ii) prior, concurrently with or subsequent to performing step (i), introducing into the cell a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting an increase in the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid; and (iii) determining whether the cell's phenotype now correlates with that of a cell from a subject in whom the disorder has been alleviated or the disorder is not evident, thereby determining whether inhibiting the expression of the target nucleic acid or the activity of its product may alleviate the disorder.",
"In a preferred embodiment the target nucleic acid is an endogenous nucleic acid or a part thereof, but it may also be an exogenous sequence or part thereof.",
"Preferably the level of the target nucleic acid is augmented by introducing into the cell additional copies of, or agents which are capable of inducing intracellular over-expression of, the target nucleic acid.",
"Over-expression can be achieved for an endogenous as well as an exogenous target nucleic acid.",
"Preferably the nucleic acid used for augmenting content of the target nucleic acid is a fragment, derivative or analogue of the target nucleic acid.",
"However it will be understood that the entire native sequence of the target nucleic acid may be employed.",
"Conveniently, the target nucleic acid may be coupled to a selectable marker.",
"The preferred cell is a eukaryotic cell and even more preferred is a mammalian cell.",
"In certain embodiments of the invention described herein the preferred cell is a Schizosaccharomyces pombe cell.",
"Preferably the antisense nucleic acid corresponds to a part only of the target nucleic acid.",
"According to a fourteenth aspect there is provided a cell having increased susceptibility to anti-sense-mediated inhibition of a target nucleic acid expression, which cell (i) expresses said target nucleic acid and (ii) comprises an elevated level of said target nucleic acid, with the proviso that the cell is to have introduced thereinto a molecule which is, or gives rise to, an anti-sense nucleic acid directed toward at least a portion of the RNA transcript of the target nucleic acid under conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid inhibits expression of the target nucleic acid.",
"The preferred cell is a eukaryotic cell and even more preferred is a Schizosaccharomyces pombe cell.",
"According to a fifteenth aspect there is provided a method of identifying a cellular factor capable of effecting and/or modulating expression of a target nucleic acid in a cell having the target nucleic acid and a nucleic acid which is an antisense of the target nucleic acid or part thereof, which method comprises over-expressing said factor in the cell and wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.",
"According to a sixteenth aspect there is provided a factor identified by the method of the fifteenth aspect.",
"The preferred factors can be selected from the group consisting of a gene, cDNA, RNA or a protein.",
"More preferred are factors selected from the group consisting of a transcriptional activator or the antisense nucleic acid, a component of the RNAi machinery, a component of the DNA replication machinery and a component of translational machinery.",
"Even more preferred is a factor having an res sequence.",
"Preferred factors can also be selected from the group consisting of ATP-dependent RNA helicase (ded1), transcriptional factor thi1, DNA replication protein sna41, ribosomal protein L7a, elongation factor EF-Tu and res1 as herein defined.",
"According to a seventeenth aspect there is provided an RNAi factor which is an res sequence obtainable from transformed cells designated herein W18, W20, W21, W23, W27, W28, W30, W32 and W47.According to an eighteenth aspect there is provided an RNAi factor which is an res sequence represented by Seq ID Nos 1 to 4.According to a nineteenth aspect there is provided a Schizosaccharomyces pombe cell having a target nucleic acid or a part thereof and a antisense nucleic acid or a part thereof which corresponds to the target nucleic acid or a part thereof, wherein the expression of the target nucleic acid is capable of being enhanced or only partially suppressed.",
"The term “inhibiting expression of a target nucleic acid” as used in the context of the present invention is intended to encompass, but not be limited to, reduction or elimination of gene expression, whether or not the target nucleic acid is a gene, or a part thereof, introduced into the cell or it is an endogenous gene.",
"The term “RNAi factor” is intended to include in its scope any naturally occurring, modified or synthetic molecule capable of enhancing RNAi activity.",
"This definition includes in its scope the factors referred to herein as RNAi enhancing sequences (res).",
"The term res may be used herein interchangeably with the term aes (anti-sense enhancing sequences).",
"It will be understood by those skilled in the art that “elevating RNAi factor level” can be achieved by transfection, upregulation or other means known in the art.",
"The term “anti-sense nucleic acid molecule” as used in the context of the present invention is intended to encompass RNA or DNA and contain region(s) related to a specific target RNA transcript or its gene.",
"It is also intended to include molecules giving rise to antisense sequences, including inverted repeat, sense RNA, etc.",
"The term “conditions permitting the RNAi factor to increase the degree to which the anti-sense nucleic acid molecule inhibits expression of the gene” is intended to include the concept of inhibition of gene expression which is high enough to be effective, but not so high as to harm the cell.",
"The effective concentration range can be determined using routine methodology known in the art.",
"Reference to a “subject” is intended to include human, animal (mouse), plant, or other life forms.",
"Reference to a “cell” is intended to encompass eukaryotic cells such as yeast, mammalian, plant, etc The term “disorder” will be understood to include viral infection (HIV, HPV, etc), cancer, autoimmune disease and other chronic and acute diseases.",
"The term “phenotype” as used in the context of the present invention is intended to include cell staining, morphology, growth, and similar characteristics.",
"BRIEF DESCRIPTION OF THE FIGURES FIG.",
"1.Target genes and antisense fragments.",
"a, The strain KC4-6 expresses low levels of lacZ RNA.",
"The lacZ expression cassette is integrated at the ura4 locus on chromosome III and is composed of the SV40 early promoter, the E. coli lacZ gene and the SV40 3′ processing signal.",
"The 5′ and 3′ flanking DNA sequences of the ura4 locus are as indicated.",
"b, The strain RB3-2, which expresses high levels of lacZ RNA, contains an expression cassette containing the S. pombe adh1 promoter, the lacZ coding region, and the S. pombe ura4 3′ processing signal.",
"c, The full-length 3.5 kb antisense lacZ fragment is shown.",
"The crippled sense fragment was generated by end-filling the ClaI cut lacZ vector and religating on itself.",
"d, The AML1 strain contains a c-myc-lacZ fusion cassette integrated at the ura4 locus.",
"Exons 2 and 3 of the human c-myc gene were employed as a target.",
"The relative position of the c-myc antisense fragment is shown.",
"Transcription initiation sites are indicated by the bent arrows.",
"The straight arrows represent the normal direction of transcription for a particular DNA fragment.",
"FIG.",
"2.dsRNA-mediated gene silencing in fission yeast.",
"a, A fission yeast strain containing the integrated lacZ gene under control of the strong adh1 promoter was transformed with lacZ antisense vectors.",
"The antisense gene was under control of the weak nmt41 promoter (low antisense), the strong nmt1 promoter in single copy (15) (medium antisense), or the nmt1 promoter in multi-copy (high antisense).",
"For the strain expressing maximum antisense RNA two episomal nmt1-driven antisense vectors containing different selectable markers were co-transformed into the target strain.",
"b, A fission yeast strain containing the integrated lacZ gene under control of the weak SV40 promoter (low target) or strong adh1 promoter (high target) was transformed with the antisense lacZ vector or antisense lacZ and sense lacZ vectors.",
"Strains were co-transformed with vectors containing appropriate selectable markers to complement auxotrophy.",
"c, The lacZ inverted repeat vector was expressed in the high-expressing lacZ target strain.",
"The strain expressing the antisense construct only is also indicated.",
"d, A fission yeast strain containing an integrated c-myc-lacZ fusion gene was transformed with the sense construct, the antisense construct, or both.",
"Antisense and sense constructs were also expressed in the high-expressing lacZ strain.",
"All strains were transformed with appropriate control plasmids to complement auxotrophy.",
"FIG.",
"3.Effect of co-expressing single copies of sense and antisense genes.",
"Strains containing target lacZ alone (RB3-2), the target and a single copy of the antisense gene (K40-7), and the target and both the antisense and frameshifted lacZ genes (M62-1) were assayed for β-galactosidase activity.",
"pREP2 and pREP4 are parental plasmids containing the LEU2 and ura4 selectable markers respectively.",
"Strains were transformed with the appropriate control plasmids to complement auxotrophy.",
"For each sample three independent colonies were assayed in triplicate.",
"FIG.",
"4.RNA expression profiles in fission yeast strains a, The target strain RB3-2 was transformed with the lacZ inverted repeat vector (pM53-1) or co-transformed with antisense (pGT2) and sense (pM54-3) vectors and grown to mid-log in the absence of thiamine.",
"Total RNA from each strain was separated on a 1% denaturing agarose gel, transferred to a nylon membrane and probed with the radioactively labeled 2.2 kb nmt1 fragment.",
"The episomal lacZ signal was normalized with the endogenous nmt1 transcript and quantitated by phosphorimage analysis.",
"The relative level of episomal lacZ expression is shown.",
"b, The strain M38-1 which contains a single copy stably integrated lacZ inverted repeat gene was grown to mid-log in the absence of thiamine, its RNA isolated and analyzed by Northern hybridization.",
"The relative steady-state level of the “panhandle” lacZ RNA is indicated in comparison to episomally expressed antisense lacZ RNA.",
"FIG.",
"5.In vivo dsRNA assay.",
"a, The constructs employed for assaying the ability of the lacZ inverted repeat to form an intramolecular RNA duplex are shown.",
"Each vector contains a functional lacZ fragment which gives the transformed strain a blue color phenotype when grown in the presence of X-GAL.",
"The predicted secondary structure of the pM81-2 generated transcript is indicated.",
"b, The strain 2037 was transformed with the vectors pM85-1, pM91-1 and pM81-2 in the absence of thiamine Single colonies were streaked on minimal media plates and overlayed with 0.5% agarose medium containing 500 μg/ml X-gal and 0.01% SDS.",
"FIG.",
"6.The effect of ded1 on dsRNA-mediated gene regulation.",
"a, The ded1 gene was co-expressed in a fission yeast strain containing the integrated lacZ gene.",
"β-galactosidase activity of the strain transformed with and without the antisense lacZ vector is shown.",
"b, The target strain was transformed with a vector expressing a short lacZ antisense gene or with both the short lacZ antisense vector and the ded1-expressing vector.",
"Strains were transformed with appropriate control plasmids to complement auxotrophy.",
"FIG.",
"7.Over-expression of thi1 in an antisense lacZ expressing strain.",
"β-galactosidase activity of the target strain RB3-2 transformed with and without the antisense lacZ vector is shown.",
"Over-expression of the thi1 gene in the antisense expressing strain is also indicated.",
"Strains were transformed with appropriate control plasmids to complement auxotrophy.",
"FIG.",
"8.Over-expression screening strategy for RNAi modulating factors.",
"A target strain containing the integrated lacZ gene under control of the adh1 promoter and the episomal vector containing the nmt1-driven lacZ antisense gene is transformed with an S. pombe cDNA library.",
"Library fragments are driven by the nmt1 promoter.",
"Transformants are individually screened for a change in the lacZ-encoded blue colony colour phenotype.",
"These transformants are then quantitatively assayed for β-galactosidase activity in the presence and absence of thiamine.",
"The antisense vector is then segregated out of transformants showing a cDNA dependent modification of lacZ suppression.",
"A tertiary β-galactosidase assay is performed to determine if the effect is dependent on the presence of dsRNA.",
"cDNA vectors are recovered from strains of interest, sequenced, and subjected to BLASTN analysis.",
"FIG.",
"9.Over-expression screen of a S. pombe cDNA library.",
"a, Transformants were grown on minimal media plates and over-layed with X-GAL-containing medium.",
"Those which showed a reduced blue colour-phenotype (black arrow) were analysed further.",
"Transformants demonstrating an enhanced blue-colour phenotype were also identified (white arrow).",
"b, Transformants which showed a visual reduction in the blue phenotype were assayed for β-galactosidase activity in liquid culture in the absence of thiamine.",
"Thiamine was added to the medium to demonstrate that the enhanced lacZ suppression was dependent on RNA expression.",
"Transformants were again assayed for β-galactosidase activity following antisense vector segregation.",
"c, Over-expression of unique res genes in dsRNA-expressing strains.",
"FIG.",
"10.lacZ panhandle-mediated gene silencing.",
"(A) The lacZ panhandle construct contains the full-length crippled lacZ gene which is followed by the inverted 5′ 2.5 kb lacZ fragment.",
"Intramolecular hybridization generates an RNA with 2.5 kb RNA duplex and a 1 kb loop.",
"(B) The relative steady-state level of the episomally expressed panhandle lacZ RNA (7.0 kb) is shown in comparison to episomally expressed antisense lacZ RNA (4.5 kb).",
"The lacZ signals were normalized to the endogenous nmt1 transcript (1.3 kb) and quantitated by phosphorimage analysis.",
"(C) The target strain was transformed with the episomally expressed lacZ panhandle and analyzed for b-galactosidase activity.",
"The appropriate plasmids were co-introduced to complement auxotrophy.",
"At least three independent colonies were assayed in triplicate for each strain.",
"Transformants were assayed in the presence of thiamine to abrogate expression of the panhandle RNA (hatched).",
"(D) The target lacZ strain was transformed with the panhandle vector only or both the panhandle and aes2 vector.",
"FIG.",
"11.Co-expression of ded1 and lacZ antisense genes.",
"b-galactosidase activity of the target strain co-transformed with the ded1 gene and the antisense lacZ vectors is shown.",
"Ded1 was expressed from the ura4-based plasmid pREP4 while the antisense genes were expressed from the LEU2-based plasmid pREP2.Strains expressing antisense RNAs or ded1 RNA alone are also indicated.",
"Strains were transformed with appropriate control plasmids to complement auxotrophy.",
"Three independent colonies were assayed in triplicate for each strain.",
"FIG.",
"12.DNA sequence for aes1 factor FIG.",
"13.DNA sequence for aes2 factor FIG.",
"14.DNA sequence for aes3 factor FIG.",
"15.DNA sequence for aes4 factor DESCRIPTION OF THE PREFERRED EMBODIMENTS Using the lacZ fission yeast model to investigate features of antisense-RNA technology in vivo (1-4), it has been shown that the degree of target gene suppression is dependent on the level of antisense RNA (FIG.",
"2A)(1).",
"During the course of developing this model it was surprisingly found that by increasing the steady-state levels of the lacZ target mRNA 20-fold in the presence of the same pool of antisense RNA that antisense-mediated suppression was not only maintained, but enhanced (FIG.",
"2B).",
"To verify that this effect was independent of the target mRNA a lacZ gene containing a frameshift mutation (leading to an inability to translate the β-galactosidase enzyme) was co-expressed in antisense-expressing strains.",
"Again, additional stimulation of lacZ inhibition was observed (FIG.",
"2B).",
"These observations are consistent with the potential formation of additional dsRNA and consequent stimulation of gene silencing.",
"They also indicate that the dsRNA-mediated gene silencing is dose-dependent in this system.",
"To further show that dsRNA was the key component in target gene inhibition in fission yeast dsRNA was generated by expressing a lacZ inverted repeat with 2.5 kb of internal complementarity in the presence of lacZ target mRNA.",
"Expression of this construct also reduced β-galactosidase activity in a lacZ expressing strain to a similar level to the full-length antisense RNA (FIG.",
"2C).",
"Overall, these data suggested that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, resulted in efficient interference of target gene expression in S. pombe.",
"To test the ability of dsRNA to specifically interfere with another target sequence in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (3).",
"A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene was previously found to suppress β-galactosidase activity within the c-myc-lacZ fusion target strain by 47% (3).",
"β-galactosidase assays demonstrated that co-expression of the antisense and sense c-myc constructs in the target strain enhanced c-myc suppression compared with the antisense c-myc vector alone (FIG.",
"2D).",
"Transformation of the strain expressing only the lacZ target with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is sequence-specific (FIG.",
"2D).",
"All of the above observations, as well as the reduction in target mRNA levels in response to additional dsRNA (data not shown), are consistent with features associated with RNAi.",
"This is the first example of dsRNA-mediated gene regulation in yeast and, unlike RNAi in all other organisms except zebrafish, appears to be dependent on the concentration of intracellular dsRNA.",
"The variation in RNAi efficacy between organisms may be due to the absence or under-expression of some components of the RNAi pathway or the presence of RNAi inhibitors.",
"We have now further developed this genetic model for identification of factors involved in RNAi.",
"Without wishing to be bound by any particular mechanism, it is probable that a multi-protein complex exists which mediates the post-transcriptional degradation of target mRNA in RNAi.",
"Several genes which are involved in the RNAi phenomenon have been identified through genetic screens in Neurospora and nematodes.",
"These genes include an RNA-dependent RNA polymerase (qde-1, ego-1) a RecQ DNA helicase (qde-3), an RNase D homologue (mut-7), and a putative translation initiation factor (rde-1, qde-2).",
"The genes mut-2, rde-2, rde-4, and rde-7 have also been identified as being involved in either the initiation or maintenance of RNAi.",
"Additionally, Drosophila cell free assays have shown that RNAi is mediated by nuclease degradation of the targeted mRNA while a 21-25 nt RNA species appears to be integral to specific post-transcriptional genetic interference.",
"RNAi has also been shown to be dependent on ATP which may be required for strand dissociation of dsRNA.",
"However, a missing component of the proposed multi-protein complex in the RNAi pathway is the implied RNA helicase.",
"The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.",
"In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.",
"Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.",
"The first gene that was tested in mediating RNAi activity in the present model was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.",
"As we have previously shown that antisense RNA-mediated gene suppression is dose dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.",
"The thi1 open reading frame was PCR amplified and subcloned into pREP4 as a BamHI fragment.",
"This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.",
"As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.",
"7).",
"This result indicated that host-encoded factors are present which affect the robustness of dsRNA-mediated gene suppression in fission yeast and also validated the overexpression strategy.",
"The second gene investigated has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.",
"According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.",
"It was therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.",
"Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.",
"6A).",
"When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.",
"6A).",
"The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (16).",
"Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.",
"6B).",
"It was thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.",
"By over-expressing a cDNA library in a dsRNA fission yeast model we have identified four novel, genes with potential roles in the RNAi pathway.",
"All of the known genes identified are essential and have natural associations with DNA, RNA or nucleic acid binding proteins, consistent with the expectation of RNAi cellular factors.",
"In addition, two of the three known cDNAs encoded proteins involved in the process of translation.",
"These findings along with our previous identification of an ATP-dependent RNA helicase (Raponi & Arndt, submitted) and reports of the involvement of a translation initiation factor in RNAi and the co-purification of ribonuclease activity with ribosomal fractions, suggests that this may be one site at which RNAi functions.",
"Alternatively, the identification of such varied proteins as DNA helicases and translational components as part of the RNAi machinery implies that particular cellular proteins may be recruited into more than one multiprotein complex.",
"Under the latter conditions, over-expression of these specific proteins may result in the generation of an RNAi complex that recognises dsRNA and mediates target gene suppression.",
"Certain of these proteins may be rate-limiting or rate-determining in RNAi and only through supplementation of these factors are their roles in RNAi uncovered.",
"It is proposed, without wishing to be bound by any particular mechanism, that in addition to the previously identified core proteins in the RNAi multiprotein complex(es), additional factors such as EF Tu, L7a, sna41 and res1 may be enlisted for a role in dsRNA-mediated gene suppression.",
"The over-expression strategy described overcomes some limitations associated with mutagenesis by identifying essential genes with a role in RNAi.",
"In addition, this strategy complements these other systems by allowing the isolation of cellular factors that modify the efficacy of RNAi in vivo.",
"The roles of these modulators of RNAi may be varied and include recognition and amplification of the dsRNA, delivery of the small 21-25 nt dsRNAs to the target mRNA, association between the antisense and target mRNA strands, and RNAi complex formation.",
"Alternatively, these modulators may control the rate of RNAi or the formation of different complexes within cell types or for different forms of post-transcriptional gene silencing.",
"The fact that over-expression of specific RNAi modulators enhanced dsRNA-mediated gene regulation in fission yeast indicates that a similar approach could be used to identify RNAi modulators in other organisms.",
"In addition, the co-expression of these factors with different forms of post-transcriptional gene silencing including co-suppression, quelling, and antisense RNA could be one way of enhancing the efficacy of these methods.",
"This may be especially important for application of RNAi to mammalian cells and tissues or to genes which have been somewhat recalcitrant to this form of regulation.",
"The present invention demonstrates for the first time the intrinsic involvement of an ATP-dependent RNA helicase as a key component in RNAi.",
"Further, it can be rate limiting, as the over-expression leads to increased RNAi activity in this system.",
"This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.",
"These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.",
"The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.",
"The present invention also provides for the first time a novel and quantitative genetic system based on S.pombe, for rapidly identifying essential cellular factors involved in RNAi.",
"The use of this model has enabled verification of RNA helicase activity as a critical contributor to efficient RNAi activity and isolated novel RNAi factors including EF Tu, L7a, sna41, and an unidentified gene res1.The present invention also demonstrates that gene silencing may be enhanced by concomitant expression of such RNAi factors.",
"The invention will now be described more particularly with reference to non-limiting examples of certain preferred embodiments of the invention.",
"Example 1 Materials and Methods Used to Exemplify the Invention S. pombe Media and Manipulations.",
"All yeast strains were maintained on standard YES or EMM media (6).",
"Repression of nmt1 transcription was achieved by the addition of thiamine to EMM media at a final concentration of 4 μM (7).",
"Yeast cells were transformed with plasmid DNA by electroporation (8) and stable integrants were identified as previously described (6).",
"A glass bead procedure (9) was used to isolate genomic DNA which was used for Southern analysis and PCR diagnosis.",
"Total RNA was extracted as previously described (10).",
"S. pombe Strain and Plasmid Construction.",
"Construction of the low expressing lacZ strain, KC4-6 (h−, ura4::SV40-lacZ, leu1-32), has been previously described (2).",
"The strain RB3-2 (h−, ura4::adh1-lacZ, leu1-32) which expresses higher levels β-galactosidase has also been previously described (1).",
"The target strain which contains the c-myc:lacZ fusion has previously been described (3).",
"The construction of the long lacZ antisense containing episomal plasmid, pGT2, and corresponding control plasmids have been described (2).",
"Plasmid pREP4-As was generated by subcloning the lacZ BamHI fragment contained in pGT2 into the plasmid pREP4 (11).",
"pREP4 is identical to pREP1 except that the S. cerevisiae LEU2 gene has been replaced with the S. pombe ura4 selectable marker.",
"To decrease the steady-state level of episomally expressed antisense lacZ, plasmids pREP42-As and pREP82-As were constructed by subcloning the lacZ BamHI fragment from pGT2 into pREP42 and pREP82, respectively.",
"These plasmids are derivatives of pREP4 with mutations in the TATA box of the nmt1 promoter (12).",
"The crippled lacZ vector, pGT62, was generated by end-filling the ClaI site of pGT2 and re-ligating (2).",
"This frameshifted fragment was then subcloned into the BamHI site of pREP4 to generate the plasmid pM54-3.The lacZ panhandle integration vector, pM30-8, was generated by first introducing a NotI site into the XmaI site of pRIP1/s (11) using the self-complementary linker 5′ CCG GGC GGC CGC 3′ to generate pL121-14.The 2.5 kb sequence of the 5′ end of the frameshifted lacZ gene (2) was then PCR-amplified to give it NotI ends using the forward primer 5′ ATGCGGCCGCAATTCCCGGGGATCGAAAGA 3′ and reverse primer 5′ ATGCGGCCGCAATGGGGTCGCTTCACTTA 3′.",
"This product was cloned using a TA cloning kit (TOPO: Invitrogen, San Diego, Calif., USA) and then subcloned into the NotI site of pL121-14 in the antisense orientation.",
"The integrating vector was introduced into target strains in single copy by using the sup3-5/ade6-704 complementation system (13).",
"The full-length frameshifted lacZ fragment was then introduced into the BamHI site of this vector in the sense orientation upstream of the 2.5 kb antisense fragment to generate pM30-8.The episomal version of this vector (pM53-1) was made by removing the Pst1 sup3-5 fragment and introducing the autonomous replicating sequence as an EcoRI fragment (11).",
"For testing the ability of the panhandle construct to form dsRNA in vivo, the frameshifted lacZ fragment was replaced with the functional lacZ fragment in the vectors pM54-3 and pM53-1, to generate the vectors pM85-1 and pM81-2, respectively.",
"The vector pM91-1, which is unable to form a lacZ panhandle transcript, was generated by removing the 2.5 kb NotI lacZ fragment from pM81-2 and reintroducing it in the sense orientation.",
"Generation of the c-myc antisense construct, pCM-17, has been described elsewhere (3).",
"The 792 by BglII c-myc fragment from pCM-17 was subcloned into the BamHI site of pREP4 in the sense orientation to generate pN12-1.The ded1, and thi1, open reading frames were amplified from fission yeast genomic DNA (strain 1913).",
"ded1 was amplified to give it BamHI ends using the forward primer 5′ ATGGGATCCCAACCAAACACTTCAACTCAG 3′ and the reverse primer 5′ ATGGGATCCTCAGAAGCCTGTGCATAACAC 3′.",
"thi1 was amplified to give it BglII ends using the forward primer 5′ ATGAGATCTGTGGTTGGTATTCTAGAGAGA 3′ and the reverse primer 5′ ATGAGATCTAACAAAGACCTGCAAAAAACC 3′.",
"PCR products were purified (Qiagen PCR purification kit), digested with either BamHI or BglII, gel-purified (Qiagen), and subcloned into the BamHI site of pREP4 in the sense orientation.",
"Southern and Northern Analysis.",
"Nucleic acid electrophoresis and membrane transfer was performed as described (14).",
"Southern and Northern blots were hybridized using ExpressHyb solution according to the manufacturer's instructions (Clontech Laboratories).",
"DNA probes were 32P-labelled using the Megaprime labelling kit (Amersham).",
"Probes included a 960 bp BamHI/ClaI lacZ fragment from pI2-1 (1), a 570 by HindIII/EcoRI ura4-3′ fragment from pGT113 (15), and a 2.2 kb PstI/SacI nmt1 fragment from pRIP1/s.",
"Radioactive signals were detected by autoradiography and quantitated by phosphorimage analysis (ImageQuant; Molecular Dynamics).",
"Plasmid Distribution Analysis.",
"Plasmid co-transformants of strain RB3-2 were grown under selective conditions to a cell density of 1-2×107 cells/ml.",
"A serial dilution of each culture was performed and cells plated for single colonies in triplicate onto each of YES, EMM, EMM+leucine, and EMM+uracil agar media.",
"The number of colonies grown on YES was taken as the total number of viable cells, while colonies growing on EMM represented the cells in the sampled population that contained both the ura4-containing (pREP4-based) and the LEU2-containing (pREP1-based) plasmids.",
"Cells containing either the ura4-containing plasmid or the LEU2-containing plasmid were identified from the EMM+leucine and EMM+uracil plates respectively.",
"The ratio of the number of colonies grown on selective media to the total number of viable colonies was used as the quantitative measure of the proportion of cells in the population, grown under selection, which contained plasmids.",
"β-galactosidase Assays.",
"The expression of the lacZ gene-encoded product, β-galactosidase, was quantitated using a cell permeabilization protocol as previously described (Raponi et al., 2000).",
"A semi-quantitative overlay assay was also employed for rapid screening of yeast transformants (3).",
"Isolation of S. pombe cDNA Clones That Alter Antisense RNA Efficacy.",
"The S. pombe cDNA library was originally constructed in pREP3Xho by Bruce Edgar and Chris Norbury (5).",
"The vector pREP3Xho is derived from pREP3 which contains the LEU2 marker and inserts are under control of the nmt1 promoter (11).",
"A total of 5 μg of library DNA was transformed into the strain RB3-2 containing the episomal antisense lacZ vector pREP4-lacZAS and grown in EMM liquid media to the mid-logarithmic phase.",
"Transformants were then plated on EMM solid media and grown at 30° C. for 3 days.",
"Colonies were over-layed with medium containing 0.5 M sodium phosphate, 0.5% agarose, 2% dimethylformamide, 0.01% SDS, and 500 μg/ml X-GAL (Progen, Australia).",
"Plates were then incubated at 37° C. for 3 hrs, colonies of interest recovered and assayed for β-galactosidase activity.",
"Plasmid Segregation.",
"Strains were plated on EMM containing limiting uracil and 1 mg/ml 5-fluoroorotic acid (6).",
"Strains were then replica plated on both selective and non-selective media.",
"Those colonies that did not grow on selective media were identified as having lost the ura4-containing antisense plasmid.",
"To determine the mitotic stability of pREP-based plasmids we used the plasmid segregation method previously described (16).",
"This method indicated that up to 30% of the cells grown in selective media did not contain the resident plasmid.",
"Example 2 Effect of Steady-State Level of Target mRNA and Antisense RNA on Antisense Efficacy To determine the role of target mRNA steady-state levels in antisense-mediated gene suppression, we investigated the ability of a long lacZ antisense RNA (2) to regulate lacZ target genes under control of both weak and strong constitutive promoters.",
"The low-level expressing strain, KC4-6, contained the lacZ gene driven by the SV40 early promoter integrated at the ura4 locus in chromosome III (FIG.",
"1A; (2)).",
"The high-level lacZ expressing strain, RB3-2, was constructed by placing the lacZ gene under control of the strong fission yeast adh1 promoter and the 3′ processing signal from the ura4 gene and integrating this expression cassette at the ura4 locus (FIG.",
"1B; (1)).",
"Characterization of both strains indicated that RB3-2 expressed 20-fold more lacZ mRNA than KC4-6 while an approximate 40-fold increase in β-galactosidase was also detected (data not shown).",
"Both strains were transformed with the lacZ antisense gene-containing plasmid (pGT2) and its corresponding sense control plasmid (pGT62).",
"β-galactosidase assays indicated that the antisense RNA suppressed β-galactosidase activity by 45% in KC4-6 while the same RNA reduced β-galactosidase activity by 52% in RB3-2 (FIG.",
"2B).",
"No reduction in β-galactosidase activity was seen in either strain when transformed with the control plasmid pGT62 which expresses a crippled version of lacZ.",
"These results indicated that despite a 20-fold increase in the steady state level of the lacZ target mRNA in RB3-2 the efficacy of antisense-mediated down regulation was not only maintained, but enhanced.",
"The increase in gene suppression demonstrated with strain RB3-2 was also seen when antisense molecules targeted to subregions of the lacZ target gene were used, but was not as pronounced.",
"These data suggested an increase in target mRNA can result in enhanced target gene suppression.",
"It has previously, been demonstrated that a stably integrated antisense lacZ gene acts in a dose dependent fashion with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number (1).",
"Here the role of the steady-state level of episomally expressed antisense RNA in both the low lacZ expressing target strain, KC4-6, and the high lacZ expressing target strain, RB3-2, was investigated.",
"The expression of lacZ antisense RNA was increased by co-transforming KC4-6 and RB3-2 with plasmids pGT2 and pREP4-As, each of which contains the lacZ antisense gene under control of the nmt1 promoter, but different selectable markers.",
"To decrease the steady-state level of antisense RNA, the plasmids pREP42-As and pREP82-AS were employed.",
"The nmt1 promoter in these vectors contain deletions in the TATA box sequence which affect the level of transcription, but have no impact on the site of transcription initiation or thiamine repressibility (12).",
"Each of the different antisense gene-containing plasmids was co-transformed with a control plasmid to complement auxotrophy where appropriate.",
"This resulted in a set of co-transformants of both RB3-2 and KC4-6 each containing the same lacZ antisense gene, but with different promoter capacities.",
"Each co-transformant was analyzed for antisense RNA steady-state levels and β-galactosidase activity.",
"Table 1 indicates that with both strains the degree of target suppression is enhanced with the increase of antisense lacZ RNA expression.",
"TABLE 1 Effects of varying the level of antisense RNA in strains expressing both low and high level target.",
"Relative β-gal.",
"activity antisense Strain Plasmids Descriptions (units) % suppressiona RNA levelb KC4-6 pREP2, pREP4 Vector 1.07 0 0 pREP2, pREP82-AS Low As 1.10 0 0.04 pREP2, pREP42-AS Medium As 1.07 0.1 0.14 pREP2, pREP4-AS Full As-ura4 0.75 30 1.0 pGT2, pREP4 Full As-LEU2 0.70 34.6 2.2 pGT2, pREP4-AS Double-As 0.54 49.2 5.6 RB3-2 pREP2, pREP4 Vector 48.5 0 0 pREP2, pREP82-AS Low As 44.0 9.4 0.05 pREP2, pREP42-AS Medium As 43.9 9.6 0.18 pREP2, pREP4-AS Full As-ura4 27.2 43.9 1.0 pGT2, pREP4 Full As-LEU2 24.9 48.7 1.9 pGT2, pREP4-AS Double-As 16.6 65.8 5.4 aThe % suppression of β-galactosidase activity was determined by expressing each β-galactosidase activity as a percentage of the activity found in the pREP2, pREP4 co-transformant.",
"bWithin each strain the steady-state level of long lacZ antisense RNA was normalized to nmt1 mRNA and then expressed relative to the level observed in the pREP2, pREP4-As co-transformant.",
"These data agree with the previous report that antisense RNA-mediated gene inhibition is dose dependent in S. pombe (1).",
"However, for each co-transformant the degree of β-galactosidase suppression was greater by 10-15% in the high lacZ expressing strain, RB3-2, than the low lacZ expressing strain, KC4-6, again demonstrating that low levels of target mRNA can limit antisense efficacy.",
"Example 3 Effect of Increasing Sense RNA on Antisense RNA-Mediated Target Gene Suppression To determine whether the increase in gene suppression was due to formation of an antisense RNA:target mRNA hybrid or an antisense RNA:sense RNA hybrid, a version of lacZ which is unable to be translated into functional β-galactosidase was co-expressed in strains expressing the lacZ antisense gene.",
"If antisense RNA was required to hybridize to target mRNA for inhibition of the gene expression pathway, then over-expression of the sense RNA would compete with the target for the available antisense molecules and a decrease in lacZ gene suppression would result.",
"Initially, both antisense and sense lacZ vectors were integrated in single copy into separate target strains and then crossed with each other.",
"In strains containing the antisense gene alone, β-galactosidase activity was reduced by approximately 40% (FIG.",
"3; (1)) while there was no reduction in the strain expressing sense lacZ alone (data not shown).",
"However, in the strain expressing both complementary transcripts (M62-1), β-galactosidase activity was reduced by 50% (FIG.",
"3).",
"These results suggested that increasing the intracellular concentration of sense RNA in the presence of antisense RNA, and therefore potentially generating more dsRNA, stimulates target gene suppression.",
"To further increase the potential formation of intracellular dsRNA an episomal sense lacZ plasmid (pM54-3; FIG.",
"2B) was co-transformed with the episomal antisense lacZ plasmid, pGT2, into the target strain R133-2.Both of these sequences were expressed by the strong conditional nmt1 promoter and RNA analysis showed that each transcript was being expressed at high levels when grown in the absence of thiamine (FIG.",
"4A).",
"When both RNAs were co-expressed in RB3-2, the target lacZ was suppressed by 65% compared to 50% in the strain expressing the antisense plasmid alone (FIG.",
"2B).",
"Northern analysis demonstrated that the total relative level of episomally expressed nmt1-driven transgenes was approximately 60% higher than when either vector was expressed alone (FIG.",
"4A).",
"Although expression of these complementary RNA transcripts is at a high level compared with single copy integrants only an additional 15% suppression was observed.",
"One explanation for the absence of higher levels of target gene suppression is plasmid segregation.",
"S. pombe undergoes asymmetric segregation, with the result that mitosis produces a daughter cell which lacks the segregating plasmid (17).",
"Therefore, when the 65% suppression level seen in the total population is corrected for only those cells containing the ura4-based and LEU2-based plasmids the level of suppression approaches approximately 100% (data not shown).",
"Overall these data suggest that increasing the potential formation of dsRNA, but not necessarily an antisense RNA:target mRNA hybrid, is required for efficient interference of target gene expression in S. pombe.",
"However, unlike the phenomenon of RNAi seen in plants and nematodes, the dsRNA-mediated gene suppression demonstrated in fission yeast seems to be dependent on the concentration of intracellular dsRNA or a threshold level of dsRNA is required to invoke potent gene silencing.",
"Example 4 Effect of a lacZ Inverted Repeat on lacZ Gene Expression To further investigate the ability of dsRNA to inhibit gene expression in fission yeast we generated a vector containing the full-length 3.5 kb frameshifted lacZ sequence with a 2.5 kb inverted repeat.",
"This construct generates a transcript of approximately 7 kb in length with 2.5 kb of self-complementarity which, predictably, will form a strong intramolecular RNA duplex.",
"This gene was initially integrated into a fission yeast strain in single copy and then crossed with the strain RB3-2.The resulting strain which contained the single copy inverted repeat gene and target lacZ showed no reduction in β-galactosidase activity when transcription of the inverted repeat was activated.",
"Southern analysis confirmed that the cassette was intact (data not shown) while RNA analysis indicated that the 7 kb transcript was being generated but approximately 10-fold less than episomally expressed antisense lacZ (FIG.",
"4A&B).",
"To increase the steady-state level of the inverted repeat RNA we generated the episomal plasmid pM53-1 containing this cassette.",
"RNA analysis demonstrated that this vector is expressed at a similar level to episomally expressed antisense lacZ (FIG.",
"4A).",
"When expressed in RB3-2 pM53-1 inhibited β-galactosidase activity by 40% (FIG.",
"2C), while addition of thiamine to the culture medium returned β-galactosidase activity to control level.",
"To confirm that gene inhibition was due to this construct forming an RNA duplex, an in vivo assay for dsRNA was developed.",
"To this end, a series of vectors were generated which contained functional lacZ sequences including lacZ alone (pM85-1), a lacZ inverted repeat (pM81-2), and the lacZ repeat with both sequences in the sense orientation (pM91-1) (FIG.",
"5A).",
"These vectors were then introduced into a strain lacking the integrated target sequence (NCYC2037; h+, ura4-D18) and resulting transformants were overlayed with X-GAL-containing agarose.",
"Both strains containing control vectors generated high levels of β-galactosidase while the strain expressing the lacZ inverted repeat did not (FIG.",
"5B).",
"In each case Northern analysis demonstrated that strains were expressing the transgene.",
"These results indicate that the inverted repeat RNA could not be efficiently translated to produce β-galactosidase, most probably due to the formation of a strong intramolecular hairpin structure.",
"Taken together, these results show that a construct which has the ability to form dsRNA by intramolecular hybridization will interfere with target gene expression when expressed at high levels, again indicating that dsRNA mediates target gene suppression in a dose-dependent manner in fission yeast.",
"Example 5 Gene-Specificity of dsRNA-Meditated Gene Silencing in Fission Yeast To test the ability of dsRNA to specifically interfere with other target sequences in fission yeast, sense and antisense c-myc sequences were co-expressed in a strain containing an integrated c-myc-lacZ fusion cassette (FIG.",
"1D; (3)).",
"A 792 bp antisense c-myc fragment from exon 2 of the human c-myc gene (named CM-17) was previously found to suppress β-galactosidase activity within a c-myc-lacZ fusion target strain (AML1) by 47% (3).",
"CM-17 was subsequently subcloned into pREP4 in the sense orientation to generate pN12-1.The antisense c-myc vector (pCM-17) and the sense c-myc vector were transformed into AML1 both independently and together.",
"β-galactosidase assays demonstrated that co-expression of the antisense and sense constructs enhanced c-myc suppression by an additional 13% compared with the antisense c-myc vector alone (FIG.",
"2D).",
"Transformation of RB3-2 with the antisense and sense c-myc constructs resulted in no down-regulation of β-galactosidase activity indicating that the action of dsRNA is gene-specific (FIG.",
"2D).",
"These results demonstrate that dsRNA can specifically interfere with multiple targets in fission yeast including one of human origin.",
"Example 6 Over-Expression of a Translation Factor can Enhance RNAi in Fission Yeast The dose-dependency of dsRNA-mediated gene silencing in fission yeast described above has allowed us to use an over-expression strategy to identify genes involved in RNAi.",
"In comparison to mutagenesis strategies, over-expression can enable the identification of genes which are otherwise essential for cell viability.",
"Also, cellular factors that quantitatively enhance or reduce RNAi activity can be determined.",
"The first gene that we have tested in mediating RNAi activity in the present model has been the S. pombe ATP-dependent RNA helicase gene, ded1.Ded1 is an essential gene which has previously been characterized as a suppressor of sterility, a suppressor of checkpoint and stress response, and a general translation initiation factor.",
"According to current models of dsRNA-mediated gene regulation an ATP-dependent RNA helicase may be required in conjunction with a dsRNA-dependent RNA polymerase for the formation of short single-stranded RNA fragments which specifically degrades target mRNA.",
"We therefore reasoned that over-expression of this gene in fission yeast could enhance the efficiency of dsRNA-mediated gene silencing by stimulating the unwinding of dsRNA.",
"Co-expression of the ded1 vector with the antisense lacZ vector significantly enhanced dsRNA-mediated lacZ inhibition by a further 50% compared to the antisense expressing strain (FIG.",
"6A).",
"When ded1 was over-expressed in the absence of the antisense lacZ vector, β-galactosidase activity was comparable to control strains indicating that its effect was dependent on the presence of dsRNA (FIG.",
"6A).",
"The ded1 vector was also co-expressed with a short antisense lacZ vector which has previously been shown to be less effective than the full-length antisense gene (2).",
"Again, the over-expression of ded1 stimulated dsRNA-mediated lacZ inhibition (FIG.",
"6B).",
"We have thus shown that over-expression of an ATP-dependent RNA helicase greatly enhances RNAi in fission yeast.",
"Although an ATP-dependent RNA helicase has recently been suggested to be a key component in RNAi, this is the first demonstration of its intrinsic involvement.",
"Further, it can clearly be rate limiting, as the over-expression leads to increased RNAi activity in this system.",
"This ability of the ded1-encoded RNA helicase is consistent with its activities as a member of the DEAD box family of helicases, with their three core domains of ATPase, RNA helicase, and RNA binding activities.",
"These could allow the enzyme to enhance gene suppression as follows: (i) in a dissociative mechanism it could mediate either the unwinding of dsRNA to generate a cRNA in conjunction with an RNA-dependent RNA polymerase or strand separation of fragmented dsRNA to enhance binding to homologous transcripts, and/or (ii) in an associative mechanism it could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.",
"The presence of ded1 homologues in other organisms displaying RNAi further supports the general involvement of this component in the RNAi machinery.",
"Example 7 Over-Expression of a Transcription Factor can Enhance RNAi in Fission Yeast The second gene investigated was the nmt1 transcription factor thi1.This gene has been shown to specifically up regulate nmt1 expression when overexpressed in fission yeast.",
"As we have previously shown that antisense RNA-mediated gene suppression is dose-dependent in S. pombe (1) it was hypothesized that over-expression of thi1 would result in increased production of nmt1-driven antisense lacZ RNA and a consequent enhancement in target gene suppression.",
"The thi1 open reading frame was PCR-amplified and subcloned into pREP4 as a BamHI fragment.",
"This vector was then transformed into RB3-2/pGT2 and β-galactosidase assays performed.",
"As predicted, lacZ suppression was enhanced when compared to a strain expressing antisense RNA alone (FIG.",
"7).",
"This result indicated that host-encoded factors are present which modulate the efficacy of dsRNA-mediated gene suppression in fission yeast and also validated the over-expression strategy.",
"Example 8 Screening for Novel RNAi Modulating Factors Over-expression of a fission yeast cDNA library (5) in an antisense lacZ expressing strain, revealed a series of transformants in which β-galactosidase activity was significantly reduced from that demonstrated in antisense RNA-expressing strains alone.",
"The screening strategy is shown in FIG.",
"8.Transformants were grown on selective plates and overlayed with X-GAL-containing media to generate a blue phenotype which could be analysed visually in a semi-quantitative manner (FIG.",
"9A)(3).",
"From 12,000 transformants screened, 48 were initially identified as having a reduced blue phenotype compared with background transformants.",
"Of these, 25 demonstrated a reproducible enhancement in antisense RNA-mediated lacZ suppression when quantified by a liquid β-galactosidase assay (FIG.",
"9B).",
"To demonstrate that the observed effects were due to transcription of the cDNA insert, transformants were grown in the presence of thiamine.",
"This repression of the nmt1 promoter resulted in a return to control levels of β-galactosidase activity in all transformants (FIG.",
"9B).",
"This indicated that the enhanced suppression was due to expression of the cDNA rather than other genetic events such as lacZ reporter mutations.",
"To determine if the effect was dependent on the presence of dsRNA, the antisense plasmid was segregated out of the transformants and the strains were again assayed for β-galactosidase activity.",
"Nine of the 25 transformants demonstrated a return to the level of β-galactosidase seen in the parental strain indicating that the effect was dsRNA-dependent (FIG.",
"9B).",
"The cDNAs being expressed in these transformants were named RNAi enhancing sequences (res).",
"All transformants displayed normal growth phenotypes indicating that over-expression of the resident cDNAs did not affect general biology of the cell.",
"In the absence of antisense RNA the remaining 16 transformants retained a reduced β-galactosidase activity indicating that the enhanced gene silencing was not dependent on the presence of dsRNA.",
"This suggests that the cDNA-encoded proteins in these strains could have been affecting lacZ at the level of transcription or the stability or function of the protein.",
"The library plasmids were recovered from the aes-containing strains (also referred to as res-containing strains) and their cDNA inserts sequenced.",
"BLASTN and BLASTP analysis identified clones W18, W20, and W30 (named aes2) as homologues of domains 2 and 3 of the mitochondrial elongation factor Tu (EF Tu).",
"EF Tu is an essential protein which plays a role in transporting tRNA to the A site in the ribosome for peptide elongation.",
"The cDNA in transformants W21, W23, and W32 (named aes3) was homologous to a putative protein that was identified in a screen for fission yeast ORFs.",
"Interestingly, the cDNA insert was also homologous to the antisense strand of the 3′ UTR of the fission yeast gene sna41 which has previously been shown to be involved in DNA replication.",
"It is thus possible that aes3 may operate through more than one mechanism in enhancing antisense RNA activity.",
"The cDNA in transformant W47 (named aes4) was homologous to the antisense strand of the ribosomal protein L7a, a component of the 60S ribosomal subunit.",
"aes4 also contained a small ORF of unknown biological function.",
"The inserts in transformants W27 and W28 (named aes1) shared homology with a putative protein from C. albicans that was identified in a screen for genes essential for cell growth.",
"A tertiary quantitative β-galactosidase assay was performed to obtain accurate levels of gene silencing augmentation in the antisense lacZ strains co-expressing these unique factors (FIG.",
"9C).",
"This analysis showed that these co-factors enhanced antisense suppression by up to 50%.",
"These results demonstrated that over-expression of a cDNA library was an effective way of identifying novel co-factors that magnify the suppressive effect mediated by antisense RNA.",
"aes1 shared 43% identity with amino acids 4 to 202 of a C. albicans hypothetical protein (AJ390519).",
"aes2 shared 99% identity with nucleotides 10452 to 9484 of the translation elongation factor EF Tu (AL049769).",
"aes3 shared 94% identity with nucleotides 776 to 1145 of D89239 S. pombe ORF (D89239) and 93% identity with nucleotides 3246 to 2876 of the antisense strand of the DNA replication factor sna41 (AB001379).",
"aes3 also contained a 220 nt stretch of a GA repeat sequence at its 3′ end.",
"aes4 shared 99% identity with nucleotides 9678 to 8897 of the antisense strand of ribosomal protein L7a (AJ001133) and 99% identity with nucleoStides 1365 to 584 of the antisense strand of ribosomal protein L4 (AB005750).",
"The reference numerals in brackets refer to accession numbers in the GeneBank database (the GeneBank database can be accessed from the following web site: http://www.ncbi.nlm.nih.gov/).",
"EF Tu is analogous to the eukaryotic EF1α and acts by transporting tRNA to the A site in the ribosome for peptide elongation.",
"EF1α is an essential protein which has also been implicated in a large array of cellular activities including actin binding, microtubule severing, cellular transformation, cell senescence, protein ubiquitination, and protein folding.",
"Detailed analysis of the EF Tu-expressing strain showed that it enhanced antisense RNA-mediated lacZ silencing by an additional 15% (FIG.",
"9C).",
"It has been proposed that dsRNA is fragmented into 21-25 nt species by dsRNA-specific nucleases, amplified by RNA-dependent RNA polymerase, and dissociated by an ATP-dependent RNA helicase.",
"The small antisense fragments are then free to attack homologous mRNA by RNA nuclease-mediated degradation.",
"It has recently been suggested that an associative mechanism could catalyse the ATP-dependent exchange of the sense strand of the short dsRNA with the target mRNA.",
"This may involve the transport of fragmented dsRNA to the ribosome where, in a competitive reaction with tRNA complexes, the complementary antisense strand binds to the mRNA.",
"This would permit the association of the antisense strand of the dsRNA with the target mRNA when the latter is in a structurally exposed state and both inhibit translation and target the mRNA for ribonuclease degradation.",
"Without wishing to be bound by any particular mechanism of action, EF Tu could act by binding to RNAi-dependent short dsRNA species and bring them to the site of action.",
"The protein encoded by sna41 has previously been shown to be involved in DNA replication.",
"sna41 has low homology with CDC45 and might have DNA helicase properties which could facilitate the expression of complementary sequences.",
"It is conceivable that the antisense plasmid and target DNA sequences may ectopically pair by intermolecular complementarity.",
"Such pairing may inhibit RNA expression and consequently reduce the level of intracellular dsRNA.",
"Similarly the intramolecular pairing of inverted repeat DNA sequences may also interfere with RNA expression.",
"The over-expression of a protein with DNA helicase properties could facilitate the generation of more dsRNA which could in turn enhance the RNAi effect.",
"Furthermore, CDC45 mutants show an increased rate of plasmid segregation.",
"If plasmid loss is inhibited by over-expression of sna41 then more dsRNA may be generated leading to more effective RNAi in this system.",
"In this light it is not unreasonable to expect that other proteins normally involved in DNA and/or RNA metabolism and function could also have a role in RNAi modulation and/or enhancement.",
"The L7a protein is part of the 60s ribosomal sub-unit.",
"Without wishing to be bound by any particular mechanism of action, RNAi augmentation by over-expression of this protein is reasonable as it is hypothesised that the short dsRNA species may undergo strand displacement with target mRNA at the ribosome.",
"The L7a ribosomal protein may act in RNAi by i) mediating docking of dsRNA or its unwound form into the A site of the ribosome, assisting in association of the antisense strand with the target mRNA, and/or shuttling of dsRNA to the ribosomal complex.",
"Nucleotide sequences representative of the aes factors referred to above are provided below and identified as Seq ID Nos 1 to 4: DNA sequence for aes1 factor (Seq ID No 1) TTTAACTTAGTTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAA GCCGCAATTGTAACAGTAGATTTTTTGCATCATTATTACTCTCCGAAACA TGACTGAACACTCATTTAAGCAAATAGACGTGTTTTCTAATAAAGGTTTT CGAGGTAATCCTGTTGCAGTTTTTTTTGATGCAGATAATTTATCACAAAA GGAAATGCAGCAGATTGCCAAGTGGACAAATTTATCTGAGACAACATTTG TTCAAAAGCCGACAATCGATAAAGCAGATTACAGACTTCGTATATTTACC CCAGAATGTGAATTAAGCTTTGCTGGTCACCCAACAATTGGATCGTGCTT TGCTGTTGTTGAAAGTGGATATTGTACTCCAAAAAACTGTAAAATTATTC AGGAATGTTTAGCCGGTTTAGTTGAATTAACTATCGATGGGGAAAAGGAT GAAGACACTTGGATTTCTTTCAAACTTCCGTATTACAAAATTTTACAGAC TTCTGAAACTGCAATTTCAGAAGTAGAAAATGCATTGGGTATTCCTCTGA ATTATAGTTCTCAAGTTTCTCCTCCTGTGTTAATAGATGATGGACCAAAG TGGCTTGTAATTCAACTTCCAAACGCTACAGATGTGCTCAACCTCGTTCC GAAATTTCAGTCCCTTTCCCAAGTTTGTAAAAACAATGATTGGATAGGCG TCACCCGTCTTTGGTGAATTAGAAAAGACTCGTTTGAAAGCCCGAAGCTT TGCGCCTTTAATACATGTCAATGAGGATCCGGCTTGCGGTAGTGGTGCAG GAGCTGTCGGTGTGTATATTGGAAGCTCTCAAAAAACTCCAACTTCTCTA TCATTTACGATTTCTCAAGGTACAAAATTAAGTAGACAAGCAATTTCCAA AGTCAGCGTAGACGTTTCCTCCAATAAATCAATTGCTGTTTTTGTCGGTG GACAGGCAAAAACTTGTATTTCTGGAAAATCGTTTATTTAATGTTTTTAT TACAAATATTCACTTGCGAGTTTATTTTCCAATACTGAAGACTTTCAATC AATAGCAAATATGCTACTCAAGGAAGTTCACTCATTCAAAAGCAATTGGT TTACTATATCGTTTTTTCTAACTAGTTACTAGTCATTGAACAATCTACCG AATGATAAAATGAAATTTTGGTTTTTCCCCGGGTAAAAGGAATGTCTCCC TTGCCAGTACTGCTAGGGTTTTTCTTTCGAACTATAAGA DNA sequence for aes2 factor (Seq ID No 2) AGTCCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAGCCTGATA TGATCGAGCTTGTCGAAATGGAAATGCGTGAGCTACTCTCCGAATACGGA TTTGATGGTGACAATACTCCAATTGTTAGCGGCAGTGCTTTATGTGCCTT AGAGGGTCGTGAGCCTGAGATTGGTCTCAATAGTATTACTAAATTGATGG AAGCTGTTGATAGTTATATTACTCTTCCTGAAAGAAAAACGGATGTCCCT TTCTTGATGGCCATCGAGGACGTTTTTTCAATTTCAGGTCGCGGAACTGT AGTCACTGGCCGTGTCGAGCGCGGTACTTTAAAGAAGGGTGCTGAAATCG AAATCGTCGGTTATGGTAGCCATTTAAAGACTACCGTTACTGGAATTGAA ATGTTCAAAAAGCAGCTTGATGCCGCCGTTGCCGGTGACAATTGTGGCCT TTTACTTCGTTCTATCAAGCGAGAGCAATTAAAACGTGGAATGATTGTCG CTCAACCAGGAACCGTTGCTCCTCATCAGAAATTCAAGGCATCATTCTAT ATTTTGACAAAAGAGGAAGGAGGTCGTCGTACCCGGTTTCGTTGACAAGT ATCGTCCCCAACTGTACAGTCCGTACTTCCGACGTTACTGTCGAACTTAC CCACCCTGATCCTAACGACTCAACAAAATGGTTATGCCTGGAGACAATGT CGAGATGATCTGTACGCTTATTCACCCCATTGTCATCGAAAAAGGACAAC GCTTCACAGTTCGTGAGGGTGGAAGCACTGTAGGCACAGCTTTGGTTACT GAACTTTTGGATTAGTGCATTTATGAACTTATTGGCTTTAAAAATTTTGC ATGCTGAATACCAATATTATGTCCCTTCTCAGAATTCTATAACTACAGTG TCATTATTGTAATAAGACTTTTGCATCCATTGACAATGGTATTTGATACT TTTATAGTTTCTACTATTGTTAGCCAAAGTTATAAAACAAATAATAAAAT AACGTTGAATCAAAAAAAAAAAAAAAAAAAGCGGCCGCGGATCCCCGGGT AAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCTTTCAAACTA TGGGA DNA sequence for aes3 factor (Seq ID No 3) ATTTCAGACGCAATTCACATGGCTTTTGACTGTATTGCTATTCTTGTCGG TTTAGTTGCTACGACGCTTGCCAAGATGCCTCTAAATTATGCTTACCCCT TTGGATTTGCAAAAATTGAGGCTCTTTCGGGTTTCACTAATGGTATTTTT TTAGTTTTGATTTCATTTTCTATCGTCGGCGAGGCATTATATAGGTTATT TCATCCGCCCCAAATGAATACCGACCAATTGTTGTTGGTTAGTTTTTTGG GCCTTGTTGTGAATTTGGTAGGTATCCTAGCGTTCAATCATGGGCATAAT CATGATCATGGGTCTCATCACCATCATTCCCATAGTAATCATAGTATGTG TCTGCCTAACACTACAAATGATATAAATATTTTTGAAGAGTTTGAAGAAG AAAAAGATAATGTTGAAGCCCAGAAAATGGGCTATACGAATGACGATCAC GTATCCCAACATGAACATACCCATGAGAATAGTCAGGAACATCACCATGA GCATAACCACAATCATGATCACATCCATAAATACAATGAAAAATGCGACC ATGAAAGCATAAGTCTCCAGAATTTAGACAATGATCATCACTGTCATCAT CACCATGAAAATCATAATATGCATGGCATATTTCTGCATATTATCGCAGA TACTATGGGCTCTGTTGGAGTTATTGTCTCTACTATATTAATACAGTGGT TTTCATGGACCGGTTTTGATCCTTCGGCATCTCTAATAATTGCTGCATTA ATATTTGTTTCTGTACTTCCATTAATTAAAGATTCGGCGAAGAATTTGCT CTCTGTGACTGATCCAGAATCGGAATATTTATTGAAGCAGTGTTTGTCGA ACATCAGTTTAAGTCACTCCGTTGTCAGTTTATCCAACCCTAAGTTCTGG ACAAACGAAAGAGGTGAAGTGTATGGAATACTCCATATTCAGGTGAGCAT AGACGGTGATTTAAACGTGGTTCGTAATGAAGTATTTAGGAAGCTCTCAA TCGCTGTACCAAATTTAAAACACATTTGTATACAATCTGAACGGCCAAAC AATTGCTGGTGTGGAAAATAGTTCTTACATCAGTTGATATCCATACTTAT TTACGTGTAATTTTAATTAGATGAATTAATATTTTCTTTATTAGC DNA sequence for aes4 factor (Seq ID No 4) TTTACTTTAGTCGCTTTGTTAAATTGGCCTCGAGGTCGACGTTAATTAAG CTTTTTTTTTAAGAGATATAACATATGTCAACGCGTCATTGATTAACTAC ATAACACGCCAATTATAAACTTCTCCCAAAAGAACTTAAGAATTTCCATT TTCAATCCAGATGAATTTATTTAAGAGACGAACAGTAGCGGCAGCAGCCT TAGCACGCTTAGCGAGCAAAGCTTGGGTCTTACCACCCATGATACCCACC ACCCCACTTACGACGGGCTTTCGTCGTACTTAGCAGAGAAGTTAGCATCA ACGGCGGAGACAATAGAAGCGAGTTCGTTCTTGTCTTCCTCACGGACCTC AGTGACAGCTAAAACAGCAGCAGTCTTTTGGTGAATGACAGTACCAAGGC GGGCCTTGTTCTTGACAATGGCATAAGGAACACCCATCTTCTTGCACAAA GCAGGCAAGAAAACGACGAGTTCAATGGGGTCGACATCGCTGGCAATGAG AACCAACTTAGCCTTCTTGGCCTCAATGAGAGCTACAACATGGTTCAAAC CATATTTAACATTGTAAGGCTTCTTAGAGACGTCTTGAGCAGACTTGCCG TTGGCAACAGCCTCGGCTTCAGCAACCAAACGTTGCTTCTTTTCAGCAGC AGTCTCAGGACGGTACTTGTTAAGCAACTTGAAGACCTGAGTAGCAGTGT TTTTGTCCAAAGTCTTCTGGAACTGAGCAATGGCAGGAGGAACCTTCAAA CGCAAGTTCAAAATCTTGCGACGGCGTTGAAGGCGGATATACTCAGGCCA CTTAACAAAACGGCTCAAGTCACGCTTAGGTTGGATGTCTTGTCCCCCGG GTAAAAGGAATGTCTCCCTTGCCAGTACTGCTAGGGTTTTTCGTTCGAAT AAGGCC Example 9 Effect of Novel RNAi Factors on Antisense RNA and dsRNA-Mediated Gene Regulation With recent studies on PTGS suggesting that antisense RNA, co-suppression, and dsRNA-mediated interference may share similar mechanisms, we wanted to determine whether over-expression of an aes factor would also enhance dsRNA-mediated regulation.",
"Having demonstrated that dsRNA could mediate gene suppression in this fission yeast model, the effect of an antisense enhancing sequence on dsRNA-mediated regulation was tested.",
"To this end, the aes2 gene was co-expressed with the lacZ panhandle construct in a yeast strain containing the lacZ target gene.",
"Under these conditions, this transformant displayed an additional 30% suppression of β-galactosidase activity when compared to the transformant expressing only the panhandle lacZ RNA (see FIG.",
"10).",
"This result shows that the aes2 gene, encoding the two domains of the mitochondrial elongation factor EF Tu, stimulated not only antisense RNA but also dsRNA-mediated gene inhibition.",
"Further it was shown that these two forms of regulation are related by over-expressing the ATP-dependent RNA helicase ded1 in the presence of lacZ antisense RNA and showing that this helicase enhanced gene suppression by a further 50% compared to the control strain (example 6).",
"ded1 was tested on both active and inactive antisense plasmids and demonstrated that ded1 augmentation of gene silencing was dependent on an active antisense RNA (see FIG.",
"11).",
"This could be due to the absence of RNA duplex formation with the inactive antisense RNA and the consequent lack of a substrate for the RNA helicase.",
"The methods of the present invention have utility in demonstrating a range of RNAi efficacies, in identifying new factors which enhance or reduce gene silencing, in inhibiting gene expression or increasing sensitivity to antisense inhibition of gene expression, in the treatment or prevention of disorders which require inhibition or down-regulation of gene expression.",
"Although the present invention has been described with reference to specific examples and preferred embodiments it will be clear to those skilled in the art that variations and modifications which do not depart from the concept and the spirit of the invention described herein are also contemplated as being within the scope of the present invention.",
"REFERENCES 1.Raponi, M., Atkins, D., Dawes, I.",
"& Arndt, G. (2000) Antisense and Nucleic Acid Drug Development 10, 29-34.2.Arndt, G., Atkins, D., Patrikakis, M. & Izant, J.",
"(1995) Molecular and General Genetics 248, 293-300.3.Arndt, G., Patrikakis, M. & Atkins, D. (2000) Nucleic Acids Research 28, e15.4.Clarke, M., Patrikakis, M. & Atkins, D. (2000) Biochemical and Biophysical Research Communications 268, 8-13.5.Moreno, S. & Nurse, P. (1994) Nature 367, 236-242.6.Moreno, S., Klar, A.",
"& Nurse, P. (1991) Methods in Enzymology 194, 795-823.7.Maundrell, K. (1990) Journal of Biological Chemistry 265, 10857-10864.8.Prentice, H. (1992) Nucleic Acids Research 20, 621.9.Hoffman, C. & Winston, F. (1987) Gene 57, 267-272.10.Rose, M., Winston, F. & Hieter, P. (1990) Methods in yeast genetics: A laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor).",
"11.Maundrell, K. (1993) Gene 123, 127-1390.12.Basi, G., Schmid, E. & Maundrell, K. (1993) Gene 123, 131-136.13.Carr, A., MacNeill, S., Hayles, J.",
"& Nurse, P. (1989) Molecular and General Genetics 218, 41-49.14.Sambrook, J., Fritsch, E. & Maniatis, T. (1989) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).",
"15.Patrikakis, M., Izant, J.",
"& Atkins, D. (1996) Current Genetics 30, 151-158.16.Brun, C., Dubey, D. & Huberman, J.",
"(1995) Gene 164, 173-177.17.Heyer, W.-D., Sipiczki, M. & Kohli, J.",
"(1986) Molecular and Cellular Biology 6, 80-89."
]
] | Patent_10297167 |
[
[
"Ligands of Integrin Receptors",
"The invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds."
],
[
"1.The use of compounds of the formula I B-G-L I as ligand of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3, together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4 RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —C1-NRG12RG13—NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11 B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.",
"2.The use as claimed in claim 1, wherein the structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally contained or the carbon atoms can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.3.The use as claimed in one of claims 1 or 2, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18, where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—Cl1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms-O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both: radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA1, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- or bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11 Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.",
"4.The use as claimed in one of claims 1 to 3, wherein the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE9RE10)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 1-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NHC1-C6-alkoxyalkyl, CO—NHC1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an, optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.",
"5.The use as claimed in one of claims 1 to 4, wherein the spacer structural-element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6—IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14 (ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical or independently of one another are in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle, which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms together with RW2 or RW2* a saturated or unsaturated C3-C7-heterocycle, which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.",
"6.The use of the structural element of the formula IGL -G-L, IGL for the preparation of compounds which bind to integrin receptors, where G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 und RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted, C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, with the proviso that in the case of this fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle substituents are excluded which contain a structural element —V—CO—R8, where V is an optionally substituted C1-C2-alkylene radical R8 is a hydroxyl group, a C1-C8-alkoxy, aryl-C0-C6-alkoxy, C1-C8-alkylcarbonyloxy-C1-C4-alkoxy or aryl-C1-C8-alkylcarbonyloxy-C1-C4-alkoxy group or an L- or D-amino acid, which is bonded by an amide bond and in which the carboxylic acid component of said amino acid is present as a free acid or esterified with C1-C6-alkyl, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—N12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and which can contain up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 and RG11* is a radical RG11 which is independent of RG11.7.The use of the compounds as claimed in one of claims 1 to 5 as ligands of the αVβ3 integrin receptor.",
"8.The use of the compounds as claimed in one of claims 1 to 5 for the production of drugs for treating diseases in which the interaction between integrins and their natural ligands is excessive or decreased.",
"9.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 8 for the treatment of diseases in which the interaction between αVβ3 ingegrin and its natural ligands is excessive or decreased.",
"10.The use of the compounds as claimed in one of claims 1 to 5 as claimed in claim 9 for the treatment of atherosclerosis, rheumatoid arthritis, restenosis after vascular injury or stent implantation, angioplasty, acute kidney failure, angiogenesis-associated microangiopathies, diabetic angiopathies, blood platelet-mediated vascular occlusion, arterial thrombosis, congestive heart failure, myocardial infarct, stroke, cancer, osteoporosis, high blood pressure, psoriasis or viral, parasitic or bacterial conditions, inflammation, wound healing, hyperparathyroidism, Paget's disease, malignant hypercalcemia or metastatic osteolytic lesions.",
"11.A compound of the formula I′ A-E′-G′-L I′ where A, E′, G′ and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G′ is a structural element of the formula IG where the structural element A-E′ is bonded to the structural element G′ via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG′12RG′13, C1-C4-alkylene-CO—NRG′12RG′13, C1-C4-alkylene-O—CO—NRG′12RG′13, C1-C4-alkylene-NRG′12RG′13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG′12RG′13, —SO2—NRG′12RG′13, —CO—NRG′12RG′13, —NRG′12RG′13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG′12, RG′13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-heterocycloalkyl, C1-C4-alkylene-heterocycloalkenyl or hetarylalkyl radical, E′ is a structural element, composed of two to four substructural elements, selected from the group E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO-o, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE8 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—C—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW12)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded, A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon, which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the ring nitrogen optionally present or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur, and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.",
"12.A compound as claimed in claim 11, wherein the structural element A used is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, q independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where both radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* is a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6 are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms Q, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.",
"13.A compound as claimed in claim 11 or 12, wherein the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 (NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon, which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group-O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G is excluded.",
"14.A compound as claimed in one of claims 11 to 13 for use as a drug.",
"15.The use of the compounds as claimed in one of claims 11 to 13 for the production of drugs for treating diseases.",
"16.A pharmaceutical preparation comprising, in addition to the customary pharmaceutical excipients, at least one compound as claimed in one of claims 11 to 13.17.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet adhesion, activation or aggregation, anticoagulants which prevent thrombin activity or formation, antagonists of blood platelet-activating compounds or selectin antagonists.",
"18.The use of the pharmaceutical preparation as claimed in claim 17 for the production of a drug for treating blood platelet-mediated vascular occlusion or thrombosis.",
"19.A pharmaceutical preparation, comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group inhibitors of blood platelet activation or aggregation, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vascular wall transmigration, fibrinolysis-modulating compounds, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists.",
"20.The use of the pharmaceutical preparation as claimed in claim 19 for the production of a drug for treating myocardial infarct or stroke.",
"21.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors.",
"22.The use of the pharmaceutical preparation as claimed in claim 21 for the production of a drug for treating congestive heart failure.",
"23.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, inhibitors of blood platelet adhesion, activation or aggregation, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants.",
"24.The use of the pharmaceutical preparation as claimed in claim 23 for the production of a drug for treating restenosis after vascular injury or stent implantation.",
"25.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, glycosylation inhibitors and AGE formation inhibitors or AGE breakers and antagonists of their receptors.",
"26.The use of the pharmaceutical preparation as claimed in claim 25 for the production of a drug for treating diabetic angiopathies.",
"27.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers.",
"28.The use of the pharmaceutical preparation as claimed in claim 27 for the production of a drug for treating atherosclerosis.",
"29.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group cytostatic or antineoplastic compounds, compounds which inhibit proliferation and heparin or low-molecular weight heparins or further GAGs.",
"30.The use of the pharmaceutical preparation as claimed in claim 29 for the production of a drug for the treatment of cancer.",
"31.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group compounds for antiresorptive therapy, compounds for hormone exchange therapy, recombinant human growth hormone, bisphosphonates, compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, interleukin-6 antagonists and Src tyrosine kinase inhibitors.",
"32.The use of the pharmaceutical preparation as claimed in claim 31 for the production of a drug for the treatment of osteoporosis.",
"33.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors.",
"34.The use of the pharmaceutical preparation as claimed in claim 33 for the production of a drug for treating rheumatoid arthritis.",
"35.A pharmaceutical preparation comprising at least one compound as claimed in one of claims 1 to 5, if appropriate pharmaceutical excipients and at least one further compound selected from the group collagenase, PDGF antagonists and MMPs.",
"36.The use of the pharmaceutical preparation as claimed in claim 35 for the production of a drug for improving wound healing."
],
[
"The present invention relates to the use of cyclic compounds as ligands of integrin receptors, in particular as ligands of the αVβ3 integrin receptor, the novel compounds themselves, their use, and pharmaceutical preparations comprising these compounds.",
"Integrins are cell surface glycoprotein receptors which mediate interactions between similar and different cells as well as between cells and extracellular matrix proteins.",
"They are involved in physiological processes, such as embryogenesis, hemostasis, wound healing, immune response and formation/maintenance of the tissue architecture.",
"Disturbances in the gene expression of cell adhesion molecules and functional disorders of the receptors can contribute to the pathogenesis of many disorders, such as tumors, thromboembolic events, cardiovascular disorders, lung diseases, disorders of the CNS, the kidney, the gastrointestinal tract or inflammation.",
"Integrins are heterodimers of an α- and a β-transmembrane subunit in each case, which are noncovalently bonded.",
"Up to now, 16 different α- and 8 different β-subunits and 22 different combinations have been identified.",
"Integrin αvβ3, also called the vitronectin receptor, mediates adhesion to a multiplicity of ligands—plasma proteins, extracellular matrix proteins, cell surface proteins, of which the majority contain the amino acid sequence RGD (Cell, 1986, 44, 517-518; Science 1987, 238, 491-497), such as vitronectin, fibrinogen, fibronectin, von Willebrand factor, thrombospondin, osteopontin, laminin, collagen, thrombin, tenascin, MMP-2, bone sialoprotein II, various viral, fungal, parasitic and bacterial proteins, natural integrin antagonists such as disintegrins, neurotoxins—mambin—and blood fluke proteins—decorsin, ornatin—and also some non-RGD ligands, such as Cyr-61 and PECAM-1 (L. Piali, J.",
"Cell Biol.",
"1995, 130, 451-460; Buckley, J.",
"Cell Science 1996, 109, 437-445, J. Biol.",
"Chem.",
"1998, 273, 3090-3096).",
"A number of integrin receptors show cross-reactivity with ligands which contain the RGD motif.",
"Thus integrin αIIbβ3, also called the platelet fibrinogen receptor, recognizes fibronectin, vitronectin, thrombospondin, von Willebrand factor and fibrinogen.",
"Integrin αvβ3 is expressed, inter alia, on endothelial cells, blood platelets, monocytes/macrophages, smooth muscle cells, some B cells, fibroblasts, osteoclasts and various tumor cells, such as melanoma, glioblastoma, lung, breast, prostate and bladder carcinomas, osteosarcomas or neuroblastomas.",
"Increased expression is observed under various pathological conditions, such as in the prothrombotic state, in vascular injury, tumor growth or metastasis or reperfusion and on activated cells, in particular on endothelial cells, smooth muscle cells or macrophages.",
"An involvement of integrin αvβ3 has been demonstrated, inter alia, in the following syndromes: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury, and angioplasty (neointima formation, smooth muscle cell migration and proliferation) (J. Vasc.",
"Surg.",
"1994, 19, 125-134; Circulation 1994, 90, 2203-2206), acute kidney failure (Kidney Int.",
"1994, 46, 1050-1058; Proc.",
"Natl.",
"Acad.",
"Sci.",
"1993, 90, 5700-5704; Kidney Int.",
"1995, 48, 1375-1385), angiogenesis-associated microangiopathies such as diabetic retinopathy or rheumatoid arthritis (Ann.",
"Rev.",
"Physiol 1987, 49, 453-464; Int.",
"Opthalmol.",
"1987, 11, 41-50; Cell 1994, 79, 1157-1164; J. Biol.",
"Chem.",
"1992, 267, 1093.1-10934), arterial thrombosis, stroke (phase II studies with ReoPro, Centocor Inc., 8th annual European Stroke Meeting), carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis) (Cell 1991, 64, 327-336; Nature 1989, 339, 58-61; Science 1995, 270, 1500-1502), osteoporosis (bone resorption after proliferation, chemotaxis and adhesion of osteoclasts to bone matrix) (FASEB J.",
"1993, 7, 1475-1482; Exp.",
"Cell Res.",
"1991, 195, 368-375, Cell 1991, 64, 327-336), high blood pressure (Am.",
"J. Physiol.",
"1998, 275, H1449-H1454), psoriasis (Am.",
"J. Pathol.",
"1995, 147, 1661-1667), hyperparathyroidism, Paget's disease (J. Clin.",
"Endocrinol.",
"Metab.",
"1996, 81, 1810-1820), malignant hypercalcemia (Cancer Res.",
"1998, 58, 1930-1935), metastatic osteolytic lesions (Am.",
"J. Pathol.",
"1997, 150, 1383-1393), pathogenic protein (e.g.",
"HIV-1 tat)-induced processes (e.g.",
"angiogenesis, Kaposi's sarcoma) (Blood 1999, 94, 663-672) inflammation (J.",
"Allergy Clin.",
"Immunol.",
"1998, 102, 376-381), cardiac insufficiency, CHF, and also in antiviral, antiparasitic, antifungal or antibacterial therapy and prophylaxis (adhesion and internalization) (J. Infect.",
"Dis.",
"1999, 180, 156-166; J. Virology 1995, 69, 2664-2666; Cell 1993, 73, 309-319).",
"On account of its key role, pharmaceutical preparations which contain low-molecular weight integrin αvβ3 ligands are of high therapeutic or diagnostic benefit, inter alia, in the indications mentioned.",
"Advantageous αvβ3 integrin receptor ligands bind to the integrin αvβ3 receptor with an increased affinity.",
"In contrast to integrin αvβ3, particularly advantageous αvβ3 integrin receptor ligands additionally have an increased selectivity and are less active with respect to the integrin αIIbβ3 by at least a factor of 10, preferably at least a factor of 100.For a multiplicity of compounds, such as anti-αvβ3 monoclonal antibodies, peptides which contain the RGD binding sequence, natural, RGD-containing proteins (e.g.",
"disintegrins) and low-molecular weight compounds, an integrin αvβ3 antagonistic action has been shown and a positive in vivo effect demonstrated (FEBS Letts 1991, 291, 50-54; J. Biol.",
"Chem.",
"1990, 265, 12267-12271; J. Biol.",
"Chem.",
"1994, 269, 20233-20238; J.",
"Cell Biol 1993, 51, 206-218; J. Biol.",
"Chem.",
"1987, 262, 17703-17711; Bioorg.",
"Med.",
"Chem.",
"1998, 6, 1185-1208).",
"Antagonists of the αVβ3 integrin receptor based on a bicyclic structural element are described in WO 9906049, WO 9905107, WO 9814192, WO 9724124, WO 9724122 and WO 9626190.EP 540 334 and WO 9308174 describe bicyclic antagonists of the αIIbβ3 integrin receptor.",
"WO 9407488 A1 describes compounds having a bicyclic molecular structure and which accelerate the release of growth hormone.",
"Further, vasopressin antagonists having a bicyclic molecular structure are described in the specifications EP 620216, WO 9534540, WO 9408582, WO 9802432, WO 9420473, JP 09221476 A1, JP 11060488 A1, WO 9404525, JP 04321669 A1, WO 9722591, as well as in Matsuhisa et al., Chem.",
"Pharm.",
"Bull.",
"1999, 47, 3, 329-339.It is an object of the present invention to make available novel integrin receptor ligands having advantageous properties.",
"We have found that this object is achieved by the use of compounds of the formula I B-G-L I as ligands of integrin receptors, where B, G and L have the following meanings: L is a structural element of the formula IL —U-T IL where T is a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH and —U— is —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1—, where a is 0 or 1, b is 0, 1 or 2 XL is CRL3RL4, NRL5, oxygen or sulfur, RL1, RL2, RL3, RL4 independently of one another are hydrogen, -T, —OH, —NRL6RL7, —CO—NH2, a halogen radical, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C3-C7-cycloalkyl, —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 or C1-C4-alkoxy radical, an optionally substituted radical C1-C2-alkylene-T, C2-alkenylene-T or C2-alkynylene-T, an optionally substituted aryl or arylalkyl radical or in each case independently of one another are two radicals RL1 and RL2 or RL3 and RL4, or optionally RL1 and RL3 together are an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three identical or different heteroatoms O, N, S, RL5, RL6, RL7 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted CO—O-alkylenearyl, SO2-aryl, CO-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, G is a structural element of the formula IG where the structural element B is bonded to the structural element G via the ring nitrogen and the structural element L is bonded via WG, YG is CO, CS, C═NRG2 or CRG3RG4, RG2 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—C3-C7-cycloalkyl radical or an optionally substituted aryl, —O-aryl, arylalkyl or —O-alkylenearyl radical, RG3, RG4 independently of one another are hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radical or both radicals RG3 and RG4 together are a cyclic acetal —O—CH2—CH2—O— or —O—CH2—O— or both radicals RG3 and RG4 together are an optionally substituted C3-C7-cycloalkyl radical, RG5 and RG6 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, an optionally substituted aryl or arylalkyl radical or both radicals RG5 and RG6 together are an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S, WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, RG1 is hydrogen, halogen, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radical, RG7, RG8, RG9, RG10 independently of one another are hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical or in each case independently of one another two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 together are an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group O, N, S and up to two double bonds, RG11 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, RG12, RG13 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11, and RG11* is a radical RG11 which is independent of RG11, B is a structural element containing at least one atom which, under physiological conditions, as a hydrogen acceptor can form hydrogen bridges, where at least one hydrogen acceptor atom has a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton, and the physiologically tolerable salts, prodrugs and the enantiomerically pure or diastereomerically pure and tautomeric forms.",
"In the structural element L, T is understood as meaning a group COOH, a radical hydrolyzable to COOH or a radical bioisosteric to COOH.",
"A radical hydrolyzable to COOH is understood as meaning a radical which changes into a group COOH after hydrolysis.",
"A group which may be mentioned by way of example as a radical T hydrolyzable to COOH is in which R1 has the following meanings: a) OM, where M can be a metal cation, such as an alkali metal cation, such as lithium, sodium, potassium, the equivalent of an alkaline earth metal cation, such as calcium, magnesium and barium, or an environmentally tolerable organic ammonium ion such as primary, secondary, tertiary or quaternary C1-C4-alkylammonium or ammonium ion, such as ONa, OK or OLi, b) a branched or unbranched, optionally halogen-substituted C1-C8-alkoxy radical, such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, in particular methoxy, ethoxy, 1-methylethoxy, pentoxy, hexoxy, heptoxy, octoxy, difluoromethoxy, trifluoromethoxy, chlorodifluoromethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2,2,2-trifluoroethoxy, 2-chloro-1,1,2-trifluoroethoxy or pentafluoroethoxy c) a branched or unbranched, optionally halogen-substituted C1-C4-alkylthio radical such as methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio or 1,1-dimethylethylthio radical d) an optionally substituted —O-alkylenearyl radical, such as —O-benzyl e) R1 is further a radical —(O)m—N(R18)(R19), in which m is 0 or 1 and R18 and R19, which can be identical or different, have the following meanings: hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl or the corresponding substituted radicals, preferably methyl, ethyl, propyl, butyl or i-butyl, C2-C6-alkenyl radical, such as vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl or the corresponding substituted radicals, C2-C6-alkynyl radical, such as ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl or the corresponding substituted radicals, C3-C8-cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl und cycloheptyl, cyclooctyl or the corresponding substituted radicals, or a phenyl radical, optionally mono- or polysubstituted, for example mono- to trisubstituted, by halogen, nitro, cyano, C1-C4-alkyl, C1-C4-halogenoalkyl, C1-C4-alkoxy, C1-C4-halogenoalkoxy or C1-C4-alkylthio such as 2-fluorophenyl, 3-chlorophenyl, 4-bromophenyl, 2-methylphenyl, 3-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-methoxyphenyl, 4-trifluoroethoxyphenyl, 2-methylthiophenyl, 2,4-dichlorophenyl, 2-methoxy-3-methylphenyl, 2,4-dimethoxyphenyl, 2-nitro-5-cyanophenyl, 2,6-difluorophenyl, or R18 and R19 together form an optionally substituted, e.g.",
"C1-C4-alkyl-substituted, C4-C7-alkylene chain closed to give a cycle, which can contain a heteroatom selected from the group consisting of oxygen, sulfur and nitrogen, such as —(CH2)4—, —(CH2)5—, —(CH2)6—, —(CH2)7—, —(CH2)2—O—(CH2)2—, —CH2—S—(CH2)3—, —(CH2)2—O—(CH2)3—, —NH—(CH2)3—, —CH2—NH—(CH2)2—, —CH2—CH═CH—CH2—, —CH═CH—(CH2)3—, —CO— (CH2)2—CO— or —CO— (CH2)3—CO—.",
"A radical bioisosteric to COOH is understood as meaning radicals which can replace the function of a group COOH in active compounds by equivalent bond donor/acceptor capabilities or by equivalent charge distribution.",
"Radicals which may be mentioned by way of example as radicals bioisosteric to —COOH are those such as described in “The Practice of Medicinal Chemistry, Editor: C. G. Wermuth, Academic Press 1996, pages 125 and 216, in particular the radicals —P═O(OH)2, —SO3H, tetrazole or acylsulfonamides.",
"Preferred radicals T are —COOH, —CO—O—C1-C8-alkyl or —CO—O-benzyl.",
"The radical —U— in the structural element L is a spacer selected from the group —(XL)a—(CRL1RL2)b—, —CRL1═CRL2—, ethynylene or ═CRL1-.",
"In the case of the radical ═CRL1—, the structural element L is linked to the structural element G via a double bond.",
"XL is a radical CRL3RL4, NRL5, oxygen or sulfur.",
"Preferred radicals —U— are the radicals —CRL1═CRL2—, ethynylene or —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a=1).",
"Particularly preferred radicals —U— are the radicals —(XL)a—(CRL1RL2)b—, where XL is preferably CRL3RL4 (a=0 or 1) or oxygen (a 1).",
"Under RL11, RL2, RL3 or RL4 in the structural element L, a halogen radical is understood as meaning, for example, F, Cl, Br or I, preferably F. Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C6-alkyl radical is understood as meaning, for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, preferably branched or unbranched C1-C4-alkyl radicals such as methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, particularly preferably methyl.",
"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkenyl radical is understood as meaning, for example, vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl or 3-methyl-2-pentenyl.",
"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C2-C6-alkynyl radical is understood as meaning, for example, ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably ethynyl, 2-propynyl, 2-butynyl, 1-methyl-2-propynyl or 1-methyl-2-butynyl.",
"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C3-C7-cycloalkyl radical is understood as meaning, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.",
"Under RL1, RL2, RL3 or RL4 in structural element L, a branched or unbranched C1-C4-alkoxy radical is understood as meaning, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy or 1,1-dimethylethoxy.",
"The radicals —CO—NH(C1-C6-alkyl), —CO—N(C1-C6-alkyl)2 are secondary or tertiary amides and are composed of the amide bond and the corresponding C1-C6-alkyl radicals such as described above for RL1, RL2, RL3 or RL4.The radicals RL1, RL2, RL3 or RL4 can furthermore be a radical C1-C2-alkylene-T, such as methylene-T or ethylene-T, C2-alkenylene-T, such as ethenylene-T or C2-alkynylene-T, such as ethynylene-T, an aryl radical, such as phenyl, 1-naphthyl or 2-naphthyl or an arylalkyl radical, such as benzyl or ethylenephenyl (homobenzyl), where the radicals can optionally be substituted.",
"Furthermore, two radicals RL1 and RL2 or RL3 and RL4 or optionally RL1 and RL3 can in each case independently of one another together be an optionally substituted 3- to 7-membered saturated or unsaturated carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. All radicals for RL1, RL2, RL3 or RL4 can be optionally substituted.",
"For the radicals RL1, RL2, RL3 oder RL4 and all further substituted radicals of the description below, suitable substituents, if the substituents are not specified in greater detail, are independently of one another up to 5 substituents, for example selected from the following group: —NO2, —NH2, —OH, —CN, —COOH, —O—CH2—COOH, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl, CF3, C2F5 or CH2F, —CO—O—C1-C4-alkyl, C3-C6-cycloalkyl, C1-C4-alkoxy, C1-C4-thioalkyl, —NH—CO—O—C1-C4-alkyl, —O—CH2—COO—C1-C4-alkyl, —NH—CO—C1-C4-alkyl, —CO—NH—C1-C4-alkyl, —NH—SO2—C1-C4-alkyl, —SO2—NH—C1-C4-alkyl, —N(C1-C4-alkyl)2, —NH—C1-C4-alkyl, or —SO2—C1-C4-alkyl radical, such as —SO2—CF3, an optionally substituted —NH—CO-aryl, —CO—NH-aryl, —NH—CO—O-aryl, —NH—CO—O-alkylenearyl, —NH—SO2-aryl, —SO2—NH-aryl, —CO—NH-benzyl, —NH—SO2-benzyl or —SO2—NH-benzyl radical, an optionally substituted radical —SO2—NR2R3 or —CO—NR2R3 where the radicals R2 and R3 independently of one another can have the meaning RL5 as below or both radicals R2 and R3 together can be a 3- to 6-membered, optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three further different or identical heteroatoms O, N, S, and optionally two radicals substituted on this heterocycle can together be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted cycle can be fused to this cycle.",
"If not specified in greater detail, in all terminally bonded, substituted-hetaryl radicals of the description, two substituents can form a fused 5- to 7-membered, unsaturated or aromatic carbocycle.",
"Preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical or the radical —NRL6RL7.Particularly preferred radicals RL1, RL2, RL3 or RL4 are independently of one another hydrogen, fluorine or a branched or unbranched, optionally substituted C1-C4-alkyl radical, preferably methyl.",
"The radicals RL5, RL6, RL7 in structural element L are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, for example as described above for RL1, C3-C7-cycloalkyl radical, for example as described above for RL1, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl or CO—C1-C6-alkyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the C1-C6-alkyl radicals described above for RL1, or an optionally substituted CO—O-alkylenearyl, SO2-aryl, SO2-alkylenearyl or CO-alkylenearyl radical, which is composed of the group CO—O, SO2 and CO and, for example, of the aryl or arylalkyl radicals described above for RL1.Preferred radicals for RL6 in structural element L are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—O—C1-C4-alkyl, CO—C1-C4-alkyl or SO2—C1-C4-alkyl radical or an optionally substituted CO—O-benzyl, SO2-aryl, SO2-alkylenearyl or CO-aryl radical.",
"Preferred radicals for RL7 in structural element L are hydrogen or a branched or unbranched, optionally substituted C1-C4-alkyl radical.",
"Preferred structural elements L are composed of the preferred radicals of the structural element.",
"Particularly preferred structural elements L are composed of the particularly preferred radicals of the structural element.",
"G is a structural element of the formula IG where the structural element B is bonded via the ring nitrogen and the structural element L is bonded via WG to the structural element G, optionally via a double bond.",
"YG in structural element G is CO, CS, C═NRG2 or CRG3RG4, preferably CO, C═NRG2 or CRG3RG4, particularly preferably CO or CRG3RG4.RG2 in structural element G is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy or C3-C7-cycloalkyl radical, for example as described above for RL1 in each case, an optionally substituted —O—C3-C7-cycloalkyl radical, which is composed of an ether group and, for example, of the C3-C7-cycloalkyl radical described above for RL1, an optionally substituted aryl or arylalkyl radical, for example as described above for RL1 in each case or an optionally substituted —O-aryl or —O-alkylenearyl radical, which is composed of a group —O— and, for example, of the aryl or arylalkyl radicals described above for RL1.Branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkoxy radicals for RG3 or RG4 in structural element G independently of one another are understood as meaning, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG3 and RG4 can together form a cyclic acetal, such as —O—CH2—CH2—O— or —O—CH2—O—.",
"Furthermore, both radicals RG3 and RG4 can together form an optionally substituted C3-C7-cycloalkyl radical.",
"Preferred radicals for RG3 or RG4 are independently of one another hydrogen, C1-C4-alkyl or C1-C4-alkoxy.",
"Branched or unbranched, optionally substituted C1-C6-alkyl or C1-C4-alkoxy radicals and optionally substituted aryl or arylalkyl radicals for RG5 and RG6 in structural element G independently of one another are, for example, the corresponding radicals in each case described above for RL1.Further, both radicals RG5 and RG6 can together form an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S. Preferred radicals for RG5 and RG6 are independently of one another hydrogen or optionally substituted aryl radicals, preferably phenyl or arylalkyl radicals, preferably benzyl, and in each case both radicals RG5 and RG6 together can contain an optionally substituted, fused, unsaturated or aromatic 3- to 10-membered carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S. In particularly preferred radicals for RG5 and RG6, both radicals RG5 and RG6 together form an optionally substituted, fused, unsaturated or aromatic 3- to 6-membered carbocycle or heterocycle, for example selected from one of the following doubly bonded structural formulae: in particular selected from one of the following, doubly bonded structural formulae: Suitable substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are in particular substituents such as generally described above.",
"Particularly preferred substituents of these fused, unsaturated or aromatic 3- to 10-membered carbocycles or heterocycles which together can form RG5 and RG6 are independently of one another up to four substituents selected from the following group: hydroxyl, —CN, F or Cl or a branched or unbranched, optionally substituted C1-C4-alkoxy or C1-C4-alkyl radical, such as methoxy, methyl, CF3, C2F5 or CH2F.",
"WG is a structural element selected from the group of structural elements of the formulae IWG1 to IWG4, where the dashed lines intersect the atomic bonds within the structural element G and the carbon atom substituted by RG7 and RG8 is bonded to YG.",
"In a preferred embodiment, WG is a structural element selected from the group of structural elements of the formulae IWG2 and IWG3, in particular the structural element of the formula IWG2.RG1 in structural element WG is hydrogen, halogen, such as Cl, F, Br or I, a hydroxyl group or a branched or unbranched, optionally substituted C1-C6-alkyl radical, preferably C1-C4-alkyl or C1-C4-alkoxy radical such as in each case described above for RL1.Particularly preferred radicals for RG1 are hydrogen, methoxy or hydroxyl.",
"RG7, RG8, RG9 and RG10 in structural element G are independently of one another hydrogen, a hydroxyl group, CN, halogen, such as F, Cl, Br, I, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl or 1-ethyl-2-methylpropyl, C2-C6-alkenyl radical, such as optionally substituted vinyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl or 1-ethyl-2-methyl-2-propenyl, C2-C6-alkynyl radical, such as optionally substituted ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl or 1-ethyl-1-methyl-2-propynyl, an optionally substituted C3-C7-cycloalkyl radical, such as optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, C3-C7-heterocycloalkyl radical, such as optionally substituted aziridinyl, diaziridinyl, oxiranyl, oxaziridinyl, oxetanyl, thiiranyl, thietanyl, pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,4-dioxanyl, hexahydroazepinyl, oxepanyl, 1,2-oxathiolanyl or oxazolidinyl, C3-C7-heterocycloalkenyl radical, such as optionally substituted azirinyl, diazirinyl, thiirenyl, thietyl, pyrrolinyls, oxazolinyls, azepinyl, oxepinyl, α-pyranyl, β-pyranyl, γ-pyranyl, dihydropyranyls, 2,5-dihydropyrrolinyl or 4,5-dihydrooxazolyl, a branched or unbranched, optionally substituted C1-C4-alkylene-C3-C7-cycloalkyl radical, which is composed, for example, of branched or unbranched C1-C4-alkylene radicals such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-cycloalkyl radicals, a branched or unbranched optionally substituted C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, which is composed of optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene and, for example, the abovementioned C3-C7-heterocycloalkyl or C3-C7-heterocycloalkenyl radicals, the radicals being preferred which in the cyclic moiety contain one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds, a branched or unbranched, optionally substituted radical C1-C4-alkylene-O—RG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13, C1-C4-alkylene-SRG11 or C1-C4-alkylene-SO—RG11, which is composed of branched or unbranched, optionally substituted C1-C4-alkylene radicals, such as methylene, ethylene, propylene, n-butylene, isobutylene or t-butylene, the corresponding groups —O—, —CO—, —S—, —N and the terminal radicals RG11, RG12 and RG13 described below, an optionally substituted aryl radical, preferably optionally substituted phenyl, 1-naphthyl or 2-naphthyl, arylalkyl radical, preferably optionally substituted benzyl or ethylenephenyl (homobenzyl), hetaryl radical, preferably optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, thiadiazolyl, oxadiazolyl or triazinyl or their fused derivatives such as indazolyl, indolyl, benzothiophenyl, benzofuranyl, indolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolinyl or isoquinolinyl, hetarylalkyl radical, preferably optionally substituted —CH2-2-pyridyl, —CH2-3-pyridyl, —CH2-4-pyridyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-thiazolyl, —CH2-4-thiazolyl, CH2-5-thiazolyl, —CH2—CH2-2-pyridyl, —CH2—CH2-3-pyridyl, —CH2—CH2-4-pyridyl, —CH2—CH2-2-thienyl, —CH2—CH2-3-thienyl, —CH2—CH2-2-thiazolyl, —CH2—CH2-4-thiazolyl or —CH2—CH2-5-thiazolyl or a radical —S—RG11, —O—RG11, —SO—RG11, —SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13, CO—RG11.Further, two radicals RG7 and RG9 or RG8 and RG10 or RG7 and RG8 or RG9 and RG10 can in each case independently of one another together form an optionally substituted, saturated or unsaturated, nonaromatic, 3- to 7-membered carbocycle or heterocycle which can contain up to 3 heteroatoms selected from the group consisting of O, N, S and up to two double bonds.",
"Preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, a hydroxyl group, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C4-alkylene-C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radical, a branched or unbranched, optionally substituted radical C1-C4-alkylene-ORG11, C1-C4-alkylene-CO—ORG11, C1-C4-alkylene-O—CO—RG11, C1-C4-alkylene-CO—RG11, C1-C4-alkylene-SO2—NRG12RG13, C1-C4-alkylene-CO—NRG12RG13, C1-C4-alkylene-O—CO—NRG12RG13, C1-C4-alkylene-NRG12RG13 or C1-C4-alkylene-SRG11, C1-C4-alkylene-SO—RG11, a radical —S—RG11, —O—RG11, —SO—RG11, SO2—RG11, —CO—ORG11, —O—CO—RG11, —O—CO—NRG12RG13, —SO2—NRG12RG13, —CO—NRG12RG13, —NRG12RG13 or CO—RG11, an optionally substituted C3-C7-cycloalkyl, C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, aryl, hetaryl, arylalkyl or hetarylalkyl radical, as described above in each case.",
"Particularly preferred radicals for RG7, RG8, RG9 and RG10 in the structural element G are independently of one another hydrogen, F or a branched or unbranched, optionally substituted C1-C4-alkyl radical, as described above.",
"A branched or unbranched, optionally substituted C1-C8-alkyl radical for RG11, RG12 and RG13 is understood as meaning independently of one another, for example, the C1-C6-alkyl radicals mentioned above for RG1, plus the radicals heptyl and octyl.",
"Preferred substituents of the branched or unbranched, optionally substituted C1-C8-alkyl radicals for RG11, RG12 and RG13 independently of one another are the radicals halogen, hydroxyl, C1-C4-alkoxy, —CN, —COOH and —CO—O—C1-C4-alkyl.",
"A branched or unbranched, optionally substituted C2-C6-alkenyl, C2-C6-alkynyl or C1-C4-alkylene-C3-C7-cycloalkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RG11, RG12 and RG13 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Preferred, branched or unbranched, optionally substituted —C1-C5-alkylene-C1-C4-alkoxy radicals for RG11, RG12 and RG13 are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.",
"Preferred, branched or unbranched, optionally substituted mono- and bis-alkylaminoalkylene or acylaminoalkylene radicals for RG11, RG12 and RG13 are independently of one another branched or unbranched, optionally substituted radicals —C1-C4-alkylene-NH(C1-C4-alkyl), —C1-C4-alkylene-N(C1-C4-alkyl)2 or —C1-C4-alkylene-NH—CO—C1-C4-alkyl.",
"Preferred optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1.Particularly preferred, optionally substituted heterocycloalkyl, heterocycloalkenyl, C1-C4-alkyleneheterocycloalkyl or C1-C4-alkyleneheterocycloalkenyl radicals for RG11, RG12 and RG13 are independently of one another the C3-C7-heterocycloalkyl, C3-C7-heterocycloalkenyl, C1-C4-alkylene-C3-C7-heterocycloalkyl or C1-C4-alkylene-C3-C7-heterocycloalkenyl radicals described above for RG1, one or two heteroatoms selected from the group consisting of N, O and S and up to two double bonds being contained in the cyclic moiety.",
"Further, RG12 and RG13 can independently of one another be a radical —SO2—RG11, —CO—O—RG11, —CO—NRG11RG11* or —CO—RG11, RG11* being a radical RG11 which is independent of RG11.Preferred structural elements G are composed of at least one preferred radical of the structural element G, while the remaining radicals are widely variable.",
"Particularly preferred structural elements G are composed of the preferred radicals of the structural element G. Very particularly preferred structural elements G are composed of the particularly preferred radicals of the structural element G. Structural element B is understood as meaning a structural element comprising at least one atom which under physiological conditions can form hydrogen bridges as a hydrogen acceptor, at least one hydrogen acceptor atom having a distance of 5 to 14 atomic bonds from structural element G along the shortest possible route along the structural element skeleton.",
"The arrangement of the structural skeleton of structural element B is widely variable.",
"Suitable atoms which under physiological conditions can form hydrogen bridges as hydrogen acceptors are, for example, atoms having Lewis base properties, such as the heteroatoms nitrogen, oxygen or sulfur.",
"Physiological conditions is understood as meaning a pH which prevails at the site in a body at which the ligands interact with the receptors.",
"In the present case, the physiological conditions have a pH of, for example, 5 to 9.In a preferred embodiment, structural element B is a structural element of the formula IB A-E- IB where A and E have the following meanings: A is a structural element selected from the group: a 4- to 8-membered monocyclic saturated, unsaturated or aromatic hydrocarbon which can contain up to 4 heteroatoms selected from the group O, N and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, or a 9- to 14-membered polycyclic, saturated, unsaturated or aromatic hydrocarbon which can contain up to 6 heteroatoms selected from the group N, O and S, where, in each case independently of one another, the optionally present ring nitrogen or the carbons can be substituted, with the proviso that at least one heteroatom selected from the group O, N and S is contained in the structural element A, a radical where ZA1 is oxygen, sulfur or optionally substituted nitrogen and ZA2 is optionally substituted nitrogen, oxygen or sulfur and a radical where RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—R11, and E is a spacer structural element which covalently bonds the structural element A to the structural element G, where the number of atomic bonds along the shortest possible route along the structural element skeleton E is 5 to 14.In a particularly preferred embodiment, the structural element A is a structural element selected from the group of structural elements of the formulae IA1 to IA18 where m, p, g independently of one another are 1, 2 or 3, RA1, RA2 independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl or CO—C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, hetarylalkyl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 or both radicals RA1 and RA2 together are a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N, and S, RA13, RA13* independently of one another are hydrogen, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, where RA14 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or C1-C6-alkylene-C3-C7-cycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RA15, RA16, independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, arylalkyl, COO-alkylenearyl, SO2-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or hetarylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, CO-aryl, CO—NH-aryl, SO2-aryl, hetaryl, CO—NH-hetaryl or CO-hetaryl radical, RA3, RA4 independently of one another are hydrogen, —(CH2)n—(XA)j—RA12, or both radicals together are a 3- to 8-membered, saturated, unsaturated or aromatic N-heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle is optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, where n is 0, 1, 2 or 3, j is 0 or 1, XA is —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1), —N(RX1)— or —N(RX1)—SO2—, RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally C1-C4-alkyl- or aryl-substituted C2-C6-alkynyl or C2-C6-alkenyl radical or a 3- to 6-membered, saturated or unsaturated heterocycle, substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, a C3-C7-cycloalkyl, aryl or hetaryl radical, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RA12, together with RX1 or RX1* forms a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RX1, RX1* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, RA6, RA6* are hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, —CO—O—C1-C4-alkyl, arylalkyl, —CO—O-alkylenearyl, —CO—O-allyl, —CO—C1-C4-alkyl, —CO-alkylenearyl, C3-C7-cycloalkyl or —CO-allyl radical or in the structural element IA7 both radicals RA6 and RA6* together are an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl, C1-C4-alkoxy, C3-C7-cycloalkyl or —O—CO—C1-C4-alkyl radical, or an optionally substituted arylalkyl, —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical, or both radicals RA6 and RA7 together are an optionally substituted, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S, RA8 is hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl, CO—C1-C4-alkyl, SO2—C1-C4-alkyl or CO—O—C1-C4-alkyl radical or an optionally substituted aryl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl, CO—O-alkylenearyl or alkylenearyl radical, RA9, RA10 independently of one another are hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, or both radicals RA9 and RA10 together in the structural element IA14 are a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA11 is hydrogen, —CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16, RA17 is hydrogen or, in the structural element IA16, both radicals RA9 and RA17 together are a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals, RA18, RA19 independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 which is independent of RG11, Z1, Z2, Z3, Z4 independently of one another are nitrogen, C—H, C-halogen or a branched or unbranched, optionally substituted C—C1-C4-alkyl or C—C1-C4-alkoxy radical, Z5 is NRA8, oxygen or sulfur.",
"In a further very particularly preferred embodiment, the structural element A is a structural element of the formula IA1, IA4, IA7, IA8 or IA9.For RA1 or RA2 independently of one another a branched or unbranched, optionally substituted C1-C6-alkyl radical is understood as meaning, for example, the corresponding radicals described above for RG1, preferably methyl or trifluoromethyl.",
"For RA1 or RA2 in the structural elements IA1, IA2, IA3 or IA17, the branched or unbranched, optionally substituted radical CO—C1-C6-alkyl is composed, for example, of the group CO and the branched or unbranched, optionally substituted C1-C6-alkyl radicals described above for RA1 or RA2.Optionally substituted hetaryl, hetarylalkyl, aryl, arylalkyl or C3-C7-cycloalkyl radicals for RA1 or RA2 independently of one another are understood as meaning, for example, the corresponding radicals described above for RG7.For RA1 or RA2, the optionally substituted radicals CO—O—RA14, O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or SO2NRA15RA16 are composed, for example, of the groups CO—O, O, S, N, CO—N or SO2—N and the radicals RA14, RA15 or RA16 described in greater detail below.",
"Further, both radicals RA1 and RA2 can together form a fused, optionally substituted, 5- or 6-membered, unsaturated or aromatic carbocycle or heterocycle which can contain up to three heteroatoms selected from the group consisting of O, N and S. RA13 and RA13* are independently of one another hydrogen, CN, halogen, such as fluorine, chlorine, bromine or iodine, a branched or unbranched, optionally substituted C1-C6-alkyl radical, such as described above for RG1, preferably methyl or trifluoromethyl or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2NRA15RA16 or CO—NRA15RA16 as in each case described above for RA1.Preferred radicals for RA13 and RA13* are the radicals hydrogen, F, Cl, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or arylalkyl or a radical CO—O—RA14, O—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16.A branched or unbranched, optionally substituted C1-C6-alkyl, C3-C7-cycloalkyl, alkylenecycloalkyl, alkylene-C1-C4-alkoxy, C2-C6-alkenyl or C2-C6-alkynyl radical for RA14 in structural element A is understood as meaning, for example, the corresponding radicals described above for RG7.Optionally substituted aryl, arylalkyl, hetaryl or alkylhetaryl radicals for RA14 in structural element A are understood as meaning, for example, the corresponding radicals described above for RG7.Preferred radicals for RA14 are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical and optionally substituted benzyl.",
"A branched or unbranched, optionally substituted C1-C6-alkyl or arylalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or hetarylalkyl radical for RA15 or RA16 independently of one another is understood as meaning, for example, the corresponding radicals described above for RA14.The branched or unbranched, optionally substituted CO—C1-C6-alkyl, SO2—C1-C6-alkyl, COO—C1-C6-alkyl, CO—NH—C1-C6-alkyl, COO-alkylenearyl, CO—NH-alkylenearyl, CO—NH-alkylenehetaryl or SO2-alkylenearyl radicals or the optionally substituted CO-aryl, SO2-aryl, CO—NH-aryl, CO—NH-hetaryl or CO-hetaryl radicals for RA15 or RA16 are composed, for example, of the corresponding groups —CO—, —SO2—, —CO—O—, —CO—NH— and the corresponding branched or unbranched, optionally substituted C1-C6-alkyl, hetarylalkyl or arylalkyl radicals or the corresponding optionally substituted aryl or hetaryl radicals described above.",
"A radical —(CH2)n—(XA)j—RA12 for RA3 or RA4 independently of one another is understood as meaning a radical which is composed of the corresponding radicals —(CH2)n—, (XA)j and RA12.Here, n can be: 0, 1, 2 or 3 and j can be: 0 or 1.XA is a doubly bonded radical selected from the group —CO—, —CO—N(RX1)—, —N(RX1)—CO—, —N(RX1)—CO—N(RX1*)—, —N(RX1)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RX1)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RX1)—, —N(RX1)— and —N(RX1)—SO2—.",
"RA12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, as described above for RG7.a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, or a 3- to 6-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, triazinyl.",
"Further, RA12 and RX1 or RX1* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radical RA12 together with the radical RX1 or RX1* forms a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl, where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.",
"A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C12-alkynyl, preferably C2-C6-alkynyl or C2-C6-alkenyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl or hetaryl radical for RX1 and RX1* independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.Preferred, branched or unbranched, optionally substituted C1-C6-alkoxyalkyl for RX1 and RX1* are independently of one another methoxymethylene, ethoxymethylene, t-butoxymethylene, methoxyethylene or ethoxyethylene.",
"Preferred, branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, SO2—C1-C6-alkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl are preferably composed of the C1-C6-alkyl, arylalkyl, aryl or hetaryl radicals and the radicals —CO—, —O—, —SO2— described above.",
"Preferred radicals for RX1 and RX1* are independently of one another hydrogen, methyl, cyclopropyl, allyl and propargyl.",
"RA3 and RA4 can further together form a 3- to 8-membered saturated, unsaturated or aromatic N heterocycle which can additionally contain two further, identical or different heteroatoms O, N or S, where the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, RA5 is a branched or unbranched, optionally substituted C1-C6-alkyl, arylalkyl, C1-C4-alkyl-C3-C7-cycloalkyl or C3-C7-cycloalkyl radical or an optionally substituted aryl, hetaryl, heterocycloalkyl or heterocycloalkenyl radical, such as described above for RG7.RA6 and RA6* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as optionally substituted methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl, —CO—O—C1-C4-alkyl or —CO—C1-C4-alkyl radical such as composed of the group —CO—O— or —CO— and the C1-C4-alkyl radicals described above, arylalkyl radical, as described above for RG7, —CO—O-alkylenearyl or —CO-alkylenearyl radical such as composed of the group —CO—O— or —CO— and the arylalkyl radicals described above, —CO—O-allyl or —CO-allyl radical, or C3-C7-cycloalkyl radical, such as described above for RG7.Further, both radicals RA6 and RA6* in structural element IA7 can together form an optionally substituted, saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. RA7 is hydrogen, —OH, —CN, —CONH2, a branched or unbranched, optionally substituted C1-C4-alkyl radical, for example as described above for RA6, C1-C4-alkoxy, arylalkyl or C3-C7-cycloalkyl radical, for example as described above for RL14, a branched or unbranched, optionally substituted —O—CO—C1-C4-alkyl radical, which is composed of the group —O—CO— and, for example, of the C1-C4-alkyl radicals mentioned above or an optionally substituted —O-alkylenearyl, —O—CO-aryl, —O—CO-alkylenearyl or —O—CO-allyl radical which is composed of the groups —O— or —O—CO— and, for example, of the corresponding radicals described above for RG7.Further, both radicals RA6 and RA7 can together form an optionally substituted unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to two further different or identical heteroatoms O, N, S. For RAS in structural element A, the branched or unbranched, optionally substituted C1-C4-alkyl radical or an optionally substituted aryl or arylalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA15, where the radicals CO—C1-C4-alkyl, SO2—C1-C4-alkyl, CO—O—C1-C4-alkyl, CO-aryl, SO2-aryl, CO—O-aryl, CO-alkylenearyl, SO2-alkylenearyl or CO—O-alkylenearyl are composed analogously to the other composed radicals of the group consisting of CO, SO2 and COO and, for example, of the corresponding C1-C4-alkyl, aryl or arylalkyl radicals described above for RA15 and these radicals can be optionally substituted.",
"In each case, for RA9 or RA10 independently of one another, a branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl or C3-C7-cycloalkyl radical is understood as meaning, for example, the corresponding radicals described above for RA14, preferably methyl or trifluoromethyl.",
"In each case, for RA9 or RA10 independently of one another, a radical CO—O—RA14, O—RA14, S—RA14, SO2—NRA15RA16, NRA15RA16 or CO—NRA15RA16 is understood as meaning, for example, the corresponding radicals described above for RA13.Further, both radicals RA9 and RA10 together in structural element IA14 can form a 5- to 7-membered saturated, unsaturated or aromatic carbocycle or heterocycle, which can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.",
"Substituents in this case are in particular understood as meaning halogen, CN, a branched or unbranched, optionally substituted C1-C4-alkyl radical, such as methyl or trifluoromethyl or the radicals O—RA14, S—RA14, NRA15RA16, CO—NRA15RA16 or —((RA8)HN)C═N—RA7.A branched or unbranched, optionally substituted C1-C6-alkyl radical or an optionally substituted aryl, arylalkyl, hetaryl, C3-C7-cycloalkyl radical or a radical CO—O—RA14, O—RA14, S—RA14, NRA15RA16, SO2—NRA15RA16 or CO—NRA15RA16 for RA11 is understood, for example, as meaning the corresponding radicals described above for RA9.Further, in structural element IA16, both radicals RA9 and RA17 together can form a 5- to 7-membered saturated, unsaturated or aromatic heterocycle which, in addition to the ring nitrogen, can contain up to three different or identical heteroatoms O, N, S and is optionally substituted by up to three identical or different radicals.",
"A branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG11 for RA18 and RA19 independently of one another is understood as meaning, for example, the radicals described above for RG12, preferably hydrogen or a branched or unbranched, optionally substituted C1-C8-alkyl radical.",
"Z1, Z2, Z3, Z4 are independently of one another nitrogen, C—H, C-halogen, such as C—F, C—Cl, C—Br or C—I or a branched or unbranched, optionally substituted C—C1-C4-alkyl radical which is composed of a carbon radical and, for example, a C1-C4-alkyl radical described above for RA6 or a branched or unbranched optionally substituted C—C1-C4-alkoxy radical which is composed of a carbon radical and, for example, a C1-C4-alkoxy radical described above for RA7.Z5 is oxygen, sulfur or a radical NRA8.Preferred structural elements A are composed of at least one preferred radical of the radicals belonging to the structural element A, while the remaining radicals are widely variable.",
"Particularly preferred structural elements A are composed of the preferred radicals of the structural element A.",
"In a preferred embodiment, the spacer structural element E is understood as meaning a structural element that consists of a branched or unbranched aliphatic C2-C30-hydrocarbon radical which is optionally substituted and contains heteroatoms and/or of a 4- to 20-membered aliphatic or aromatic mono- or polycyclic hydrocarbon radical which is optionally substituted and contains heteroatoms.",
"In a further preferred embodiment, the spacer structural element E is composed of two to four substructural elements, selected from the group consisting of E1 and E2, where the sequence of linkage of the substructural elements is arbitrary and E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where c, d, f, g, h independently of one another are 0, 1 or 2, k1, k2, k3, k4, k5, k6 independently of one another are 0 or 1, XE, QE independently of one another are an optionally substituted 4- to 11-membered mono- or polycyclic, aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O and S, where the ring carbons and/or the ring nitrogens can optionally be substituted, YE, ZE independently of one another are CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9, RE10 independently, of one another are hydrogen, halogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical, a radical —(CH2)x—(WE)z—RE17, an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, or independently of one another in each case two radicals RE1 and RE2 or RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 together are a 3- to 7-membered, optionally substituted, saturated or unsaturated carbocycle or heterocycle which can contain up to three heteroatoms selected from the group O, N and S, x is 0, 1, 2, 3 or 4, z is 0 or 1, WE is —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, RW2, RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3- to 8-membered, saturated or unsaturated heterocycle substituted by up to three identical or different radicals, which can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S and the cycle can optionally be substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, or the radical RE17 forms, together with RW2 or RW2*, a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group O, S and N, RE11, RE11* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl radical, RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl radical, an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical or a radical CO—RE16, COORE16 or SO2—RE16, RE13, RE14 independently of one another are hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE15 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, RE16 is hydrogen, a hydroxyl group, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical.",
"The coefficient c is preferably 0 or 1, the coefficient d is preferably 1 or 2, and the coefficients f, g, h independently of one another are preferably 0 or 1.An optionally substituted 4- to 11-membered mono- or polycyclic aliphatic or aromatic hydrocarbon which can contain up to 6 double bonds and up to 6 identical or different heteroatoms selected from the group N, O, S, where the ring carbons or ring nitrogens can optionally be substituted, for QE and XE independently of one another is preferably understood as meaning optionally substituted arylene, such as optionally substituted phenylene or naphthylene, or optionally substituted hetarylene such as the radicals and their substituted or fused derivatives, or radicals of the formulae IE1 to IE11, where the incorporation of the radicals can take place in both orientations.",
"Aliphatic hydrocarbons are understood as meaning, for example, saturated and unsaturated hydrocarbons.",
"Z6 and Z7 are independently of one another CH or nitrogen.",
"Z8 is oxygen, sulfur or NH.",
"Z9 is oxygen, sulfur or NRE20.r1, r2, r3 and t are independently of one another 0, 1, 2 or 3.s and u are independently of one another 0, 1 or 2.Particularly preferably, XE and QE independently of one another are optionally substituted phenylene, a radical and their substituted or fused derivatives, or radicals of the formulae IE1, IE2, IE3, IE4 and IE7, where the incorporation of the radicals can take place in both orientations.",
"RE18 and RE19 are independently of one another hydrogen, —NO2, —NH2, —CN, —COOH, a hydroxyl group, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C4-alkoxy, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical, as in each case described above.",
"RE20 is independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C3-C12-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, hetaryl, CO-hetaryl or SO2-alkylenearyl radical, preferably hydrogen or a branched or unbranched, optionally substituted C1-C6-alkyl radical.",
"YE and ZE are independently of one another CO, CO—NRE12, NRE12—CO, sulfur, SO, SO2, SO2—NRE12, NRE12—SO2, CS, CS—NRE12, NRE12—CS, CS—O, O—CS, CO—O, O—CO, oxygen, ethynylene, CRE13—O—CRE14, C(═CRE13RE14), CRE13═CRE14, —CRE13(ORE15)—CHRE14— or —CHRE13—CRE14(ORE15)—, preferably CO, SO2 and oxygen.",
"RE12 is hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C8-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, hetaryl, arylalkyl or hetarylalkyl radical, such as correspondingly described above for RG7 or a radical CO—RE16, COORE16 or SO2—RE16, preferably hydrogen, methyl, allyl, propargyl and cyclopropyl.",
"A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE13, RE14 or RE15 independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.A branched or unbranched, optionally substituted C1-C4-alkoxy radical for RE13 or RE14 independently of one another is understood as meaning, for example, the C1-C4-alkoxy radicals described above for RA14.Preferred alkylenecycloalkyl radicals for RE13, RE14 or RE15 independently of one another are, for example, the C1-C4-alkylene-C3-C7-cycloalkyl radicals described above for RG7.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical, or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkylene-C3-C7-heterocycloalkyl, C1-C4-alkylene-C3-C7-heterocycloalkenyl or hetarylalkyl radical for RE16 is understood as meaning, for example, the corresponding radicals described above for RG11.A branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or alkylenecycloalkyl radical or an optionally substituted C3-C7-cycloalkyl, aryl, arylalkyl, hetaryl or hetarylalkyl radical for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 independently of one another is understood as meaning, for example, the corresponding radicals mentioned above for RG1.Further, two radicals RE3 and RE4 or RE5 and RE6 or RE7 and RE8 or RE9 and RE10 can in each case independently of one another together form a 3- to 7-membered, optionally substituted, saturated or unsaturated carbo- or heterocycle which can contain up to three heteroatoms from the group O, N and S. The radical —(CH2)x—(WE)z—RE17 is composed of a C0-C4-alkylene radical, optionally a bonding element WE selected from the group —CO—, —CO—N(RW2)—, —N(RW2)—CO—, —N(RW2)—CO—N(RW2*)—, —N(RW2)—CO—O—, —O—, —S—, —SO2—, —SO2—N(RW2)—, —SO2—O—, —CO—O—, —O—CO—, —O—CO—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, preferably selected from the group —CO—N(RW2)—, —N(RW2)—CO—, —O—, —SO2—N(RW2)—, —N(RW2)— or —N(RW2)—SO2—, and the radical RE17, where RW2 and RW2* independently of one another are hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C2-C6-alkenyl, C2-C8-alkynyl, CO—C1-C6-alkyl, CO—O—C1-C6-alkyl or SO2—C1-C6-alkyl radical or an optionally substituted hetaryl, hetarylalkyl, arylalkyl, C3-C7-cycloalkyl, CO—O-alkylenearyl, CO-alkylenearyl, CO-aryl, SO2-aryl, CO-hetaryl or SO2-alkylenearyl radical, preferably independently of one another are hydrogen, methyl, cyclopropyl, allyl, propargyl, and RE17 is hydrogen, a hydroxyl group, CN, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, an optionally substituted C3-C7-cycloalkyl, aryl, hetaryl or arylalkyl radical, a C2-C6-alkynyl or C2-C6-alkenyl radical optionally substituted by C1-C4-alkyl or aryl, an optionally substituted C6-C12-bicycloalkyl, C1-C6-alkylene-C6-C12-bicycloalkyl, C7-C20-tricycloalkyl or C1-C6-alkylene-C7-C20-tricycloalkyl radical, or a 3 to 8-membered, saturated or unsaturated heterocycle which is substituted by up to three identical or different radicals and can contain up to three different or identical heteroatoms O, N, S, where two radicals together can be a fused, saturated, unsaturated or aromatic carbocycle or heterocycle which can contain up to three different or identical heteroatoms O, N, S, and the cycle can be optionally substituted or a further, optionally substituted, saturated, unsaturated or aromatic cycle can be fused to this cycle, such as optionally substituted 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-furyl, 3-furyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 6-pyrimidyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-(1,3,4-thiadiazolyl), 2-(1,3,4)-oxadiazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl or triazinyl.",
"Further, RE17 and RW2 or RW2* can together form a saturated or unsaturated C3-C7-heterocycle which can optionally contain up to two further heteroatoms selected from the group consisting of O, S and N. Preferably, the radicals RE17 and RW2 or RW2* together form a cyclic amine as the C3-C7-heterocycle in the case where the radicals are bonded to the same nitrogen atom, such as N-pyrrolidinyl, N-piperidinyl, N-hexahydroazepinyl, N-morpholinyl or N-piperazinyl where in heterocycles which carry free amine protons, such as N-piperazinyl, the free amine protons can be replaced by customary amine protective groups, such as methyl, benzyl, Boc (tert-butoxycarbonyl), Z (benzyloxycarbonyl), tosyl, —SO2—C1-C4-alkyl, —SO2-phenyl or —SO2-benzyl.",
"Preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, halogen, a branched or unbranched, optionally substituted C1-C6-alkyl radical, optionally substituted aryl or the radical —(CH2)x—(WE)z—RE17.Particularly preferred radicals for RE1, RE2, RE3, RE4, RE5, RE6, RE7, RE8, RE9 or RE10 are independently of one another hydrogen, F, a branched or unbranched, optionally substituted C1-C4-alkyl radical, in particular methyl.",
"A branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxyalkyl, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical or an optionally substituted aryl, hetaryl or C3-C7-cycloalkyl for RE11 and RE11* in structural element E independently of one another is understood as meaning, for example, the corresponding radicals described above for RG7.The branched or unbranched, optionally substituted radicals CO—C1-C6-alkyl, CO—O—C1-C6-alkyl, CO—NH—C1-C6-alkoxyalkyl, CO—NH—C1-C6-alkyl or SO2—C1-C6-alkyl radical or the optionally substituted radicals CO—O-alkylenearyl, CO—NH-alkylenearyl, CO-alkylenearyl, CO-aryl, CO—NH-aryl, SO2-aryl, CO-hetaryl, SO2-alkylenearyl, SO2-hetaryl or SO2-alkylenehetaryl for RE11 and RE11* independently of one another are composed, for example, of the corresponding groups CO, COO, CONH or SO2 and the corresponding radicals mentioned above.",
"Preferred radicals for RE11 or RE11* are independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C6-alkyl, C1-C6-alkoxy, C2-C6-alkenyl, C2-C12-alkynyl or arylalkyl radical, or an optionally substituted hetaryl or C3-C7-cycloalkyl radical.",
"Particularly preferred radicals for RE11 or RE11* are hydrogen, methyl, cyclopropyl, allyl or propargyl.",
"In a particularly preferred embodiment of structural element E1, structural element E1 is a radical —CH2—CH2—CO—, —CH2—CH2—CH2—CO— or a C1-C5-alkylene radical.",
"In a particularly preferred embodiment of structural element E, the spacer structural element E used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where the structural elements E2 and E1 have the meanings described above.",
"Preferred structural elements E are composed of at least one preferred radical of the radicals belonging to structural element E, while the remaining radicals are widely variable.",
"Particularly preferred structural elements E are composed of the preferred radicals of structural element E. Preferred structural elements B are composed either of the preferred structural element A, while E is widely variable, or of the preferred structural element E, while A is widely variable.",
"In a further preferred embodiment, the structural element E used is the structural element E′ described below for the novel compounds of the formula I′.",
"In a further preferred embodiment, the structural element G used is the structural element G′ described below for the novel compounds of the formula I′.",
"The compounds of the formula I, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.",
"The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.",
"The use of an enantiomerically pure compound as the active compound is preferred.",
"The compounds of the formula I can also be present in other tautomeric forms.",
"The compounds of the formula I can also be present in the form of physiologically tolerable salts.",
"The compounds of the formula I can also be present as prodrugs in a form in which the compounds of the formula I are liberated under physiological conditions.",
"By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.",
"Derivatized structural elements B or A are also suitable which liberate the structural element B or A respectively under physiological conditions.",
"In preferred compounds of the formula I, in each case one of the three structural elements B, G or L has the preferred range, while the remaining structural elements are widely variable.",
"In particularly preferred compounds of the formula I, in each case two of the three structural elements B, G and L have the preferred range, while the remaining structural elements are widely variable.",
"In very particularly preferred compounds of the formula I, in each case all three structural elements B, G and L have the preferred range, while the remaining structural element is widely variable.",
"Preferred compounds of the formula I contain, for example, the preferred structural element G, while the structural elements B and L are widely variable.",
"In particularly preferred compounds of the formula I, for example, B is replaced by the structural element A-E- and the compounds contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.",
"Further particularly preferred compounds of the formula I contain, for example, the preferred structural element G and the preferred structural element A, while the structural elements E and L are widely variable.",
"The invention further relates to the use of the structural element of the formula IGL -G-L IGL for the preparation of compounds which bind to integrin receptors.",
"The compounds of the formula I bind to integrin receptors.",
"The compounds of the formula I are therefore preferably suitable as integrin receptor ligands and for the production of drugs for treating diseases in which an integrin receptor is involved, in particular for treating diseases in which the interaction between integrins and their natural ligands is dysregulated, i.e.",
"excessive or decreased.",
"Integrin receptor ligands are understood as meaning agonists and antagonists.",
"An excessive or decreased interaction is understood as meaning both an excessive or decreased expression of the natural ligand and/or of the integrin receptor and thus an excessive or decreased amount of natural ligand and/or integrin receptor or an increased or decreased affinity of the natural ligand for the integrin receptor.",
"The interaction between integrins and their natural ligands is dysregulated compared with the normal state, i.e.",
"excessive or decreased, if this dysregulation does not correspond to the physiological state.",
"An increased or decreased interaction can lead to pathophysiological situations.",
"The level of dysregulation which leads to a pathophysiological situation is dependent on the individual organism and on the site and nature of the disorder.",
"Preferred integrin receptors for which the compounds of the formula I according to the invention can be used are the α5β1, α4β1, αVβ5 and αVβ3 integrin receptors.",
"The compounds of the formula I particularly preferably bind to the αVβ3 integrin receptor and can thus be particularly preferably used as ligands of the αVβ3 integrin receptor and for the treatment of illnesses in which the interaction between αVβ3 integrin receptor and its natural ligand is excessive or reduced.",
"The compounds of the formula I are preferably used for the treatment of the following illnesses: cardiovascular disorders such as atherosclerosis, restenosis after vascular injury or stent implantation, and angioplasty (neointima formation, smooth muscle cell migration and proliferation), acute kidney failure, angiogenesis-associated microangiopathies such as diabetic angiopathies or retinopathy or rheumatoid arthritis, blood platelet-mediated vascular occlusion, arterial thrombosis, stroke, reperfusion damage after myocardial infarct or stroke, carcinomatous disorders, such as in tumor metastasis or in tumor growth (tumor-induced angiogenesis), osteoporosis (bone resorption after chemotaxis and adhesion of osteoclasts to the bone matrix), high blood pressure, psoriasis, hyperparathyroidism, Paget's disease, malignant hypercalcemia, metastatic osteolytic lesions, inflammation, wound healing, cardiac insufficiency, congestive heart failure CHF, as well as in antiviral, antimycotic, antiparasitic or antibacterial therapy and prophylaxis (adhesion and internalization).",
"Furthermore, the invention relates in particular to the use of the compounds of the formula I as ligands of the αVβ3 integrin receptor.",
"The invention furthermore relates to the novel compounds of the formula I′ A-E′-G′-L I′ where the structural elements A and L have the meanings described above.",
"Preferred structural elements A and L are described.",
"Structural element E′ is composed of two to four substructural elements selected from the group consisting of E1 and E2, the linkage sequence of the substructural elements being arbitrary and E1 and E2 having the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)g—(X)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case in which YE or ZE=CO and a radical XE or QE or an aromatic or heteroaromatic radical of the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.",
"In a further preferred embodiment of structural element E′, the structural element E′ used is a structural element of the formula IE1E2 -E2-E1- IE1E2 where E1 and E2 have the following meanings: E1 is a substructural element of the formula IE1 —(YE)k1—(CRE1RE2)c-(QE)k2-(CRE3RE4)d— IE1 and E2 is a substructural element of the formula IE2 —(NRE11)k3—(CRE5RE6)f-(ZE)k4-(CRE7RE8)—(XE)k5—(CRE9RE10)h—(NRE11*)k6— IE2, where all radicals and coefficients of structural element E′ have the meanings of structural element E described above, with the proviso that in the case where YE═CO, k1 and k5=1 and h and k6=0 the sum of the indices c, k2 and d must be other than 0 and in the case where an aromatic or heteroaromatic radical from the structural element A is bonded directly to YE or ZE, a direct atomic bond from YE or ZE to the structural element G′ is excluded.",
"Further preferred embodiments of structural element E′ correspond to the preferred embodiments of structural element E with the proviso described above.",
"Structural element G′ is identical to structural element G, as described above, except for the radicals RG12 and RG13.In structural element G′, the radicals RG12 and RG13 of structural element G are replaced by the radicals RG′12 and RG′13.The radicals RG12 and RG′13 in structural element G′ have the following meanings: independently of one another hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, C1-C5-alkylene-C1-C4-alkoxy, mono- and bis-alkylaminoalkylene or acylaminoalkylene radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical, or a radical —SO2—RG11, —CO—ORG11, —CO—NRG11RG11* or —CO—RG14, where RG14 is hydrogen, a branched or unbranched, optionally substituted C1-C8-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C1-C5-alkylene-C1-C4-alkoxy radical or an optionally substituted aryl, heterocycloalkyl, heterocycloalkenyl, hetaryl, C3-C7-cycloalkyl, C1-C4-alkylene-C3-C7-cycloalkyl, arylalkyl, C1-C4-alkyleneheterocycloalkyl, C1-C4-alkyleneheterocycloalkenyl or hetarylalkyl radical.",
"Preferred radicals of RG′12 and RG13 are the corresponding radicals described above for RG12 and RG13.Further preferred embodiments of the structural element G′ correspond to the preferred embodiments of structural element G. The compounds of the formula I′, and also the intermediates for their preparation, can have one or more asymmetric substituted carbon atoms.",
"The compounds can be present as pure enantiomers or pure diastereomers or as a mixture thereof.",
"The use of an enantiomerically pure compound as active compound is preferred.",
"The compounds of the formula I′ can also be present in other tautomeric forms.",
"The compounds of the formula I′ can also be present in the form of physiologically tolerable salts.",
"The compounds of the formula I′ can also be present as prodrugs in a form in which the compounds of the formula I′ are liberated under physiological conditions.",
"By way of example, reference may be made here to the group T in structural element L, which in some cases contains groups which are hydrolyzable to the free carboxylic acid group under physiological conditions.",
"Derivatized structural elements A which liberate the structural element A under physiological conditions are also suitable.",
"Analogously to the compounds of the formula I, the following applies for the compounds of the formula I′ as mentioned above: In preferred compounds of the formula I′, one of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.",
"In particularly preferred compounds of the formula I′, two of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural elements are widely variable.",
"In further particularly preferred compounds of the formula I′, three of the four structural elements A, E′, G′ or L in each case have the preferred range, while the remaining structural element is widely variable.",
"In very particularly preferred compounds of the formula I′, all four structural elements A, E′, G′ or L have the preferred range.",
"Preferred compounds of the formula I, have, for example, the preferred structural element G′, while the structural elements A, E′ and L are widely variable.",
"Further particularly preferred compounds of the formula I′ have, for example, the preferred structural element G′ and the preferred structural element A, while the structural elements E′ and L are widely variable.",
"Very particularly preferred compounds of the formula I′ are listed below, the number before the text block being the number of an individualized compound of the formula I′, and in the text block A-E′-G′-L the abbreviations separated by a bonding dash in each case being an individual structural element A, E′, G′ or L and the meaning of the abbreviations of the structural elements being explained after the table.",
"No.",
"A-E′-G′-L 1 bhs-edia3-phen-es 2 2py-inda2-phen-es 3 bhs-35thirna2-meph-es 4 bim-dibema2-dmeph-es 5 2py-bam2-4-clph-es 6 2py-dibema2-dmeph-es 7 bhs-a24thima2-dmeph-es 8 bhs-aaf-phen-es 9 bhs-a24thima2-4-clph-es 10 bim-me42thiaz2-phen-es 11 2py-edia2-phen-es 12 bim-a24thima2-hdb-es 13 2py-apma2-4-clph-es 14 gua-chex2-phen-es 15 bhs-dibema2-4-clph-es 16 bhs-bam2-phen-es 17 bim-a23thima2-4-clph-es 18 bim-dibema2-phen-es 19 bim-bam2-4-clph-es 20 2py-pipa2-4-clph-es 21 2py-me25thima2-phen-es 22 gua-a24thima2-ioph-es 23 imhs-apma2-phen-es 24 2py-apma2-ioph-es 25 gua-edia3-phen-es 26 bhs-a23thima2-4-clph-es 27 2py-a24thima2-2pyph-es 28 2py-bam2-thoph-es 29 gua-apma2-phen-es 30 2py-a24thima2-reph-es 31 gua-hexa-phen-es 32 dimethpym-apma2-phen-es 33 2py-dibema2-meph-es 34 bhs-apma2-phen-es 35 bim-edia3-phen-es 36 gua-apma2-meph-es 37 bim-apma2-reph-es 38 2py-a24thima2-dmeph-es 39 gua-aaf-phen-es 40 gua-apma2-yrph-es 41 gua-pipeme2-phen-es 42 2py-35thima2-phen-es 43 gua-a24thima2-reph-es 44 bim-bam2-phen-es 45 gua-a24thima2-phen-ms 46 gua-apma2-reph-es 47 mam2py-a24thima2-phen-es 48 2py-a24thima2-phen-gs 49 2py-apma2-phen-nes 50 2py-a24thima2-4-clph-es 51 bhs-penta-phen-es 52 gua-35thima2-dmeph-es 53 bim-dibema2-thoph-es 54 bim-a24thima2-reph-es 55 2py-a23thima2-4-clph-es 56 gua-a23thima2-phen-es 57 dhim-a24thima2-phen-es 58 gua-penta-phen-es 59 bhs-a24thima2-reph-es 60 2py-a23thima2-meph-es 61 gua-prodia2-phen-es 62 bhs-apma2-reph-es 63 2py-apma2-meph-es 64 gua-bam2-4-clph-es 65 2py-me35thima2-phen-es 66 gua-apma2-2pyph-es 67 2py-35thima2-thoph-es 68 clim-a24thima2-phen-es 69 2py-buta-phen-es 70 am2py-apma2-phen-es 71 gua-a24thima2-dmeph-es 72 bhs-apma2-hdb-es 73 bhs-dibema2-thoph-es 74 bim-dibema2-meph-es 75 bhs-bam2-meph-es 76 bhs-apma2-yrph-es 77 bhs-apma2-dmeph-es 78 gua-me25thima2-phen-es 79 2py-a24thima2-meph-es 80 gua-inda2-phen-es 81 bhs-mepipe2-phen-es 82 bhs-a24thima2-phen-as 83 bim-pipa2-thoph-es 84 bhs-bam2-4-clph-es 85 bhs-a24thima2-phen-es 86 bhs-35thima2-phen-es 87 bim-penta-phen-es 88 bhs-apma2-dbph-es 89 gua-42thiaz2-phen-es 90 bhs-a24thima2-24pym-es 91 2py-dibema2-phen-es 92 bim-a24thima2-dm-es 93 bhs-pipa2-4-clph-es 94 2py-apma2-phen-ps 95 2py-apma2-dmeph-es 96 2py-mepipe2-phen-es 97 bhs-35thima2-4-clph-es 98 gua-apma2-phen-mals 99 gua-35thima2-4-clph-es 100 mam2py-apma2-phen-es 101 gua-buta-phen-es 102 2py-a23thima2-thoph-es 103 2py-pyma2-phen-es 104 gua-apma2-phen-gs 105 bim-apma2-phen-as 106 bhs-apma2-4-clph-es 107 bhs-a24thima2-4-pyph-es 108 thpym-apma2-phen-es 109 gua-pipa2-dmeph-es 110 amim-a24thima2-phen-es 111 bim-aepi2-phen-es 112 bim-a23thima2-thoph-es 113 bhs-a23thima2-thoph-es 114 gua-pdagk-phen-es 115 2py-hexa-phen-es 116 bhs-a24thima2-meph-es 117 gua-bam2-meph-es 118 2py-35thima2-meph-es 119 2py-pipa2-meph-es 120 bhs-a23thima2-phen-es 121 bim-pipeme2-phen-es 122 bhs-buta-phen-es 123 bhs-pipa2-dmeph-es 124 bhs-pipa2-meph-es 125 2py-35thima2-dmeph-es 126 bim-bam2-thoph-es 127 gua-35thima2-thoph-es 128 bim-edia2-phen-es 129 bim-apma2-phen-nes 130 gua-bam2-thoph-es 131 gua-bam2-phen-es 132 pippy-a24thima2-phen-es 133 gua-35thima2-phen-es 134 bim-a23thima2-phen-es 135 gua-dibema2-dmeph-es 136 bhs-apma2-phen-gs 137 bhs-apma2-thoph-es 138 2py-apma2-phen-es 139 im-a24thima2-phen-es 140 gua-aepi2-phen-es 141 2py-mea2-phen-es 142 gua-a24thima2-phen-es 143 2py-a24thima2-thaph-es 144 gua-apma2-4-clph-es 145 bhs-apma2-phen-f2es 146 bhs-inda2-phen-es 147 bim-a24thima2-dbph-es 148 bim-apma2-phen-ms 149 gua-a23thima2-thoph-es 150 pippy-apma2-phen-es 151 bhs-apma2-meph-es 152 2py-apma2-phen-as 153 gua-mea2-phen-es 154 bhs-me35thima2-phen-es 155 bhs-pdagk-phen-es 156 bim-42thiaz2-phen-es 157 2py-pipeme2-phen-es 158 bim-me25thima2-phen-es 159 gua-dibema2-phen-es 160 2py-apma2-oxph-es 161 bhs-a24thima2-3clph-es 162 bim-pyma2-phen-es 163 bhs-edia2-phen-es 164 imhs-a24thima2-phen-es 165 gua-dibema2-thoph-es 166 bim-a24thima2-4-clph-es 167 bim-apma2-phen-ps 168 gua-apma2-dm-es 169 2py-a24thima2-phen-mals 170 2py-dibema2-4-clph-es 171 bhs-dibema2-meph-es 172 bim-aof-phen-es 173 bhs-pipeme2-phen-es 174 gua-35thima2-meph-es 175 bim-aaf-phen-es 176 bim-dibema2-4-clph-es 177 bim-a24thima2-2pyph-es 178 bim-a24thima2-yrph-es 179 bim-35thima2-4-clph-es 180 bhs-aepi2-phen-es 181 bim-pipa2-phen-es 182 gua-a23thima2-dmeph-es 183 2py-a24thima2-yrph-es 184 gua-a24thima2-dmeoph-es 185 bhs-42thiaz2-phen-es 186 bim-mepipe2-phen-es 187 2py-chex2-phen-es 188 bhs-prodia2-phen-es 189 gua-bam2-dmeph-es 190 bhs-me25thima2-phen-es 191 thpym-a2.4thima2-phen-es 192 bim-pipa2-dmeph-es 193 bhs-dibema2-phen-es 194 gua-a24thima2-thoph-es 195 2py-pipa2-thoph-es 196 clim-apma2-phen-es 197 bhs-chex2-phen-es 198 gua-a24thima2-phen-nes 199 gua-a24thima2-meph-es 200 bhs-a24thima2-dmeoph-es 201 2py-apma2-24pym-es 202 2py-a24thima2-phen-pms 203 gua-a24thima2-4-piph-es 204 bim-apma2-3clph-es 205 gua-apma2-hdb-es 206 2py-bam2-meph-es 207 bhs-a24thima2-phen-nes 208 gua-pyma2-phen-es 209 bim-a24thima2-thoph-es 210 gua-mepipe2-phen-es 211 bim-apma2-4-clph-es 212 2py-42thiaz2-phen-es 213 bim-apma2-24pym-es 214 bhs-a23thima2-dmeph-es 215 gua-apma2-dbph-es 216 gua-me35thima2-phen-es 217 bim-35thima2-phen-es 218 gua-apma2-thaph-es 219 bim-35thima2-thoph-es 220 gua-apma2-phen-f2es 221 2py-apma2-3clph-es 222 gua-apma2-thoph-es 223 bim-pipa2-meph-es 224 2py-aof-phen-es 225 bhs-35thima2-dmeph-es 226 bhs-hexa-phen-es 227 bim-pipa2-4-clph-es 228 bhs-apma2-phen-pms 229 bim-a23thima2-dmeph-es 230 2py-me42thiaz2-phen-es 231 bim-a24thima2-phen-gs 232 bim-apma2-dmeph-es 233 gua-a23thima2-4-clph-es 234 bim-a24thima2-phen-mals 235 2py-apma2-phen-ms 236 bhs-a24thima2-ioph-es 237 bim-a24thima2-phen-es 238 2py-pdagk-phen-es 239 gua-a24thima2-phen-ps 240 2py-pipa2-phen-es 241 2py-aepi2-phen-es 242 2py-a24thima2-thoph-es 243 bhs-bam2-dmeph-es 244 bim-hexa-phen-es 245 bim-a24thima2-meph-es 246 bhs-me42thiaz2-phen-es 247 am2py-a24thima2-phen-es 248 bim-apma2-meph-es 249 bim-me35thima2-phen-es 250 gua-pipa2-phen-es 251 bhs-a24thima2-oxph-es 252 2py-pipa2-dmeph-es 253 2py-apma2-4-pyph-es 254 bhs-35thima2-thoph-es 255 gua-me42thiaz2-phen-es 256 bim-a24thima2-thaph-es 257 gua-a24thima2-phen-as 258 2py-a24thima2-phen-f2es 259 2py-a24thima2-dbph-es 260 bim-35thima2-meph-es 261 bim-apma2-phen-es 262 bim-a24thima2-phen-pms 263 bim-chex2-phen-es 264 bim-a24thima2-dmeph-es 265 bim-mea2-phen-es 266 2py-apma2-thoph-es 267 dimethpym-a24thima2-phen-es 268 2py-dibema2-thoph-es 269 2py-apma2-dmeoph-es 270 gua-dibema2-4-clph-es 271 bhs-pipa2-phen-es 272 gua-edia2-phen-es 273 gua-apma2-dmeph-es 274 2py-edia3-phen-es 275 gua-a23thima2-meph-es 276 bim-pdagk-phen-es 277 gua-apma2-phen-pms 278 bhs-mea2-phen-es 279 bim-35thima2-dmeph-es 280 bhs-aof-phen-es 281 2py-prodia2-phen-es 282 bim-inda2-phen-es 283 bhs-bam2-thoph-es 284 bim-apma2-4-pyph-es 285 2py-aaf-phen-es 286 2py-bam2-phen-es 287 bhs-apma2-dm-es 288 2py-penta-phen-es 289 gua-aof-phen-es 290 im-apma2-phen-es 291 gua-pipa2-4-clph-es 292 bim-apma2-ioph-es 293 bim-bam2-meph-es 294 gua-pipa2-meph-es 295 bhs-apma2-thaph-es 296 bhs-apma2-2pyph-es 297 bim-apma2-dmeoph-es 298 amim-apma2-phen-es 299 dhim-apma2-phen-es 300 bhs-a24thima2-phen-ps 301 2py-a23thima2-dmeph-es 302 gua-pipa2-thoph-es 303 bim-a23thima2-meph-es 304 2py-bam2-dmeph-es 305 bhs-a24thima2-thoph-es 306 bhs-apma2-phen-mals 307 bhs-a23thima2-meph-es 308 bim-buta-phen-es 309 2py-apma2-reph-es 310 gua-dibema2-meph-es 311 2py-a24thima2-hdb-es 312 gua-a24thima2-4-clph-es 313 bhs-pipa2-thoph-es 314 gua-a24thima2-3clph-es 315 gua-a24thima2-oxph-es 316 bim-bam2-dmeph-es 317 bim-apma2-thoph-es 318 bim-apma2-oxph-es 319 2py-a24thima2-phen-es 320 bhs-a24thima2-phen-ms 321 bim-prodia2-phen-es 322 2py-a24thima2-dm-es 323 bhs-pyma2-phen-es 324 bim-a24thima2-phen-f2es 325 2py-a23thima2-phen-es 326 gua-a24thima2-24pym-es 327 2py-35thima2-4-clph-es 328 bhs-dibema2-dmeph-es In the above list, the following abbreviations are used for the structural units A, E′, G′ and L. A= Abbreviation 2py dhim bim imhs dimethpym mam2py am2py thpym bhs gua amim clim im pippy E′= Abbreviation edia2 pyma2 pipa2 aepi2 me35thima2 dibema2 edia3 buta aaf 42thiaz2 chex2 bam2 apma2 pdagk mepipe2 prodia2 inda2 35thima2 me25thima2 penta aof hexa mea2 pipeme2 me42thiaz2 a23thima2 a24thima2 The bond to the structural unit L should be understood as meaning a double bond in the compound where L=as.",
"G′= Abbreviation 24pym dmeph dm thoph thaph 2pyph 4clph phen dbph hdb dmeoph meph 3clph oxph ioph yrph 4pyph reph L= Abbreviation es gs pms f2es mals ps ms nes as The compounds of the general formula I and thus also the compounds of the formula I′ as well as the starting substances used for their preparation can be prepared by methods of organic chemistry known to the person skilled in the art, such as are described in standard works such as Houben-Weyl, “Methoden der Organischen Chemie” [Methods of Organic Chemistry], Thieme-verlag, Stuttgart, or March “Advanced Organic Chemistry”, 4th Edition, Wiley & Sons.",
"Further preparation methods are also described in R. Larock, “Comprehensive Organic Transformations”, Weinheim 1989, in particular the preparation of alkenes, alkynes, halides, amines, ethers, alcohols, phenols, aldehydes, ketones, nitrites, carboxylic acids, esters, amides and acid chlorides.",
"The synthesis of compounds of the formula I can be carried out either according to the “classical” method in solution or on a polymeric support, in each case reaction conditions as are known and suitable for the respective reactions being used.",
"Use can also be made in this case of variants which are known per se but not mentioned here.",
"The general synthesis of compounds of the formula I is described in schemes 1-7.If not stated otherwise, all starting materials and reagents are commercially available, or can be prepared from commercially obtainable precursors by customary methods.",
"Fused 2,3,4,5-tetrahydro-1H-azepinediones of type II are known and can be prepared by known methods, e.g.",
"starting from anthranilic acid esters or the corresponding heterocyclic analogs via Dieckmann condensation and subsequent decarboxylation, as is described in the following publications: J.",
"Am.",
"Chem.",
"Soc.",
"80, 1958, 2172-2178; J. Chem.",
"Soc.",
"1959, 3111; J. Chem.",
"Soc.",
"1934, 1326; Arch.",
"Pharm.",
"324, 1991, 579-581.The preparation of 3,4-dihydro-1H-azepine-2,5-dione is described in Heterocycles 8, 1977, 345-350.The conversion into compounds of the type III is generally carried out by methods known to the person skilled in the art, such as are described, for example, in Larock, “Comprehensive Organic Transformations”, Weinheim 1989, pp.",
"167ff, although methods which are not mentioned here can also be used.",
"Preferably, compounds of the general formula III can be prepared by reaction of the ketones II with a phosphonic ester of the general formula (EtO)2P(═O)(XL)a(CRL1RL2)b—COOSG1 in the presence of a base.",
"The reaction preferably takes place in a polar aprotic solvent, such as tetrahydrofuran, dioxane; dimethylformamide (DMF), dimethylacetamide or acetamide; dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; in a temperature range depending on the nature of the solvent used from −40° C. up to the boiling point of the corresponding solvent.",
"The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.",
"sodium carbonate or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such lithium diisopropylamide, or lithium, sodium or potassium bis(trimethylsilyl)amide.",
"The reaction to give IV is carried out by hydrogenation of the double bond under standard conditions.",
"Use can also be made here of variants which are known per se but not mentioned.",
"Preferably, the hydrogenation is carried out in the presence of a noble metal catalyst, such as Pd on activated carbon, Pt, PtO2, Rh on Al2O3 in an inert solvent at a temperature of 0-150° C. and a pressure of 1-200 bar; the addition of an acid such as acetic acid or hydrochloric acid can be advantageous.",
"Hydrogenation in the presence of 5-10% Pd on activated carbon is particularly preferred.",
"Solvents which can be used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; sulfoxides such as dimethyl sulfoxide, sulfolane; pyridine, N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, water or mixtures of the solvents mentioned.",
"Compounds of type V are prepared by reaction with compounds of the general formula A-E-X1 (VI), the radical X1 being a customary leaving group, for example halogen such as chlorine, bromine, iodine or aryl- or alkylsulfonyl optionally substituted by halogen, alkyl or haloalkyl, such as toluenesulfonyl, trifluoromethanesulfonyl and methylsulfonyl or another equivalent leaving group.",
"The reaction preferably takes place in an inert solvent (such as previously described) with addition of a suitable base, i.e.",
"of a base which brings about deprotonation of the intermediate IV, in a temperature range from −40° C. up to the boiling point of the corresponding solvent.",
"The base used can be an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride or calcium hydride, a carbonate such as alkali metal carbonate, e.g.",
"sodium or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide or potassium hydroxide, an alkoxide such as sodium methoxide, potassium tert-butoxide, an organometallic compound such as butyllithium or alkali metal amides such as lithium diisopropylamide, lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide or potassium bis(trimethylsilyl)amide.",
"Removal of the protective group SG1 according to standard conditions (see below) leads to the compounds of the general formula I.",
"If SG1 is C1-C4-alkyl or benzyl, the compounds of the general formula V correspond directly to the compounds of type I. Alternatively to this synthesis strategy, compounds of type I can also be prepared via VII as an intermediate, here too reaction conditions being used such as are known to the person skilled in the art and described in standard works.",
"Compound VII is prepared by reaction of compounds of type IV having radio of the general formula DE-E-X2 (VIII) under reaction conditions such as have already been described for the preparation of V from IV and VI.",
"X2 here is a suitable leaving group, as has already been described for X1, and DE is CN, N3 or a protected amino or acid function of the general formula NSG3 or COOSG2.The synthesis of the fragments DE-E or A-E is carried out—depending on the actual structure of E—by removal of the protective groups and coupling of the remaining fragments according to standard methods, e.g.",
"amide couplings.",
"The introduction of A is then carried out analogously to the reactions described in schemes 3-7.Compounds of the formula I in which WG in structural element G is a structural element of the formula IWG1 can be prepared according to scheme 2.The starting point of the synthesis is compounds of type IX, which are either known or are accessible by methods known to the person skilled in the art, such as are described, for example, in J.",
"Am.",
"Chem.",
"Soc.",
"71, 1949, 1985.Alkylation with a compound of the general formula XIII (X3, X4=a customary leaving group) under customary reaction conditions leads to X.",
"The further reactions to give I then proceed analogously to scheme 1 via compounds of type XI.",
"In the case in which WG in structural element G is a structural element of the formula IWG3, compounds of type III can be converted into compounds of type XII and then into I analogously to the preparation of V (scheme 2).",
"The coupling of the individual fragments and the removal of the protective groups can be carried out according to known processes (see Larock, “Comprehensive Organic Transformations; protective groups: Greene, T., “Protective Groups in Organic Synthesis”, New York 1991), in the case of amide bonds also analogously to the methods of peptide synthesis, such as are described in standard works, e.g.",
"in Bodanszky “The Practice of Peptide Synthesis”, 2nd Edition, Springer-Verlag 1994, and Bodanszky “Principles of Peptide Synthesis”, Springer-Verlag 1984.A general survey of the customary methods for peptide synthesis and a listing of suitable reagents can furthermore be found in NOVABIOCHEM 1999 “Catalog and Peptide Synthesis Handbook”.",
"The amide couplings mentioned can be carried out with the aid of customary coupling reagents using suitably protected amino and carboxylic acid derivatives.",
"Another method consists in the use of preactivated carboxylic acid derivatives, preferably of carboxylic acid halides, symmetrical or mixed anhydrides or so-called active esters, which are customarily used for the acylation of amines.",
"These activated carboxylic acid derivatives can also be prepared in situ.",
"As a rule, the couplings can be carried out in inert solvents in the presence of an acid-binding agent, preferably of an organic base such as triethylamine, pyridine, diisopropylethylamine, N-methylmorpholine, quinoline; the addition of an alkali metal or alkaline earth metal hydroxide, carbonate or hydrogencarbonate or of another salt of a weak acid of the alkali metals or alkaline earth metals, preferably of potassium, sodium, calcium or cesium, can also be favorable.",
"Depending on the conditions used, the reaction time is between minutes and 14 days, the reaction temperature between −40° C. and 140° C., preferably between −20° C. and 100° C. Suitable inert solvents are, for example, hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone, nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate; water; or mixtures of the solvents mentioned.",
"Protective groups SG which can be used are all protective groups which are known from peptide synthesis and customary to the person skilled in the art, such as are also described in the abovementioned standard works.",
"The protective groups in the compounds of the formulae V, VII, XI and XII are likewise removed according to conditions such as are known to the person skilled in the art and are described by Greene and Wuts in “Protective Groups in organic Synthesis”, 2nd Edition, Wiley & Sons, 1991.Protective groups such as SG3 are so-called N-terminal amino protective groups; Boc, Fmoc, benzyloxycarbonyl (Z), acetyl, Mtr are preferred here.",
"SG1 and SG2 are acid protective groups; C1-C4-alkyl is preferred here, such as methyl, ethyl, tert-butyl, or alternatively benzyl or trityl, or alternatively polymer-bonded protective groups in the form of the commercially available polystyrene resins such as 2-chlorotrityl chloride resin or Wang resin (Bachem, Novabiochem).",
"Acid-labile protective groups (e.g.",
"Boc, tert-butyl, Mtr, trityl) can be removed—depending on the protective group used—using organic acids such as trifluoroacetic acid (TFA), trichloroacetic acid, perchloric acid, trifluoroethanol; but also inorganic acids such as hydrochloric acid or sulfuric acid, sulfonic acids such as benzene- or p-toluenesulfonic acid, the acids generally being employed in an excess.",
"HCl or TFA is preferably used.",
"In the case of trityl, the addition of thiols such as thioanisole or thiophenol can be advantageous.",
"The presence of an additional inert solvent is possible, but not always necessary.",
"Suitable inert solvents are preferably organic solvents, for example carboxylic acids such as acetic acid; ethers such as THF or dioxane; amides such as DMF or dimethylacetamide; halogenated hydrocarbons such as dichloromethane; alcohols such as methanol, isopropanol; or water.",
"Suitable solvents are also mixtures of those mentioned.",
"The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Base-labile protective groups such as Fmoc are cleaved by treatment with organic amines such as dimethylamine, diethylamine, morpholine or piperidine as 5-50% solutions in CH2Cl2 or DMF.",
"The reaction temperature for these reactions is between −10° C. and 50° C.; the reaction is preferably carried out in a range between 0° C. and 30° C. Acid protective groups such as methyl or ethyl are preferably cleaved by basic hydrolysis in an inert solvent.",
"The bases used are preferably alkali metal or alkaline earth metal hydroxides, preferably NaOH, KOH or LiOH; the solvents used are all customary inert solvents, such as hydrocarbons such as hexane, heptane, petroleum ether, toluene, benzene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, carbon tetrachloride, chloroform, dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, methyltert-butyl ether, diisopropyl-ether, tetrahydrofuran, dioxane; glycol ethers such as ethylene glycol monomethyl ether or monoethyl ether, ethylene glycol dimethyl ether; ketones such as acetone, butanone; amides such as dimethylformamide (DMF), dimethylacetamide or acetamide; nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide, sulfolane; N-methylpyrrolidone, 1,3-dimethyltetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone; nitro compounds such as nitromethane or nitrobenzene; water or mixtures of the solvents mentioned.",
"The addition of a phase-transfer catalyst—depending on the solvent or solvent mixture used—may be advantageous.",
"The reaction temperature for these reactions is generally between −10° C. and 100° C. Hydrogenolytically removable protective groups such as benzyloxycarbonyl (Z) or benzyl can be removed, for example, by hydrogenolysis in the presence of a catalyst (e.g.",
"of a noble metal catalyst on activated carbon as support).",
"Suitable solvents are those mentioned above, in particular alcohols such as methanol, ethanol; amides such as DMF or dimethylacetamide; esters such as ethyl acetate.",
"As a rule, the hydrogenolysis is carried out at a pressure of 1-200 bar and temperatures between 0° and 100° C.; the addition of an acid such as acetic acid or hydrochloric acid may be advantageous.",
"The catalyst used is preferably 5-10% Pd on activated carbon.",
"The synthesis of structural units of type E is generally carried out by methods known to the person skilled in the art; the structural units used are either commercially available or accessible by methods known from the literature.",
"The synthesis of some of these structural units is described by way of example in the experimental section.",
"In the case in which the fragments QE or XE contained in the compounds of type VI and VIII are a hetaryl radical, the structural units used are either commercially available or accessible by methods known to the person-skilled in the art.",
"A multiplicity of preparation methods are described in detail in Houben-Weyl's “Methoden der organischen Chemie” (Vol.",
"E6: furans, thiophenes, pyrroles, indoles, benzothiophenes, -furans, -pyrroles; Vol.",
"E7: quinolines, pyridines, Vol.",
"E8: isoxazoles, oxazoles, thiazoles, pyrazoles, imidazoles and their benzo-fused representatives, as well as oxadiazoles, thiadiazoles and triazoles; Vol.",
"E9: pyridazines, pyrimidines, triazines, azepines and their benzo-fused representatives as well as purines).",
"The linkage of these fragments to E, depending on the structure of E, can also take place via the amino or acid function according to methods which are known to the person skilled in the art.",
"Structures of the general formula A-E-DE are synthesized according to methods known to the person skilled in the art, which are described, for example, in WO 97/08145.Examples of these are the conversion of compounds of the general formula: HNRE11EA1-DE (XIV) NC-EA2-DE (XV) into compounds of the general formula: A-HNRE11-EA1-DE (XVI) A-E-DE (XVII) The groups EA1 and EA in the formulae XIV-XVIII represent structural fragments which, after an appropriate modification (e.g.",
"reaction with suitable reagents or coupling with appropriate structural units) as a whole form the structural fragment A-E.",
"These structural units can then be reacted either directly—in the case of the corresponding free amines or carboxylic acids—or after removal of the protective groups—to give compounds of the general formula I (scheme 1 and 2).",
"In principle, A, however, can also be introduced into compounds of the type IV, as described in scheme 1, where the reaction conditions mentioned can be used just as variants which are not described here.",
"In schemes 3-7, a number of the methods for the introduction of A are described by way of example, in each case reaction conditions being used which are known and suitable for the respective reactions.",
"Use can also be made in this case of variants which are known per se but not mentioned here.",
"Ureas and thioureas (AE-1 to AE-3) can be prepared by customary methods of organic chemistry, e.g.",
"by reaction of an isocyanate or of an isothiocyanate with an amine, if appropriate in an inert solvent with heating (Houben-Weyl Volume VIII, 157ff.)",
"(scheme 3): Scheme 4 shows, by way of example, the preparation of compounds of the type AE-4, as is described, for example, by Blakemoore et al.",
"in Eur.",
"J. Med.",
"Chem.",
"1987 (22) 2, 91-100, or Misra et al.",
"in Bioorg.",
"Ned.",
"Chem.",
"Lett.",
"1994 4 (18), 2165-2170.Unsubstituted or cyclic guanidine derivatives of the general formulae AE-5 and AE-6 can be prepared by means of commercially available or simply accessible reagents, such as are described, for example, in Synlett 1990, 745, J. Org.",
"Chem.",
"1992, 57, 2497, Bioorg.",
"Ned.",
"Chem.",
"1996, 6, 1185-1208; Bioorg.",
"Med.",
"Chem.",
"1998, 1185, or Synth.",
"Comm.",
"1998, 28, 741-746 (scheme 5).",
"The preparation of compounds of the general formula AE-7 can be carried out analogously to U.S. Pat.",
"No.",
"3,202,660, compounds of the formulae AE-9, AE-10, AE-11 and AE-12 analogously to WO 97/08145.Compounds of the formula AE-8 can be prepared, as shown in scheme 6, e.g.",
"according to the method described by Perkins et al., Tetrahedron Lett.",
"1999, 40, 1103-1106.Scheme 5 gives a survey of the synthesis of the compounds mentioned, the circle in AE-8 representing a fused cycle, such as aryl or hetaryl.",
"Compounds of the general formula AE-13 can be prepared analogously to Froeyen et al., Phosphorus Sulfur silicon Relat.",
"Elem.",
"1991, 63, 283-293; AE-14 analogously to Yoneda et al., Heterocycles 1998, 15 N′-1, Spec.",
"Issue, 341-344 (scheme 6).",
"The preparation of corresponding compounds can also be carried out analogously to WO 97/36859.Compounds of the general formula AE-15 can be prepared according to Synthesis 1981, 963-965 or Synth.",
"Comm.",
"1997, 27 (15), 2701-2707; AE-16 analogously to J. Org.",
"Chem.",
"1991, 56 (6), 2260-2262 (scheme 7), the circle being a fused cycle, such as aryl, hetaryl or cycloalkyl.",
"The invention further relates to pharmaceutical preparations, comprising at least one compound of the formula I′ in addition to the customary pharmaceutical excipients.",
"The compounds according to the invention can be administered orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally) in the customary manner.",
"Administration can also be carried out through the nasopharynx using vapors or sprays.",
"Further, the compounds according to the invention can be introduced by direct contact with the affected tissue.",
"The dose depends on the age, condition and weight of the patient and on the manner of administration.",
"As a rule, the daily dose of active compound is between approximately 0.5 and 50 mg/kg of body weight in the case of oral administration and between approximately 0.1 and 10 mg/kg of body weight in the case of parenteral administration.",
"The novel compounds can be administered in solid or liquid form in the customary pharmaceutical administration forms, e.g.",
"as tablets, film-coated tablets, capsules, powders, granules, coated tablets, suppositories, solutions, ointments, creams or sprays.",
"These are prepared in a customary manner.",
"The active compounds can in this case be processed using the customary pharmaceutical excipients such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, release-delaying agents, antioxidants and/or propellants (cf.",
"H. Sucker et al.",
": Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991).",
"The administration forms thus obtained normally contain the active compound in an amount from 0.1 to 90% by weight.",
"The invention further relates to the use of compounds of the formula I′ for the production of drugs for treating illnesses.",
"The compounds of the formula I′ can be used for treating human and animal illnesses.",
"The compounds of the formula I′ which represent the novel compounds of the formula I bind, as mentioned above, to integrin receptors.",
"They are therefore suitable, as mentioned above, preferably as integrin receptor ligands and for the production of drugs for treating illnesses in which an integrin receptor is involved, in particular for the treatment of illnesses in which the interaction between integrins and their natural ligands is dysregulated, i.e.",
"excessive or reduced, as described above.",
"Advantageously, the compounds of the formula I, preferably the compounds of the formula I′, can be administered in combination with at least one further compound in order to achieve an improved curative action in a number of indications.",
"These further compounds can have the same or a different mechanism of action as/from the compounds of the formula I.",
"In addition to the compounds of the formula I, preferably in addition to the compounds of the formula I′ and the customary pharmaceutical excipients, the pharmaceutical preparations can therefore contain at least one further compound, depending on the indication, in each case selected from one of the 10 groups below.",
"Group 1: inhibitors of blood platelelet adhesion, activation or aggregation, such as acetylsalicylic acid, lysine acetylsalicylate, piracetam, dipyridamol, abciximab, thromboxane antagonists, fibrinogen antagonists, such as tirofiban, or inhibitors of ADP-induced aggregation such as ticlopidine or clopidogrel, anticoagulants which prevent thrombin activity or formation, such as inhibitors of IIa, Xa, XIa, IXa or VIIa, antagonists of blood platelet-activating compounds and selectin antagonists for the treatment of blood platelet-mediated vascular occlusion or thrombosis, or Group 2: inhibitors of blood platelet activation or aggregation, such as GPIIb/IIIa antagonists, thrombin or factor Xa inhibitors or ADP receptor antagonists, serine protease inhibitors, fibrinogen-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 inhibitors of leukocyte adhesion inhibitors of vessel wall transmigration, fibrinolysis-modulating compounds, such as streptokinase, tPA, plasminogen-activating stimulants, TAFI inhibitors, XIa inhibitors or PAI-1 antagonists, inhibitors of complement factors, endothelin receptor antagonists, tyrosine kinase inhibitors, antioxidants and interleukin 8 antagonists for the treatment of myocardial infarct or stroke, or Group 3: endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists, endopeptidase inhibitors, beta-blockers, calcium channel antagonists, phosphodiesterase inhibitors and caspase inhibitors for the treatment of congestive heart failure, or Group 4: thrombin inhibitors, inhibitors of factor Xa, inhibitors of the coagulation pathway which leads to thrombin formation, such as heparin or low-molecular weight heparins, inhibitors of blood platelet adhesion, activation or aggregation, such as GPIIb-IIIa antagonists or antagonists of the blood platelet adhesion and activation mediated by vWF or GPIb, endothelin receptor antagonists, nitrogen oxide synthase inhibitors, CD44 antagonists, selectin antagonists, MCP-1 antagonists, inhibitors of signal transduction in proliferating cells, antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF and antioxidants for the treatment of restenosis after vascular injury or stent implantation, or Group 5: antagonists of the cell response mediated by EGF, PDGF, VEGF or bFGF, heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, selectin antagonists, endothelin antagonists, ACE inhibitors, angiotensin receptor antagonists and glycosylation inhibitors or AGE formation inhibitors or AGE breakers and antagonists of their receptors, such as RAGE, for the treatment of diabetic angiopathies, or Group 6: lipid-lowering compounds, selectin antagonists, antagonists of ICAM-1 or VCAM-1 heparin or low-molecular weight heparins or further GAGs, inhibitors of MMPs, endothelin antagonists, apolipoprotein A1 antagonists, cholesterol antagonists, HMG CoA reductase inhibitors, ACAT inhibitors, ACE inhibitors, angiotensin receptor antagonists, tyrosine kinase inhibitors, protein kinase C inhibitors, calcium channel antagonists, LDL receptor function stimulants, antioxidants LCAT mimetics and free radical scavengers for the treatment of atherosclerosis, or Group 7: cytostatic or antineoplastic compounds, compounds which inhibit proliferation, such as kinase inhibitors and heparin or low-molecular weight heparins or further GAGS for the treatment of cancer, preferably for the inhibition of tumor growth or metastasis, or Group 8: compounds for antiresorptive therapy, compounds for hormone exchange therapy, such as estrogen or progesterone antagonists, recombinant human growth hormone, bisphosphonates, such as alendronates compounds for calcitonin therapy, calcitonin stimulants, calcium channel antagonists, bone formation stimulants, such as growth factor antagonists, interleukin-6 antagonists and Src tyrosine kinase inhibitors for the treatment of osteoporosis, or Group 9: TNF antagonists, antagonists of VLA-4 or VCAM-1, antagonists of LFA-1, Mac-1 or ICAMs, complement inhibitors, immunosuppressants, interleukin-1, -5 or -8 antagonists and dihydrofolate reductase inhibitors for the treatment of rheumatoid arthritis, or Group 10: collagenase, PDGF antagonists and MMPs for improved wound healing.",
"A pharmaceutical preparation comprising at least one compound of the formula I, preferably comprising at least one compound of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, is understood as meaning a combined administration of at least one of the compounds of the formula I, preferably of one of the compounds of the formula I′, with at least one further compound in each case selected from one of the groups described above and, if appropriate, pharmaceutical excipients.",
"Combined administration can be carried out by means of a substance mixture comprising at least one compound of the formula I, preferably of the formula I′, if appropriate pharmaceutical excipients and at least one further compound, depending on the indication, in each case selected from one of the above groups, but also spatially and/or chronologically separate.",
"In the case of the spatially and/or chronologically separate administration, the administration of the components of the pharmaceutical preparation, the compounds of the formula I, preferably of the formula I′ and the compounds selected from one of the abovementioned groups, takes place spatially and/or chronologically separately.",
"For the treatment of restenosis after vascular injury or stenting, the administrations of the compounds of the formula I, preferably of the formula I′, can be carried out locally at the affected sites, on their own or in combination with at least one compound selected from group 4.It may also be advantageous to coat the stents with these compounds.",
"For the treatment of osteoporosis, it may be advantageous to carry out the administration of the compounds of the formula I, preferably of the formula I′, in combination with an antiresorptive or hormone exchange therapy.",
"The invention accordingly relates to the use of the abovementioned pharmaceutical preparations for the production of drugs for treating illnesses.",
"In a preferred embodiment, the invention relates to the use of the abovementioned combined pharmaceutical preparations for the production of drugs for treating blood platelet-mediated vascular occlusion or thrombosis when using compounds of group 1, myocardial infarct or stroke when using compounds of group 2, congestive heart failure when using compounds of group 3, restenosis after vascular injury or stent implantation when using compounds of group 4, diabetic angiopathies when using compounds of group 5, atherosclerosis when using compounds of group 6, cancer when using compounds of group 7, osteoporosis when using compounds of group 8, rheumatoid arthritis when using compounds of group 9, wound healing when using compounds of group 10.The following examples illustrate the invention, the selection of these examples being non-limiting.",
"I. SYNTHESIS EXAMPLES I.A Precursors Example 1 t-Butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) 22.3 g (80 mmol) of t-butyl diethylphosphonate (95%) were added dropwise at 0° C. to a suspension of 3.27 g of NaH (60%; deoiled) in ml of DMF.",
"The mixture was stirred until a clear solution was formed and then 12.4 g (70.9 mmol) of 3,4-dihydro-1H-1-benzazepine-2,5-dione (preparation according to Arch.",
"Pharm.",
"1991, 324, 579) in 90 ml of DMF were added dropwise at 0° C. The reaction mixture then remained standing at RT for about 3 days.",
"For workup, the mixture was poured into 700 ml of cold 5% NaCl solution, and the resulting yellow precipitate was filtered off with suction and washed with H2O.",
"The moist residue was taken up in CH2Cl2, washed with 5% NaHCO3 solution and dried over Na2SO4.The residue which remained after evaporation was treated with 150 ml of cyclohexane in the presence of heat, and after cooling, filtering off with suction and washing with n-hexane 17.5 g (90.5%) of white crystals remained; m.p.",
": 136-138° C. Example 2 t-Butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetate (2) A suspension of 3 g of 10% Pd/C in 50 ml of ethanol was prehydrogenated, then a solution of compound 1 (14.7 g; 53.8 mmol) in 125 ml of ethanol and 75 ml of dioxane was added, and the mixture was hydrogenated under standard conditions until the absorption of hydrogen was complete.",
"After filtering off the catalyst with suction and washing it with ethanol, the filtrate was concentrated in vacuo, the oily residue was dissolved in diethyl ether and the crystallization commencing was completed by addition of n-hexane.",
"After filtering off the precipitate with suction and washing it with n-hexane, 14.2 g (96%) of white crystals remained; m.p.",
": 101-103° C. Example 3 [5-(2-t-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) a.)",
"A solution of compound 2 (16.8 g; 61.1 mmol) in 60 ml of DMF was added dropwise at 10-20° C. to a suspension of 2.6 g of NaH (60%, deoiled) in 35 ml of DMF and the mixture was stirred until the appearance of an almost clear yellowish solution.",
"t-Methyl bromoacetate (10 g; 63.4 mmol) was then added dropwise and the mixture was stirred overnight.",
"For workup, the reaction mixture was poured into 400 ml of 5% cold NaCl solution and extracted 3× with 100 ml each of a diethyl ether/n-hexane mixture.",
"The combined extracts were then washed with H2O, 10% NaHCO3 solution and NaCl solution, dried over Na2SO4, filtered and evaporated.",
"The residual yellowish oil was reacted further without further purification; FAB-MS: 348 [M−H+].",
"b.)",
"Crude product 3a was dissolved in 100 ml of dioxane and 65 ml of 1N NaOH were added dropwise at RT with stirring.",
"After about 45′, the reaction mixture was adjusted to pH 7 using 1N KHSO4 solution, the dioxane was largely distilled off in vacuo, and the residue was diluted with H2O, adjusted to pH 9 using 1N NaOH and extracted 3× with diethyl ether.",
"The aqueous phase was then rendered acidic using 1N KHSO4 solution, the acid precipitating was extracted with a mixture of diethyl ether/n-hexane 4:1, and the organic phase was washed with H2O, 1N NaOH solution and NaCl solution and dried over Na2SO4.Filtration and evaporation afforded an oily residue, which could be crystallized by treatment with diethyl ether/n-hexane 1:4 (water-saturated).",
"Filtering with suction, washing with n-hexane and drying afforded 17.8 g (87.5%) of white crystals: m.p.",
": 117-119° C. Example 4 N-[4-(Aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) a.)",
"20 g of tert-butyl-4-aminobenzyl carbamate (89.97 mmol)—dissolved in 100 ml of CH3CN—were added dropwise at 0° C. to a solution of 24.5 g of thiocarbonyldiimidazole and 1.56 g of imidazole in 600 ml of CH3CN and the mixture was stirred at RT overnight.",
"19.5 g of 1,2-phenylenediamine were then added and the mixture was again stirred at RT for 2 h. For workup, the reaction mixture was evaporated in vacuo, the residue was taken up in CH2Cl2, and the solution was washed 7× with 10% citric acid solution and 2× with satd.",
"NaCl solution, dried over Na2SO4, filtered and concentrated.",
"The crude product thus obtained (31.78 g; brown foam) was reacted directly without further purification; ESI-MS [M+H+]=373.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.5 and 9.05 (each s, 1H), 7.45 (d, 2H), 7.35 (m, 1H), 7.20 (d, 1H), 7.15, 6.95, 6.75, 6.60 (each m, 1H), 4.85 (s, 2H), 4.10 (d, 2H), 1.35 (s, 9H).",
"b.)",
"Crude product 4a was dissolved in 750 ml of ethanol together with 36.7 g of HgO (yellow) and 0.4 g of sulfur and heated to reflux for 2 h. The reaction mixture was then filtered twice through Celite and evaporated to dryness; 20.7 g, ESI-MS [M+H+]=339.15.c.)",
"7 g of the crude product 4b were introduced into 70 ml of CH2Cl2, 35 ml of HCl in diethyl ether (satd.",
"at 0° C.) were added and the mixture was stirred at RT for 2 h. The resulting precipitate was filtered off with suction, washed with CH2Cl2 and dried.",
"6.7 g of brown amorphous solid; ESI-MS [M+H+]=239.15 1H-NMR (360 MHz, DMSO) δ ppm: 11.6 (s broad, 1H), 8.4 (s broad, 3H), 8.25 (s broad, 1H), 7.65 and 7.55 (each d, 2H), 7.45 and 7.3 (each m, 2H), 4.19 (m, 2H).",
"Example 5 N-[5-(Aminomethyl)-1,3-thiazol-2-yl]guanidine (dihydrochloride) (5) a.)",
"31 g (130 mmol) of 2-chloro-3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal (preparation according to THL 39 (1998), 8085-8088) and 15.4 g of amidinothiourea were heated at 110° C. for 75′ in 200 ml of n-butanol, then the mixture was evaporated and the residue was treated with CH2Cl2 and conc.",
"NH3.Evaporation of the organic phase, purification of the residue by chromatography on silica gel (CH2Cl2/CH3OH 0-5%) and crystallization from acetone afforded 12.3 g of N-{5-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]-1,3-thiazol-2-yl}guanidine.",
"b.)",
"1 g of 5a in 20 ml of CH3OH was treated with 0.81 ml of hydrazine hydrate and stirred at RT for 2 h. The mixture was then cooled to 0° C., filtered, and the filtrate was concentrated and stirred with dilute HCl.",
"This process was repeated a number of times, and the crude product obtained in this way was then stirred with ethanol; 0.92 g of white solid, ESI-MS [M+H+]=172.05.Example 6 N-[4-(Aminomethyl)phenyl]-N′-benzylurea (6) a.)",
"4-Aminobenzylamine (10.0 g, 81.85 mmol) in 150 ml CH2Cl2 was treated with triethylamine (6.8 g, 67.12 mmol) and then treated at 0° C. with di-t-butyl dicarbonate (18.6 g, 85.0 mmol).",
"The mixture was stirred at 0° C. for 1 h and then at RT for 2 h. For workup, 150 ml of a 1%-aqueous citric acid solution were added, the phases were separated and the aqueous phase was reextracted 2 times with CH2Cl2 (150 ml).",
"Fresh washing with H2O, drying of the combined organic phases using Na2SO4 and evaporation afforded a solid, which was washed with stirring with a little diisopropyl ether, filtered off with suction and dried.",
"13.0 g; ESI-MS [M+H+-tBu]=167.05.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (2H, d), 6.61 (2H, d), 4.78 (1H, s br.",
"), 4.17 (2H, d), 3.67 (2H, s br.",
"), 1.46 (9H, s).",
"b.)",
"Benzyl isocyanate (2.40 g, 18.0 mmol) was added with ice-cooling to a solution of the protected amine 6a (4.0 g, 17.99 mmol) and triethylamine (1.82 g, 18.0 mmol) in 220 ml of toluene/DMF 10:1.The reaction mixture was stirred at RT overnight.",
"It was possible to filter off some of the urea formed directly as a precipitate and dry it.",
"The filtrate was washed 2× with H2O, with dilute tartaric acid to pH 3 and again 2 times with H2O to pH 5, and the organic phase was then dried and evaporated.",
"Altogether, 6.0 g were thus obtained; ESI-MS [M+H+-tBu]=300.15.c.)",
"The urea 6b thus obtained was introduced in 90 ml of CH2Cl2, and TFA (2.24 g, 196.25 mmol)—dissolved in 90 ml of CH2Cl2—was added dropwise at 0° C. After 3 h, 1 ml of TFA was added again, then the mixture was stirred at RT overnight.",
"After fresh addition of 1 ml of TFA, the mixture was stirred for a further 5 h, then poured onto ice water and extracted with ethyl acetate (2×50 ml).",
"The water phase was rendered basic with 2N NaOH solution and extracted with CH2Cl2 (2×50 ml).",
"The insoluble portion between the phases was filtered off and dried.",
"4 g; ESI-MS [2M+H+]=511.35 1H-NMR (200 MHz, DMSO) δ (ppm): 8.52 (1H, s), 7.39-7.07 (9H, m), 6.62 (1H, t), 4.27 (2H, d), 3.61 (2H, s).",
"Example 7 [4-(1H-Benzimidazol-2-yl)phenyl]methaneamine (hydrochloride) (7) a.)",
"Di(tert-butyl) 4-cyanobenzylimidodicarbonate (10 g, 30.08 mmol; preparation according to Synth.",
"Comm.",
"28, 23, 1998, 4419ff) in 200 ml of pyridine was treated with 45 ml of triethylamine and saturated at 0° C. with H2S for 1.5 h. The reaction mixture was allowed to stand at RT overnight and then evaporated.",
"The residue thus obtained was then stirred with diethyl ether, filtered off with suction and dried (8.5 g).",
"b.)",
"6 g of the thioamide 7a (16.37 mmol) in 40 ml of dry CH2Cl2 were alkylated at RT overnight using 23.2 g of CH3I and the mixture was then evaporated.",
"The residue thus obtained was taken up in 40 ml of CH3OH, 1.95 g of 1,2-phenylenediamine were added and it was again stirred overnight.",
"Evaporating the reaction mixture and stirring the solid with n-pentane afforded 6.9 g of the desired benzimidazole.",
"M.p.",
": >170° C. (decomposition); ESI-MS: [M+H+]=424.25 c.) 1 g of the bis-Boc compound 7b was dissolved in 5 ml of CH2Cl2, 5 ml of TFA were added at 0° C. and the mixture was stirred at room temperature for 1 h. Evaporating the reaction mixture, treating with HCl in diethyl ether and stirring the isolated solid with diethyl ether afforded 0.6 g of the amine as the hydrochloride; ESI-MS: [M+H+]=224.05.Example 8 N1-(1H-Benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) Preparation was carried out analogously to the preparation of compound 4 starting from 7 g of N-Boc-1,5-diaminopentane hydrochloride (29.3 mmol).",
"After reaction analogously to 4a, 10.3 g of N-Boc 5-{[(2-aminoanilino)carbothioyl]amino}pentane-1-amine were obtained; ESI-MS [M+H+]=353.25.Cyclodesulfurization and subsequent removal of the Boc group with TFA afforded an oily crude product, which was taken up in CH3OH and converted into the corresponding hydrochloride using 250 ml of ethereal HCl (saturated at 0° C.).",
"Stirring the solid obtained with a mixture of CH3OH/methyl t-butyl ether afforded 1.8 g of a reddish amorphous solid.",
"1H-NMR (360 MHz, DMSO) δ ppm: 9.30 (t, 1H), 8.15 (s broad, 3H), 7.40 and 7.25 (each m, 2H), 3.35 (m, 2H superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).",
"Example 9 N1-(1H-Benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) Preparation was carried out analogously to the preparation of compound 4 starting from 9.87 g of N-Boc-1,4-diaminobutane (52.3 mmol).",
"After reaction analogously to 4a, 17.08 g of N-Boc 4-{[(2-aminoanilino)carbothioyl]amino}butane-1-amine were obtained; ESI-MS [M+H+]=338.99.Subsequent cyclodesulfurization and Boc removal using TFA afforded a brown solid, which was stirred a number of times with n-pentane and then recrystallized from a mixture of CH3OH/methyl t-butyl ether; 14.35 g, ESI-MS [M+H+]=205.15.1H-NMR (360 MHz, DMSO) δ ppm: 9.20 (t, 1H), 7.80 (s broad, 3H), 7.35 and 7.20 (each m, 2H), 3.40 (m, 2H partially superimposed with H2O peak), 2.80 (m, 2H), 1.65 (m, 4H).",
"Example 10 trans-N-[(4-Aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) Preparation was carried out analogously to compound 4 starting from 5.4 g of tert-butyl-4-(aminomethyl)cyclohexylamine carbamate (WO 9603374; Bioorg.",
"Med.",
"Chem.",
"Lett.",
"1997, 7 (1), 67).",
"After removal of the Boc group, 3.3 g of white dihydrochloride were obtained; FAB-MS [M+H+]: 245.Example 11 trans-N-{[4-(Aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) Preparation was carried out analogously to compound 4 starting from 10 g of benzyl {4-[(tert-butoxycarbonyl)amino]cyclohexyl}-methylcarbamate (EP 669317) by removal of the Boc group using 4N HCl in dioxane, synthesis of the benzimidazole and subsequent hydrogenolysis.",
"3.6 g of white dihydrochloride were isolated; FAB-MS [M+H+]: 245.Example 12 [6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) a.)",
"Preparation was carried out analogously to 7 starting from tert-butyl (6-cyanopyridin-3-yl)methylcarbamate (6.0 g, 25.72 mmol); crystallization of the crude product from ethanol afforded 5.15 g; ESI-MS [M+H+]=325.b.)",
"0.55 g of the Boc-protected amine 12a in 10 ml of CH2Cl2 was treated with 5 ml of TFA and stirred at RT for 2 h. Evaporation of the reaction mixture afforded 0.95 g of a white solid; ESI-MS [M+H+]: 225.25.Example 13 N-[4-(Aminomethyl)phenyl]-2-pyridineamine (13) tert-Butyl 4-aminobenzylcarbamate (2 g; 9 mmol) was heated to reflux for 32 h with 8.74 g of 2-fluoropyridine.",
"The reaction mixture was evaporated in vacuo and the residue obtained was stirred with n-pentane (1.9 g).",
"The Boc group was cleaved using TFA, and the crude product obtained was precipitated from diethyl ether as the hydrochloride and then converted into the free-base (0.8 g) using NH3; ESI-MS [M+H+]: 200.25.N-[4-(Aminomethyl)benzyl]-2-pyridineamine (14) a.)",
"20 g 2-Aminopyridine were dissolved in 100 ml CH3OH, adjusted to pH 6 with isopropanolic HCl and 36 g p-cyanobenzaldehyde were added.",
"9.35 g Sodium cyanoborohydride were added portionwise over one hour and stirred overnight.",
"For workup, the suspension was evaporated, the residue taken up in 100 ml water and with KOH adjusted to pH>10.The watery phase was saturated with NaCl and extracted 3× with diethylether.",
"The ether phase was washed after filtration of a precipitate 3× with a FeSO4 solution, dried and evaporated.",
"Purification of the residue by chromatography on silica gel (heptane/ethyl acetate 1:1) afforded 28.15 g 4-[2-pyridinyl-amino)methyl]benzonitrile.",
"b.)",
"10 g 4-[2-Pyridinyl-amino)methyl]benzonitrile were dissolved in 280 ml ammonia-alkali CH3OH, 10 g Raney-nickel added and it was hydrogenated for 24 h. It was filtered, evaporated and the residue purified by chromatography on silica gel (ethyl acetate/ethyl alcohol 1:3).",
"5.18 g, ESI-MS: [M+H+]=214.",
"[5-(1H-Benzimidazole-2-yl(thiene-2-yl]methaneamine (15) Preparation was carried out starting from 5-(aminomethyl)thiophene-2-carbonitrile (preparation according to WO 95/23609), which was reacted to the respective Boc-derivative according to standard methods.",
"1.1 eq.",
"Sodium methanolate solution was added to t-butyl-5-cyanothiene-2-ylcarbamate (25 g; 104.9 mmol) in 330 ml CH3OH and stirred overnight at RT, then for 2 h at 40-50°.",
"18.95 g Phenylenediaminebihydrochloride were then added and again stirred at RT.",
"For workup, water was added, the resulting precipitate filtered off and carefully dried.",
"19.6 yellow solid; ESI-MS: [M+H+]=330.The following cleavage of the Boc-group with TFA afforded a raw product, which was dissolved in water, 2× extracted with diethyl ether, the watery phase adjusted to pH 10-11 and then extracted 2× with ethyl acetate.",
"The watery phase was saturated with NaCl and again extracted with ethyl acetate.",
"The combined organic phases were dried and evaporated (6.3 g); ESI-MS [M+H+]=230.1.tert-Butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) Preparation was carried out analogously to the preparation of [4-(1H-benzimidazole-2-yl)phenyl]methaneamine (hydrochloride) (7) starting from tert-butyl-2-(4-cyanophenyl)ethylcarbamate.",
"The raw product obtained after reaction with H2S, alkylation with CH3I and reaction with 1,2-phenylenediamine was purified by chromatography on silica gel (CH2Cl2/CH3OH 4-50%) (4.8 g); ESI-MS [M+H+]=338.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with TFA (under standard conditions); the isolated TFA-salt was then directly utilized in the respective couplings.",
"N-(Piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoroacetate) (17) a) A solution of tert-butyloxycarbonyl-4-(aminomethyl)-1-piperidine (5.39 g; 25 mmol) in 25 ml CH3CN was added dropwise to 6.75 g thiocarbonyldiimidazole and 0.5 g imidazole in 100 ml CH3CN at 0° C. and then stirred for 3 h at RT.",
"1,2-Phenylenediamine (5.5 g; 50.86 mmol) was then added and heated for about 1 h to 60° C. The solid obtained upon cooling was filtered off with suction and dried.",
"6.79 g; ESI-MS [M+H+-tBu]=309.15 b) tert-Butyoxycarbonyl-4-({[(2-aminoanilino)carbothioyl]amino}methyl)1-piperidine (5 g; 13.72 mmol), 5.94 g HgO (yellow) and 0.6 g sulfur in 150 ml ethyl alcohol were heated under reflux for 1 h. The mixture was filtered 2× over Celite, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 5-25%).",
"2.65 g; ESI-MS [M+H+]=331.25 1H-NMR (360 MHz, DMSO) δ ppm: 7.15 and 6.9 (each m, 2H), 3.95 (d, 2H) 3.2 (m, 2H, 2.7 (br m; 2H), 1.8 (m, 1H), 1.7 (m, 2H), 1.35 (s, 9H), 1.05 (m, 2H).",
"c) tert-Butyloxycarbonyl-4-[(1H-benzimidazole-2-ylamino)methyl]-1-piperidine (2.65 g; 8.02 mmol) was treated with 10 ml TFA according to standard conditions.",
"Evaporation and mixing the raw product with n-pentane afforded 2.39; ESI-MS [M+H+]=231.15.1H-NMR (360 MHz, DMSO δ ppm: 13.25 (s, 1H), 9.35 (m, 1H), 8.8 and 8.5 (each br s, 1H), 7.4 and 7.20 (each m, 2H), 3.3 (m, 4H), 2.85 (m, 2H), 1.9 (m, 3H), 1.35 (m, 2H).",
"N-{[5-(Aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoroacetate) (18) a) A solution of tert-butyl-(4-cyanothiene-2-yl)methylcarbamate (7 g; 29.4 mmol) in 120 ml ethyl alcohol was saturated with NH3 and then hydrogenated under standard conditions in the presence of Ra—Ni (9 g watery suspension; decanted with ethyl alcohol).",
"Filtration of the reaction mixture, evaporation and chromatography of the obtained residue on silica gel (CH2Cl2/CH3OH plus watery NH3) afforded 4.4 g of the amine as a yellow oil.",
"b) 1.2.of the amine 18a (4.3 mmol), 06.g ethyldiisopropylamine and 15 g 2-fluoropyridine were heated for 20 h to reflux.",
"The residue obtained after evaporation of the mixture was taken up in CH2Cl2, washed with 0.1n HCl and saturated NaCl solution, dried and again evaporated.",
"1 g; ESI-MS [M+H+]=320.15 c) 0.9 of the Boc-protected amine 18b were dissolved in 10 ml CH2Cl2, 5 ml TFA was added at 0° C. and it was stirred at room temperature for 1 h. Evaporation of the reaction mixture afforded 1.65 g of a brownish oil which was reacted directly without further purification (ESI-MS [M+H+]=220.05).",
"3-Amino-N-(1H-imidazole-2-yl)propaneamide (19) a) Z-β-Alanine (10 g; 44.8 mmol) was dissolved in 200 ml DMF and 15.86 g (3.5 eq) N-methylmorpholine and 5.9 g (0.5 eq) 2-aminoimidazolesulfate were added.",
"7.87 g (1.3.eq) HOBt and 11.16 g (1.3 eq) N′-(dimethylaminopropyl)-N-ethylcarbodiimide were added at −10° C. and stirred for 1 h whilst to RT and then for 18 h. 150 ml diethyl ether were added whereupon a wide residue precipitated which was filtered with suction.",
"The residue was washed with cold diethyl ether, suspended in ethyl acetate and 1n HCl was added up to an acid reaction.",
"The watery solution was extracted 1× with ethyl acetate, the watery phase was then adjusted to a basic pH with 10% NaOH at 4° C. The resulting precipitate was filtered with suction and washed with water.",
"5.4 g; ESI-MS [M+H+]=289.05.b) 5.3 g of the Z-compound 19a were suspended in 250 ml ethyl alcohol and 530 mg 10% Pd on activated carbon was added.",
"It was hydrogenated with H2 for 18 h at RT, then diluted with CH3OH and the suspension was boiled up whereon the precipitate of the product disintegrated.",
"Filtering and evaporation of the solution afforded 1.5 g; ESI-MS [M+H+]=155.05.N-[4-(Aminomethyl)phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) tert-Butyl-4-aminobenzylcarbamate (2 g; 9 mmol), 2.2 g ethyldiisopropylamine and 4.4 g (2-(3,5-dimethylpyrazole)-4,5-dihydroimidazole×HBr in 15 ml DMF were stirred at 110° C. After the reaction had completed, the mixture was evaporated, the residue taken up in ethyl acetate, each 2× washed with saturated NaHCO3 and NaCl solution, dried and again evaporated.",
"The basic watery phase was also evaporated, mixed with acetone, the precipitate filtered with suction and the mother liquor evaporated.",
"The combined residues were purified by means of MPLC (silica gel: Fa.",
"Bischoff Prontoprep 60-2540-C18E, 32 μm; solvent: CH3CN/H2O+0.1% acetic acid).",
"0.22 g; ESI-MS [M+H+]=291.15.The product thus obtained was treated with 4n HCl in dioxane for 2 h at RT.",
"The resulting precipitate was filtered with suction, washed with pentane and dried.",
"110 mg; ESI-MS [M+H+]=191.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.85 (s, 1H), 8.45 (s broad, 3H), 8.40 (s, 1H), 7.60 and 7.30 (each d, 2H), 4.05 (m, 2H), 3.70 (s, 4H).",
"N-{[5-Aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) Amine 18a (6.5 g; 23.31 mmol) was reacted to the respective aminobenzimidazole by reaction with thiocarbonyldiimidazole, imidazole and then phenylenediamine analogously to the preparation of 17.1.6 g: ESI-MS [M+H+]=359.15.The subsequent cleavage of the Boc-group by means of 4n HCl in dioxane afforded 1.3 g of slightly yellow solids: ESI-MS [M+H+]=191.15.N1-Pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) Preparation analogously to 18b by reaction of N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 20.3 g 2-fluoropyridine, 5.64 g clear oil; ESI-MS [M+H+]=280.15.Cleavage of the Boc-group with 4n HCl in dioxane afforded 3.46 g white solids; ESI-MS [M+H+]=180.20.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (s broad, 1H), 8.05 (s broad, 3H), 7.85 (m, 2H), 7.20 (m, 2H), 6.80 (m, 1H), 3.45 (m, superimposed by H2O), 2.80 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H).",
"N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol), 5.4 g ethyldiisopropylamine and 5.11 g 2-(methylsulfanyl)-4,5-dihydro-1H-imidazole×HI in 30 ml DMF were stirred overnight at RT.",
"The reaction mixture was evaporated, taken up in CH2Cl2, washed with water and saturated NaCl solution, dried and again evaporated.",
"5.05 g clear oil, ESI-MS [M+H+]: 271.18.Cleavage of the Boc-group with 4n HCl in dioxane and purification of the raw product by means of MPLC afforded 2.57 g; ESI-MS [M+H+]: 171.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 160.3, 43.3 (2 signals superimposed).",
"42.85, 39.70, 28.90, 27.2, 23.60.N-[4-(Aminomethyl)phenyl]-1H-imidazole-2-amine (24) a) 5.24 g BrCN—dissolved in 50 ml CH3OH-were added dropwise to a mixture of tert-butyl-4-aminobenzylcarbamate (10 g; 44.99 mmol) and 11.07 g sodium acetate in 100 ml CH3OH at 0° C., and it was stirred for 3 hours at 0° C. and overnight at RT.",
"The mixture was evaporated, the obtained residue taken up in water and 2× extracted with methyl-tert-butylether.",
"Drying and evaporation of the organic phases afforded 12.99 of a yellow-orange oil.",
"b) 28.6 g triethylamine were added to 7 g tert-butyl-4-[(iminomethylene)amino]benzyl carbamate in 50 ml pyridine, H2S was introduced for 1 hour at 0° C. and the mixture was allowed to stand for 48 hours.",
"Evaporation afforded 9.79 g of a rosa foam; ESI-MS [M+H+]: 282.05.1H-NMR (360 MHz.",
"D6DMSO) δ ppm: 9.70 (s, 1H), 7.35 (m, 4H), 7.20 (m, 2H), 4.15 (d, 2H), 1.45 (s, 9H).",
"c) 5 g of the thioamide in 50 ml CH3OH where methylated with 5.05 g CH3I and the obtained raw product was directly reacted with 1.73 g aminoacetaldehyde-diethylacetal in 7.5 ml CH3CN for 3 hours at RT.",
"Evaporation of the reaction mixture afforded 6.36 g of a reddish oil (ESI-MS [M+H+]: 381.25) which was dissolved in 50 ml 6n HCl and stirred for 3 hours at 0° C. A pH of 12 was then adjusted with a 25% NaOH solution and it was again stirred for 48 hours at RT.",
"The mixture was extracted 4× with ethyl acetate, the combined organic phases dried and evaporated.",
"The oil thus obtained was stirred 2× with methyl-tert-butylether, the obtained solids filtered with suction and dried.",
"0.6 g red solid; ESI-MS [M+H+]: 189.15.13C-NMR (90.55 MHz, d6-DMSO) δ ppm: 145.5, 141.60, 130.75, 128.4, 119.8, 115.6, 44.85.N1-(1,4,5,6-Tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (25) Preparation was carried out analogously to 23 starting from N-1-Boc-1,5-diaminopentane×HCl (5 g; 20.94 mmol) and 5.4 g 2-(methylsulfanyl)-1,4,5,6-tetrahydropyrimidine×HI.",
"After workup, 1.3 g of a yellowish oil was obtained; [M+H+]: 282.2.Cleavage of the Boc-group with 4n HCl in dioxane and purification by means of MPLC afforded 0.46 g; ESI-MS [M+H+]: 185.15.N-[4-(Aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26) Benzyl (4-aminocyclohexyl)methylcarbamate (TFA-salt) (5 g; 13.28 mmol)-preparation by TFA cleavage starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317)-was heated to reflux analogously to 18 with 1.71 g ethyldiisopropylamine in 50 ml 2-fluoropyridine.",
"Usual workup and crystallization of the raw products from methyl-tert-butylether/methanol afforded 4.15 g; ESI-MS [M+H+]: 340.29.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.75 (d broad, 1H), 7.85 (m, 2H), 7.35 (m, 5H), 7.05 (d, 1H), 6.85 (m, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.75 (each m, 2H), 1.45-0.90 (m, 6H).",
"Cleavage of the Z group under standard conditions (H2; Pd-activated carbon), precipitation of the resulting amine as hydrochloride and drying of the obtained precipitates afforded 1.5 g; ESI-MS [M+H+]: 206.15.tert-Butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-ylacetate (27) 75 ml of a 1.0 m BH3-THF solution were added to a solution of tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10 g; 36.32 mmol) in 100 ml THF and it was stirred at RT.",
"For workup, water was carefully added, it was 2× extracted with diethyl ether and then the organic phases were washed 2× with water.",
"Evaporation and drying afforded 9.3 g; ESI-MS [M+H+]: 262.04.",
"[5-(2-tert-Butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) a) 3 g K2CO3, 0.05 g KI and 5 g methylbromoacetate were added to a solution of tert-butyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl acetate (27) in 50 ml DMF and it was stirred for 12 hours at 90° C. Thereafter, 4 g methylbromoacetate were added and it was stirred for further 5 hours at 120° C. For workup, the mixture was concentrated, diluted with CH2Cl2, washed with saturated NaCl solution, dried and again evaporated.",
"Chromatography on silica gel (CH2Cl2/CH3OH 1-5%) afforded 4.6 g of a bright yellow oil; ESI-MS [M+H+]: 334.12.b) 4.19 of the methylester in 20 ml dioxane/15 ml H2O were saponified with 19 KOH at RT.",
"For workup, the mixture was concentrated, adjusted to a pH of 2 with 2n HCl and extracted with CH2Cl2.The combined organic phases were dried, evaporated and the obtained raw product purified by chromatography on silica gel (CH2Cl2/CH3OH 2-7%).",
"2.4 g oil, ESI-MS [M+H+]: 320.15.1H-NMR (360 MHz, CDCl3) δ ppm: 7.20-7.10 (m, 1H), 6.90 and 6.80 (each m, 1H), 4.0 (s, 2H), 3.55 (m, 1H), 3.10 (m, 2H), 2.85 and 2.70 (each m, 1H), 2.90-2.50 (m, 4H), 1.35 (s, 9H).",
"N-[4-(Aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) Preparation analogously to the preparation of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (23) starting from benzyl-(4-aminocyclohexyl)methylcarbamate (TFA salt) (3 g; 7.97 mmol).",
"a) Reaction with BrCN and subsequent purification (1.52 g; ESI-MS [M+H+]: 288.15).",
"1H-NMR (360 MHz, d6-DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.75 (d, 1H), 5.05 (d, 2H), 2.85 (m, 3H), 1.85 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.20 and 0.95 (each m, 2H).",
"b) Conversion to the corresponding thiourea and subsequent methylation (1.489; ESI-MS [M+H+]: 336.15.c) Reaction with aminoacetaldehyde-diethylacetal and subsequent cyclization afforded 0.79 g; ESI-MS [M+H+]: 329.15.1H-NMR (360 MHz, DMSO) δ ppm: 7.45-7.25 (m, 5H), 6.45 (s, 2H), 5.35 (d, 1H), 5.05 (s, 2H), 2.90 (m, 2H), 1.95 and 1.70 (each m, 2H), 1.35 (m, 1H), 1.15 and 0.95 (each m, 2H).",
"d) For cleavage of the Z-group it was dissolved in 30 ml HBr/glacial acidic acid and stirred for 3 h at RT.",
"For workup, the mixture was evaporated and several times co-evaporated with acetone.",
"0.89 g; ESI-MS [M+H+]: 195.15.tert-Butyloxycarbonyl-4-[(2-pyridinylamino)methyl]-1-piperidine (30) tert-Butyloxycarbonyl-4-(aminomethyl)-1-piperidine (3 g; 14 mmol) and 10 ml 2-fluoropyridine were heated for 4 h to reflux.",
"Evaporation and mixing the raw product in n-pentane afforded 3 g of a wide solid, mp: 126-130° C.; ESI-MS [M+H+]=292.15.The amine required for the further reaction was obtained by cleavage of the Boc-group with HCl in dioxane (under standard conditions); the isolated HCl-salt was then directly utilized.",
"Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-oxo-2,3,4,6-tetrahydro H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) a) A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) in 20 ml DMF was added dropwise to a suspension of 1.28 g NaH (60%; deoiled) in 10 ml DMF at 5° C. and it was stirred for 1 h. 3.49 g Bromoacetonitrile-dissolved in 20 ml DMF-were then added dropwise and it was stirred for 4 h at RT.",
"For workup, water was added carefully, it was diluted with CH2Cl2, washed several times with H2O and saturated NaCl solution, dried and evaporated.",
"Purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 5%) afforded 7.61 g; ESI-MS [M+H+-tBu]: 259.05.b) 5 g tert-Butyl-[1-(cyanomethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate in 70 ml pyridine were saturated for 1 h with H2S and then allowed to stand overnight at RT.",
"Evaporation of the mixture and mixing of the obtained residue with pentane afforded 5.5 g of a rosa solid which was directly reacted.",
"c) A mixture of 2 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.62 g ethylpyruvate and 0.86 g KHCO3 in 30 ml dioxane was stirred for 2.5 h at RT.",
"Dilution with CH2Cl2, washing with saturated NaCl solution, drying and evaporation afforded 2.65 g of a yellow oil; ESI-MS [M+H+]: 445.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.50 (s, 1H), 7.50 and 7.35 (each m, 1H), 7.30-7.20 (m, 2H), 7.35 (d, 1H), 5.20 (broad, 1H), 4.30 (q, 2H), 4.20 (m, 1H), 3.65-3.50 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 3H), 1.70-1.50 (m, 2H), 1.30 (s, 9H; superimposed by t, 3H).",
"2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) 2.6 g Ethyl-2-{[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carboxylate (31) was provided in a mixture of 31 ml dioxane and 4 ml H2O, 1.5 eq.",
"KOH was added and it was heated to reflux.",
"After 5 h, again 1 eq.",
"KOH was added and it was further stirred for 12 h at RT.",
"For workup, it was concentrated, the residue was taken up in water, a pH of 4-5 was adjusted with 2n HCl and it was extracted several times with CH2Cl2.The combined org.",
"phases were washed with saturated NaCl solution, dried and evaporated.",
"Mixing with n-pentane afforded 2.1 g of a white solid; ESI-MS [M+H+]: 417.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 7.5 (m, 1H), 7.35 (m, 1H), 7.30-7.20 (m, 3H), 5.25 (m, 2H), 2.70 (m, 2H), 2.35-2.10 (m, 6H), 1.70 (m, 1H), 1.30 (s, 9H).",
"2-Ammonio-6-(ammoniomethyl)pyridinium trichloride (33) a) 2-Amino-6-methylpyridine (0.14 mol, 15.0 g) and phthalanhydride (0.14 mol, 20.55 g) were heated to 190° C. at the water separator.",
"Distributing between H2O and CH2Cl2, evaporation of the org.",
"phase and recrystallisation of the residue (diethyl ether) afforded 28.25 g of a slightly yellowish solid; ESI-MS [M+H+]=239.15.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.95 (2H, m), 7.78 (3H, m), 7.23 (2H, m), 2.64 (3H, s).",
"b.)",
"N-bromosuccimide (25.18 mmol, 4.48 g) was added portionwise to a boiling suspension of 2-(6-methyl-2-pyridinyl)-1H-isoindole-1,3(2H)-dione (33a, 20.99 mmol, 5.0 g), AIBN (2.10 mmol, 0.35 g) and dibenzoylperoxide (2.10 mmol, 0.51 g) in CCl4.The reaction mixture was boiled for 20 h, filtered and the filtrate evaporated.",
"Chromatography (CH2Cl2) afforded 3.12 g of the target product and 1.20 g of the dibromo compound; ESI-MS 318.95, 316.95.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98 (2H, m), 7.95 (1H, t), 7.81 (2H, m), 7.58 (1H, d), 7.35 (1H, d), 4.60 (2H, s).",
"c) Potassium phthalimide (9.46 mmol, 1.75 g) was added to a solution of 2-[6-(bromomethyl)-2-pyridinyl]-1H-isoindole-1,3(2H)-dione (33b, 6.31 mmol, 2.0 g) in DMF (30 ml), the reaction mixture was heated for 15 h to 60° C., stirred for 24 h at RT, water (60 ml) was added and it was stirred for 2 h at 0° C. The residue was filtrated and washed with a mixture of H2O-DMF and then with diethyl ether; 2.12 g; ESI-MS [M+H+]=384.05.1H-NMR (270 MHz, CDCl3) δ (ppm): 7.98-7.91 (4H, m), 7.88 (1H, t), 7.85-7.75 (4H, m), 7.38-7.32 (2H, m), 5.10 (2H, s).",
"d) 2-{6-[(1,3-Dioxo-1,3-dihydro-2H-isoindole-2-yl)methyl]-2-pyridinyl}-1H-isoindole-1,3(2H)-dione (33c, 5.22 mmol, 2.09) was heated to reflux for 3 h with hydrazinium hydroxide (13.04 mmol, 0.65 g) in methanol (50 ml).",
"For workup, water was added, it was evaporated and the watery phase acidified with conc.",
"HCl.",
"Anew evaporation and recrystallising (ethyl alcohol) afforded 1.20 g of a white solid; ESI-MS [M+H+]=124.05.1H-NMR (270 MHz, D2O) δ (ppm): 7.94 (1H, t), 7.08 (1H, d), 6.99 (1H, d), 4.33 (2H, s).",
"trans-N-[4-(Aminomethyl)cyclohexyl]-N′-benzylurea (34) Preparation was carried out analogously to compound 6 starting from benzyl-{4-[(tert-butoxycarbonyl)amino]cyclohexyl}methylcarbamate (EP 669317) by cleavage of the boc-group with 4n HCl in dioxane.",
"Build-up of the benzyl urea by reaction with benzyl isocyanate and triethylamine in DMF and subsequent hydrogenolysis afforded 0.55 g of the target product; ESI-MS [M+H+]=262.20.7-(4-Aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoroacetate) (35) a.)",
"A solution of 5-tert-butoxycarbonylaminovaleric acid (50.0 mmol, 10.86 g), O,N-dimethylhydroxylamine hydrochloride (50 mmol, 4.88 g), N-Methylmorpholine (0.30 mol, 30.35 g), HOBT (53.90 mmol, 8.42 g) and EDCI*HCl (55.0 mmol, 10.54 g) in CH3CN (200 ml) were stirred for 2 days at RT.",
"After evaporation the residue was taken up in ethyl acetate, and then washed with water, a 10% KHSO4-solution, a saturated watery NaHCO3 solution and a saturated watery NaCl-solution, subsequently.",
"Drying and evaporation of the organic phase afforded 6.96 g of a yellowish oil; ESI-MS: [2M+Na+]=543.3, [M+Na+]=283.1, 205.1, 161.1.1H-NMR (270 MHz, CDCl3) δ (ppm): 4.63 (1H, s.",
"br.",
"), 3.68 (3H, s), 3.21-3.05 (3+2H, m), 2.44 (2H, t), 1.76-1.48 (2+2H, m), 1.43 (9H, s).",
"b.)",
"Methyl magnesia bromide (60.0 mmol, 17.30 ml of a 3M solution in Et2O) at 0° C. was added dropwise to a solution of tert-butyl 5-[methoxy(methyl)amino]-5-oxopentylcarbamate (35a, 30.0 mmol, 6.9 g) in THF (120 ml) and stirred for 5 h at 0° C. The reaction mixture was then carefully acidified with a 10% KHSO4-solution, extracted with ethyl acetate and the organic phase then washed with saturated watery NaHCO3— and saturated watery NaCl-solution, dried and evaporated: 5.5 g yellowish oil; ESI-MS: [M-BOC+H+]=116.15.c.)",
"A mixture of tert-butyl 5-oxohexylcarbamate (35b, 9.29 mmol, 2.0 g), 2-aminonicotinaldehyde (Heterocycl.",
"1993, 36, 2518; 11.20 mmol, 1.379) and KOH (0.37 ml of a 20% watery solution) was heated to reflux for 8 h. Evaporation and column chromatography afforded 1.60 g of the target product; ESI-MS: [M+H+]=302.15.d.)",
"A suspension of tert-butyl 4-[1,8]naphthyridine-2-ylbutylcarbamate (35c, 5.31 mmol, 1.60 g) and Pd/C (10%, 1.59) in ethyl alcohol (40 ml) were stirred overnight under H2 atmosphere, then filtered over celite and washed with ethyl alcohol.",
"Column chromatography afforded 290 mg; ESI-MS: [M+H+]=306.25.1H-NMR (360 MHz, CDCl3) δ (ppm): 7.04 (1H, d), 6.29 (1H, d), 4.97 (1H, s.br.",
"), 4.81 (1H, s.br.",
"), 3.37 (2H, m sym.",
"), 3.12 (2H, q br.",
"), 2.65 (2H, t), 2.53 (2H, t), 1.89 (2H, quint.",
"), 1.67 (2H, quint.",
"), 1.51 (2H, quint.",
"), 1.43 (9H, s).",
"e.) TFA (18.30 mmol, 2.099) was added to a solution of tert-butyl 4-(5,6,7,8-tetrahydro[1,8]naphthyridine-2-yl)butylcarbamate (35d, 0.92 mmol, 0.289) in CH2Cl2 (8 ml), the solution was stirred for 20 h and evaporated: 380 mg; ESI-MS: 206.1, 130.7.1H-NMR (400 MHz, CDCl3) δ (ppm): 7.07 (1H, d), 6.31 (1H, d), 5.58 (1H, s.br.",
"), 3.39 (2H, m sym.",
"), 2.96 (2H, s br.",
"), 2.76 (2H, t), 2.68 (2H, t), 2.56 (2H, t), 1.88 (2H, quint.",
"), 1.69 (2H, quint.",
"), 1.51 (2H, quint.).",
"tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36) Tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (10.9 mmol, 3.0 g)—dissolved in THF—was added at 0° C. to a solution of diisopropylamine (11.0 mmol, 1.119) and butyl lithium (11.0 mmol, 6.91 ml of a 15% solution in hexane) in THF (100 ml) and the solution further stirred for 1 h. About 100 ml ethylene oxide were then added and the mixture was stirred overnight at RT.",
"The solution was distributed between saturated NH4Cl and ethyl acetate, the organic phase was washed with water and evaporated; 2.7 g; ESI-MS: [2M+Na+]=661.3, [M+K+]=358.1, 321.1, [M+H+]=320.1, 264.0.tert-Butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37) tert-Butyl [1-(2-hydroxyethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (36, 6.26 mmol, 2.00 g) dissolved in CH2Cl2 was added dropwise within 10 minutes to a solution of oxalyl chloride (7.93 mmol, 1.0 g) and DMSO (16.59 mmol, 1.26 g) in little CH2Cl2.After 30 min.",
"triethyl amine (38.22 mmol, 3.87 g) was added, stirred for 5 min., left to reach RT and stirred overnight at RT.",
"For workup, water was added, the mixture extracted with CH2Cl2 and the organic phase washed with saturated NaCl—, 1%-H2SO4— and with 5%-NaHCO3-solution.",
"Evaporation afforded 1.8 g of the target product; ESI-MS: 693.2, [M+K+]=358.1, 319.1, [M+H+]=318.1, 262.0.Methyl-2-amino-5-chlorobenzoic acid (38) Thionyl chloride (0.47 mmol, 55.46 g) was added dropwise at 0° C. to a solution of 2-chloro-5-aminobenzoic acid (0.23 mmol, 40.09) in methanol (400 ml) and the mixture heated to 50-60° C. After the reaction had finished, water was added and it was extracted with ethyl acetate.",
"Then the organic phase was washed with 1n NaOH and diluted HCl solution (pH 1-2), subsequently, and evaporated; 23.0 g; ESI-MS: [M+H+]=186.05.Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39) Ethyl succinic acid-chloride (0.14 mol, 22.44 g) in toluen (15 ml) was added dropwise at 0° C. to a solution of methyl 2-amino-5-chloro-benzoic acid (38, 123.9 mmol, 23.0 g) and pyridine (0.26 mol, 20.58 g) in toluen (40 ml).",
"The solution was stirred overnight at RT, water was added and it was extracted with ethyl acetate.",
"The organic phase was washed with 1N HCl-solution, with water, with a saturated NaHCO3-solution and with a saturated NaCl-solution.",
"Evaporation, recrystallisation (methanol) afforded 34.1 g of the target product; ESI-MS: [2M+Na+]=649.0, [M+K+]=352.0, [M+H+]=314.05; 1H-NMR (270.MHz, CDCl3) δ (ppm): 11.06 (1H, s br.",
"), 8.68 (1H, d), 7.99 (1H, m), 7.47 (1H, dd), 4.16 (2H, q), 3.92 (3H, s), 2.74 (4H, m), 1.24 (3H, t).",
"Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40) Methyl 4-chloro-2-[(4-ethoxy-4-oxobutanoyl)amino]benzoic acid (39, 0.16 mol, 50.20 g) in DMSO (250 ml) was added dropwise at 15° C. to a suspension of deoiled NaH (0.27 mol) in THF (50 ml) and DMSO (80 ml) and the mixture was stirred for 2 h at RT.",
"At 0° C. glacial acetic acid (24 ml) was added and stirred for 20 min.",
"Water (25 ml) was added, the resulting precipitate filtered off, washed with water, taken up in CH2Cl2, extracted with water and the organic phase evaporated.",
"The residue was then stirred with diethyl ether (50 ml), filtered and dried; 32 g of a 6:4-mixture methyl/ethyl ester, which was not separated; ESI-MS (Me-ester): [M+K+]=307.9, [M+Na+]=290.0, [M+H+]=268.0; ESI-MS (Et-ester): [M+K+]=321.9, [M+Na+]=304.0, [M+H+]=282.0.7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41) Ethyl-7-chloro-5-hydroxy-2-oxo-2,3-dihydro-1H-1-benzazepine-4-carboxylate (40, 0.11 mol, 32.0 g) was heated to 150° C. in DMSO (500 ml) and water (0.23 mol, 4.09 g) and stirred for 2 h. Water was added at 100° C., the mixture cooled to 0° C. and the resulting precipitate filtered off.",
"Drying afforded 19.09; ESI-MS: [M+Na+]=251.1, [M+H+]=211.9, 209.95, 130.1.tert-Butyl (2E,Z)-(7-Chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene) ethane acid (42) t-Butyl diethylphosphono acetic acid (0.10 mol, 25.849) was added dropwise at 0° C. to a solution of deoiled NaH (0.10 mol) in DMF (40 ml) and stirred until a clear solution develops.",
"7-Chloro-3,4-dihydro-1H-1-benzazepine-2,5-dione (41, 90.63 mmol, 19.09) in DMF (185 ml) was added dropwise at 0° C. and stirred at RT.",
"Water was added to the reaction mixture, it was stirred for 1 h and the resulting yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.",
"Recrystallisation afforded 23.5 g of the target product; ESI-MS: [2M+H+]=615.2, [M+Na+]=330.0, 293.0, 254.1, 252.1.1H-NMR (400 MHz, CDCl3) δ (ppm): 9.59 (1H, s br.",
"), 7.45 (1H, m), 7.25 (1H, m), 7.06 (1H, m), 5.99 (1H, t), 3.43 (2H, s), 2.84 (2H, d), 1.32 (9H, s).",
"t-Butyl (7-chloro-2-oxo-2,3,4,4-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43) tert-Butyl (2E,Z)-(7-chloro-2-oxo-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)ethane acid (42, 75.0 mmol, 23.089) in ethyl alcohol/dioxane (250 ml/100 ml) was hydrogenated for 4 days with Pt/carbon (5%, 4.1 g) under standard conditions.",
"Water was added to the reaction mixture, stirred for 1 h, the yellow residue filtered off with suction, washed with water and taken up in CH2Cl2.The organic phase was washed with a 5%-NaHCO3-solution and evaporated.",
"Recrystallisation afforded 23.5 g of a solid (mixture of target product and the corresponding dechlorinated compound; the mixture was reacted directly); ESI-MS: [2M+H+]=618.94, [M+K+]=350.66, 309.75, 254.11.Methyl [5-(2-tert-butoxy-2-oxo ethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44) tert-Butyl (7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (43, 60.00 mmol, 18.59 g) in DMF (10 ml) was added dropwise at 15° C. to deoiled NaH (69.00 mmol) in DMF (5 ml) and the mixture was stirred overnight at RT.",
"Ice water was added to the reaction mixture, extracted (2×) with ethyl acetate and the organic phase washed with a 10% CH3COOH-solution, with water and then with 1n NaOH.",
"Evaporation afforded 20.4 g of a raw product, which was reacted without further purification.",
"[5-(2-tert-Butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) KOH (80.45 mmol, 4.51 g) in water (150 ml) was added to methyl [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (44, 50.28 mmol, 19.29) dissolved in dioxane (250 ml) and stirred for 1 h at RT.",
"The reaction mixture was evaporated, water (100 ml) was added and it was extracted with ethyl acetate (2×).",
"After the evaporation, the residue was taken up in diethyl ether and precipitated by addition of n-pentane.",
"Recrystallisation (diisopropyl ether, 2×) afforded 4.8 g (comprises about 15% of the corresponding dechloro compound); ESI-MS: [M+Na+]=390.0, 314.0, 312.0.",
"[4-(Aminomethyl)phenyl]guanidine (Bihydrochloride) (46) p-Aminobenzylamine (6.7 g; 54.84 mmol) was suspended in 20 ml 6n HCl and 5.3 g cyanamide—dissolved in 5 ml H20—added slowly under reflux.",
"After the reaction had completed, 50% NaOH-solution was added at 0° C. to the solution, the resulting precipitate filtered with suction, boiled up in 50 ml ethyl alcohol and filtered off.",
"Evaporation of the mother liquor and mixing of the obtained residue with diethyl ether afforded 1.4 g of yellow solids; Fp.",
": 255° C. 7,8-Dimethoxy-3,4-dihydro-1H-1-benzazepine-2,5-dione (47) Analogously to the preparation of the corresponding building blocks 39, 40 and 41 first ethyl-2-amino-4,5-dimethoxybenzoate (20 g; 88.8 mmol) was reacted with ethyl succinic acid-chloride to the respective amide.",
"After mixing with n-pentane 30.4 g of a solid was obtained; ESI-MS [M+H+]: 354.15.1H-NMR (400 MHz, DMSO) δ (ppm): 10.65 (s, 1H), 8.10 and 7.40 (each s, 1H), 4.30 and 4.10 (each q, 2H), 3.85 and 3.80 (each s, 3H), 2.70 (m, 4H), 1.30 and 1.20 (each t, 3H).",
"Subsequent cyclization with 25 g of the obtained amide under utilization of NaH analogously to 40 and usual workup afforded 19.5 g of a white solid; ESI-MS [M+H+]: 308.05.1H-NMR (400 MHz, DMSO) δ (ppm): 13.3 (s, 1H), 10.10 (s, 1H), 7.25 and 6.75 (each s, 1H), 4.30 (q, 2H), 3.80 (s, 6H), 2.95 (s, 2H), 1.35 (t, 3H).",
"Decarboxylation analogously to 41 starting from 17 g afforded 10.5 g of the target product as a solid; ESI-MS [M+H+]: 236.15.1H-NMR (400 MHz, DMSO) δ (ppm): 9.90 (s, 1H), 7.35 and 6.80 (each s, 1H), 3.80 and 3.75 (each s, 3H), 2.85 and 2.65 (each m, 2H).",
"tert-Butyl (7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate (48) Preparation analogously to building blocks 42 and 43 by Wittig-Homer-reaction and subsequent hydrogenation; after mixing with n-pentane 8.16 g of solids were obtained; ESI-MS [M+H+-tBu]: 280.15.",
"[5-(2-tert-Butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (49) Analogously to 44 and 45, starting from 4 g tert-butyl-(7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 2.6 g of the target product were isolated as a bright foam; [M+H+-tBu]: 338.15.1H-NMR (400 MHz, DMSO) δ (ppm): 6.85 and 6.75 (each s, 1H), 4.35 (s broad, 2H), 3.80 and 3.75 (each s, 3H), 3.60 (s, 2H), 2.70 (m, 2H), 2.25 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H), 1.35 (s, 9H).",
"[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) a.)",
"56.1 g (406 mmol) powdered K2CO3 were added at room temperature to a solution of 37 g (135.3 mmol) t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl)acetic acid (1) and 3.7 g tetrabutylammoniumbromide in 370 ml DMF, 22.7 g (148.9 mmol) methylbromoacetic acid was added dropwise, then it was stirred for 3 h at 40° C. and overnight at room temperature.",
"The reaction mixture was poured into 1000 ml of a water-ice-mixture, and 2× extracted with each 200 ml methyl-tert.butyl ether.",
"The combined extracts were washed with H2O, 5% NaHCO3— and NaCl-solution, dried over Na2SO4, filtered off and evaporated.",
"The remaining yellow oil (about 52 g, purity about 90%) was reacted without further purification; ESI-MS [M+H+]: 346.b.)",
"9.2 g (26.6 mmol) of the raw product 50a were dissolved in 66.6 ml 4n HCl in dioxane and stirred for 24 h at 50° C., then the dioxane was extensively distilled off, 5% NaHCO3-solution and diethyl ether added to the residue, the watery phase again washed with diethyl ether and acidified with 1n KHSO4-solution.",
"The precipitating acid was extracted with diethyl ether, the ether phase washed with a NaCl-solution, dried over Na2SO4, filtered off with suction and evaporated.",
"It remained 3.2 g of a slightly yellow oil; ESI-MS [M+H+]: 290.trans-[4-(1H-Benzimidazole-2-yl)cyclohexyl]methaneamine (hydrochloride) (51) 39.3 g (0.25 mol) trans-4-(Aminomethyl)cyclohexanecarbonic acid and 27.0 g (0.25 mol) 1,2-phenylene diamine were heated to reflux for 18 h in a mixture of 167 ml conc.",
"hydrochloric acid and 333 ml H2O analogously to J. Heterocycl.",
"Chem.",
"26, 541 (1989).",
"The green reaction solution was concentrated until the occurrence of a yellow crystalline pulp and stirred with 400 ml isopropanol, filtered off, washed with 90% isopropanol and finally with diethyl ether.",
"After 2 times recrystallisation from a isopropanol-water mixture (70/30), 30 g of a white monohydrochloride remained; ESI-MS [M+H+]: 230.Methyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5benzodiazepine-1-yl)acetic acid (52) 1.99 (79.8 mmol) NaH (60% dispersion in mineral oil) were added to an ice cooled solution of 12.2 g (75.3 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-One (preparation: J.",
"Am.",
"Chem.",
"Soc.",
"1949, 71, 1985) in 350 ml DMF, stirred for 30 min.",
"at 0-5° C. and for 10 min.",
"at room temperature.",
"Then 11.5 g (75.3 mmol) methyl bromo acetic acid were added dropwise at 0° C. and it was then stirred for 30 min.",
"at the same temperature.",
"The reaction solution was poured onto 600 ml ice water and 3× extracted with each 150 ml ethyl acetate.",
"The organic phase was washed with a NaCl-solution, dried over MgSO4 and ethyl acetate was distilled off.",
"The residue was purified by column chromatography (eluent: CH2Cl2/CH3OH 9/1).",
"9.6 g of a yellowish oil were isolated; ESI-MS [M+H+]: 235.tert-butyl (2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) Preparation was carried out analogously to building block 52 by reaction of 11.99 (73 mmol) 1,3,4,5-tetrahydro-2H-1,5-benzodiazepine-2-one with 14.3 g (73 mmol) tert-butyl bromo acetic acid.",
"17 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 277.",
"[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) a.)",
"14.2 g (102 mmol) powdered K2CO3 were added at 0° C. to a solution of 9.6 g (41 mmol) compound 52 and 8.09 (41 mmol) tert-butyl bromo acetic acid in 90 ml DMF, and stirred for 1 h at 0° C. and then for 14 h at room temperature.",
"The reaction mixture was poured onto 300 ml ice water and 3× extracted with each 100 ml methyl-tert.butyl ether.",
"The combined organic phases were washed several times with a NaCl-solution, dried over MgSO4 and evaporated to dryness.",
"The residue was purified by column chromatography (eluent: ethyl acetate/cyclohexane 7/3).",
"7.0 g of a slightly yellowish oil were isolated; ESI-MS [M+H+]: 349.b.)",
"The alkaline saponification of the methyl ester was carried out analogously to 3b.",
"3.8 g white crystals were obtained; Fp.",
": 140-142° C.; ESI-MS [M+H+]: 335.",
"[5-(2-tert-Butoxy-2 oxoethyl)-4-oxo-2,3,4,6-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) Analogously to building block 54a tert-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl)acetic acid (53) was reacted with methyl bromoacetic acid and then alkaline saponified analogously to 3b.",
"After purification by column chromatography 2.9 g white crystals were obtained; Fp.",
": 82-84° C.; ESI-MS [M+H+]: 335.",
"[1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) 14.5 g (42 mmol) building block 3a were dissolved in 105 ml 4n HCl in dioxane and stirred for 2 days at 50° C. After evaporation of the solvent, the residue was dissolved in 5% NaHCO3-solution, 2× extracted with methyl-tert.butyl ether, then the watery phase was acidified with a 1n KHSO4-solution and the precipitating acid extracted with methyl-tert.butyl ether.",
"After drying over MgSO4, evaporation of the solvent and purification by column chromatography (eluent: CH2Cl2/CH3OH/glacial acetic acid 451511) 1.6 g of a viscous, slightly yellowish oil remained; ESI-MS [M+H+]: 292.",
"[(5R)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (57) 14.2 g (42.6 mmol) [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) were suspended in 170 ml diethylether and dissolved by addition of 7.3 g (42.64 mmol) (1S)-(−)-1-napthyl)ethylamine.",
"The yellow solution was inoculated with (1S)-1-napthyl)ethaneaminium[(5R)-5-(2-tert.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetate—prepared by preparative HPLC-separation of compound 3 by means of a chiral column (Chiralpak AD 500×; 50 mm; 20 μm) and subsequent salification-, the deposited precipitate filtered off with suction after 3.5 h and 3× recrystallized from a ethyl acetate/isopropanol mixture.",
"The purity of enantiomers was checked by means of a chiral HPLC.",
"3.5 g. of the salt were suspended in 30 ml of a diethylether/hexane-mixture 10/3 and after addition of 50 ml of a 5% watery amidosulfonic acid solution stirred until occurrence of a clear phase.",
"After separation of the watery phase, the organic phase was washed 3× with 5 ml amidosulfonic acid- and then with a NaCl-solution, dried over Na2SO4 and evaporated.",
"2.25 g amorphous residue; ESI-MS [M+H+]: 505.",
"[(5S)-5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (58) From the combined mother liquors of building block 57, 8 g of the acid were isolated as a yellowish amorphous residue as described with a amidosulfonic acid solution, said acid was reacted with (1R)-(+)-1-napthyl)ethyl amine into the diastereomeric salt and recrystallized until the achievement of enantiomeric purity.",
"Analogously to example 57, 2.5 g of the dextrorotatory acid were isolated as an amorphous solid, ESI-MS [M+H+]: 505.A sample of the acid was reacted with 4-bromobenzylamine into the good crystallizing amide and the absolute configuration figured out by means of a x-ray structure analysis.",
"tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate (59) A solution of 5.3 g (29.2 mmol) 6,7-dihydro-4H-thieno[3,2-b]azepine-5,8-dione (Arch.",
"Pharm.",
"1991 324, 579) and 12 g (32.2 mmol) (tert-butoxycarbonylmethylene)-triphenyl phosphorane in 15 ml toluen was heated to reflux for 10 h, then toluen was distilled off and the black residue purified by chromatography (eluent: ethyl acetate/cyclohexane 7/3).",
"The consistent fraction was again digested with 40 ml boiling cyclohexane, cooled and filtered with suction.",
"3 g yellowish crystals; ESI-MS [M+H+]: 280.tert-Butyl-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl)acetate (60) 39 (11 mol) tert-Butyl-(5-oxo-5,6-dihydro-4H-thieno[3,2-b]azepine-8-yl)acetate were hydrogenated analogously to building block 2 in the presence of 10% Pd/C.",
"Since educt was still present after 6 h according to HPLC, the catalyst was filtered with suction and after addition of new catalyst it was again hydrogenated for 6 h. After workup and chromatographic purification (eluent: ethyl acetate/cyclohexane 7/3), 1.4 g yellowish crystals were isolated, which comprised according to HPLC still about 2.5% educt; ESI-MS [M+H+]: 282.",
"[8-(2-tert-Butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) Preparation was carried out analogously to building block 50.Ester stage: 1.5 g yellowish crystals; ESI-MS [M+H+]: 354.Target product: 1.2 g yellowish crystals; ESI-MS [M+H+]: 340.I.B Compounds of the Formula I or I′ Example I t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetate 1.1 g of N-methylmorpholine were added dropwise at 0° C. to 1.5 g (4.65 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) and 1.22 g (4.8 mmol) of N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) in 20 ml of DMF, and 1.55 g (4.7 mmol) of TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetrafluoroborate) were then introduced in portions over the course of 35′.",
"The yellow reaction solution was stirred at 0° C. for 1 h and then largely evaporated in vacuo.",
"The residue was then digested a number of times with H2O, taken up in a mixture of 120 ml of ethyl acetate and 40 ml of diethyl ether, washed with 10% K2CO3 and NaCl solution, dried over Na2SO4 and concentrated, the crude product thoroughly crystallizing.",
"Purification by chromatography on silica gel (CH2Cl2/CH3OH/NH3 42:8:0.1) and crystallization from ethyl acetate/n-hexane afforded 1.45 g (65%) of white crystals.",
"M.p.",
": 190-193° C. (dec.); FAB-MS [M+H+]: 487.Example II [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid 1.2 g of the t-butyl ester from Example I were suspended in 70 ml of CH2Cl2, treated with 45 ml of 4N HCl in dioxane and stirred overnight at RT.",
"The solution was evaporated, and the residue was digested a number of times with CH2Cl2 and then dried.",
"In this way, 1.07 g of a slightly yellowish amorphous powder were isolated; FAB-MS [M−H+]: 432.Example III [1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine hydrochloride (4) and subsequent removal of the t-butyl group analogously to Example II.",
"A slightly yellowish amorphous residue was obtained, FAB-MS [M−H+]: 554.Example IV t-Butyl [1-(2-{[(2-{[amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetate Analogously to the preparation of compound 3a, 0.9 g (3.3 mmol) of t-butyl (2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl)acetate (1) was alkylated with methyl bromoacetate (FAB-MS [M−H+]: 346) and then hydrolyzed analogously to 3b (0.44 g; FAB-MS [M−H+]: 332).",
"Coupling of 0.57 g (1.72 mmol) of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-1-yl]acetic acid with N-[5-(aminomethyl)-1,3-thiazol-2-yl]guanidine dihydrochloride (5) analogously to Example I afforded the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 485.Example V [1-(2-{[(2-{[Amino(imino)methyl]amino}-1,3-thiazol-5-yl)methyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepin-5-yl]acetic acid The t-butyl ester was removed analogously to Example II and afforded 0.42 g of the title compound as a slightly yellowish powder; FAB-MS [M−H+]: 429.Example VI (1-{2-[(4-{[(Benzylamino)carbonyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl)acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-N′-benzylurea (6) and subsequent removal of the t-butyl group analogously to Example II.",
"Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 27 mg as an amorphous solid; ESI-MS [M+H+]: 515.2; [M+K+]: 553.2.Example VII [1-(2-{[4-(1H-Benzimidazol-2-yl)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [4-(1H-benzimidazol-2-yl)phenyl]methaneamine (7) and subsequent cleavage of the t-butyl group analogously to Example II.",
"Purification of the crude product by elution through a silica gel cartridge (Chromasorb; CH2Cl2/CH3OH 0-20%) afforded 9 mg as an amorphous solid; ESI-MS [M+H+]: 485.2.Example VIII [1-(2-{[4-(1H-Benzimidazol-2-ylamino)butyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)butane-1,4-diamine (trifluoroacetate) (9) and subsequent cleavage of the t-butyl group analogously to Example II.",
"After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.5 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 464.Example IX [1-(2-{[5-(1H-Benzimidazol-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N1-(1H-benzimidazol-2-yl)pentane-1,5-diamine (hydrochloride) (8) and subsequent cleavage of the t-butyl group analogously to Example II.",
"After chromatographic purification on silica gel (eluent: CH2Cl2/CH3OH/50% acetic acid 42:8:0.7), 0.48 g was isolated as a slightly yellowish amorphous powder; FAB-MS [M−H+]: 478.Example X {1-[2-({4-[(1H-Benzimidazol-2-ylamino)methyl]cyclohexyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with trans-N-[(4-aminocyclohexyl)methyl]-1H-benzimidazole-2-amine (dihydrochloride) (10) and subsequent cleavage of the t-butyl group analogously to Example II.",
"After chromatographic purification on silica gel, 0.7 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XI {1-[2-({[4-(1H-Benzimidazol-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-5-yl]acetic acid (3) with trans-N-{[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) and subsequent cleavage of the t-butyl group analogously to Example II.",
"After chromatographic purification on silica gel, 0.5 g of slightly yellowish amorphous powder was isolated; FAB-MS [M+H+]: 504.Example XII [2-Oxo-1-(2-oxo-2-{[4-(pyridin-2-ylamino)benzyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl]acetic acid Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-2-pyridineamine (13) and subsequent cleavage of the t-butyl group analogously to Example II (14 mg); ESI-MS [M+H+]: 459.15.Example XIII {1-[2-({[6-(1H-Benzimidazol-2-yl)pyridin-3-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-5-yl}acetic acid (bishydrochloride) Preparation was carried out analogously to Example I by reaction of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepin-1-yl]acetic acid (3) with [6-(1H-benzimidazol-2-yl)pyridin-3-yl]methaneamine (trifluoroacetate) (12) and subsequent cleavage of the t-butyl group analogously to Example II.",
"(13 mg); ESI-MS [M+H+]: 484.15.Compounds of the general formula I analogously to example II were prepared: Example 14 {2-Oxo-1-[2-oxo-2-({4-[(pyridine-2-ylamino)methyl]benzyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of N-[4-(aminomethyl)benzyl]-2-pyridine amine (14) as educt, 207 mg as an amorphous solid were obtained according to MPLC 207 mg; ESI-MS [M+H+]: 473.28.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.5 (m, 1H), 7.95 (d, 1H), 7.45 (m, 1H), 7.35-72.0 (m, 9H), 6.60 (m, 2H), 4.45 (m, 3H), 4.25 (m, 3H), 3.55 (m, 1H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m 1H).",
"Example 15 {1-[2-({[5-(1H-Benzimidazole-2-yl)thiene-2-yl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of [5-(1H-benzimidazole-2-yl)thiene-2-yl]methaneamine (15) as educt, 210 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 489.27.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 7.70 (m, 1H), 7.65 (m, 2H), 7.35-7.15 (m, 6H), 7.05 (m, 1H), 4.55 (m, 3H), 4.20 (m, 1H), 3.55 (m, 1H), 2.75 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H).",
"Example 16 {1-[2-({2-[4-(1H-Benzimidazole-2-yl)phenyl]ethyl}amino)-2-oxoethyl]-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Under utilization of tert-butyl-2-[4-(1H-benzimidazole-2-yl)phenyl]ethylcarbamate (16) as educt, 190 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 497.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.15 (d, 2H), 7.65 (d, 2H), 7.45 (m, 2H), 7.30-7.15 (m, 6H), 4.45 and 4.15 (each m, 1H), 3.6-3.25 (m, superimposed by H2O), 2.80 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.70 (m, 1H).",
"Example 17 [1-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of (3 N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) as educt, 134 mg as amorphous white solids were obtained according to MPLC; ESI-MS [M+H+]: 490.29.Example 18 [2-Oxo-1-(2-oxo-2-{[2-(pyridine-2-ylamino)ethyl]amino}ethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N1-pyridine-2-ylethane-1,2-diamine, the subsequent MPLC afforded 278 mg as amorphous white solids; ESI-MS [M+H+]: 397.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.95 (m, 1H), 7.70 (m, 2H), 7.35-7.20 (m, 4H) 6.80 and 6.70 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.0 (m, superimposed by H2O), 2.70 (m, 2H), 2.30 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).",
"Example 19 (2-Oxo-1-{2-oxo-2-[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]ethyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18), the subsequent MPLC afforded 135 mg as amorphous white solids; ESI-MS [M+H+]: 479.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.65 (m, 1H), 8.0 (m, 1H), 7.45 (m, 1H), 7.35-7.15 (m, 6H), 6.95 (s, 1H), 6.60 and 6.55 (each m, 1H), 4.60-4.30 (m, 5H), 4.15 (m 1H), 3.50 (m, 1H), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.70 (m, 1H).",
"Example 20 [1-(2-{[3-(1H-imidazole-2-ylamino)-3-oxopropyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of 3-amino-N-(1H-imidazole-2-yl)propane amide (19) and following purification of the raw product by MPLC 50 mg were obtained as amorphous white solids; ESI-MS [M+H+]: 414.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.20 (m, 1H), 7.30-7.20 (m, 7H), 4.45 and 4.15 (each m, 1H), 3.75-3.25 (m, superimposed by H2O), 2.80-2.65 (m, 4H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60 (m, 1H).",
"Example 21 [1-(2-{[4-(4,5-Dihydro-1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Under utilization of N-[4-(aminomethyl)-phenyl]-4,5-dihydro-1H-imidazole-2-amine (hydrochloride) (20) as educt, 12 mg were obtained as amorphous white solids after MPLC; ESI-MS [M+H+]: 450.3.Example 22 (1-{2-[({4-[(1H-Benzimidazole-2-ylamino)methyl]thiene-2-yl}methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-6-yl)acetic acid Utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}-1H-benzimidazole-2-amine (hydrochloride) (21) as educt; purification by MPLC afforded 90 mg; ESI-MS [M+H+]: 518.29.Example 23 [2-Oxo-1-(2-oxo-2-{[5-(pyridine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-pyridine-2-ylpentane-1,5-diamine (hydrochloride) (22) as educt; after MPLC, 210 mg white solids were obtained; ESI-MS [M+H+]: 439.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (m, 1H), 7.95 (m, 1H), 7.75 (m broad, 1H), 7.65 (m, 1H), 7.30-7.15 (m, 4H), 6.75 and 6.60 (each m, 1H), 4.45 and 4.15 (each m, 1H), 3.70-3.20 (m, superimposed by H2O), 2.70 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.60, 1.45 and 1.30 (each m, 2H).",
"Example 24 N-[5-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)pentyl]-4,5-dihydro-1H-imidazole-2-amine (acetate) Utilization of N1-(4,5-Dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) as educt; 22 mg were obtained after MPLC; ESI-MS [M+H+]: 430.15.Example 25 [1-(2-{[4-(1H-imidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N-[4-(aminomethyl)phenyl]-1H-imidazole-2-amine (24) as educt; purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 448.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.95 (s, 1H), 8.45 (m, 1H), 7.35-7.10 (m, 8H), 6.75 (s, 2H), 4.50 (m, 1H), 4.20 (m, 3H), 3.5-3.1 (m, superimposed by H2O).",
"2.70 (m, 1H), 2.35 (m 1H), 2.20 (m, 2H), 1.65 (m, 1H).",
"Example 26 [2-Oxo-1-(2-oxo-2-{[1-(1,4,6,6-tetrahydropyrimidine-2-ylamino)pentyl]amino}ethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Utilization of N1-(1,4,5,6-tetrahydropyrimidine-2-yl)pentane-1,5-diamine (hydrochloride) (26) as educt; 40 mg were obtained after MPLC; ESI-MS [M+H+]: 444.9.Example 27 {2-Oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Utilization of N-[4-(aminomethyl)cyclohexyl]pyridine-2-amine (hydrochloride) (26), purification by means of an elution over a Chromabond-C18-cartridge afforded 85 mg; ESI-MS [M+H+]: 465.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.55 (s broad, 1H), 8.05 (m, 1H), 7.90 (d, 1H), 7.85 (m, 1H), 7.35 (m, 2H), 7.25 (m, 2H), 6.95 (d, 1H), 6.80 (m, 1H), 4.40 and 4.20 (each m 1H), 3.55 (m, superimposed by H2O), 2.95 (m, 2H), 2.70 (m, 2H), 2.35 (m, 1H), 2.15, 1.95 and 1.75 (each m, 2H), 1.60 and 1.40 (each m, 1H), 1.25 (m, 2H), 1.05 (m, 2H).",
"Example 28 Ethyl-{2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 30 ml 4n HCl in dioxane were added to 100 mg {2-oxo-1-[2-oxo-2-({[4-(pyridine-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid in 10 ml EtOH and it was stirred for 48 h at RT.",
"Evaporation and elution over a Chromabond-C8-cartridge afforded 26 mg; ESI-MS [M+H+]: 493.25.Example 29 (1-{4-[4-(1H-Benzimidazole-2-ylamino)phenyl]butyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid a.)",
"A solution of t-butyl (2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (2) (0.8 g; 2.91 mmol) in 20 ml DMF was added dropwise at 0° C. to a suspension of 0.169 NaH (60%; deoiled) in 10 ml DMF and stirred for 1 h. A spatula tip full of KI was then added likewise at 0° C., 1 g 1-(4-bromo butyl)-4-nitrobenzene (preparation according to J. Med.",
"Chem.",
"38, 13 (1995), 2418-2420)—dissolved in 10 ml DMF—was slowly added dropwise and stirred for 2 h at RT.",
"For workup, water was carefully added to the reaction mixture, it was diluted with CH2Cl2 and several times extracted with a saturated NaCl-solution.",
"After drying and evaporation, the obtained raw product was purified by chromatography on silica gel (CH2Cl2/CH3OH 2-10%); 0.429 yellow oil, ESI-MS [M+H+-Boc]: 397.15.b.)",
"Hydrogenation of the nitro group in 75 ml CH3OH with 100 mg 10% Pd on activated carbon under standard conditions afforded 0.36 mg of a bright oil; ESI-MS [M+H+]: 423.25.c.)",
"The build-up of the corresponding aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and then phenylene diamine.",
"Chromatography on silica gel (CH2Cl2/CH3OH 2-4%) afforded 220 mg of a bright foam; ESI-MS [M+H+]: 539.25.d.)",
"200 mg tert-Butyl-(1-(4-[4-(1H-benzimidazole-2-ylamino)phenyl]butyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate was treated at RT with 10 ml TFA.",
"Evaporation and lyophilizing the obtained oil afforded 213 mg of a solid; ESI-MS [M+H+]: 483.25.Example 30 2-(4-{[{[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}(methyl)amino]methyl}anilino)-1H-benzimidazole (trifluoro acetate) a.)",
"Standard coupling of [5-(2-t.Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (2.2 g; 6.6 mmol) with 1.32 g N-methyl(4-nitrophenyl)methane amine (J.",
"Am.",
"Chem.",
"Soc.",
"65 (1943), 1984-1990) in 40 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.",
"Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 2-4%) afforded 2.269 of a slightly yellow oil; ESI-MS [M+H+]: 482.03.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 8.25/8.20 (d, 2H), 7.60/7.50 (d, 2H), 7.35-7.20 (m, 4H), 4.85-4.5 (m, 4H), 3.05/2.85 (s, 3H), 2.70 (m, 1H), 2.30 (m, 1H), 2.15 (m, 2H), 1.65 (m, 1H), 1.30 (s, 9H).",
"b.)",
"5 ml Hydrazine hydrate were added at 60° C. to 1.26 g nitro compound a and 15 mg FeCl3×6H2O in 20 ml CH3OH and for 1 h stirred at 60° C. The mixture was filtered over Celite and the filtrate evaporated.",
"1.17 g; ESI-MS [M+H+]: 452.17.c.)",
"The build-up of the respective aminobenzimidazole was carried out analogously to the preparation of 17 by reaction with thiocarbonyldiimidazole, imidazole and phenylene diamine, subsequently.",
"Chromatography on silica gel (CH2Cl2/CH3OH 4-7.5%) afforded 0.9 g of a slightly beige foam; ESI-MS [M+H+]: 568.25.d.)",
"Cleavage of the t-butylester with 50 ml TFA and subsequent purification by chromatography on silica gel (CH2Cl2/CH3OH 8-10%) afforded 0.88 g; ESI-MS [M+H+]: 512.25.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.05 (broad, 1H), 7.60-7.20 (m, 1.2H), 4.85 (m, 1H), 4.75-4.45 (m, 3H), 3.65 (m, 1H), 3.20 (s, 3H), 2.70 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).",
"Example 31 tert-Butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Coupling of [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) analogously to I and purification by chromatography on silica gel (CH2Cl2/CH3OH 2-3%) afforded 200 mg of a beige foam; ESI-MS [M+H+]: 540.25.Example 32 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) TFA-cleavage of tert-butyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate afforded 240 mg; ESI-MS [M+H+]: 484.36.1H-NMR (360 MHz, d6-DMSO) δ ppm (Rotamers): 11.10 (s, 1H), 8.30 (t, 1H), 7.5-7.25 (m, 8H), 7.15 (m, 2H), 6.85 (m, 2H), 4.40 (m, 2H), 3.75-3.55 (m, superimposed by H2O), 3.5 (m, 1H), 3.0 (m, 1H), 2.70 (m, 2H), 1.70 (m, 2H), 1.45 (m, 1H).",
"Example 33 tert-Butyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).",
"410 mg of a white foam; ESI-MS [M+H+]: 546.35.Example 34 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) Analogously to example 32; 420 mg of white solids; ESI-MS [M+H+]: 490.47.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.0 (d, 1H), 7.65 (m, 1H), 7.40 and 7.30 (each m, 2H), 7.15 and 6.85 (each m, 2H), 3.80 (m, 2H), 3.50 (m, 1H), 3.0 (m, 4H), 2.75 (m, 2H), 2.0 (m, 2H), 1.75-1.50 (m, 5H), 1.50-1.20 (m, 5H), 0.95 (m, 2H).",
"Example 35 tert-Butyl-[1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation analogously to example 31 starting from [5-(2-tert-butoxy-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (28) and N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8).",
"300 mg of a foam; ESI-MS [M+H+]: 520.39.Example 36 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butyl ester from example 35 with 15 ml 4nHCl in dioxane afforded 300 mg solid; ESI-MS [M+H+]: 464.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 9.10 (m, 1H), 7.70 (m, 1H), 7.35, 7.25, 7.10 and 6.85 (each m, 2H), 4.75 (m 2H), 3.30, 3.15, 2.95, 2.70 (each m, 2H), 1.65 (m, 5H), 1.45 and 1.30 (each m, 2H).",
"Example 37 tert-Butyl-{1-[2-({[4-(1H-imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate Preparation analogously to example I under utilization of N-[4-(aminomethyl)cyclohexyl]-1H-imidazole-2-amine (hydrobromide) (29) as educt; chromatography on silica gel (CH2Cl2/CH3OH 4-15%) afforded 530 mg of a solid foam; ESI-MS [M+H+]: 510.35.Example 38 {1-[2-({[4-(1H-Imidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Cleavage of the t-butyl ester from example 37 mit TFA and subsequent purification by means of chromatography on silica gel (CH2Cl2/CH3OH 10-15%+2% glacial acetic acid) afforded 570 mg; ESI-MS [M+H+]: 454.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.0 (broad, 1H), 7.30 and 7.25 (each m, 2H), 6.80 (broad, 1H), 6.70 (s, 2H), 4.40 and 4.20 (each broad, 1H), 4.75-4.25 (superimposed by H2O), 3.0 (m, 2H), 2.65 (m, 2H), 2.35 (m, 1H), 2.15 (m, 2H), 1.95 (m, 1H), 1.75 (m, 2H), 1.60 and 1.35 (each m, 1H), 1.20 (m, 4H), 0.95 (m, 2H).",
"Example 39 tert-Butyl-(2-oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate Standard coupling of 2-{[5-(2-tert-Butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]methyl}-1,3-thiazole-4-carbonic acid (32) (0.5 g; 1.2 mmol) with N-(piperidine-4-ylmethyl)pyridine-2-amine (hydrochloride; accessibly from 29) in 15 ml CH2Cl2 under utilization of HATU as a coupling reagent and ethyldiisopropylamine as a base.",
"Subsequent chromatography of the raw product on silica gel (CH2Cl2/CH3OH 3-20%) afforded 0.44 g of a slightly yellow oil; ESI-MS [M+H+]: 590.35.Example 40 (2-Oxo-1-{[4-({4-[(pyridine-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 31 with TFA afforded 385 mg; ESI-MS [M+H+]: 534.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.05 (s, 1H), 7.90 (m, 2H), 7.55 (m, 1H), 7.35 (m, 1H), 7.25 (m, 2H), 7.05 and 6.80 (each m, 1H), 5.40-5.20 (m, 2H), 4.20 and 4.0 (each m, 1H), 3.25 (m, 2H), 2.95 (m, 1H), 2.80-2.60 (m, 3H), 2.30 (m, 1H), 2.25-2.05 (m, 2H), 1.80 and 1.65 (each m, 2H), 1.20-1.10 (m, 3H).",
"Example 41 tert-Butyl-[1-({4-[({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino) carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetate Analogously to example 29 under utilization of trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11) as educt; chromatography of the raw product on silica gel (CH2Cl2/CH3OH 5-8%) afforded 350 mg of a bright yellow oil; ESI-MS [M+H+]: 643.45.Example 42 [1-({4-[({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)carbonyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example 31 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 300 mg; ESI-MS [M+H+]: 587.35.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (broad, 1H), 8.20 (m, 1H), 8.20 (s, 1H), 7.35-7.20 (m, 5H), 7.15 (m, 2H), 5.40 and 5.25 (each d, 1H), 3.15 (m, superimposed by H2O), 2.80-2.60 (m, 2H), 2.35 (m, 1H), 2.20 (m, 2H), 2.05 and 1.80 (each m, 2H), 1.65, 1.30 and 1.15 (each m, 2H).",
"Example 43 tert-Butyl-(1-{[4 ({4-[(1H-benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetate 430 mg were obtained analogously to example 29 under utilization of N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17) and subsequent purification; ESI-MS [M+H+]: 629.45.Example 44 (1-{[4-({4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}carbonyl)-1,3-thiazole-2-yl]methyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Cleavage of the t-butylester of example 43 and purification by elution over Chromabond-C18 (acetonitrile/H2O 10-80%+0.1% glacial acetic acid) afforded 340 mg; ESI-MS [M+H+]: 573.35.1H-NMR (360 MHz, d-DMSO) δ ppm: 9.20 (m, 1H), 7.95 (s, 1H), 7.45 (m, 1H), 7.40 (m, 2H), 7.30-7.15 (m, 5H), 5.35-5.10 (m, 2H), 4.45 (m, 1H), 4.0-3.25 (m, superimposed by H2O), 2.95 (m, 1H), 2.8-2.60 (m, 2H), 2.30 and 2.20 (each m, 1H), 2.0-1.75 (m, 3H), 1.70-1.50 (m, 2H), 1.20 (m, 3H).",
"Example 45 tert-Butyl-{2-oxo-1-[(4-{[({4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 330 mg were obtained analogously to example 39 under utilization of N-{[5-(aminomethyl)thiene-3-yl]methyl}pyridine-2-amine (trifluoro acetate) (18) and purification; ESI-MS [M+H+]: 618.25.Example 46 {2-Oxo-1-[(4-{([{4-[(pyridine-2-ylamino)methyl]thiene-2-yl}methyl)amino]carbonyl}-1,3-thiazole-2-yl)methyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Cleavage of the t-butylesters of example 45 and purification by means of MPLC afforded 150 mg; ESI-MS [M+H+]: 562.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.80 (m, 1H), 8.20 (m, 1H), 8.0 (d, 1H), 7.50 (d, 1H), 7.30-7.20 (m, 4H), 7.10 and 6.95 (each s, 1H), 6.85 (m, 1H), 6.50 (m, 2H), 5.40 and 5.20 (each d, 1H), 4.55 and 4.35 (each m, 2H), 2.80-2.55 (m, 2H), 2.30 (m, 1H), 2.20 (m, 2H), 1.65 (m, 1H).",
"Example 47 tert-Butyl-[1-({4-[4-(1H-benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate a.)",
"A mixture of 1.5 g tert-butyl-[1-(2-amino-2-thioxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate, 1.36 g 2-bromo-4-nitroacetophenone and 0.65 g KHCO3 in 30 ml dioxane were stirred for 12 h at RT.",
"Workup analogously to building block 30c and mixing of the raw product with n-pentane afforded 2.1 g of a brown solid; ESI-MS [M+H+]: 494.15.1H-NMR (360 MHz, d6-DMSO) δ ppm: 8.40 (s, 1H), 8.30 and 8.20 (each m, 2H), 7.60 and 7.35 (each m, 1H), 7.25 (m, 2H), 5.50 and 5.30 (each d, 1H), 2.70 (m, 2H), 2.30 (m, 1H), 2.20 (m, 3H), 1.65 (m, 1H), 1.25 (s, 9H).",
"b.)",
"Reduction of the nitro group analogously to example 30b (1.6 g; ESI-MS [M+H+]: 464.15) and build-up of the aminobenzimidazole as already described afforded after chromatography on silica gel (CH2Cl2/CH3OH 24%) 0.58 g of a slightly yellow foam; ESI-MS [M+H+]: 614.35.Example 48 [1-({4-[4-(1H-Benzimidazole-2-ylamino)phenyl]-1,3-thiazole-2-yl}methyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester of example XXXXVII and purification by means of MPLC afforded 40 mg; ESI-MS [M+H+]: 524.25.1H-NMR (360 MHz, d6-DMSO) δ ppm: 11.95 and 9.60 (each broad, 1H), 7.85-7.75 (m, 4H), 7.65 (m, 1H), 7.40-7.25 (m, 5H), 7.0 (m, 2H), 5.40 and 5.30 (each d, 1H), 3.50 (m, 1H), 2.75 (m, 1H), 2.45 (m, 2H), 2.20 (m, 2H), 1.70 (m, 1H).",
"Example 49 tert-Butyl (1-{2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Ethyldiisopropylamine (0.42 mmol, 54.50 mg) and HATU (0.50 mmol, 190.11 mg) were added at 0° C. to a solution of [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.42 mmol, 140.59 mg) in CH2Cl2 (10 ml), the mixture was stirred for 1 h at 0° C. and [4-(aminomethyl)phenyl]guanidine (bihydrochloride) (46) (0.42 mmol, 100 mg), ethyldiisopropylamine (0.63 mmol, 81.76 mg) added.",
"The mixture was stirred for 1 h at 0° C. and overnight at RT.",
"After evaporation, the residue was taken up in CH2Cl2/water, washed with watery NaHCO3—, 5%-citric acid- and finally with watery saturated NaCl-solution.",
"Evaporation and chromatography on silica gel (CH2Cl2/CH3OH 0-100%) afforded 6.0 mg target product; ESI-MS [M+H+]: 426.1, 425.1.Example 50 {4-[({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]anilino}(imino)methaneamine (trifluoro acetate) tert-Butyl-({2-[(4{[amino(imino)methyl]amino}benzyl)amino]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (example 49, 0.01 mmol, 6 mg) was dissolved in 5 ml CH2Cl2, TFA (1.25 mmol, 142.53 mg) added at 0° C. and stirred overnight at room temperature.",
"After evaporation, the residue was taken up in CHCl3/water and the watery phase washed with CHCl3; evaporation of the watery phase affords 4.8 mg target product; ESI-MS [M+H+]=425.1.Example 51 tert-Butyl [1-(2-{[(6-amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49, under utilization of 2-ammonio-6-(ammonio methyl)pyridinium trichloride (33) (0.30 mmol, 69.76 mg) and following purification and subsequent purification (chromatography; CH2Cl2/MeOH 0-100%) afforded 120 mg of the target product; ESI-MS: [M+H+]=439.28, 383.36.Example 52 [1-(2-{([(6-Amino-2-pyridinyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (hydrochloride) Cleavage of the t-butylester, chromatography of the raw product (CHCl3/MeOH 0-5%) and ester interchange with HCl in diethyl ether afforded 15.0 mg; the target product as hydrochloride; ESI-MS [M+H+]=383.1.Example 53 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 2 Drops of a solution of HCl in diethylether were added to N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (0.63 mmol, 196.10 mg) in methanol (40 mL) and stirred for 5 min.",
"at RT.",
"tert-Butyl.",
"[2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) was added, NaBH3CN (3 mmol, 188.5 mg) portionwise added after 10 min.",
"and the mixture stirred for 17 h at RT.",
"After evaporation, the residue was taken up in ethyl acetate and washed with NaHCO3— (pH 8-9), saturated NaCl— (1×) and a 10% FeSO4-solution (2×).",
"Chromatography (CHCl3/MeOH 0-5%) afforded 84 mg of the target product; ESI-MS [M+H+]=540.24, 270.75.Example 54 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}ethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Cleavage of the t-butylester and chromatography (CHCl3/MeOH 0-100%) afforded 4 mg of the target product; ESI-MS [M+H+]=484.1.Example 55 tert-Butyl [1-(2-{[(4-{[(benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogously to example 49 under utilization of N-[4-(aminomethyl)cyclohexyl]-N′-benzyl urea (34) (0.50 mmol, 130 mg) afforded 320 mg; ESI-MS [M+H+]=577.11.Example 56 [1-(2-{[(4-{[(Benzylamino)carbonyl]amino}cyclohexyl)methyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Analogous cleavage of the t-butylester afforded a raw product which after evaporation was distributed between ethyl acetate/water.",
"The watery phase was then adjusted to pH 10 with 0.1n NaOH and extracted with ethyl acetate.",
"It was adjusted then with 1nN HCl to pH4, extracted with CH2Cl2, washed and evaporated; 80 mg; ESI-MS [M+H+]=521.3.Example 57 tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 53 under utilization of N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (0.63 mmol, 183.47 mg) and tert-butyl [2-oxo-1-(2-oxoethyl)-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (37, 0.63 mmol, 0.20 g) afforded 50.0 mg of the target product; ESI-MS [M+H+]=520.41, 260.79.Example 58 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) tert-Butyl [1-(2-{[5-(1H-benzimidazole-2-ylamino)pentyl]amino}ethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (example 57, 0.04 mmol, 20.00 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.",
"Evaporation of the reaction mixture, chromatography (CHCl3/MeOH 0-100%) afforded 10 mg of the target product; ESI-MS [M+H+]=464.25.Example 59 tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 53 under utilization of ethyldiisopropylamine and EDCI*HCl starting from trans-N-[[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (0.63 mmol, 0.209) afforded 84 mg; ESI-MS [M+H+]=546.32, 273.79.Example 60 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)ethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (trifluoro acetate) tert-Butyl {2-oxo-1-[2-({[4-({4-[(1Z)-1-propenyl]-5-vinyl-1H-imidazole-2-yl}amino)cyclohexyl]methyl}amino)ethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (example 59, 0.01 mmol, 8 mg) was dissolved in 5 ml CH2Cl2, TFA (65.34 mmol, 7.45 g) added at 0° C. and stirred overnight at room temperature.",
"The reaction mixture was evaporated, the watery phase washed with a 3:1 mixture CHCl3/EtOH.",
"Evaporation of the watery phase afforded 6.0 mg of the target product; ESI-MS [M+H+]=490.25.Example 61 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) (1.70 mmol, 467.08 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).",
"TFA (1.73 mmol, 197.72 mg) was added at 0° C. to the obtained raw product and it was stirred overnight at room temperature.",
"After evaporation, the residue was distributed between ethyl acetate and water, the watery phase adjusted to pH 10 with 2n NaOH and extracted with ethyl acetate; purification by means of MPLC afforded 90 mg of the target product as TFA-salt; ESI-MS [M+K+]=570.2, 534.2, 532.2, 266.5, 101.1.Example 62 [1-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) (1.7 mmol, 433.10 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600.00 mg).",
"Treatment of the raw product with TFA analogously to example 61 afforded 48 mg as a TFA-salt; ESI-MS: 514.2, 512.2, 101.2.Example 63 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)butyl]amino}-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Coupling analogously to example 49 starting from N1-(1H-benzimidazole-2-yl)butane-1,4-diamine (trifluoro acetate) (9) (1.7 mmol, 541.1 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).",
"Treatment of the raw product with TFA analogously to example 61 afforded 15 mg as a TFA-salt; ESI-MS: 536.2, 500.1, 498.2, 105.1, 101.2.Example 64 [7-Chloro-1-(2-{[5-(4,5-dihydro-1H-imidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (trifluoro acetate) Coupling analogously to example 49 starting from N1-(4,5-dihydro-1H-imidazole-2-yl)pentane-1,5-diamine (hydrochloride) (23) (1.70 mmol, 351.43 mg) and [5-(2-tert-butoxy-2-oxoethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (1.63 mmol, 600 mg).",
"Treatment of the raw product with TFA analogously to example 61 afforded 85 mg as a TFA-salt; ESI-MS: 464.15.Example 65 tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate Ethyldiisopropylamine (2.08 mmol, 268.41 mg) and EDCI*HCl (0.78 mmol, 150.44 mg) were added at 0° C. to a solution of [5-(2-t.butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) (0.46 mmol, 0.15 g) in CH3CN (45 ml).",
"After 1 h at 0° C., 7-(4-aminobutyl)-1,2,3,4-tetrahydro[1,8]naphthyridine (bitrifluoro acetate) (35) (0.46 mmol, 0.2 g) was added, stirred for 1 h at 0° C. and overnight at RT.",
"Since by chromatography a purification could not be achieved, the obtained was reacted to the corresponding Boc-derivative according to standard methods.",
"Chromatography (heptane/CH2Cl2 0-100%, CH2Cl2/MeOH 0-100%) afforded 15 mg of the target product (about 95% purity); ESI-MS [M+Na+]: 643.5, 622.5, [M+H+]=621.5.Example 66 7-[4-({[5-(Carboxymethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-1,2,3,4-tetrahydro[1,8]naphthyridine (trifluoro acetate) tert-Butyl 7-[4-({[5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)butyl]-3,4-dihydro[1,8]naphthyridine-1(2H)-carboxylate (example 65; 0.02 mmol, 15 mg) was dissolved in 2 ml CH2Cl2, TFA (0.53 mmol, 60.7 mg) added at 0° C. and stirred overnight at RT.",
"Usual workup afforded 9.3 mg of the target product as TFA-salt; ESI-MS: 466.2, [M+H+]=465.2, 233.3.Example 67 N-{4-[({[5-(Carboxymethyl)-7-chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetyl}amino)methyl]cyclohexyl})-1H-benzimidazole (acetate) Coupling analogously to example 49 starting from trans-N-[4-(aminomethyl)cyclohexyl]-1H-benzimidazole-2-amine (dihydrochloride) (11) (2.99 mmol, 948.78 mg) and [5-(2-tert-butoxy-2-oxoethyl) 7 chloro-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (45) (2.72 mmol, 1.00 g) afforded 760 mg raw product.",
"Treatment of the raw product with TFA analogously to example 61 and purification of the raw product by means of MPLC afforded 400 mg; ESI-MS: [M+H+]=540.3, 538.25, 269.6, 101.1.Example 68 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 15 ml HCl in diethyl ether (saturated at 0° C.) were added to 300 mg {1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2,3,4,5-tetrahydro-1H-1-1-benzazepine-5-yl}acetic acid in 30 ml ethyl alcohol and allowed to stand for 3 days at room temperature.",
"The mixture was evaporated, the remaining residue purified by chromatography on silica gel (CH2Cl2/CH3OH 5%+1% glacial acetic acid) and lyophilized.",
"230 mg; ESI-MS: [M+H+]=518.35.Example 69 {1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid (acetate) Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (1 g; 2.54 mmol) with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); after chromatography on silica gel (CH2Cl2/CH3OH 5-7%), 1.03 g were isolated as a white glass; ESI-MS: [M+H+]=620.35.Cleavage of the ester under utilization of TFA and subsequent purification of the raw product by chromatography on silica gel (CH2Cl2/CH3OH 10%+2% glacial acetic acid) afforded 0.8 g of the target product; ESI-MS: [M+H+]=564.25.Example 70 [1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Standard coupling of [5-(2-tert-butoxy-2-oxoethyl)-7,8-dimethoxy-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetate (building block 49) (0.5 g; 1.27 mmol) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); after chromatography on silica gel (CH2Cl2/CH3OH 3-5%) 0.67 g were isolated as a foam; ESI-MS: [M+H+]=614.35.Cleavage of the ester under utilization of TFA and lyophilization of the obtained product afforded 0.61 g; ESI-MS: [M+H+]=558.35.Example 80 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl}acetic acid 0.4 g (1.4 mmol) [1-(2-Methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.37 g (1.4 mmol) [4-(1H-benzimidazole-2-yl)cyclohexyl]methane amine (hydrochloride) (51) were reacted analogously to example I, the reaction product then purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.",
"NH3 45/5/0.2) and then the methyl ester cleaved with 0.8 ml 1n NaOH in 8 ml dioxane at room temperature.",
"After neutralization with 1n HCl, evaporation of the solvent, extraction with ethyl acetate and drying with Na2SO4, the raw product was purified by chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1).",
"After lyophilization, 0.22 g remained as a white amorphous residue; ESI-MS: [M+H+]=487.Example 81 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) and 0.36 g (1.4 mmol) N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8) were reacted.",
"Purification of the raw product, alkaline saponification of the methyl ester and purification of the end stage was conducted analogously to example 80.0.3 g of a white amorphous residue; ESI-MS: [M+H+]=476.Example 82 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1H-benzazepine-1-yl]acetic acid Analogously to example I 0.49 (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) was reacted with 0.63 g (1.4 mmol) N-(piperidine-4-ylmethyl)-1H-benzimidazole-2-amine (trifluoro acetate) (17).",
"After purification, alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.3 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=488.Example 83 [5-(2-{4-[(1H-Benzimidazole-2-ylamino)methyl]piperidine-1-yl}-2-oxoethyl)-2-oxo 2,3-dihydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3-dihydro-1H-1-benzazepine-5-yl]acetic acid (50) were reacted with 0.43 g trans-N-J[4-(aminomethyl)cyclohexyl]1H-benzimidazole-2-amine (dihydrochloride) (11).",
"After purification of the ester (eluent: CH2Cl2/CH3OH/25% NH3 45/5/0.2), alkaline ester hydrolysis and chromatographic purification of the end product (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) 0.18 g of a white amorphous powder were obtained; ESI-MS: [M+H+]=502.Example 84 [5-(2-{[5-H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Analogously to example I 0.65 g (2 mmol) [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (54) were reacted with N1-(1H-benzimidazole-2-yl)pentane-1,5-diamine (hydrochloride) (8), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.",
"NH3 45/5/0.2); ESI-MS: [M+H+]=535.Then the tert-butyl ester was cleaved with 4n HCl in dioxane, and after chromatographic purification 40 mg of a white amorphous powder were isolated; ESI-MS: [M+H+]=479.Example 85 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11); ester stage ESI-MS: [M+H+]=561.Purification after ester cleavage was conducted by means of preparative thick-layer chromatography (CH2Cl2/CH3OH/50% glacial acetic acid 12/3/1); 0.35 g of a white amorphous powder; ESI-MS: [M+H+]=505.Example 86 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (hydrochloride) Preparation and purification was conducted analogously to example 84 by reaction with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4); ester stage ESI-MS: [M+H+]=555; target product: 0.4 g of a white amorphous powder; ESI-MS: [M+H+]=499.Example 87 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid Analogously to example I 1.2 g (3.6 mmol) [5-(2-tert-butoxy-2-oxoethyl)-4-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl]acetic acid (55) was reacted with 0.86 g (3.6 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4), the reaction product purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.",
"NH3 45/5/0.1); ESI-MS: [M+H+]=: 555.Subsequent cleavage of the tert-butylester with 4n HCl in dioxane afforded 0.8 g of a white amorphous powder; ESI-MS [M+H+]=499.Example 88 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-yl}acetic acid Preparation and purification analogously to example 87; ester stage ESI-MS: [M+H+]=561; 0.9 g of the target product were obtained as a white amorphous powder; ESI-MS [M+H+]=505.Example 89 (1-{2-[4-(1H-Benzimidazole-2-yl)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (acetate) Analogously to example I 0.5 g (1.5 mmol) [5-(2-t-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-benzazepine-1-yl]acetic acid (3) and 0.36 g (1.5 mmol) 2-(4-piperidinyl)-1H-benzimidazole (J. Heterocycl.",
"Chem.",
"1989, 26, 541) were reacted, purified by means of preparative thick-layer chromatography (eluent CH2Cl2/CH3OH/conc.",
"NH3 45/5/0.2); ESI-MS: [M+H+]=517.Subsequent cleavage of the tert-butyl ester with 4n HCl in a mixture of dioxane/glacial acetic acid and chromatographic purification afforded 0.259 of a white amorphous powder; ESI-MS [M+H+]=: 461.Example 90 (1-{2-[[3-(1H-Benzimidazole-2-yl)propyl](methyl)amino]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid (hydrochloride) Preparation analogously to example I by reaction with N-[3-(1H-benzimidazole-2-yl)propyl]-N-methylamine (WO 9849148); after chromatographic purification 0.69 were obtained as a white amorphous powder; ESI-MS [M+H+]=: 449.Example 91 (1-{2-[4-(1H-Benzimidazole-2-ylamino)piperidine-1-yl]-2-oxoethyl}-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-5-yl)acetic acid Preparation analogously to example I by reaction with N-piperidine-4-yl-1H-benzimidazole-2-amine (J. Med.",
"Chem.",
"1985, 28, 1925); after chromatographic purification 0.45 g were obtained as a white amorphous powder; ESI-MS [M+H+]=: 476.Example 92 [5-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Analogously to example I 0.4 g (1.4 mmol) [1-(2-methoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (56) and 0.33 g (1.4 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted and purified by means of preparative thick-layer chromatography (eluent: CH2Cl2/CH3OH/conc.",
"NH3 45/5/0.2); ESI-MS: [M+H+]=512.Subsequent cleavage of the methyl ester with 1n NaOH in dioxane and chromatographic purification of the residue (CH2Cl2/CH3OH/50% glacial acetic acid 20/5/1) afforded 0.15 g, isolated as a white amorphous powder; ESI-MS [M+H+]=498.Example 93 {5-[2-({[4-(1H-Benzimidazole-2-yl)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=503; chromatographic purification after cleavage of the ester afforded 0.3 g of the target product as a white amorphous powder; ESI-MS [M+H+]=489.Example 94 [5-(2-{[5-(1H-Benzimidazole-2-ylamino)pentyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-1-yl]acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=492.Chromatographic purification after cleavage of the ester afforded 20 mg as a white amorphous powder ESI-MS [M+H+]=478.Example 95 {5-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl}acetic acid Preparation and purification analogously to example 92; ester stage ESI-MS [M+H+]=518.Chromatographic purification after cleavage of the ester afforded 0.15 g as a white amorphous powder; ESI-MS [M+H+]=504.Example 96 Ethyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.6 g (2.4 mmol) N,N-Bis(2-oxo-3-oxazolidinyl)phosphoryl chloride were added to a solution of 0.8 g (1.6 mmol) of the acid of example XI, 0.15 g (3.2 mmol) ethyl alcohol, 0.2 g (1.6 mmol) 4-dimethylaminopyridine and 0.4 g (4 mmol) triethylamine in 60 ml CH2Cl2 and stirred overnight.",
"After the reaction had completed 2 g glacial acetic acid and 3.7 g ethyl alcohol were added to the reaction solution and stirred for another 20 h at room temperature.",
"For workup, 50 ml H2O were added to the mixture, the organic phase washed with 10% K2CO3-solution and H2O, dried over MgSO4 and evaporated.",
"Chromatographic purification of the residue (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 15/5/1) afforded 0.35 g of a white amorphous powder; ESI-MS [M+H+]=532.Analogously to example 96 were prepared: Example 97 Cyclohexyl-{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.26 g; ESI-MS [M+H+]=586.Example 98 Neopentyl{1-[2-({[4-(1H-benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetate 0.21 g; ESI-MS [M+H+]=574.Example 99 tert-Butyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate Preparation was carried out analogously to example I by reaction of [5-(2-tert-butoxy-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-1-yl]acetic acid (3) with N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4).",
"After chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% glacial acetic acid 45/5/0.3) the residue was dissolved in 70 ml CH2Cl2 and 5 ml CH3OH, washed with 5% K2CO3-solution and H2O, dried over Na2SO4 and evaporated to dryness.",
"The amorphous residue was mixed with 25 ml CH3OH in heat.",
"0.51 g of white crystals; Fp.",
": 231° C. (decomposition); ESI-MS [M+H+]=554.Example 100 Cyclohexyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.64 g (1.28 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml cyclohexanol, 1.0 g HCl-gas introduced with stirring and the resulting yellow solution allowed to stand for 4 days at room temperature.",
"Since an acid was still detectable by chromatography, it was heated for 7 h to 40-50° C. For workup, 100 ml diethylether were added, washed with K2CO3-solution and H2O, dried over Na2SO4 and the solvent—finally for removal of cyclohexanol under oil pump vacuum and at a bath temperature of 50° C.—distilled off.",
"The residue was purified chromatographically (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45151410.3).",
"0.659 of a slightly beige coloured powder; ESI-MS [M+H+]=580.Example 101 Ethyl-[1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,6-tetrahydro-1H-1-benzazepine-6-yl]acetate 0.619 (1.23 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (acid of example III) was suspended in 30 ml ethyl alcohol, 0.4 g HCl-gas introduced and allowed to stand for 4 days at room temperature.",
"The ethyl alcohol was distilled off in vacuum, the residue taken up in ethyl acetate, washed with 5% NaHCO3- and NaCl-solution, dried over Na2SO4 and evaporated.",
"After thick-layer chromatographic purification (eluent: CH2Cl2/ethyl alcohol/50% acetic acid 43/7/0.5) the eluate was diluted with some CH2Cl2 and for removal of the acetic acid washed with 50% NaHCO3-solution.",
"After drying over Na2SO4 it was evaporated and the residue converted to an amorphous powder filterable with suction by means of diethylether/n-hexane; 0.55 g; ESI-MS [M+H+]=526.Example 102 1-{[(Cyclohexyloxy)carbonyl]oxy}ethyl [1-(2-{[4-(1H-benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetate 0.3 g K2CO3 were added at 3° C. to a solution of 0.6 g (1.2 mmol) of [1-(2-{[4-(1H-benzimidazole-2ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid (add of example III) in 10 ml DMF with stirring, some crystals 18-crown-6, 0.4 g cyclohexyl-1-iodo ethylcarbonate (preparation from cyclohexyl-1-chloroethylcarbonate and NaI analogously to J. Antibiot.",
"1987, 40 (1), 81-90) dissolved in 5 ml CH3CN were added dropwise and stirred for 20 min.",
"After addition of 100 ml cold NaCl-solution it was extracted several times with ethyl acetate, the combined organic phases washed with NaCl-solution, dried over Na2SO4 and evaporated.",
"The residue was purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/5/0.3), after evaporation of the solvent taken up in 50 ml CH2Cl2, washed with 5% NaHCO3-solution, dried over Na2SO4 and again evaporated.",
"90 mg of a white amorphous powder; ESI-MS [M+H+]=668.Example 103 [(5R)-1-(2-{[4-(1H-Benzimidazol-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid 1.0 g (3.0 mmol) of the left-rotating acid (building block 57) and 0.72 g (3.0 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) were reacted analogously to example I and the reaction product purified by column chromatography (eluent: CH2Cl2/acetone/methanol/50% acetic acid 45/5/4/0.3); ester stage ESI-MS [M+H+]=554.Cleavage of the tert-butyl ester with 4n HCl in dioxane afforded 0.82 g of a white amorphous powder; ESI-MS [M+H+]=498; [α]D20=−107.7° (K+-salt, c=1 in H2O).",
"Example 104 [(5S)-1-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 1.5 g of a amorphous powder; ESI-MS [M+H+]=554.Target product: 0.79 g of a white amorphous powder; ESI-MS [M+H+]=498.Example 105 {(5R)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example I from the left-rotating acid building block 57 and trans-N-{[4-(aminomethyl)cyclohexyl]}-1H-benzimidazole-2-amine (dihydrochloride) (11).",
"Ester stage: 0.9 g of a white amorphous powder; ESI-MS [M+H+]=560.Target product: 0.67 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=−104° (K+-salt, c=1 in H2O).",
"Example 106 {(SS)-1-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl}acetic acid Preparation analogously to example 103 starting from the dextrorotatory acid building block 58.Ester stage: 0.72 g of a amorphous powder; ESI-MS [M+H+]=560.Target product: 0.56 g of a white amorphous powder; ESI-MS [M+H+]=504; [α]D20=+101.60 (K+-salt, c=1 in H2O).",
"Example 107 [4-(2-{[4-(1H-Benzimidazole-2-ylamino)benzyl]amino}-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl]acetic acid Analogously to example I 0.6 g (1.8 mmol) [8-(2-tert-butoxy-2-oxoethyl)-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-4-yl]acetic acid (61) were reacted with 0.42 g (1.8 mmol) N-[4-(aminomethyl)phenyl]-1H-benzimidazole-2-amine (hydrochloride) (4) and the reaction product purified by thick-layer chromatography (eluent: CH2Cl2/methanol/conc.",
"NH3 451510.2); ESI-MS [M+H+]=560.Cleavage of the t-butyl group with 4n HCl in dioxane afforded 0.34 g of a slightly yellowish powder; ESI-MS [M+H+]=504.Example 108 {4-[2-({[4-(1H-Benzimidazole-2-ylamino)cyclohexyl]methyl}amino)-2-oxoethyl]-5-oxo-5,6,7,8-tetrahydro-4H-thieno[3,2-b]azepine-8-yl}acetic acid Analogously to example 107.Ester stage: 100 mg of a white amorphous powder, ESI-MS [M+H+]=566.Target product: 98 mg of a white amorphous powder; ESI-MS [M+H+]=510.II.",
"BIOLOGICAL EXAMPLES Example 1 Integrin αvβ3 Assay For the identification and assessment of integrin αvβ3 ligands, a test system was used which was based on competition between the natural integrin αvβ3 ligand vitronectin and the test substance for binding to solid phase-bound integrin αvβ3.Procedure Microtiter plates coated with 250 ng/ml of integrin αvβ3 in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturation with 1% powdered milk/assay buffer; 0.3 ml/well; 0.5 h/RT 3× washing with 0.05% Tween 20/assay buffer test substance in 0.1% powdered milk/assay buffer, 50 μl/well+0 μg/ml or 2 μg/ml of human vitronectin (Boehringer Ingelheim T007) in 0.1% powdered milk/assay buffer, 50 μl/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 1 μg/ml of anti human vitronectin antibody coupled to peroxidase (Kordia SAVN-APHRP) in 0.1% powdered milk/assay buffer; 0.1 ml/well; 1 h/RT 3× washing with 0.05% Tween 20/assay buffer 0.1 ml/well of peroxidase substrate stop reaction with 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Integrin αvβ3: Human placenta is solubilized with Nonidet and integrin αvβ3 affinity-purified on a GRGDSPK matrix (elution with EDTA).",
"Impurities due to integrin αIIbβ3 and human serum albumin, and the detergent and EDTA are removed by anion-exchange chromatography.",
"Assay buffer: 50 mM Tris pH 7.5; 100 mM NaCl; 1 mM CaCl2; 1 mM MgCl2; 10 μM MnCl2 Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M sodium acetate pH 4.9), then add 14.7 μl of 3% H2O2.Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the ligand at which 50% of the ligand is displaced).",
"The compound from Example VII showed the best result here.",
"Example 2 Integrin αIIbβ3 Assay The assay is based on competition between the natural integrin αIIbβ3 ligand fibrinogen and the test substance for binding to integrin αIIbβ3.Procedure Coat microtiter plates with 10 μg/ml of fibrinogen (Calbiochem 341578) in 0.05 M NaHCO3 pH 9.2; 0.1 ml/well; saturate with 1% BSA/PBS; 0.3 ml/well; 30 min/RT 3× washing with 0.05% Tween 20/PBS test substance in 0.1% BSA/PBS; 50 μl/well+200 μg/ml of integrin αIIbβ3 (Kordia) in 0.1% BSA/PBS; 50 μl/well; 2 to 4 h/RT 3× washing as above biotinylated anti-integrin αIIbβ3 antibody (Dianova CBL 130 B); 1:1000 in 0.1% BSA/PBS; 0.1 ml/well; 2 to 4 h/RT.",
"3× washing as above streptavidin-peroxidase complex (B.M.",
"1089153) 1:10000 in 0.1% BSA/PBS; 0.1 ml/well; 30 min/RT 3× washing as above 0.1 ml/well of peroxidase substrate stop reaction using 0.1 ml/well of 2 M H2SO4 measurement of the absorption at 450 nm Peroxidase substrate: mix 0.1 ml of TMB solution (42 mM TMB in DMSO) and 10 ml of substrate buffer (0.1 M Na acetate pH 4.9), then add 14.7 μl of 3% H2O2 Various dilutions of the test substances are employed in the assay and the IC50 values are determined (concentration of the antagonist at which 50% of the ligand is displaced).",
"By comparison of the IC50 values in the integrin αIIbβ3 and integrin αvb3 assay, the selectivity of the substances can be determined.",
"Example 3 CAM Assay The CAM (chorioallantoic membrane) assay serves as a generally recognized model for the assessment of the in vivo activity of integrin αvβ3 antagonists.",
"It is based on the inhibition of angiogenesis and neovascularization of tumor tissue (Am.",
"J. Pathol.",
"1975, 79, 597-618; Cancer Res.",
"1980, 40, 2300-2309; Nature 1987, 329, 630).",
"The procedure is carried out analogously to the prior art.",
"The growth of the chicken embryo blood vessels and of the transplanted tumor tissue can be readily monitored and assessed.",
"Example 4 Rabbit Eye Assay In this in vivo model, the inhibition of angiogenesis and neovascularization in the presence of integrin αvβ3 antagonists can be monitored and assessed analogously to Example 3.The model is generally recognized and is based on the growth of rabbit blood vessels starting from the edge in the corner of the eye (Proc.",
"Natl.",
"Acad.",
"Sci.",
"USA.",
"1994, 91, 4082-4085; Science 1976, 193, 70-72).",
"The procedure is carried out analogously to the prior art."
]
] | Patent_10297202 |
[
[
"Method, apparatus and system for building a compact language model for large vocabulary continous speech recognition (lvcsr) system",
"According to one aspect of the invention, a method is provided in which a set of probabilistic attributes in an N-gram language model is classified into a plurality of classes.",
"Each resultant class is clustered into a plurality of segments to build a code-book for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process.",
"A probabilistic attribute in each class is then represented by the centroid of the corresponding segment to which the respective probabilistic attribute belongs."
],
[
"1.A method comprising: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.",
"2.The method of claim 1 wherein a probabilistic attribute is represented by a first number of data bits before clustering.",
"3.The method of claim 2 wherein the respective probabilistic attribute is represented by an index value having a second number of data bits after clustering, the second number being smaller than the first number, the respective index value being used to reference a corresponding codeword in the respective codebook.",
"4.The method of claim 1 wherein a probabilistic attribute is a probability value associated with an n-gram node in the N-gram language model.",
"5.The method of claim 1 wherein a probabilistic attribute is a back-off weight associated with an n-gram node in the N-gram language model.",
"6.The method of claim 1 wherein the N-gram language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.",
"7.The method of claim 1 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.",
"8.The method of claim 7 wherein computing the total deviation includes: computing a deviation of each segment based upon the corresponding centroid of the respective segment; and summing the deviations of all segments to generate the total deviation.",
"9.The method of claim 7 wherein performing the set of operations includes: determining, for each segment in the plurality of segments, whether the new edge value equals the previous edge value; and indicating that the one or more optimization criteria are met if the new edge value equals the previous edge value.",
"10.The method of claim 7 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more optimization criteria are met if the predetermined number of iterations is reached.",
"11.The method of claim 7 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more optimization criteria are met if the changing rate in the total deviation is less than the predetermined threshold.",
"12.A method comprising: clustering a set of probabilistic values associated with each data class of a plurality of data classes of an N-gram language model into a plurality of clusters using a modified K-means clustering process which dynamically adjusts the size and centroid of each respective cluster during each iteration in the modified K-means clustering process until one or more predetermined conditions are satisfied, the clustering including: initializing the plurality of clusters such that all clusters have equivalent areas; setting the centroid of each resultant cluster such that the area of each respective cluster is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more predetermined conditions are satisfied, including: computing a total deviation of the clusters; replacing the edge of each cluster by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.",
"13.The method of claim 12 wherein performing the set of operations includes: determining, for each cluster in the plurality of clusters, whether the new edge value equals the previous edge value; and indicating that the one or more predetermined conditions are satisfied if the new edge value equals the previous edge value.",
"14.The method of claim 12 wherein performing the set of operations includes: determining whether a predetermined number of iterations is reached; and indicating that the one or more predetermined conditions are satisfied if the predetermined number of iterations is reached.",
"15.The method of claim 12 wherein performing the set of operations includes: determining whether a changing rate in the total deviation is less than a predetermined threshold; and indicating that the one or more predetermined conditions are satisfied if the changing rate in the total deviation is less than the predetermined threshold.",
"16.An apparatus comprising: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in a language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria.",
"17.The apparatus of claim 16 wherein the language model is a tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding tp a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.",
"18.The apparatus of claim 16 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.",
"19.The apparatus of claim 18 wherein logic to compute the total deviation includes: logic to compute a deviation of each cluster based upon the corresponding centroid of the respective cluster; and logic to sum the deviations of all clusters to generate the total deviation.",
"20.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value.",
"21.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a predetermined number of iterations is reached.",
"22.The apparatus of claim 18 wherein logic to perform the set of operations includes: logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.",
"23.A system comprising: a feature extraction unit to convert an input signal representing an input speech into a set of feature vectors each representing a corresponding frame of the input signal; an acoustic model comprising a set of phonetic statistical models each representing a distinct phonetic unit; a language model including: a plurality of codebooks each being built based upon a corresponding class of probabilistic values that represent conditional probabilities of a set of words in the language model, each codebook including a plurality of codewords each representing one or more corresponding probabilistic values, wherein each codebook of the plurality of codebooks is built using a modified K-means clustering process which dynamically adjusts the size and the centroid of each cluster during each iteration in the modified K-means clustering process until a total deviation of the clusters is optimized based upon one or more optimization criteria; and a decoder coupled to the feature extraction unit, the acoustic model, and the language model, the decoder to recognize the input speech based, at least in part, upon the feature vectors, the acoustic model, and the language model.",
"24.The system of claim 23 wherein the language model is an tri-gram language model and wherein the plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigram back-off weights, and a fifth class corresponding to a set of trigram probability values.",
"25.The system of claim 23 wherein each codebook for the corresponding class of probabilistic values is built by the modified K-means clustering process which includes: logic to initialize a plurality of clusters such that all clusters have equivalent areas; logic to set the centroid of each cluster such that the area of the respective cluster is approximately equally split based on the corresponding centroid; and logic to perform a set of operations iteratively until one or more optimization criteria are met, including: logic to compute a total deviation of the plurality of clusters; logic to replace the edge of each cluster by an arithmetic average of the nearest two centroids; and logic to set a new centroid for each resultant cluster.",
"26.The system of claim 25 wherein logic to perform the set of operations includes: logic to determine, for each cluster, whether the new edge value equals the previous edge value; logic to determine whether a predetermined number of iterations is reached; and logic to determine whether a changing rate in the total deviation is less than a predetermined threshold.",
"27.A machine-readable medium comprising instructions which, when executed by a machine, cause the machine to perform operations including: classifying a set of probabilistic attributes in an N-gram language model into a plurality of classes; clustering each resultant class into a plurality of segments to build a codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and centroid of each segment during each iteration in the modified K-means clustering process; and representing a probabilistic attribute in each class by the centroid of the corresponding segment to which the respective probabilistic attribute belongs.",
"28.The machine-readable medium of claim 27 wherein the N-gram language model is a tri-gram language model and wherein thy plurality of classes includes a first class corresponding to a set of unigram probability values, a second class corresponding to a set of unigram back-off weights, a third class corresponding to a set of bigram probability values, a fourth class corresponding to a set of bigrarn back-off weights, and a fifth class corresponding to a set of trigram probability values.",
"29.The machine-readable medium of claim 27 wherein clustering each resultant class includes: initializing the plurality of segments such that all segments have equivalent areas; setting the centroid of each segment such that the area of the respective segment is approximately equally split based on the corresponding centroid; and performing a set of operations iteratively until one or more optimization criteria are met, including: computing a total deviation of the plurality of segments; replacing the edge of each segment by an arithmetic average of the nearest two centroids; and setting a new centroid for each resultant segment.",
"30.The machine-readable medium of claim 29 wherein performing the set of operations includes: determining, for each cluster, whether the new edge value equals the previous edge value; determining whether a predetermined number of iterations is reached; and determining whether a changing rate in the total deviation is less than a predetermined threshold."
],
[
"<SOH> BACKGROUND OF THE INVENTION <EOH>Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.",
"In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.",
"In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).",
"In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.",
"In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.",
"The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.",
"Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.",
"N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).",
"In a typical LVCSR system, the size of the language model is usually very large.",
"For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.",
"Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.",
"One solution is to use a memory-map file format to access language model files in LVCSR system.",
"Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.",
"In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.",
"In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed."
],
[
"<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.",
"1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.",
"2 is a diagram illustrating an HMM-based phone model; FIG.",
"3 is a diagram illustrating an N-gram language model; FIG.",
"4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.",
"5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.",
"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.",
"7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.",
"8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.",
"9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.",
"detailed-description description=\"Detailed Description\" end=\"lead\"?"
],
[
"FIELD OF THE INVENTION The present invention relates to the field of speech recognition.",
"More specifically, the present invention relates to a method, apparatus, and system for building a compact language model for a large vocabulary continuous speech recognition (LVCSR) system.",
"BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words) to determine the most probable sentence or word sequence given the input sequence of observations.",
"In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.",
"In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).",
"In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMBs.",
"In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.",
"The purpose of a language model is to provide a mechanism for estimating the probability of a word in an utterance given the preceding words.",
"Most modern LVCSR systems typically employ some forms of N-gram language model which assumes that the probability of a word depends only on the preceding (N-1) words.",
"N is usually limited to 2 (for a bi-gram model) or 3 for a tri-gram model).",
"In a typical LVCSR system, the size of the language model is usually very large.",
"For example, for a Chinese dictation system having a vocabulary size of about 50,000 words, the size of a typical tri-gram language model file is about 130 Mbytes and the size of a typical bi-gram look-ahead language model file is about 109 Mbytes.",
"Since the language models files are usually very large, it is difficult to load such files directly into memory because of insufficient physical memory in most of the desktop machines.",
"One solution is to use a memory-map file format to access language model files in LVCSR system.",
"Accessing the language model files using memory-map file format is slower than loading the language model files directly into memory.",
"In addition, because a LVCSR system accesses language model files randomly, searching such a big space randomly also costs much time.",
"In short, the large size of language model files in a LVCSR system can negatively impact the system performance in terms of memory requirement and run-time speed.",
"BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.",
"1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.",
"2 is a diagram illustrating an HMM-based phone model; FIG.",
"3 is a diagram illustrating an N-gram language model; FIG.",
"4 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; FIG.",
"5 shows a block diagram of one embodiment of a method according to the teachings of the present invention; FIG.",
"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process according to the teachings of the present invention; FIG.",
"7 provides a graphical illustration of the segment/cluster initialization according to the teachings of the present invention; FIG.",
"8 provides a graphical illustration of a centroid (central point) of a segment; and FIG.",
"9 provides a graphical illustration of a segment refinement according to the teachings of the present invention.",
"DETAILED DESCRIPTION In the following detailed description numerous specific details are set forth in order to provide a thorough understanding of the present invention.",
"However, it will be appreciated by one skilled in the art that the present invention may be understood and practiced without these specific details.",
"In the discussion below, the teachings of the present invention are utilized to implement a method, apparatus, system, and machine-readable medium for building a compact language model for a speech recognition system.",
"In one embodiment, a set of N-gram probabilistic attributes that represent conditional probabilities of a set of words in an N-gram language model is classified into a plurality of classes based upon a relative order of the respective N-gram probabilistic attributes.",
"Each resultant class is then clustered into a plurality of segments or clusters to build a corresponding codebook for the respective class using a modified K-means clustering process which dynamically adjusts the size and the centroid of each segment during each iteration in the modified K-means clustering process.",
"In one embodiment, the modified K-means clustering process is performed until one or more optimization criteria are met.",
"In one embodiment, after the codebook for the respective class has been built, each N-gram probabilistic attribute in the respective class can be represented by the centroid of the corresponding segment to which the respective N-gram probabilistic attribute belongs.",
"For example, after a corresponding codebook has been built for a class of uni-gram probability values, a probability value associated with a given uni-gram node can be represented by a corresponding codeword in the respective codebook which can be referenced by a corresponding index value.",
"In one embodiment, an N-gram probabilistic attribute is represented by a first number of data bits prior to the construction of the corresponding codebook.",
"After the construction of the corresponding codebook, the respective N-gram probabilistic attribute can be represented by a corresponding index value having a second number of data bits which is smaller than the first number of data bits.",
"In one embodiment, an N-gram probabilistic attribute corresponds to an N-gram probability value or an N-gram back-off weight.",
"In one embodiment, the N-gram language model can be a tri-gram language model and the plurality of class can include a first class corresponding to a set of uni-gram probability values, a second class corresponding to a set of uni-gram back-off weights, a third class corresponding to a set of bi-gram probability values, a fourth class corresponding to a set of bi-gram back-off weights, and a fifth class corresponding to a set of tri-gram probability values.",
"In one embodiment, the clustering of each class of probabilistic attributes is performed as follows.",
"First, the plurality of segments are initialized such that all segments have equivalent areas.",
"The centroid for each segment is set such that the area of the respective segment is approximately equally split based on the corresponding centroid.",
"A set of clustering operations is then performed iteratively until one or more optimization criteria are met to create a corresponding codebook for the respective class of probabilistic attributes.",
"During each iteration, a total deviation of the plurality of segments is computed.",
"The edge of each segment is then replaced by the arithmetic average of the nearest two centroids.",
"A new centroid is then set for each resultant segment such that each resultant segment is approximately equally split by the corresponding centroid.",
"A new total deviation of the resultant segments is then computed.",
"In one embodiment, the one or more optimization criteria are met (e.g., the total deviation becomes steady) if one of the following conditions is satisfied: a) For each segment, the new edge value equals the previous edge value.",
"In other words, if the arithmetic average of the nearest two centroids equals the previous edge value then the total deviation is considered steady; b) A predetermined number of iterations is reached; or c) A change of total deviation is less than a predetermined threshold.",
"The teachings of the present invention are applicable to any method, apparatus, and system for speech recognition that employs N-gram language models.",
"However, the present invention is not limited to N-gram language models in speech recognition systems and can be applied to the other types of language modeling in speech recognition.",
"In addition, the present invention can also be applied to data modeling in other fields or disciplines including, but not limited to, image processing, signal processing, geometric modeling, computer-aided-design (CAD), computer-aided-manufacturing (CAM), etc.",
"FIG.",
"1 illustrates a block diagram of one embodiment of a speech recognition system 100 according to the teachings of the present invention.",
"The system 100, as shown in FIG.",
"1, includes an analog to digital converter (A/D) 110, a feature extractor or spectral analysis unit 120, a decoder 130, an acoustic model 140, and a language model 150.An input signal 105 representing the input speech is first digitized using the A/D 110.In one embodiment, the digital signal is then sliced up into frames typically of 10, 15, or 20 ms. Each frame of the signal is then converted into a corresponding feature vector which is used to characterize the spectral properties of the input signal.",
"In one embodiment, the feature vector is multi-dimensional and has a plurality of feature components.",
"The feature vectors generated by the feature extractor unit 120 are then inputted into the decoder 130 which determines the sentence or sequence of words that has the highest probability given the acoustic events characterized by the feature vectors, based upon the acoustic model 140 and the language model 150.The language model 150, in one embodiment, is an N-gram language model which includes a plurality of codebooks.",
"Each codebook corresponds to a class of probabilistic attributes (e.g., uni-gram probability values, uni-gram back-off weights, bi-gram probability values, etc.)",
"that are used to represent various conditional probabilities of a set of words.",
"For example, one of the codebook may correspond to the uni-gram probability class, another codebook may correspond to the uni-gram back-off weight class, and so on.",
"The language model 150 and the construction of the corresponding codebooks are described in greater detail below.",
"FIG.",
"2 illustrates a diagram of one embodiment of an HMM-based phone model structure used in the acoustic model 140 to model a phonetic unit (e.g., a phoneme or a subword unit, etc.).",
"Each individual phonetic unit is represented or modeled by a corresponding HM.",
"As shown in FIG.",
"2, an “MM has a set of sequence of states (1-5) that are connected by a set of transition probabilities (a12, a23, a34, a45), and a set of observation probabilities or likelihoods (b2 (o1),b2(o2),b3(o3),b4(o4),b4(o5),b4(o6)).",
"Each transition probability aij represents the probability of transitioning from a state i to a state j.",
"Each observation probability or distribution bi(oj) represents the probability of an observation vector oj being generated from a state i.",
"Thus, the transition probabilities model the durational variability in speech and the output probabilities model the spectral variability.",
"Accordingly, the set of states, the set of transition probabilities and the set of output probabilities are the parameters that are used to define an HMM.",
"The HMM shown in FIG.",
"2 has a left-right topology.",
"In many modern speech recognition systems, each state output distribution or observation probability function is modeled by a multivariate mixture Gaussian as follows: b j ( o t ) = ∑ k = 1 M c jk N ( o t , m jk , V jk ) where cjk is the weight of mixture component k in state j and N(oi, mjk, Vjk) denotes a multivariate Gaussian of mean mjk and covanance Vjk for the kth mixture component in state j.",
"FIG.",
"3 shows a structure diagram of one embodiment of an N-gram language model.",
"As described above, the purpose of a language model is to provide a mechanism for estimating the probability of some word, wk, in an utterance given the preceding words, W1k-1=w1 .",
".",
".",
"wk1.An N-gram language model assumes that wk depends only on the preceding N-1 words, that is P ( w k ❘ W 1 k - 1 ) = P ( w k ❘ W k - n + 1 k - 1 ) .",
"Because of the very large amount of models and associated data that need to be stored and processed, N is usually limited to two (for a bi-gram or 2-gram language model) or three (for a tri-gram or 3-gram language model).",
"A back-off mechanism can be used to compute a conditional probability of a given word when some N-gram context (e.g., a tri-gram or a bi-gram) does not exist or cannot be found for the given word.",
"For example, for given words “a” “b” “c”, there may be no bi-gram probability for “b→a” or tri-gram probability for “c→b→a” in certain contexts.",
"In this case, a back-off mechanism is used to compute the bi-gram or tri-gram probability for the given word “a”.",
"For example, for a tri-gram of words “c→b→a” (meaning word “a” preceded by word “b” preceded by word “c”), the corresponding tri-gram and bi-gram probabilities of this word sequence can be computed as follows using a back-off mechanism: P ( a / b , c ) = { P ( a / b , c ) , if P ( a / b , c ) exists , P ( a / b ) × w ( b , c ) , if w ( b , c ) exists , P ( a / b ) otherwise } P ( a / b ) = { P ( a / b ) , if P ( a / b ) exists , P ( a ) × w ( b ) otherwise } where: P(a/b,c) represents the tri-gram probability of the tri-gram c→b→a; P(a/b) represents the bi-gram probability of the bi-gram b→a; P(a) represents the uni-gram probability of word a; w(b,c) represents the back-off weight for the bi-gram c→b; and w(b) represents the back-off weight for the word b.",
"In various LVCSR systems, language model look-head techniques can be used to speed up the search in the corresponding N-gram language model.",
"The look-head techniques are used to incorporate the language model probabilities as early as possible in the pruning process of the beam search.",
"In general, look-head probabilities are pre-computed and stored in certain file formats or data structures.",
"Referring to FIG.",
"3, an N-gram language model in a typical LVSR system can be logically represented as a set of nodes 300 which includes a set of uni-gram nodes 310, a set of bi-gram nodes 320, and so on.",
"The uni-gram nodes are used to represent single words and their associated probability data (e.g., probability value and back-off weight).",
"The bi-gram nodes are used to represent word pairs and their associated probability data, etc.",
"In one embodiment, the second words in the bi-gram nodes that follow the same first word in a uni-gram node can be grouped together and point them to the corresponding first word in the uni-gram node.",
"Thus, only the second words and the corresponding bi-gram probability data need to be stored in the bi-gram nodes.",
"Tri-gram nodes can be structured similarly.",
"For example, all the third words in the tri-gram nodes that follow the same bi-gram node can be grouped together and point to the corresponding bi-gram node.",
"Thus, only the third words and their associated probability data need to be stored in the tri-gram nodes.",
"As shown in FIG.",
"3, each uni-gram node can be linked to its corresponding bi-gram nodes via a pointer.",
"Similarly, each bi-gram node can be linked to its corresponding tri-gram nodes via a pointer, and so on.",
"In other words, each uni-gram node points to the head of a set of bi-gram nodes whose words follow the word stored in the respective uni-gram node, each bi-gram node points to the head of a set of tri-gram nodes, and so on.",
"Each n-gram node (e.g., uni-gram node, bi-gram node, tri-gram node, etc.)",
"typically has the following components: the word identity of the word that the respective node represents (which can be the word itself or a word index corresponding to the word), an n-gram probability value, a back-off weight, and a pointer to the head of the corresponding (n+1)-gram nodes.",
"For example, a uni-gram node typically contains an index value of a particular word that is represented by the respective uni-gram node, a uni-gram probability value, a uni-gram back-off weight, and a pointer to a corresponding set of bi-gram nodes that are linked with the respective uni-gram node.",
"In one embodiment, as described above, each n-gram node only needs to store the last word of the corresponding n-gram.",
"For example, a bi-gram node only needs to store the second word of the corresponding bi-gram that the respective bi-gram node represents, a tri-gram node only needs to store the third word of the corresponding tri-gram that the respective tri-gram node represents, and so on.",
"In general, for computational efficiency and to avoid numerical underflow, probability values and back-off weights associated with the n-gram nodes can be represented in logarithmic space.",
"A n-gram node may be represented by various data structures depending upon the particular implementations and applications of the language model.",
"For example, an n-gram node may be represented by a corresponding data structure as shown below: typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID is an index of the word W that this node represents; P and W are the corresponding probability value and back-off weight, respectively; PTR is a pointer that points to the (n+1)-gram area linked by this node.",
"If there is no (n+1)-gram linked with this node, PTR can be set to a pre-defined value.",
"In this example, the word index WID is an unsigned 16-bit value (e.g., unsigned short data type); the probability value P and the back-off weight are 32-bit float values (e.g., 32-bit float data type); and the PTR is a 32-bit integer value.",
"The whole size of the data structure in this example is therefore 112 bits or 14 bytes.",
"An example of a data structure that can be used to represent a look-head node is shown below: typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; In this example, the NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.",
"It can be understood from the above description that a typical language model for a LVCSR system can contain a very large number of nodes each of which may contain corresponding probability value and/or backup weight that are represented by very large number of data bits (e.g., probability value and back-off weight are 32-bit float values in the above examples).",
"Thus, a language model for a typical LVCSR requires a very large amount of memory space.",
"As discussed above and described in more details below, the present invention provides a method and mechanism for reducing the size of the language model and the access time while maintaining a high level of recognition accuracy.",
"According to the teachings of the present invention, a method is introduced and described in detail herein to reduce the size of a language model in a LVCSR system.",
"In one embodiment, the probability values and back-off weights (also referred to as probabilistic attributes herein) associated with the respective n-gram nodes in the respective language model are quantized or clustered by a modified K-means clustering algorithm to compress the size of the respective language model.",
"In one embodiment, the probability values or back-off weights are divided or grouped into separate classes or groups based on the relative n-th order of the corresponding n-gram nodes and a modified K-means clustering algorithm is applied to each class to cluster the probability values or back-off weights in each class to build a corresponding codebook for the respective class.",
"For example, uni-gram probability values associated with uni-gram nodes are grouped into one class, uni-gram back-off weights are grouped into another class, bi-gram probability values are grouped into yet another class, and so on.",
"Thus, for a tri-gram language model (which includes uni-gram nodes, bi-gram nodes, and tri-gram nodes), there can be five separate classes (also referred to as data classes herein) that can be quantized or clustered to reduce the size of the language model.",
"These classes include a uni-gram probability class, a uni-gram back-off weight class, a bi-gram probability class, a bi-gram back-off weight class, and a tri-gram probability class.",
"For each class, the field of probability values (also referred to as the range of probability values) is quantized or clustered into a plurality of clusters or segments using a modified K-means clustering algorithm described in more detail below.",
"A centroid is defined for each cluster or segment.",
"Thus, a separate codebook can be constructed for each class of probability values or each class of back-off weights.",
"After quantization or clustering, a given probability value or back-off weight can be represented by the centroid (also called codeword) of the corresponding segment or cluster to which the respective probability value or back-off weight belongs.",
"By quantizing or clustering the field of probability values in each class, the probability values or back-off weights (which originally were represented by large-size data types such as 32-bit float type) can now be represented by smaller-size index values such as 8-bit char type that points to the corresponding codeword (centroid) in the corresponding codebook.",
"In one embodiment, the field of probability value for each class is divided into 256 segments or clusters using the modified K-means clustering process described in greater details below.",
"As such, the corresponding codebook will have 256 codewords.",
"Each codeword can be referenced by an 8-bit index value.",
"Thus, assuming that the original probability values or back-off weights use 32-bit float type, these probability values or back-off weights can now be replaced by 8-bit index values which are used to reference the corresponding codewords in the codebook.",
"It should be appreciated and understood by one skilled in the art that the teachings of the present invention are not limited to any particular number of segments or clusters.",
"Similarly, the teachings of the present invention are not limited to any particular data types that are used to represent the probability values and back-off weights before and after the quantization.",
"The number of segments or clusters for each class (hence the size of the corresponding codebook) and the data types that are used to represent the probability values, the back-off weights, or the indices to the codebook can vary depending upon the particular applications and implementations of the present invention.",
"FIG.",
"4 illustrates a flow diagram of one embodiment of a method 400 according to the teachings of the present invention.",
"At block 410, the probabilistic attributes (e.g., the probability values and back-off weights) in a N-gram language model are classified or grouped into a plurality of separate classes.",
"For example, in a tri-gram language model that includes uni-gram nodes, bi-gram nodes, and tri-gram nodes, the probabilistic attributes can be classified into five separate classes as follows: uni-gram probability, uni-gram back-off weight, bi-gram probability, bi-gram back-off weight, and tri-gram probability.",
"At block 420, the field of value for each class is clustered or quantized using a modified K-means clustering process to build a corresponding codebook for the respective class.",
"The modified K-means clustering process is described in more details below.",
"At block 430, each probabilistic attribute (e.g., a probability value or a back-off weight) can be represented by the corresponding codeword in the respective codebook.",
"In one embodiment, as described herein, after quantization or clustering, the probability value or back-off weight for any given node in the language model can then be replaced by a corresponding index value that is used to reference the corresponding codeword.",
"For example, assuming that the original probability value or back-off weight for a given node was 32-bit data type (e.g., 32-bit float value) and that there are 256 codewords in the corresponding codebook, then after quantization or clustering, the respective probability value or back-off weight for the given node can be represented by an 8-bit index value which is used to reference or point to the corresponding codeword in the codebook.",
"FIG.",
"5 shows a block diagram of one embodiment of a method according to teachings of the present invention.",
"As shown in FIG.",
"5, the probabilistic attributes (e.g., probability values and back-off weights) 510 in an N-gram language model are classified or grouped into separate classes based on the n-gram order of the respective nodes.",
"In this example, the classes include: uni-gram probability 520A, uni-gram back-off weight 520B, bi-gram probability 520C, bi-gram back-off weight 520D, tri-gram probability 520E, etc.",
"Each class is then clustered or quantized using a modified K-means clustering process to create a corresponding codebook (e.g., 530A-E) for each respective class.",
"As described herein, the size of the codebook for each class may or may not be the same, depending on the particular applications and implementations of the present invention.",
"Each probabilistic attribute (e.g., a uni-gram probability value associated with a uni-gram node) can then be represented by a corresponding codeword in the respective codebook (e.g., the codebook for the uni-gram probability class).",
"FIG.",
"6 illustrates a flow diagram of one embodiment of a modified K-means clustering process 600 to create a corresponding codebook for a probabilistic class (e.g., uni-gram probability class or bi-gram probability class, etc.",
"), according to the teachings of the present invention.",
"The process starts at block 601 and proceeds to block 605 to initialize the codebook as follows: At block 605: initialization of the process is performed by clustering the field of values for the respective class into a plurality of segments (e.g., n segments in this example) and initialize the segments such that all the segments have equivalent areas.",
"The segments are also called clusters herein.",
"The centroid of each segment is then set such that the respective segment is about equally split (i.e., the segment can be split into two equivalent sub-areas by the corresponding centroid).",
"As shown in FIGS.",
"7 and 8, the segment initialization is performed according to the following conditions: E 1 = min ( x ) , E 2 … E n , E n + 1 = max ( x ) , C 1 , C 2 … C n - 1 , C n ∫ E 1 E 2 P ( x ) ⅆ x = ∫ E 2 E 3 P ( x ) ⅆ x = … = ∫ E n - 1 E n P ( x ) ⅆ x = ∫ E n E n + 1 P ( x ) ⅆ x ∫ E i C i P ( x ) ⅆ x = ∫ C i E i + 1 P ( x ) ⅆ x i = 1 , 2 … n Where: x is a possible probability value in the respective class; P(x) is the probability that x occurs; the i segment is defined as [Ei, Ei+1; and Ci is defined as the centroid of the i segment.",
"At this point, there are n segments or clusters which have equivalent areas and each i segment has a corresponding centroid Ci which can split the respective segment into two equivalent sub-areas.",
"Set the number of iterations R=0 At block 610: compute the total deviation Dev as sum of the deviation of each segment Devi as follows: Dev=ΣDevi.",
"for the i segment, Devi is defined as ∫ E i E i + 1 P ( x ) * abs ( x - C i ) ⅆ x , (illustrated in FIG.",
"8) At block 615: replace the edge Ei of each segment by the arithmetic average of the nearest two centroids as shown below: Ei=(Ci-1+Ci)/2 (illustrated in FIG.",
"9) set the new centroid Ci for each resultant segment compute the new total deviation Dev increment the number of iterations R=R+1 At block 625: if the total deviation is not steady, then the process 600 loops back to block 615.Otherwise, the corresponding codebook is obtained at block 635 and the process 600 proceeds to ends at block 691.In one embodiment, the total deviation is considered steady if one of the following conditions (also called optimization criteria) is met: a.",
"For each i, Ei=Ei (the new edge value is the same as the previous edge value); b.",
"The number of iterations performed reaches a predetermined number (R=Rmax); or c. The change of total deviation is less than a pre-determined threshold value Δth, |Dev−Dev|/Dev<Δth Significant reduction in memory requirement and access time for a language model can be attained according to the teachings of the present invention.",
"For example, assuming that before compression or quantization, the following data structures were used for n-gram nodes and look-head nodes, respectively: n-gram node data structure (before compression): typedef struct _asNGram{ unsigned short WID; float P; int PTR; float W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (32-bit float value) and W (32-bit float value) are the corresponding probability value and back-off weight, respectively, PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.",
"If there is no (n+1)-gram linked with this node, PTK is a pre-defined value.",
"Thus the whole size of an-gram data structure in this example is 112 bits or 14 bytes.",
"look-head node data structure (before compression): typedef struct _asTreeBi-gramt{ int NODENO; float P; } asTreeBi-gram; where: NODENO is a 32-bit index of the corresponding node and P is a 32-bit probability value.",
"Thus the whole size of a look-head data structure is 64 bits.",
"After compression, an n-gram node and a look-head node can be represented by the following data structures, respectively.",
"It is assumed in this example that 8-bit codebooks are used (e.g., each codebook has 256 codewords): n-gram node data structure (after compression): typedef struct _asNGram{ unsigned short WID; unsigned char P; int PTR; unsigned char W; } asNGram; where: WID (16-bit unsigned value) is an index of the word W that this node represents; P (8-bit unsigned char type) is the index value used to reference the corresponding quantized probability value (codeword) in the corresponding codebook; W (8-bit unsigned char type) is the index value used to reference the corresponding quantized back-off weight (codeword) in the corresponding codebook; PTR (32-bit integer value) is a pointer that points to the (n+1)-gram area linked by this node.",
"If there is no (n+1)-gram linked with this node, PTR is a pre-defined value.",
"Thus the whole size of an n-gram data structure after compression in this example is 64 bits or 8 bytes.",
"look-head node data structure (after compression): typedef struct _asTreeBi-gramt{ int NODENO; unsigned P; } asTreeBi-gram; where: NODENO is now an 8-bit index value that is used to reference the corresponding quantized probability value in the corresponding codebook.",
"Thus the size of the look-head data structure after compression is 40 bits.",
"From the above example, it can be seen that the size of the language model can be significantly reduced according to the teachings of the present invention.",
"In this example, the size of each n-gram node is reduced from 112 bits to 64 bits and the size of each look-head node is reduced from 64 bits to 40 bits.",
"For a language model in a typical LVCSR system which usually has a very large number of nodes, the reduction in memory requirement can be very substantial.",
"The additional memory requirement for the codebooks is minimal or insignificant compared to the reduction in the memory requirement for the whole language model.",
"For example, the memory requirement for an 8-bit codebook which has 256 codewords and each codeword is a 32-bit float value is only: 256(codewords)*32(bits/codeword)=8192 bits Tables 1 and 2 below show some experimental results based upon various experiments conducted according to the teachings of the present invention.",
"These experiments were conducted on a 51K Mandarin LVCSR system and using a testing machine such as a double-PIII866 with 512M memory and 256K L2 Cache.",
"Table 1 illustrates the improvements with respect to memory requirement and decoding speed using 8-bit codebooks (e.g., each codebook has 256 codewords).",
"It can be seen from table 1 that the memory requirement and decoding speed have been reduced after compression while the increase in the word error rate (WER) of the system after compression is minimal or insignificant.",
"The XRT notation in table 1 is used to mean times of real time.",
"TABLE 1 System using BaseLine modified K-Means System (before clustering (after compression) compression) WER 10.0% 10.1% Tri-gram file size 130M 75M Treebi-gram file size 109M 68M MapMode XRT 1.18XRT 1.08XRT Loading directly mode 1.11XRT 1.02XRT XRT As shown in table 1, the experiments and comparisons were done to show the improvements with respect to two different embodiments: one embodiment using memory-map file format for the language model files and the other embodiment loading the language model files directly into memory.",
"After quantization the size of the language model file is reduced by 40% from 239M(130M+109M) to 143M (75M+68M).",
"In addition, the decoding speed has been improved by 8% from 1.18×RT to 1.08×RT using memory-map file format while WER only increases from 10.0% to 10.1%.",
"Furthermore, if language model files are loaded directly to memory, the decoding speed can be improved by another 6%.",
"Table 2 shows the experimental results using smaller-size codebooks.",
"In this example, 6-bit codebooks and 4bit codebooks were used.",
"TABLE 2 System using modified K-means clustering (after compression) 6-bit 4-bit codebooks codebooks WER 10.1% 12.8% MapMode XRT 1.07XRT 1.03XRT Loading directly 1.01XRT 0.98XRT mode XRT The invention has been described in conjunction with the preferred embodiment.",
"It is evident that numerous alternatives, modifications, variations and uses will be apparent to those skilled in the art in light of the foregoing description."
]
] | Patent_10297354 |
[
[
"Refrigeration unit",
"The refrigerating unit can be operated by means of a thermal solar system as energy source.",
"Therein it is provided that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.",
"The refrigerating unit has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to form a closed fuel circuit with one another."
],
[
"1-12.Cancelled.",
"12.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit, comprising: a closed fuel circuit having a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for rectification of a mixture of fuel and water vapor.",
"13.The refrigerating unit according to claim 12, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.",
"14.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"15.The refrigerating unit according to claim 13, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"16.The refrigerating unit according to claim 13, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.",
"17.The refrigerating unit according to claim 13, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.",
"18.The refrigerating unit according to claim 17, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.",
"19.The refrigerating unit according to claim 13, further comprising a rising tube having an inner structure promoting the formation of bubbles.",
"20.The refrigerating unit according to claim 12, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).",
"21.The refrigerating unit according to claim 13, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.",
"22.The refrigerating unit according to claim 13, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 23.The refrigeration unit of claim 12, where the closed fuel circuit comprises an expeller condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit.",
"24.The refrigerating unit according to claim 23, further comprising a thermal solar system actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.",
"25.The refrigerating unit according to claim 23, wherein the the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"26.The refrigerating unit according to claim 23, wherein the the expeller has at leapt one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"27.The refrigerating unit according to claim 12, wherein the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.",
"28.The refrigerating unit according to claim 23, further comprising a rising tube and wherein the expeller is actively connected, on an inlet side with respect to the rising tube, to a supply line, coming from the triple heat exchanger, for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line, leading to the triple heat exchanger, for the conveyance of the solution which is low in fuel.",
"29.The refrigerating unit according to claim 28, wherein the fuel vapor supply line leads essentially coaxially through the recycling line.",
"30.The refrigerating unit according to claim 23, further comprising a rising tube having an inner structure promoting the formation of bubbles.",
"31.The refrigerating unit according to claim 23, wherein the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).",
"32.The refrigerating unit according to claim 23, wherein the thermal solar system is actively connected over an entire longitudinal extension of the rising tube of the expeller.",
"33.The refrigerating unit according to claim 23, further comprising at least one of an absorber and an evaportator having conveyance lines, wherein the conveyance lines have upper free ends with a corresponding capillary sleeve 34.A refrigerating unit, which is formed as a diffusion-absorption refrigerating unit and can be operated by means of a thermal solar system as energy source, with an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir which are actively connected to one another to form a closed fuel circuit, wherein the refrigerating unit has, in the fuel circuit, a triple heat exchanger for preheating a solution which is high in fuel and precooling a solution which is low in fuel, and for the rectification of a mixture of fuel and water vapor.",
"35.A method for refrigerating a unit, the method comprising: forming a fuel circuit having a triple head exchanger; and preheating a solution which is high in fuel, precooling a solution which is low in fuel, and rectifying a mixture of fuel and water vapor."
],
[
"<SOH> BACKGROUND <EOH>The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1 .",
"Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.",
"For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.",
"In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.",
"Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.",
"These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.",
"For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.",
"A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.",
"This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.",
"A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.",
"Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.",
"from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.",
"A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.",
"The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.",
"The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.",
"It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.",
"Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.",
"Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.",
"Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.",
"A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.",
"Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).",
"According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.",
"of the still not vaporized fuel.",
"Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.",
"If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.",
"In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.",
"This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.",
"Instead of ribbing, deflecting plates can also be provided.",
"Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.",
"This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.",
"The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.",
"Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.",
"The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.",
"In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.",
"Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.",
"An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.",
"Advantageously, the rising tube has an inner structure promoting the formation of bubbles.",
"An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.",
"The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.",
"Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H 2 O).",
"NH3 as fuel and a mixture of NH3 and H 2 O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.",
"Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).",
"According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.",
"By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.",
"In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).",
"Additional advantageous developments of the invention follow from the description."
],
[
"RELATED APPLICATIONS The present application is the U.S. National Phase of PCT Application PCT/EP01/06412, filed Jun.",
"6, 2001, which claims priority to German Patent Application No.",
"10028543.0, filed Jun.",
"8, 2000.BACKGROUND The invention relates to a refrigerating unit, which can be operated by means of a thermal solar system as energy source, according to the preamble of claim 1.Refrigerating units of the type addressed here, such as, for example, absorption refrigerating units or compression refrigerating units, are known.",
"For solar operation, these absorption refrigerating units disadvantageously require a high outlay with regard to control technology and investment, in particular with regard to a continuous reheating or heat storage.",
"In view of this, absorption refrigerating units are limited to a relatively narrow temperature and flow range.",
"Also the known, photovoltaically operated compression refrigerating units are characterized by a high outlay with regard to investment and have relatively low efficiency.",
"These known refrigerating units are not suited to problem-free and economical operation with thermal solar systems, in particular in the average capacity range.",
"SUMMARY OF THE INVENTION It is the objective of the invention to provide a refrigerating unit of the type stated initially which can be operated in a reliable, effective, and operation-friendly manner by means of a thermal solar system, in particular in the lower to average refrigerating capacity range.",
"For the realization of this objective, a refrigerating unit with the features of claim 1 is proposed which is distinguished by the fact that the refrigerating unit is formed as a diffusion-absorption refrigerating unit.",
"A diffusion-absorption refrigerating unit can be operated in a particularly reliable and efficient manner by means of a thermal solar system as an energy source.",
"This behavior, which is favorable to operation, can also be achieved in a smaller to average refrigerating capacity range.",
"A diffusion-absorption refrigerating unit is advantageously suitable to be operated by means of various energy sources.",
"Among these, a thermal solar system as well as another heat transfer medium circuit, e.g.",
"from a heat recovery process, can be used for the alternative or enhancing energizing of the refrigerating unit.",
"A diffusion-absorption refrigerating unit is thus advantageously suitable, in a manner which is flexible and favorable for operation, to be energized with thermal energy by means of a thermal solar system as well as, if needed or desired, additional, different energy sources.",
"The refrigerating unit advantageously has an expeller, a triple heat exchanger, a condenser, an evaporator, a gas heat exchanger, an absorber, and a fuel reservoir, which are actively connected to one another to form a closed fuel circuit.",
"The refrigerating unit characterized by a closed fuel circuit can be formed as a hermetically closed, compact unit which is distinguished by an advantageous independence of site.",
"It is operated merely by means of thermal energy and thus advantageously needs no electrical power supply.",
"Since no components working mechanically within the refrigerating unit, such as, for example, pumps, are required to operate the refrigerating unit, the unit is maintenance-friendly, relatively favorable from the standpoint of cost, and can be operated, at least nearly, without noise.",
"Furthermore, it is possible to develop the refrigerating unit so that the mounting of several refrigerating units in parallel can be realized in a relatively simple manner.",
"Preferably, the thermal solar system can be actively connected to an expeller formed as a gas bubble pump for the desorption and vaporization of a fuel contained in a solution.",
"A gas bubble pump is particularly suitable for desorbing and vaporizing, in a manner which is effective and favorable for operation, a fuel contained in a solution such as, for example, ammonia (NH3) in an ammonia-rich solution.",
"Furthermore, a gas bubble pump permits an efficient heat transfer accomplished by means of a thermal energy source, which is a prerequisite for reliable and effective desorption and vaporization of the fuel (ammonia).",
"According to a preferred form of embodiment, the expeller has at least one rising tube which on the inlet side is provided with a ribbing promoting the vaporization of the fuel contained in the rising tube.",
"The rising tube of the expeller formed as a gas bubble pump contains a solution which is high in fuel and which, due to the vaporization of the fuel in the rising tube, experiences an increase in volume so that it assumes an operating volume which is a multiple of the original volume value, i.e.",
"of the still not vaporized fuel.",
"Due to this increase in volume, the level of the vaporous fuel, or of the fuel mixture in the rising tube, rises to a corresponding operational conveyance height.",
"If this operational conveyance height exceeds the length of the rising tube due to a corresponding increase in volume of the fuel mixture or a vaporization of the fuel, the conveyance of the vaporous fuel, or a remaining solution which is low in fuel, to additional functional units of the refrigerating unit is started, said functional units being actively connected to one another in the form of a closed fuel circuit so that the refrigerating unit is activated at this moment in its operation.",
"In so doing, an effective heat transfer from a thermal energy source (solar system) into the expeller or into the rising tube serves for a reliable and rapid activation of the refrigerating unit.",
"This is guaranteed by means of a suitable ribbing of the rising tube whereby an enlargement of the heat transfer surface to improve the heat transfer into the rising tube is obtained.",
"Instead of ribbing, deflecting plates can also be provided.",
"Advantageously, the expeller has a plurality of parallel rising tubes spaced at a distance from one another for the conveyance of a solution which is high in fuel, where a parallel recycling line for the conveyance of the solution which is low in fuel is disposed between at least two rising tubes.",
"This makes possible an expeller formation which is compact and has the form of a bundle of tubes, where advantageously the rising tubes disposed externally can be energized with thermal energy, in a manner favorable to efficiency and operation, by means of a thermal energy source (solar system) while the interposed, parallel recycling line, through which a heated solution which is low in fuel is conveyed, makes possible an additional heating of the outer rising tubes, or counteracts a cooling of the same.",
"The amount of heat radiating, and thus not actively utilized, is reduced in an effective manner due to a compact arrangement of the outer rising tubes as a bundle of tubes and a centrally disposed recycling line.",
"Preferably, the expeller is actively connected, on the inlet side with respect to the rising tube, to a supply line coming from the triple heat exchanger for the conveyance of the solution which is high in fuel, on the outlet side with respect to the rising tube, to a fuel vapor supply line leading to the triple heat exchanger, and, on the outlet side with respect to the recycling line, to a supply line leading to the triple heat exchanger for the conveyance of the solution which is low in fuel.",
"The expeller preferably having a compact bundle of tubes is thus actively connected, on the inlet side as well as on the outlet side, to the triple heat exchanger which in turn is actively connected to additional functional units of the refrigerating unit to form a closed fuel circuit.",
"In so doing, a solution which is high in fuel, a solution which is low in fuel, and fuel vapor are conveyed in the expeller after activation of the refrigerating unit.",
"Advantageously, the fuel vapor supply line leads essentially coaxially through the recycling line.",
"An expeller of this type is formed particularly compactly and is distinguished by a particularly effective and reliable desorption and vaporization of a fuel in a solution which is high in fuel, said solution being contained in the respective rising tube, since an undesirable radiation of heat from the expeller into the environment can be reduced to a significant extent due to its compact arrangement as a bundle of tubes.",
"Advantageously, the rising tube has an inner structure promoting the formation of bubbles.",
"An inner structure of this type in the rising tube can, for example, be achieved by means of a coating and/or a roughening of the inner surface and serves for the desired bubble formation in a solution which is high in fuel in a definite area in the rising tube due desired turbulence of the same solution, said turbulence being caused by the inner structure.",
"The expeller formed as a gas bubble pump is suitable, with the use of rising tubes of this type, for a particularly effective desorption, vaporization, and conveyance of fuel contained in a solution which is high in fuel and for the conveyance of a solution which is low in fuel.",
"Advantageously, the fuel is ammonia (NH3) and the solution a mixture of ammonia and water (NH3/H2O).",
"NH3 as fuel and a mixture of NH3 and H2O as solution are particularly suited to an effective operation of the refrigerating unit according to the invention.",
"Along with this, an auxiliary gas, used, if needed or desired, for the operation of the refrigerating unit, can be helium (He).",
"According to a preferred form of embodiment, the thermal solar system is actively connected over the entire longitudinal extension of the rising tube of the expeller to the same.",
"By means of heating of the rising tube over its entire longitudinal extension, it is avoided in a reliable manner that, due to an undesired cooling in an unheated area of the rising tube, vaporous NH3, already expelled, condenses and thus cancels an intended increase in volume of the fuel contained in the rising tube, where the increase in volume would lead to the starting of the conveyance of the fuel, or the solution which is low in fuel.",
"In this, the rising tubes are preferably each provided on the inlet side with a ribbing for the heating of the triple heat exchanger, of a fuel reservoir, and of a supply line leading to the condenser (vapor line).",
"Additional advantageous developments of the invention follow from the description.",
"BRIEF DESCRIPTION OF THE DRAWINGS The invention is explained below in several embodiment examples with the aid of the accompanying drawings.",
"Shown are: FIG.",
"1 shows a block diagram of a refrigerating unit according to the invention; FIG.",
"2a shows a schematic representation of a first part of the refrigerating unit according to FIG.",
"1; FIG.",
"2b shows a schematic representation of a second part of the refrigerating unit according to FIG.",
"1; FIG.",
"3 shows an alternative form of embodiment of an expeller for the refrigerating unit according to FIG.",
"2; FIG.",
"4 shows a schematic representation of a conveyance line having a capillary sleeve on an enlarged scale.",
"DETAILED DESCRIPTION FIG.",
"1 shows a block diagram of a refrigerating unit 10.Furthermore, FIGS.",
"2a and 2b, which complement one another as partial figures, show the refrigerating unit 10 according to FIG.",
"1 in schematic representation.",
"The refrigerating unit 10 has an expeller 12 which is actively connected to a thermal solar system 11 by means of heat transfer medium lines represented as arrows 13 and 14 (FIG.",
"2a: 43, 43a, 43b, 44).",
"A fuel vapor supply line represented as an arrow 16 (FIG.",
"2a: 51) and a supply line, represented as an arrow 17 (FIG.",
"2a: 47) and serving to convey a solution which is low in fuel, leads from the expeller 12 to a triple heat exchanger 15, from which a supply line, represented as an arrow 18 (FIG.",
"2a: 48) and serving to convey a solution which is high in fuel, leads to the expeller 12.The triple heat exchanger 15 is actively connected to a condenser 19 by means of a supply line, represented as an arrow 20 (FIGS.",
"2a/2b: 55), for fuel vapor and, if needed or desired, with low percentages of water (H2O).",
"The condenser 19 is actively connected to a refrigerating medium circuit 21 by means of cold transfer medium lines, represented as arrows 22, 23 (FIG.",
"2b: 56, 57).",
"A supply line, represented as an arrow 25 (FIG.",
"2b: 58) for liquid fuel leads from the condenser 19 to an evaporator 24 which is actively connected to a refrigerating medium circuit 26 by means of cold transfer medium lines, represented as arrows 27, 28 (FIG.",
"2b: 60).",
"A supply line, represented as an arrow 30 (FIG.",
"2b: 59) for a gaseous mixture of vaporized fuel and an auxiliary gas leads from the evaporator 24 to a gas heat exchanger 29 which, in operation, is actively connected to the evaporator 24 by means of a supply line, represented as an arrow 31 (FIG.",
"2b: 76), for the precooled auxiliary gas.",
"By means of a supply line, for the heated gas mixture consisting of fuel and auxiliary gas and represented as an arrow 33 (FIG.",
"2b: 77), the gas heat exchanger 29 is actively connected to an absorber 32, from which a supply line, for warm auxiliary gas and represented as an arrow 34 (FIG.",
"2b: 61), leads to the gas heat exchanger 29.The absorber 32 is actively connected to a refrigerating medium circuit 35 by means of cold transfer medium lines represented as arrows 36, 37 (FIG.",
"2b: 63, 64).",
"A supply line, for a solution which is high in fuel and represented as an arrow 38 (FIGS.",
"2a/2b: 68), leads from the absorber 32 to a triple gas heat exchanger 15 and from the latter a supply line, for a solution which is low in fuel and represented as an arrow 39 (FIGS.",
"2a/2b: 69), leads to the absorber 32.The refrigerating unit 10 is divided, by means of a dotted line 40 according to arrow 42, into an upper area in which the total operational pressure is equal to the fuel pressure and, according to arrow 41, into a lower area in which the total operational pressure is equal to the sum of the fuel pressure and the auxiliary gas pressure.",
"Preferably the fuel is ammonia (NH3), the auxiliary gas is helium (He), and each of the solution which is high in fuel and the solution which is low in fuel is a mixture of ammonia and water (NH3/H2O).",
"NH3 vapor with low percentages of H2O is thus conveyed from the expeller 12 to the triple heat exchanger 15 (arrow 16), from which NH3 vapor flows into the condenser 19 (arrow 20) and is cooled to liquid NH3 where the liquid NH3 subsequently flows into the evaporator 24 (arrow 25) in which NH3 diffuses into a precooled He atmosphere forming a “heavy” cold He-NH3 gas mixture.",
"This cold gas mixture flows into the gas heat exchanger 29 (arrow 30) in which the same is heated due to a heat transfer from a warm He stream (arrow 34) flowing in the opposite direction through the gas heat exchanger 29 and subsequently flows from the absorber 32 to the absorber 32 (arrow 33).",
"The solution which is high in NH3/H2O flows on the contrary from the absorber 32 to the triple heat exchanger 15 (arrow 38) and from there to the expeller 12 (arrow 18).",
"The solution which is low in NH3/H2O flows on the contrary to the triple heat exchanger 15 (arrow 17) and from there to the absorber 32 (arrow 39).",
"Furthermore, the withdrawal of cooling capacity of the refrigerating unit 10 is done by means of the refrigerating medium circuit 26, which is actively connected to the evaporator 24.FIG.",
"2a shows in addition the expeller 12 preferably formed as a gas bubble pump, said expeller being actively connected to the thermal solar system 11 and to the triple heat exchanger 15.The thermal solar system 11 has a solar cell unit 72 in the form of a solar collector cell unit which is actively connected, by means of the heat transfer medium lines 43, 43a, 43b, and 44, to a plurality of rising tubes 45 (in FIG.",
"2a two rising tubes 45 are represented) of the expeller 12.The heat transfer medium lines 43, 43a, and 43b are connected to one another in operation by means of a change-over valve 78 in such a manner that a heat transfer medium is conveyed according to arrow 13 from the solar cell unit through the heat transfer medium line 43 to the change-over valve 78, which conducts the heat transfer medium through the heat transfer medium line 43a and/or 43b according to arrow 13a or 13b to the expeller 12.The rising tubes 45 have on one side a ribbing 50 (or deflecting plate not represented) in order to insure a rapid, spatially defined, and reliable heat transfer from the heat carrier of the solar cell unit 11 to the solution which is high in fuel, said solution being located in the rising tubes 45.Between the rising tubes 45 disposed in parallel at a distance from one another, a parallel recycling line 49 is disposed to convey a solution which is low in fuel.",
"The expeller 12 is thus formed as a compact bundle of tubes (rising tubes 45 for solution which is high in fuel, recycling line 49 for solution which is low in fuel).",
"The solution which is low in fuel is conducted, by means of a supply line 47, from the expeller 12 to the triple heat exchanger 15 and conducted from there to the absorber 32 by means of the supply line 69 (see also FIG.",
"2b).",
"The fuel vapor is conducted, by means of a conducting line 51, from the expeller 12 to the triple heat exchanger 15 in which water is condensed out from the mixture of fuel and water vapor and conducted, by means of the line 52, according to the arrow 53 to the supply line 48 which serves to convey the solution which is high in fuel into the expeller 12.Pure, or nearly pure, fuel is conducted, by means of the supply line 55, from the triple heat exchanger 15 to the condenser 19 (see also FIG.",
"2b).",
"The supply line 48 for conveying the solution which is high in fuel is actively connected to a fuel reservoir 54.A dotted line 70 denotes the level of liquid in the fuel reservoir 54 while a dotted line 71 represents the level of liquid in the inlet of the absorber 32 (see also FIG.",
"2b).",
"The expeller 12 is formed as a compact bundle of tubes with a plurality of cold transfer medium lines 65 and a plurality of auxiliary gas conveyance lines 62.Likewise, the gas heat exchanger 29, the evaporator 24, and the condenser 19 are each formed as a compact bundle of tubes.",
"The absorber 32 can however also be formed in such a manner that the cold transfer medium lines 65 are provided with intermediate spaces for the auxiliary gas conveyance lines 62 and preferably with deflecting plates.",
"To expel the fuel from the solution which is high in fuel as well as to convey the same, the expeller 12 needs a certain minimum supply of energy from the solar cell unit 72.A supply of energy into the expeller 12 below this limit would lead, when conveying the heat transfer medium into the expeller 12 through the heat transfer medium line 43a below the liquid level denoted as the dotted line 70, to undesirable reduction of the solution which is high in fuel without the same being conveyed.",
"With longer operation of the refrigerating unit in a state of this type, the solution which is high in fuel, said solution being located in the expeller 12, would be reduced still further in fuel so that higher and higher operating temperatures would be required in order to expel additional fuel from the solution which is high in fuel.",
"This has as a consequence that the refrigerating unit could no longer be put in operation without additional effort.",
"Providing a second heat transfer medium line 43b, which is actively connected to the expeller 12 above the liquid level denoted as the dotted line 70, advantageously makes it possible that, on starting the refrigerating unit, that energy which is not sufficient to expel the fuel from the solution which is high in fuel can still be used to preheat the expeller 12.Thereby it is avoided that a “high-value” energy has to be used to heat the expeller 12 (material of the tubes) so that this energy can be used exclusively for the conveyance of the solution which is high in fuel, said solution being located in the expeller 12, and to expel the fuel from the same.",
"Furthermore, the solution which is high in fuel can be preheated in the triple heat exchanger 15, the fuel reservoir 54, and in the supply line 55 (vapor line) which leads to the condenser 19 by means of “low-value” solar energy.",
"FIG.",
"3 shows an expeller 12 according to an alternative development.",
"This expeller 12 corresponds essentially to that of FIG.",
"2 and is different in that according to FIG.",
"3 the fuel vapor supply line 51 leads coaxially through the recycling line 49.In order to guarantee reliable separation in the expeller 12 of the fuel vapor (supply line 51) and the solution which is low in fuel (recycling line 49), an encircling, conically tapering baffle plate 73 is provided, disposed on the outlet side with respect to the rising tubes 45.The baffle plate 73 serves to conduct the solution which is high in fuel according to the arrows 75 into the recycling line 49 while the fuel vapor is conducted through a gap formed between the baffle plate 73 and the supply line 51 according to the arrow 74 into the supply line 51.The expeller 12 according to FIG.",
"3 is thus formed as a compact bundle of tubes with a plurality of rising tubes 45, a central recycling line 49, and a supply line 51 guided coaxially through the recycling line 49.The additional active connections of the expeller 12, or of the corresponding supply lines, to the corresponding functional units of the refrigerating unit 10 correspond to the above-described form of embodiment according to FIGS.",
"1 and 2.The individual functional units of the refrigerating unit 10 are actively connected to one another and disposed spatially so as to form three gravity-based gas or liquid circuits.",
"In so doing, only the external circuits for cooling (cooling circuits 21, 26, and 35) and for the thermal solar system 11 are maintained by means of conventional pumps.",
"Within the refrigerating unit, each of the solution circuit, refrigerating medium circuit, and auxiliary gas circuit is closed.",
"The solution circuit begins in the expeller 12 formed as a gas bubble pump, where, by means of the solar system 11, thermal energy is supplied to convert a solution which is high in fuel into a mixture of vaporous fuel and water and into a solution which is low in fuel.",
"The solution which is low in fuel is conveyed from the expeller 12 to the absorber 32 in which the solution which is low in fuel backs up in the supply line 69 up to a certain level of liquid.",
"The solution which is low in fuel is thus raised from the level of liquid in the fuel reservoir 54 (dotted line 70) to the level of the inlet of the absorber 32 (dotted line 71).",
"In the process, the conveyance height and the mass flow of the solution which is low in fuel depend on the geometric configuration of the expeller 12, the level of liquid in the fuel reservoir 54 (dotted line 70), and thus the immersion depth of the rising tubes 45, and the recycling line 49 of the expeller 12 as well as on the number of conveyance tubes (rising tubes 45, recycling line 49) of the expeller 12.The solution which is low in fuel conveyed to the absorber 32 absorbs the fuel supplied by the evaporator 24 to form a solution which is high in fuel, said solution being conducted for preheating through the triple heat exchanger 15 and flowing back into the expeller 12.The solution which is high in fuel flowing back into the expeller 12 is thus available once again to expel the fuel in the same.",
"In the refrigerating medium circuit, the mixture of hot fuel and water vapor, said mixture having been vaporized in the expeller 12, flows into the triple heat exchanger 15 and is precooled and rectified there by the cold solution which is high in fuel coming from the absorber 32 and flowing in the opposite direction.",
"The pure fuel vapor, near to the condensation temperature, rises from the triple heat exchanger 15 into condenser 19 in which, through additional withdrawal of heat by means of the refrigerating medium circuit 21, the pure fuel is cooled to under its condensation temperature so that the fuel vapor condenses.",
"The liquid fuel subsequently flows from the condenser 19 into the evaporator 24 in which the liquid fuel diffuses into an atmosphere of auxiliary gas and draws heat from the refrigerating medium of the refrigerating medium circuit 26.Subsequently the relatively heavy mixture of auxiliary gas and fuel sinks into the absorber 32.In the auxiliary gas circuit, the atmosphere of auxiliary gas in the evaporator 24 is constantly pressurized with pure auxiliary gas in order to avoid the atmosphere of auxiliary gas reaching its saturation limit through the diffusing fuel and thus sufficient diffusion of liquid fuel into the atmosphere of auxiliary gas not taking place.",
"The relatively heavy mixture of auxiliary gas and fuel sinks through the lines 59 from the evaporator 24 into the gas heat exchanger 29 and flows from it through the supply line 77 into the absorber 32.In the absorber 32 the fuel contained in the mixture is absorbed by the solution which is low in fuel, where the then lighter, pure auxiliary gas rises through the lines 62 from the absorber 32, flowing in the opposite direction, and with precooling by means of the refrigerating medium circuit 35 in the gas heat exchanger 29.From there, the pure auxiliary gas flows through the supply lines 61 and 76 into the upper area of the evaporator 24 in which it is enriched with diffusing fuel and subsequently sinks back into the absorber 32 once again as a mixture of fuel and auxiliary gas.",
"Advantageously, the triple heat exchanger 15 simultaneously fulfills three different functions, namely preheating of the solution which is high in fuel coming from the expeller 12, precooling of the low solution flowing to the absorber 32, and rectification of the NH3-H2O vapor coming from the expeller 12.By conducting the hot vapor mixture along the solution which is high in fuel cooled in the absorber 32, the water vapor contained condenses and falls back as condensate into the solution which is high in fuel.",
"The heat of condensation or rectification being released is thus also used to preheat the rich solution.",
"The triple heat exchanger 15 thus provides for a nearly complete recovery of the heat of rectification which advantageously no longer has to be supplied to the expeller 12 from outside.",
"FIG.",
"4 shows a detail of the absorber 32 on an enlarged scale.",
"According to this preferred form of embodiment, the given auxiliary gas conveyance line 62 of the absorber 32 is provided with a capillary sleeve 79 which is actively connected to the line 62, on the outlet side with respect to the auxiliary gas conveyance line (arrow 82).",
"The capillary sleeve 79 forms, together with the line 62, a capillary gap 81, extending circumferentially, through which the solution which is low in fuel (liquid) can flow according to the arrows 83 and 84.The solution which is low in fuel is thus conducted by means of the supply line 69 into the absorber 32 and, due to the capillary action present, conveyed according to the arrows 83 and 84 into the given auxiliary gas conveyance line 62 of the absorber 32 in which the same slides downwards along its walls (arrow 84).",
"The auxiliary gas is conducted according to the arrows 82 through the auxiliary gas conveyance line 62 and through a central longitudinal hole 80 of the capillary sleeve 79 and flows according to the arrows 34 to the supply line 61 which actively connects the absorber 32 to the gas heat exchanger 29.A separating plate 85 connected to a jacket tube 86 of the absorber 32 makes it possible that the solution which is low in fuel, said solution being located in the absorber 32, can be backed up to a certain level of liquid (FIG.",
"2b: dotted line 71).",
"A given auxiliary gas conveyance line 62 equipped with a capillary sleeve 79 is advantageous since, due to the capillary action present at the time in the corresponding capillary gap 81, a uniform distribution of the solution which is low in fuel to the individual auxiliary gas conveyance line 62 in the absorber 32 is guaranteed independently of the level, present at the time, of the solution which is low in fuel in the absorber 32 or on the amount of the solution which is low in fuel flowing through the supply line 69 into the absorber 32 according to the arrows 39.In particular in the case of an oblique position (not represented) of the absorber 32, it is avoided by means of the capillary sleeves 79, due to capillary action setting in, that several auxiliary gas conveyance lines 62 are completely filled with solution which is low in fuel and the remaining auxiliary gas conveyance lines 62 are not pressurized with solution which is low in fuel so that the absorber 32 would not be functional, at least not completely.",
"According to a preferred form of embodiment not represented, each of the auxiliary gas conveyance line 62 can be provided with a plurality of projections acting as space holders at its free end projecting into the interior of the auxiliary gas conveyance line 62, by means of which projections a coaxial, with respect to the corresponding auxiliary gas conveyance lines 62, positioning of the respective capillary sleeve 79 is possible in a relatively simple manner.",
"Projections of this type at the free end of the respective capillary sleeve 79 are preferably distributed uniformly over its circumference and spaced sufficiently far from one another that a free flow of the solution which is low in fuel through the capillary gap according to arrows 83 and 84 is guaranteed.",
"Corresponding to another additional form of embodiment not represented, the lines 59 of the evaporator 24 are provided at their upper free end with a corresponding capillary sleeve 79 according to FIG.",
"4."
]
] | Patent_10297462 |
[
[
"Acetyl glucosaminyl inositol deacetylase, a mycothiol biosynthetic enzyme, and methods of use",
"The present invention provides a family of bacterial acetyl glucosaminyl inositol deacetylases (MshB) with deacetylase activity against acyl glucosaminyl inositol and which play a key role in mycothiol biosynthesis.",
"The invention deacetylases are characterized by a conserved 100 amino acid N-terminal region and three highly conserved histidine-containing regions and by having deacetylase activity as well as amide hydrolase activity.",
"The invention further provides methods for using the invention deacetylases in drug screening assays to determine compounds that inhibit activity.",
"The invention provides for treatment of actino-mycete infections in mammals using antibiotics that inhibit production or activity of MshB and thereby reduce the production of mycothiol and the virulence of the infecting bacteria."
],
[
"1.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, b) three domains of conservation, wherein two of the domains contain conserved histidine residues, and c) deacetylase activity against acetyl glucosaminyl inositol.",
"2.The purified deacetylase of claim 1, wherein the deacetylase hydrolyzes a C2-amide bond of the glucosaminyl inositol moiety.",
"3.The purified deacetylase of claim 1, wherein the acetyl glucosaminyl inositol is a precursor of mycothiol.",
"4.The purified deacetylase of claim 1, wherein the acetyl glucoasaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).",
"5.The purified deacetylase of claim 1, wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6, conservative variations thereof, and any combination of two or more thereof.",
"6.The purified deacetylase of claim 1, wherein the deacetylase is derived from an actinomycetes.",
"7.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis.",
"8.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. tuberculosis.",
"9.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. leprae.",
"10.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. bovis.",
"11.The purified deacetylase of claim 6, wherein the deacetylase is derived from M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum.",
"M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.",
"12.The purified deacetylase of claim 6, wherein the bacterium is selected from the group consisting of Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.",
"13.The purified deacetylase of claim 1, wherein the deacetylase has amidase activity against acyl glucosaminyl inositols.",
"14.The purified deacetylase of claim 1, wherein the deacetylase has an amino acid sequence as set forth in SEQ ID NO:1 15.The purified deacetylase of claim 1, wherein the deacetylase is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO:7.16.A purified acetyl glucosaminyl inositol deacetylase, characterized as having: a) an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, b) one or more domains of conservation containing conserved metal chelating residues, and c) deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.",
"17.The purified deacetylase of claim 16, wherein the metal ion is selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+.",
"18.The purified deacetylase of claim 17 wherein the metal ion is Zn2+.",
"19.The purified deacetylase of claim 16, wherein the metal chelating residues are selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.",
"20.An antibody, or functional fragment thereof, that binds specifically to an deacetylase of claim 1.21.An isolated polynucleotide that encodes a deacetylase of claim 1.22.A vector containing a polynucleotide that encodes an deacetylase of claim 1.23.A cell transformed with a vector of claim 22.24.A method for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase, said method comprising: a) contacting a candidate compound with a deacetylase of claim 1 in the presence of an acyl glucosaminyl inositol under suitable conditions and b) determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity or amidase activity, wherein the substantial absence of the deacetylase activity or the amidase activity is indicative of a candidate compound that inhibits activity of the deacetylase.",
"25.The method of claim 24, wherein the deacetylase is an acetyl glucosaminyl inositol deacetylase.",
"26.The method of claim 24, wherein a breakdown product is a free amine.",
"27.The method of claim 26, wherein the free amine is GlcN-Ins.",
"28.The method of claim 24, wherein a breakdown product is acetate 29.The method of claim 24, wherein a breakdown product is a derivative of cysteine.",
"30.The method of claim 24, wherein the acyl glucosaminyl inositol is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside.",
"31.The method of claim 24, wherein the acyl glucosaminyl inositol is an S-conjugate of mycothiol.",
"32.The method of claim 31, wherein the S-conjugate of mycothiol is the monobromobimane derivative of mycothiol (MSmB).",
"33.The method of claim 24, wherein the three domains in the deacetylase that contain conserved histidine residues have amino acid sequences selected from SEQ ID NOs: 3, 5, 6 and any combination of two or more thereof.",
"34.The method of claim 24, wherein the deacetylase is produced in an actinomycete.",
"35.The method of claim 34, wherein the actinomycete is M. smegmatis.",
"36.The method of claim 34, wherein the actinomycete is M. tuberculosis.",
"37.The method of claim 34, wherein the actinomycete is M. leprae.",
"38.The method of claim 34, wherein the actinomycete is M. bovis.",
"39.The method of claim 34, wherein the actinomycete is M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, or M. scrofulaceum.",
"40.The method of claim 24, wherein candidate compound is a polypeptide, polynucleotide or small molecule.",
"41.A high throughput screening method for identifying inhibitors of the deacetylase of claim 1, said method comprising: a) contacting each of a plurality of candidate compounds with a deacetylase of claim 1 in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and b) determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine, wherein the substantial absence of binding of the marker to a reaction mixture is indicative of a candidate compound that inhibits activity of the deacetylase.",
"42.The method of claim 41, wherein the plurality of reaction mixtures are formed in the wells of a microtiter plate.",
"43.The method of claim 41, wherein the detectable marker is colorometric or fluorometric.",
"44.The method of claim 43 wherein the fluorometric marker is fluorescamine or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.",
"45.An inhibitor of the deacetylase of claim 1 wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.",
"46.The inhibitor of claim 45 wherein the inhibitor additionally chelates one or more metal ions metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.",
"47.A derivative of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.",
"48.The derivative of claim 47, wherein the reactive residue is a nitroso residue.",
"49.The derivative of claim 47, wherein the reactive residue is a nitroalkyl residue comprising 1 to 3 carbon atoms.",
"50.The derivative of claim 47, wherein the reactive residue is a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms.",
"51.The derivative of claim 47, wherein the reactive residue is a peroxyalkyl residue comprising 2 to 4 carbon atoms.",
"52.A live mutant actinomycete, whose genome comprises a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein said disruption results in said mutant actinomycetes exhibiting transient survival in mammalian white blood cells for an immune response-raising period of time.",
"53.The live mutant actinomycete of claim 52, wherein the period of time is from 1 to 30 days.",
"54.The live mutant actinomycete of claim 52, wherein the survival of the mutant actinomycetes in mammalian white blood cells does not exceed 30 days.",
"55.The live mutant actinomycete of claim 52, wherein the mutant actinomycete is derived from a pathogen selected from the group consisting of M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, and M. scrofulaceum.",
"56.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes.",
"is derived from M. smegmatis.",
"57.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. tuberculosis.",
"58.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. leprae.",
"59.The live mutant actinomycete of claim 52, wherein the mutant actinomycetes is derived from M. bovis and said mammal is bovine.",
"60.A method for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells, said method comprising: introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity, wherein the intracellular presence of the inhibitor decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.",
"61.The method of claim 60, wherein the inhibitor inhibits intracellular production of the deacetylase.",
"62.The method of claim 60, wherein the inhibitor inhibits intracellular deacetylase activity of the deacetylase.",
"63.The method of claim 60, wherein the introducing comprises culturing the bacterium in the presence of the inhibitor.",
"64.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.",
"65.The method of claim 60, wherein the inhibitor is an anti-sense oligonucleotide that hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.66.The method of claim 60, wherein the bacterium is an actinomycete and the inhibitor inhibits intracellular production of mycothiol.",
"67.The method of claim 60, wherein the bacterium is selected from the group consisting of the pathogenic bacteria M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M abscessus, and M. scrofulaceum.",
"68.The method of claim 60, wherein the bacterium is an actinomycete.",
"69.A method for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, said method comprising administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.",
"70.The method of claim 69, wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates a metal ion selected from the group consisting of Mn2+ and Ni2, Cd2+, Co2+, and Zn2+, or otherwise binds the enzyme active site in the deacetylase.",
"71.The method of claim 70, wherein the moiety is ClCH2CONH—.",
"72.The method of claim 70 wherein the moiety is HONHCONH—.",
"73.The method of claim 70, wherein the moiety is HONHCOCH2—.",
"74.The method of claim 70, wherein the moiety is HOPO(CH3)NH—.",
"75.The method of claim 70, wherein the moiety is HOPO(CH3)nCH2—; wherein n=1-5.76.The method of claim 70 wherein the moiety is HSCH2(CH2)nNH—; wherein n=2-5.77.The method of claim 70 wherein the moiety is HS(CH2)nCONH—; wherein n=1-3.78.The method of claim 69 wherein the acetyl glucosaminyl inositol-producing bacterium is a mycothiol-producing bacterium.",
"79.A process for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), said method comprising: contacting an acyl glucosaminyl inositol with an deacetylase of claim 1 under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.",
"80.A method for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample, said method comprising: a) contacting a sample containing GlcNAc-Ins with a deacetylase of claim 1 under suitable conditions and b) determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.",
"81.The method of claim 80, wherein the amount of GlcN-Ins is determined by HPLC after labeling thereof with a fluorometric or calorimetric reagent.",
"82.The method of claim 81, wherein the fluorometric reagent is fluorescamine.",
"83.The method of claim 81, wherein the fluorometric reagent is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate."
],
[
"<SOH> BACKGROUND INFORMATION <EOH>Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.",
"However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.",
"Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.",
"Actinomycetes, including Streptomyces and Mycobacteria , do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).",
"The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.",
"However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.",
"First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.",
"(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)",
"pp 517-551, John Wiley & Sons, et al.).",
"An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.",
"(1997) FEBS Lett.",
"409:221-222).",
"This enzyme has been sequenced (A. Norin, et al.",
"(1997) Eur.",
"J. Biochem.",
"248:282-289).",
"The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.",
"(1999) J. Amer.",
"Chem.",
"Soc.",
"120:11538-11539; M. P. Patel et al.",
"(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).",
"The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.",
"Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.",
"Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis , is a well-known problem faced by medical practitioners in treatment of bacterial diseases.",
"Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals."
],
[
"<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.",
"In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.",
"Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.",
"In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.",
"Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.",
"In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.",
"The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.",
"Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.",
"In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.",
"In still another embodiment, the present invention provides live mutant actinomycetes.",
"The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.",
"In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.",
"The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.",
"By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.",
"For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.",
"In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn 2+ , or otherwise binds the enzyme active site of the deacetylase.",
"In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.",
"In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins."
],
[
"FIELD OF THE INVENTION The present invention generally relates to a family of enzymatic compounds produced by bacteria and methods of their use in drug discovery and disease control, and more specifically to acetyl glucosaminyl inositol deacetylases and methods of their use.",
"BACKGROUND INFORMATION Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins.",
"However, most gram positive bacteria, including many strict aerobes, do not produce glutathione.",
"Yet aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader.",
"Actinomycetes, including Streptomyces and Mycobacteria, do not make GSH but produce instead millimolar levels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).",
"The biochemistry of mycothiol appears to have evolved completely independently of that of glutathione.",
"However, it has already been established that the metabolism of mycothiol parallels that of glutathione metabolism in two enzymatic processes.",
"First, formaldehyde is detoxified in glutathione-producing organisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L. Uotila, et al.",
"(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part A (D. Dolphin, et al., Eds.)",
"pp 517-551, John Wiley & Sons, et al.).",
"An analogous process involving NAD/mycothiol-dependent formaldehyde dehydrogenase has been identified in the actinomycete Amycolatopsis methanolica (M. Misset-Smits, et al.",
"(1997) FEBS Lett.",
"409:221-222).",
"This enzyme has been sequenced (A. Norin, et al.",
"(1997) Eur.",
"J. Biochem.",
"248:282-289).",
"The second enzymatic process involves a mycothiol homolog of glutahione reductase recently cloned from M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.",
"(1999) J. Amer.",
"Chem.",
"Soc.",
"120:11538-11539; M. P. Patel et al.",
"(1999) Biochemistry 38:11827-11833; M. P. Patel et al (2001) Biochemistry 40:3119-3126).",
"The reductase is reasonably specific for the disulfide of mycothiol, but is also active with the disulfide of AcCys-GlcN, the desmyo-inositol derivative of mycothiol.",
"Therefore, there is a need in the art for methods and compounds useful for investigation of the details of the metabolism of mycothiol and comparison with the established roles for the metabolism of glutathione.",
"Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases.",
"Therefore, there is a need in the art for new antibiotics, drugs and vaccines and for screening techniques to discover antibiotics, drugs and vaccines effective to treat or prevent bacterial infections in humans and in other mammals, such as domestic and farm animals.",
"SUMMARY OF THE INVENTION The present invention solves these and other problems in the art by providing, in one embodiment, purified acetyl glucosaminyl inositol deacetylases, characterized as having an N-terminal region with an amino acid sequence with 40% or more sequence identity to SEQ ID NO:2 and conservative variations thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.",
"In another embodiment, the invention provides purified acetyl glucosaminyl inositol deacetylases characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.",
"Antibodies and functional fragment thereof, that bind specifically to invention deacetylases, isolated polynucleotide that encode the invention deacetylases, vectors containing a polynucleotide that encodes an invention deacetylase and cells containing such vectors are also provided.",
"In another embodiment, the invention provides methods for identifying an inhibitor of acetyl glucosaminyl inositol deacetylase by contacting a candidate compound with an invention deacetylase in the presence of an acyl glucosaminyl inositol under suitable conditions and determining the presence or absence of breakdown products of the acyl glucosaminyl inositol indicative of deacetylase activity.",
"Substantial absence of the breakdown product of deacetylase activity is indicative of a candidate compound that inhibits activity of the deacetylase.",
"In yet another embodiment, the present invention provides high throughput screening methods for identifying inhibitors of the invention deacetylases.",
"The screening methods involve contacting each of a plurality of candidate compounds with an invention deacetylase in the presence of acyl glucosaminyl inositol under suitable conditions to form a plurality of reaction mixtures and determining the presence or absence of binding to the reaction mixtures of a detectable marker that binds to free amine.",
"Substantial absence of binding of the marker to a reaction mixture in the invention screening methods is indicative of a candidate compound that inhibits activity of the deacetylase.",
"In yet another embodiment, the present invention provides derivatives of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress and wherein the derivative is selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.",
"In still another embodiment, the present invention provides live mutant actinomycetes.",
"The invention mutant actinomycetes have a genome with a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene, wherein said disruption prevents function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids are substantially unaffected, and wherein the disruption results in the mutant actinomycetes exhibiting survival in mammalian white blood cells for an immune response raising period of time.",
"In yet another embodiment, the present invention provides methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium in mammalian cells by introducing into the bacterium an inhibitor of acetyl glucosaminyl inositol deacetylase activity.",
"The intracellular presence of the inhibitor in the bacterium decreases activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.",
"By virulence is meant the relative power and degree of pathogenicity possessed by organisms to produce disease as measured by clinical symptoms particular to the disease under consideration.",
"For example, the virulence of a M. tuberculosis is measured with reference to the manifestation in an infected individual of the clinical symptoms recognized by a medical practitioner as indicative of tuberculosis.",
"In another embodiment, the present invention provides inhibitors of the invention acetyl glucosaminyl inositol deacetylases wherein the inhibitor is derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+, or otherwise binds the enzyme active site of the deacetylase.",
"In still another embodiment, the present invention provides methods for inhibiting growth of an acetyl glucosaminyl inositol-producing bacterium in a mammal, by administering to the mammal an effective amount of an inhibitor of intracellular acetyl glucosaminyl inositol deacetylase, thereby inhibiting growth of the bacterium in the mammal.",
"In yet another embodiment, the present invention provides processes for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) by contacting an acyl glucosaminyl inositol with an invention deacetylase under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.",
"BRIEF DESCRIPTION OF THE DRAWINGS FIGS.",
"1A and B show the nucleotide sequence of M. tuberculosis deacetylase gene mshB (Rv1170) (SEQ ID NO:7).",
"FIG.",
"1C shows the deduced amino acid sequence of M. tuberculosis GlcNAc-Ins deacetylase (SEQ ID NO:1) encoded by mshB.",
"FIG.",
"2 is a schematic drawing showing a map of plasmid pYA1170b employed to express Rv1170 in E. coli.",
"FIG.",
"3 is a chart showing the chemical structures of the substrates for assaying deacetylase and amidase activities of the invention acetyl glucosaminyl inositol deacetylases (MshB), such as MshB Rv1170 and homologs.",
"FIG.",
"4 is a graph showing growth of a mutant M. smegmatis (mutant 49) in 7H9 Middlebrook medium containing 17 μM GlcNAc-Ins.",
"Mycothiol (MSH) and MSH precursor contents in cells are expressed in micromoles per gram of residual dry weight (RDW).",
"FIG.",
"5 is a schematic representation of the proposed MSH biosynthesis pathway.",
"FIG.",
"6 is a chart showing alignment of the amino acid sequences of four homologs of MshB of M. tuberculosis H37Rv (Rv1170) GenBank accession number B70875 and M. leprae GenBank accession number CAC30445.Sequences for M. avium (The Institute for Genomic Research (TIGR)), and M. smegmatis (Sanger Center) are from unfinished genomes.",
"FIG.",
"7 is a graph showing MshB deacetylase activity with 0.1 mM GlcNAc-Ins in the presence of 1,10-phenanthroline.",
"FIG.",
"8A is a graph showing MshB deacetylase reaction specific activity with 0.8 mM GlcNAc-Ins as substrate in the presence of increasing concentrations of thiol inhibitors.",
"FIG.",
"8B is a graph showing MshB amidase reaction specific activity with 0.55 mM MSmB as substrate in the presence of increasing concentrations of inhibitors.",
"FIG.",
"9 is a map of the plasmid pYA1170C used to express the recombinant His-tagged acetyl glucosaminyl inositol deacetylase encoded by M. tuberculosis Rv1170.FIG.",
"10 is a graph showing percent survival of strains of M. smegmatis versus hydrogen peroxide concentration after treatment as described in Example 11 herein.",
"Open triangles=mutant TN3, which produces no MSH; open circles=chemical mutant 164, which produces a low residual MSH content; and darkened squares=high MSH content mc2 155.FIG.",
"11 is a graph showing percent survival over time of strains of M. smegmatis in murine macrophages as described in Example 11 herein.",
"Open circles=chemical mutant TN3 (0% MSH content), Experiment 1; open triangles=mutant TN3 (0% MSH content), Experiment 2; and darkened squares=mc2155 (100% MSH content).",
"DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there are provided a family of purified acetyl glucosaminyl inositol deacetylase polypeptides with enzymatic deacetylase activity for acetyl glucosaminyl inositol.",
"The invention acetyl glucosaminyl inositol deacetylases are characterized by having an N-terminal region with an amino acid sequence with 40% or greater sequence identity to an amino acid sequence as set forth in SEQ ID NO:2 and conservative variation thereof, four domains of conservation, wherein three of the domains contain conserved histidine residues, and deacetylase activity against acetyl glucosaminyl inositol.",
"SEQ ID NO:2 corresponds to amino acid residues 1-100 of amino acids of SEQ ID NO:1 (FIG.",
"1C).",
"The members of the family of invention deacetylases hydrolyze a C2-amide bond of acetyl glucosaminyl inositol moiety, such as N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).",
"Preferably the acetyl glucosaminyl inositol is a precursor of mycothiol (e.g., in a mycothiol producing bacterium).",
"In preferred embodiments the three histidine-containing conserved domains in the invention deacetylases are selected from VHAHPDDE (SEQ ID NO:3), which corresponds to amino acids 10 through 17 of SEQ ID NO:1; VTCTLGEXGEV (SEQ ID NO:4), which corresponds to amino acids 38 through 49 of amino acid SEQ ID NO:1; YDPXGGYGHPDH (SEQ ID NO:5), which corresponds to amino acids 136 through 147 of SEQ ID NO:1; and AXXAHATQ (SEQ ID NO:6), which corresponds to amino acids 240 through 247 of amino acid SEQ ID NO:1 (FIG.",
"1C) wherein X is any amino acid; and conservative variations thereof, or any combination of any two or more thereof (See FIG.",
"6).",
"Members of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides are metalloproteases.",
"Accordingly, the invention further provides purified acetyl glucosaminyl inositol deacetylases that are characterized as having an N-terminal region with an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, one or more domains of conservation containing conserved metal chelating residues, and deacetylase activity against acetyl glucosaminyl inositol in the presence of metal ion.",
"The invention deacetylases have been shown to contain an active site that contains a metal ion selected from the group consisting of Mn2+, Ni2, Cd2+, Co2+, and Zn2+, with the preferred metal ion being Zn2+.",
"The metal chelating residues in the invention deacetylases are preferably selected from the group consisting of histidine, aspartic acid and glutamic acid, and combinations thereof.",
"In preferred embodiments, the invention deacetylases have additional activity as amidases, catalyzing the hydrolysis of acyl glucosaminyl inositols.",
"As used herein, the term “acyl glucosaminyl inositol” means substrate compounds for the invention family of deacetylases having the chemical formula R—CONH-GlcN-Ins wherein R═CH3(CH2)n— where n=0-6.Included in the group of substrate compounds is the natural substrate, acetyl-GlcN-Ins, where n=0.Members of this group of substrate compounds are derived from an alkanoic acid.",
"In addition, compounds having the chemical structure R—CONH-GlcN-Ins wherein R=aryl-(CH2)n, where n=0-6 are encompassed by the term.",
"Members of this group of substrate compounds are derived from an aryl alkanoic acid such as phenyl-(CH2)nCOOH.",
"The acyl glucosaminyl inositols of the above chemical formula can also be derived from the reaction of GlcN-Ins with any commercial acid chloride in the form RCOCl, wherein R is: o-tolyl-, 4-ethylphenyl-, 4-propylphenyl-, 4-biphenyl-, 3,4-dimethoxyphenyl-, 3,4,5-trimethoxyphenyl-, 2-furyl-, and the like.",
"The acyl glucosaminyl inositols of the above chemical formula can also be derived from an amino acid or an N-acetyl amino acid.",
"Preferred amino acids include N-acetylcysteinyl-S—R′ or cysteinyl-S—R′, wherein R′ is an organic group attached to the cysteine sulfur, such as may be derived from commercial thiol labeling reagents like 2-bromoacetophenone, monobromobinane, N-ethylmaleimide, cerulenin, 7-diethylamino-3-(4′-maleimidyl phenyl)-4-methyl coumarin, 3(N-maleimidopropionyl)biocytin, and the like.",
"The studies described herein further elaborate the pathway involved in MSH biosynthesis.",
"The present studies demonstrate that GlcNAc-Ins is a major intracellular MSH component in M. smegmatis and is converted to GlcN-Ins by GlcNAc-Ins deacetylase.",
"This conversion defines the second step in MSH biosynthesis.",
"The discovery described herein that Rv1170 is the gene encoding the deacetylase in the M. tuberculosis genome represents the first gene of the MSH biosynthesis pathway to be identified.",
"Assuming that a single enzyme produces GlcNAc-Ins, it is proposed that the genes for the MSH biosynthesis pathway be designated mshA, mshB, mshC, and mshD, with the corresponding enzymes labeled as shown in FIG.",
"5.Identification of mshA, mshC, and mshD would be greatly simplified if they were clustered with mshB in a single operon, but inspection of the gene assignments and open reading frames surrounding mshB (Rv1170) in the M. tuberculosis genome indicates that this is not the case.",
"All mycobacteria thus far examined have high MSH content (G. L. Newton et al.",
"(1996) J. Bacteriol.",
"178:1990-1995) and, based on the studies described herein, are expected to have an ortholog of the MSH biosynthesis enzyme MshB (Rv1170).",
"The available unfinished mycobacterial genome databases yield homologs close to Rv1170 for Mycobacterium leprae (Sanger Centre), Mycobacterium bovis (Sanger Centre), M tuberculosis CDC1551 (The Institute for Genomic Research [TIGR]), M. smegmatis (TIGR), and Mycobacterium avium (TIGR).",
"This sequence homology (FIG.",
"6) indicates that MSH biosynthesis in these organisms utilizes a GlcNAc-Ins deacetylase (MshB) in the same manner as that described here for M. smegmatis.",
"Other actinomycetes that produce MSH are also expected to have a GlcNAc-Ins deacetylase (MshB) gene homologous to Rv1170.Sequence BLAST searches to determine molecules with amino acid homology to the Rv1170 sequence of SEQ ID NO:1 (FIG.",
"1C) also show M. tuberculosis homolog Rv1082 (mycothiol S-conjugate deacetylase) and the Rv1082 orthologs from M. leprae, M. bovis (Sanger Centre), M. tuberculosis CDC1551 (TIGR), M. smegmatis (TIGR), and M. avium (TIGR) to be close homologs.",
"Sequences with partial homology to Rv1170 were also found in the yeast, rat, and human genome databases.",
"These genes code for PIG-L, the second enzyme in glycosylphosphatidylinositol (GPI) anchor biosynthesis (Nakamura et al., (1997) J. Biol.",
"Chem.",
"272:15834-15840), and catalyze the deacetylation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI).",
"The studies described in the Examples herein indicate that the invention deacetylase is rather specific for GlcNAc-Ins and is not a broad spectrum deacetylase.",
"For example, removal of the inositol residue from the substrate GlcNAc-Ins resulted in a more than 300-fold decrease in reactivity (i.e., GlcNAc-Ins versus GlcNAc) (Table 1).",
"Also, the studies described herein (Table 1) show that neither MSH nor MSmB is deacetylated by the invention deacetylases.",
"Therefore, the AcCys moiety of MSH is inert to the deacetylase.",
"This finding shows that the invention deacetylase has no significant activity for the degradation of MSH.",
"The invention deacetylase appears to be the control point for MSH biosynthesis.",
"In cells tested, a very large endogenous pool of its substrate GlcNAc-Ins (12 to 15 μmol per g of residual dry weight (RDW)) was found (Table 2), but quite low levels were found of its product GlcN-Ins (˜0.2 μmol per g of RDW) (Table 2) and of the final intermediate Cys-GlcN-Ins (<0.006 μmol per g of RDW) leading to MSH.",
"This finding shows that substantial quantities of MSH can be produced upon demand from the endogenous pool of GlcNAc-Ins under control of the invention family of acetyl glucosaminyl inositol deacetylases.",
"If a particular member of the invention family of polypeptide deacetylases hydrolyzes its substrate (GlcNAc-Ins), cleavage of the substrate molecule will form breakdown products wherein one product is a free amine (e.g., 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins)) and the other product is acetate.",
"If a particular member of the invention family of polypeptide deacetylases has activity as an amide hydrolase (amidase), cleavage of the substrate acyl glucosaminyl inositol will form breakdown products wherein one product is a carboxylic acid, and the other product is a free amine (e.g., GlcN-Ins).",
"If the substrate amide is a mycothiol-derived amide, one of the breakdown products will be GlcN-Ins and the other breakdown product will be a sulfur-containing carboxylic acid, such as an AcCys S-conjugate.",
"AcCys S-conjugates are termed mercapturic acids, the final excreted product in the mercapturic acid pathway of glutathione-dependent detoxification in mammals.",
"Mycothiol (1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside) (MSH) is present in a variety of actinomycetes and plays an essential role in a pathway of detoxification in such bacteria.",
"Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes.",
"In the mycothiol-dependent detoxification process in actinomycetes, an alkylating agent is converted to a S-conjugate of mycothiol, the latter is cleaved to release a mercapturic acid, and the mercapturic acid is excreted from the cell (Newton et al.",
"(2000) Biochemistry 39:10739-10746).",
"It has been discovered that invention acetyl glucosaminyl inositol deacetylases participate in the pathway of mycothiol biosynthesis in which substrate GlcNAc-Ins is an intermediate.",
"In the mycothiol pathway as shown schematically in FIG.",
"5, GlcNAc-Ins is converted to GlcN-Ins by the invention deacetylase.",
"In fact, the studies disclosed herein have shown that the invention deacetylases are the critical step in the production of mycothiol in actinomycetes.",
"For example, it has been shown by the studies described herein (Example 10) that MshB is inhibited by mycothiol in a manner consistent with a negative feedback loop dependent upon the level of mycothiol for control of mycothiol biosynthesis in the cell.",
"Thus, a falling level of mycothiol in the cell available for detoxification of intracellular toxins increases activity of the deacetylase so that substrate GlcNAc-Ins will be cleaved by the expressed deacetylase, in turn, providing the GlcN-Ins moiety necessary for mycothiol production.",
"A member of the invention family of polypeptide deacetylases shown to be responsible for cleavage of GlcNAc-Ins has been cloned from M. tuberculosis gene Rv1170 and has an amino acid sequence as set for in SEQ ID NO:1 (shown in FIG.",
"1C; see also GenBank Accession No.",
"B70875) and a nucleotide sequence as set forth in SEQ ID NO:7 (shown in FIG.",
"1; see also nucleic acid residues 4222-5133 of GenBank Accession No.",
"AL010186).",
"The N-terminal region 100 residues of this newly discovered GlcNAc-Ins deacetylase has the amino acid sequence as shown in SEQ ID NO:2.Members of invention family of acetyl glucosaminyl inositol deacetylases are formed in vivo by bacteria as part of a mycothiol biosynthesis pathway, most usually in bacteria characterized by intracellular production of mycothiol.",
"Additional bacteria from which the invention deacetylase polypeptides can be derived include actinomycetes, such as M. smegmatis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC) (Holzman, in Tuberculosis ed.",
"by Rom and Gary (Little, Brown, and Company, 1996) Chapter 56), M. ulcerans, M. abscessus, or M. scrofulaceum, and the like.",
"Homologous non-mycobacterial deacetylase proteins can also be derived from the antibiotic producers Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei, and the polyketide erythromycin antibiotic producer Saccharopolyspora erythraea.",
"Inhibitors of the invention acetyl glucosaminyl inositol deacetylases are particularly well suited as antibiotics since mycothiol production will cease in the absence of the product GlcN-Ins produced by activity of the invention deacetylases.",
"Accordingly, in another embodiment according to the present invention, there are provided methods for identifying an inhibitor of an invention acetyl glucosaminyl inositol deacetylase, MshB.",
"The invention methods comprise contacting a candidate compound with a member of the invention family of deacetylases in the presence of a substrate for the enzyme under suitable conditions and determining the presence or absence of breakdown products of the substrate indicative of deacetylase (or optionally S-conjugate amidase) activity.",
"For example, if the test compound is a putative inhibitor of amide hydrolase activity of the invention polypeptide deacetylase, the absence of acetate or other carboxylic acid and/or free amine, such as GlcN-Ins, as breakdown products indicates the candidate compound is an inhibitor of the activity of the invention polypeptide as a deacetylase.",
"The preferred substrate for testing for deacetylase activity of the invention polypeptides is the acetyl glucosaminyl inositol (GlcNAc-Ins).",
"On the other hand, if the test compound is a putative inhibitor of MshB, the appearance of free amine, such as GlcN-Ins, and/or acetate or carboxylate indicates that the candidate compound is not an inhibitor of the activity of the invention deacetylase as a deacetylase or as an amidase of an acyl glucosaminyl inositol.",
"A preferred substrate for use in screening for inhibitors of deacetylase activity is N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins).",
"Substantial absence of the breakdown products of enzymatic activity is indicative of a candidate compound that inhibits enzymatic activity of the deacetylase.",
"In a preferred embodiment, the deacetylase used in the invention method is an acetyl glucosaminyl inositol deacetylase and the method is designed to assay for production of free amine.",
"Such assays can be conducted en masse using a high throughput screening method for identifying inhibitors of the invention deacetylase.",
"For example, a plurality of candidate compounds can be contacted with an invention deacetylase in the presence of a substrate for the enzyme (e.g.",
"an acyl glucosaminyl inositol) under suitable conditions (e.g., as shown in the Examples herein) for activity of the deacetylase.",
"Binding of a marker selected to bind to a breakdown product can then be assayed using any technique known in the art for high throughput screening.",
"For example, a colorometric or fluorometric marker that binds to free amine can be used to screen a plurality of reaction mixtures formed in the wells of a microtitre plate (Wong, et al.",
"(2001) Anal.",
"Biochem.",
"290:338-346).",
"A preferred fluorometric marker for use in the invention high throughput screening methods is fluorescamine, which binds to free amine.",
"In another embodiment of the invention, methods are provided for determination of acetyl glucosaminyl inositol (GlcNAc-Ins) in a sample by contacting a sample containing GlcNAc-Ins with an invention deacetylase under suitable conditions and determining the amount of GlcN-Ins produced, wherein the amount of GlcN-Ins produced is a measure of the GlcNAc-Ins in the sample.",
"The amount of GlcN-Ins in the sample can be determined by HPLC after labeling thereof with a fluorometric or colorimetric reagent, such as 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (sold as AccQFluor® reagent by Waters), and the like.",
"In an alternative embodiment of the invention, methods are provided for decreasing the virulence in mammalian cells of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium, such as an actinomycete.",
"In the invention method for decreasing the virulence of pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria, an inhibitor of acetyl glucosaminyl inositol deacetylase (for example, one identified by the above-described screening method) is introduced into the bacterium.",
"Intracellular uptake by the treated bacterium of the inhibitor results in decreased activity of the deacetylase, thereby decreasing mycothiol biosynthesis by the bacterium as compared with untreated control bacterium.",
"For example, for treatment of isolated mammalian cells, the introducing can comprise culturing the bacterium in the presence of the inhibitor.",
"Alternatively, for treatment of mammalian cells contained in a living organism, the inhibitor may be administered systemically to the living organism.",
"Pathogenic acetyl glucosaminyl inositol deacetylase-producing bacteria whose virulence can be reduced according to the invention methods include such actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis particularly in bovine subjects), M. intracellulare, M. africanum, and M. marinarum.",
"M. chelonai, Corynebacterium diphtheriae, Actinomyces israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.",
"The inhibitors used in the invention methods for decreasing the virulence of a pathogenic acetyl glucosaminyl inositol deacetylase-producing bacterium may either inhibit intracellular production of the deacetylase or inhibit intracellular deacetylase activity of the deacetylase.",
"In one embodiment according to the present invention, there are provided inhibitors of the members of the invention family of deacetylase polypeptides.",
"The invention inhibitors are characterized by being derived from GlcN-Ins by replacing the amino group therein with a moiety that chelates Zn2+ or otherwise binds the enzyme active site.",
"The invention inhibitors may also chelate one or more metal ions selected from the group consisting of Mn2+ and Ni2, Cd2+, and Co2+.",
"The following are examples of moieties that can substitute for the amino group in GlcN-Ins in the invention inhibitors: ClCH2CONH— HONHCONH— HONHCOCH2— HOPO(CH3)NH— HOPO(CH3)nCH2—; wherein n=1-5 HSCH2(CH2)nNH—; wherein n=2-5 HS(CH2)nCONH—; wherein n=1-3.In another embodiment according to the present invention, there are provided derivatives of GlcN-Ins, wherein the derivative contains a reactive residue attached to the amino group that promotes oxidative stress (e.g., including nitrosative stress) so as to be selectively toxic to a mycothiol-producing actinomycete by being concentrated in the actinomycete.",
"Examples of reactive residues that can be attached to the amino group of GlcN-Ins to cause the compound to be selectively toxic to mycothiol-producing actinomycetes by intracellular concentration include, but are not limited to a nitroso residue, a nitroalkyl residue comprising 1 to 3 carbon atoms, a S-nitrosomercaptoalkyl residue comprising 2 to 4 carbon atoms, or a peroxyalkyl residue comprising 2 to 4 carbon atoms.",
"In another embodiment according to the present invention, the invention inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with 40% or more sequence identity to the amino acid sequence of SEQ ID NO:2 or 80% or more sequence identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6 and conservative variations thereof.",
"In another embodiment the anti-sense oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.For example, in one embodiment, the candidate compound inhibits intracellular production or activity of the acetyl glucosaminyl inositol deacetylase.",
"A presently preferred drug candidate for screening in live bacteria for activity that inhibits intracellular production or activity of acetyl glucosaminyl inositol deacetylase is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence with 40% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:2, or a conservative variation thereof, for example, 50%, 55%, 60% or 65% sequence identity.",
"Suitable conditions for conducting invention drug screening methods are well known in the art and are described, for example, in the Examples hereinbelow.",
"Alternatively, an anti-sense oligonucleotide can be designed to hybridize under in vivo conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2, or contains an amino acid segment as set forth in SEQ ID NOs:3, 4, 5, and 6, or a conservative variation thereof.",
"The anti-sense oligonucleotide can comprise from about 10 to about 60 nucleic acid residues, for example from 10 to about 50, or from 10 to about 40, 30 or 20 nucleic acid residues.",
"“Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing.",
"Hybridization reactions can be sensitive and selective so that a particular sequence of interest will join with a complementary strand even in samples in which it is present at low concentrations.",
"Suitable intracellular conditions for hybridization of an anti-sense oligonucleotide to messenger RNA will be determined by the particular bacterium used in the invention method.",
"In general, the pH, temperature and salt concentration must be comparable to intracellular conditions in the test bacterium.",
"In yet another embodiment according to the present invention, there are provided live mutant actinomycetes, whose genomes comprise a disruption in an endogenous acetyl glucosaminyl inositol deacetylase gene and thereby prevents mycothiol synthesis.",
"Disruption of genes for mycothiol biosynthesis in mycobacteria reduces their survival in mammalian macrophages (FIG.",
"11).",
"Disruption of the endogenous acetyl glucosaminyl inositol deacetylase gene will prevent function of an endogenous acetyl glucosaminyl inositol deacetylase while cell surface proteins and lipids should be substantially unaffected.",
"As a result, invention live mutant actinomycetes exhibit the phenotype of transient survival in mammalian white blood cells, such as murine or human white blood cells, for an immune response raising period of time.",
"Such genetically engineered live mutant actinomycetes will survive in mammalian white blood cells for a period of time from 1 to 30 days, for example from 4 to 25 days or from 5 to 20 days, but in no event for more than 30 days.",
"Due to lack of intracellular acetyl glucosaminyl inositol deacetylase the invention live mutant bacterium will fail to produce mycothiol.",
"Hence, the mutant live bacterium is unable to survive the oxidative stress inherent in the intracellular environment of mammalian white blood cells long enough to establish infection in the cells or to establish infection in an immunocompetent mammal containing such white blood cells.",
"Thus, invention live mutant actinomycetes possess a combination of features desired for a vaccine effective in mammals against infection by such pathogenic actinomycetes as M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomycetes israelii, M. avium complex (MAC), M. ulcerans, M. abscessus, M. scrofulaceum, and the like.",
"An individual (e.g., an animal, such as a mouse, a farm animal or a human) to which the live mutant is administered according to a protocol appropriate for inducing a protective immune response and who has not previously been infected by the counterpart wild type will mount an immune response to the vaccine, for example an immune response sufficient to protect the individual against future infection by the corresponding wild-type live pathogen.",
"Alternatively, the invention live mutant actinomycetes are useful as a research tool to investigate the properties desirable in a live mutant vaccine.",
"In still another embodiment according to the present invention, there are provided processes for preparation of GlcN-Ins by contacting an N-acyl glucosaminyl inositol under suitable conditions with an acetyl glucosaminyl inositol deacetylase so as to hydrolyze the amide bond therein to obtain stereochemically pure α(1-1) GlcN-Ins (1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside).",
"For example, the N-acyl glucosaminyl inositol can be a mycothiol S-conjugate, such as the bimane derivative of mycothiol.",
"The deacetylase cleaves the N-acyl glucosaminyl inositol, freeing GlcN-Ins as one of the cleavage breakdown products.",
"GlcN-Ins has utility in conducting research regarding deacetylase activity and mycothiol biochemistry in bacteria, development of products and procedures for overcoming the antibiotic resistance of pathogenic bacteria, such as actinomycetes, and as a precursor for formation of acyl glucosaminyl inositol derivatives and inhibitors of deacetylases thereof as disclosed herein.",
"A “conservative variation” in an amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.",
"A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).",
"One or more amino acids can be deleted, for example, from an deacetylase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.",
"For example, carboxyl-terminal amino acids that are not required for deacetylase or deacetylase activity of the invention polypeptides can be removed.",
"Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.",
"Such vectors are used to incorporate DNA sequences of the invention.",
"In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted eukaryotic genetic sequence are used in connection with the host.",
"The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells.",
"In addition to expression vectors known in the art such as bacterial, yeast and mammalian expression systems, baculovirus vectors may also be used.",
"One advantage to expression of foreign genes in this invertebrate virus expression vector is that it is capable of expression of high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts.",
"Baculovirus vectors and the appropriate insect host cells used in conjunction with the vectors will be known to those skilled in the art.",
"The term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the invention acetyl glucosaminyl inositol deacetylase genetic sequences.",
"Such expression vectors contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host.",
"The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.",
"Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol.",
"Chem., 263:3521, 1988) and baculovirus-derived vectors for expression in insect cells.",
"The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).",
"The vector may include a phenotypically selectable marker to identify host cells which contain the expression vector.",
"Examples of markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin (β-lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase).",
"Examples of such markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and xanthine guanine phosphoribosyltransferse (XGPRT, gpt).",
"The isolation and purification of host cell expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody.",
"Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art.",
"Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by electroporation or the CaCl2 method using procedures well known in the art.",
"Alternatively, MgCl2 or RbCl could be used.",
"Where the host used is a eukaryote, various methods of DNA transfer can be used.",
"These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or the use of virus vectors.",
"Eukaryotic cells can also be cotransformed with DNA sequences encoding the polypeptides of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.",
"Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.",
"(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).",
"Examples of mammalian host cells include COS, BHK, 293, and CHO cells.",
"Eukaryotic host cells may also include yeast.",
"For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells.",
"Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose, M. et al., Gene, 60:237, 1987).",
"The invention provides antibodies that are specifically reactive with invention deacetylase polypeptides or fragments thereof.",
"Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.",
"Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).",
"Monoclonal antibodies specific for acetyl glucosaminyl inositol deacetylase polypeptide can be selected, for example, by screening for hybridoma culture supernatants that react with acetyl glucosaminyl inositol deacetylase polypeptides, but do not react with other bacterial deacetylases.",
"Antibody that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.",
"Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).",
"The term “antibody” as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2 and Fv, which are capable of binding the epitopic determinant.",
"These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.",
"Methods of making these fragments are known in the art.",
"(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference).",
"As used in this invention, the term “epitope” means any antigenic determinant on an antigen to which the paratope of an antibody binds.",
"Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.",
"Antibodies that bind to acetyl glucosaminyl inositol deacetylase polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.",
"The polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis and can be conjugated to a carrier protein, if desired.",
"Such commonly used carriers, which are chemically coupled to the peptide, include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.",
"The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).",
"If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.",
"Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated herein by reference).",
"It is also possible to use the anti-idiotype technology to produce monoclonal antibodies that mimic an epitope.",
"For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the “image” of the epitope bound by the first monoclonal antibody.",
"In yet other preferred embodiments, the recombinant acetyl glucosaminyl inositol deacetylase polypeptide is a fusion protein further-comprising a second polypeptide portion having an amino acid sequence from a protein unrelated to the acetyl glucosaminyl inositol deacetylase.",
"Such fusion proteins can be functional in a two-hybrid assay.",
"Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an acetyl glucosaminyl inositol deacetylase polypeptide, or a fragment thereof, having an amino acid sequence at least 60% homologous to one of SEQ ID NOs:2, 3, 4 or 5.In a more preferred embodiment, the nucleic acid encodes a protein having an amino acid sequence 40% or more homologous to SEQ ID NO:2, more preferably at least 50% homologous to SEQ ID NO:2, and most preferably at least 65% homologous to SEQ ID NO:2.In another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:3; more preferably to at least 24 consecutive nucleotides encoding SEQ ID NO:3.In a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:4; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:4.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:5; more preferably to at least 30 consecutive nucleotides encoding SEQ ID NO:5.In yet a further embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides encoding SEQ ID NO:6; more preferably to at least 20 consecutive nucleotides encoding SEQ ID NO:6.Furthermore, in certain embodiments, the acetyl glucosaminyl inositol deacetylase nucleic acid will comprise a transcriptional regulatory sequence, e.g.",
"at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the acetyl glucosaminyl inositol deacetylase-gene sequence so as to render the recombinant acetyl glucosaminyl inositol deacetylase gene sequence suitable for use as an expression vector.",
"The present invention also features transgenic non-human organisms, e.g.",
"live mutant actinomycetes, which either express a heterologous acetyl glucosaminyl inositol deacetylase gene, or in which expression of their own acetyl glucosaminyl inositol deacetylase gene is disrupted.",
"In addition to the other utilities of such organisms disclosed herein, such a transgenic organism has utility for overproduction of acetyl glucosaminyl inositol needed for screening (particularly high throughput screening) for compounds that inhibit acetyl glucosaminyl inositol deacetylase activity in mycothiol-producing bacteria.",
"The present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or anti-sense sequence encoding one of the amino acid sequences encompassed by SEQ ID NOs:2, 3, 4 5, or 6, or naturally occurring mutants thereof.",
"Yet another aspect of the invention pertains to a peptidomimetic that binds to or interferes with an acetyl glucosaminyl inositol deacetylase polypeptide and inhibits its binding to or cleavage of substrate acyl glucosaminyl inositols.",
"For example, a preferred peptidomimetic is an analog of a peptide having the sequence of one of the SEQ ID NOs.",
"1, 2, 3, 4, 5, or 6.Non-hydrolyzable peptide analogs of such residues can be generated using, for example, benzodiazepine, azepine, substituted gama-lactam rings, keto-methylene pseudopeptides, beta-turn dipeptide cores, or beta-aminoalcohols.",
"Other features and advantages of the invention will be apparent from the detailed description herein, and from the claims.",
"The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.",
"Such techniques are explained fully in the literature.",
"See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.",
"by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al.",
"U.S. Pat.",
"No.",
"4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.",
"1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.",
"1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.",
"154 and 155 (Wu et al.",
"eds.",
"), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).",
"As used herein, the term “actinomycetes” and “an actinomycete” encompasses any bacterium of the order Actinomycetales.",
"As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).",
"The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or anti-sense) and double-stranded polynucleotides.",
"As used herein, the terms “gene”, “recombinant gene” and “gene construct” refer to a nucleic acid comprising an open reading frame encoding an invention acetyl glucosaminyl inositol deacetylase, including both exon and (optionally) intron sequences.",
"The term “intron” refers to a DNA sequence present in a given acetyl glucosaminyl inositol deacetylase gene which is not translated into protein and is generally found between exons.",
"“Homology” refers to sequence similarity between two peptides or between two nucleic acid molecules.",
"Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.",
"When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position.",
"Percent homology between sequences is a function of the number of matching or homologous positions shared by the sequences.",
"The sequence data of a test clone is aligned to the sequences in the database or databases using algorithms designed to measure homology between two or more sequences.",
"Sequence alignment methods include, for example, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), and FASTA (Person & Lipman, 1988).",
"For example, optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.",
"The term “transfection” or “transforming” and grammatical equivalents thereof, refers to the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.",
"“Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of one of the invention family of acetyl glucosaminyl inositol deacetylases.",
"“Cells” or “cell cultures” or “recombinant host cells” or “host cells” are often used interchangeably as will be clear from the context.",
"These terms include the immediate subject cells that express the acetyl glucosaminyl inositol deacetylases of the present invention, and, of course, the progeny thereof.",
"It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment.",
"However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell.",
"In the present case, such a characteristic might be the ability to produce a recombinant acetyl glucosaminyl inositol deacetylase polypeptide.",
"As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.",
"The term “expression vector” includes plasmids, cosmids or phages capable of synthesizing the subject acetyl glucosaminyl inositol deacetylase polypeptide encoded by the respective recombinant gene carried by the vector.",
"Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.",
"In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.",
"Moreover, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.",
"“Transcriptional regulatory sequence” is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, as well as polyadenylation sites, which induce or control transcription of protein coding sequences with which they are operably linked.",
"In preferred embodiments, transcription of a recombinant acetyl glucosaminyl inositol deacetylase gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in a cell-type in which expression is intended.",
"It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences that control transcription of the naturally occurring form of the regulatory protein.",
"As used herein, a “transgenic organism” is any organism, preferably a bacteria in which one or more of the cells of the organism contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.",
"The nucleic acid is introduced into the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or a vector.",
"The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.",
"This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.",
"In the typical transgenic organisms described herein, the transgene causes cells to express a recombinant form of the subject acetyl glucosaminyl inositol deacetylase polypeptides.",
"As used herein, the term “transgene” means a nucleic acid sequence (encoding, e.g., an acetyl glucosaminyl inositol deacetylase polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic organism or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic organism or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the organism's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).",
"A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.",
"The term “evolutionarily related to”, with respect to nucleic acid sequences encoding acetyl glucosaminyl inositol deacetylase polypeptides, refers to nucleic acid sequences that have arisen naturally in an organism, including naturally occurring mutants.",
"The term also refers to nucleic acid sequences which, while derived from a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide, have been altered by mutagenesis, as for example, combinatorial mutagenesis, yet still encode polypeptides which have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide.",
"One aspect of the present invention pertains to an isolated nucleic acid comprising the nucleotide sequence encoding an acetyl glucosaminyl inositol deacetylase polypeptide, fragments thereof encoding polypeptides having acetyl glucosaminyl inositol deacetylase activity, and/or equivalents of such nucleic acids.",
"The term-nucleic acid as used herein is intended to include such fragments and equivalents.",
"The term equivalent is understood to include nucleotide sequences encoding functionally equivalent acetyl glucosaminyl inositol deacetylase polypeptides or functionally equivalent peptides having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide such as described herein.",
"Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will also include sequences that differ from the nucleotide sequence encoding native acetyl glucosaminyl inositol deacetylases due to the degeneracy of the genetic code.",
"Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27° C. below the melting temperature of the DNA duplex formed in about 1 M salt) to the nucleotide sequence of an acetyl glucosaminyl inositol deacetylase gene, such as that as set forth in SEQ ID NO:7, particularly those segments encoding the polypeptides shown in one of SEQ ID NOs.",
"2, 3, 4, 5 or 6, and conservative variations thereof.",
"In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to such nucleotide sequences.",
"The term “isolated” or “purified” as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.",
"For example, an isolated nucleic acid encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the acetyl glucosaminyl inositol deacetylase gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence.",
"The term isolated or purified as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.",
"Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state.",
"In yet another embodiment, the nucleic acid of the invention encodes a peptide having an amino acid sequence as shown SEQ ID NO:1.Preferred nucleic acids encode a peptide having acetyl glucosaminyl inositol deacetylase polypeptide activity and being 40% or more homologous, more preferably 50% homologous and most preferably 65% homologous with an amino acid sequence as set forth in SEQ ID NO:2 (encoded by a nucleic acid sequence as set forth in SEQ ID NO:7).",
"Nucleic acids that encode peptides having an activity of an invention acetyl glucosaminyl inositol deacetylase polypeptide are also within the scope of the invention.",
"Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes an acetyl glucosaminyl inositol deacetylase polypeptide having all or a portion of an amino acid sequence shown in one of SEQ ID NOs.",
"2, 3, 4, 5 or 6.Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Isolated nucleic acids that differ from the nucleotide sequences disclosed herein due to degeneracy in the genetic code are also within the scope of the invention.",
"For example, more than one triplet designates a number of amino acids.",
"Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “silent” mutations that do not affect the amino acid sequence of the protein.",
"However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject acetyl glucosaminyl inositol deacetylase polypeptides will exist among prokaryotic cells.",
"One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding a particular member of the acetyl glucosaminyl inositol deacetylase polypeptide family may exist among individuals of a given species due to natural allelic variation.",
"Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.",
"Fragments of the nucleic acid encoding a biologically active portion of the subject acetyl glucosaminyl inositol deacetylase polypeptides are also within the scope of the invention.",
"As used herein, a fragment of the nucleic acid encoding an active portion of an acetyl glucosaminyl inositol deacetylase polypeptide refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, for example, the deacetylase polypeptides represented in nucleic acid SEQ ID NO:1, and which encodes a peptide which retains at least a portion of the biological activity of the full-length protein (i.e., a peptide capable of acetyl glucosaminyl inositol deacetylase activity) as defined herein, or alternatively, which is functional as an antagonist of the deacetylase activity of the full-length protein.",
"Nucleic acid fragments within the scope of the invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species, e.g.",
"for use in screening protocols to detect homologs of the subject acetyl glucosaminyl inositol deacetylase polypeptides.",
"Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant peptides.",
"As indicated by the examples set out below, a nucleic acid encoding a peptide having an activity of an invention deacetylase polypeptide may be obtained from mRNA or genomic DNA present in any of a number of antibiotic-producing or pathogenic bacteria, particularly actinomycetes, in accordance with protocols described herein, as well as those generally known to those skilled in the art.",
"A cDNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide, for example, can be obtained by isolating total mRNA from a bacterial cell.",
"Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.",
"A gene encoding an acetyl glucosaminyl inositol deacetylase polypeptide can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.",
"Another aspect of the invention relates to the use of an “anti-sense” isolated nucleic acid.",
"As used herein, an “anti-sense” inhibition of endogenous production of an acetyl glucosaminyl inositol deacetylase molecule is carried out by administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g.",
"bind) under intracellular conditions, with the cellular mRNA and/or genomic DNA encoding an acetyl glucosaminyl inositol deacetylase polypeptide so as to inhibit expression of that protein or a constituent thereof, e.g.",
"by inhibiting transcription and/or translation.",
"The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.",
"In general, “anti-sense” therapy refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.",
"An anti-sense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the transformed cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an acetyl glucosaminyl inositol deacetylase polypeptide.",
"Alternatively, the anti-sense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one of the subject acetyl glucosaminyl inositol deacetylase proteins.",
"Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g.",
"exonucleases and/or endonucleases, and is therefore stable in vivo.",
"Exemplary nucleic acid molecules for use as anti-sense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.",
"Nos.",
"5,176,996; 5,264,564; and 5,256,775).",
"Additionally, general approaches to constructing oligomers useful in anti-sense techniques have been reviewed, for example, by van der Krol et al.",
"(1988) Biotechniques 6:958-976; and Stein et al.",
"(1988) Cancer Res 48:2659-2668.In addition, the oligomers of the invention may be used as reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind.",
"Such diagnostic tests are described in further detail below.",
"This invention also provides expression vectors comprising a nucleotide sequence encoding a member of the invention family of acetyl glucosaminyl inositol deacetylase polypeptides and operably linked to at least one regulatory sequence.",
"Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence.",
"Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.",
"Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements.",
"Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).",
"For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the acetyl glucosaminyl inositol deacetylase polypeptides of this invention.",
"Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.",
"It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.",
"Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.",
"As will be apparent, the subject gene constructs can be used to cause expression of the subject acetyl glucosaminyl inositol deacetylase polypeptides in cells propagated in culture, e.g.",
"to produce proteins or peptides, including fusion proteins or peptides, for purification.",
"This invention also pertains to a host cell transfected with a recombinant acetyl glucosaminyl inositol deacetylase gene in order to express a polypeptide having an activity of an acetyl glucosaminyl inositol deacetylase polypeptide.",
"The host cell may be any prokaryotic or eukaryotic cell.",
"For example, an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells.",
"Other suitable host cells are known to those skilled in the art.",
"Another aspect of the present invention concerns recombinant acetyl glucosaminyl inositol deacetylase polypeptides that have the deacetylase activity of an acetyl glucosaminyl inositol deacetylase polypeptide, or which are naturally occurring mutants thereof.",
"The term “recombinant protein” refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the acetyl glucosaminyl inositol deacetylase polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.",
"Moreover, the phrase “derived from”, with respect to a recombinant gene encoding the recombinant acetyl glucosaminyl inositol deacetylase polypeptide, is meant to include within the meaning of “recombinant protein” those proteins having an amino acid sequence of a native acetyl glucosaminyl inositol deacetylase polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions of a naturally occurring acetyl glucosaminyl inositol deacetylase polypeptide of a organism.",
"The present invention further pertains to methods of producing the subject acetyl glucosaminyl inositol deacetylase polypeptides.",
"For example, a host cell transfected with expression vector encoding one of the subject acetyl glucosaminyl inositol deacetylase polypeptide can be cultured under appropriate conditions to allow expression of the peptide to occur.",
"The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide.",
"Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.",
"A cell culture includes host cells, media and other byproducts.",
"Suitable media for cell culture are well known in the art.",
"The peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the subject acetyl glucosaminyl inositol deacetylase polypeptides.",
"Thus, a nucleotide sequence derived from the cloning of an acetyl glucosaminyl inositol deacetylase polypeptide of the present invention, encoding all or a selected portion of the protein, can be used to produce a recombinant form of the protein via microbial cellular processes.",
"The recombinant acetyl glucosaminyl inositol deacetylase polypeptide can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in bacterial cells.",
"Expression vehicles for production of a recombinant acetyl glucosaminyl inositol deacetylase polypeptide include plasmids and other vectors.",
"For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.",
"A number of vectors exist for the expression of recombinant proteins in yeast.",
"For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al.",
"(1983) in Experimental Manipulation of Gene Expression, ed.",
"M. Inouye Academic Press, p. 83, incorporated by reference herein).",
"These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.",
"In addition, drug resistance markers such as ampicillin can be used.",
"The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.",
"For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.",
"by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.In some instances, it may be desirable to express the recombinant acetyl glucosaminyl inositol deacetylase polypeptide by the use of a baculovirus expression system.",
"Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III).",
"This invention further contemplates a method of generating sets of combinatorial mutants of the present acetyl glucosaminyl inositol deacetylase polypeptides, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g.",
"homologs) that are functional in cleaving acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide molecules.",
"In a representative embodiment of this method, the amino acid sequences for a population of acetyl glucosaminyl inositol deacetylase polypeptide homologs are aligned, preferably to promote the highest homology possible.",
"Such a population of variants can include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation.",
"Amino acids that appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.",
"The presence or absence of amino acids from an aligned sequence of a particular variant is relative to a chosen consensus length of a reference sequence, which can be real or artificial.",
"In order to maintain the highest homology in alignment of sequences, deletions in the sequence of a variant relative to the reference sequence can be represented by an amino acid space (*), while insertional mutations in the variant relative to the reference sequence can be disregarded and left out of the sequence of the variant when aligned.",
"Further expansion of the combinatorial library can be made by, for example, by including amino acids that would represent conservative mutations at one or more of the degenerate positions.",
"Inclusion of such conservative mutations can give rise to a library of potential acetyl glucosaminyl inositol deacetylase sequences.",
"Alternatively, amino acid replacement at degenerate positions can be based on steric criteria, e.g.",
"isosteric replacement, without regard for polarity or charge of amino acid sidechains.",
"Similarly, completely random mutagenesis of one or more of the variant positions can be carried out.",
"In a preferred embodiment, the combinatorial acetyl glucosaminyl inositol deacetylase library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential acetyl glucosaminyl inositol deacetylase polypeptide sequences.",
"For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential acetyl glucosaminyl inositol deacetylase nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g.",
"for phage display) containing the set of acetyl glucosaminyl inositol deacetylase polypeptide sequences therein.",
"There are many ways by which the library of potential acetyl glucosaminyl inositol deacetylase homologs can be generated from a degenerate oligonucleotide sequence.",
"Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression.",
"The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential acetyl glucosaminyl inositol deacetylase sequences.",
"The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.",
"(1981) Recombinant DNA, Proc 3rd Cleveland Sympos.",
"Macromolecules, ed.",
"A G Walton, Amsterdam: Elsevier pp.",
"273-289; Itakura et al.",
"(1984) Annu.",
"Rev.",
"Biochem.",
"53:323; Itakura et al.",
"(1984) Science 198:1056; Ike et al.",
"(1983) Nucleic Acid Res.",
"11:477.A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and, for that matter, for screening cDNA libraries for gene products having a certain property.",
"Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of acetyl glucosaminyl inositol deacetylase homologs.",
"The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene product was detected.",
"Each of the illustrative assays described below are amenable to high throughput analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.",
"The invention also provides for reduction of the subject acetyl glucosaminyl inositol deacetylase polypeptides to generate mimetics, e.g.",
"peptide or non-peptide agents, which are able to mimic binding of the authentic acetyl glucosaminyl inositol deacetylase polypeptide to a substrate molecule.",
"Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of an acetyl glucosaminyl inositol deacetylase polypeptide which participate in protein-protein interactions involved in, for example, binding of the subject acetyl glucosaminyl inositol deacetylase polypeptide to a substrate.",
"To illustrate, the critical residues of a subject acetyl glucosaminyl inositol deacetylase polypeptide which are involved in molecular recognition of substrate can be determined and used to generate acetyl glucosaminyl inositol deacetylase-derived peptidomimetics which cleave acetyl glucosaminyl inositol or other acyl glucosaminyl inositol amide substrates and, like the authentic acetyl glucosaminyl inositol deacetylase polypeptide, cleave the substrate molecule, for example by amide hydrolase activity.",
"By employing, for example, scanning mutagenesis to map the amino acid residues of a particular acetyl glucosaminyl inositol deacetylase polypeptide involved in binding to a substrate, peptidomimetic compounds (e.g.",
"diazepine or isoquinoline derivatives) can be generated which mimic those residues cleaving substrate.",
"For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al; in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al.",
"in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al.",
"in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al.",
"(1986) J Med Chem 29:295; and Ewenson et al.",
"in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), beta-turn dipeptide cores (Nagai et al.",
"(1985) Tetrahedron Lett 26:647; and Sato et al.",
"(1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols (Gordon et al.",
"(1985) Biochem Biophys Res Commun 126:419; and Dann et al.",
"(1986) Biochem Biophys Res Commun 134:71).",
"The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.",
"EXAMPLE 1 Preparation of Reagents and Assay Methods Used.",
"Mycothiol (MSH) was isolated from M. smegmatis and derivatized with monobromobimane to form the MSH-bimane derivative (MSmB).",
"Mycothiol can be purified from Streptomyces clavuligerus, M. smegmatis, or from cells of any other mycothiol containing bacterium.",
"Early stationary phase cells (25 gm wet weight) were extracted with 250 ml of 50% acetonitirile containing 25 mM methanesulfonic acid and 1 mM DTT at 60° C. for 15 min.",
"The cell debris was removed by centrifugation for 30 min (4° C.) at 20,000×g.",
"The excess acetonitrile was removed using a rotary evaporator.",
"The aqueous extract was neutralized with Tris base to pH 8.0 and clarified by centrifugation for 30 min (4° C.) at 20,000×g The extract was loaded on a 25×70 mm propyl thiol Sepharose 6B resin activated with 2-mercaptopyridine with 1 mmole total thiol binding capacity.",
"The column was washed with 10 column volumes of 20 mM Tris-HCl pH 8.0 to remove unbound extract components.",
"The bound thiols were eluted with 200 ml of 20 mM Tris-HCl pH 8.0 containing 3 mM DTT.",
"The eluate was extracted 5 times with an equal volume of ethyl acetate to remove excess DTT.",
"The ethyl acetate washed aqueous extract was reduced to dryness by lyophilization.",
"The extract was suspended in 5 ml of aqueous 0.1% trifluoroacetic acid (TFA).",
"The mycothiol was purified by preparative HPLC using a 10×250 mm Vydac C-18 (Cat.",
"# 218TP152022) reverse phase column (or equivalent).",
"Mycothiol eluted at 26 min using an isocratic elution in aqueous 0.1% TFA at 5 ml per min.",
"The volatile solvent was removed by lyophilization to yield pure mycothiol.",
"Mycothiol is stable for years when stored as a mildly acidic aqueous solution of 30-100 mM at −70° C. The bimane derivative of mycothiol is readily made using monobromobimane (Molecular Probes) at 2 mM excess over the thiol concentration.",
"The derivatization is carried out in aqueous 20 mM Tris-HCl pH 8.0 for 15 min in the dark at room temperature.",
"The derivatization mixture was acidified to pH<3 with TFA and extracted 2 times with equal volumes of dichloromethane to remove excess monobromobimane.",
"The derivatization mixture was purified on the preparative reverse phase column described above using a gradient elution.",
"The A-buffer was 0.1% aqueous TFA and the B buffer was methanol.",
"The linear gradient was 15-30% methanol over 30 min with a flow rate of 5 ml per min.",
"The MSmB was detected at 385 nm and eluted as a symmetrical peak at 33 min.",
"The volatile solvent was removed by lyophilization to yield>98% pure MSmB.",
"This derivative was stored as a 10-30 mM solution in water at −70° C. The purified M. smegmatis mycothiol S-conjugate amidase was used to quantitatively hydrolyze MSMB to stereochemically pure GlcN-Ins, and the latter was purified from the other hydrolysis product, AcCySmB, using a Sep-Pak® C18 (Waters) cartridge.",
"A 10 mM stock solution of GlcNAc-Ins was prepared by addition of a tenfold excess of acetic anhydride (Fisher) to 10 mM GlcN-Ins in 10 mM NaHCO3 over 20 min while adjusting the pH to 8.5 with NaOH.",
"The reaction was monitored for GlcN-Ins loss as described below to insure that the reaction was complete and that no residual GlcN-Ins was present.",
"This stock solution of GlcNAc-Ins was assayed as described below and used without further purification.",
"Preparation of Mycothiol S-conjugates.",
"Mycothiol S-conjugates were prepared by reaction of excess electrophile with mycothiol followed by removal of excess electrophile.",
"Stock solutions (100 mM) of iodoacetamide (Sigma) or bromoacetophenone (Sigma) were prepared in acetonitrile.",
"Reaction of these electrophiles at 2 mM in 25 mM HEPES chloride pH 7.5 was initiated by addition of mycothiol to 1 mM from a 32 mM stock solution in H2O (pH˜3) and was allowed to proceed 15 min at 22° C. Excess reagent was removed by extracting 3 times with an equal volume of H2O-saturated dichloromethane.",
"Prior to extraction a 1 μl aliquot was withdrawn for reaction with mBBr and analysis of residual mycothiol by HPLC (see above) to verify complete reaction.",
"This showed that>99% of the mycothiol had reacted.",
"The MS-acetophenone was purified to homogeneity by reverse phase preparative HPLC as described above for MSmB, except that MS-acetophenone was detected at 260 nm.",
"Assay of MSH and MSH precursors.",
"Mycothiol and its precursors cysteine, and Cys-GlcN-Ins were assayed as the bimane derivative by high performance liquid chromatography (HPLC) on a Ultrasphere® ODS IP (Beckman) reversed phase column.",
"The bimane derivatives were detected by fluorescence using a Fluoromonitor III® monitor (Thermo Separation products) with excitation filters 340-380 nm and emission filters 418-700 nm.",
"The A buffer was 0.1% TFA and the B buffer was 7.5% methanol in acetonitrile.",
"The separation was achieved at 22° C. and a flow rate of 1 ml/min with linear gradients as follows: time 0 min 0% B, 10 min 0% B, 30 min 20% B, 33 min 100% B, 36 min 100% B, 38 min 0% B, 50 min reinject.",
"CySmB-GlcN-Ins, CySmB and mycothiol-mB (MSmB) eluted at 28, 31 and 33 min, respectively.",
"GlcN-Ins was assayed by precolumn derivatization using AccQFluor® (Waters) reagent followed by HPLC analysis.",
"A sample, 7.5 μl of 50% acetonitrile sample containing 5 mM NEM or 5 mM NEM and 1 mM 1,10 phenanthroline was mixed with 39.4 μl of buffer stock.",
"The buffer stock was composed of 30% acetonitrile in aqueous 160 mM HEPES pH 7.5.This was mixed with 15.6 μl of AccQ-Fluor® reagent (Waters) and incubated for 1 min at 22° C. followed by 10 min at 60° C. The sample was diluted with 188 μl of water and the sample was analyzed by HPLC.",
"The sample was centrifuged for 3 min 14,000×g in a microcentrifuge.",
"The HPLC separation conditions were the same as described above for bimane derivatives, except the B buffer was 0.1% TFA in methanol.",
"The product was detected by fluorescence using 254 nm excitation and 370-700 nm emission filters.",
"AccQ-GlcN and AccQ-GlcN-Ins eluted at 12 and 24 min respectively.",
"Mycothiol S-conjugate amidase assay.",
"The enzymatic activity was routinely assayed by quantitation of the bimane derivative of N-acetylcysteine (AcCySmB) produced from the bimane derivative of mycothiol (MSmB), prepared from purified mycothiol (see above).",
"To conduct an amidase assay a sample (2-10 μL) of extract was mixed with 40 μL of 30 μM MSmB in 3 mM 2-mercaptoethanol, 25 mM HEPES chloride pH 7.5 and reacted 10-30 min at 30° C. before quenching the reaction with 50 μL of 40 mM methanesulfonic acid on ice.",
"The mixture was centrifuged for 3 min at 14,000×g in a microcentrifuge at room temperature and the supernatant analyzed by HPLC without dilution.",
"Separation of MSmB and AcCySmB was achieved by HPLC on a 0.46×250 mM Ultrasphere®ODS column (Beckman) using a linear gradients: time 0 min 10% B, 15 min 18% B, 30 min 27% B, 32 min 100% B, 34 min 10% B, 45 min reinject.",
"Buffer A (aqueous 0.25% acetic acid titrated to pH 3.6 with NaOH) and buffer B (methanol) were pumped at 1.2 ml/min at ambient temperature (22° C.).",
"The bimane derivative of mycothiol (MSmB) eluted at 23.5 min and AcCySmB eluted at 27 min.",
"EXAMPLE 2 Cloning and Expression of the Rv1170 gene from M. tuberculosis and demonstration of its GlcNAc-Ins deacetylase activity.",
"Since the invention deacetylases and the mycothiol S-conjugate amidase both cleave an amide bond involving glucosamine, further studies were conducted to determine whether these proteins might be structurally related.",
"A search was conducted of the Sanger Centre database for homologs of the 288 amino acid M. tuberculosis deacetylase, Rv1082.The closest homolog found was Rv1170, with a length of 304 residues and 36% identity to Rv1082 with homology throughout the sequence.",
"To isolate the Rv1170 gene from M. tuberculosis H37Rv genomic DNA was prepared using known methods.",
"The open reading frame Rv1170 was amplified from this DNA with the following primers: 5′-TAGCCATGGTGTCTGAGACGCCGCG-3′ (SEQ ID NO:8) and 5′-GGATCCCGGGGTGAAGCCCAGAC-3′ (SEQ ID NO:9) These primers contain NcoI and BamHI restriction sites, respectively.",
"PCR was performed with Taq polymerase (Gibco BRL), using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.",
"The 30 cycles of PCR included denaturation at 94° C. for 40 s, annealing at 55° C. for 1 min, and amplification at 72° C. The PCR products were separated on a 1% agarose gel.",
"The appropriate PCR product was ligated into vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into Escherichia coli DH5α by standard chemical transformation procedures.",
"Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml), and plasmid DNA was digested with the restriction endonucleases NcoI and BamHI (Fermentas).",
"Restriction enzyme-digested plasmids were isolated with a QIAquick® gel extraction kit (Qiagen Ltd.).",
"A corresponding digestion was applied to the plasmid pET-22b, and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170.In order to express Rv1170 in E. coli without the pelB leader sequence, the gene from pYA1170 was excised using NcoI and Bpu1102I (Fermentas) and ligated to an aliquot of pET16b cut with NcoI and Bpu1102I to generate the plasmid pYA1170b (FIG.",
"2).",
"This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg ampicillin/ml.",
"Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).",
"After overnight incubation at 37° C. with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37° C. with shaking.",
"Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.4 mM when the A600 reached 0.6, and incubation was continued overnight at 25° C. before harvesting by centrifugation at 5000×g for 15 min at room temperature.",
"The pellets were lysed by sonication.",
"Proteins were separated by centrifugation (15,000×g, 4° C., 30 min) into soluble and insoluble fractions.",
"Total proteins were separated by sodium dodecyl sulfate 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride membranes (Bio-Rad).",
"The N-terminal amino acid sequence was verified using Edman degradation after separation of samples by SDS-PAGE and electroblotting to a polyvinylidene difluoride membrane.",
"E. coli BL21 (DE3) was transformed with the blank cloning vector (pET16b) or with vector containing Rv1170 (pYA1170b).",
"SDS-PAGE was used to separate desalted 20% to 50% saturated ammonium sulfate extracts of these isopropyl-β-D-thiogalactopyranoside induced E. coli.",
"Bio-Rad Broad Range molecular mass standards and purified M. smegmatis mycothiol S-conjugate deacetylase were also assayed for controls.",
"The results of the SDS-PAGE showed that the 20% to 50% saturated ammonium sulfate fraction from E. coli carrying the Rv 1170 gene contained elevated levels of a protein of the expected size (36 kDa) as compared to those from E. coli prepared with a blank cloning vector.",
"Another, smaller protein (˜26 kDa) was also present at elevated levels and is believed to be a degradation product of the deacetylase.",
"The identity of the 36-kDa protein was confirmed by determining the N-terminal amino acid sequence and finding it identical to that predicted for the protein encoded by open reading frame Rv1170.The amidase and deacetylase activity of the 20 to 50% saturated ammonium sulfate extracts of E. coli transformed with pYA1170b or with the blank cloning vector pET16b after desalting and concentrating was assayed as described above.",
"The results of these assays are shown in Table 1 below.",
"TABLE 1 Substrate specificity of crude deacetylase from E. coli transformed with pYA1170b Activity (nmol min−1 mg of protein−1)(n = 3) Substrate Rv1170 Blank vector MsmBa 1.2 ± 0.1 <0.002 MSmBb <10−4 NDf GlcNAc-Insc 27 ± 2 <0.002 GlcNAcc 0.087 ± 0.005 <0.005 MSHd 0.002 ± 0.001 ND MSHe <10−4 ND aAnalysis for AcCySmB produced by amidase cleavage.",
"bAnalysis for production of the deacetylase product CySmB-GlcN-Ins.",
"cAnalysis for corresponding amine resulting from deacetylase reaction.",
"dAnalysis for AcCys produced by amidase cleavage.",
"eAnalysis for production of the deacetylase product Cys-GlcN-Ins.",
"fNot determined.",
"As shown by the results summarized in Table 1, extract from cells expressing Rv1170 exhibited substantial deacetylase activity with GlcNAc-Ins as the substrate, over 300-fold greater than the deacetylase activity determined with GlcNAc and 23-fold greater than the amidase activity measured with MSMB (FIG.",
"2).",
"Thus, Rv1170 was putatively identified as the GlcNAc-Ins deacetylase gene in the MSH biosynthesis pathway of M. tuberculosis.",
"M. smegmatis mycothiol S-conjugate amidase lacks deacetylase activity.",
"Mycothiol S-conjugate amidase from M. smegmatis, a homolog of the protein encoded by M. tuberculosis Rv1082, cleaves the amide bond linking Cys to GlcN in MSH derivatives having an alkylated sulfur residue (G. L. Newton et al., Biochemistry 39:10739-10746, 2000).",
"Tests were conducted to determine whether the mycothiol S-conjugate amidase also cleaves the corresponding bond in GlcNAc-Ins, functioning as a deacetylase (FIG.",
"3).",
"GlcNAc-Ins was prepared from GlcN-Ins and the ability of the amidase purified from M. smegmatis to hydrolyze GlcNAc-Ins (i.e., to function as a deacetylase) was compared with its ability to function as an amidase by cleaving the amide bond in the monobromobimane conjugate of MSH (MSmB).",
"The assays utilized are described above.",
"For a test with 100 μM substrate, the activity measured with deacetylase substrate MSmB was 4.5±1.0 μmol min−1 mg of protein−1, whereas that measured with the invention deacetylase substrate GlcNAc-Ins was <1 nmol min−1 mg of protein−1 (n=4).",
"The comparative assays showed that the mycothiol S-conjugate deacetylase does not exhibit measurable deacetylase activity and cannot serve this function in the MSH biosynthesis pathway of M. smegmatis.",
"EXAMPLE 3 Extracts of M. smegmatis and mutant 49 have deacetylase activity.",
"If the deacetylase is involved in MSH biosynthesis and if mutant 49 is blocked at an earlier step in the MSH-production pathway, then strains mc2155 and 49 should both produce deacetylase activity.",
"To test this hypothesis, centrifuged extracts of exponentially growing cells were assayed in duplicate for GlcN-Ins production with and without the addition of 100 μM GlcNAc-Ins.",
"The background rate for production of GlcN-Ins measured without added GlcNAc-Ins was high for strain mc2155 (6.1±0.1 pmol min−1 mg of protein−1) presumably reflecting the substantial endogenous level of GlcNAc-Ins present in the undialyzed extract.",
"Addition of 100 μM GlcNAc-Ins increased the rate to 19.7±0.4 pmol min−1 mg of protein−1, giving a net rate increase of 13.6±0.5 pmol min−1 mg of protein−1.For mutant 49, which does not contain GlcNAc-Ins, the background rate was <0.06 pmol min−1 mg of protein−1 and the rate with 100 μM GlcNAc-Ins was 16.4±1.4 pmol min−1 mg of protein−1.Thus, the deacetylase activity of strain 49 is essentially the same as that of the parent strain.",
"EXAMPLE 4 Assay for GlcNAc-Ins.",
"To confirm that GlcNAc-Ins is present in mycobacteria, an assay for this component was needed.",
"The availability of cloned Rv1170 allowed the development of such an assay.",
"Cell extracts were prepared in warm 50% acetonitrile containing NEM, as employed previously for an assay of GlcN-Ins (Anderberg et al.",
"(1998) J. Biol.",
"Chem.",
"273:30391-30397).",
"One portion of the extract was assayed directly for GlcN-Ins content while a second portion was assayed after treatment with a preparation of expressed Rv1170 under conditions sufficient to convert all GlcNAc-Ins to GlcN-Ins.",
"The difference in these two assay results yields the GlcNAc-Ins content for the sample.",
"In most cases, GlcN-Ins levels were at least an order of magnitude lower than those of GlcNAc-Ins, so this method gives reliable results.",
"Recovery experiments for GlcNAc-Ins were conducted using a M. smegmatis MSH-deficient mutant (“mutant 49”), derived from mc2155 by chemical mutagenesis.",
"Mutant 49 was cultured in Middlebrook 7H9 medium at 37° C. as previously described (Newton, et al.",
"(1999) Biochem.",
"Biophys.",
"Res.",
"Comm.",
"255:239-244).",
"In the uptake and metabolism assay, 50% acetonitrile-NEM extracts were analyzed after the addition of 0.7 and 7.0 μM GlcNAc-Ins.",
"Extracts of mutant 49 contained no detectable GlcN-Ins or MSH, so that any GlcN-Ins found in the extracts after treatment with the deacetylase preparation was derived solely from the deacetylation of GlcNAc-Ins.",
"The relationship between A600 and RDW was determined for aliquots of mc2155 cells producing ˜30 mg of RDW and found to be 0.42±0.02 mg of RDW per ml of cells with A600 at 1.00.No variation of >15% in this value was seen between log- and stationary-phase cells.",
"The method was tested with extracts prepared from log-phase cells of mutant 49 by spiking the extract with GlcNAc-Ins at levels equivalent to 0.13 and 0.013 μmol per g of RDW.",
"The recoveries from triplicate determinations were 97%±5% and 132%±3%, respectively.",
"At the lowest level of added GlcNAc-Ins, a significant correction had to be applied for background peaks present in unspiked controls that overlapped the GlcN-Ins peak.",
"The greater than 100% recovery found for the samples spiked at lowest level indicates that this correction produces a systematic error and thus defines the lower limit of useful sensitivity for this assay.",
"EXAMPLE 5 M. smegmatis mc2155 produces GlcNAc-Ins but MSH-deficient mutant 49 does not.",
"M. smegmatis mc2155 cells were examined in exponential and stationary phases for their content of MSH and its precursors using the methods described above.",
"The results of these studies are summarized in Table 2 below: TABLE 2 Levels of MSH and precursors in M. smegmatis mc2155 Content (μmol [g of RDW]−1) ( n = 3) Growth phase A600 MSH GlcNAc-Ins GlcN-Ins Log 0.54 7.8 ± 0.6 14.5 ± 1.2 0.2 ± 0.1a Stationary 3.3 6.6 ± 0.6 12.6 ± 0.6 <0.03 aBest estimate after correction for overlapping peak in HPLC analysis.",
"These results show that M. smegmatis accumulates GlcNAc-Ins to a substantial level, almost twice the level of MSH content.",
"The GlcN-Ins content was very much lower in log phase and declined further in stationary phase, as had been observed previously (Anderberg et al; (1998) Supra.",
"EXAMPLE 6 MSH-deficient mutant 49 can import GlcNAc-Ins and utilize it to sythesize MSH.",
"The foregoing results imply that mutant 49 should be able to synthesize MSH if supplied with GlcNAc-Ins.",
"The concentration dependence of the uptake of GlcNAc-Ins was examined with cultures of M. smegmatis mutant 49.For this study mutant 49 was cultured at 37° C. for 23 hr in 5 ml of 7H9 Middlebrook medium as described below, supplemented with 0, 4, 8, 21 μM GlcNAc-Ins.",
"The cells were collected by centrifugation and extracted for amine and thiol analysis as described above.",
"The absence of GlcNAc-Ins in mutant 49 shows that it is defective in an initial step of MSH biosynthesis.",
"When mutant 49 was grown in media supplemented with GlcNAc-Ins, the cells did show the presence of MSH and mycothiol precursors in increasing concentrations with the media content of GlcNAc-Ins.",
"These results established that GlcNAc-Ins is indeed imported intact, deacetylated and utilized for the production of MSH in a concentration dependent manner (Table 3).",
"TABLE 3 Levels of MSH and precursors in MSH-deficient strain 49 (M. smegmatis) Content (μmol [g of RDW]−1) [GlcNAc-Ins]a (μM) MSH GlcNAc-Ins GlcN-Ins 0 <0.04 <0.01 <0.02 4 0.47 0.39 <0.02 8 0.91 0.80 0.026 21 2.1 1.4 0.047 aIn Middlebrook 7H9 medium.",
"; [GlcNAc-Ins] = 0).",
"In another experiment mutant 49 was cultured in 7H9 Middlebrook medium with 0.4% glucose and 0.5% TWEEN 80 detergent to log phase (A600=0.6) and 10-ml aliquots transferred to sterile 25 ml erlenmeyer flasks.",
"Duplicate flasks contained cells only (control), 20 μM inositol, 20 μM glucosamine, 20 μM N-acetylglucosamine, or 17 μM GlcNAc-Ins and were cultured at 37° C., shaking at 225 rpm.",
"Samples (2.3×108 cells) were taken at 2, 6, 19 and 46 hr of culture.",
"the cells were pelleted by centrifugation for 2 min at 14,000×g and were extracted in 50% acetonitrile containing 20 mM HEPES (pH 8.0) and either 2 mM monobromobimane for thiol analysis or 5 mM NEM for amine analysis and thiol control samples.",
"Cells from duplicate cultures were analyzed for GlcN-Ins, GlcNAc-Ins, Cys-GlcN-Ins, cysteine, and MSH.",
"No significant MSH was found in any sample except for the cultures supplemented with GlcNAc-Ins (FIG.",
"4).",
"Strain 49 cultured in 17 μM GlcNAc-Ins showed a cysteine level that remained unchanged at 0.36±0.08 μmol per gm of residual dry weight over the experiment.",
"Cells contained 2.9 μmole per gm of residual dry weight of GlcNAc-Ins at the first measurement (2 hr), which corresponds to a level of GlcN-Ins approaching millimolar.",
"This occurred prior to the appearance of significant GlcN-Ins in the cell, showing that strain 49 is able to import and concentrate GlcNAc-Ins intact prior to its deacetylation.",
"The level of GlcNAc-Ins appeared to fall at 6 hr before rising to an even higher level in stationary phase.",
"The MSH content was measurable as early as 2 hr, but was about 10-fold less than the GlcNAc-Ins content at that time.",
"The MSH content increased thereafter, and the level at 48 hr was about 60% of that measured for the parent strain (Table 2).",
"The level of GlcN-Ins ranged from 0.025 (2 hr) to 0.1 (48 hr) μmole per gm of residual dry weight, values in the range of those measured for mc2155 (Table 2).",
"These results show that exogenous GlcNAc-Ins is efficiently imported by strain 49 and substantially restores its defective biosynthesis of MSH.",
"The intracellular level of GlcN-Ins found in mutant 49 at 48 hours indicates that GlcNAc-Ins has been concentrated more than 100 fold from the supplemented medium.",
"This implies that toxic analogs of GlcNAc-Ins, such as those with oxidizing or nitrosating moieties, will be concentrated inside pathogenic mycobacteria or other actinomycetes and function as antimicrobial agents.",
"EXAMPLE 7 Cloning, expression and purification of M. tuberculosis His-tagged MshB.",
"To facilitate the purification of the M. tuberculosis deacetylase, Rv1170 was cloned to contain a C-terminal HisTag permitting its easy isolation by affinity chromatography on a Ni2+ chelate resin.",
"Genomic DNA of M. tuberculosis H37Rv was prepared as described previously (Av-Gay et al., Infect.",
"Immun.",
"67:5676-5682, 1999).",
"The open reading frame Rv1170 was amplified from this DNA with the primers 5′-TTCATATGGTGTCTGAGACGCCG-3′; (SEQ ID NO:10 and 5′-ATAAGCTTGTAGCCGGACGCGGTG-3′ (SEQ ID NO:11) containing NdeI and HindIII restriction sites, respectively.",
"PCR was performed with Taq polymerase obtained from Gibco BRL, using 1.5 mM MgCl2 and 5% dimethyl sulfoxide.",
"The 30 cycles of PCR included denaturation at 94° C. for 40 seconds, annealing at 55° C. for 1 min and amplification at 72° C. The PCR products were separated on a 1% agarose gel.",
"The appropriate PCR product was ligated into the vector pCR2.1 of the TA cloning kit (Invitrogen) and transformed into E. coli DH5a by standard chemical transformation procedure.",
"Clones containing the vector were selected on plates of Luria-Bertani (LB) agar plus ampicillin (100 μg/ml) and plasmid DNA was digested with restriction endonucleases NdeI and HindIII (Fermentas).",
"Restriction enzyme-digested plasmids were isolated with a QIAquick gel extraction kit (Qiagen Ltd.).",
"A corresponding digestion was applied to the plasmid pET-22b and the two products were ligated together with T4 DNA ligase to obtain the plasmid pYA1170c (shown in FIG.",
"9).",
"This provides a recombinant protein without a pelB leader sequence, but with a c-terminal His tag.",
"This plasmid was transformed by the heat shock method to competent E. coli BL21(DE3) prepared according to the CaCl2 method and plated on LB agar containing 100 μg/ml ampicillin.",
"Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml).",
"After over-night incubation at 37° C. with shaking, 5 1-liter cultures were inoculated to 1% with the starter culture and incubated at 37° C. with shaking.",
"Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM when the A600 reached 0.6 and incubation was continued overnight at 22° C. before harvesting by centrifugation at 5000×g for 20 minutes at 4° C. The pellets were lysed by sonication in 3 mM 2-mercaptoethanol with 50 mM HEPES pH 7.4 (lysis buffer) containing 35 μM each protease inhibitors (N-α-p-tosyl-L-lysinechloromethyl ketone and N-α-p-tosyl-L-phenylalaninechloromethyl ketone).",
"Proteins were separated by centrifugation (1-5,000×g, 4° C., 30 min) into-soluble and insoluble-fractions.",
"The soluble proteins were applied to a 2.5×11 cm Fast Flow® chelating resin (Pharmacia) in the Ni2+ form in lysis buffer containing 10 mM imidazole.",
"The column was washed with 10 volumes of the loading buffer and then step eluted with lysis buffer containing 150 mM imidazole followed by lysis buffer containing 500 mM imidazole.",
"Fractions were collected and the purity of the eluted protein was assayed by SDS PAGE gel electrophoresis.",
"The early 150 mM imidazole fractions were contaminated with E. coli proteins, but the later fractions of the 150 mM and the early 500 mM imidazole fractions were judged>95% pure and pooled.",
"The yield of pure recombinant MshB (Rv1170) was estimated to be ˜125 mg of protein.",
"The protein was concentrated and the buffer exchanged repeatedly with 50 mM HEPES containing 50 mM NaCl (assay buffer) in a Centricon® 10 spin ultra filter, adjusted to 10% glycerol and frozen at −70° C. The enzyme as purified was assayed for deacetylase activity with 0.1 mM GlcNAc-Ins as substrate and was found to be sensitive to chelating agents such as 1,10-phenanthroline.",
"The deacetylase reaction was substantially inhibited by the presence of 0.1 mM 1,10-phenanthroline in the reaction mixture (FIG.",
"7).",
"1 mM 1,10-phenanthroline completely inhibited the deacetylase reaction, indicating a dependence of the reaction on divalent metal ions.",
"The apoenzyme was generated by treatment of the concentrated enzyme with 5 mM 1,10-phenanthroline in assay buffer for 30 min on ice.",
"The chelator was removed by repeated dilution with assay buffer and concentration until the 1,10-phenanthroline concentration was estimated to be below 1 μM.",
"All solutions were treated with Chelex-100® chelator to remove extraneous divalent cations.",
"The apoenzyme was stored at 10 mg/ml in 50 mM NaCl+50 mM HEPES pH 7.5 with 10% glycerol at −70° C. The zinc form (Zn2+) of the deacetylase was prepared by treating the apoenzyme with 0.1 mM ZnCl2 in the assay buffer for 30 min on ice.",
"The excess Zn2+ was removed by repeated dilution and concentration with assay buffer until the Zn2+ was estimated to be below 1 μM.",
"The Zn2+ form of the enzyme was stored as described above for the apoenzyme and is herein referred to as “His-tagged MshB”.",
"EXAMPLE 8 Metal ion dependence of the deacetylase.",
"The metal ion dependence of the deacetylase activity of MshB was assayed using GlcNAc-Ins in the presence of 0.1 mM divalent metal ion.",
"Previous studies with ZnCl2 have shown that excess Zn2+ is not inhibitory at 0.1 mM and only slightly inhibitory at 1 mM (see above).",
"The metal salts tested, ZnCl2, CuSO4, MgCl2, MnCl2, CaCl2, COCl2, CdCl2 and NiCl2, were of reagent grade or higher purity and solutions of the metals were prepared as 10 mM stock solutions in water treated with Chelex-100 chelator.",
"Enzymatic activity was assayed in Chelex-100® treated 50 mM NaCl with 50 mM HEPES pH 7.4 (assay buffer) containing 0.1 mM of the desired metal salt and 0.1 mM GlcNAc-Ins as substrate.",
"Buffer and 10 μg of purified apoenzyme were preincubated the divalent metal ion in an 80 μl total reaction volume for 10 min at 37° C. and the assay initiated by the addition of the substrate.",
"Duplicate reactions were sampled at 3, 10, and 30 min by removing a 20 μl sample of the reaction mixture and adding it to 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.",
"The samples were incubated for 10 min at 60° C., placed on ice, and subsequently reacted with AccQ Fluor® (Waters) reagent as described above assayed for AccQ-GlcN-Ins by HPLC as described above.",
"The MshB apoenzyme was able to catalyze both the deacetylase (GlcNAc-Ins as substrate) and the amidase (MSmB as substrate) reactions at substantially reduced rates, as shown in Table 4 below.",
"TABLE 4 M. tuberculosis MshB (Rv1170) metal ion dependencea Amidase Specific Deacetylase Activityb Amidase Specific Deacetylase Metalloenzyme nmole/ Activity Activityc Activity Form min/mg relative rate nmole/min/mg Relative rate Apoenzyme 3.4 ± 0.5 1.0 35 ± 6 1.0 Untreated 15 ± 0.2 4.4 70 ± 19 2.0 Zn2+ 17 ± 0.7 5.0 81 ± 16 2.3 Ni2+ 13 ± 0.7 3.8 62 ± 23 1.8 Mn2+ 22 ± 0.7 6.5 69 ± 5 2.0 Ca2+ 3.3 ± 0.1 0.97 34 ± 5 0.97 Mg2+ 3.5 ± 0.2 1.0 41 ± 4 1.2 Cu2+ 3.2 ± 0.1 0.94 32 ± 10 0.9 Cd2+ 21 ± 1.5 6.2 34 ± 2 0.97 Co2+ 29 ± 0.3 8.5 236 ± 23 6.7 Zn2+ + Mg2+ ND — 75 ± 14 2.1 Ni2+ + Mg2+ ND — 64 ± 15 1.8 aRecombinant Rv1170 purified to homogeneity over Ni2+ Sepharose.",
"b0.1 mM MSmB as substrate with 0.1 mM metal ion.",
"c0.1 mM GlcNAc-Ins as substrate with 0.1 mM metal ion.",
"The low metal ion content of the apoenzyme reduced the amidase activity to a greater extent than the deacetylase activity relative to the untreated purified protein.",
"The activity of the Ni2+ Sepharose purified (untreated) form of the MshB appears to be consistent with the Ni2+ form of the enzyme.",
"The Zn2+, Mn2+ and Ni2+ forms appear to have about the same activity for both the amidase and deacetylase reactions.",
"The MshB apoenzyme was not activated by the presence of Ca2+, Mg2+ or Cu2+ for either the amidase or deacetylase reactions.",
"The MshB deacetylase and amidase reactions were differently affected by the presence of the Cd2+ ion.",
"The amidase reaction was enhanced to the level of Zn2+ while the deacetylase reaction remained at the apoenzyme activity level.",
"Similarly anomalous activity with Cd2+ was observed for the peptidase and esterase activity of carboxypeptidase, a zinc metalloprotease (R. C. Davis et al Biochemistry 7, 1090-1099 (1968)).",
"The highest level of activation for both the amidase and deacetylase reactions was observed with Co2+, as has been reported for numerous zinc metalloproteases (D. S. Auld, Chapter 14, Methods in Enzymology, 248:229-243, 1995).",
"Some zinc metalloproteases have been reported to require the presence of Mg2+ for optimal activity (Vallee and Auld (1993) Biochemistry 32:6493-6500) so activity assays were conducted with 0.1 mM each Zn2+ and Mg2+.",
"No additional activation over that of Zn2+ alone for either the amidase or deacetylase reactions was observed.",
"Thus, the metal ion dependence of MshB is similar to that found for members of the metalloprotease superfamily and is therefore likely that MshB is a zinc metalloenzyme in its native state.",
"EXAMPLE 9 Kinetics and substrate specificity of His-tagged MshB.",
"The kinetic constants were determined for GlcNAc-Ins, the deacetylase substrate for MshB, and for MSmB, an amidase substrate.",
"The rates were determined with His-tagged MshB in 50 mM NaCl containing 50 mM HEPES pH 7.5 (assay buffer).",
"The enzyme (10 μg) was preincubated with 80 μl of assay buffer for 10 min at 37° C. and the reaction was initiated with the addition of the substrate.",
"The deacetylase substrate was assayed at 9 different concentrations between 10 μM and 2 mM.",
"Duplicate reactions were sampled at 3, 10, and 30 min by removing 20 μl of the sample, and mixing with 20 μl of acetonitrile containing 10 mM NEM and 2 mM 1,10-phenanthroline.",
"The samples were quenched by incubation for 10 min at 60° C. The quenched sample was reacted with AccQ Fluor® reagent (Waters) and analyzed for GlcN-Ins by HPLC as described above.",
"The amidase reaction was assayed similarly using 10 concentrations of MSmB between 10 μM and 10 mM.",
"The amidase reaction was sampled at 3, 10, and 30 min by removing 20 μl of sample and mixing it with 60 μl of 40 mM methanesulfonic acid.",
"The mercapturate product, AcCySmB was analyzed by HPLC as described above.",
"Initial rates were obtained by extrapolation of the rates calculated at 3, 10, and 30 min to zero time.",
"The kinetic constants were obtained from an Eadie-Hofstee plot.",
"The amidase and deacetylase substrates have Km values near of 0.5 mM as shown below in Table 5 below.",
"TABLE 5 Kinetic characterization of M. tuberculosis MshB (Rv1170) Km Vmax kcat Substrate Activity μM nmol min−1mg−1 s−1 GlcNAc-Ins deacetylase 400 470 0.25 MSmB amidase 560 220 0.12 The intracellular concentration of GlcN-Ins is roughly twice the mycothiol level, or about 2-6 mM (Newton et al, J.",
"Bacteriol., 182: 6958-6963, 2000) and is sufficient to saturate the enzyme at physiological concentrations.",
"The substrate specificity of MshB was examined with various amidase and deacetylase substrates at a concentration of 0.1 mM.",
"The product analyzed will vary with the substrate used.",
"The deacetylase substrates tested were GlcNAc, GlcNAc-Ins, MSH, and MSmB, and the products assayed were GlcN, GlcN-Ins, Cys-GlcN-Ins, and CySmB-GlcN-Ins, respectively.",
"The amidase substrates tested were MSH, MSmB, AcCySmB-GlcN, CySmB-GlcN-Ins, MS-acetamide, and MS-acetophenone and the product assayed was GlcN-Ins, except for AcCySmB-GlcN were GlcN was assayed.",
"The amines were analyzed as the AccQ-derivative and thiols as the bimane derivative as described above.",
"The results of these substrate specificity studies are summarized in Table 6 below.",
"TABLE 6 Substrate specificity of M. tuberculosis MshB (Rv1170)a Amidase Specific Deacetylase Activityb Specific Activityb Relative Substratea nmol min−1mg−1 nmol min−1mg−1 Rate GlcNAc-Insc 70 ± 3 1.0 GlcNAcd 1.6 ± 1 0.023 MSHe <0.025 ± 0.012 <0.00036 MSHc 0.3 ± 0.1 0.0043 MSmBf <0.006 <0.00009 MSmBc 20 ± 2 0.29 AcCySmB-GlcNd <0.1 <0.001 CySmB-GlcN-Insc 265 ± 10 3.8 MS-acetamidec 0.14 ± 0.013 0.002 MS-acetophenonec 88 ± 8 1.3 aHis tagged MshB (Rv1170) in the Zn2+ form.",
"b0.1 mM substrate assayed in 50 mM NaCl + 50 mM HEPES pH 7.4 at 37° C. cReaction assayed for GlcN-Ins.",
"dReaction assayed for GlcN.",
"eReaction assayed for CyS-GlcN-Ins.",
"fReaction assayed for CySmB-GlcN-Ins.",
"As shown by the results in Table 6, the deacetylase activity of MshB is dependent on the presence of the inositol moiety as the GlcNAc substrate is 44 fold less active than GlcNAc-Ins.",
"Interestingly MSH and MSmB are not substrates for the deacetylase activity of MshB.",
"However, mycothiol S-conjugates with reasonably large groups on the cysteine sulfur, such as acetophenone and bimane S-conjugates, are good substrates for the amidase activity of the MshB.",
"The smaller mycothiol S-conjugate acetamide is a poor substrate for the MshB amidase activity, as was observed previously for mycothiol S-conjugate amidase from M. smegmatis (Newton et al Biochemistry, 39, 10739-10746, 2000).",
"The importance of the inositol moiety for the amidase activity of MshB is also apparent by the lack of activity with AcCySmB-GlcN.",
"One difference between the amidase activity of M. tuberculosis His-tagged MshB and the amidase from M. smegmatis is the ability to use CySmB-GlcN-Ins as substrate.",
"The M. smegmatis amidase required the acetylated amine on the cysteine moiety (or MSmB) for activity.",
"Another difference between MshB and the M. smegmatis amidase is that the amidase will not deacetylate GlcNAc-Ins (Newton et al J.",
"Bacteriol., 182, 6958-6963, 2000).",
"EXAMPLE 10 Inhibition of His-tagged MshB.",
"Naturally occurring inhibitors of the His-tagged MshB deacetylase and amidase activities have been examined.",
"The inhibition assays are similar to those described above in Example 9.For the amidase reaction of His-tagged MshB 10 μg enzyme was preincubated for 10 min at 37° C. in 80 μl of buffer containing 50 mM NaCl, 50 mM HEPES pH 7.5, 0.1 mM ZnCl2, 0.2 mM DTT and 0.1-1.0 mM inhibitor.",
"The inhibitors examined were mycothiol, glutathione and GlcN-Ins.",
"The purpose of the excess Zn+2 is to prevent depletion of the protein bound Zn2+ by thiols.",
"The DTT is added to limit the oxidation of the thiol inhibitors, which can be significant at the lower concentrations.",
"The uninhibited enzyme activity was determined using the same buffer and preincubation conditions without inhibitor.",
"The assays were initiated with the addition of MSmB to 0.55 mM and sampled at 3, 10, and 30 min by removing a 20 μl sample and quenching the reaction by mixing with 60 μl of 40 mM aqueous methanesulfonic acid.",
"This sample was assayed directly by HPLC for AcCySmB as described above.",
"The deacetylase reaction was assayed with GlcNAc-Ins using 0.1-1.0 mM glutathione and mycothiol as inhibitors.",
"The reaction was initiated by the addition of GlcNAc-Ins to 0.8 mM and was sampled at 3, 10, and 30 min.",
"The reaction was quenched and analyzed for GlcN-Ins as described in Example 9.Mycothiol appears to be an effective inhibitor of both the amidase (FIG.",
"8A) and the deacetylase (FIG.",
"8B) activities of His-Tagged MshB in the range of 0.1 to 1 mM.",
"This is not simply due to disruption of the enzyme metal binding site by a thiol as shown by the lack of inhibition by glutathione in this concentration range.",
"The lack of inhibition of the amidase reaction by GlcN-Ins (FIG.",
"8A) also shows that the immediate product of the reaction is not inhibitory.",
"Mycothiol is the terminal product in the biosynthesis scheme shown in FIG.",
"5 and it appears to completely inhibit the deacetylase (MshB) at mycothiol concentrations above 1 mM.",
"The estimated intracellular concentration of mycothiol in mycobacteria is about 1-3 mM and thus the deacetylase is in a substantially inhibited state until the mycothiol level falls below 1 mM, as may happen during cell division or cell stress.",
"Inhibition of the deacetylase reaction was analyzed at 0.8 mM GlcNAc-Ins, a concentration nearing saturation, but still below the estimated cellular concentration of 2-6 mM for M. smegmatis.",
"Thus, in M. smegmatis the deacetylase is exposed to saturating GlcNAc-Ins concentrations but is inhibited when the cellular mycothiol level is above 1 mM.",
"If mycothiol level falls substantially below 1 mM, the enzyme will catalyze the deacetylation of GlcNAc-Ins from a large cellular pool at near maximal rates to GlcN-Ins and ultimately to mycothiol.",
"It has previously been shown that mycothiol levels are quite constant from logarithmic through stationary phase growth of M. smegmatis (Newton et al, J.",
"Bacteriol., 178: 1990-1995, 1996).",
"It appears that the intracellular mycothiol level is regulated by feedback inhibition of the GlcNAc-Ins deacetylase by mycothiol itself.",
"The amidase substrate, MSmB, served as a useful model substrate for finding inhibitors of the GlcNAc-Ins deacetylase.",
"The amidase and deacetylase activities are both completely inhibited by mycothiol at concentrations over 1 mM.",
"These results identify mycothiol as an inhibitor for the deacetylase and amidase reactions with similar inhibitory concentrations.",
"Thus, the substrates for either the amidase or deacetylase activity of GlcNAc-Ins deacetylase can be used to screen for inhibitors of the enzyme.",
"EXAMPLE 11 Oxidant sensitivity of M. smegmatis MSH-deficient mutants.",
"The importance of mycothiol biosynthesis for the survival of mycobacteria under oxidative stress is illustrated by results obtained with chemical and transposon mutants deficient in mycothiol biosynthesis.",
"Chemical mutants were produced by mutagenesis of M. smegmatis mc2155 (˜2.5×109 colony forming units per ml Middlebrook 7H9 liquid medium) with N-methyl-N-nitro-N-nitrosoguanidine (4 μg per ml) for 30 min at 37° C. Cells were washed three times with 9 ml of fresh medium, resuspended in 10 ml of fresh medium, and incubated 8.5 h at 37° C. with shaking.",
"After dilution in fresh medium and passage through a 22-guage needle, the cells were plated on Middlebrook 7H9 agar containing 0.05% Tween 80 and 0.4% glucose.",
"After 9 days in a humidified incubator, the plates were scored and individual colonies replated in duplicate using a grid layout in 100 mm culture dishes.",
"After colony development, one plate was used to screen for MSH-deficient mutants using a membrane-based immunoassay specific for MSH (Unson, et al.",
"(1998) J. Immunol.",
"Methods 214:29-39).",
"MSH-deficient candidates were grown in bulk in liquid medium and analyzed for MSH content as described in Example 1.Two MSH-deficient mutants (strains 49 and 164) were identified and strain 49 was complemented using a M. tuberculosis library (pYUB412::H37Rv) of ˜18 kb DNA fragments cloned in the integrative shuttle plasmid pYUB412 to produce the strain 49::H37Rv A18 with partially restored MSH biosynthesis.",
"Transposon mutants of M. smegmatis mc2155 were prepared using the pCG79 plasmid carrying transposon Tn611 following the methods of Perez, et al.",
"(1998) Meth.",
"Molecular Biol.",
"101:187-198).",
"Plasmid pCG79 was introduced into M. smegmatis mc2155 by electroporation after which the cells were incubated 2 h at 32° C. in 7H9 medium without antibiotic.",
"They were then plated on Middlebrook 7H10 agar plates with ADC enrichment and 20 μg/mL of kanamycin and incubated 6 days at 32° C. Single colonies were restreaked on 7H10km plates and incubated 4 days at 32° C. A loopful of each streaked transformant was resuspended, serially diluted, and plated in duplicate on 7H10 Km plates.",
"One set of plates was incubated 36 h at 32° C. and then shifted to 42° C. for 5 days to select transposon mutants.",
"The other set of plates was incubated at 32° C. for 7 days and served as positive controls.",
"The transposon mutants were replated on 7H10Km plates at 1000-2000 colonies per plate and incubated 5d at 42° C. The cells were scraped into 10 ml of 7H9 medium, mixed with 1 mL of glycerol, and stored at −70° C. This library contained ˜10,000 mutants.",
"Screening of the library for MSH-deficient mutants was accomplished using as ELISA assay method to detect MSH production in cultures grown in microtiter plates (Unson, et al.",
"(1999) J. Clin.",
"Microbiol 37:2153-2157).",
"The first 1000 mutants screened produced three nearly identical MSH-deficient mutants (TN1, TN2 and TN3) that accumulated GlcN-Ins to high levels but failed to produce detectable levels of Cys-GlcN-Ins or MSH.",
"Since these clones seemed likely to be daughter cells, TN3 was selected for more detailed studies.",
"Results obtained with the parent and mutant strains of M. smegmatis are summarized in Table 7 below.",
"TABLE 7 Characterization of M. smegmatis mutant strains MSH Content H2O2 IC50a Strain μmol per g RDW Enzyme Deficiency mM mc2155 10 None 12 49b <0.004 no MshAc 1 49::H37Rv A18b 3.3 partial MshAd 1.8 I64 0.1 low MshCe 3.5 TN3 <0.004 no MshCe 0.5 aConcentration producing 50% loss of viability following 2 h incubation in Middlebrook 7H9 liquid medium containing H2O2 at 37° C., dilution and plating on 7H10 agar, incubation for 10 d, and counting of colony formation.",
"bData from Newton, et al.",
"(1999) Biochem.",
"Biophys.",
"Res.",
"Commun.",
"255: 239-244.cBased upon failure to produce GlcNAc-Ins or GlcN-Ins.",
"dBased upon restoration of ability to produce GlcN-Ins to ˜15% that of the parent strain.",
"eBased upon absence of MshC activity in cell extracts and accumulation of GlcN-Ins to 2-3 μmol per g RDW (˜25-fold above the level of the parent strain).",
"The results of these experiments show that the parent strain (mc2 155) has a high MSH content and exhibits a marked resistance to killing by hydrogen peroxide.",
"By contrast, strain 49, which is blocked in the first step of MSH biosynthesis and produces no detectable MSH, is 12-fold more sensitive to H2O2.Partial restoration of MSH biosynthesis in strain 49 through complementation (strain 49::H37Rv A18) reduced the peroxide sensitivity of mutant 49 by a factor of two.",
"Chemical mutant 164, blocked at the ligase biosynthetic enzyme MshC, has a low residual MSH content and is three-fold more sensitive to peroxide than the parent strain.",
"However, transposon mutant TN3, also blocked at MshC but producing no detectable MSH, was >20-fold more sensitive to H2O2 than the parent strain (FIG.",
"10).",
"These results show that blocking MSH biosynthesis before or after the deacetylase step (MshB) produces a dramatic increase in peroxide sensitivity.",
"It logically follows that blocking-MSH biosynthesis at MshB would produce a comparable increase in peroxide sensitivity in mycothiol-producing bacteria.",
"The sensitivity of strains mc2155, 164, and TN3 to superoxide was also examined (courtesy of Drs.",
"H. Buchmeier and D. Piddington).",
"Superoxide sensitivity was determined according to the method of De Groote, et al.",
"(1997) Proc.",
"Natl.",
"Acad.",
"Sci.",
"94: 13997-14001 using timed exposure of cells to 250 μM hypoxanthine in the presence of 0.1 unit/ml of xanthine oxidase followed by dilution and plating to determine survival.",
"Under these conditions, strain mc2155 exhibits no significant killing whereas mutant TN3 is >90% killed in 1 hour.",
"By contrast, chemical mutant 164, with a low residual MSH content, exhibited an intermediate level of killing.",
"Survival of mycothiol-deficient mutant in macrophage.",
"Transposon mutant TN3 was compared with the parent strain for survival in murine macrophages (data courtesy of Drs.",
"N. Buchmeier and D. Piddington).",
"Following the method of De Groote, et al.",
"(1997) Supra, periodate activated, γ-interferon treated murine peritoneal exudate cells were infected with TN3 and mc2155, and extracellular bacteria were washed free of the macrophages with medium.",
"At timed intervals the macrophages were resuspended in PBS and lysed with 0.5% deoxycholate; serial dilutions of the lysate were plated on Middlebrook 7H9 agar for determination of colony forming units.",
"The results of two independent experiments performed on different days (FIG.",
"11, experiments 1 and 2) show no loss of viability of the parent strain mc2155 in the macrophages over the period of 24 hours; whereas over 60% of the MSH-deficient mutant TN3 was killed by the macrophages within 6 hours.",
"These results suggest that mycobacteria deficient in MSH biosynthesis will exhibit significantly enhanced killing by mammalian macrophages.",
"While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed."
]
] | Patent_10297512 |
[
[
"Process for the electromagnetic modelling of electronic components and systems",
"Process for the electromagnetic modelling of electronic components and systems, for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes.",
"A test machine of the commercial type is used for these measurements, by generating stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer.",
"The measurement phase is followed by a simulation phase during which the electric parameters used for modelling electronic components and systems are extracted."
],
[
"1.Process for the electromagnetic modelling of electronic components and systems, in particular for the extraction of certain electrical parameters, such as the static V-I characteristics and the input and output impedance, the output switching times in particular conditions of load and the transition times of the protection diodes of said electronic components and systems, to ports of these components and systems suitable stimulus signals are sent and the correlated signals are measured, reconstructing the respective time patterns, characterised in that a test machine of the commercial type is used for generating the stimulus signals and measuring the correlated signals, the test machine being suitable for parametric direct current measurements, functional tests and digital integrated circuit timing and also being used as a time domain reflectometer, in which case the test machine sends said stimulus signals to the component or system inputs or outputs, fitted on a suitable support and connected by means of suitable interconnection channels, and detects the reflection due to impedance offset, providing the respective time patterns, which are then compared with those provided by a simulated measurement system model, comprising the test machine signal generation and measurement part, the interconnection channels and the support, to verify the coincidence between the measure time pattern and that provided by the simulated model obtained by setting different values with respect to the electric parameters to be determined.",
"2.Process according to claim 1, characterised in that the model simulated by the measurement system is obtained by the reflectometer response of each channel of the test machine in the situation in which the support of the component or system is not connected to the machine.",
"3.Process according to claim 1, characterised in that the reflected signals are reconstructed by means of the periodical sampling method, by fine tuning the sampling pulse phase and operating on the comparison threshold of the measurement part of the test machine to identify the succession of values assumed in time by the reflected signal, so as to obtain the time pattern.",
"4.Process according to claim 1, characterised in that for measuring the electronic component and system input and output impedance, a variable load impedance is introduced in the simulated model, so as to obtain various patterns of the reflected signal, following the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated impedance value corresponding to the impedance of the input or output to be determined.",
"5.Process according to claim 1, characterised in that for measuring the switching time of the outputs or transition time of the protection diode, a variable time is introduced in the simulated model, so as to obtain different patterns of the reflected signal, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation, the simulated switching or transition time corresponding to the value to be determined."
],
[
"This invention relates to measurement methods for evaluating electromagnetic compatibility, and particularly relates to a process for the electromagnetic modelling of electronic components and systems.",
"With the increased level of integration and operating frequency of electronic components and systems, the need to check the electromagnetic compatibility between different parts of any one system, or between different systems in conditions of mutual interference, and the integrity of signals exchanged in electronic systems between the various components is becoming increasingly important.",
"The integrity of signals is particularly important in digital systems, where the treated signals are essentially binary signals.",
"For the good operation of systems, these signals must satisfy certain minimum quality requirements.",
"Particularly, two voltage levels, corresponding to bit 1 and 0, and the transition times between the levels must be preserved.",
"Furthermore, interference between adjacent conductors, skin effect attenuation and spurious signals originated from reflection of the original signals in the presence of discontinuous impedance must be minimised.",
"In digital systems, the increase in signal switching front steepness and dock frequency increases the intensity of the electromagnetic field radiated by the various component parts, such as conductors and active and passive components, with the consequent possibility of exceeding the limits established in the emission standard.",
"As a consequence, the device may not be type-approved or, consequently, marketed.",
"These problems can be obviated by an accurate system design, which accounts for the electromagnetic performance of the various components employed.",
"The validity of design can be verified before system construction by exploiting the possibilities offered by simulation programs readily available on the market.",
"Electromagnetic compatibility data, which can be provided by such programs, will be closer to reality the more the component and system models employed for describing the entire device to be simulated are accurate.",
"Standard model definition processes have existed for several years.",
"These processes guide technicians in identifying the parameters of the model and describing the model Of such processes, the most common is that proposed by the IBIS (Input-output Buffer Information Specification) Association, which consists in describing the static V-I characteristics of all component inputs and outputs, the output transition times in particular conditions of load, the parasite parameters associated to all inputs and outputs, such as the capacitance with respect to a reference potential, and other parameters.",
"At this time, there are not many available electronic component models.",
"The few available models essentially refer to integrated circuits and are widely incomplete and not very accurate.",
"These models are obtained by simulation employing known methods (SPICE), using technological parameters, in some cases provided by the manufacturers, or by using the scarce information provided by component data sheets.",
"In other cases, the models are made by means of measurements using single instruments, with considerable waste of time for preparing the bench and for making settings and measurements.",
"The process illustrated in this invention obviates these disadvantages and solves the technical problem described above by allowing automatic electronic component and system modelling using normal electronic test machines in an original way, without requiring costly modifications.",
"All access points of the device under test can be characterised rapidly also when dealing with very complex stimulation signals.",
"The test machine can be used in a conventional way for conducting functional tests or, according to this invention, for modelling in the same test session, without the need for additional manual interventions on behalf of the operator.",
"Specifically, object of this invention is a process for the electromagnetic modelling of electronic components and systems the characteristics of which are described in claim 1.This invention will be better explained by the following detailed description with reference to the accompanying drawings as non-limiting example, wherein: FIG.",
"1 is a flow chart illustrating a typical measurement session; FIGS.",
"2 and 3 are flow charts illustrating the process for conducting the measurements by means of the test machine; FIG.",
"4 is a flow chart illustrating the process which is used to extract the output switching time and the capacitance of the input or output ports of the component or system under test.",
"The process for the electromagnetic modelling of electronic components and systems employs a test machine of the commercial type, designed for conducting parametric measurements in direct current, functional tests and digital integrated circuit timing.",
"The characteristics required for the machine to be used in this process are: the possibility of generating digital signals with sufficiently steep switching fronts, typically lower than 300 ps; impedance controlled along all interconnections in the device under test; possibility of monitoring all signals output by the device under test by sampling, with time resolution lower than 300 ps.",
"Various types of such machines are made by various manufacturers, e.g.",
"the machine identified by code ITS9000 IX, made by Schlumberger.",
"In normal use for integrated circuit functional tests, signals with a suitable pattern are sent to the device inputs and the output signals are measured, checking that such signals are as required in a precise instant of the clock cycle.",
"The sampling method is used to measure the signals.",
"Specifically, at input port level transition, the signal at the output port is compared with a pre-programmed reference threshold and the time instant in which the threshold was exceeded is recorded.",
"By using a sufficiently high sampling frequency, the signal can be closely reconstructed.",
"A subsequent study can be conducted to evaluate the response of the device, checking that the parametric, functional and timing characteristics, the output signal time delays and the input and output signal threshold values are within the tolerance range permitted in specifications.",
"The test machine according to this invention is used in a different way with respect to that described above: it is used as a time domain reflectometer, i.e.",
"the test machine is used to send specific signals to the component ports and the reflections due to impedance offset are measured.",
"This method is not exploited in normal test machine use.",
"Only in the initial machine channel calibration and characterisation phase, a signal is sent to the device support and the delay of the corresponding reflected signal is measured to determine the delay introduced by the device interconnection channel.",
"The consequent compensations can be implemented, if required.",
"However, the time pattern of the reflected signal is not studied in detail, to the extent that the integrated circuit under test is not present: in other words, the conditions are those of total reflection.",
"By extending the possibility of reconstructing signals, also to involve the signals reflected by the access ports of the device under test, the respective electric parameters can be obtained.",
"These are essential for the electromagnetic modelling of the entire device according to the IBIS standard process mentioned above.",
"In detail, the following parameters can be obtained: the static voltage-current (V-I) characteristics at inputs and outputs; the output switching times in the specific conditions of load; the input and output capacitance; the tripping times of the protection and clamp diode circuits, normally present in input ports; various parasite parameters.",
"For the use according to this invention, the stimulus signals generated by the test machine must be sufficiently steep and present a sufficient amplitude, so as to allow high frequency measurements, preserving a good signal/noise ratio.",
"For example, to measure the typical input capacitance of a digital integrated circuit, an amplitude of approximately 1 V is conveniently selected for obtaining a transition time of approximately 400 ps, which suitably adapts to the amplitude resolution of the test machine comparators.",
"The signal offset can be set at a suitable level for the device, e.g.",
"2.5 V for an integrated circuit powered at 5 V. Since impedance offset is measured, naturally the output impedance of the test machine must be controlled and fixed at a suitable value, e.g.",
"50 Ohms, as the impedance of all the interconnections between the generator and the access port of the device.",
"As mentioned above, the reflected signals are reconstructed using the periodical sampling method.",
"The sampling input phase can be very finely tuned, e.g.",
"with a resolution of 20 ps.",
"After stablising the sampling phase, the comparison threshold is used to identify the internal comparator switching value, by using a suitable search algorithm, eg.",
"dichotomy, linear etc., according to the morphology of the signal.",
"After reconstructing the pattern of the reflected signal, it is compared with the pattern obtained by simulating the measurement configuration in the time domain with a circuit simulator.",
"The simulated model must include the same stimulus signal, the same impedance and the same interconnection channel delay and a variable load impedance, which is the unknown quantity to be determined.",
"By varying the simulated impedance parameters (e.g.",
"capacitance), various reflected signal patterns are obtained, given the same stimulus signal, to find the pattern which best reproduces the measured pattern by subsequent approximation.",
"The value of the simulated impedance parameters thus corresponds to that associated to the device port.",
"Other parasite effects in the interconnection channel between generator and device, due to discontinuous impedance between support and connection cable, between support and device, etc., or loss due to skin effect in the cable and in the support, which are intrinsic to the test machine, can be accounted for in the model used for simulation and consequently compensated.",
"A similar process can be applied to the measurement of protection circuit and clamps on device access ports, by suitably arranging the voltage levels of the stimulus signal.",
"For example, to characterise the protection circuit at input of an integrated circuit to the earth terminal, a stimulus signal passing from 0 V to −1 V and then from −1 V to 0 V can be used.",
"By measuring the waveform reflected by the diode, which firstly conducts and then cuts off the stimulus signal, the characteristic transition time of the diode can be determined.",
"The subsequent approximation process herein described can also be applied to more conventional types of measurements, i.e.",
"measurements based on the use of the test machine as a stimulus signal generator and respective output signal sampler, instead of as a time domain reflectometer.",
"The component is programmed, by means of the test machine, so to generate repetitive output signals, which can be measured by means of the sampling procedure.",
"The measured waveforms are not those of the component under test but those effected by the distortion introduced in the entire measurement system, consisting of the machine, the interconnections, the physical component support, etc.",
"Consequently, a system model is required, for simulating the measurement providing the output waveform to be compared with those resulting from the measurements.",
"In this case, in the model used for the simulation, the unknown quantity to be identified consists of the intrinsic switching times of the device.",
"A sequence of increasing switching times within a predefined range is set in the model for each output port so that the simulated waveform output by the measurement system model better approximates the measured waveform.",
"The set switching time leading to the best approximation of the waveforms, is the unknown quantity sought.",
"The entire procedure, comprising the measurement phases, the simulation phases and the comparison phases, can be easily automated by implementing a specific test machine management and result processing application.",
"A typical measurement session according to the process of this invention is illustrated in the flow chart in FIG.",
"1.Test device 101, with respective data sheet 102 and JTAG data 103, where relevant, form the starting data of phase 100.As known, JTAG is a standard according to which some component pins are dedicated to functional tests.",
"For example, thanks to this standard, some parts of the device, deemed particularly critical, can be isolated and individually operated, during testing or trouble-shooting.",
"Consequently, the test support is prepared during phase 104.The test support is specific for the device under test and suitable to the test machine.",
"The device is fitted in phase 105.According to the type of parameters to be extracted, suitable stimulus signals 106, to be used in the test machine in phase 107 for the measurements, must be prepared.",
"According to the resulting measurements, the device input and output voltage-current static characteristics are extracted in phase 108, the output switching times in certain conditions of load are extracted in phase 109, and the input or output port capacitance reactance is extracted in phase 110.Particularly, the switching time measurements are made according to the measured waveforms and the capacitance on the basis of reflected signals.",
"During the subsequent phase 111, the IBIS model containing all the measurement parameters is finally created.",
"The measurement process implementing the test machine, indicated with numeral 107, is illustrated in greater detail in FIGS.",
"2 and 3.In FIG.",
"2, the various operations, indicated with numerals 201, .",
".",
".",
"206, consist in: measuring the input static characteristics; output conditioning by means of JOAG functions; measuring output static characteristics; generating and measuring input reflectometer signals; output conditioning by means of JTAG functions; measuring the output fronts.",
"The reflectometer and output waveform front measurement process is illustrated in FIG.",
"3.In phase 301 “Generate one input or output waveform front”, the test machine controls the device under test.",
"If measurements are carried out on the output signals, the machine programs the component to switch the outputs.",
"In the next phase 302, the starting point of the switching front is established.",
"This determines the start instant of the level measurement.",
"This measurement is conducted in phase 303, by comparing the signal with a variable threshold by means of a machine comparator to check correspondence of values.",
"Consequently, the measurement instant is increased by one time unit defined in phase 304 and, if the measurement time window is not over because the switching transient is not complete, the cycle is repeated to measure the new signal level in phase 305.The succession of levels obtained, which reproduces the pattern of the waveform, is finally stored in a file during phase 306.During reflectometer measurements, the output signal of the circuit under test is no longer considered in phase 301.In this case, the signal is reflected on an input port The process is similar and the result is the pattern of the reflected signal.",
"The process in which the output switching times and the input and output port capacitance, indicated with numerals 109 and 110, are extracted is illustrated in greater detail in FIG.",
"4.In phase 410 “setup model”, an electric simulator model representing the measurement configuration is created, specifically including the interconnection cables with respective loss and impedance discontinuity, where relevant, the physical support and the stimulus signals.",
"This model can be created on the basis of the reflectometer response obtained from the test machine in the configuration in which the support plate of the device under test is not inserted and is specific for each measurement channel available on the machine.",
"Said responses contain all the information (characteristics impedance levels, propagation delay, etc.)",
"required for the description by means of circuit simulation primitives.",
"The circuit model completed in phase 402 can be obtained by connecting the measurement system model to the device model containing one or more circuit parameters on circuit simulation level (for example capacitance, switching time, etc.)",
"which value is to be extracted.",
"In phase 402, the model is updated with a first hypothesis of parameter, for example the switching time, which is then simulated in phase 404 and compared with the measurements provided by the test machine in phase 405.If the error exceeds a predefined threshold, the parameter is changed in phase 403 and the approximation cycle is repeated to obtain the minimal discrepancy between the measurement and the simulation.",
"Finally, the resulting parameter is used for constructing the IBIS model.",
"The description herein is provided as a non-limiting example and obviously variations and changes are possible within the scope of protection of this invention."
]
] | Patent_10297634 |